Decontamination of Particulate Foods Using Intense Pulsed

DECONTAMINATION OF PARTICULATE FOODS USING INTENSE PULSED

LIGHT AND OTHER NON-THERMAL TECHNOLOGIES

A DISSERTATION

SUBMITTED TO THE FACULTY OF THE

UNIVERSITY OF MINNESIOTA

Dongjie Chen

IN PARTIAL FULFILLMENT OF THE REQUIERMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Primary advisor: Roger Ruan, Co-advisor: Paul Chen

January 2020

Acknowledgement

Firstly, I sincerely thank to my advisor Dr. Roger Ruan for his instruction and encouragement during my doctorial period at University of Minnesota. He kept driving me to make progress for my research and thesis dissertation. His high efficiency and passions towards research motivated and inspired me all the time.

Secondly, I would like to express my gratitude to my co-advisor Dr. Paul Chen, who also guided me in my research and helped tremendously with my experiment planning and design, data analysis, and writing. I would also thank my other committee members: Dr.

A. Saari Csallany, Dr. Joellen Feirtag, and Dr. Yanling Cheng. They gave me a lot of constructive suggestions on my research and thesis dissertation.

Thirdly, I would like to show my appreciation to Justin R. Wiertzema, Erik Anderson,

Min Min, Chi Chen, David J. Baumler, Zata Vickers, Laurence Lee, Peng Peng, Nan

Zhou, Qingqing Mao, Wes Mosher, Jun An, Kun Li, Pengfei Cheng, and Shuhao Huo.

Their help with my research, experimental design, system developments, and valuable feedbacks to my thesis proposal mean a lot to me.

Finally, I am indebted to my parents Lixin Chen and Hongjue Fu for their support and care. They gave me a lot of inspiration and encouragement when I felt frustrated and confused.

Abstract

Low-moisture particulate foods comprise a wide range of food products such as milk powder, protein powder, egg powder, whey powder, spice, flour, grain, and seeds. Various pathogens or toxins such as C. sakazakii, Salmonella spp., Bacillus cereus spores, and deoxynivalenol (DON) were infected in particulate food matrices. These contaminants are physiologically dormant and metabolically quiescent in low moisture particulate foods and are therefore resistant to conventional thermal process. Conventional heating processes used to eliminate foodborne pathogens may cause some degrees of undesirable flavor and quality changes on particulate food products that are unacceptable for uses by consumers and food industries. This dissertation research focuses on developing non-thermal microbiocidal technologies for dry particulate foods.

Lack of knowledge related to continuous nonthermal techniques on low-moisture particulate foods prevents the technology from applications in food industry. In this study, an intense pulsed light (IPL) treatment system was developed, processing parameters such as relative humidity, temperature, water activity, pulsed duration, voltage, pulsed frequency, and residence time, etc., were evaluated on different low-moisture particulate foods. After several generation-improvement of the IPL system and processes, the results showed a maximum of 4 log10 CFU/g reduction of microbe could be obtained after 60s IPL treatment on the conditions of 1 Hz and 3000 voltage. Furthermore, additional one log10

CFU/g microbial reduction could be achieved when combining IPL with TiO2 based catalysts. Food products such wheat, wheat kernels, and NFDM could be potentially subjected to IPL with minimized quality loss. For milk powder process, the IPL step can be fitted after spray drying.

On the other hand, cold atmospheric plasma was able to inactivate be used to inactivate ~3 log10CFU/g of C. sakazakii in non-fat dry milk after only 120 s. However, the throughput of the system was limited and thereby, difficult to scale up. With respect to plasma activated water, the system was effective in degrading DON (34.6 %) in germinating barley samples while maintaining sample quality after 5 min. For microwave or catalytic microwave treatments, the results indicated microwave treatment below 60 °C was feasible to inactivate pathogens in wheat kernels up to 5 log10CFU/g at the water activity level of

~0.8. Therefore, the process can be fitted in a step after tempering.

