Penggunaan Pgfpuv Mutan Untuk Mempelajari Pengaruh Pengeringan Pada Ketahanan Hidup Cronobacter Sakazakii Dalam Jagung.

USING pGFPuv MUTANTS TO STUDY THE INFLUENCE
OF DRYING ON THE SURVIVAL OF Cronobacter sakazakii IN
MAIZE

ALAELDIN MOHAMMED AHMED MUSA

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

STATEMENT OF ORIGINALITY
I hereby declare that this thesis entitled “USING pGFPuv MUTANTS
TO STUDY THE INFLUENCE OF DRYING ON THE SURVIVAL OF
Cronobacter sakazakii IN MAIZE” and the work reported herein were composed
by and originated entirely from me and my supervisors. I there declare that, this is
a true copy of my thesis as approved by my supervisory committee and has not
been submitted for a higher degree to any other University or Institution.
Information derived from the published and unpublished work of others has been
duly acknowledged in the text as well as references given in the list of sources and
in view of this, I therefore confer the copyright of this thesis to Bogor Agricultural

University.
Bogor, October 2015
Alaeldin Mohammed Ahmed Musa
Registration No: F251138501

RINGKASAN
ALAELDIN MOHAMMED AHMED MUSA. Penggunaan pGFPuv Mutan
Untuk Mempelajari Pengaruh Pengeringan Pada ketahanan hidup Cronobacter
sakazakii Dalam Jagung. Dibimbing oleh RATIH DEWANTI- HARIYADI dan
ELVIRA SYAMSIR.
Cronobacter sakazakii adalah bakteri patogen emerging penyebab
penyakit meningitis dan necrotizing enterocolitis pada kelompok bayi tertentu.
Beberapa C. sakazakii telah diisolasi dari sumber pangan di Indonesia dan
transformasi dengan plasmid yang menyandi Green Fluorescent Protein (GFP)
telah menghasilkan mutan C. sakazakii pGFPuv dengan pola pertumbuhan serupa
dengan galur wild type-nya.
Penelitian ini bertujuan untuk memanfaatkan C. sakazakii pGFPuv untuk
mempelajari pengaruh pengeringan terhadap kadar air dan aktivitas air jagung
serta sintasan (survival) C. sakazakii pada jagung. Jagung pipil dengan kadar air
40% (b.k) diinokulasi dengan C. sakazakii pGFPuv sehingga konsentrasi awalnya

CFU/g dan dikeringkan pada suhu 42 ºC, 46 ºC dan 50 ºC selama sepuluh
hari. Setiap hari, sampel jagung diambil untuk diukur kadar airnya dengan
menggunakan metode oven, aktivitas airnya dengan Aw meter, serta dienumerasi
Total Plate Count dan C. sakazakii yang bertahan dengan metode pemupukan agar
cawan. Disamping itu, sampel terpilih dari pengeringan 50oC diamati dengan
Scaning Electron Microscopy (SEM).
Tingkat sintasan C.sakazakii selama pengeringan ditentukan dari
kemiringan regresi linier kurva sintasan C.sakazakii. Hasil penelitian
menunjukkan bahwa jumlah C. sakazakii turun dengan cepat pada fase laju
penurunan k.a/aw cepat dan lebih lambat pada laju saat penurunan ka/Aw lambat.
Pada fase lambat, C. sakazakii cenderung lebih mampu bertahan dibandingkan
dengan total bakteri.. Aw kritis untuk bakteri patogen adalah antara 0.85-0.86.
Pengamatan dengan SEM menunjukkan bahwa C. sakazakii pGFPuv
membentuk koloni pada permukaan dan pada bagian tip cap jagung. Penelitian
ini menunjukkan bahwa mutan C. sakazakii pGFPuv dapat digunakan untuk
mempelajari sintasan C. sakazakii selama pengeringan jagung. C. sakazakii dapat
bertahan pada pengeringan jagung suhu 42 ºC, 46 ºC dan 50 ºC selama sepuluh
hari.
Kata Kunci: Cronobacter sakazaki, pGFPuv, jagung, sintasan, pengeringan,
aktivitas air.

SUMMARY
ALAELDIN MOHAMMED AHMED MUSA. Using pGFPuv Mutants To Study
The Influence Of Drying On The Survival Of Cronobacter sakazakii In Maize.
Supervised by RATIH DEWANTI- HARIYADI and ELVIRA SYAMSIR.
Cronobacter sakazakii is an emerging Gram-negative foodborne bacterial
pathogen regarded as the causative agent of meningitis and necrotizing
enterocolitis in certain groups of infants. Several isolates of C C. sakazakii have
been obtained from food samples in Indonesia and transformation with a Green
Fluorescent Protein (GFP) plasmid has produced C. sakazakii pGFPuv mutants
with growth pattern similar to that of the wild-type strains. Therefore, they are
potential to be used for studying C. sakazakii behaviour without the need to
suppress other microorganisms and the use of diagnostic media.
The research activities focused on the following objectives : to study the
effect of drying on water content and water activity of maize and to evaluate the
survival of C. sakazakii pGFPuv as well as other naturally occurring
microorganisms in maize during drying. Maize kernels with moisture content of
40% (d.b) were inoculated with C. sakazakii pGFPuv mutant such that the initial
CFU/g and drying was performed at
concentration in the maize was

temperatures of 42 ºC, 46 ºC and 50 ºC for ten days in a drying chamber.
Every day the maize samples were taken and the water content were
analyzed using oven method while water activity by Aw meter. Total Plate Count
and C. sakazakii surviving in the maize were enumerated by standard plate count.
The survival rate of C. sakazakii during drying was determined by the slope of
linear regression from C. sakazakii survival curve.
The results showed that the fate of decrease of C. sakazakii during the rapid
decline of water content and Aw can be divided into three phases, i.e. logarithmic
decrement, static growth and the final decline. The critical water activity for these
pathogenic bacteria was in the range of 0.85-0.86. Observation by SEM showed
that C. sakazakii GFPuv form colonies on the surface of corn and the tip cap. This
study showed that of C. sakazakii pGFPuv can be used to study the survival of C.
sakazakii during corn drying.
Keywords: Cronobacter sakazaki, pGFPuv, maize, survival, drying, water
activity

