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EFFECTS OF PULSED LIGHT ON MICROBIAL DECONTAMINATION OF DRY FOOD POWDER AND FOOD PROCESSING AIDS
By
YING GUO
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2013
© 2013 Ying Guo
To my family and friends
4
ACKNOWLEDGMENTS
There are several people I would like to thank because all that I learned and
accomplished with this thesis is in great part of them. I would like to thank Dr. Wade
Yang, my major advisor, for bringing me into his research group and being supportive
for my study and research. I would also like to thank my other committee members Dr.
Paul Sarnoski and Dr. J. Glenn Morris for their assistance and time dedicated to my
project. Additionally, I would like to thank Dr. Soohyoun Ahn for generously letting me
use her lab facilities.
I also want to express my gratitude to Dr. Anita Wright, Braulio Macias and Lei
Fang for teaching me all the techniques in microbiology; Senem Guner and Akshay
Anugu for being such great tutors, who guided me through using all the facilities and
experimental procedures. And my close friends Syed Abbas, Shuang Wu and Jennifer
Lee, who have always been a huge emotional support for me, without them, I could
hardly have accomplished this. Moreover, I would like to thank Dr. Yavuz Yagiz and
Changmou Xu for helping acquire the data from the color analysis.
I would also like to thank my labmates, Xingyu Zhao, Bhaskar Janve, Tara
Faidhalla, Samet Ozturk, Abeer Alhendi and Kane Smith for being there as an
inspiration.
Finally, I would love to thank my parents, for their unconditional love and support
throughout my life.
5
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
LIST OF ABBREVIATIONS ............................................................................................. 9
ABSTRACT ................................................................................................................... 11
CHAPTER
1 INTRODUCTION .................................................................................................... 13
Food Powders ......................................................................................................... 13
Nutritional Values ............................................................................................. 13 Safety Concern ................................................................................................. 14
Decontamination Method ........................................................................................ 16
Pulsed Light Technology......................................................................................... 16 Inactivation Mechanisms .................................................................................. 17
Advantages and Disadvantages ....................................................................... 17
2 JUSTIFICATION OF STUDY .................................................................................. 19
3 OBJECTIVES ......................................................................................................... 22
4 REVIEW OF LITERATURE .................................................................................... 23
Food Powders ......................................................................................................... 23
Problems and Issues .............................................................................................. 24 Decontamination Methods ...................................................................................... 26
Chemical Methods ............................................................................................ 27 Ethylene Oxide and Derivatives ................................................................. 27 Ozone ........................................................................................................ 29
Thermal Methods ............................................................................................. 30
Infrared heating .......................................................................................... 30 Microwave .................................................................................................. 31 High temperature short time process ......................................................... 32
Non-Thermal Methods ...................................................................................... 33 Pulsed light ................................................................................................ 33 Decontamination of food-contact surface. .................................................. 36 Allergen mitigation ..................................................................................... 37 Irradiation ................................................................................................... 37
6
High hydrostatic pressure .......................................................................... 38
5 MATERIALS AND METHODS ................................................................................ 45
Sample Preparation ................................................................................................ 45
Growth Study .......................................................................................................... 45 Preparation of Inoculum .......................................................................................... 46 Inoculation .............................................................................................................. 47 Pulsed Light Treatment ........................................................................................... 47 Temperature Profile Measurements ....................................................................... 48
Microbial Recovery procedure and enumeration .................................................... 48 Total Aerobic Count .......................................................................................... 48 Yeast and Mold Counts .................................................................................... 49 Bacillus subtilis Count ...................................................................................... 49
Color Analysis ......................................................................................................... 50 Water Activity Measurements ................................................................................. 51
Statistical Analysis .................................................................................................. 51
6 RESULTS AND DISCUSSION ............................................................................... 54
Growth curve study of B. subtilis ............................................................................. 54
Efficiency of Microbial Decontamination ................................................................. 54 Evaluation of PL Efficiency in Inactivate B. Subtilis on Wheat Flour and
Black Pepper ................................................................................................. 54 Yeast Molds Inactivation and Total Aerobic Count ........................................... 57
Temperature Measurement .................................................................................... 59
Water Activity Measurement ................................................................................... 61
Color Analysis ......................................................................................................... 62
7 CONCLUSIONS AND RECOMMENDATIONS ....................................................... 76
LIST OF REFERENCES ............................................................................................... 78
BIOGRAPHICAL SKETCH ............................................................................................ 90
7
LIST OF TABLES
Table page 4-1 Concentration of B. cereus SIK 340 spores and natural background flora. ........ 40
4-2 Survival of natural microbial contaminations in black pepper powder following gamma radiation and microwave treatments. Legend: - No Growth; + Growth; ++ More Growth; +++ Heavy growth (Omar and others 1995). ............. 41
4-3 Characterization of different UV components (Krishnamurthy 2006). ................. 42
6-1 Influence of two different distance and treatment time on two different samples .............................................................................................................. 64
6-2 Temperature profile of two different distance (6 cm and 9 cm) at various exposure time ..................................................................................................... 65
6-3 Effects on water activity of wheat flour and black pepper ................................... 66
6-4 Effects on color values and color difference of wheat flour with PL treatment .... 67
8
LIST OF FIGURES
Figure page 4-1 The HTST process procedure for seed and food powder decontamination ........ 43
4-2 Schematic of pulsed light equipment .................................................................. 44
5-1 Schematic diagram of continuous PL system (Model LH840-LMP-HSG) ........... 52
5-2 Energy dose (fluence) during PL treatment as measured by a radiometer ......... 53
6-1 Growth curve of Bacillus subtilis ATCC 6633 in TSB. ......................................... 68
6-2 Bacillus subtilis (●) growth curve measured by spectrophotometer at 600 nm . 69
6-3 Bacillus subtilis vegetative cells a). untreated samples; b) treated by PL at 3000 V, 1 Hz, 10 J cm2 / flash ............................................................................ 70
6-4 Survivor curves for B. subtilis treated with PL at distances of 6 cm and 9 cm from the quartz window for wheat flour sample .................................................. 71
6-5 Survivor curves for B. subtilis treated with distance of 6 cm and 9 cm from the quartz window for black pepper sample ....................................................... 72
6-6 Temperature profile of wheat flour under different distance (6 cm and 9 cm) and exposure times ............................................................................................ 73
6-7 Temperature profile of black pepper under different distance (6 cm and 9 cm) and exposure times ............................................................................................ 74
6-8 Black pepper after under different treatment time at 6 cm (distance from sample to quartz window .................................................................................... 75
9
LIST OF ABBREVIATIONS
ANOVA Analysis Of Variance
AOAC Association of Official Agricultural Chemists
CDC Centers for Disease Control and Prevention
CMVS Color Machine Vision System
DHA Docosahexaenoic Acid
DNA Deoxyribonucleic acid
E-beam Electron beam
EBH Ethylene bromohydrin
ECH Ethylene chlorohydrin
EO Ethylene oxide
ERH Equilibrium relative humidity
FDA Food and Drug Administration
HHP High Hydrostatic Pressure Processing
HIV Human Immunodeficiency Virus
HTST High Temperature Short Time
HUS Hemolytic Uremic Syndrome
IR Infrared
kGy kilo Grays
PL Pulsed Light
PW Peptone Water
RF Radio Frequency
SAS Statistical Analysis System
10
TAC Total Aerobic Count
TSA Tryptic Soy Agar
USDA United States Department of Agriculture
UV Ultraviolet
11
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
EFFECTS OF PULSED LIGHT ON MICROBIAL DECONTAMINATION OF DRY FOOD
POWDER AND FOOD PROCESSING AIDS
By
Ying Guo
December 2013
Chair: Wade Yang Major: Food Science and Human Nutrition
Food powders provide convenience and nutritional value for consumers while
also providing manufacturers a manageable food form with a long shelf life and easy
transportation. Food powders, however, carry food safety risks because of their high
nutritional value, which has led to food outbreaks in the past. Due to the high nutritional
and aroma requirements of food powders, thermal decontamination methods are not
suitable. Furthermore, it is not easy to remove the chemical residue because of the
special physical properties of dry food powder. Pulsed light (PL) technology effectively
inactivate the spoilage microorganisms while maintaining the quality of the products. In
the present study, the efficacy of PL inactivation on Bacillus subtilis and investigated the
molds and yeast and total aerobic counts (TAC) has been evaluated on wheat flour and
black pepper. Wheat flour and black pepper were inoculated with B. subtilis and treated
with PL (3 pulses/s) at distances of 6 cm and 9 cm from the quartz window of the PL
system. Wheat flour samples were treated for 15 s, 30 s, 60 s and 90 s. For black
pepper, reduced times were viable and therefore the times were 5 s, 10 s, 15 s and 30
s. Natural flora were used in the molds and yeast counts and TAC. All the trials were
conducted at the room temperatures (25 °C), and the temperature was monitored during
12
PL treatment. The quality parameters assessed were changes in color and water
activity. Results reveal significant reduction (p<0.05) in microbial load after PL
treatment. Maximum reduction attained in TAC of wheat flour is 2.5 ± 0.8 log CFU/g;
while for black pepper, TAC can reach 3.2 ± 0.2 log CFU/g reduction. Furthermore, the
maximum inactivation attained for Bacillus subtilis is as high as 4.1 ± 0.2 log CFU/g on
wheat flour and 4.3 ± 0.1 log CFU/g on black pepper after 90 s and 30 s, respectively,
with a distance from quartz window to sample at 6 cm. Likewise, temperature increased
significantly (p<0.05) after the treatment, especially on black pepper. Meanwhile, a
dramatic decrease in water activity, which enhanced the effects on further storage and
decreased the potential microbial contamination. There were, however, no significant
differences (p>0.05) observed in color. In conclusion, PL may be an effective and
practical method for enhancing the safety of food powder and dry ingredients while
preserving the food quality, which would have a positive impact on food safety and
public health.
13
CHAPTER 1 INTRODUCTION
Food Powders
Food powders, such as dried milk powder, protein isolates, seasoning powders,
and baking soda, have many benefits for both consumers and manufacturers. For
consumers, food powders can provide a conveniently packaged product as well as
nutritional value; for manufacturers, they offer an easy and manageable food form with
a longer shelf life and can be easily transported. These food powders have been
traditionally used in cakes, cookies, doughnuts, and crackers while newer applications
involve use of food powders in spring roll wrappers, steamed breads, and instant
noodles (Alan 2011).
