-
Ultraviolet (UV) Radiation Inactivation of Cronobacter sakazakii
in
Dry Infant Formula Observed Using Fourier Transform Infrared
Spectroscopy and Electron Microscopy
By
Qian Liu
A thesis submitted in partial fulfillment of
The requirements for the degree of
Master of Science in Food Science
WASHINGTON STATE UNIVERSITY
School of Food Science
May 2011
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ii
To the Faculty of Washington State University:
The members of the Committee appointed to examine the thesis of
QIAN LIU find it
satisfactory and recommend that it be accepted.
Barry G. Swanson, Ph.D., Chair
Dong Hyun Kang, Ph.D.
Barbara Rasco, Ph.D.
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iii
Acknowledgements
First, I would like to express my gratitude to Dr. Dong-Hyun
Kang for his support and
encouragement during my study in Washington State University. He
gave me an opportunity
to work in food science and led me to scientific research.
Besides the guidance on my
research work, he enabled me to obtain many possibilities in my
life. I am a very lucky
person to have him as my first advisor. His philosophy and
enthusiasm on life and work
enlightens me to establish a career in science.
I am also very grateful to Dr. Barry Swanson and Dr. Barbara
Rasco at Washington
State University who care about me as their own child. Without
great support and
encouragement from them, this work could not have been
completed. I would also express
my thanks to Dr. Gulhan Unlu who helped me in both course and
research work, and Dr.
Shyam Sablani who allowed me to use the radiometer. I also want
to thank Dr. Valerie Lynch-
Holm at Franceschi Microscopy and Imaging Center, who did not
only provide valuable
information in electron microscopy works but also share with me
her research experience to
encourage me to go through the depressed part in my life.
I would like thank staff members in our department, Jodi
Anderson who took care of me
as a mama bear and helped me get used to life in America. I also
thank Barbara Smith and
Carolee Armfield, who helped me with financial materials. A
special thank goes to Frank
Younce for providing help on usage of equipment in the Pilot
Plant.
I would like to express my appreciation to my lab members who
consider me as a family
member: Peter Gray, A Reum Han and Tahir Zahoor. They
contributed their patience and
hard work to teach me important lab skills in the food safety
area. This work could not have
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iv
been done without their help and support. I also thank my dear
friends in Pullman, Beata
Vixie and her family, Yuhui Chao, Peichi Yang, Donglei Luan and
his wife. They made my
life in Pullman easy and happy.
Last, I would express my great gratitude to my family and my
boyfriend Xiaonan Lu.
No matter how far away they are, they are always ready to
support me and help me to fulfill
my dreams. I cannot live in this world without them.
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v
Ultraviolet (UV) Radiation Inactivation of Cronobacter sakazakii
in
Dry Infant Formula Observed Using Fourier Transform Infrared
Spectroscopy and Electron Microscopy
Abstract
By Qian Liu, M.S. Washington State University
May, 2011
Chair: Barry Swanson
Cronobacter sakazakii is an opportunistic pathogen associated
with dry infant formula
which poses a high risk to low birth weight neonates. In the
current study, inactivation of C.
sakazakii in dry infant formula by ultraviolet (UV) radiation
alone and in combination with
heat treatment at temperatures of 55, 60 and 65oC were applied.
UV radiation with doses in a
range of 12.1 to 72.8 kJ/m2 at room temperature had a
significant effect on the inactivation of
C. sakazakii in dry infant formula (p
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS…………………………………………………………………..iii
ABSTRACT…………………………………………………………………………………...v
LIST O TABLES…………………………………………………………………………….viii
LIST OF FIGURES……………………………………………………………………………x
1 INTRODUCTION
..................................................................................................................
1
1.1 Infant formula processing
.................................................................................................
1
1.2 Biochemical characteristics and taxonomy of C. sakazaki
............................................... 2
1.3 Environment and food
sources..........................................................................................
4
1.4 Growth requirements of C.
sakazakii................................................................................
5
1.5 Biofilm formation
.............................................................................................................
6
1.6 Resistance of C. sakazakii to environmental stress
........................................................ 10
1.6.1 Heat resistance
.......................................................................................................
10
1.6.2 Osmotic and desiccation resistance
.......................................................................
11
1.6.3 Antibiotics resistance
.............................................................................................
13
1.7 Virulence factors
.............................................................................................................
14
1.8 Detection methods
..........................................................................................................
16
1.9 UV radiation treatments
..................................................................................................
20
1.10 Fourier Transform Infrared Spectroscopy
....................................................................
23
2 MATERIAL AND METHODS
.............................................................................................
26
2.1 Bacterial strains, culture methods and preparation of stock
cultures .............................. 26
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vii
2.2 Sample preparation and inoculation
................................................................................
26
2.3 Ultraviolet radiation treatment
........................................................................................
27
2.4 Combined UV radiation and hot water treatment
........................................................... 27
2.5 Bacterial enumeration.
....................................................................................................
28
2.6 Membrane filtration and FT-IR spectroscopy
.................................................................
28
2.7 Electron microscopy analysis
.........................................................................................
29
2.8 Data preprocessing and chemometrics
............................................................................
30
2.9 Statistical analysis
...........................................................................................................
31
3 RESULTS AND DISCUSSION
............................................................................................
32
3.1 C.sakazakii inactivation effect of UV radiation
..............................................................
32
3.2 Combined treatments of heat and UV radiation
.............................................................
34
3.3 FT-IR spectroscopy decoding and cluster analysis.
........................................................ 36
3.4 Electron microscopy analysis.
........................................................................................
38
4 CONCLUSION
.....................................................................................................................
39
References
................................................................................................................................
40
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viii
LIST OF TABLES
Table 1: Biochemical traits suitable for differentiation between
C. sakazakii and E. cloacae 56
Table 2. DNA-DNA relatedness of Coronobacter strains
....................................................... 57
Table 3. Food and environmental sources of C. sakazakii
....................................................... 58
Table 4. D values of C. sakazakii strains in Artificial media
and reconstituted infant formula
..............................................................................................................................................
59
Table 5. z values of C. sakazakii strains in different media
..................................................... 65
Table 6. UV radiation treatment time and dose.
......................................................................
67
Table 7. D values of C.sakazakii inoculated in infant formula
for UV radiation in combination
with hot water treatments and hot water treatments only
..................................................... 68
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ix
LIST OF FIGURES
Figure 1. FDA standard protocol for the isolation of C.
sakazakii from infant formula ......... 69
Figure 2. Microbial survival curve of C. sakazakii inoculated in
infant formula and treated
with UV radiation (254 nm) for 5 min, 10 min, 15min, 20 min, 25
min and 30 min. ......... 70
Figure 3. Population reduction of C. sakazakii inoculated in
infant formula after UV radiation
exposure with a sample layer of 0.032 cm, 0.16 cm and 0.32 cm
........................................ 71
Figure 4(a). Microbial survival population of C. sakazakii
inoculated in infant formula after
UV radiation 20 min and 55oC hot water treatment and 55oC heat
treatment only ............. 72
Figure 4(b). Microbial survival population of C.sakazakii
inoculated in infant formula after
UV radiation 20 min and 60oC hot water treatment and 60oC hot
water treatment. ............ 73
Fig.4(c). Microbial survival population of C.sakazakii
inoculated in infant formula after UV
radiation 20 min and 65oC hot water treatment and 65oC hot water
treatment only ............ 74
Figure. 5. Raw infrared spectra of non-fat milk powder and
non-fat milk powder inoculated
with C.sakazakii.
..................................................................................................................
75
Figure 6 (a). FT-IR spectroscopy: second derivative of spectra
of UV-treated C. sakazakii and
intact C. sakazakii in the fingerprint region (1800 – 900 cm-1)
........................................... 76
Figure 6 (b). FT-IR spectroscopy: second derivative of spectra
of UV-treated C.sakazakii and
intact C.sakazakii between 3400 – 2800 cm-1
......................................................................
77
Figure 7. Principal component analysis of C. sakazakii treated
with UV and control. The
cluster analysis model was validated by new treatments
..................................................... 78
Figure 8(a). Scanning electron micrograph of C. sakazakii in
infant formula at 29,978×. ..... 79
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x
Figure 8(b). Scanning electron micrograph of C. sakazakii in
infant formula treated by UV
radiation for 20 min at 30,017×.
...........................................................................................
80
Figure 8(c). Scanning electron micrograph of infant formula at
29,987×. .............................. 81
Figure 9(a). Transmission electron micrograph of thin section of
C. sakazakii and infant
formula mixture.
...................................................................................................................
82
Figure 9(b). Transmission electron micrograph of thin section of
UV radiation of 20 min
treated C. sakazakii and infant formula mixture.
.................................................................
83
Figure 9(c). Transmission electron micrograph of thin section of
infant formula.. ................. 84
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1 Introduction
1.1 Infant formula processing
Infant formula is an important breast milk substitute formulated
to resemble
nutrient composition of human breast milk. Bovine’s milk is a
primary protein source for
infant formula production, although soy protein is also used. To
simulate breast milk, fat is
removed from milk. Whey protein, carbohydrates, fat, minerals
and vitamins are then added
back. In the process of infant formula manufacture, skim milk is
pasteurized and evaporated
followed by addition and blending of dry ingredients. Addition
of these ingredient leaves the
possibility for microbial contamination, thus a heat treatment
is required either before or after
evaporation and is a critical control point in the production
process. Some heat sensitive
nutrients must be added after drying to avoid quality change and
nutrition retention.