© Copyright, owned by IPB, 2015
All rights reserved
No part of this document may be reproduced or transmitted in any form or
by any means, electronic, mechanical, photocopying, recording, or otherwise,

without prior written permission from Bogor Agricultural University (IPB)
No part of this document may be reproduced or transmitted in any form
without prior written permission from Bogor Agricultural University (IPB)

USING pGFPuv MUTANTS TO STUDY THE INFLUENCE
OF DRYING ON THE SURVIVAL OF Cronobacter sakazakii IN
MAIZE

ALAELDIN MOHAMMED AHMED MUSA

A Thesis
Submitted in partial fulfillment of the requirements for the degree of
Master of Science
In
Food Science

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

External examiner: Dr. Siti Nurjanah, STP, M.Si

Approved by,
Supervisory Committee

Endorsed by,

ACKNOWLEDGEMENT
First and foremost I praise and acknowledge Allah, the most beneficent and
the most merciful. Secondly, my humblest gratitude to the Holy Prophet
Muhammad (Peace be upon him) whose way of life has been a continuous
guidance for me. I would like to express my deepest gratitude to my advisors, Prof.
Dr. Ratih Dewanti - Hariyadi and Dr. Elvira Syamsir as well as Dr. Siti Nurjanah
as an external examiner whose invaluable supervisions, guidance, caring,
patience, and providing me with an excellent atmosphere for doing research.
I also wish to acknowledge the Government of Indonesia through the
Ministry of Education and Culture (DIKTI) for the grant of scholarship. I also
wish to thank the all staff Department of Food Science and Technology, Faculty
of Agricultural Technology, Bogor Agricultural University (IPB). I would like to

thank the financial support of this research, which was provided through Riset
Unggulan Nasional scheme from the Directorate General of Higher Education,
Ministry of Education, Republic of Indonesia.
I further wish to thank all colleagues in the Department of Food Science and
Technology (IPN 2012, 2013) Bogor Agricultural University for the contributions
and help. Assistance from the laboratory experts of Southeast Asian Food and
Agricultural Science and Technology (SEAFAST) Center, Bogor Agricultural
University.
I also wish to acknowledge the Government of Sudan, Ministry of Higher
Education and Scientific Research through the Omdurman Islamic University. I
am very grateful for this prestigious opportunity given me to study for my
master‟s degree.
Furthermore, I would like to express my profound love and special thanks to
my loving parents; Mr. Mohammed Ahmed and Mrs. Mofidah Abdellah for their
constant pieces of advice, guidance and prayers.
Finally, I appreciate each person that directly and/or indirectly contributed
to the completion of this work piece. Indeed, you have all played significant roles
to this work and may God richly bless you all.
Bogor, October 2015
Alaeldin Mohammed Ahmed Musa

TABLE OF CONTENTS
LIST OF TABLE

vi

LIST OF FIGURE

vi

LIST OF APPENDICCES

vi

1 INTRODUCTION
Background
Problem Statement
Research Objective
Benefits of Research
Research Hypothesis

1
1
2
2
3
3

2 LITERATURE REVIEW
Drying
Maize
Cronobacter Sakazakii
The Growth of C.sakazakii
Contamination Source of C.sakazakii
Diseases Caused by C. sakazakii
Infantile or Neonatal Infection
Necrotizing Enteroclititis (NEC)
CNS Infection
C. sakazakii Thermal Resistance
C. sakazakii Resistance to Dry Condition

Accumulation of Trehalose
Capsule Formation
Oxidative Stress

3
3
4
5
6
6
7
7
7
8
8
9
11
11
11

3 MATERIALS AND METHODS
Place and Time of Research
Materials
Equipment
Methods
Confirmation of C. sakazakii pGFPuv Mutants
Preparation of Maize
Inoculum Preparation
Inoculation
Drying
Enumeration of C. Sakazakii
Enumeration of TPC
Analysis of Water Content
Analysis of Water Activity
SEM Observation
Statistical Analysis

13
12
12
12
12
14
14
14
14
14
14
15
15
15
15
15

4 RESULTS AND DISCUSSION
Changes in Water Content of Maize During Drying
Changes in Water Activity of Maize During Drying
Changes in Total Plate Count of Maize During Drying
Survival of C.sakazakii During Drying
Colonization of C.sakazakii on the Surface of Maize
Penetration to the Tip Cap of Maize

16
16
17
17
18
20
21

5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation

22
22
22

REFERENCES

23

APPENDICES

29

LIST OF TABLES
1 Cronobacter sakazakii isolated from Indonesia
2 D and Z values for the various strains of Cronobacter spp

6
9

LIST OF FIGURES
1
2
3
4
5
6
7
8
9

C.sakazakii
The drying chamber for maize
The flow diagram of research
Changes in water content of maize during drying
Changes in water activity of maize during dying
Changes in total plate count of maize during drying
C. sakazakii survival curve during drying
SEM observation Colonization of C. sakazakii on the surface of corn
SEM observation. Penetration of C. sakazakii in cavity of tip cap corn

5
12
13
16
17
18
19
21
21

LIST OF APPENDICES
Appendix 1 Change in water content of maize during drying (wb)

29

Appendix 2 Change in water activity of maize during drying

30

Appendix 3 Change in TPC of maize during drying

31

Appendix 4 C.sakazakii isolate FWHc3 during drying

32

Appendix 5 C.sakazakii isolate E2 during drying

33

Appendix 6 Change in water activity ( liner regression)