Nutritional Values
Food powders contain nutrients, such as lactose, collagen, vitamins, and fiber,
which are available in a condensed form and can be easily assimilated and absorbed by
the gastrointestinal tract (Samonis and others 1990). For instance, powdered infant milk
formula provides the main supplementary resource of nutrients (such as
docosahexaenoic acid (DHA) and vitamins B1, B2 and B12) for infants and children
(Camilla and others 2008). In bread making, wheat flour is commonly used as the major
flour and provides essential nutrients, such as carbohydrates, proteins, vitamins,
minerals, and fiber (Giannou and others 2003). Moreover, onion powder can provide
one of the major sources for flavonoids and is used as a nutraceutical, foods that
function both as medical and health foods (Debnath 2002).
14
Safety Concern
Despite their beneficial characteristics, however, several safety issues are
associated with dry food powders. For instance, in the last two years there have been a
few outbreaks related to contaminated food powders, particularly red pepper (Capsicum
anmum L.), black pepper (Piper nigrum L.) , and dry milk powder (CDC 2007; CDC
2010a; CDC 2010b). In April 2013, the Centers for Disease Control and Prevention
(CDC) reported an outbreak related to the bread crust, frozen pasta products and pizza
dough that infected 35 persons with Escherichia coli O121 (STEC O121) (CDC 2013).
As a results, nine people were hospitalized, and two of them developed hemolytic
uremic syndrome (HUS). In 2010, the CDC reported that approximately 250 individuals
had contracted Salmonellosis attributed to meat contaminated by black and red pepper
in at least 44 states and the District of Columbia (CDC 2010b). According to the Food
and Drug Administration (FDA) the contamination of the meat led to over 15 related
recalls (FDA 2010a). Such problems with food powder safety are not new. Goepfert and
others (1972) pointed out that the high frequency poisoning in Hungary in the 1970s
was due to the consumption of meat seasoned with black pepper and other spices.
Based on their investigation, it is clear that spices need to be inactivated before
consumption (Goepfert and others 1972).
Moreover, since food powder may be high in nutrients, such as protein and
vitamins, it is a great medium for microbial growth, especially in products (such as
paprika powder and seasoning) (Staak and others 2007). Cross-contamination is also
possible because the processing of food powders in other foods or beverages may
result in illness. For example, reconstituted infant foods are susceptible to E. coli
O157:H7 when factors, such as water activity, pH, and temperature are not controlled.
15
Cross-contamination can also occur when hygiene is inadequate and can lead to
contaminated foods in unexpected places, such as homes or day-care centers (Deng
1998). While low moisture and water activity can extend the shelf-life of dry food
powders, most food powders are used as ingredients and will be rehydrated or
combined with other ingredients that have high water activity, such as milk powder and
spices.
This research focuses as on wheat flour and black pepper. Wheat flour is
traditionally sold in a pre-packaged form at retail and has a shelf life ranging from 6 to 8
weeks. Wheat flour is derived from wheat, which is one of the major crops produced in
the world. In the United States, wheat products are the third most frequently consumed
products, just behind soybean and corn production (USDA ERS 2012). Although the
wheat industry faces growth challenges due to competition from other subsidized crops
and cheaper foreign production, there remains a demand for wheat in the domestic
market. In 2010, the United States Department of Agriculture (USDA) reported that U.S.
production reached as high as 2,218.06 million bushels (USDA ERS 2012).
Several classifications of wheat exist, depending on the gluten content, protein
content, and hardness (Giannou 2003). Regardless, the preparation for wheat flour is
the same. As the wheat crop matures and reaches harvest, the kernel is removed from
the stalks and the chaff. Next, the wheat kernel is ground to give the wheat flour and
then is packaged for sale. The final product has a shelf life of approximately two
months, after which it becomes susceptible to insect spoilage (Marathe and others
2002).
16
Decontamination Method
Several technologies have been employed in order to decontaminate food
powders, including wheat flour. Technologies range from traditional heating methods to
newer, non-thermal methods. These include the following: gamma radiation (Nagy and
others 2002), ultraviolet (UV) radiation (Sharma and others 2003), steam processing
(Schneider 1993), and infrared (IR) heating (Hamanaka and others 2000). Pulsed Light
methods are the focus of this research.
Pulsed Light Technology
Pulsed light (PL) is an emerging technology and is made up of 54% UV light,
26% infrared light, and 20% visible light from the electromagnetic spectrum
(Krishnamurthy 2007). The UV light can be emitted either as a continuous wave
(continuous light) or in short duration pulses of 1 – 20 per second as in the case of
pulsed light. Continuous UV illumination is a lower energy source, while pulsed light
illumination functions at higher energies and is therefore more potent for microbial
inactivation. By storing electrical energy in capacitors, that energy is released through
arcing an inert gas, such as Xenon or Krypton, which intensifies the energy of the light
and causes the light to have up to 20,000 times the energy of the sun ray at sea level
(Kaack 2007).
Pulsed light has been proven to be effective in pathogen inactivation because it
can provide intense momentary electromagnetic energy at wavelengths of 100 - 1000
nanometers (nm) (Gemma and others 2010). It has been shown that PL not only
inactivates vegetative bacteria but also spores, which are hard to diminish (Nicorescu
and others 2013; Smelt 1998; Warth 1978). In addition, PL has been used to modify
compounds in foods and reduce allergens. For instance, PL was found effective in
17
reducing food allergens, such as soy (Yang and others 2010), peanut (Chung and
others 2008), shrimp (Yang and others 2012), and almonds (Chung 2008).
Inactivation Mechanisms
Three mechanisms are responsible for the effect of PL on foods: photochemical,
photothermal and photophysical (Robert and others 2000). The photothermal effect is
the thermal component of PL and heats the surface of the material due to PL’s high
energy. The photophysical effect is the vibration of the molecules within the material or
bacterial cell. The photophysical effect can cause the rupture of the bacterial
membrane, thereby releasing intra-cellular contents into the external environment.
Lastly, there is the photochemical effect. It is suggested that the photochemical effect is
the main mechanism of the PL treatment on microbial inactivation. In most
microorganisms, such as bacteria, viruses and other pathogens, PL can cause
structural changes in deoxyribonucleic acid (DNA). Thymine dimers form within the DNA
that can inhibit DNA transcription and replication in the bacterial cell, which can lead to
cell death, also known as apoptosis (Miller and others 1999; Wang and others 2005).
And similar to UV light, oxygen in the air can generate ozone after PL exposure.
Advantages and Disadvantages
With respect to the beneficial advantages observed in other foods, the
application of PL can provide a medium to decontaminate food powders, particularly
wheat flour. Several associated risks are present in contaminated wheat flour that poses
a foodborne danger. PL suggests an effective remedy for wheat flour and black pepper
based on its effectiveness. Benefits would include a safer food, a longer shelf life for the
product, and lower costs for both consumers and manufacturers, including health costs
and processing costs when compared with gamma irradiation. The major disadvantages
18
of PL are still penetration and overheating after a longer exposure time unlike the
conventional chemical methods.
19
CHAPTER 2 JUSTIFICATION OF STUDY
From a health perspective, contaminated food has resulted in approximately
1,000 reported diseases, 48 million illnesses, 128,000 hospitalizations, and 3,000
deaths annually in the United States (CDC 2010). It is estimated that there are
approximately 76 million cases of foodborne illnesses each year in the United States
alone, which presents both a major public health threat and economic burden (United
States Department of Agriculture (USDA 2010). The pathogen Salmonella has resulted
in an expenditure of $365 million dollars in direct medical costs annually (USDA 2010).
For E. coli O157 H7, the societal cost of a single fatal case can be as high as $7 million
(Frenzen and others 2005). Similarly, this amount is derived from direct and also non-
direct medical costs. Direct medical costs include physician and hospital services,
supplies, medications, and procedures required to alleviate the foodborne illness.
Indirect medical costs include transportation to health care, relocation expenses,
specialized education, and employment compensation.
Despite the potential health risk and economic costs of contamination, the
application of decontamination techniques in the industry is a deterrent parameter due
to its inconvenience. Initially, the industry adopted chemical methods as the primary
method for decontaminate dry food powders, spices, and seeds. These methods used
compounds, such as ethylene oxide (EO), ethylene glycol, and ethylene chlorohydrin
(ECH) (Wesley 1965). In 1977, according to the American Spice Trade Association
(ASTA), more than 80 million pounds (lbs) of spices were treated by EO (Gerhardt and
Ladd 1982). The major problems with chemical treatments were uneven treatment and
the absorption of residues (Thorén 1995). Thermal methods were also used, but led to
20
other issues regarding the quality of the food including aroma, flavor, and texture
changes that were undesirable (Nazarowec-White 1997).
This study evaluated B. subtilis as our target bacteria. Not only has B. subtilis
has been used as a Gram-positive model for more than a century, but also because B.
subtilis is one of the commonly encountered spore-forming bacteria in spices (Barbe
and others 2009). Based on a study in Nigeria, the natural contamination can reach as
high as 8 log CFU/g (Antai 1988). The white and whole flour loaves contain up to 106
CFU/g of Bacillus absent any preservation following two days of storage at modest
summer temperatures of 25-30°C (Rosenkvist and Hansen 1995). Specifically, 70% of
the contamination in the loaves arises from B. subtilis. Furthermore, it is reported that B.
subtilis is a common bacterium in dough and flour products and cause rope spoilage of
bread at 108 CFU/g (and only 105-106 CFU/g is needed for foodborne illness) (Kramer
and Gilbert 1989). This indicates a relationship between B. subtilis and the consumption
of ropy bread (Toddy 1982; Scokett 1991). Although B. Subtilis is usually non-
pathogenic bacteria at low concentrations, it can cause special toxi-infections including
severe vomiting, abdominal cramps and diarrhea at higher concentrations (Nicorescu
and others 2013). Therefore, there is a need to control the growth and survival of B.
subtilis in foods.
Previous studies have shown that PL can be effective in reducing and preventing
the proliferation of both spoilage and pathogenic microorganisms (Wang and others
2005). This may play an essential role in decreasing the risk of foodborne illnesses in
food powders, such as wheat flour. Consequently, this can bring about reduced
21
healthcare costs for consumers and provide manufacturers a potential processing
method to decontaminate wheat flour.
22
CHAPTER 3 OBJECTIVES
The overall objective of this study was to evaluate the effectiveness of PL on
the microbial decontamination of wheat flour and black pepper.
Specific Objectives: The following specific objectives were assessed in this study:
1. Evaluate the effects of PL treatment on the reduction of total aerobic microorganism counts, mold, and yeast counts in wheat flour and black pepper samples treated at different times and distances to the PL source.
