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
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 Information derived from the published and unpublished work of others has been duly acknowledged in the text as well as references given in the list of sources and in view of this, I therefore confer the copyright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
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.
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 concentration in the maize was CFU/g and drying was performed at 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
© 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)
A Thesis
Submitted in partial fulfillment of the requirements for the degree of Master of Science
In Food Science
USING pGFPuv MUTANTS TO STUDY THE INFLUENCE
OF DRYING ON THE SURVIVAL OF
Cronobacter sakazakii
IN
MAIZE
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
2015
Approved by,
Supervisory Committee
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.
TABLE OF CONTENTS
LIST OF TABLE vi
LIST OF FIGURE vi
LIST OF APPENDICCES vi
1 INTRODUCTION 1
Background 1
Problem Statement 2
Research Objective 2
Benefits of Research 3
Research Hypothesis 3
2 LITERATURE REVIEW 3
Drying 3
Maize 4
Cronobacter Sakazakii 5
The Growth of C.sakazakii 6
Contamination Source of C.sakazakii 6
Diseases Caused by C. sakazakii 7
Infantile or Neonatal Infection 7
Necrotizing Enteroclititis (NEC) 7
CNS Infection 8
C. sakazakii Thermal Resistance 8
C. sakazakii Resistance to Dry Condition 9
Accumulation of Trehalose 11
Capsule Formation 11
Oxidative Stress 11
3 MATERIALS AND METHODS 13
Place and Time of Research 12
Materials 12
Equipment 12
Methods 12
Confirmation of C. sakazakii pGFPuv Mutants 14
Preparation of Maize 14
Inoculum Preparation 14
Inoculation 14
Drying 14
Enumeration of C. Sakazakii 14
Enumeration of TPC 15
Analysis of Water Content 15
Analysis of Water Activity 15
SEM Observation 15
4 RESULTS AND DISCUSSION 16 Changes in Water Content of Maize During Drying 16 Changes in Water Activity of Maize During Drying 17 Changes in Total Plate Count of Maize During Drying 17
Survival of C.sakazakii During Drying 18
Colonization of C.sakazakii on the Surface of Maize 20
Penetration to the Tip Cap of Maize 21
5 CONCLUSION AND RECOMMENDATION 22
Conclusion 22
Recommendation 22
REFERENCES 23
LIST OF TABLES
1 Cronobacter sakazakii isolated from Indonesia 6
2 D and Z values for the various strains of Cronobacter spp 9
LIST OF FIGURES
1 C.sakazakii 5
2 The drying chamber for maize 12
3 The flow diagram of research 13
4 Changes in water content of maize during drying 16 5 Changes in water activity of maize during dying 17 6 Changes in total plate count of maize during drying 18
7 C. sakazakii survival curve during drying 19
8 SEM observation Colonization of C. sakazakii on the surface of corn 21 9 SEM observation. Penetration of C. sakazakii in cavity of tip cap corn 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
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 birth-weight 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).
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;
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).
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).
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).
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-Forsythe-Iversen (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 Cronobactersakazakii
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 Isolate DES c7/JF800180 C.sakazakii Corn starch Dewanti
Hariyadi et al. 2010 DES b10/JF800181 C.sakazakii Powdered infant
formulae YR c3 a/JF800183 C.sakazakii Weaning Food
Meutia et al. 2008
YR K2 a/JF800187 C.sakazakii Weaning Food YR t2a/JF800182 C.sakazakii Powdered infant
formulae
FWHb15 C.sakazakii Powder Sugar
Hamdani 2012
Estuningsih et al. 2006
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
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 life-threatening 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)
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 low-birth-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).
9 Table 2. D value (min) and Z value (°C) for Cronobacter spp local isolates.
Media Strains D value (min)
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
1.72-1.90 ATCC 51329 104.17-172.41 68.03-83.33 13.64-17.24 9.0-9.0 - 4.54-5.14 Infant
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).
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).
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
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 LV-SEM.
Methods
13
Figure 3 The flow diagram of research 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
SEM Observation
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 expected to contain cells/g.
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.
15
Where:
N = Number of colonies per ml or g of product.
∑C = Sum of all colonies on all plates counted. n1 = Number of plates in first dilution counted.
n2 = Number of plates in second dilution counted.
d = 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
16
3
RESULTS AND DISCUSSIONChanges 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 the maize was quickly removed. Water was removed from the maize surface by evaporation during the initial day of drying which causes the water content level to drop accordingly. After the first day, slower removal of water occurred which was a typical trend during the slow drying rate. During the slow drying rate, water was eliminated from inside the maize. This became more and more difficult as the water percolate further through the maize from the center to the outer part of the seed from where the evaporation took place.