34

Appendix 7 The maize field

34

Appendix 8 Inoculum preparation

34

Appendix 9 Enumeration of C.sakazakii

34

1 INTRODUCTION
Background
Drying is one of the main techniques for preserving agricultural and food
product and takes place in the processing of many products, as the main operation
or as a consequence of other processing step. Drying of a food material occurs
when water vapour is removed from its surface into the surrounding space,
resulting in a relatively dried form of the material (Krokida et al. 2003). It can be
as an industrial preservation method in which water content and activity of fruits
and vegetables are decreased by heated air to minimize biochemical, chemical and
microbiological deterioration (Doymaz & Pala 2003). The objective of drying in
food products is to remove the water content to a certain level, at which microbial
spoilage is greatly minimized. C.sakazakii, however, was shown to have a
remarkable capability to survive in a dry environment for a long time period
(Krokida et al. 2003).
Cronobacter spp. (formerly Enterobacter sakazakii) is a Gram-negative,
rod, motile, non-sporulating, bacterium with peritrichous flagella. Cronobacter
spp. was recently proposed to consist of six genomospecies (Iversen et al. 2008)
and they are regarded as emerging opportunistic human pathogens. The bacteria
are the aetiological agents of life-threatening bacterial infections in low birthweight neonates and infants. The clinical presentation includes meningitis, brain
abscess, bacteraemia and necrotizing entercolitis (Mullane et al. 2006). The
International Commission of Microbiological Specifications for Foods has ranked
this pathogen as a „severe hazard for restricted population‟ life threatening or
substantial chronic sequelae or long duration (ICMSF 2002). Cronobacter spp can
be found in environment, and has been isolated from a variety of foods such as
UHT milk, cheese, meat, vegetables, grains, sorghum, rice, herbs, spices,
fermented bread, fermented drinks, tofu, and sour tea (Iversen & Forsythe 2004;
Iversen et al. 2004).
Powder Infant Formula (PIF) has been the only food epidemiologically
linked to the cases of infant infections with C. sakazakii. However, C. sakazakii
has been isolated from a variety of foods such as UHT milk, cheese, meat,
vegetables, grains, sorghum, rice, herbs, spices, fermented bread, fermented
drinks, tofu, and sour tea (Iversen & Forsythe 2004; Iversen et al. 2004). An
international survey of dry infant formula from 35 countries found that
approximately 14% of the 141 cans examined had detectable levels of C.
sakazakii (Edelson-Mammel & Buchanan 2004). Survival of C. sakazakii in such
a dry environment largely depends on the osmotic or dry stress resistance of the
bacteria and the dried infant formulas has low water activity (Aw). In case of
drying, which can be seen as an extreme form of osmotic stress, the cells need to
preserve their biological integrity in the absence of liquid water (Breeuwer 2003).
C. sakazakii is known to survive for at least two years in powdered infant formula
at Aw as low as 0.2 (Beuchat 2009; Adekunte et al. 2010).
Ivesen and Forsythe (2004) isolated C. sakazakii from corn flour;
Restaino et al. (2006) isolated it from grits, while Dewanti-Hariyadi et al. (2010)
isolated it from corn starch. Maize and maize product are commonly used as

2
ingredients in various foods including infant formula and common food for
weaning infants at the age of 4–6 months. In Indonesia, C. sakazakii has been
isolated from several PIF, weaning food and corn starch (Gitapratiwi et al. 2012).
Studies on the behavior of C. sakazakii in food using wild type bacteria
has been found to be difficult because target organisms are indistinguishable from
naturally occurring microorganisms. In maize for example, various moulds
(Aspergillus, Fusarium, Penicillium, Rhizopus), yeasts (Kodamaea and Candida),
and bacteria (Pediococcus & Lactoobacillus) (Rahmawati et al. 2013). Labelling
techniques become an alternative to study the behaviour of these bacteria without
killing or suppressing the growth or killing other microbes.
This research uses C. sakazakii labeled with plasmid containing Green
Fluorescent Protein (pGFPuv), which produces green fluorescent colony under
ultraviolet light. A number of studies involving GFP-labeled strains of bacteria
have revealed that GFP expression does not alter the biochemical, morphological,
or survival characteristics of the labeled bacteria ( Bloemberg et al .1997)
Ma et al (2011) reported that E.coli O157:H7, Salmonella and Listeria
strains can be effectively labeled with the plasmid-borne gfp gene which, in
certain isolates, can be stable for many generations without adversely affecting
growth rates. C.sakazakii pGFPuv mutant was reported to have a growth curve
similar to that of the wild type (Nurjanah et al. 2013). Green Fluorescent Protein
(GFP) is a protein aequorin from the jellyfish ( Acquorea victoria ) that can
fluoresce (Nifosi et al. 2005). This protein was first isolated in 1961 by
Shimomura (2005) and then developed by several researchers. Inserted plasmid is
expressed in both eukaryotic and prokaryotic on the host (Ehrenberg 2008).
Problem Statement
Cronobacter sakazakii is a group of gram-negative bacteria that exists in the
environment and which can survive in very dry conditions. The natural habitat for
Cronobacter is not known. It has been found in a variety of dry foods, including
powdered infant formula, skimmed milk powder, herbal teas, and starches. It has
also been found in wastewater. C. sakazakii illnesses are rare, but they are
frequently lethal for infants and can be serious among people with immune
compromising conditions and the elderly. As early stages, this research conducted
searches C. sakazakii in maize, through the study of survival during drying, the
ability of these bacteria to colonize and penetration the maize.
Research Objectives
The general objective of this research to understand the behavior of
Cronobacter sakazakii pGFPuv during maize drying.The specific objective of
this study therefore are;
1. To study the effect of drying on water content and water activity of maize.
2. To evaluate the survival of C.sakazakii pGFPuv and other naturally occurring
bacteria /microorganism in maize during drying.

3
Benefits of Research
1. Obtaining information about the effect of drying on water content and water
activity.
2. Provides information about the survival, colonization and penetration capability
of C.sakazakii during maize drying for control application.
3. C.sakazakii isolates labeled with GFP mutant can be applied to studying the
behavior of these bacteria in other foodstuffs beside maize.
Research Hypothesis
1. Moisture content and water activity affect the survival of C.sakazakii
pGFPuv during drying.
2. C.sakazakii pGFPuv can survive during maize drying process.