2. Investigate the effects of PL treatment in disinfecting samples inoculated with Bacillus subtilis.
3. Measure the resultant color changes and water activity of samples after PL treatment for product quality purpose.
4. Monitor the temperature changes of the samples at different PL treatment times.
23
CHAPTER 4 REVIEW OF LITERATURE
Food Powders
Food powders, such as wheat flour, dairy, egg, gelatin, vitamins, starch, fruits
and vegetables powders, spices, and some food aids like coloring or flavoring agents
are commonly used in many food applications (Woo and others 2009). Generally, these
food powders are obtained from raw agricultural materials processed by different
methods, such as fragmentation, separation, drying, fermentation, milling and
stabilization (Cuq and others 2011). In addition, food powders are easy to preserve,
store, transport, weigh, and most importantly, they can have a relatively long shelf life
without losing their nutritional values (Cuq and others 2011).
It is well known that the major aim of consuming food is to obtain nutrition, which
can support the growth, development and productivity of animals (Brody and Lardy
1946). Nutritional value is one of the most important determinants and criteria in food
and provides consumers an estimate as to whether the food can satisfy the customer’s
nutritional needs. The higher the nutritional content of the food, the better it is. However,
while nutritional food can provide growth, development, and productivity to animals, it
also provides microorganisms a perfect media to grow at the same time (Bjornson
1982). Some powders, such as infant formulas and protein powders are at high risk for
microbial contamination.
Moreover, in addition to nutritional value, other factors that enhance the value of
the food are convenience and extended shelf life. Food powders provide these benefits
by giving an easy, transportable, and convenient product and also by providing a longer
shelf life. Food powders also preserve their nutritional value (Gonzales and others
24
2010). For example, infant formulas are a popular choice for young infants. Infant
formulas have many purposes, from providing a balanced, nutritional source that is
convenient, to giving a lactose free food for babies that are lactose-intolerant in the form
of soy infant formula, and are the source of other nutrients, such as docosahexaenoic
acid (DHA) and arachidonic acid (ARA), which are vital for proper visual and cognitive
development (FDA 2012).
However, as stated earlier, the downside to all the nutritional gains is that a lot of
food powders are vulnerable to microbial growth. In dry food powders, microorganisms
can exist mainly as spores, which are not able to germinate when the water activity is
low. However, when the seasonings are readjusted to higher moisture foods with a
higher water activity, this can trigger the growth of microorganisms due to this rich
media (Jaquette and Beuchat 1998). It is also the case that in some food powders, such
as spices and seasonings, heating is avoided since this can cause the loss of flavor and
aroma, and therefore no heating is applied. Most of the seasonings are delivered
without decontamination and therefore the products may yield a high level of
microorganisms and spores (Deng and others 1998; Staack and others 2008). For such
reasons, decontamination methods are being developed in this area to avoid these
shortcomings, and newer, novel methods are needed (Chee 2012).
Problems and Issues
In the last 30 years, there have been a few cases of severe incidents in the U.S.,
leading to more than 4 deaths from consuming microbial contaminated powder food
(Padhye and Doyle 1992; CDC 2007; CDC 2009; CDC 2010a; CDC 2010b). In 2008, an
outbreak of Salmonella Rissen infections was transmitted though white pepper (CDC
2010b). In 2009, an outbreak occurred in the District of Columbia and 44 states, of
25
which 272 cases were identified. The cause of the outbreak was black and red pepper
contaminated with Salmonella Montevideo associated with salami products. In August
2010, 44 individuals were infected with Salmonella Chester in California, Georgia, and
16 other states (CDC 2010a). However, for spices, which are known to be harbors for
fungi and bacteria, few reports have been recorded connecting spices with human
illnesses (Vij and others 2006).
For infant formula, more than 40 infants were infected with Enterobacter
sakazakii in the last 20 years because of contaminated infant formula (Drudy 2006).
Once infants are infected with Enterobacter sakazakii, the outcome can be severe.
Seizures, brain abscesses, hydrocephalus, developmental delay, and death rate can be
as high as 80% and premature infants are at higher risk than mature infants and
toddlers (Arseni 1987; Nazarowec-White 1997).
Part of the reason for the outbreaks is due to incomplete sterilization, which is
determined by the special properties of the dry food ingredients (FDA 2012a; FDA
2012b). First, the difficulty with sterilization is correlated with the specific microflora
present, such as vegetative cells and spores. The microflora can adapt to a low water
activity (aw) environment (Fine and Gervais 2005). Second, the heat resistance of the
microorganism depends on the surrounding water activity within its environment. Murrell
and Scoot (1966) reported that the thermal resistance of spores is more significant in
dry media the liquid media. For example, the cells’ heat resistance in Salmonella will
increase from 25% to 75%, as demonstrated by the D value when aw decreased
(Goepfert and others 1970).
26
Additionally, companies order recalls whenever a product is found be harmful or
defective. The product is withdrawn from the market to avoid potential risks or to make
corrections to the product, if possible (FDA 2010b). In order to protect the interest of
most consumers and the reputation for the company, companies will recall their own
products once problems are detected. Other times, FDA will raise concerns and the
company will have to recall their products (FDA 2010b). From 2004 to 2012, 271
products related to food powder have been recalled, with the most recent recall
occurring in May 2012. Over 13 tons of Select Roast Meat Type Flavor was recalled as
a result of Salmonella contamination (FDA 2012).
Decontamination Methods
A variety of chemical, thermal, and non-thermal methods have been applied to dry
food powders, including dry heating (Baron 2003), high temperature short time treatments
(Faubion and Hoseney 1982), steam processing (Schneider 1993), microwave heating
(Gerhardt and others 1985), and infrared (IR) heating (Hamanaka and others 2000).
Food powders that can be affected include natural spices, dry food ingredients,
and vegetable powders. Natural spices, such as mace, sage, allspice, nutmeg, paprika,
ginger, coriander, cinnamon, and cloves, are all susceptible to microbial contamination
with spores of yeast, molds, and bacteria. Rice flour, soybean flour, wheat flour, and corn
flour cocoa, all of which are grain powders and are used in comparatively larger amounts,
are also at risk. Dry vegetable powders including asparagus powder, garlic powder, and
onion powder also are liable to microbial contamination, as are their dry fruit equivalents,
such as apples, raspberries, dates, figs, apricots, peaches, prunes, and raisins.
Traditionally, chemical methods were used to decrease the amount of microbes
in dry food powders. Chemical methods involved fumigation with ethylene oxide (EO) or
27
other chemicals, but these chemicals are believed to cause chronic or carcinogenic
effects (Farkas 1988). Treatment by heat application was also used as it could reduce
the risk of contamination by modifying the DNA and protein profile of microorganisms.
However, it would lead to a loss of nutritional quality at the same time, which is not
desired. Meanwhile, since the subtle flavor and aroma elements of spices and herbs are
sensitive to heat, traditional heat decontamination methods would affect the functional
characteristics of the enzymes and the texturing agents found within the food powder.
Gradually, non-thermal methods have been favored over thermal methods. Non-thermal
methods give the advantage of preserving the quality of the food. In addition, since dry
food ingredients have a higher heat resistance than moist food ingredients, it avoids
extended thermal treatments (Murrell and Scott 1966). A survey of these methods is
provided below and gives an overview of the different decontamination methods used to
preserve and ensure safety in dry food powders.
Chemical Methods
Chemical methods were first adopted for spices decontamination (Gerhardt and
Ladd 1982). Chemical methods have been proved can effectively inactivate microbes
for decades. Ethylene Oxide is the method used first in industry which has been
prohibited in many countries due to carcinogenicity (Fowles and others 2001).
Ethylene Oxide and Derivatives
In the 1980s, treatment with ethylene oxide (EO) was considered the most widely
applied method for decontamination of dry ingredients. According to the American Spice
Trade Association (ASTA), the U.S. had consumed over 800,000 lbs of EO in 1977 to
treat 80,000,000 lbs of spices (Gerhardt and Ladd 1982). Ethylene chlorohydrin (ECH),
which has a longer effect than EO, and ethylene bromohydrin (EBH) also were
28
considered as alternatives in the period during ethylene oxide fumigation (Wesleyet and
others 1965). EBH is a potential metabolite of EO and ethylene dibromide fumigation.
However, both EO and ECH were later discovered to be mutagens and also were highly
suspected of inducing other chronic or delayed toxic effects (Ehrenberg and Hussain
1981; Gerhardt and Ladd 1982; Kligerman and others 1983). EO would form toxic 2-
hydroxyethyl compounds with lysine and cysteine and up to 30 parts per million (ppm)
had been found in dried egg and milk powder (Gerhardt and Ladd 1982). Additionally, it
had been found that there was 260 ppm of 2-chloroethanol in EO-sterilized flour and an
average of 805 ppm was present in a spice mixture (Wesley and others 1965). In curry
powder, 160 ppm of chloroethanol could be tested 182 days after fumigation
(Scudamore and Heuser 1971). In wheat, levels of 20 ppm of EO were detected after
wheat sterilization. However, according to the report, during the storage and milling
procedures, any residues of EO were virtually removed (Pfeilsticker and Rasmussen
1968). In the U.S., the Environmental Protection Agency (EPA) (EPA 1977) had defined
the tolerated level of propylene oxide in foodstuffs, such as cocoa and spices to 300
ppm. Thus, in the 1980s, EO fumigation was highly represented as a significant
occupational health hazard and regulatory restrictions followed (Gerhardt and Ladd
1982). Subsequently, scientists acknowledged that in many experiments, EO caused
harmful effects. It was shown that EO induced tumors in laboratory mice through via
oral and inhalation routes formed adducts to proteins and DNA in humans and animals
by direct genotoxic mechanisms (IARC 1994).
EO can significantly reduce microbial population, which has been proven by
multiple researchers(De Boer and others 1985; Farkas 1985). However, other than
29
aroma and color changed a lot by using this chemical method, volatile compounds are
lost because of the low pressure that is needed for sanitizing agent removing (Farkas
and Andrassy 1988). Besides that, mutagen and carcinogen after inhalation is also a
huge problem for this method to be more diversely apply (Steenland and others 2003).
Ozone
Ozonation, which is considered as an oxidation method, was developed in the
1990s to reduce the toxicity of aflatoxins in dry foods (Samarajeewa and others 1990).