In summary, several nonthermal technologies specifically used for particulate food pasteurization were developed in the current research, optimized conditions for disinfection and particulate sample preservation were comprehensively investigated. The findings from this research has filled the key knowledge gaps of preventing the technology from commercialization.

iii

Table of Contents

List of Tables ...... ix

List of Figures ...... ix

Chapter 1. Introduction ...... 1

1.1. Background ...... 1

1.1.1. Outbreaks of particulate foods ...... 1

1.1.2. Conventional pasteurization ...... 3

1.2. Intense pulsed light...... 5

1.2.1. Intense pulsed light generation and spectral distribution ...... 5

1.2.2. The feasibility of intense pulsed light in particulate foods ...... 7

1.3. Cold atmospheric plasma ...... 8

1.4. Plasma activated water ...... 9

1.5. Microwave system...... 10

1.5.1. Mechanisms involved in inactivating pathogens by microwave ...... 10

1.5.2. Microwave-enhanced photocatalysis of TiO2 for inactivating microorganisms

...... 10

1.5.3. The use of microwave in food processing ...... 11

1.6. Objective ...... 13

Chapter 2. Develop and evaluate an experimental intense pulsed light system for processing of particulate foods ...... 14

2.1. Introduction ...... 14

2.2. Materials and methods ...... 15

2.2.1. Bacteria inoculum preparation and enumeration ...... 15

2.2.2. Intense pulsed light treatment ...... 15

2.2.3. Measurements of the sample physicochemical properties ...... 16

2.3. Results and discussion ...... 16

2.3.1. Inactivation kinetics of IPL treatment of C. sakazakii ...... 16

2.3.2. IPL energy distribution on the surface of the vibratory feeder...... 18

2.3.3. Study of IPL efficiency as a function of IPL spectrum range ...... 19

2.3.4. Study of IPL efficiency as a function of sample thickness and lamp height. .. 21

2.3.5. Study of IPL efficiency as a function of different water activity levels and

sample temperature ...... 23

2.3.6. Analysis of surface appearance and amino acid composition ...... 26

2.3.7. Evaluation of IPL effect on different powdered foods after multiple passes .. 27

Chapter 3. Develop and evaluate the prototype intense pulsed light on particulate foods 33

3.1. Introduction ...... 33

3.2. Materials and methods ...... 35

3.2.1. The intense pulsed light system design and development ...... 35

3.2.2. Bacteria inoculum preparation, water activity adjustment, and enumeration . 38

3.2.3. Coating TiO2 on substrates ...... 39

3.2.4. Measurements of the IPL fluence, temperature, and flow rate ...... 39

3.2.5. Particle size measurements ...... 40

3.2.6. Gamma radiation and IPL treatment of mesquite flour ...... 40

3.2.7. Physicochemical property analysis of treated mesquite flour ...... 41

3.2.8. Cytotoxicity analysis ...... 41

3.3. Results and discussion ...... 42

3.3.1. Optimizing processing parameters for pathogen disinfection ...... 42

3.3.2. Effect of photocatalyst ...... 50

3.3.3. Comparison of the intense pulsed light and gamma radiation on Bacillus

cereus spores in mesquite pod flour ...... 52

3.3.4. Effects of the intense pulsed light on microbes in seeds ...... 62

3.3.5. C. sakazakii inactivation kinetics in NFDM ...... 65

3.4. Conclusion ...... 68

Chapter 4. Effects of cold atmospheric plasma on Cronobacter sakazakii inactivation and physicochemical property changes of non-fat dry milk powder ...... 70