In infant formula industry, efforts to reduce contamination with
C. sakazakii focus on
environmental monitoring, hygiene practice and end-product
testing for the organisms
(Farber, 2003). Control measures are necessary on receipt of raw
materials, such as milk.
Development of validated detection methods improves detection
efficiency of the organism in
the raw materials.
Heat treatment is a traditional method to reduce C. sakazakii
contamination in the
industry. C. sakazakii exhibited greater heat and desiccation
resistance compared to other
bacteria in Enterobacter family. Thus, C. sakazakii can survive
for long periods in spray dried
infant formula. Because infant formula is not a sterile product,
there is potential risk of
contamination in packaging, storage and distribution. For
example, bacteria can be introduced
into spray dried products by addition of non-heat-treated
ingredients. Raw bovine milk has to
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undergo short time high temperature sterilization to eliminate
all viable microorganisms. Raw
ingredients which cannot undergo heat treatment must be stored
in a sterilized room
separated from ingredients pasteurized. Heat treatment either
before or after drying will
improve control of microbial contamination. The dry area of
manufacturing facilities must be
hygienic to avoid undesirable entry of contaminants. The low
moisture area is also a critical
control point for post pasteurization contamination. The
packaging area must also be
separated from the processing and storage area to avoid exposure
of finished products to
contamination. The blenders, utensils and equipments used in
processing should be
disinfected and sanitary to avoid biofilm growth. Alternative
sanitizing methods such as
radiation should be studied and applied to infant formula
industry to reduce the
contamination, particularly the formation of biofilms on food
contact surfaces that may be a
source of contamination.
When preparing and handling reconstituted infant formula,
professionals should follow
preparation and hygiene recommendations provided by public
health officials and
organizations such as American Dietetic Association (Farber,
2003) and instruct parents and
infant caregivers accordingly. Particularly, controlling the
temperature of hot water used to
reconstitute infant formula and the “holding time” (amount of
time a formula is at room
temperature in the feeding bag or bottle and accompanying lines
during enteral tube feeding)
of reconstituted infant formula are good strategies to prevent
C. sakazakii contamination of
milk (Farmer, 2003; Chen et al., 2009).
1.2 Biochemical characteristics and taxonomy of C. sakazakii
C. sakazakii is motile peritrichous, Gram negative rod (Farmer,
1980). C. sakazakii was
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first characterized as yellow pigment producing Enterobacter
cloacae in the 8th edition of
Bergey’s Manual of Determinative Bacteriology (Sakazaki, 1974).
To classify this organism
into Enterobactericeae is based upon DNA-DNA hybridization,
biochemical characterization,
pigment production, and antibiotic susceptibility. By DNA-DNA
hybridization, the type
strain of E. sakazakii was 83 to 89% related to other strains of
E. sakazakii but only 31 to 49%
related to strains of E. clocae. This result provided the
fundamental information that C.
sakazakii is a new species rather than a phenotypically distinct
subgroup within the existing E.
cloacae species (Farmer et al, 1980). In the same study, Farmer
et al. (1980) provided details
on biochemical traits and antibiotic susceptibility of C.
sakazakii (Table 1). Unlike E.
cloacae, C. sakazakii produces yellow-pigmented colonies on
trypticase soy agar, brain heart
infusion (BHI) agar and blood agar during incubation of 48 to 72
h. The yellow pigments are
more pronounced after incubation at 25 than at 36oC, and the
intensity of the pigmentation
varies from strain to strain (Lehner, 2004). E. sakazakii cannot
ferment D-sorbitol and
exhibits delayed extracellular DNase activity. In enzyme
activity studies, Muytjens et al.
(1984) reported that α-glucosidase activity can be used to
differentiate C. sakazakii from
other Enterobacter species. Detection of α-glucosidase activity
enables the development of
selective and differential media for C. sakazakii, such as Oh
and Kang media (Oh and Kang,
2004), Leuschner, Baird, Donald and Cox media (Leuschner, 2004),
and Druggan-Forsythe-
Iversen media (Iversen et al., 2004b). The absence of
lecithinase and production of Tween
esterase are also distinctive characteristics of C. sakazakii.
(Farmer, 1980).
Iverson et al. (2008) reclassified the isolates described as C.
sakazakii and proposed a
novel genus. The formal description of these organisms is
completed by using Biotype 100
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4
and Biolog Phenotype MicroArray data (Iversen et al., 2008).
Based on further DNA-DNA
hybridization experiments, phenotypically different strains
within the formal species C.
sakazakii were reclassified as Cronobacter sakazakii gen. nov.,
comb. nov. Cronobacter
malonaticus sp. nov., Cronobacter turicensis sp. nov.;
Cronobacter muytjensii sp. nov.
Cronobacter dublinensis sp. nov.; Cronobacter dublinensis sub
sp. Dublinensis subsp. nov.;
Cronobacter dublinensis subsp. Lausannensis subsp. nov. and
Cronobacter dublinensis subsp.
Lactaridi subsp. Nov. (Iverson et al, 2008). The DNA-DNA
relatedness values for species
presented below in Table 2.
1.3 Environment and food sources
The natural habitat or reservoir of C. sakazakii is unknown. The
organism is not found in
surface water but is found in mud, grind, rotting wood, bird
dung, rodents, domestic animals,
cattle, and raw bovine’s milk (Muytjens and Kollee, 1990). A
possible environmental
reservoir of C. sakazakii was the gut of insects such as the
Mexican fruit fly Anastrepha
ludens and the stable fly Stomoxys calcitrans (Kuzina et al.,
2001; Hamilton et al., 2003.).
Flies serve as vehicles in C. sakazakii transmission because
they are fed on blood of warm-
blooded animals and are found wherever cattle, pigs and horses
are kept (Iversen and
Forthyse, 2004).
Kandha et al. (2004) investigated the presence of C. sakazakii
in food manufacturing
factories and households by adaptive cultivation methods,
biochemical characterization and
ribotyping. The environmental samples from factories were
obtained by scraping or sweeping
surfaces in the production-line environment or by sampling
vacuum-cleaner bags. Samples
from households were taken from vacuum-cleaner bags. Kandhai
(2004) found 8 out of 9
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5
environmental samples from factories and 5 out of 16 household
samples contained C.
sakazakii. The organism was isolated from a range of foods
including cheese, fermented
bread, tofu, sour tea, cured meats, minced beef and sausage
meat. C. sakazakii may occur in a
large range of food ingredients due to its wide distribution in
soil, however the detail is not
reported. The efficiency and accuracy of isolation and detection
methods involved in these
studies are unclear so estimations of the prevalence of C.
sakazakii may not be precise.
Members of the family Enterobacteriaceae were cultured from
52.5% of 141 milk
substitute infant formulas which were obtained in 35 countries
(Muytjens et al., 1988b). C.
sakazakii was one of the most frequently isolated species
(Muytjens et al., 1988b). Postupa
and Aldova (1984) isolated six C. sakazakii strains from
powdered milk. Both Simmons et al.
(1989) in the United States and Bierling et al. (1989) in
Iceland also isolated C. sakazakii
from dried infant formula.
1. 4 Growth requirements of C. sakazakii
C. sakazakii grows on media such as MacConkey, eosin methylene
blue and violet red
bile agar used to isolate enteric organism Two morphologically
different colony types are
observed when pure culture of C. sakazakii are streaked on agar
plate: scallop-edged rubbery
colonies and smooth colonies (Farmer et al., 1980). C. sakazakii
can grow to ca 109 CFU/ml
overnight from 104 at 37 or 44oC in trypticase soy broth
(Iversen et al., 2004a). C. sakazakii
can grow at a temperature range of 6-47oC with an optimum of
37-43oC depending on the
medium. The doubling time at 37oC varied from 14 to 29 min in
whitley impedance broth,
brain heart infusion medium and trpticase soy broth medium. In
infant formula milk, the
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6
doubling times at 6 and 21oC are 13.7 and 1.7 h (Iversen et al.,
2004a).
Survival and growth of C. sakazakii in dry infant formula powder
was investigated in the
last decade. C. sakazakii is reported to survive for at least
two years in powdered infant
formula at a aw of 0.14 (Edelson-Mammel et al., 2005; Barron and
Forsythe, 2007).
Population reduction of C. sakazakii at aw of 0.43-0.40 is
greater than reduction in powders at
aw of 0.25-0.30 at 4oC for 6 months. Decreases in populations
were greater in dry infant
formula stored at 30oC than at 21 or 4 oC. Survival of C.
sakazakii does not demonstrate
significant differences (p
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Leberkuhne and Wagner, 1986). The proposed mechanism of enhanced
bacteria resistance to
environmental stress in biofilms is extracellular polymeric
substances produced in biofilm
formation provide a protective barrier against environmental
stresses (Kim et al., 2007). C.
sakazakii cells at or near the surface of biofilm also act as
protective barriers for the cells
deeply in the biofilm. Although cells deep in the biofilm matrix
cannot access oxygen and
nutrients in the environment, the cells undergo starvation
leading to increased resistance to
environmental stress. Biofilms formed by Listeria monocytogenes
on stainless steel and
Teflon coupons adapt to sanitizing agents while cells removed
from biofilms individually are
not resistant to sanitizing agents. The development of
resistance to environmental stress may
be attributed to extracellular substances instead of layering
the cells in biofilms (Pan et al.,
2006). Lehner et al. (2005) observed calcofluor stained fibrils
in biofilm matix formed by C.
sakazakii in Luria-Bertani broth and brain heart infusion broth
suggesting the presence of
cellulose as an extracellular compound in this type of biofilm.