Ozone, or O3, is a powerful oxidizing and disinfectant agent (McKenzie and others
1997). It reacts against the 8,9 double bond of the furan ring in aflatoxin via an
electrophilic attack and causes the formation of primary ozonides, followed by its
rearrangement into monozonide derivatives (Proctor and others 2004). In 2001, O3
received approval in the U.S. for its use as an antimicrobial agent in food
decontamination, especially for dried foods (such as pistachios, black pepper, grains,
and cereals) (FDA 2001, Naitoh and others 1987, 1988, Zagon and others 1992, Kim
and others 1999, Liangji 1999, Khadre and others 2001, Al-Haddad and others 2005,
Akbas and Ozdemir 2006). Based on several studies, gaseous O3 treatment methods
gave 6 log CFU/g reductions of Micrococcus and Bacillus spp. counts in dried foods
(Naitoh and others 1987, Naitoh and others 1988; Zhao and Cranston 1995; Khadre
and Yousef 2001), which was more effective than hydrogen peroxide. Other studies
showed that high ozone concentration (>18 ppm) with adequate exposure time (>8 h)
could cause significant changes in food powders and bee pollen and also could
decrease the pH value of Korean red ginseng powder (Byun and others 1997, 1998;
Yook and others 1997).
30
Ozone is suitable for advanced oxidation processes (APOs) with appropriate
inhibitors results in potential effectiveness on most resistant microorganisms.
Meanwhile, ozone is able to apply on both liquid and solid food material with a quick
decomposes leaving no residues (Khadre and others 2001). However, based on its
strong oxidase ability, ozone removes the color and amour of spices a lot, which make
the product less desirable for customers.
Thermal Methods
Infrared heating
Infrared heating has been applied in the inactivation of microorganisms. Infrared
refers to wavelengths of light that lay between microwave and ultraviolet radiation in the
electromagnetic spectrum. The wavelength ranges from 0.76 to 1000 μm and interacts
with food components due to absorption, reflection, scattering, and transmission of the
light (Staack and others 2008). The diffraction of radioactive energy as heat results in
surface penetration and temperature depths specific to the products treated, depending
on the food product, IR wavelength, water activity (aw), and the thickness of sample.
Compared with other conventional heating methods, IR light heats the sample
directly and therefore the samples will not be influenced by the airflow around the
powder. Meanwhile, the treatment times are shorter (Ranjan and others 2002). Staack
and others (2008) showed a chart depicting the number change in flora in order to
assess the effectiveness of IR heating, which is shown below in Table 4-1.
To summarize briefly, the initial population was measured at 7.48 log10 CFU/g
for B. cereus spores in the inoculated samples and 4.90 log10 CFU/g for the
background flora in the non-inoculated samples.
31
Staack and others (2008) also concluded that IR could be used to lower the
microbial numbers in paprika powder. However, a higher heat flux degraded the product
quality on the surface of the powder bed. Using this method could reduce the treatment
time significantly. However, the industrial implementation of this process should be
designed in order to prevent overheating and over drying. Furthermore, it is possible
that sometimes the thermal methods may not work that well because of the heat
sensitivity of seasoning powders and herbs. Meanwhile, the higher temperatures
influence the quality and sensory attributes of the food product.
Microwave
Microwaves are radio waves with wavelengths of 1 meter (m) to 1 millimeter
(mm) that have frequencies that span from 300 MHz to 300 GHz (Pozar 2009). Omar
and others (1995) used an electric microwave oven with a frequency of 2450 MHz (± 20
MHz) and treated samples of black pepper powder for 20 seconds (s), 40 s, and 75 s.
They compared microwave exposure with gamma irradiation to measure the
effectiveness of each treatment on microbial inactivation. The comparative results are
shown below in Table 4-2.
Based on the results, Omar and others (1995) concluded that gamma rays are
more effective than microwave treatments in reducing Enterobacter spp. and
Clostridium spp. The concentration of the microbes decreased significantly after 75 s of
microwave treatment whereas for the 40 s treatment, the results were not satisfactory.
Even though, microwave seems to be efficient in treating other samples,
however, the efficiency of microwave treatment is based on the movement of polar
molecular in food sample, thus water molecular as a polar molecular can influence the
efficient of microbial inactivation. And it can be measured by quantified by water activity.
32
Instead of the explanation expressed by Omar and others (1995), in their study, they
explain this phenomenon as oven’s frequency.
High temperature short time process
High temperature short time (HTST) treatment is based on a very high
temperature (200-600 °C) treatment within a short duration (0.1-30s) followed by an
instantaneous cooling procedure. As Fine and Gervais (2005) have reported that HTST
can cause less chemical modifications in the product while maintain the same
inactivation effect as relatively lower temperature and longer time.
The Fig 4-1 shows the schematic structure of the HTST machine, after the
treatment, they reached a 5.2 log CFU/g reduction after 120s treatment, however, the
log reduction is already 5 log CFU/g after 60s. And as some scientists mentioned , the
decontamination effect is strongly influenced by water activity of the samples, the drier
the sample is, the higher the resistance for the thermal treatment(Laroche and Gervais
2003).
In Almela and other’s study (2002), a temperature range of 130 to 170 °C
temperature range was chosen, and the treatment time varies from 4 to 6 s respectively,
all these combination were investigated in this study. Almela and others observed a 3-4
log CFU/g inactivation rate combined with up to 40% color loss (Almela and others
2002).
Some scientists have pointed out that process used for the decontamination of
dried products, such as steam, microwaves, or Joule-effect treatments, can induce
organoleptic degradation or achieve a relatively low destruction rate. Meanwhile,
majority of the thermal methods can also lead to color modification, loss of essential oil,
33
flavor change and aroma changes (Almela and others 2002; Schweiggert and others
2005; Calucci and others 2003; Nicorescu and others 2013).
Non-Thermal Methods
Pulsed light
The electromagnetic spectrum is comprised of several wavelengths, including
ultraviolet radiation, radio waves, microwaves, infrared radiation, visible light, X-ray and
gamma radiation (Diffey 2002). One particular type of light, pulsed light, is an emerging
technology and makes up 54% UV light, 26% visible light, and 20% infrared light
(Krishnamurthy 2007). The UV wavelength is the largest component of PL and makes
up the wavelength from 100 nm to roughly 400 nm, the parameter of UV light is shown
in the Table 4-3.
Within UV light, there are further distinctions including UV-A, UV-B, UV-C, and
Vacuum UV. Out of these, UV-C is considered to be the most efficient germicide against
microbes, such as viruses, bacteria, protozoa, yeast, algae and molds (Bintsis 2000).
Furthermore, UV treatment is considered as one of the most efficient methods for
decontamination of the surface and has been found to be effective in several products,
such as water and fruit juice (Guerrero-Beltrán and Barbosa-Cánovas 2004, 2005).
Moreover, since UV light has a low running cost and uses less energy than
thermal pasteurizers and saves space and time, it has been widely applied in many
areas. UV light applications will likely become more widely accepted in time, considering
that UV is a simple, trustable and low-cost treatment. In addition, UV light is a clean
technology that does not produce any residual irradiation, even at a high level exposure
(Yaun and others 2003).
34
PL can be emitted either as a continuous wave or in short duration pulses of 1 –
20 per second. Continuous wave illumination is of a lower energy while pulsed light
illumination is at a higher energy and is therefore used for microbial inactivation.
The mechanism for PL is that it releases electrical energy that is multiplied
several folds by first storing the energy in a capacitor and then releasing it as a short
duration intermittent pulse using a lamp filled with inert gases, such as Xenon or
Krypton gas (Krishnamurthy 2006). This can cause the light to have 20,000 times the
energy of the sun (Kaack 2007) as it comes into contact with the gas. The energy then
is absorbed by the surface of the material, which can create changes within the
material. Considerable research has been done to investigate the mechanism of PL
technology. The changes in the material can be attributed due to the photochemical,
photophysical, and photothermal effects of PL (Robert and others 2000).
The photothermal effect is the thermal component of pulsed light that can heat
the surface of the material being treated due to PL’s high energy. It is a kind of heat
damage caused to the bacterial cell because of the differences in the heating rates of
the bacteria and the surrounding media. This results in a localized heating of the
bacterial cell. Next, there is the photochemical effect. It has been suggested that the
photochemical effect is the main mechanism of the PL treatment on microbial
inactivation. It can cause structural changes in deoxyribonucleic acid (DNA) in most
microorganisms, such as bacteria, viruses, and other pathogens. Thymine dimers can
form within the DNA that can inhibit DNA transcription and replication in the bacterial
cell, which can lead to cell death, also known as apoptosis (Wang and others 2005,
Miller and others 1999). Lastly, there is the photophysical effect, which is the vibration of
35
the molecules within the material or bacterial cell that can cause the rupture of the
bacterial membrane, therefore releasing intra-cellular contents into the external
environment. However, the fatal action of pulsed light is mainly attributed to the
photothermal and the photochemical effects of PL. It is possible that both mechanisms
cooperate and the comparative importance of each one would depend on the fluency
and target microorganism. However, most of the authors explain their results based on
the photochemical effect. In practice, the application of PL on the decontamination of
foods has three broad categories: (a) inhibition of microorganisms on the food surface;
(b) destruction of microbes in the air; (c) and sterilization of liquids (Bintsis and others
2000).
As early as 1975, Bachman successfully applied UV rays to sterilization of air
and water, which ended up giving almost no suspended subjects after treatment.
Bachman also used this method for surface decontamination and found great success
in this application. Furthermore, he demonstrated excellent potential in application of UV
light on packaging materials (Bachman 1975).
UV light also has been applied in water decontamination for many years, since
UV application will not influence color, pH, odor, or flavor (Bintsis and others 2000). As
a result of these special properties of UV light, Wright and others (2000) decided to
apply this technology into decontamination of apple cider. The results from the
experiment showed a reduction of nearly 4 logs CFU/g of Escherichia coli O157:H7 in
unpasteurized apple cider. In another study on apple cider, a decrease of 2.67 logs
CFU/g of the entire microbial population was seen (Harrington and Hills 1968).
36
Maricel and others (2002) applied UV radiation to inactivate the microorganisms
in fruit juice. They concluded that UV radiation successfully decreased the microbiology
load in various kinds of juices. The required dosage of UV light varies depending on the
transparency of the juice. The clearer the juice is, the lower the dosage level needed.
The reason a higher dosage is needed for less transparent juices is that the amount of
suspended components, such as fruit cells and fiber in the fruit juice can protect the
microorganisms against the UV illumination by preventing the passage of the light to the
microbial surface. Another study showed that a 20 - 100 times higher dosage
illumination solved the coverage and shading problem, giving sterilization of spores and
other microorganisms (Wallhauser 1978).
Furthermore, Blalka and Demirci (2008) applied pulsed light to decontaminate
E.coli and Salmonella populations in raspberries and strawberries. They found that most
reduction of E.coli and Salmonella happened when they applied the PL treatment for 60
seconds (s). However, they did not evaluate the sensory aspects of the fruits following
treatment. Such sensory evaluation would help determine whether PL could be applied
within the industry.