2 LITERATURE REVIEW
Drying
Drying is one of the oldest methods of food preservation. Drying
preserves foods by removing enough moisture from food to prevent decay and
spoilage. Water content of properly dried food varies from 5 to 25 percent
depending on the food. Successful drying depends on enough heat to draw out
moisture without cooking the food, dry air to absorb the released moisture and
adequate air circulation to carry off the moisture. When drying foods, the key is to
remove moisture as quickly as possible at a temperature that does not seriously
affect the flavor, texture and colour of the food. If the temperature is too low in
the beginning, microorganisms may survive and even grow before the food is
adequately dried. If the temperature is too high and the humidity too low, the food
may harden on the surface. This makes it more difficult for moisture to escape and
the food does not dry properly (Chen 2009).
In general, drying refers to the removal of moisture from solids, gases or
liquids. For drying gases and liquids, adsorption is normally used. The food
technology industry is an example of where drying solids on a large scale is
important. Thermal drying of solids involves removing moisture from
the material by vaporisation or evaporation. The drying characteristics depend
on how the moisture is retained within the material. In the first instance,
the liquid adhering to the surface of the material to be dried can be
removed by vaporisation or evaporation. Once this liquid has been
removed, drying of the moisture contained within the capillaries and pores
of the material begins. The drying speed reduces due to the need to
overcome capillary forces and diffusion resistance. Crystal water which is
bonded into the crystal structure of the material, can only be removed by
intense heating in addition to low drying speed (Chen & Mujumdar 2009).
In the global food market, plant based food materials hold a significant
proportion, and numerous research are being conducted to improve new food

4
products and efficient processing techniques, food drying is used to process about
20% of the world‟s perishable crops and is therefore can be considered as one of
the key plant food processing techniques (Grabowski et al. 2003). Since plant
food materials usually contain very high moisture, even up to 90% by weight, are
highly subjected to spoilage (Karunasena et al. 2015).
Powdered Infant Formula is the result of processing preservation by
reducing the water content of 87% milk (fresh milk) to 3% ( powder milk ),
Production of powdered infant formula using different processes, among others,
dry procedure, wet procedure, or a combination of both. In the dry procedure,
skim milk is pasteurized and then evaporated. All materials ingredient the fat,
whey, vitamins, emulsifiers and stabilizers are then added and mixed.
The mixture is then pasteurized at 110° C for 60 seconds after it
do spray dryer while The wet form of the mixing procedure carried out in wet
conditions before drying so that the liquid skim milk, skim milk before mixing,
as well as the fat component is treated at 80-82° C for 20 seconds and then
the mixture was heated at 107-110 ° C for 60 seconds and the liquid mixture was
concentrated by falling film evaporator. Concentrate was treated with heat
back at a temperature of 80° C and a final spray drying.
PIF has been fed to millions of infants for years, and it constitutes the
majority of infant formula used worldwide. This product is formulated to mimic
the nutritional profile of human breast milk. Because PIF is not a sterile product, it
is an excellent medium to support bacterial growth. Bovine milk is an essential
ingredient of PIF and a potential source of bacteria that are pathogenic to humans
(Drudy et al. 2006).
Maize
Maize is an important staple food in many countries and the productions
of maize in the world have been increasing steadily. Maize has a wide variety of
usages, ranging from food and feed to industrial products, and more recently, as
an alternate fuel. The area planted with maize is continuously increasing over the
years. Indonesia is the sixth largest maize producer in the world, contributing 2%
to the global production. Maize is grown almost all year round, during both rainy
season and dry season in irrigated land. In a farming system, maize has changed
its status from a companion catch crop toa main cash crop. Maize is one of the
main agricultural products in Indonesia, is an important industrial raw material in
the starch industry. Traditionally fruits, vegetables and cereals such as grape, red
pepper, apricot and maize are dried in an open atmosphere by exposing them to
the sun. During this period, the product may get contaminated due to soil, sand
particles and other matter in the drying environment (Doymaz & Pala 2003).
FAO-WHO (2004) survey results indicate that the contamination of C.
sakazakii in powdered milk ingredient mostly from starch. This reinforces the
notion that bacterial contamination in powder milk can be derived from ingredient.
Currently corn is used as one of the manufacture of products
Dry food. Some corn-based dry food products are reported to be contaminated
Cronobacter spp. Ivesen and Forsythe (2004) has isolated C.sakazakii from corn
flour; Restaino et al. (2006) isolated from grits, while Dewanti-Hariyadi et al.
(2010) isolated from corn starch.

5
Cronobacter sakazakii
Cronobacter sakazakii is a Gram-negative, rod, motile, non-sporulating
bacterium with peritrichous flagella (Iversen et al. 2008). The bacteria are the a
etiological agents of life-threatening bacterial infections in low birth-weight
neonates and infants (Mullane et al. 2006). Cronobacter sakazakii has been
identified as an infrequently isolated opportunistic pathogen based on neonatal
illnesses associated with contaminated powered infant formula (PIF).
Cronobacter spp. formerly known as Enterobacter sakazakii, was first called
“yellow pigmented Enterobacter cloacae” by Pangalos in a case of septicemia in
an infant in the late 1929.

Figure 1 C.sakazakii (SEM x4800) (Kunkel 2009).
Only after 1980, C. sakazakii was considered to be a distinct species and
was named in honour of the Japanese bacterial taxonomist and microbiologist
Riichi Sakazaki (1920–2002), who discovered a distinct yellow-pigmented variant
of Enterobacter cloaca. It has been implicated in outbreaks of neonatal illness
(premature infants), in isolated cases of severely immune compromised
individuals and in the elderly, but it rarely causes disease in healthy adults. More
than 120 cases of C. sakazakii related illness have been reported, and most are
presented as life threatening infections (FAO/WHO 2008).
Classification of the genus Cronobacter was proposed for revision in the
year 2007, based on a detailed polyphasic taxonomical approach; a method that
incorporates all available molecular, biochemical, morphological, and
physiological data into a consensus classification. Cronobacter sakazakii was
reclassified into the six species: C. sakazakii, C. malonaticus, Cronobacter
turicensis, Cronobacter muytjensii, Cronobacter dublinensis, and Cronobacter
genomospecies along with three subspecies of C. dublinensis, namely, dublinensis,
lausannensis, and lactaridi. Although frequently utilized, 16s rRNA gene
sequencing has been found not to be an ideal method of distinguishing C.
sakazakii and C. malonaticus, due to their close relatedness and since both of
these species are defined according their biotype – biotype 1. DNA–DNA
hybridization and biochemical tests reveal that C. sakazakii consists of 15
biogroups biotype 1 being the most common. Yellow-pigmented C. sakazakii
strains were only 41 and 54% homologous to non pigmented Citrobacter freundii
and E. cloacae, based on DNA–DNA hybridization data analysis (Iversen et al.
2007).