Extracellular polysaccharides
produced by 24 C. sakazakii isolates analyzed with high
performance liquid chromatography
revealed the presence of glucose, galactose, fucose and
glucuronic acid (Lehner et al., 2005).
Dancer et al. (2009) determined that a polysaccharide capsule
may not be a necessary
determinant of biofilm density by transmission electron
microscopy. Hartmann et al. (2010)
conducted research to investigate the genetic basis of biofilm
formation by Cronobacter spp.
on polystyrene surfaces by screening a library of random
transponson mutant strain ES 5 for
decreased biofilm formation. Genes associated with flagellar
structure and flow cell biofilm
architecture rather than genes associated with cellulose
biosynthesis contributed to the
attachment of Cronobacter to polystyrene surfaces.
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Cell-to-cell signaling, also known as quorum sensing, is
demonstrated to play a role in
biofilm formation by foodborne pathogens (Annous et al., 2009).
In Gram negative bacteria,
the homologues for Vibrio fischeri LuxI-LuxR regulatory genes
are the key factor of quorum
sensing system. The auto-inducer acylated homoserine lactones
(AHLs) are synthesized by
the LuxI homologues in the cell and secreted to the external
environment. When the AHLs
reach a threshold concentration, AHLs enter bacteria cells and
bind to the LuxR homologues
to activate or repress target gene transcription. Thus, the
quorum sensing system can regulate
biofilm formation. In Gram positive bacteria, the auto-inducer
is secreted external to the cells
similarly, but the auto-inducer regulates target gene
transcription by binding to the receptors
on the cell surface instead of reentering the cells (Miller and
Bassler, 2001). Quorum sensing
systems of a large number of bacteria also depend on molecules
other than autoinducer-1 (AI-
1). For example, autoinducer-2 (AI-2) is a byproduct of the
activated methyl cycle catalyzed
by LuxS enzyme (Smith et al., 2004; Vendeville et al., 2005).
Certain food pathogens such as
Escherichia, Shigella, Salmonella, Yersinia and other Gram
negative bacteria possess the
auto-inducer-3/epinephrine/norepinephrine (AI-3/epi/norepi)
signaling system (Walters and
Sperandio, 2006). Salmonella, Escherichia, Shigella, and
Klebsiella that do not produce
AHL detect AHLs produced by other bacteria (Michael et al.,
2001). In C. sakazakii strains,
the presence of AHLs is determined by thin-layer chromatography.
Lehner et al. (2005)
analyzed ethyl acetate extracts of cell supernatants for 56
selected C. sakazakii strains and
observed production of AHLs by C. sakazakii.
Attachment and biofilm formation on the abiotic surface of C.
sakazakii is influenced by
temperature, nutrient availability and humidity. In one study,
stainless steel coupons and
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9
enteral feeding tubes were immersed in phosphate-buffer saline
cell suspension of five strains
of C. sakazakii grown to stationary phase (7 log CFU/ml) to gain
the attachment of 5.33 to
5.51 and 5.03 to 5.12 log CFU/cm2 at 4oC. Then coupons and tubes
attached by C. sakazakii
were immersed into tryptic soy broth, infant formula and lettuce
juice broth followed by
incubation for 10 days at 12 or 25oC. Biofilms were not observed
at 12oC, and the cells
increased 1.42 to 1.67 and 1.16 to 1.31 log CFU/cm2 on stainless
steel coupons and enteral
feeding tubes in infant formula at 25oC (Kim et al., 2006).
Biofilms were formed on abiotic
surfaces immersed in infant formula, but not on abiotic surfaces
immersed in typtic soy broth
and lettuce juice suggesting that nutrient availability play a
role in biofilm formation (Kim et
al., 2006). Dancer et al. (2009) concluded that nitrogen source
is a more important
determinant than carbohydrate source in biofilm formation of C.
sakazakii in milk compared
with carbohydrates (Dancer et al., 2009). Quantification of
biofilm formation on plastic
surfaces by 72 seletected C. sakazakii strains in brain heart
infusion broth, nutrient broth,
tryptic soy broth, 1/20 tryptic soy broth and reconstituted
infant formula revealed that
reconstituted infant formula supported biofilm formation more
effectively than artificial
media (Oh et al., 2007). Survival of C. sakazakii cells in
biofilms on stainless steel coupons
immersed in M9 medium and reconstituted infant formula were
investigated when exposed to
23, 43, 68, 85 and 100% relative humidity. The overall order of
survival as affected by
humidity was 100 > 23 = 43 = 68 > 85% relative humidity
regardless of matrices (Kim et al.,
2008). Temperature control of infant feeding preparation and
storage conditions choice is
important for preventing C. sakazakii biofilm formation.
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10
1.6 Resistance of C. sakazakii to environmental stress
1.6.1 Heat resistance
Preliminary studies of D values of C. sakazakii strains were
conducted in artificial
medium and reconstituted infant formula from 50 to 70oC. D55 of
selected strains in
reconstituted infant formula varied from 1.51 to 14.21 min
according to Al-Holy et al. (2009).
D55 range selected C. sakazakii strains was 3.27 min to 17.07
min in phosphate buffer pH 7.0
reported by Dancer et al. (2009). Considerable variations in the
heat resistance of the tested
C.sakazakii strains were observed. Physiological properties of
the tested strains are an
important factor that influences heat resistance. C. sakazakii
ATCC 29544 exhibited a D55
value of 14.83 min while the D55 value of C. sakazakii 55 is
1.51 min in reconstituted infant
formula. Edelson-Mammel and Farmer (2004) concluded that C.
sakazakii may have a set of
genetic determinants for heat resistance based on two distinct
phenotypes presented in D
value profile. Williams (2005) reported a protein only expressed
in heat resistant strains and
identified as homologous to a hypothetical protein found in the
heat resistant bacteria,
Methylobacillus flagellatus KT by a top-down proteomics
approach. Asakura (2007) studied
the genetic heat resistance of C. sakazakii, and concluded that
infB gene which encodes for a
translation initiation factor is expressed in a higher amount in
heat resistant strains than in
heat sensitive strains.
Iversen et al. (2004a), Dancer et al. (2009) and Al-Holy et al.
(2008) reported D60 values of
C. sakazakii strains. Iversen et al. (2004a) compared heat
resistance of type and capsulated
strains in typtone soy broth and reconstituted infant formula.
The medium used for bacteria
culture influences the heat resistance of selected strains. D60
value of type strain of C.
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11
sakazakii was 0.9 min in tryptone soy broth and 1.1 min in
reconstituted infant formula
(Iversen et al., 2004a). Moreover, C. sakazakii 55 demonstrated
a D55 of 1.51 min in
reconstituted infant formula (Al-Holy et al., 2008) and 3.27 min
in phosphate buffer with pH
7.0 (Dancer et al., 2009). D60 values ranged from 0.17 min to
2.71 min in both reconstituted
infant formula and liquid medium except C. sakazakii 607 with a
D60 value of 264.4 min in
reconstituted infant formula (Edelson-Mammel and Buchanan, 2004;
Al-Holy et al., 2008;
Dancer et al., 2009). D65 value of C. sakazakii 607 was 35.2 min
in reconstituted infant
formula reported by Edelson-Mammel and Buchanan (2004) (Table
4).
Overall z value for selected food and clinical strains of C.
sakazakii calculated by
Nazarowec-White and Farmer (1997) was 5.82oC based on D values
at 52, 54, 56, 58 and
60oC in reconstituted infant formula. z value for C. sakazakii
1387-2 was 3.1oC calculated
with D values of 53, 54, 56, 58oC in phosphate buffer pH 7.0.
The most heat resistant C.
sakazakii 607 strain reviewed exhibited a z value of 5.6oC in
reconstituted infant formula
based on D values of 56, 58, 60, 65, 70oC (Edelson-Mammel and
Buchanan, 2004). Iversen et
al. (2004a) presented different z values of C. sakazakii NCTC
11467 and capsulated strain C.
sakazakii 823 at 5.8oC and 5.7oC in typtone soy broth with D
values of 54, 56, 58, 60, 62 oC
(Iversen et al., 2004a). Al-Holy et al. (2009) reported z values
of tested C. sakazakii strains in
reconstituted infant formula varied from 3.76oC to 10.11oC based
on D values of 55, 60, 63oC.
The selected C. sakazakii strains exhibited a range of z values
from 6.26oC to 10.86oC in
phosphate buffer pH 7.0 based on D values of 50, 55, 60oC
(Dancer et al., 2009) (Table 5).
1.6.2 Osmotic and desiccation resistance
Dried infant formula powder has a water activity about 0.2. To
survive under this
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12
extreme condition, C. sakazakii possesses osmotic and
desiccation resistant mechanisms.