Belgin and others (2011) used UV light to decontaminate cumin seeds. Since
cumin seeds contain all the proteins and nutrients that a plant needs to grow, it happens
to be a great habitat for microorganisms. From the experiments, Belgin and others
discovered that a combination of UV-C and FIR application would decrease the
microorganism count to a lower level without damaging the quality of the seed.
Decontamination of food-contact surface.
Since microbial contamination on food-contact surface, especially on food
processing equipment surface is a major concern in food industry. Surface
37
contamination of equipment can cause severe cross-contamination of pathogenic
microorganisms (Kusumaningrum and others 2003). Cause of the limitation of majority
of contamination methods, it is better to use PL in order to achieve a better penetration.
Rowan and others (1999) found that after 300 pulses on food contact surface, it can
reach a 6 log CFU/g reduction.
Allergen mitigation
PL has been reported can significantly reduce peanut, peanut butter, soybean,
milk, shrimp, egg and wheat allergens. Chung and others (2008) noted that for liquid
peanut butter, after PL treatment, the Immunoglobulin E (Ig E) binding can reduced to
approximately 20%. And PL also been noticed be able to reduce soybean protein, such
as glycinin, and it shows a 50% reduction in Ig E binding with ELISA after 6 min PL
treatment (Yang and others 2010).
Irradiation
Gamma ray, e-beam (electron beam) and x-ray all belong to irradiation. The most
commonly used method is still gamma irradiation. Doses from 3-10 kGy proved to be
reliable for improving microbial safety for spices (Reddy and others 2009). Lastly,
despite the controversy of using gamma irradiation, this technology has been found to
be effective in decontamination of foods while assuring food safety and improved shelf
life (Reddy and others 2009, Bhat and others 2010). In a study mentioned earlier, Omar
and others (1995) conducted a set of experiments to compare the effectiveness of
gamma rays and microwave irradiation on black pepper. The results from the
experiment showed a reduction in microbial count when the sample was exposed to
average doses of 5.0 kilo Grays (kGy) and 10 kGy using Cobalt-60 at a dose rate of 1.2
Gy/s. Compared with other microwave decontamination methods, gamma irradiation,
38
when applied in shorter durations, could eliminate almost all of the microbial organisms.
The effect of gamma irradiation on T. foenum-graecum seed has also been studied by
Gupta and others, it shows a 2 log reduction in Total Aerobic Count (TAC), when the
dosage is 2 kGy. (Gupta and others 2009)
Irradiation, has been proven to be reliable for improving the quality of spices
product, but also herbs, vegetable seasonings with maximum 10kGy average
dosage(Farkas 2006). Even though gamma irradiation has been proved as an efficient,
environmentally friendly, energy-effective official approved method. However, the lack of
application is mainly due to customer concern and high cost on equipment and
maintain. Meanwhile, the flavor and aroma will be slightly alternated (Farag and others
1995). Other than the sensorial changes, the irradiation used on packaged food
material, may results in the formation of small volatile particles or non-volatile radiolysis
products from the packaging material (Goulas and others 2004).
High hydrostatic pressure
High hydrostatic pressure processing uses a pressure that varies from 100 to
1000 Mpa. Hite and others (1914) investigated the application of high pressure as a milk
preserving methods, and later the study has been focus on providing shelf-stable fruit
and vegetable products (Hite and others 1914). HHP applied on wheat flour as a
method of inactivate vegetative cells and enzyme in wheat flour, the quality of wheat
flour start to become a concern. Some scientists investigate the modification of structure
and mixing properties after HHP treatment, based on their research, when pressure is
higher 400 MPa, starch granule structure lost occur, also gluten protein will aggregate
after the treatment (McCann and others 2013). However, the inactivation effect is mainly
based on equilibrium relative humidity (ERH) of product. Dry food ingredients usually
39
show no reduction in microbial count when water activity lower than 0.66 (Butz and
others 1994). Based on that respect, HHP can hardly make an efficient method for dry
ingredients.
40
Table 4-1. Concentration of B. cereus SIK 340 spores and natural background flora (±SD) in different location in paprika powder with aw 0.5, 0.8, and 0.96 after exposure to IR heat flux from medium-IR of 5 kW/m2 and near IR of 11kW/m2 (Staack and others 2008; Bialka and Demirci 2008).
Heat flux
(kW/m2)
Location in powder
Microbial counts after IR heating(Log10 CFU/g)
Bacillus cereus Natural flora Total Plate count Total plate
count Spore plate count
aw 0.50 5 med-IR Surface 7.12 - 4.67
Overall 7.15(±0.04) 7.16(±0.08)
4.45(±0.11)
11 near-IR Surface 6.78 - 4.59
Overall 7.05(±0.06) 6.95(±0.05)
4.39(±0.07)
aw 0.80 5 med-IR Surface 6.87 - 4.02
Overall 7.17(±0.09) 7.01(±0.45)
4.14(±0.14)
11 near-IR Surface 6.77 - 3.65
Overall 6.34(±0.13) 6.23(±0.30)
3.24(±0.15)
aw 0.96 5 med-IR Surface 7.02(±0.25) 6.98(±0.19)
4.72(±0.08)
Inside 2.21(±0.59) 2.11(±0.38)
2(±0.24)
Overall 6.19(±0.49) 6.05(±0.27)
3.49(±0.19)
11 near-IR Surface 6.58(±0.21) 6.50(±0.42)
<1
Inside <1 <1 <1
Overall 5.77(±0.24) 5.71(±0.17)
<1
41
Table 4-2. Survival of natural microbial contaminations in black pepper powder following gamma radiation and microwave treatments. Legend: - No Growth; + Growth; ++ More Growth; +++ Heavy growth (Omar and others 1995).
Contaminant Gamma irradiation does (kGy) Microwave exposure (s)
0 5 10 20 40 75
Bacterial flora
Escherichia coli + - - + + -
Enterobacter sp. ++ - - ++ + -
Bacillus subtilis +++ +++ + ++ + +
B. megaterium +++ ++ + ++ + +
Clostridium sp. +++ +++ ++ + + +
Pseudomonas sp. ++ + - + + -
Micrococcus sp. +++ +++ ++ ++ ++ ++
Fungal Flora
Alternaria sp. ++ - - + + -
Aspergillus sp. +++ - - ++ + -
Penicillium sp. ++ + - + + -
Cladosprium sp. ++ + - ++ + -
42
Table 4-3. Characterization of different UV components (Krishnamurthy 2006).
Region Wavelength (nm) Frequency (Hz) Photon Energy (eV)
Vacuum UV 100-200 3×1016-3×1015 24-12.4
UV-C 200-280 3×1015-1.07×1015 12.4-4.43
UV-B 280-315 1.07×1015-9.52×1014 4.43-3.94
UV-A 315-400 9.52×1014-7.49×1014 3.94-3.10
43
Figure 4-1. The HTST process procedure for seed and food powder decontamination (Fine and Gervais 2005)
44
Figure 4-2. Schematic of pulsed light equipment (Bialka and Demici 2008).
45
CHAPTER 5 MATERIALS AND METHODS
Sample Preparation
Wheat flour and black pepper were purchased from a local grocery store
(Gainesville, FL, U.S.A) and stored at 20°C at 55% relative humidity. Following storage,
the wheat flour samples were aseptically transferred to sterile foil boxes for future
inoculation and cultivation. While, for black pepper sample, was kept in controlled Aw
chamber until inoculation.
To create optimal conditions for yeast and mold growth, the flour was moisturized
to a water activity of 0.77 using 100 mL of distilled water in a sterilized aluminum plate
combined with 1000 g of wheat flour. The moistened samples were incubated for 7 days
at 35°C.
Growth Study
The growth curve of bacteria varies a lot, however, majority of them follow the
same pattern of growth as: lag phase, logarithmic, stationary and death phase. The
reason why growth curve study was performed in my research is to make sure that
bacterial vegetative cells can maintain in stationary phase. In which, it can reach to its
highest quantity and relatively bigger resistance to a range of stresses (e.g., thermal,
oxidative, starvation) due to a large number of phonotypic and genotypic changes.
Yoshikawa and Sueoka (1963) conducted a research on B. subtilis chromosome, they
concluded that in the stationary phase, B. subtilis has the complete genetic sequence. It
has been proved that bacterial are more resistance to extreme environment, such as
temperature, pH. Besides that, the stationary phase is less likely to death regarding to
the growing concentration of toxin generate by themselves.
46
After B. subtilis colonies were rehydrated in the original container with PW and
the cells were transferred from bottle to TSB tubes for incubation. The strain grew at
30°C for 24 h with gentile agitation (180 rpm). Next, Transferred the cultures in 10ml
TSB-Rif (200 µg/L) tubes and incubated the tubes under 30°C for 6 h two times to
acheive uniform vegetative cell. On the same day, the cultures were serially diluted
(1:10) in peptone water (PW) (0.1%) and yeilded cell populations of approximately 102
CFU/ml. The sample were then ransferred into 100 ml TSB flasks.
Bacterial growth was monitered at every 50 to 10 mins by direct and viable cell
counts methods. The measuring time interval started with every 50 mins, after 300
mins, the time interval between two measurments reduced to every 10 mins. Viable
cells counts were conducted simultaneously by a series of dilutions of the PW. Dilutions
were pour-plate into TSA, incubated for 24 hours at 30°C. After enumeration, the
counting of the plates was expressed as CFU/ml. A growth curve was obtained by
creating scatter plots of Log of counts against time (min).
Preparation of Inoculum
The B. subtilis strain cells were obtained from the American Type Culture
Collection (ATCC 6633) and maintained at -80°C on Columbia Agar (Franklin Lakes,
NJ) (Arantxa and others 2011). The inoculum was prepared as described by Arantxa
and others (2011) by streaking a loop-full of frozen culture onto Typtone agar and
incubating anaerobically at 37°C for 24 h. After incubation, a 250 mL conical flask
containing 100 mL brain-heat infusion (BHI) broth (Franklin Lakers, NJ) was inoculated
with a single colony and incubated at 37°C with agitation (115 rpm). After the second
incubation for 24 h, a new 250 mL conical flask containing 100mL of TSB was
inoculated with 1mL of the pre-culture and incubated under the same conditions. Next, 2
47
mL of the cellular suspension were inoculated in a sporulation agar medium and
incubated for 24 h at 37°C. The medium was autoclaved at 121 °C for 20 min. Then,
spores and vegetative cells were collected by flooding the agar plates with sterile
double-distilled water and scratching the surfaces with a glass spatula. After harvesting,
cells were purified and washed three times with centrifugation at 4000 rpm for 20 min
(Fine and Gervais 2005). The last step was to place the inoculum into the freeze-dryer
for 24 hours and then dry the bacteria vegetative cells and spores.