6
The Growth of Cronobacter sakazakii
Cronobacter sakazakii can grow on an agar medium selective for
organisms Enteric such as MacConkey, Eosin Methylene Blue, Deoxycholate
order, as well as on a selective chromogenic medium that is Druggan-ForsytheIversen (DFI). In addition Cronobacter sakazakii can be grown on non-selective
media such as Tryptic Soy Agar (TSA). Some broth selectively reported to inhibit
the growth for some strains of Cronobacter sakazakii. As much as 3 of the 70
strains obtained from various sources cannot be grown in broth lauryl sulfate or
broth brilliant green bile incubated at a temperature of between 7° C and 57 ° C
for 48 hours, although viability has been confirmed in Tryptic Soy Agar (Iversen
et al. 2004). The optimum growth of Cronobacter sakazakii affected by
temperature Cronobacter sakazakii able to grow in a temperature range between
8° C to 47° C (Kandhai et al. 2006). The optimum growth temperature between
37° C to 43°C dependent on the growth medium (Iversen et al. 2004).
Contamination Sources of Cronobacter sakazakii
C. sakazakii has been isolated from a wide spectrum of environmental
sources including water, waste, thermal spring water, soil, dust from households
and food production-lines, animals especially from birds, lizards, rats and piglets
(Freidemann 2007; Shaker et al. 2007). C. sakazakii has been also isolated from
a wide range of foods including ultra high-temperature treated milk (UHT milk),
cheese, meat, vegetables, grains, sorghum seeds and rice (Shaker et al. 2007).
Powder Infant Formula (PIF) is the only food epidemiologically linked to
the cases of infant infections with C. sakazakii. However, C. sakazakii has
isolated from local foods in Indonesia including infant formula, cornmeal cocoa
powder and corn starch (Dewanti-Hariyadi 2011; Gitapratiwi 2011; Estuningsih
2006; Meutia 2008). Table (1) shows the local isolats of C. sakazakii.
Table 1. Cronobacter sakazakii isolated from Indonesia
Isolation
Isolate
Sources
Codes/GenBank
Species
(From
Accession Number
Indonesia)
DES c7/JF800180
C.sakazakii Corn starch
Powdered infant
DES b10/JF800181
C.sakazakii
formulae
YR c3 a/JF800183
C.sakazakii Weaning Food
YR K2 a/JF800187
C.sakazakii Weaning Food
Powdered infant
YR t2a/JF800182
C.sakazakii
formulae
FWHb15
C.sakazakii Powder Sugar
FWH d2u
C.sakazakii Chili powder
FWH d 11
C.sakazakii Caraway powder
FWH b6
C.muytjensii Flour
FWH d16/JX535016
C.sakazakii Pepper powder
FWHc3
C.sakazakii Tapioca
E2

C.sakazakii

Weaning Food

Reference
Dewanti
Hariyadi
et al. 2010
Meutia et al.
2008

Hamdani 2012
Estuningsih et al.
2006

7
Skradal et al. (1993) states that Cronobacter spp. is one of bacterial
contaminants in milk cartons ultra high temperature (UHT), indirectly, these
microorganisms can survive temperatures as UHT or contamination after the
process. In a comparison of the D-values of several members of the
Enterobacteriaceae in dairy product, C. sakazakii appeared to be one of the most
thermotolerant organisms (Nazarowec-White & Farber 1997). An international
survey of dry infant formula from 35 countries found that approximately 14% of
the 141 cans examined had detectable levels of C. sakazakii (Edelson-Mammel &
Buchanan 2004).
Factors affecting growth of C. sakazakii in reconstituted infant formulae
have been studied by others. Nazarowec- White and Farber (1997) reported that
five-strain mixtures of clinical and food isolates had similar generation times in
three brands of infant formula powders reconstituted with water. Average
generation times were 40 min at 23° C and 4.98 h at 10° C. Iversen and Forsythe
(2004) reported a doubling time of 75 min for C. sakazakii in infant formula
stored at 21° C.
Diseases Caused by Cronobacter sakazakii
Infantile or Neonatal Infections
These organisms are regarded as opportunistic pathogens linked to lifethreatening infections predominantly in neonates (infants 4 weeks old) (Mullane
et al. 2006). Clinical presentation of Cronobacter infections in infants include
NEC, bacteraemia and meningitis, with case fatality rates ranging between 40 and
80% being reported (Friedemann 2007). Infections in older infants have also been
noted. The Food and Agriculture Organization of the United Nations/World
Health Organization statistics for 2008 estimated that the annual incidence rate in
the USA among low-birth-weight infants who weighed ,2500 g and were ,1 year
old was 8.7 per 100 000. Globally, there is no active surveillance system for
tracking this pathogen; however, in their 2008 report, the WHO expert panel
tracked cases from 1961 to 2008, and found 120 recorded cases of Cronobacter
among infants and children, 3 years old. Although only~120 cases have been
reported worldwide, the actual number of cases is considered far higher
(FAO/WHO 2008).
Necrotizing Enterocolitis (NEC)
It is generally thought that Cronobacter gain entrance to the human body
through the gastrointestinal tract (GIT) where they may cause NEC. Point out that
the development of NEC requires a susceptible host, typically a premature infant
with physiological impairment (i.e. hypoxia, hypothermia and intestinal
ischaemia), administration of enteral formula feeds (which lack the beneficial
protective components normally found in breast milk) and uncontrolled bacterial
colonization. These conditions lead to an increased degree of mucosal
inflammation, which subsequently results in the production of high levels of host
inflammatory factors, including cytokines, nitric oxide, platelet-activating factor
and prostanoids, which further damage the apical GIT epithelium (Liu & Beuchat
2007).