4.1. Introduction ...... 70

4.2. Materials and methods ...... 70

4.2.1. Sample inoculation and enumeration ...... 70

4.2.3. Physicochemical property evaluation after CAP ...... 72

4.3. Results and discussions ...... 72

4.3.1. Effects of CAP on C. sakazakii inoculated in NFDM ...... 72

4.3.3. Effects of CAP on the color of NFDM ...... 76

4.3.4. Effects of CAP on NFDM crystallinity ...... 79

4.3.5. Effects of CAP on the amino acid composition of NFDM ...... 80

4.3.6. Effects of CAP on total phenolic content ...... 82

4.3.7. Characterization of CAP through optical emission spectroscopy ...... 83

4.4. Conclusions ...... 86

Chapter 5. Decontamination of Deoxynivalenol in Raw and Germinating Barley using

Plasma-Activated Water and Intense Pulsed Light ...... 87

5.1. Introduction ...... 87

5.2. Methods and materials...... 87

5.2.1. Sample preparation ...... 87

5.2.2. PAW generation and treatment...... 87

5.2.3. ORP, conductivity, and pH value measurements ...... 88

5.2.4. Laboratory germination test...... 88

5.3. Results and discussions ...... 89

5.3.1. Effect of PAW on DON Reduction and Germination Rate ...... 89

5.3.2. Effects of IPL on DON Reduction and Germination Rate ...... 96

5.4. Conclusion ...... 101

vii

Chapter 6. Decontamination of wheat kernels and wheat flour using catalytic microwave system ...... 102

6.1. Introduction ...... 102

6.2. Materials and methods ...... 102

6.2.1. Sample preparation and inoculation ...... 102

6.2.2. Microwave and catalytic microwave treatments ...... 103

6.3. Results and discussions ...... 104

6.3.1. Disinfection of wheat/wheat flour using microwave ...... 104

6.3.2. The synergistic effect of TiO2 and microwave on wheat kernel disinfection 106

6.3.3. Photocatalyst modification ...... 108

6.4. Conclusions ...... 111

Chapter 7. Summary and future work ...... 112

7.1. Summary of the dissertation ...... 112

7.2. Future work ...... 113

Bibliography ...... 115

Appendix A: Effects of IPL on different microbes in filter papers ...... 133

Appendix B: Sensory evaluation of IPL treated wheat flour ...... 136

viii

List of Tables

Table 2.1. C. sakazakii inactivation as a function of different spectrum range ...... 20

Table 2.2. Composition of major amino acids (% w/w) in control and IPL-treated NFDM.

...... 27

Table 2.3. Processing parameters of each IPL treatment (28s)...... 30

Table 2.4. The product mean particle size as a function of different number of passes. .. 31

Table 3.1. Temperature and water activity profiles of different treatments...... 46

Table 3.2. Particle diameters of treated and untreated samples...... 47

Table 3.3. Color difference (ΔE) of IPL treated samples ...... 49

Table 3.4. Disinfections of 60s-IPL and 60s-CIPL...... 51

Table 3.5. The L*, a*, b* values, and color difference (ΔE) of mesquite flour subjected to

IPL treatment of 28 s and different dose of gamma radiation ...... 55

Table 3.6. Chemical markers separating IPL and γ-irradiation treatments from Control . 62

Table 3.7. Energy consumption and microbial inactivation for different seeds under IPL, the data is mean values of duplicated experiments...... 64

Table 3.8. Chemical Statistical parameters of four models for kinetic inactivation of C. sakazakii in NFDM ...... 67

Table 4.1. The L*, a*, b* values, and color difference (∆E) of NFDM subjected to CAP treatment at gas flow rate from 8-20 L/min up to 120 s...... 78

Table 4.2. Composition of major amino acids (% w/w) as a function of CAP treatment time ...... 81

Table 4.3. Total phenolic content as a function of CAP treatment time ...... 83

Table 5.1. Germination score based on the number of kernels with rootlet over one kernel length...... 88

Table 5.2. Physicochemical properties (ORP, conductivity, and pH) of control (DI water) and PAW after 20 min treatment without samples ...... 90

Table 5.3. The values of (a) ORP, (b) electrical conductivity, and (c) pH value of control

(PAW without samples), raw barley, and germinating barley, respectively, from 0-20 min plasma activation time...... 91