Breeuwer et al. (2003) reported that C. sakazakii strains
inoculated in BHI with 40% sorbitol
(aw = 0.934) decreased about 1 log after 2 months, making the
most resistant strain to osmotic
stress compared to the nine microorganisms within
Enterobactericeae tested as part of this
study. At a sorbitol concentration of 75% (aW = 0.811), C.
sakazakii strains 1387-2 and 1360
were detectable after four weeks. The addition of trehalose,
proline or glycine betaine to
sorbitol stressed C. sakazakii did not improve survival. In
contrast, C. sakazakii cannot grow
in M9 medium with 1 mol/L NaCl, but addition of glycine betaine
to medium enhance
growth (Breeuwer et al., 2003).
In the study of desiccation resistance, the C. sakazakii cell
populations in dry infant
formula at day 46 exhibited a 1.0-1.5 log10 cfu/g reduction in
stationary phase in dry infant
formula with a aw of 0.14 at 20oC. Similarly, Edelson-Mammel et
al. (2005) reported a 2.4
log10 cfu/g reduction was observed at the initial 5 months and
an additional 1 log10 cfu/g
reduction during the subsequent 19 months. Barron and Forsythe
(2007) also reported that
some capsulated C. sakazakii strains were recoverable after 2.5
year storage in dry infant
formula powder while other selected strains belonged to
Enterobacteriaceae were not
recovered after 6, 15 and 24 months.
Research to explain the osmoadapation of bacteria suggests
organisms can protect
themselves from a NaCl concentration stress environment by
pumping out sodium and
accumulating potassium through a proton gradient that impart
enzymes with more negative
charged amino acid residues than those in normal environment. To
survive in an anhydrous
environment, a compatible solute such as trehalose is
accumulated by the microorganism to
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13
stabilize dry cell membranes by interaction with –OH groups on
phospholipid membrane.
Moreover, trehalose also stabilizes dry proteins by interaction
with –OH groups on polar
residues in dry protein. The hydrogen bonds formed between
trehalose and phosphate or
amino acid residues provide a water shell for these structure
compounds (Crowe, 1992).
C. sakazakii can survive in desiccation and high osmotic
pressure environments by
stabilizing macromolecules.
1.6.3 Antibiotics resistance
Muytjens et al. (1988a) determined the activities of 29
antimicrobial agents against C.
sakazakii and compared antibiotic resistance with seven other
Enterobacter species. C.
sakazakii was resistant to 27 out of 29 antibiotics, except for
cephalothin and
sulfamethoxazole. The minimum inhibitory concentration (MIC) for
C. sakazakii was at least
twofold higher than the MIC for E. cloacae. C. sakazakii also
exhibited exclusive resistant to
ampicillin in the tested strains with MIC of 8 µg/ml and MIC for
cephalothin in a
concentration range from 256 to 512 µg/ml (Muytjens, 1988a).
Farmer et al. (1980) reported
that C. sakazakii is susceptible to gentamicin, kanamycin,
chloramphenicol and ampicillin;
More than 87% were susceptible to nalidixic acid, streptomycin,
tetracycline and
carbenicillin. All selected 24 strains of C. sakazakii were
resistant to penicillin (Farmer,
1980). Nazarowec-White and Farber (1999) reported that 13 out of
24 strains were
susceptible to ampicillin, cefotaxime, chloramphenicol,
gentamicin, kanamycin, polymyxin B,
trimethoprimsulphamethoxazole, tetracycline and streptomycin,
but resistant to
sulphisoxazole and cephalotin. One of the food strains was
resistant to sulphisoxazole,
cephalothin, chloramphenicol and ampicillin. Kuzina et al.
(2003) reported that C. sakazakii
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14
isolated from M fruit flies is resistant to ampicillin,
cephalothin, erythromycin, novobiocin,
and penicillin. Lai (2001) reported that C. sakazakii clinical
isolates examined exhibited
uniform resistance to ampicillin, cefazolin and extended
spectrum penicillins and variable
resistance to the 3rd generation cephalosporines and the
quinolones. Compared with the
results previously reported, the C.sakazakii isolates from M
fruit flies were susceptible to
ampicillin, tetracycline, chloramphenicol, gentamycin and the
3rd generation cephalosporines.
All selected isolates were susceptible to aminoglycosides and
trimethoprim-sulfamethoxazole.
Denisson and Morris (2002) reported that an isolate from an
infected vascular graft and thigh
wound was resistant to ampicillin, gentamicin and cefotaxime.
The prevalence of antibiotic
resistance among isolates of C. sakazakii may relate to the
increasing trend of antibiotic
resistance among Enterobacter species. The intensive units of
antibiotics applied to patients
impart selective pressure on the isolates in infection sites and
result in increasing antibiotic
resistance among those isolates. Moreover, transmission of
antibiotic resistance is considered
through a transpron among antibiotic resistant isolates and
antibiotic susceptible isolates
(Lehner, 2004).
1. 7 Virulence factors
C. sakazakii causes life threatening bacterial infections in low
birth weight, premature and
immunosuppressed infants. There were at least 111 cases and 26
reported deaths resulting
from C. sakazakii infection worldwide reported 1958. Infant
formula contaminated by C.
sakazakii was associated with at least five infections in the US
and Europe from 1958 to 2010
(Anonymous, 2010).
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15
Potential virulence factors are required to evaluate the
pathogenicity of C. sakazakii to
humans. Successful colonization, establishment and ultimately
production of disease are
properties necessary for a pathogenic microorganism to cause a
disease epidemically. To date
only a few studies were conducted to examine the mechanisms of
pathogenicity for C.
sakazakii.
Pagotto et al. (2003) used 18 clinical and foodborne isolates of
C. sakazakii and the
suckling mice assay to evaluate enterotoxin production. The
tested strains were lethal to
suckling mice at a dose of 108 CFU. In in vitro assays, CHO,
Vero and Y-1 cells exhibited
lysis and loss of cell morphology in the presence of C.
sakazakii strain LA filtrates, which
may contain enterotoxin produced by the bacteria. Four out of
eighteen C. sakazakii strains
were positive for enterotoxin production. The adherence of 50 C.
sakazakii strains to
epithelial cell lines HEp-2, Caco-2 and brain microvascular
endothelial cell line (HBMEC)
were examined by Mange et al. (2008). Diffusion and formation of
localized clusters of
C.sakazakii on the cell surfaces were observed for three cell
lines. C. sakazakii infection is
linked with outbreaks of neonatal meningitis and necrotizing
enterocolitis (Anonymous,
2010). The mortality rates of meningitis and necrotizing
enterocolitis caused by C. sakazakii
infection are 10-55% and 40-80%, respectively. (Iversen and
Forsythe, 2003).
Necrotizing enterocolitis (NEC) resulting from consumption of
powdered infant formula
is common in neonates and characterized by intestinal necrosis
and pneumatosis intestinalis.
In an NEC outbreak in 1998, a total of 11 C. sakazakii strains
were isolated from stomach
aspirate, anal swabs, and blood (Van Acker, 2001).
The first reported case of meningitis due to “yellow pigmented
E. clocae” occurred in
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16
England in 1961 (Iversen and Forsythe, 2003). Like most
microorganisms, in order to cause
meningitis, C. sakazakii must adhere to and colonize intestine
surfaces, translocate into the
blood stream, escape from host immune system, cross the
blood/brain barrier, and survive in
the cerebral spinal fluid. Most reported outbreaks of meningitis
due to C. sakazakii are
resulting from hospital nurseries and neonatal intensive care
units (Iversen and Forsythe,
2003). Muytjens et al. (1983) reported eight cases of neonatal
meningitis occurred in the
Netherlands during the last six years with 75% fatality. It is
probable that C. sakazakii
exhibits developmental dependence on access to the central
nervous system (Iversen and
Forsythe, 2003).
1.8 Detection methods
The FDA recommended detection protocol is a presumptive, most
probable number
(MPN) assay based on three tubes of enrichment broth to allow
the detection of small
population of C. sakazakii present in reconstituted infant
formula. The approved method
requires five days to complete. Figure 1 illustrates a summary
of the method. This method
was first used by Mutjyens (1988a). Mutjyens (1988a)
reconstituted infant formula powder
with buffered peptone water rather than distilled water, and
subcultured growth in sheep
blood agar and eosin-methylene blue agar rather than trypticase
soy agar before the API
biochemical tests. The FDA recommended method involves three
main elements:
preenrichment, enrichment and selection. In the preenrichment
step, a total of 333 g infant
formula powder is reconstituted with distilled water overnight
at 36oC, followed by
enrichment in Enterobacteriaceae Enrichment (EE) broth for
overnight at 36oC. Point one ml
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17
or loopful culture is spread on violet red bile glucose (VRBG)
agar and incubated at 36oC
overnight to select presumptive Enterobacteriaceae. The
presumptive positive clonines are
subcultured on trypticase soy agar (TSA) and incubated for 42 to
72 h at 25oC to encourage
the yellow pigment production. The yellow pigmented colonies are
inoculated into the API
20E system and incubated at 36oC for 18 – 24 h to subject to the
biochemical identification
(U.S. FDA, 2002; Figure 1). API 20E system can determine
determine the metabolic
capabilities, the genus, and species of enteric bacteria in the
family Enterobacteraceae by
reactions in miniaturized test couples with different agents The
detection limit of this method
is < 1 cell in 25 g (how can it be less than 1 cell? )infant
formula powder (Mansfield, 2000).