8
CNS Infections
Once the organism has entered the systemic circulation, it has a tropism
toward the CNS, thus increasing the propensity to cause meningitis among lowbirth-weight neonates and infants, whilst causing bacteraemia or sepsis among
slightly higher-birth-weight infants or adults. Once the pathogen crosses and
enters the brain, it might cause ventriculitis and form cysts or brain abscesses,
which later may develop into hydrocephalus– a condition where excessive
accumulation of cerebrospinal fluid (CSF) in the brain occurs. The
disproportionate accumulation of CSF results in an abnormal enlargement of the
spaces in the brain called ventricles. The enlarged CSF-filled ventricles create a
situation where the balance between CSF production and absorption is disturbed,
which potentially leads to an increase of cranial pressure on the tissues of the
brain (Yan et al. 2012).
Cronobacter sakazakii Thermal Resistance
Thermal treatment of foods just prior to consumption has long been used
as a primary means of reducing the risks associated with foodborn pathogen. It
has been identified as a practical means of reducing the risk of C.sakazakii in
dehydrated infant formulas. The effective use of thermal treatments requires
accurate information on the heat resistance of the target microorganism; the
thermal treatment should be sufficient to inactivate the microorganism of concern
while minimizing the loss of nutrients. Assessment of the adequacy of heat can be
estimated based on log reduction of bacteria through the concept of D and Z value
(Edelson- Mammel 2004). The susceptibility of bacteria to heat at a
specific temperature is characterized by the value of D, the time (min)
required to obtain one log reduction (tenfold reduction) in the bacterial
population (Tang et al .2000), Z value was defined as the temperature change
needed to change microbial inactivation rate by a factor of 10( Toledo 2007).
Iversen et al. (2004) reported The D-values for C. sakazakii at 58° C was
2.4 min. The Z-value for the organism 5.7° C. C.sakazakii is one of the members
of the Enterobacteriaceae. The most heat resistant studies in tryptic soy broth
(TSB), infant formula, and phosphate buffer was concluded that not all strains of
Cronobacter spp. has the same heat resistance (Breeuwer et al. 2003; Iversen et al.
2004). Based on the D and Z value, Seftiono (2012) reported the kinetics of
inactivation of Cronobacter spp during the heating process in the infant formula,
the range of D & Z values of Cronobacter spp local isolates based on the study
done by Ardelino (2011) and Seftiono (2012) in the infant formula medium show
in Table 2.

9
Table 2. D value (min) and Z value (°C) for Cronobacter spp local isolates.
Media

Strains

Infant
Formulab

Infant
Formula c

50 °C

D value (min)

Z value

(min – Max)

(°C)

52 °C

54 °C

56 °C

58 °C

DES c13

48.03-117.65

17.92-49.50

16.39-20.12

6.06-11.36

2.55

5.04-5.69

DES b10

69.44-111.11

34.60-66.22

13.05-16.00

4.04-5.48

2.65

4.04-5.69

DES b7a

104.17-117.65

33.00-49.75

20.00-25.32

5.40-8.55

1.721.90

3.54-4.36

YR c3a

103.09-243.90

35.48-46.73

21.41-21.74

3.61-4.10

-

4.20-4.92

YR t2a

119.05-169.40

43.10-80.00

31.64-33.67

5.83

-

4.20-4.92

DES d7

68.97-79.36

49.50-64.93

16.92-22.07

3.45-3.73

-

4.49-4.58

E6

200-256.41

87.72114.94

26.81-30.55

8.79-9.73

ATCC 51329

104.17-172.41

68.03-83.33

13.64-17.24

9.0-9.0

3.043.55

4.18-4.31

-

4.54-5.14

ATCC 51329

n.t.

n.t.

8.66±0.16

4.10±0.16

1.39

5.65±0.2
3

YR ta2

n.t.

n.t.

3.61±0.12

1.34±0.03

0.90±0.
03

6.08±0.0
8

YR c3a

n.t.

n.t.

3.83±0.33

1.39±0.03

0.89±0.
02

5.8±0.43

E9

n.t.

n.t.

4.24±0.05

1.39

0.71±0.
05

5.58±0.0
2

a

n.t. not tested
From Seftiono 2012
c
From Ardelino 2011
b

C.sakazakii Resistance to Dry Conditions
C. sakazakii is known to survive at least two years in powdered infant
formula at low Aw 0.2 (Beuchat 2009; Adekunte et al. 2010). To establish
appropriate methods to control the cross-contamination, the survival behavior of
Cronobacter spp. on food contact surfaces is needed (Kuo et al. 2013). C.
sakazakii, initially at populations as low as 0.31 log CFU/g (2 CFU/g), can
survive at 4, 21, or 30° C for up to 12 months in infant cereals in an (Aw) range of
0.30–0.83. Very small numbers of C. sakazakii if present in infant cereals can
survive for up to 12 months in cereals exposed to conditions under which they
may be held during distribution and storage. Tolerance of the pathogen to these
conditions enhances the probability of its survival in cereals at the time of
reconstitution and feeding to infants (Lin & Beuchat 2007).
C. sakazakii are also resistant to osmotic and dry stress compared with
other strains of the Enterobacteriaceae group such as Salmonella and E.coli. The
good survival of dried C.sakazakii cells at elevated temperatures 45°C and the