Inoculation
Prior to the PL treatments, microbial cell and spores were prepared and stored at
-5°C for less than 24 h. The dried inoculum and the samples were mixed in a mixing
bowl and blended for 20min at 25°C. The mixture was then stored at room temperature
for 24 hours under a laminar flow hood to enhance the adherence (Akbas and Ozdemir
2008). Four different spot in the blender have been researched and the results shown
that there is no significant differences among those spots.
Pulsed Light Treatment
Wheat flour and black pepper samples were weighed and placed on separate,
aluminum weighing dishes (70 mL, Fisher Scientific, IL, U.S.A). The samples were then
transferred to the Xenon continuous PL system (Model LH840-LMP-HSG, Xenon Corp.,
Wilmington, MA, U.S.A.), which is shown as a schematic diagram in Figure 3-1. The
equipment can generate a spectrum wavelength ranging from 100 to 1100 nanometers
with frequencies from 1 - 20 pulses per second. For this experiment, the settings were
longer time pulses of 360 µs (three times/second) and spectrum wavelengths (100-1100
nm) where the white light is rich in UV (200-400 nm). The output source was the Xenon
lamp. Different energies (PL fluence or PL dose) were adopted in this study. Based on a
48
previous study, the PL doses ranged from 16.2 J/cm2 to 1.08×102 J/cm2 and is
conducted on the similar model showed in Figure 3-2 (Krishnamurthy 2006).
For the PL treatment, samples with natural flora and inoculated wheat flour were
exposed under PL for different times and distances. The treatment parameters were 3
pulses per second for 15 s, 30 s, 60 s, and 90 s using a distance of about 6 cm, 9 cm
from the lamp source, respectively for wheat flour samples. A different treatment time
was used, however, for the black pepper samples, darker color increased the
temperature and led to higher readings. Based on that fact, the treatment time was
adjusted to 5 s, 10 s, 15 s and 30 s, respectively 6 cm, 9 cm. At the end of each
treatment, following 5-10 s delay, the samples were removed from the PL chamber in
order to record the surface temperatures.
Temperature Profile Measurements
The initial temperature of each sample was measured and recorded using a non-
contact infrared thermometer (Omega OS423-LS, Omega Technologies, Stamford, CT,
U.S.A.). This gave the surface temperature of each sample. After the PL treatment, the
temperature was recorded again following a 5 – 10 s delay due to sample removal from
the treatment chamber. Measurements for the natural flora and inoculated wheat flour
samples were recorded at different treatment times and different distances.
Microbial Recovery Procedure and Enumeration
Total Aerobic Count
AOAC 966.23 C is cited as a reference procedure. From each PL treated wheat
flour and black pepper sample, serial dilution (1/10 ml) was made up to 10-4. Next, 1 ml
of each diluted sample was placed into sterile petri dishes using a sterile pipette. A
Molten Plate Count Agar (at 45°C) was poured into each of petri dishes containing the
49
1ml inoculum. The plates were allowed to solidify on a flat surface. Then, the plates
were inverted and incubated at 35°C for 48 hours. After incubation, reduction of bacteria
in tenfold was calculated for different treated wheat flour samples. This method was
validated and approved as the official methods as prescribed by the Association of
Official Analytical Chemists (AOAC).
Yeast and Mold Counts
According to AOAC Method 995.21, Yeast and Mold Counts (YMC) in foods, the
powder needs to be thoroughly mixed by sterile spoon. The samples were diluted to
1:10 by aseptically weighing 1g of the samples into a sterile wide-mouth, screw-top
bottle and then mixed with 9 ml peptone water (0.1%). Next, the bottle was shaken
rapidly on a vortex for 25 s. Next, the homogenate was filtered using a vacuum source
and placed on the surface of a pre-dried YM-11 plate. YM-11 agar is made from 20 g
soy peptone mixed with 20 g tryptone, 5 g D-glucose, 5 g NaCl, 2.4 g anhydrous
K2HPO4, 0.03 g trypanblue, 0.1 g chloramphenicol, 15 g of agar and 1 L distilled water.
It was heated to boiling and stirred it before autoclave. The mix was autoclaved at
121°C for 15 min and then tempered to 45-50°C. Next, we aseptically poured larger
than 3 mm layer of agar and let it solidify. Finally, the samples were cultured for 48 h at
26 °C.
Bacillus subtilis Count
For the B. subtilis analysis, agar Columbia agar was used for the detection ( Fine
and Gervais 2005; Levy and others 2011). Take out 10 g Columbia powder. Add further
distilled water in a 1 L cylinder after suspension to ensure the accuracy. After the
mixture was autoclave at 121 °C for 20 min, Treated samples and control groups were
transferred to sterile bags. Each sample was hydrolyzed by 9 ml of sterile peptone
50
water (0.1%, w/v) in the sterile bags and homogenized with a vortex mixture at a
medium speed for 2min. Further decimal dilutions were prepared in peptone water
solution and were plated out on the media for B. subtilis (Akbas and Ozdemir 2008).
Viability was determined by counting “colony-forming units” (CFU). Experiments were
performed no less than 3 times and mean values were calculated, as well as 95%
confidence interval (CI) for the means. These CIs were found to be no more than 15%
of the mean viability.
Color Analysis
The color of the wheat flour samples was measured using a color machine vision
system (CMVS). This equipment consists of a Nikon D 200 digital color camera (Nikon
Corp, Japan) housed in a light box [42.5 cm (W) x 61.0 cm (L) x 78.1(H)] (Wallat and
others 2002). The camera (focal light, 35 mm; polarization, 18.44 mm), which was
connected to a computer, was used to capture the images prior to color analysis.
LensEye software (Engineering and Cybersolutions Inc., Gainesville, FL, USA) was
used to analyze the wheat flour color based on the values of L (lightness), a* (redness)
and b* (yellowness). The sample analyses are based on 3-dimensional color values
with the following scale: L* value for whiteness (100 white, 0 black); a* value for red-
green chromaticity (+60 red, −60 green); and b* value for yellowness (+60 yellow, −60
blue) (Labsphere, North Sutton, NH, USA). Color coordinates for with the untreated
samples were L=81.22, a=2.75, and b=10.92. In this study, the color differences
between the treated and untreated samples were assessed by three parameters shown
as: total color differences ∆E, C* (chroma) and h° (hue angle), which is shown in
Equation 1, 2 and 3 below. All the samples were aligned in the center of the light box
before acquiring the images.
51
∆𝑬 = √(∆𝑳 ∗)𝟐 + (∆𝒂 ∗)𝟐 + (∆𝒃 ∗)𝟐 (1)
𝒄 ∗= √𝒂𝟐 + 𝒃𝟐 (2)
𝒉° = 𝑡𝑎𝑛−1 (𝑏
𝑎) (3)
Water Activity Measurements
Water activity measurements were performed at various treatment times (t = 15,
30, 60, and 90s). During the treatment, changes in the water activity of the samples
were monitored to maintain the low water activity to prevent any potential post-
contamination (Laroche and others 2003). The water activities of the samples were
checked using a dew-point osmometer (Decagon Devices, WA, U.S.A) before and after
PL treatment. The control group wheat flour sample was placed in the water activity
chamber to initiate water activity measurements and provided a baseline to compare
against all the other treatment groups.
Statistical Analysis
Statistical analysis was carried out using JMP Version 9. Paired t-test was
conducted to determine the different effectiveness among different distance.Multiple
comparison of mean value were determined using the Turkey’s methods. The data was
tabulated as the average of the 3 to 9 replicates± standard deviation and the level of
significance was set at α=0.05.
52
Figure 5-1. Schematic diagram of continuous PL system (Model LH840-LMP-HSG)
53
Figure 5-2. Energy dose (fluence) during PL treatment as measured by a radiometer
(Adapted from Krishnamurthy 2006)
54
CHAPTER 6 RESULTS AND DISCUSSION
Growth curve study of B. subtilis
The purpose of the growth curve study is to monitor bacterial vegetative cells
during the stationary phase, where the cells reach peak population and develop
resistance to a range of stresses (e.g., thermal, oxidative, starvation) due to phenotypic
and genotypic changes (Hall and Foster 1996). Yoshikawa and Sueoka (1963)
conducted research on the B. subtilis chromosome and concluded that in the stationary
phase, B. subtilis has the complete genetic sequence. In addition, the bacteria are less
likely to die in the stationary phase due to the growing concentration of toxins that
bacteria generate after stationary phase (Gardner and others 1998).
We employed the growth study curve on B. subtilis to determine the parameters
for reaching a stationary phase prior to apply PL treatment. Figure 6-1 displays B.
subtilis starting the stationary phase after approximately a 300 min incubation and the
stationary phase lasts up to 600 min. Gao and others (2014) present similar results
illustrating that B. subtilis reaches its stationary phase after 5 hours incubation and
remains in the stationary phase until 14 hours. Based on the work of Gao and others
(2014), the bacterial show a similar on the growth curve that after 5 h incubation, the
flora entered the stationary phase. For this reason, the incubation time of B. subtilis
inoculum for inactivation was optimized to 6±1 h.
Efficiency of Microbial Decontamination
Evaluation of PL Efficiency in Inactivate B. Subtilis on Wheat Flour and Black Pepper
As mentioned in the literature review, PL inactivation is based on three working
mechanisms, which are photochemical, photophysical and photothermal and have
55
major effects in inactivating microbial cells. According to Wang and others (2005), for
the inactivation study, wavelengths ranging from 200-300 nm align with photochemical
effects and have the most lethal effect for bacterial vegetative cells. It has been well
documented that formation thymine dimers on microbial DNA is the most lethal reaction,
because it prevents the cell from replication by distorting the DNA helix, namely,
causing the inability to replicate or clonogenic death (Bank and others 1990; Jay 1997).
On bacterial spores, UVC results in forming 5-thyminyl-5, 6-dihydrothymine (spore
photoproduct) and also forming single and double strand breaks and cyclobutance
pyrimidine dimers (Slieman and Nichololson 2000). In addition, UV light leads to double
bond cross-linking of aromatic amino acids, which results in membrane depolarization
(Lado and Yousef 2002). Also, photochemical effects can generate ozone and
peroxides after PL exposure. For example, hydroperoxide radicals can be generated by
long wavelength UV treatment, which may react with membranes and lead to changes
in permeability. Short wavelength UV can turn oxygen into ozone that has a strong
oxidation potential which can be applied on inactivating bacteria (Nordhauser and Olson
1998).