In this protocol, VRBG and TSA are not specific for C.
sakazakii, time consuming and is not
sensitive.
Muytjens et al. (1984) first demonstrated C. sakazakii isolates
produce α-glucosidase
while other isolates of E. cloacae, E. aerogenes and E.
agglomerans do not produce α-
glucosidase by performing α-glucosidase test. The special
α-glucosidase activity indicates
that the detection of α-glucosidase can be used as a biomarker
of rapid detection and
differentiation of C. sakazakii from other Enterobacter species.
Several selective media are
formulated based on α-glucosidase production. Iversen, Druggan
and Forsythe developed a
chromogenic medium (Druggan-Forsythe-Iversen, DFI) for the
selective detection of C.
sakazakii. DFI medium detects α-glucosidase activity by using
5-bromo-4 chloro-3-indolyl-α,
D-glucopyranoside (XαGlc). C.sakazakii hydrolyzes XαGlc to an
indigo pigment, producing
blue-green colonies on this medium (Iversen et al., 2004b).
Compared to FDA recommended
methods, the DFI medium requires three days to complete
detection instead of five days
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18
(Iversen et al., 2004b). DFI medium exhibits improved accuracy
because it is specific for C.
sakazakii and differentiates C. sakazakii from other
Enterobacter species. Restaino et al.
(2005) used two chronogenic substrates,
5-bromo-4-chloro-3-indoxyl-β-D-cellobioside and
5-bromo-4-chloro-3-indoxyl- α-D-glucopyranoside as indicators of
α-glucosidase activity.
Pure cultures of C. sakazakii strains yield blue-black raised
colonies, and other enteric
organisms or Pseudomonas aeruginosa yield white, yellow, green
or clear colonies with or
without clear halos. The medium may give false positive readings
for Shigella sonnei or
Pantoea strain (Restaino, 2005). This new detection method
exhibited 100% sensitivity and
96.6% specificity for 240 samples (Restaino et al., 2005).
Oh and Kang (2004) developed a fluorogenic medium using
4-methylumbelliferyl-α-D
glucoside as a marker to exhibit α-glucosidase activity.
4-methylumbelliferyl-α-D glucoside
is digested by α-glucosidase into a free 4-methylumbelliferyl
moiety and is fluororecent when
exposed to UV radiation with a wavelength of 365 nm.
A cationic-magnetic-bead capture technique is used to detect C.
sakazakii by
combination charged paramagnetic beads and chromogenic medium.
The positively charged
magnetic beads interact with negatively charged
lipopolysaccharide on surface of Gram
negative bacteria. Capture by ionic interaction collects C.
sakazakii cells to achieve a
detectable concentration. This method needs 6-h enrichment at
42oC in buffered peptone
water, a 30 min cationic bead capture and selective culture on
DFI agar. This method can
detect 1 to 5 CFU cells in 500 g infant formula powder within 24
h (Mullane, 2006 a&b).
Detection by culturing methods is labor intensive, time
consuming and can have a low
sensitivity. Molecular based methods are developing to overcome
the limitations of
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19
phenotypic detection. Molecular based methods include
chromosomal DNA restriction
analysis, plasmid typing, ribotyping, pulsed field gel
electrophoresis (PFGE) and random
amplification of polymorphic DNA (RAPD).
Malorny developed a real time PCR targeting 16 rRNA gene for
specific detection of C.
sakazakii. This real time PCR assay is applied to diagnostic
detection of C. sakazakii in
infant formula with a 100% detection probability when the C.
sakazakii cell concentration is
103 CFU/ml (Malorny, 2005). Seo and Brackett (2005) developed a
real time PCR targeted
specific sequence within the macromolecular synthesis (MMS)
operon. The detection limit of
this newly developed assay is 100 CFU/ml in pure culture and in
reconstituted infant formula.
The assay can detect C. sakazakii with highly specificity
without any enrichment steps (Seo
and Brackett, 2005).
Bacterial typing systems are based on the principle that
clonally related isolates share
characteristics by which they can be differentiated from
unrelated isolates. Nazarowec-White
and Farber (1999) clustered C. sakazakii infant formula and
clinical isolates by using
phenotypic (biotype and antibiograms) and genotypic (ribotyping,
RAPD and PFGE)
methods. Clark et al. (1990) used a combination of typing
methods (plasmid analysis,
antibiograms, chromosomal restriction endonuclease analysis,
ribotying and multilocus
enzyme electrophoresis) to evaluate the isolates from patients
and infant formulas and
determine their relatedness. These methods trace the isolates
from patients back to the source
infant formula by comparing the typing patterns of the
isolates.
Molecular based methods are valuable analytical tools to
identify and trace the source of
contamination of infant formula. These methods are required to
be standardized and verified
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20
to apply in epidemiological research of C. sakazakii
infection.
1.9 UV radiation treatments
Ultraviolet radiation is well developed for water treatment, air
disinfection and surface
decontamination. The US Food and Drug Administration (FDA)
approved UV light as an
alternative treatment to thermal pasteurization of fresh juice
products (US FDA, 2000). The
performance criterion defined by FDA for fruit and vegetable
juice processing is a five log 10
reduction in the number of the target pathogen of concern (US
FDA, 2000).
UV radiation processing involves the use of radiation from the
UV region of the
electromagnetic spectrum for purposes of disinfection (US FDA,
2009). The wavelength
range from 100 to 400 nm is classified as UV radiation. UV
radiation is subdivided into
vacuum UV (100 to 200 nm), UVA (315 to 400 nm), UVB (280-315 nm)
and UVC (200 to
280 nm) (Srikanth, 1998). The vacuum UV has high energy and
produces reactive radicals,
but most radiation with wavelengths in this range is blocked by
ozone. UVA is the largest
contributor of UV in sunlight and responsible for skin tanning.
UVB penetrates the skin and
leads to skin cancer. UVC effectively inactivates bacteria and
viruses by damaging DNA
within defined germicidal range. Inactivation of microorganism
by UV radiation is achieved
through absorption of UV radiation by DNA molecules and the
dimerization of thymine bases.
A limitation of UV radiation to food processing is its poor
transmissivity within a food
product. The geometric configuration of reactors, the power,
wavelength and arrangement of
UV sources, and the geometric and chemical properties of
products influence the effective
inactivation of microorganisms. UV radiation is used with other
processing technologies,
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21
such as chemical disinfectants (Ha, 2010), oxidizing catalysts
(Kim, 2009) and powerful
oxidizing agents (Hadjok, 2008) that may exhibit synergistic
effects for inactivating
microbial pathogens.
Ha et al. (2010) reported that combined treatments of UV
radiation and ethanol
inactivate Bacillus cereus, C. sakazakii, Staphlococcus aureus,
Escherichia coli and
Salmonella enteric Typhimurium in bovine albumin solutions.
Hadjok et al. (2009)
demonstrated that treating fresh lettuce with combined aqueous
hydrogen peroxide and UV
radiation inactivates Salmonella, E.coli O157:H7, Pectobacterium
carotovora and
Pseudomonas fluorescens on the surfaces and within fresh produce
without quality
deterioration (Hadjok, 2008). Jung (2008) applied combined UV
radiation and ozone
treatments to inactivate Bacillus subtilis spores and obtain a
synergistic effect, because ozone
absorbs UV radiation and produces radicals, which exhibit a
powerful oxidizing effect. Kim
et al. (2009) investigated the efficacy of titanium dioxide – UV
photocatalytic disinfection
treatment on the shelf life of iceberg lettuce. Titanium dioxide
produces hydroxyl radicals by
photocatalytically reacting with UV radiation, providing a
germicidal effect. The TiO2-UV
system inactivates more pathogens than UV treatments or NaOCl
treatments alone and
inhibits the growth of survivors at 4 and 25 oC (Kim, 2009).
UV radiation is well established in liquid food processing.
Parts of water receive a UV
radiation exposure of at least 400 J/m2 at 254 nm to reduce
human pathogen by at least four
logs (Bernhardt, 1994). UV radiation sensitivity of
microorganisms is a key factor affecting
efficacy of UV treatment on microorganisms in liquid foods.
Microorganism sensitivity is
variable due to cell wall structure and composition, proteins
and nucleic acid structures.
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22
Generally, bacterial spores are the most resistant forms. Gram
positive bacteria are more
resistant than Gram negative bacteria (Koutchma, 2009).
Physico-chemical parameters are
also considered in UV reactor design to increase bacteria
inactivation efficacy including
fluid dynamic parameters, transmissivity and absorptive
properties.
Chemical components in a liquid system reduce antimicrobial
activity of UV
treatments. For example, dissolved organic solutes and compounds
such as fructose,
sucrose, glucose and malic acid, attenuate bacteriacidal effects
by absorbing UV radiation
(Fan and Geveke, 2007). Suspended solids scatter, absorb and
block UV radiation as well
as protect bacteria by providing particulate surfaces for
aggregation (Christenen and
Linden, 2001). Thus, the homogeneity of the flow pattern should
be controlled to achieve
uniform inactivation. Product composition, solids content,
color, starches and other
chemical properties of foods are also major factors that
influence bacterial inactivation.