The drying process is a delicate operation that achieves an equilibrium between two different mass transfer process, i.e. diffusion and evaporation. During fast drying rate, evaporation rate is equal to the diffusion rate of water from the inside to the surface of corn. Meanwhile during slow drying rate, the rate of evaporation is greater than the rate of diffusion of water from the inside to the surface (Simpson et al. 2012). It was noted that the drying rate slowed down during the slow drying rate period and reached the state of hygroscopic
equilibrium (Schlunder 2004). Eventually no more moisture can be removed from
the maize and was concluded to be in equilibrium with the drying air. Fig 4 showed that drying at 42° C and 46° C results in relative similar decrease in the water content , while that at 50° C recorded the lowest water content.
17
Changes in Water Activity of Maize during Drying
During drying the water activity (Aw) of various perishable materials generally decreases, thus enable storage at ambient temperature. Decrease in water activity is important for controlling the shelf-life of foods by suppressing the growth of microorganisms (Bonazii & Dumoulin 2011). Fig 5 showed that drying at 42° C and 46° C results in similar decrease in the water activity, while that at 50° C resulted in the lowest water activity.
Similar to changes in moisture content, changes in water activity during maize drying also consisted of two phases i.e. fast rate phase and slow rate phase. Fast rate phase occurred in the first day at 50 °C while in the Second day of drying at 42 °C and 46 °C. Meanwhile the slow rate phase happened at day 3 in which water activity continued to decrease slowly until the final day of drying. Higher drying temperature had a significant effect on the decrease in water activity, as observed by the slope of the linear regression 0.0109, 0.0125 and 0.0127 for the temperature 42 °C, 46 °C and 50 °C respectively during slow rate phase (Appendix 6).
Figure 5 Changes in water activity of maize during dying at 42 °C, 46 °C, and 50 °C
Changes in Total Plate Count of Maize during Drying
The total plate count in maize observed during drying reflected the microbial resistance to drying. In this research it was observed that the number of microorganisms decreased during drying at the three temperatures. During the first two days of drying the number of microorganisms decreased rapidly (1.65 log CFU/g) at the three drying temperatures.
The results also suggested that when the water activity was removed at a fast rate, the microorganisms had no time to adapt themselves either through genetic expression or adjustment of their metabolism (Guergoletto et al. 2012). After two days, the bacteria started to adapt to the high temperatures, thus the curve showed a relatively more resistant bacteria up to the eighth day. At the eighth day, in addition to temperature, the low water activity was suspected to play role in the fast decline of the number of microorganisms. At the last day of
18
drying, microorganisms had reached the lowest microbial population regardless of the drying temperatures. Drying of maize for 10 days at 50 °C has resulted in an undetectable level of microbial counts. In general, the microorganisms were slightly more resistant during drying at 42 °C and 46 °C than those at 50 °C Fig 6
Figure 6 Changes in total plate count of maize during drying at 42 °C, 46 °C and 50°C
Survival of C. Sakazakii during Drying
Both C. sakazakii pGFPuv isolates decreased in number during drying. The results also showed that the fate of decrease of C. sakazakii can be divided into three phases, i.e. logarithmic decrement, static phase and the final decline. For isolate FWHc3, the logarithmic decrement occurred at day 0-4, static phase at day 4-7 and the final decline at day 7-10. Meanwhile, isolate E2 dried at 50°C experienced logarithmic decrement at day 0-5, static phase at day 5-8 and the final decline at day 8-10. However, drying temperatures of 42° C and 46° C has resulted in the logarithmic decrement at day 0-6, static phase at day 6-8 and the final decline at day 8-10. C. sakazakii is more sensitive to drying temperature of 50°C, while the effect of drying at temperatures 42° C and 46° C are relatively similar. With high initial load ( CFU/g), the number of surviving C. sakazakii after 10 day of drying at 50o C was CFU/g.
Richardson et al. (2009) reported that the infectious dose of C. sakazakii was CFU. The above results suggested that it was possible to have C. sakazakii in maize after 10-day drying at 50° C in the number that might be infective. In dry products such as PIF or weaning foods, C. sakazakii should not be not able to grow, but after the addition of water, reconstituted PIF or weaning food is a good medium for growth. Once reconstituted, the only barriers for infection to occur are short storage and low temperature storage to prevent bacterial growth (Huertas et al. 2015). Seftiono (2012) reported the kinetics of inactivation of Cronobacter spp during the heating process of infant formula and the D values obtained ranged from 3.61-11.36 minutes at a temperature of 56° C and 68.97-256.41 minutes at a temperature of 50° C. Meanwhile the Z value for all isolates studied ranged from 3.54-5.69° C.
The rate of decline of C. sakazakii number (log/day) during drying was indicated by plotting the number of the bacteria log CFU/g on the Y axis and the
19 time interval (days) drying on the X axis. At the three drying temperatures (42°C, 46°C and 50°C) Fig 7, the rate of decline in the number of FWHc3 isolates was faster than isolates E2.