There is also evidence provided by Hiramoto (1984) that rays absorbed into
bacterial are expected to heat the bacteria, providing the same effect as thermal
inactivation. Dunn and others (1989) explained that the light pulses heat a superficial
layer of food product, and the heat will transfer to the center. For the photophysical
effect, Wekho and others (2003) provided evidence that spores have an obvious
deformation and rupture on top of their spores, which provides escape of an overheated
56
spore content. Later, Nicorescu and others (2013) showed an electron-microscope
photograph that provided strong evidence for the theory in Figure 6-3.
In the first step, the inactivation effectiveness of PL was studied on B. subtilis,
which was grown to a stationary phase, to make sure the resistance of B. subtilis is
uniform. The results shown in Table 6-1 shows a significant difference from 0.1 log
reduction to 4.1 log. As indicated by the results, the highest reduction achieved was at a
distance of 6 cm for 90 s. The log reduction is 4.1 ±0.3 log on wheat flour with after 90 s
treatment when 6 cm away from quartz window. As illustrated in Figure 6-4, survivor
curves were characterized by a tail. Yaun and others (2003) explained several reasons
for this phenomenon: running out of homogenous population, multi-hit phenomena,
accumulation of suspended solids, and shading effects of Petri dish used in experiment.
Accordingly, McDonald and others (2000) stated that decreasing population density
reduces the likelihood of exposing the biological element under PL.
There are a few factors that can influence the efficiency of PL. The most crucial
factor is fluence incident on the sample. Gómez-López and others (2007) stated that
the energy emitted from the lamp is different from the energy incident on the food
samples. The distance from the lamp to food sample can be a crucial factor, as shown
in Table 6-1. A paired t-test was conducted to test the null hypothesis that there is no
obvious difference between the inactivation effects of different distances from the
sample to the lamp under the same exposure time. Based on the inactivation results
(p<0.05), there are significant differences on inactivation effects between 6 cm and 9
cm. A tukey’s test was also conducted. Based on Table 6-1, bacterial counts dropped
dramatically at 15 to 30 s (p<0.05) on wheat flour samples and lasted consistently for an
57
additional 60s as the fluence increases. The same phenomenon is also detected in
black pepper samples. However, a tailing effect could be detected in this research. The
results matches the finding in Hillegas and Demirci’s (2003) paper that a closer distance
to the lamp would be more effective. The module used to describe this phenomenon
was built by Sharma and Demirci (2003). In their research, the module was built based
on the data they acquired from inactivating E. coli O157:H7 on inoculated alfalfa seed.
The thickness of the sample is another factor can alter the results since it can affect
penetrability. Overlapping opaque obstacle shield the surface from light exposure
(Sharma and Demirci 2003). Lastly, another important factor for PL inactivation effect is
the color of food sample. The details will be illustrated in the temperature profile
analysis.
Yeast Molds Inactivation and Total Aerobic Count
For each treatment, six replicates were used on three days. The initial count of
molds and yeast were measured as high as 6.8 × 104 CFU/ g. Total aerobic count
contamination in the untreated sample was found to be about 107 CFU/g. According to
our observations, yeast and mold counts exhibited a 3.5 log reduction, which is higher
than that reported in another study by Fine and Gervais (2004), who observed 0.7 log
reduction in their wheat flour samples. However, in a study by Kazuko and others’,
when they inoculate the yeast cells on agar, a 6 log CFU/ ml reduction can be achieved.
The huge difference between the results may be attributable to the contamination
surface. For agar, the surface is smooth and can barely influenced by the shadowing
effect. In the food powder samples, the small particles shadowed each other, which
could lead to uneven exposure. Another possible reason that could explain the
difference in reduction may be the higher moisture content of agar, which makes the
58
heat transfer more rapidly, leading to the temperature being amassed at a higher level.
Two distances of 6 cm and 9 cm were used as the distance from the sample to the
quartz window of the pulsed light lamp. Starting at 45 pulses, reduction could be seen
up to 270 pulses for wheat flour. When black pepper samples were analyzed, a strong
overheating effect, accompanied with a darker color was observed. Therefore, the
pulsed light dosages used to treat black pepper was reduced to no more than 90
pulses. In the study by Ito and Islam (1993), a 3 log CFU/g reduction has been achieved
by using both gamma irradiation and electron-beam irradiation on the contaminated
black pepper samples. When the dosage is higher than 7 kGy, it may achieve the 3 log
reduction. An even higher dosage of (8 kGy) is required for the same reduction effect
when the black pepper was treated with e-beam. The results achieved by Ito and Islam
(1993) was quite close to the results for this study, however, according to Ito and Islam
(1993), the dosage is still high, and penetration is the major limiting factor for those two
irradiation methods.
Microbial distribution has always been treated as a critical parameter in PL
applications in food industry (Fine and Gervais 2004). Microorganisms are not
concentrated in the center and they can be present on the outer edges of the plates. In
that case, the distribution of microorganism make it difficult for the PL to properly treat
microorganisms disseminated at the edges. In addition to the challenge of uniform
energy distribution of PL, the penetration of PL can vary depending on the vertical
position of the microorganisms. Microbes that are closer to the lamp will experience
more penetration than those which are beneath and those are shielded by microbes
above. This allows microbes below to avoid being placed directly underneath the PL
59
treatment. Consequently, shielding may dramatically influence the reduction results
(Dunn and others 1995)
Other reasons exist that may lead to the inefficiency of the decontamination
process, one of the reasons is energy fluence (also called energy intensity). When the
distance from the sample to the lamp was adjusted to 6cm, the intensity of energy for
the 15 s treatment was 1.45 J/cm2. Accordingly, as the treatment time extended to 90 s,
the energy measurement increased to 28.8 J/ cm2. Despite the increase, the energy is
still below the threshold for inactivation of microorganisms that exhibit a stronger
resistance (Sharma and Demirci 2003; Kreitner and others 2001). Based on the data we
acquired, it is obvious that there was a significant difference (P<0.05) between the
effects when applied on different distances, which might mainly be caused by the
differences among the fluence.
Temperature Measurement
Pulsed light has generally been considered as a non-thermal inactivation method
when applied for short exposure times. As the treatment time is prolonged, more energy
is absorbed by the food sample, resulting higher temperatures characteristic of a
photothermal effect. As shown in Figure 6-2, the temperature varies for the treatment
times at different distances on both wheat flour and black pepper samples.
Temperatures were measured by an infrared noncontact thermometer. The
average temperature for control group wheat flour was 22.7±0.1 °C and for untreated
black pepper, it was 22.6±0.1°C. Based on the results, wheat flour has up to a
25.1±1.7°C and 15.9±1.9°C temperature increase at 6 cm and 9 cm, respectively. While
for black pepper, it has a larger temperature increase from 65.0±4.7 °C (6 cm) to
44.7±4.6 °C (9 cm). Comparison of the temperature rises in Table 6-2 between 6 cm
60
and 9 cm distance from quartz window illustrated tat shorter distance generated greater
photothermal impact during illumination given the same duration.
As illustrated in the literature, as the distance increases from the lamp source,
temperature increase drops and the food samples receive a less intensive treatment
(Krishnamurthy 2006). In Oms-Oliu and others’ (2010), the researchers pointed out that
the temperature increase after PL treatment is strongly dependent on the color of the
food products. Thus, the darker the product’s color, more energy will be absorbed and
the product’s temperature will increase. According to Oms-Oliu and others (2010), the
differences between fish’s colors can results in different temperature increases after the
treatment. The darker the fish, the greater the temperature increase. Since black pepper
has a generally darker color, it displays higher temperature increases. In Fine and
Gervais’ work (2005), the researchers reached the same conclusion on wheat flour and
black pepper temperature change. They found that black pepper has a more dominant
temperature increase than wheat flour, which results in an overheating effect on black
pepper.
In the statistical results, there was a significant difference among the samples
treated at different distances based on the paired t-test for wheat flour. In addition, the
Tukey HSD test showed that at the first 60 s, there were no dramatic temperature
changes, but after 60 s, there is a 17°C temperature increase. For black pepper, a
significant differences (P<0.05) in inactivation effect between 6 cm and 9 cm treatment
distance can be observed through a paired t-test. This P value indicated that the
different distances could lead to different temperature increases under the same
treatment time. This phenomenon was also explained by Krishnamurthy and others
61
(2008), i.e., temperature increase is proportional to treatment time, but it is inversely
proportional to the sample’s distance from lamp.
Water Activity Measurement
Water activity has always been considered an important factor in microbial
inactivation because water activity alone can nourish or not nourish microorganisms.
Fine and Gervais (2005) pointed out in their study that the highest inactivation rate
under the same conditions was achieved when water activity is 0.15, and the valley is
found when water activity is 0.4 and goes up again. Moreover, Larchos and Gervin
(2003) mentioned that heat resistance of dried vegetative cells is many times higher
than the resistance of the same microbes in an aqueous environment. Thus, water
activity is a crucial factor for the future quality regards to longer storage time. In the
study, for 6 cm treatment on wheat flour, there was a dramatic decrease after 90 s of
treatment on wheat flour. The highest water activity reduction was encountered in the
samples at 6 cm distance to the quartz window, after 90 s treatment on wheat flour. The
water activity was reduced to 0.14, which makes it almost impossible for microbial
growth. While for the same sample, wheat flour, when the distance from the quartz
window increased to 9 cm, the water activity was 0.42 after 90 s of exposure. The same
phenomenon occurred in black pepper samples too, when the distance from the quartz
window became shorter, the water activity decrease was more obvious. Given the same
phenomenon observed in those two groups of samples, it was more probably due to
higher instantaneous temperatures incurred on the samples.
In this study, there were significant differences (p<0.05) among water activities
with different PL treatments. Meanwhile, an obvious decrease could be detected as the
treatment time prolonged. Among all the treatments, the color parameters were
62
relatively consistent. However, there were significant differences (P<0.05) among the
two distances. This provided assurance that PL treatment would positively influence the
future storage of food powder and dry ingredients by decreasing the water activity.
Color Analysis
As mentioned previously, color is a major sensory characteristic associated with
quality in the food industry and market, determining the acceptability of spices and
wheat flour. The CIELAB system is defined by the Commission of International
del’Eclairage (CIE) as a widely used color chromaticity system, using L*, a*, b* to
describe a color. Additionally, hue and chrome also used in the color measurement.
Color parameters of wheat flour before and after PL treatments are summarized
in Table 6-4. The L* value ranged from 62.77 ± 7.72 to 82.30 ± 3.22 for eggs exposed
90s at 6 cm and 60s at 9 cm from the PL lamp respectively, suggesting the lightness of
wheat flour. The sudden drop of L* value indicated a strong over heating effect, which
make wheat flour darker. a* value ranged from 11.04 ±1.03 to 11.79 ± 1.04 for wheat
flour treated 60s at 6cm and 90s at 9 cm respectively, indicating the redness of
samples. Lastly, b* value ranged from 17.83 ±1.49 to 20.98 ±3.04 in sample subjected
to 15s and 9 cm and 90s at 6 cm, suggesting a yellow pattern on wheat flour surface.