Table 5.4. Effect of PAW treatment on the DON level (%) in raw and germinating barley samples...... 94

Table 5.5. Raw and germinating barley samples germination rate score as a function of

PAW treatment time...... 96

Table 5.6. DON concentration of raw and germinating barley during IPL treatment...... 98

Table 5.7. Raw and germinating barley samples germination rate score as a function of

PAW treatment time...... 100

Table 6.1. Microwave disinfection as a function of different treatment conditions ...... 105

List of Figures

Figure 1.1. The schematic diagram of intense pulsed light generation ...... 6

Figure 1.2. The typical spectral distribution of intense pulsed light ...... 6

Figure 2.1. A schematic diagram of the IPL system ...... 16

Figure 2.2. Inactivation of C. sakazakii using IPL treatment ...... 18

Figure 2.3. Energy distribution and fluence received during the original IPL illumination.

...... 19

Figure 2.4. Response surface diagram of IPL on log10 reduction of C. sakazakii ...... 23

Figure 2.5. Log reduction (CFU/g) of NFDM treated with IPL at different water activity levels ...... 25

Figure 2.6. The NFDM mean particle size as a function of water activity, ...... 25

Figure 2.7. Effect of multiple passes of IPL on the inactivation of C. sakazakii and E. faecium...... 30

Figure 3.1. Schematic diagram illustrating the principle of TiO2 photocatalysis ...... 34

Figure 3.2. (a) Schematic diagram of original IPL system; (b) the modified IPL system;

Figure 3.3. Inactivation of C. sakazakii and E. faecium in wheat flour and NFDM at different peak voltage and feed rate ...... 45

Figure 3.4. Microbial inactivation of mesquite flour under different treatments ...... 55

Figure 3.5. Chemometric analysis of potential chemical changes in mesquite flour after

IPL- and -irradiation treatments ...... 60

Figure 3.6. Concentration of short-chain fatty acids in mesquite flour after IPL- and - irradiation treatments...... 61

Figure 3.7. Direct effect of flour with different treatments on Caco-2 cell viability ...... 62

Figure 3.8. Mean surviving population of C. sakazakii during prototype IPL ...... 68

Figure 4.1. Schematic diagram of the CAP system ...... 72

Figure 4.2. Log reduction (CFU/g) of NFDM treated with CAP at different flow rates and treatment times ...... 75

Figure 4.3. Surface temperature of NFDM samples during atmospheric cold plasma treatment from 0-40 s ...... 76

Figure 4.4. XRD results for the untreated samples and the CAP treated NFDM samples from flow rate of 8-20 L/min for 120 s ...... 80

Figure 4.5. Optical emission spectra of CAP discharge (6.5 cm above the nozzle) at the flow rate of 20 L/min ...... 85

Figure 5.1. Surface temperature of raw and germinating barley during IPL treatment .... 98

Figure 6.1. A schematic diagram of catalytic microwave/microwave system ...... 104

Figure 6.2. Effect of C. sakazakii population inoculated in wheat kernels as a function of different microwave treatments ...... 107

Figure 6.3. Temperature of wheat kernel during microwave treatment combined with

TiO2 ...... 108

Figure 6.4. Log10 reduction of C. sakazakii in wheat kernels as a function of different catalytic treatments ...... 110

xii

Chapter 1. Introduction

1.1. Background

1.1.1. Outbreaks of particulate foods

Particulate food products consist a wide range of food products such as milk powder, whey powder, egg white powder, protein powder, wheat flour, spice, and seeds (Beuchat,

Komitopoulou, Beckers, Betts, Bourdichon, Fanning, et al., 2013). They remain resistant to microorganism contamination in the condition of relatively low water activity levels

(Cordier, 2014). However, inappropriate environmental conditions or food processing may lead to foodborne hazards. Many outbreaks associated with pathogens including

Salmonella spp., Cronobacter sakazakii, Bacillus cereus, Clostridium spp., E. coli

O157:H7, and Staphylococcus aureus, etc., were reported recently in particulate foods

(Beuchat, et al., 2013).