10
capacity to grow up to 47°C illustrate that in warm and dry environments such as
in the vicinity of drying equipment in factories, the bacteria have a competitive
advantage when compared with other members of the Enterobacteriaceae. The
high tolerance to desiccation provides a competitive advantage for C. sakazakii in
dry environments, as found in milk powder factories, and thereby increases the
risk of post pasteurization contamination of the finished product (Breeuwer et al.
2003).
Dried infant formulas by their nature have a low water activity (Aw) 0.2.
As a result, for C. sakazakii to survive in such an environment, the bacterium
must possess osmotic and dry resistance mechanisms that allow the organism to
withstand these extreme conditions. Following a storage period at 25°C, analysis
of the cell population at day 46 showed the death of 1–1.5 log10 CFU/ml of
stationary phase cells. Reduction in cell numbers of the order of 1 log10 CFU/ml
after 100 days followed by continued reductions up to and including day 700.
Some C. sakazakii isolates appeared to be more resistant than others. When
trehalose was added to the medium, however, cells remained viable for a longer
period (Mullane et al. 2006). Studied by Ipan (2012) report that the Spray drying
at temperatures killed substantial numbers of C. sakazakii but did not yield C.
sakazakii free powder at the levels of contamination used. At an inlet temperature
of 160°C, log reduction of C. sakazakii ranged from 2.54 to 3.07 depending upon
C. sakazakii. Survival of C. sakazakii decreased as spray drying temperature
increased.
Nurjanah et al. (2013) reported that the ability of the bacterial colonization
and penetration during drying decreased the amount of corn for drying at a
temperature of 40 ° C, 45 ° C and 50 ° C and there are colonies before the corn
moisture content reaches 14% . Drying temperature of 40 ° C is not effective to
reduce the number of C. sakazakii. Isolates were more resistant to the toxic
FWHd16 third drying temperature compared with non-toxic isolates YRt2a with
the rate of decline for each temperature was 0.7, 0.9 and 1.1 log cycles / day. Both
isolates were able colonizes the surface of the corn and the corn penetrate through
the wound or through the cavities at the tip cap. Low drying temperature, moisture
content of corn 14%, the presence of C. sakazakii colonization and penetration in
corn may contribute to the bacterial contamination in dried corn products. In case
of drying, which can be seen as an extreme form of osmotic stress, the cells need
to preserve their biological integrity in the absence of liquid water. According to
the water replacement theory, poly hydroxyl compounds such as trehalose can
replace the shell of water around macromolecules, thus preventing damage to the
cells (Breeuwer et al. 2003).
The inoculation test prior to heating, drying, and filling, Cronobacter
cannot survive the heat treatment. However, in the process of drying and filling,
the organism was inhibited slightly in the high temperature drying and survived in
the final products. Therefore, all isolates that survived even at low levels are
considered to be a potential risk. The number of Cronobacter only decreased from
6 log CFU/ml to 5 log CFU/ml after drying (Fu et al. 2011). Survival of heatsensitive bacteria during spray drying has been reported previously survival could
be improved using an industrial-scale dryer, rather than the laboratory-scale Buchi
mini-dryer. The major intrinsic factors that govern microbial survival during spray
drying are the ability of organisms to withstand high temperatures and extreme

11
dehydration (Arku et al. 2008). C.sakazakii ability to survive in the long term is
suspected because of the ability to accumulate trehalose and a capsule form
(extracellular polysaccharides) below are some of the mechanisms and bacterial
cell response to dry conditions and osmotic stress:
a. Accumulation of Trehalose
C.sakazakii can produce trehalose which is a compatible solute that
protects the bacteria from dry conditions by stabilizing phospholipid membranes
and proteins. The accumulation of trehalose in the cell may play an important role
in the high heat tolerance of C. sakazakii (Rashidat et al. 2013). In general,
bacteria protect themselves from increasing osmolarity by the rapid intracellular
accumulation of ions, mainly K+, followed by the accumulation of compatible
solutes such as proline, glycine betaine, and trehalose. In case of drying, which
can be seen as an extreme form of osmotic stress, the cells need to preserve their
biological integrity in the absence of liquid water (Breeuwer 2003).
The protective effects of trehalose during desiccation appear to be due to
its stabilising influence on membrane structure, its chemically inert nature and the
propensity of trehalose solutions to form glasses upon drying, properties which
are not shared by glycine betaine. E.coli also synthesises trehalose to high
intracellular concentrations as a compatible solute when subjected to osmotic
stress in simple glucose mineral salts media, as trehalose accumulation is central
to its osmoadaptive strategy (Welsh & Herbet 1999).
b. Capsule Formation
The formation of extracellular polysaccharide can provide protection
against physical stress conditions and environmental stress experienced by
C.sakazakii during 18 months of storage in dry conditions not shown a correlation
between the formation of the capsule with cell recovery, but after storage for 2
years, 4 of the 5 strains showed C.sakazakii capsule formation, while after storage
for 2.5 years only 2 of 25 stain forming capsules (lehner et al. 2006). The capsule
could be involved in the organism‟s ability to survive the long shelf-life (24
months). It may also enable the organism to attach to surfaces and form a biofilm
that is more resistant to cleaning and disinfectant agents. The unique biophysical
properties of the capsule have lead to patents being filed for the exploitation of C.
sakazakii capsule as a thickening agent in foods to replace xanthan gum (Iversen
and Forsythe 2003).
C. Oxidative Stress
One of the most important injuries during dehydration, free radical
damage. Several studies with laboratory yeast strains Saccharomyces cerevisiae
have shown an important accumulation of ROS ( Reactive Oxygen Species)
during dehydration resulting in denaturation of proteins, nucleic acid damage and
lipid peroxidation . As a result, these environmental injuries affect negatively the
fermentative capacity, the viability and the vitality of cells. Biochemical
indicators of oxidative stress is a reaction redo, namely the level of glutathione
and lipid peroxidation. The main genes involved are TRRI and GRX5 genes
associated with two main redox balance system, the thioredoxin and glutathione /
glutaredoxin. An increase in the amount of glutathione and lipid peroxidation
significantly the oxidative stress on cellular components (Garre et al. 2010).

12

2 MATERIAL AND METHODS
Place and Time of Research
This research was conducted in the Food Microbiology Laboratory at
Southeast Asian Food and Agricultural Science and Technology (SEAFAST)
Center, as well as in the Food Microbiology Laboratory, Department of Food
Science and Technology, Bogor Agricultural University from September 2014
until February 2015.
Materials
The main materials used in this research are C. sakazakii isolates and
maize. C. sakazakii pGFPuv FWHc3 and E2 were mutants obtained by
transformation of cytotoxic C. sakazakii using GFP labelled plasmid (Nurjanah et
al. 2013). Media and other materials used include Brain Heart Infusion medium
(BHI, Difco), Buffer Phosphate Water (BPW, Oxoid), Tryptic Soy Agar (TSA,
Oxoid), Tryptic Soy Agar (100 µg/mL) Ampicillin (TSAA), tert-butanol, Plate
Count Agar (PCA, Oxoid), and 0.22 µm membrane filter, alcohol 95% .
Equipment
The drying chamber used in this research is made up of glass frame,
network of aluminium, based silica gel, and filtered paper for covering the
chamber and placed in incubator set at special temperature Fig2.