The L*, a*, b* values obtained suggested a relative consistent color after different PL
treatment in general, except for the L* value after 90 s PL treatment with a 6 cm
distance that was extremely low. Based on the observation, the overheating effect is
believed to have a considerable influence on ∆E.
Hue and chrome is also used to determine the difference varies the color,
however, based on the results, there is no significant differences (p>0.05) among
different treatment. This might resulted from the hue angle is decided by real color of
63
food product, which can only be altered slightly. The similar results were shown by Ulu
(2004).
Overall, the color results suggest that the PL treatment as a potential inactivation
technique was capable of maintaining the original color of wheat flour and black pepper,
if we set the treatment time less than 90 s with a 6 cm distance from quartz window.
64
Table 6-1. Influence of two different distance and treatment time on two different samples
Wheat flour Black pepper
Treatment
time (s)
Distance from
quartz window
(cm)
Log remaining
(Log10 CFU/g
[mean ± SD])a
Treatment
time (s)
Distance from
quartz window
(cm)
Log remaining
(Log10 CFU/g
[mean ± SD])a
0(control) 6.3 ± 0.3 0 (Control) 5.9 ± 0.5
15 6 5.5 ± 0.4 A 5 6 5.4 ± 0.1 A
30 6 3.6 ± 0.3 B 10 6 4.6 ± 0.2 B
60 6 3.3 ± 0.2 C 15 6 3.7 ± 0.3 C
90 6 2.2 ± 0.2 D 30 6 1.7 ± 0.1 D
15 9 5.7 ± 0.1 A 5 9 5.8 ± 0.2 A
30 9 4.4 ± 0.3 B 10 9 5.2 ± 0.3 B C
60 9 3.8 ± 0.3 C 15 9 4.8 ± 0.2 C D
90 9 3.4 ± 0.2 D 30 9 4.3 ± 0.2 D
a Measured as log10 CFU per gram, where counts are average of 6 replicate trials. Value followed by
sample letter do not differ at the P<0.05 level, whereas values followed by different letters differ at P<0.05 level
65
Table 6-2. Temperature profile of two different distance (6 cm and 9 cm) at various exposure time
Wheat flour Black pepper
Treatment
time (s)
Distance from
quartz window
(cm)
Temperature (°C) Treatment
time (s)
Distance from
quartz window
(cm)
Temperature (°C)
0(control) 22.7 ± 0.1 A 0 (Control) 22.6 ± 0.1 A
15 6 26.9 ± 1.3 A 5 6 35.5 ± 2.1 B
30 6 31.6 ± 2.3 A B 10 6 50.7 ± 4.0 C
60 6 39.5 ± 2.1 B C 15 6 65.2 ± 4.7 D
90 6 48.8 ± 1.7 C 30 6 87.6 ± 3.7 E
15 9 26.7 ± 1.5 A 5 9 31.3 ± 0.8 B
30 9 29.4 ± 3.6 A B 10 9 44.3 ± 1.4 C
60 9 33.2 ± 2.7 B C 15 9 56.7 ± 2.7 D
90 9 38.6 ± 1.9 C 30 9 67.3 ± 4.6 E
a Temperature was recorded after taking out of chamber, where temperature are average of 3 replicate
trials to avoid the error cause by different time delay. Value followed by sample letter do not differ at the P<0.05 level, whereas values followed by different letters differ at P<0.05 level
66
Table 6-3. Effects on water activity of wheat flour and black pepper Wheat flour Black pepper
Treatment
time (s)
Distance from
quartz window
(cm)
Water activity Treatment
time (s)
Distance from
quartz window
(cm)
Water activity
0(control) 0.68 ± 0.01 A 0 (Control) 0.67 ± 0.01 A
15 6 0.52 ± 0.01 B 5 6 0.57 ± 0.01 B
30 6 0.46 ± 0.02 C 10 6 0.53 ± 0.04 BC
60 6 0.32 ± 0.01 D 15 6 0.47 ± 0.01 C
90 6 0.14 ± 0.02 E 30 6 0.40 ± 0.02 C
15 9 0.63 ± 0.01 B 5 9 0.59 ± 0.01 B
30 9 0.61 ± 0.02 B 10 9 0.54 ± 0.01 BC
60 9 0.44 ± 0.01 C 15 9 0.50 ± 0.01 C
90 9 0.42 ± 0.01 C 30 9 0.43 ± 0.01 D
a Water activity was recorded after taking out of chamber, where water activity are average of 3 replicate
trials to avoid the error cause by different level of rehydration. Value followed by sample letter do not differ at the P<0.05 level, whereas values followed by different letters differ at P<0.05 level
67
Table 6-4. Effects on color values and color difference of wheat flour with PL treatment Treatment
time (s)
Distance
from quartz
window
(cm)
L* a* b* ∆E h° C*
0(control) 81.29±0.22A 10.92±1.26A 17.90±1.83A 58.7±0.4A 21.02±0.21A
15 6 81.82±2.68A 11.15±1.11A 17.96±1.64A 2.09±0.06A 58.5±0.4A 20.90±0.22A
30 6 81.89±3.00A 11.06±1.12A 18.89±1.65A 1.97±0.97A 57.2±0.6A 20.07±0.18A
60 6 81.09±2.61A 11.04±1.03A 18.79±1.57A 3.27±0.95A 59.8±0.9A 22.22±0.19A
90 6 62.77±7.72B 11.59±1.58A 20.98±3.04A 22.95±0.94B 59.8±0.3A 20.12±0.17A
15 9 81.37±3.69A 11.14±1.08A 17.83±1.49A 1.01±0.87A 58.3±0.2A 20.66±0.21A
30 9 81.22±2.75A 11.16±1.08A 19.21±1.82A 1.37±0.20A 58.1±0.3A 21.66±0.19A
60 9 82.77±2.97A 11.14±1.12A 19.11±1.61A 2.64±0.26A 59.6±0.5A 20.63±0.18A
90 9 80.81±2.60A 11.79±1.04A 20.06±1.69A 1.99±0.10A 60.8±0.3A 22.12±0.17A
a Color parameters are average of 3 replicate trials to avoid the error cause by different. Value followed by sample letter do not differ at the P<0.05
level, whereas values followed by different letters differ at P<0.05 level
68
Figure 6-1. Growth curve of Bacillus subtilis ATCC 6633 in TSB.
0
1
2
3
4
5
6
0 100 200 300 400 500 600
Log
CFU
/ml
Time (min)
Bacillus subtilis Growth Curve
69
Figure 6-2. Bacillus subtilis (●) growth curve measured by spectrophotometer at 600 nm (Gao and others 2014)
70
Figure 6-3. Bacillus subtilis vegetative cells a). untreated samples; b) treated by PL at
3000 V, 1 Hz, 10 J cm2 / flash (Nicorescu and others 2013)
71
Figure 6-4. Survivor curves for B. subtilis treated with PL at distances of 6 cm and 9 cm from the quartz window for wheat flour sample
0
1
2
3
4
5
6
7
0 20 40 60 80 100
Surv
ivo
rs (
Lo
g C
FU/g
)
Treatment Time(s)
6 cm
9 cm
72
Figure 6-5. Survivor curves for B. subtilis treated with distance of 6 cm and 9 cm from the quartz window for black pepper sample
0
1
2
3
4
5
6
7
0 5 10 15 20 25 30 35
Surv
ivo
r (L
og
CFU
/g)
Treatment Time (s)
6 cm
9 cm
73
Figure 6-6. Temperature profile of wheat flour under different distance (6 cm and 9 cm)
and exposure times
0
10
20
30
40
50
60
0 20 40 60 80 100
Tem
pe
ratu
re (
°C)
Treatment time (s)
6 cm
9 cm
74
Figure 6-7. Temperature profile of black pepper under different distance (6 cm and 9
cm) and exposure times
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35
Tem
per
atu
re (
°C )
Treatment (s)
6 cm
9 cm
75
Figure 6-8. Black pepper after under different treatment time at 6 cm (distance from sample to quartz window
76
CHAPTER 7 CONCLUSIONS AND RECOMMENDATIONS
PL provides a viable method against thermal methods for the decontamination of
dry food powders and food ingredients. Based on our results, pulsed light has an effect
on the inactivation of yeast and molds in wheat flour and black pepper.
PL can significantly reduce the total number of B. subtilis in both wheat flour and
black pepper. The color does not show significant changes (p>0.05) after treatment.
This non-thermal technology may be a good choice for the pasteurization of wheat and
black pepper in the industry due to the short treatment time and effectiveness on
microbial reduction on wheat flour and black pepper.
The PL treatment time and distance from the quartz window (also regarded as
the PL fluence or intensity), number of pulses, thickness of the sample, and duration of
illumination are considered as crucial factors for optimizing a PL system. In order to
optimize the treatment for wheat flour, both inactivation effect and food quality should be
taken into consideration. Based on those qualifiers, for wheat flour at 6 cm distance
from the light source with 60 s treatment would be recommended. While for black
pepper, a 30 s treatment with the distance from quartz window at 9 cm would be
recommended to achieve the optimal inactivation effect while avoid the overheating.
There are, however, some other factors that deserve major consideration in the
present and future studies. In this study, only non-pathogens were used, whereas the
real concern in the food industry are real pathogens. It is recommended the inactivation
of Bacillus cereus should be studied. Although gamma irradiation and microwave can
impair bacteria, some bacteria have the ability to recover after the treatment. Based on
that, a longer term storage is recommended to ensure that the inactivate effect can last.
77
Meanwhile, different effects on the inactivation of Bacillus subtilis in wheat flour and
black pepper have not been fully elucidated. Therefore, a single control factor
experiment should be conducted in order to confirm the hypothesis that color is the
major factor for different inactivation effects in the two products. Besides, the influence
of PL treatment on nutritional values and composition also need to be studied.
78
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BIOGRAPHICAL SKETCH
Ying “Kelsey” Guo was born in 1989 in China. She graduated from Northwest
University with a Bachelors of Science in environmental science. In her undergraduate
studies, she become involved in laboratory research and interned with three beverage
companies, where she eventually discovered an interest in food science.
Ying began her graduate studies in the Department of Food Science and Human
Nutrition at the University of Florida in 2011. Outside academics, she partakes in
several activities including volunteering for Graduate Information Day, Habitat for
Humanity, American Cancer Society, and Gators for Heaven Hospice. During summer
2013, she interned with Paca Foods, Inc. as a technical intern under both the R&D
Department and Quality Assurance Department.