Cronobactor sakazakii was linked to a severe life-threatening neonatal meningitis

(Nazarowec-White & Farber, 1997). C. sakazakii is likely to infect humans of all ages especially for the immunocompromised people such as premature infants and elderly people. Powdered infant formula (PIF) and starches are a main route of C. sakazakii contamination. The number of C. sakazakii infected cases are tripled as in 2010 and continue to rise according to a report by the Centers for Disease Control and Prevention

(Feeney, Kropp, O’Connor, & Sleator, 2014). Because C. sakazakii is resistant to dryness and relatively high temperature, C. sakazakii can survive in extremely low moisture foods up to several years. (Asakura, Morita‐Ishihara, Yamamoto, & Igimi, 2007). As evidenced by a previous study, which indicated that the decimal reduction times of C. sakazakii strains in saline solution were 12–16 min at 52 °C (S.-H. Kim & Park, 2007).

Salmonella spp. is another important foodborne pathogen in particulate foods, the pathogen is usually contaminated in infant formula, almonds, and spices (Angulo, Cahill,

Wachsmuth, Costarrica, & Embarek, 2008; CDC, 2010). In 2009, several cases of outbreaks related to the Salmonella occurred in eggs and egg products (EFSA, 2009).

Similar to other microorganisms, the major challenge faced by many researchers is that

Salmonella spp. is resistant in dehydration conditions and high temperature (D. Miller,

Goepfert, & Amundson, 1972). A study found that Salmonella in egg white powder capable of surviving at 54 ºC for one week as longs as relative low moisture conditions are maintained (Archer, Jervis, Bird, & Gaze, 1998). Inappropriate or insufficient food processes led to severe outbreaks in wheat flour recently. In 2019, CDC reported 17 people from United Stated was infected with E. coli. O26 in wheat flour. The strain was isolated from sealed flour bag. Similarly, 63 cases from 24 states were infected with Shiga Toxin-

Producing E. coli. O26 or O121 from wheat flour in 2016. Among them, 19 patients had raw dough or raw batter (CDC, 2016).

In addition, mycotoxins such as deoxynivalenol, aflatoxins, trichothecenes, ochratoxins, and fumonisins, etc., produced by Aspergillus spp., Penicillium spp., and

Fusarium spp. are critical issues in grains especially for those in insufficient dry storage conditions, or due to sudden climate changes (aw> 0.7) (Garrett, Thomas-Sharma, Forbes,

Nopsa, Ziska, & Dukes, 2014; Magan, Hope, Cairns, & Aldred, 2003). Many studies reported mycotoxins could cause production and quality loss in wheat or barley (Pereyra

& Dill-Macky, 2008; Trail, 2009). The symptoms such as emesis, diarrhea, anorexia, hemorrhage and digestive disorders can be induced by digesting mycotoxins contaminated grain products (Wan, Huang, Pan, Wu, Chen, Tao, et al., 2013). Heating sterilization was

2 used to eliminate mycotoxins in grains (Abramson, House, & Nyachoti, 2005; Bretz, Beyer,

Cramer, Knecht, & Humpf, 2006). However, susceptibility of barley to high temperature is still a main challenge that limits the application (Hoang, Sechet, Bailly, Leymarie, &

Corbineau, 2014).

B. cereus is an aerobic or anaerobic gram‐positive, spore‐forming bacterium, the bacterium usually presents in particulate foods such as grains, raw and cooked rice, egg white powder, and milk powder (Bottone, 2010; Stenfors Arnesen, Fagerlund, & Granum,

2008). B. cereus can reproduce at both low and high temperatures (4 to 50 °C) and both wet and dry environments. Moreover, two types of toxins, namely an emetic toxin and diarrheal enterotoxins can be produced by B. cereus. B. cereus spores are very resistant to chemical stress, radiation, and UV light (Borge, Skeie, Sørhaug, Langsrud, & Granum,

2001; Moeller, Raguse, Reitz, Okayasu, Li, Klein, et al., 2014). B. cereus spores can survive up to several months in milk powder, flour, or spice (Richards, Gurtler, & Beuchat,

2005).