Both isolates are associated with toxicity. However, there are numerous reports of toxicological relationship with heat resistance. Toxic gene linkage studies vapBC (virulence-associated protein) in the thermophilic bacterium Sulfolobus solfataricus shows that those genes significant play a role in the stress response to heat (Cooper et al. 2009).
Nurjanah (2014) study the cytotoxic activity of C.sakazakii and reported that E2 had 78% while fwhc3 had 76% cytotoxic activity, this factor explain why isolate E2 more resistance than FWHc3. The presence of high-quality nutrients in the growth medium results in high intracellular nucleoside triphosphate concentrations hence, this model unifies the idea that quality of the growth medium dictates the growth rate of the cell (Tao et al .1999). In addition survival of microorganisms on food is affected by nutrient availability. Microorganisms require certain basic nutrients for growth and maintenance of metabolic functions (Olaimat & Holley 2012).
(a)
(b)
20
This results was similar to Nurjanah (2014) who showed that C. sakazakii was resistant to heat during corn drying at 50° C for four days. C. sakazakii was present in the spray drying process of formula milk (Arku et al. 2008). Spray drying of skimmed milk reduced C. sakazakii from 3.84 x CFU/ g to 2.5 x CFU/ g (Larasati 2012). The above studies also concluded that drying at 40 °C was not effective to reduce C. sakazakii in infant formula reconstitution process. C. sakazakii resistance to heat associated with the temperature and duration of heating exposure has not been explored in previous studies. Exposure to sub-lethal temperature or a few degrees above the optimum growth temperature may increase heat resistance of the bacteria. Heating in TSB medium at a temperature of 47 °C for 15 minutes increased the survival of C. sakazakii against heat, dry conditions (Chang et al. 2009b), and spray drying of skim milk (Larasati 2012). Survival of the bacteria decreased when bacteria were exposed to temperature above 48°C in Tryptic Soy Broth (Chang et al. 2009a).
Colonization of C. sakazakii on the Surface of Maize
The presence of bacteria in the dried product can also be caused by ability to carry out the attachment or colonization of bacteria on the surface of the food material. The results of microscopic observation with scanning electron microscopy (SEM) showed colonization of C. sakazakii on the surface of the corn on the second day of drying (Fig 8). However, with a TPC of CFU/g and CFU/g of C. sakazakii, it was not possible to differentiate C. sakazakii from other naturally occurring bacteria. According to the shape, we assumed that appearance observed with Scanning Electron Microscope was C. sakazakii (rod shape), while TPC shall show other shape of bacteria like coccus or bacillus. Water activity in the second day of drying at 50° C was recorded as 0.34. This was in accordance with Beuchat et al. (2009) who reported that C. sakazakii survived better in dried formula and cereal at low aw (0.25–0.30) than at high aw (0.69–0.82) during storage. Iversen et al. (2008) studied biofilm formation of C. sakazakii and reported that, these bacteria have the ability to stick to latex and polycarbonate and bulk in stainless steel, the biofilm formation affected by various conditions such as the composition of nutrients in the media and the relative humidity environment (Jung 2013). Breeuwer et al. (2003) reported that C. sakazakii have resistant to drying process with a temperature range of 25° C to 45° C, it is suspected that the biofilm formation was performed by C. sakazakii. The appearance of C. sakazakii pGFPuv on corn at second day of drying (Fig 6) is similar to biofilm observation with scanning electron microscopy by Chang et al. (2009a) and Hurrell et al. (2009).
21
Figure 8SEM observation Colonization of C. sakazakii on the surface of corn on second day of drying (5000X)
Penetration to the Tip Cap of Maize
SEM observation performed on the tip cap and bacteria found in the cavity which indicates that the wound became one of the entry points of this bacterium into corn. In addition through the wound, this bacterial penetration into the corn kernels is thought to have occurred through cavities contained in section of the tip cap corn (Fig 9).
(a) (b)
22
4
CONCLUSION AND RECOMMENDATION
Conclusion
Drying of maize kernel at 42° C , 46° C and 50° for 10 days causes a decrease in the water content to 6.9,5.8,4.2%, respectively which correlate to water activities of 0.33,0.30,0.23, respectively. The decrease in Aw can be differentiated into three phases which correspond to the decrease in total microbial numbers and C. sakazakii pGFPuv. With an initial inoculation of C. sakazakii at 108 CFU/g, after 10-days drying at 50 °C no microorganisms was detected but C. sakazakii pGFPuv was found at CFU /g. Isolate E2 was more resistant at three drying temperatures as compared to isolate FWHc3. The ability of C. sakazakii to colonize and possibly form biofilm corn surface as well as ability to penetrate into the corn through the wound or through cavities at the tip cap may have helped their survival during drying. Cronobacter sakazakii E2 and FWHc3 labeled with GFP can be applied to study the survival of C. sakazakii during drying. Drying for 1 day at 50 °C can achieve < 12 % water content, < 0.4 water content and the decrease in the bacteria approximately 2 log FU/g.
Recommendation
23
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