Compared to water, a food matrix is a complex system and many
factors must be
manipulated to obtain ideal bacteria inactivation similar to
bacterial inactivation in water.
Some components in food sensitive to UV radiation must be
considered to minimize
quality changes. Vitamin A, carotenes, cyanobalamin (vitamin
B12), vitamin D, folic acid,
vitamin K, riboflavin (vitamin B2), tocopherols (vitamin E),
tryptophan, unsaturated fatty
acids and phospholipids are “light sensitive” (Spikes, 1981).
These chemicals in foods easily
absorb photons and promote reactions to break or form bonds.
Milk and milk products are
highly light sensitive. UV radiation applied in milk industry
may result in off-flavors and
nutrients loss.
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23
1.10 Fourier Transform Infrared Spectroscopy
The infrared light originates from laser source is absorbed by
analyzing samples. The
functional groups in macromolecules change vibrational
stretching structures and can be
observed in the mid-infrared (4,000 to 400 cm-1) spectral
regions. Thus, the chemical
composition of bacterial membranes is derived from FT-IR
spectrum.
Application of vibrational spectroscopy for rapid determination
of food contamination is
increasing in recent years due to short and non-destructive
sample preparation steps and
quick analysis times. Several types of vibrational spectroscopy
are used to detect microbial
contamination in food, including mid-infrared (mid-IR, 4000-650
cm-1) spectroscopy and
Fourier transform infrared (FT-IR, 4000 – 400 cm-1)
spectroscopy. Compared to classical
microbiology methods, PCR-based molecular methods and
antigen-antibody immunology
reaction based methods, FT-IR can detect and potentially
quantify bacteria at populations of
105 CFU/g powder in food powders within 5 min instead of days.
Furthermore, spectroscopic
methods are being explored to study inactivation mechanism by
identifying changes in
composition of the cell membranes and DNA.
These spectroscopic techniques are employed to study bacterial
injury after inactivation
treatments such as sonication (Lin et al., 2004), heat
(Al-Qadiri et al., 2008) or antibiotics
(Neugebauer et al., 2007). Lin et al. (2004) employed FT-IR
(4000-600 cm-1) to discriminate
intact and sonication-injured Listeria monocytogenes ATCC 19114
by segregation of intact
and injured strains through principal component analysis.
Al-Qadiri et al. (2008) detected
sublethally heat-injured microorganisms Salmonella enterica
Typhimurium and Listeria
monocytogenes by FT-IR spectroscopy (4000-600 cm-1). The
analysis of spectra also revealed
different cell injury mechanisms for Gram positive and Gram
negative bacteria. FI-IR and
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24
Raman spectroscopy is applied to elucidate the action mode of
drugs in new drug
development. The cell composition changes of Gram positive
bacteria Staphylococcus
epidermidis occurring in the present of moxifloxacin drugs were
investigated by vibrational
spectroscopy (Neugebauer, 2007).
Although FT-IR spectroscopy is an attractive method to
differentiate biochemical
composition before and after disinfection, many factors must be
controlled to increase
sensitivity. Cell recovery methods and filtration techniques are
key factors to minimize
detection limits (Lu and Rasco, 2010). A filtration membrane
that can remove spectral
interference produced by media residue and obtain a uniform
layer of bacteria cells is ideal.
Application 500 μl of the bacterial suspension onto an aluminum
oxide membrane filter of
0.2 μm pore size and 25 mm outer diameter is widely used
filtration technique (Lu and Rasco,
2010).
Davis isolated Samonella enteric serovars from chicken breast by
filtration and
immunomagnetic separation and collected the FT-IR spectra
(Davis, 2010). The physiological
state of the microorganisms and culture medium also contribute
variability in the FT-IR
spectra (Lu and Rasco, 2010). Use of microbes in the stationary
phase is generally
recommended for FT-IR spectroscopy because spectral features
will be more consistent due
to the stable physiological state (Lu and Rasco, 2010, al-Qadiri
et al. 2007). Van der Mei et
al.(2004) and Filip et al. (2008) reported that the culture age,
composition of the media and
quantity of nutrients can influence spectral features.
In conclusion, dry infant formula contaminated by C. sakazakii
is of great concern to the
public. It is extremely important to explore novel methods to
inactivate C. sakazakii in dry
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25
infant formula. In this study, the efficacy of UV radiation for
inactivating C. sakazakii in dry
infant formula was evaluated. The efficacy of combined treatment
was evaluated by
reconstituting dry infant formula exposed to UV radiation in hot
water at 55, 60 and 65oC.
The changes in cell membranes and morphology of the bacterial
pathogen were investigated
by FT-IR and electron microscopy.
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26
2 Materials and Methods
2.1 Bacterial strains, culture methods and preparation of stock
cultures
Three strains of C. sakazakii (American Type Culture Collection
[ATCC] 51329, 29544
and 12868) were obtained from the School of Food Science culture
collection at Washington
State University (Pullman, WA, USA). All bacteria strains were
stored at -80oC in tryptic soy
broth (TSB, Difco Laboratories, Detroit, MI) with 15% glycerol.
Frozen cultures of the three
strains were streaked onto tryptic soy agar (TSA, Difco
Laboratories, Detroit, MI) and
incubated 24h at 37oC. The grown cultures were stored at 4oC for
routine use. Cultures of
each strain were combined together to construct a culture
cocktail and harvested by
centrifugation at 4,000 rpm for 20 min at room temperature
(centra CL2, 4675×g,West
Chester, PA). Cultures were washed twice with buffered peptone
water (Difco). The pellets
were resuspended in buffered peptone water, corresponding to
~108 to 109 CFU/ml.
2.2 Dry infant formula sample preparation and inoculation
Dry infant formula powder (Enfamil, Mead Johnson & Company,
Evansville, IN) was
purchased from a local grocery store. Five grams of dry infant
formula powder was placed
into a Petri dish (150×15 mm, Becton Dickinson & Co.,
Franklin Lakes, NJ) and spread into
a layer of ca 0.032 mm. One hundred microliters of the cocktail
was inoculated to dry infant
formula powder by depositing droplets in five locations with a
micropipette. The dry infant
formula was dried in a laminar flow biosafety hood with the fan
running for 30 min. After
drying, clumps of inoculated infant formula powder were crushed
with a sterile spatula and
thoroughly shaken to produce a homogeneous dispersal of
inoculums throughout the dry
infant formula.
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27
2.3 Ultraviolet radiation treatment
The UV light chamber for treatment of dry infant formula was
custom built at the School
of Food Science, Washington State University (Pullman, WA). The
chamber contains two
45.4 cm-long UV lamps 10 cm apart (Sun Ray Technologies, Inc.
Killington, VT). The lamps
are suspended across the chamber with a distance of 15 cm from
the base of the chamber. UV
intensity at 253.7 nm was determined by a UV radiometer
(Steril-Aire, Burbank, CA) at the
surface of the Petri dish. The radiometer sensor was placed
under the UV lamp with the lamp
on for 30 min to obtain a consistent reading. The interior of
chamber is lined with a highly
reflective metal foil (Sun Ray Technologies) to increase UV
intensity and to minimize any
shadowing effect on irregular surface shaped dry infant formula
samples. Plates of inoculated
dry infant formula were individually subjected to UV treatment.
The intensity was kept
constant and selected exposure times were applied to allow
respective doses (Table 6). The
Petri dish was shaken at selected time intervals to expose dry
infant formula granules to UV
radiation more evenly. To analyze the influence of layer
thickness on UV radiation
inactivation efficacy, 5, 15 and 25 g of inoculated dry infant
formula were placed onto Petri
dishes to obtain layer thickness of 0.032, 0.16 and 0.32 cm.
2.4 Combined UV radiation and hot water treatment.
Five grams of inoculated and air dried infant formula was placed
into a Petri dish and
spread into a thin layer. After exposure to UV radiation for 20
min at room temperature, UV
radiation exposed infant formula was reconstituted in 10 ml
sterile distilled water at room
temperature in a stomacher bag and homogenized for 2 min with a
Seward stomacher
(Stomacher 80, Seward, London, UK). Hot water treatment was
conducted in a water bath
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28
(VWR Scientific, West Chester, PA) for selective time intervals
at 55oC, 60oC and 65oC. The
temperature of the dry infant formula samples was monitored by a
type omega thermocouple
(OMEGA Engineering Inc., Stamford, CT) and temperature
controlled within ±0.5oC. The
treatment time was controlled by a stopwatch (Thermo Fisher
Scientific Inc., Waltham, MA).
The reconstituted infant formula was removed from the water bath
and immersed in ice
immediately after each treatment to stop heating.
2.5 Bacterial enumeration.
UV treated dry infant formula powder was rehydrated as described
previously. One
milliliter sample aliquots of reconstituted infant formula were
serially diluted tenfold in 9 ml
of sterile 0.2 % peptone water (Difco) and 0.1 ml of samples
were spread-plated onto Petri
plates of OK Medium (OK; Acumedia, Lansing, MI), a medium for
selective recovery and
enumeration of C. sakazakii. The bacteria were enumerated on to
TSA to recover the injured
cells for the combined treatments of UV radiation and hot water.