1.1.2. Conventional pasteurization

Thermal treatments are commonly used because of easy operation. Conventional heating pasteurization methods such as high temperature short time (HTST) or ultra-high temperature (UHT) pasteurization can only be used to inactivate liquid milk (Ramirez,

Patel, & Blok, 2006). On the other hand, microorganisms such as Salmonella spp. and C. sakazakii are resistant to thermal treatment. Therefore, high population of microbial residues may potentially cause risk even after thermal pasteurizations (S.-H. Kim & Park,

2007). Moreover, essential nutrient or quality attributes can be degraded in infant formula

3 during heating processes. The quality loss is exhibited in the form of discolor, off flavor, protein denaturation, etc (Rosset, Noel, & Morelli, 2007). Heating treatment was proved very low efficiency in DON degradation, DON was degraded from 4.3 to 2.9 ppm at 80 °C for 8 days (Abramson, House, & Nyachoti, 2005).

Ionizing irradiation including electron beam, X-rays, and gamma rays have been used to address safety issues for dry foods. A study showed 3-log10 CFU/g reductions of aerobic bacteria was obtained in malted flour using 10 kGy dose of gamma irradiation

(Mukisa, Muyanja, Byaruhanga, Schüller, Langsrud, & Narvhus, 2012). However, ionizing radiation beyond a dose level of 10 kGy was not approved because of safety risks (FDA,

2004b). High dose of irradiation also brought adverse impacts on the aroma of red pepper powder (Lee, Sung, Lee, & Kim, 2004). Moreover, many food processing facilities are not installed with irradiation facilities because of high costs.

Ozone is highly reactive and can be used as antimicrobial agent because of high redox potential of 2.07 V (Varga & Szigeti, 2016). Bacterial cell components including cell envelopes, cytoplasm, and cell membranes can be oxidized by ozone (Khadre, Yousef,

& Kim, 2001). An experiment was carried out to eliminate C. sakazakii in skim milk powder with gaseous ozone, it took 120 min to reduce C. sakazakii by 3.28 log10 CFU/g with 5.3 mg/L gaseous ozone (Torlak & Sert, 2013). It took 60 min to degrade DON in wheat kernels by 39.16% with 75 mg/L ozone (the initial DON concentration was 1.69 ppm) (L. Wang, Shao, Luo, Wang, Li, Li, et al., 2016).

Continuous UV light can be used to treat liquid because of relatively high transparency capability of liquid. For examples, nearly 3 log10CFU/g reduction of C. sakazakii could be achieved in liquid infant formula after 20 min of continuous UV-C

4 combined with 60 °C water treatments (Q. Liu, Lu, Swanson, Rasco, & Kang, 2012). It has limited penetration depth for solid materials. UV light has been used as a surface treatment to reduce the microbial loads on fresh produces such as fruits and vegetables, as well as to induce food matrices resistance to the microbial contamination through hormesis

(Guerrero-Beltr· n & Barbosa-C· novas, 2004).

1.2. Intense pulsed light

1.2.1. Intense pulsed light generation and spectral distribution

Intense pulsed light is also called pulsed light. It consists of intense and short flashes ranging from 10-6 to 10-1 s with broad spectral ranges from ultraviolet (UV) to near infrared

(IR) (Oms-Oliu, Martin-Belloso, & Soliva-Fortuny, 2010). The components are composed of UV-A (315-400 nm), UV-B (280-315 nm), UV-C (180-280 nm), visible light (400-700 nm), and infrared light (700-1100) (N Elmnasser, Guillou, Leroi, Orange, Bakhrouf, &

Federighi, 2007). Intense pulsed light is generated from a flash lamp through ionization of xenon. The generation process was shown in Figure 1.1. (1) Alternating current is firstly converted into direct current by a rectifier since intense pulsed light generation is required a direct current supply; (2) The electrical energy is then stored in a high-energy electrical storage capacitor. (3) The electrical energy is then discharged into a flash lamp filled with xenon gas. (4) Xenon atoms absorbs the electrical energy, electrons are excited and

5 elevated to an excited state, to return to the ground state, the electrons release a discrete amount of energy as photons (Keener & Krishnamurthy, 2014).