Figure 2 The drying chamber for maize
Other equipment are digital scales, glass objects, autoclave, incubator,
shaker, oven, laminar flow cabinet, syringe filter, digital cameras, , UV light
sources, spectrophotometer, aseptic room centrifuge, Ion Coater, JEOL 5310 LVSEM.
Methods
Every day the samples of maize were taken from three drying
temperatures (42 °C, 46 °C, and 50 °C) for analysis of water content, water
activity and enumeration of TPC and C. sakazakii. Selected samples from second
day and tenth day of drying at 50° were observed by SEM.

13

Confirmation of C. sakazakii
pGFPuv Mutan E2 and FWHc3

Preparation of Maize harvest at
water content of 33-35 % w.b

Inoculum preparation

Inoculation C.sakazakii to Maize

Drying at three temperature
(24°C, 46°C and 50° C) for
10 days

Enumeration of
C.sakazakii

Enumeration
of TPC

Analysis
of Water
Content

Analysis of
Water
Activity

Figure 3 The flow diagram of research

SEM
Observation
( selective )

14
a. Confirmation of C. sakazakii pGFPuv Mutants
Confirmation of C. sakazakii pGFPuv mutants was done by transferring 1
loop of culture into TSAA media and incubating it at 37 °C for 24 hours. The
mutants observed under UV light (Desaga 'UVIS 131100' UV Lamp UV 254nm),
showed green fluorescent colonies (Nurjanah et al. 2013).
b. Preparation of Maize
Maize used in this study is a hybrid from Pioneer 12 planted in Bogor.
The maize were harvested when the water content reached 33-35% (w.b) and the
kernels were manually extracted and stored in sterile plastic bags at room
temperature.
c. Inoculum Preparation
Green fluorescent colonies growing on TSAA were transferred to BPW
and placed in a 15 ml centrifuge tube and centrifuged (Hermle Z 383 k) at 3500
rpm (2,600 x g) for 10 min at 4°C to separate the cell pellet. The cells pellet was
washed twice using BPW and then resuspended in BPW to achieve an Optical
Density of 0.4 as measured with spectrophotometer (Shimadzu UV-2450) at 590
nm. This Optical Density corresponds to a concentration approximately of
CFU/g.
d. Inoculation
30 ml of the above inoculum was added to a sterile plastic bag containing
600 g of corn. The corn and inoculum were manually mixed after that which it
was left for 15 minute until the corn totally absorbed the inoculum. The corn is
cells/g.
expected to contain
e. Drying
Drying was carried out at three temperatures (42 °C, 46 °C, and 50 °C) for
10 days. On daily basis, corn samples were taken for measurement of moisture
content, water activity, and enumeration of Total Plate Count and C. sakazakii.
The period of drying until day tenth explain why we took high initial load of
inoculum because we need to understand the behavior of this bacteria during
drying.
f. Enumeration of C. Sakazakii
C. sakazakii pGFPuv surviving in the corn after drying was enumerated by
placing 10 g of corn in 90 ml of BPW and appropriately diluted and plated on the
TSAA media using surface method. The plates were incubated at 37°C for 24
hours and C. sakazakii pGFPuv seen as green fluorescent colonies under UV light
were enumerated. Enumerated of the number of colonies is done to see isolates
that survival after drying. The numbers of colonies were calculated in a range
between 25-250 colonies, the Number of colonies of bacteria can be calculated by
the Standard Plate Count formula as follows BAM 2001.
N=

15
Where:
N
∑C
n1
n2
d

=
=
=
=
=

Number of colonies per ml or g of product.
Sum of all colonies on all plates counted.
Number of plates in first dilution counted.
Number of plates in second dilution counted.
Dilution from which the first counts were obtained.

g. Enumeration of TPC
Total plate count was enumerated by placing 10 g of corn in 90 mL BPW
and serially diluted to achieve 25-250 colonies and incubated for 24-48 hours at
35°C and the number of colonies was calculated with the Standard Plate Count
formula (BAM 2001).
h. Analysis of Water Content
Water content was measured by oven method at 105 °C for at least 6
hours. Accurately, 2 g of the sample were weighed in covered dish previously
dried at 98-100 °C and cooled in a desiccator before weighing soon after reaching
room temperature (AOAC 2005).
. The moisture content of the sample is calculated using the following
equation
W =
X 100
Where:
W = Percentage of moisture in the sample.
A = Weight of wet sample (grams).
B = Weight of dry sample (grams).
i. Analysis of Water Activity
Water activity was measured using an Aw meter (Ro-Tronic) at 30 °C
(Passot et al. 2012).
j. SEM Observation
Colonization of C. sakazakii pGFPuv on the corn was observed by
taking samples from second and tenth day of drying at 50° C, corn kernel were
placed in sterile petri dish. The samples soaked in tert-butanol , after that freeze in
the freezer until frozen then enter to vacuum dryer until dry ( Pathan et al., 2010).
Samples were cut into small slices and coated with gold-Palladium using Ion
Coater (Mattox & Mattox, 2003).The sample was then observed with a JEOL
5310 LV-SEM.
Statistical Analysis
The experimental data were verified statistically with regression analysis using
Microsoft Excel 2007.

16

3 RESULTS AND DISCUSSION
Changes in Water Content of Maize during Drying
Drying of corn under the sun is commonly done by farmers. In this
research corn drying was done at three temperatures; 42 °C, 46 °C, and 50 °C,
putting into consideration that these drying temperatures provides ambient
environment for corn grains not to wrinkle. The results of this study confirmed
other studies by Schlunder (2004) and Chen at el. (2012) who reported that there
are two phases during drying, i.e. fast drying rate and slow drying rate. During the
first day of drying the initial moisture of