Agar plates were incubated
at 37°C for 24 h and colonies were enumerated. Colonies
fluorescing when illuminated with
365 nm UV light were counted.
2.6 Membrane filtration and FT-IR spectroscopy.
Non fat milk powder was used as a surrogate for dry infant
formula for UVC treatments to
eliminate the background noise raised by large fat molecules.
Two one hundredths of a gram
of non fat milk was dissolved in 50 ml of sterile saline water
(0.85% w/v) and filtered
through an aluminum oxide membrane filter (0.2 μm pore size, 25
mm OD) (Anodisc,
Whatman Inc., Clifton, NJ) using a Whatman vacuum glass membrane
filter holder
-
29
(Whatman Catalog No. 1960-032) to harvest bacterial cells. The
anodisc filters were removed
from the filtration apparatus and air-dried under laminar flow
at room temperature (ca. 22°C)
for 10 min to allow a homogeneous film of bacterial cells to
form.
FT-IR spectra were collected using a Nicolet 380 FT-IR
spectrometer (Thermo Electron
Inc., San Jose, USA). The aluminum oxide membrane filter coated
with a uniform and thin
layer of bacterial cells was placed in direct contact with the
diamond crystal cell (30,000 –
200 cm-1) of attenuated total reflectance (ATR) detector.
Infrared spectra were recorded from
4002 to 399 cm-1 at a resolution of 8 cm-1. Each spectrum was
acquired by adding together 32
interferograms. Five spectra were acquired for intact and UV
treated C. sakazakii at different
locations on the aluminum oxide filter for a total of 15 spectra
for each group of cells.
Triplicate experiments (N=3) were conducted and spectra from the
first two experimental
runs were used to establish chemometric models while the spectra
from the third experiment
were used for model validation.
2.7 Electron microscopy analysis.
Scanning electron microscopy (SEM) was performed to examine
morphological changes
of C. sakazakii cells before and after UV irradiation treatment
of dry infant formula powder.
The dry infant formula sample was reconstituted in sterile water
at room temperature and C.
sakazakii cells were obtained by centrifuge. First, C. sakazakii
cells were fixed with 2%
glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer
overnight. The fixed cells
were rinsed with double distilled water, followed by freeze
drying in a Virtis Lyophilizer (The
Virtis Co., Inc., Gardiner, NY, USA). The freeze dried cells
were mounted onto SEM stubs
and sputter-coating with gold. The coated cells were observed
under a FEI Quanta 200F Field
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30
Emission scanning electron microscope (Field Emission
Instruments, Hillsboro, OR, USA)
using an accelerating voltage of 30 kV.
Transmission electron microscopy (TEM) was employed to study the
influence of UV
radiation on the inner structures of C. sakazakii cells.
Uninoculated infant formula powder
was designated as a control. C. sakazakii cells in dry infant
formula untreated and treated
with UV radiation were placed into the primary fixative
overnight at 4°C. The fixed cells
were rinsed three times with 0.1M phosphate buffer (10 min each)
and post-fixed by 2%
osmium tetroxide for 2 h at room temperature. The post fixed
cells were quickly rinsed twice
with 0.1 M of buffer (10 min each), followed by dehydration with
sequential ethanol
solutions (30%, 50%, 70%, 95%, and 100%), and 100% propylene
oxide twice (10 min each).
The bacteria cells were infiltrated by propylene oxide: Spurr’s
(1:1) overnight and 100%
Spurr’s twice (overnight each). The samples were embedded in
Spurr’s resin. The
composition of Spurr’s is vinyl ten gram of cyclohexene dioxide,
six gram of diglycidyl ether
of polypropylene glycol, twenty six gram of nonenyl succinic
anhydride, and four tenth gram
of dimethylaminoethanol (Spurr, 1969). The cells were observed
in Philips electron
microscope (Field Emission Instruments, Hillsboro, OR, USA)
operating at 200 kV.
2.8 Data preprocessing and chemometrics.
Infrared spectra were first pre-processed by EZ OMNIC 7.1 a
(Thermo Electron Inc.).
Relevant background was subtracted from each raw spectrum.
Automatic baseline correction
was employed to flatten the baseline, following by a smooth of
five (Gaussian function of
9.643 cm-1). The pre-processed spectra were read by Matlab 2010a
(Math Works Inc., Natick,
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31
MA, USA) with .xls format by Excel (Microsoft Inc., Redmond, WA,
USA). The
reproducibility of vibrational spectra from four independent
experiments (N=3) was
investigated by calculating Dy1y2 according to equations (1) and
(2) (Wang et al., 2010). In the
equations, y1i and y2i are signal intensities of two selected
spectra while 1y and 2y are mean
values of signal intensities of two selected spectra; n
represents the data points in the selected
wavenumber region. The Dy1y2 ranges from 0 to 2000. The smaller
values define good
reproducibility of spectra. Zero means the two spectra are
identical; One thousand means that
the two spectra are totally unrelated. Two thousand means the
two spectra are negatively
related.
ry1y2=∑∑
∑
==
=
−−
−
n
ii
n
ii
n
iii
ynyyny
yynyy
1
22
22
1
21
21
12121
(1)
Dy1y2 =(1- ry1y2)1000 (2)
Second derivative transforms (with a gap value of 10 cm-1) were
performed for spectral
processing in Matlab. Principal component analysis (PCA) was
employed . PCA is an
unsupervised chemometric tool to reduce the dimensionality of
multivariate data while
preserving most of the variances and provides a two or three
dimensional cluster of results for
group segregation (Lu et al., 2010).
2.9 Statistical analysis.
All experiments were repeated three times with duplicate
samples. Data were analyzed by
one-way analysis of variance (ANOVA, P≤0.05) followed by T-test
using Matlab.
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32
3 Results and Discussion
3.1 C. sakazakii inactivation effect of UV radiation
UV radiation for 25 min of dry infant formula was the most
effective treatment with a
1.38 log10 CFU reduction per gram. A curve of C.sakazakii
survival population versus UV
radiation treatment time was plotted (Figure 2). An S-shaped
survival curve was observed
with initial rapid inactivation in the first five minutes
followed by a period of rapid
inactivation at 25-30 min. The initial rapid inactivation
resulted from the UV radiation
exposure and the second rapid inactivation may result from the
raised temperature in the
chamber after 20 min. After 20 min of UV radiation treatment,
the temperature in UV
chamber was about 37oC. Complete inactivation was not be
achieved because the UV
radiation did not completely penetrate the dry infant
formula.
The germicidal effect of UV radiation is influenced by food
matrices and the
physiological state of bacteria. Although UV radiation is well
established for sanitation of air,
water, liquid food pasteurization and surface decontamination,
UV radiation of powdered
food was investigated. U.S. FDA recommendations state that to
achieve a four-log microbial
inactivation, the UV radiation exposure must be at least 400
J/m2 for all parts of the product
(U.S.FDA, 2009). In this experiment, a 25 min UV radiation
treatment led to a radiation dose
of ca 60.7 kJ/m2 (Table 6). The radiation dose required for
liquid food listed by FDA is too
small for food powders. Compared to water, powdered food
exhibits a range of optical and
physical properties and diverse chemical composition that
influences UV transmittance, dose
delivery, momentum transfer and consequently microbial
inactivation (Koutchma, 2009). The
transmittance of UV radiation in infant formula is much smaller
compared to transmittance in
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33
liquid food. Infant formula particles absorb and scatter UV
radiation due to chemical
composition, optical properties and shape. Moreover, the large
clumps formed during
inoculation can provide a site for the aggregation of bacteria
in the core of the clump
surrounded by infant formula particles (Koutchma, 2009). The
clumps protect C. sakazakii
cells from UV radiation. In infant formula processing, UV
radiation is preferred as a post
pasteurization process to avoid C. sakazakii contamination
resulting from poor environment
hygiene (Johler et al., 2010)
Sensitivity of microorganisms to UV radiation also influences
the efficacy of bacteria
inactivation. Cell wall structure, thickness and composition,
absorbent compounds and
nucleic acid structure are important to bacterial sensitivity
exposed to UV radiation
(Koutchma, 2009). C. sakazakii in the stationary phase produce
carotenoids known to
stabilize cellular membranes, influence cellular membrane
fluidity, scavenge reactive oxygen
species, and play a role in the survival of C. sakazakii in
stressful environments (Gruszecki &
Strzalka, 2005). Johler et al. (2005) identified genes involved
in pigment expression in C.
sakazakii strain ES 5 and reported mutants that cannot produce
yellow pigmentation were
more sensitive to UVB radiation than wild type C. sakazakii.
Lehner et al. (2006) applied
bacterial artificial chromosome approach and heterologous
expression of the yellow pigment
in Escherichia coli to elucidate the molecular structure of the
genes responsible for pigment
production in C. sakazakii ES 5. Lehner et al. (2006)
ascertained the carotenogenic nature of
the pigment by in situ visible microspectroscopy and resonance
Raman microspectroscopy.
Besides, trehalose, which improves desiccation tolerance of C.
sakazakii may also contribute
to UV radiation. Trehalose acts as a counterbalance to
extracellular osmotic pressure,
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34
stabilizing phospholipid membranes and proteins by replacing the
shell of water surrounding
the membranes and proteins preventing irreversible damage of the
cells (Mullane, 2006a).