Figure 1.1. The schematic diagram of intense pulsed light generation

Wavelength range can vary with lamp sources. A typical spectral distribution of this xenon intense pulsed light spectrum contains around 54% of UV light, 26% of visible light, and 20% of infrared light (H. Q. Zhang, Barbosa-Canovas, Balasubramaniam, Dunne,

Farkas, & Yuan, 2011). The exact region is shown in Figure 1.2.

Figure 1.2. The typical spectral distribution of intense pulsed light

The intense pulsed light has characters of high intensity and extremely short duration (100-400 µs averagely) (N Elmnasser, Guillou, Leroi, Orange, Bakhrouf, &

Federighi, 2007). The unit of total energy accumulation is “joules per square-centimeter”.

The light energy is accumulated over a long period of time and discharged in extremely short time. Therefore, this high intensity/energy density of intense pulsed light (IPL)

6 contributes to a significantly higher microbial inactivation than continuous UV light with equivalent energy consumption (J. E. Dunn, Clark, Asmus, Pearlman, Boyer, Painchaud, et al., 1989). A study was conducted to investigate L. monocytogenes and Escherichia coli

O157:H7 inactivation and found intense pulsed light was more efficient than continuous

UV light. A type of IPL is capable to discharge around 35 MW of impulsive power at a fluence level of 3 J/cm2 (Bhavya & Umesh Hebbar, 2017). In contrast, continuous UV system only dissipate 100-1000 W (Hillegas & Demirci, 2003; MacGregor, Rowan,

McIlvaney, Anderson, Fouracre, & Farish, 1998). The main mechanism regarding microbiocidal effect by IPL light is photochemical effect, which contributes to the formation of pyrimidine dimers in the DNA of different pathogens, leading to the prevention of cell replication. Photothermal effect was induced when the temperature of the sample exceeds the fluence threshold by exposing to pulsed light. Cell disruption and protein denaturation occur in this stage. Photo-physical effect is caused by disturbances of intermittent high energy pulses. Cell exhibits in the form of cellular content leakage and cytoplasmic membrane shrinkage (Oms-Oliu, Martin-Belloso, & Soliva-Fortuny, 2010).

1.2.2. The feasibility of intense pulsed light in particulate foods

Numerous studies showed that IPL technology was effective to inactivate pathogens in the powdered food at lab scale. Around 1.5-log10 reduction of C. sakazakii in

PIF on a petri dish was obtained using IPL treatment (Choi, Cheigh, Jeong, Shin, Park,

Song, et al., 2009). Furthermore, a research measured amino acid composition of proteins and lipid oxidation in milk powder (Noura Elmnasser, Dalgalarrondo, Orange, Bakhrouf,

Haertle, Federighi, et al., 2008). And it reported that no significant change was detected after short time pulsed light treatment (10 pulses). On the other hand, the safety of egg

7 products could be also improved using IPL because of its high effectiveness in inactivating

Salmonella spp. No obvious alteration in quality of egg products was induced. For instance, a reduction of 5 log CFU/g microbe was reached with fluence of 2.1 J/cm2 IPL

(Lasagabaster, Arboleya, & De Maranon, 2011). Moreover, the researcher indicated IPL did not affect the sensory and rheological properties of egg white. This technology can also be adopted in mycotoxins elimination (Moreau, Lescure, Agoulon, Svinareff, Orange, &

Feuilloley, 2013).