After UV radiation treatments of 20 min with UV dose of 48.6
kJ/m2, the reduction of viable
microorganism on OK medium was 0.69, 0.25 and 0.07 log 10 CFU/g
infant formula with an
infant formula layer of 0.032, 0.16 and 0.32 cm, respectively
(Figure 3). Layer thickness is
an important factor influencing UV radiation inactivation
efficacy. The thincker the layer was,
the more bacteria survived through UV radiation treatment.
Oteiza et al. (2005) explained the
effect of apple juice thickness on efficacy of UV radiation as a
bactericide with the Lambert-
Beer Law:
P= P0 exp(-abc).
When UV radiation power of P0 penetrates a liquid, the food
matrix absorbs and scatters UV
radiation so the transmitted radiation power is reduced to P.
The transmittance of a solution is
the fraction of incident radiation transmitted by the solution,
T=P/ P0 (Oteiza, 2005). In this
equation, a is the specific absorptivity (l /mol/ cm), b is the
distance the radiation travels
through the sample (cm), c is the particle concentration of the
solution (mol/l) (Oteiza, 2005).
Due to the exponential nature of the Lambert-Beer law, the
transmittance power is reduced
rapidly with an increase in the travel distance of UV radiation.
As a result of UV radiation
power attenuation, the bactericidal effect of UV radiation is
reduced dramatically as the
clumping of the infant formula increased.
3.2 Combined treatments of UV radiation and heat inactivation C.
sakazakii in reconstituted infant formula
Inactivation effect of C. sakazakii in dry infant formula using
UV radiation and
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35
reconstituted in 55oC, 60oC or 65oC sterilized hot water was
evaluated. Table 7 presents D
values of C.sakazakii contaminating infant formula subjected to
hot water treatment only and
C. sakazakii contaminating infant formula subjected to UV
radiation and hot water treatment.
D values for C. sakazakii culture cocktail contaminating infant
formula reconstituted with 55,
60 or 65oC water were 3.99±1.99, 0.87±0.11, 1.10±0.55 min,
respectively. D values obtained
in this experiment were smaller than D values in previous
studies. Al-Holy et al. (2009)
reported D55 values for C. sakazakii ATCC 12868 and ATCC 29544
in reconstituted infant
formula were 14.21±3.58 and 14.83±3.50 min. D60 values for C.
sakazakii ATCC 12868 and
ATCC 29544 in reconstituted infant formula were 0.53±0.08 and
2.71±0.32 min in
reconstituted infant formula (Al-Holy et al., 2009). Inoculating
C. sakazakii cocktail culture
into dry infant formula and air drying may result in death and
injury of desiccation sensitive
cells (Edelson-Mammel, 2005).
D values for C.sakazakii in reconstituted infant formula after
UV radiation treatment are
1.88±1.05, 1.00±0.06 and 0.89±0.10 at 55, 60 and 65oC. Compared
to D55 and D65 values for
C. sakazakii in reconstituted infant formula subjected to hot
water treatments only, D55 and
D65 values decreased (Figure 4 a c).
Reconstituting dry infant formula with 70-90oC water can
contribute a four to six log
reduction of C. sakazakii (Chen et al., 2009). The highest water
temperature tested was less
than 70oC and was not achieved ideal C. sakazakii inactivation
effect before cooling down.
UV radiation treatment before reconstitution may be a method to
increase the hot water
treatment effect.
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36
3.3 FT-IR spectroscopy decoding and cluster analysis.
Due to interference of fat in dry infant formula, non-fat milk
powder was used for
spectral analysis. The fat content of dry infant formula is
24.9% w/w and 0 in non fat milk
powder. Traditional microbiological experiment was performed to
validate that there was no
significant difference (P < 0.05) of bacterial inactivation
by UV radiation treatment between
infant formula powder and non-fat milk powder. Figure 5 presents
the raw FT-IR spectral
features of non-fat milk powder and non-fat milk powder
inoculated with C. sakazakii.
Distinct differences on raw spectra were observed and provided
prerequisite for subtraction
control spectra form sample spectra (Lu and Rasco, 2010). The
intra-group variation of
spectral features is significantly (p < 0.05) smaller than
the inter-group variation of spectral
features. D values were calculated for each group and ranged
between 36.93 ± 15.13 and
76.32 ± 31.4, which demonstrates good reproducibility of
spectral features of each sample.
The differences of raw FT-IR spectra for UV radiation treated
and non-treated C.
sakazakii contaminating dry infant formula were not visually
discernible. Second derivative
transformation of raw spectra was applied to magnify small
variability (Figure 6 a, b). The
peak at 970 cm-1 is assigned to the symmetric stretching mode of
dianionic phosphate
monoesters in cellular nucleic acids (Argov et al., 2004). The
peak at 1080 cm-1 is assigned to
symmetric PO2- of nucleic acids (Wood et al., 1998). The peak at
1240 cm-1 is assigned to
P=O stretching (asymmetric) of > PO2- phosphodiesters from
nucleic acids (Naumann, 2001).
Spectral bands at 1080 and 1240 cm-1 reflect the functional
groups information of DNA. The
peak at 1317 cm-1 is assigned to amide III components of
proteins (Yang et al., 2005). The
peak at 1515 cm-1 is assigned to amide II (Lu et al., 2010a).
The peak at 1637 cm-1 is assigned
to amide I of β-pleated sheet structures (Naumann, 2001). The
peak at 1655 cm-1 is assigned
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37
to amide I of α-helical structures (Lu and Rasco, 2010). The
amide bands provide information
about α-helix, β sheet and random coil conformations in
proteins. The peak at 1400 cm-1 is
assigned to C=O symmetric stretching of COO- in proteins
(Naumann, 2001). The peak at
1455 cm-1 is assigned to symmetric bending modes of methyl
groups in skeletal proteins
(Fung et al., 1996). The peak at 1740 cm-1 is assigned to
>C=O stretching of esters (Naumann,
2001). Those bands at 1317, 1400, 1455, 1515, 1637, 1655 and
1740 cm-1 are related to
protein secondary structure (Figure 6a). The peak at 2850 cm-1
is assigned to C-H
symmetric stretching of >CH2 in fatty acids (Naumann, 2001).
The peak at 2918 cm-1 is
assigned to C-H asymmetric stretching of >CH2 in fatty acids
(Lu and Rasco, 2010). These
peaks at 2850 and 2918 cm-1 are related to fatty acids in
bacterial cell membranes (Figure
6b). Spectral variations indicated that the function of protein,
lipid and DNA are closely
associated with bacterial survival under treatment of UV
readiation.
Principal component analysis (PCA) was employed to segregate
control C. sakazakii
extracts from UV treated extracts (Figure 7). Cells from the
control treatment were tightly
clustered, but variations were observed among UV treatments
indicating differences in the
degree of cell injury.
Infrared spectroscopy was useful to monitor variability in cell
membrane composition
that is associated with bacterial injury and survival. Lin et
al. (2004) discriminated intact L.
monocytogenes cells from sonication-injured cells by ATR
spectroscopy using PCA
segregation clusters, noting that injury was attributed to
macromolecular shearing and
subsequent redistribution of cell wall components along with
possible denaturation of
intracellular proteins. Lu et al. (2010b) employed FT-IR coupled
with loading plot analysis
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38
and PCA segregation to sort bacterial injury populations after
cold and freeze treatments and
demonstrated that pathogens produce oligosaccharides and
potentially other components in
response to stress. Alvarez-Ordóñez and Prieto (2010) used FT-IR
and studied the
ultrastructure changes of Salmonella enterica cells under acid,
alkaline, heat and oxidative
stressed conditions. A wide variety of cellular compounds
involved in bacterial resistance to
unfavorable conditions (Al-Holy et al., 2006; Al-Qadiri et al.,
2008).
3.4 Electron microscopy analysis.
No detectable changes of cell surface were clearly delineated in
scanning electron
micrographs of UV radiation treated C. sakazakii (Figure 8). The
lack of clearly delineated
differences on cell surfaces was indicated little UV damage
following UV radiation treatment.
Cells were not clearly delineated in transmission electron
micrographs based on treatment
(Figure 9). The lack of no clearly delineated cells may be
attributed to decomposition of cells
from UV radiation. Casein particles were visible in C. sakazakii
micrographs.
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39
4 Conclusions
In this study, UV radiation dose in a range of 12.1 to 72.8
kJ/m2 inactivated C.
sakazakii in dry infant formula. UV radiation of 60.7 kJ/m2
resulted in a 1.38 log10 CFU/g
reduction at room temperature. UV dose required by FDA to
inactivate bacteria in liquid food
is too low for desiccation food powder. The UV radiation in
combination with 55oC and 65oC
hot water treatment decreases D values of C. sakazakii in
reconstituted infant formula. FT-IR
analysis suggested UV radiation results in structural changes in
protein, DNA and lipids of
cell injury. Electron microscopy is not an effective method to
investigate the cell injury
resulting from UV radiation. A high energy radiation is
considered as a pasteurization method
to inactivate C. sakazakii in dry infant formula (Lee et al.,
2006). Photocatalyst can be added
into food to increase UV radiation inactivation effect on
foodborne pathogen (Benabbou et al.,
2007). UV penetration material should be developed to increase
UV transmittivity.
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40
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