1.3. Cold atmospheric plasma

Nonthermal plasma (NTP) technologies have been broadly studied in many types of foods. Various studies have been conducted to inactivate pathogens such Salmonella, aflatoxins, E. coli O157: H7, and B. cereus (Moisan, Barbeau, Crevier, Pelletier, Philip, &

Saoudi, 2002). NTP can be generated in the forms of dielectric barrier, cold atmospheric plasma (CAP), plasma activated water (PAW), etc. Therefore, CAP and PAW on particulate foods will be investigated and discussed in this chapter.

Plasma corona discharge, a type of CAP generation technique was employed in the current study, which capable of generating a variety of antimicrobial agents such as reactive species (RS), electron radicals, and ions. The resulting antimicrobial agents vary depend on types of gas composition, operating temperatures, excitation properties (Chen,

Peng, Zhou, Cheng, Min, Ma, et al., 2019). The antimicrobial impacts of plasma treatment are based on the RS damage to the deoxyribonucleic acid (DNA) in the chromosomes, volatilization of compounds, and the cell surfaces (Korachi & Aslan, 2011). UV spectrum from CAP also took an important part in microbicidal effect by disrupting DNA transcription and translation. A previous research has investigated low temperature

8 atmospheric plasma on particulate foods such as milk powder. However, only resulting in

0.910 log10 CFU/g microbial reduction in milk powder. Similarly, a study reported that

1.34 log10 CFU/g of E. coli O157:H7 in almonds was inactivated after a 20-s treatment

CAP (Brendan A Niemira, 2012).

1.4. Plasma activated water

Plasma activated water (PAW) was formed by through non-thermal plasma (NTP) and deionization (DI) water. Several RS generated in PAW can be used to inactivate some pathogens (Oehmigen, Hähnel, Brandenburg, Wilke, Weltmann, & Von Woedtke, 2010).

High positive oxidation reduction potential (ORP) and low pH are major contributors of

PAW disinfection (Tian, Ma, Zhang, Feng, Liang, Zhang, et al., 2015; Q. Zhang, Liang,

Feng, Ma, Tian, Zhang, et al., 2013). Acidic (pH=2-3) solutions that contain hydrogen peroxide, nitrate, nitrite anions, and other reactive species were able to be produced from air plasma created by gliding arc or dielectric barrier discharge (DBD) at atmospheric pressure (Oehmigen, Hähnel, Brandenburg, Wilke, Weltmann, & Von Woedtke, 2010).

This solution was more effective in microbial inactivation at acidic condition (Burlica,

Grim, Shih, Balkwill, & Locke, 2010). However, the microbicidal effects decreased significantly at pH beyond 3 (Oehmigen, Hähnel, Brandenburg, Wilke, Weltmann, & Von

Woedtke, 2010). PAW had very slight or no significant impacts on food quality, it was found to eliminate S. aureus in strawberries without inducing any physicochemical changes in skin color, texture, and pH value of the strawberries (Ma, Wang, Tian, Wang, Zhang, &

Fang, 2015).

1.5. Microwave system

1.5.1. Mechanisms involved in inactivating pathogens by microwave

Microwave heating of foods was based on the presence of water and fat. The moisture content of particulate foods such as wheat and wheat flour ranges from 13% to

18%. The moisture content of bacteria is from 50-60%. Bacteria were likely to absorb more microwave energy than the food matrix if bacteria on the surface of particulate foods to microwave. Therefore, a momentous overheating is a major cause of microbial inactivation while maintaining the food quality. Microwave destruction of microbes or enzymes could be explained by the following order: selective heating, electroporation, cell membrane damage, and magnetic field coupling (Kozempel, Annous, Cook, Scullen, & Whiting,

1998). The selective heating theory indicates that the microorganisms are selectively heated as discussed above. Electroporation, membrane rupture, and magnetic field coupling cause cellular membrane damages including the leakage, rupture, or disruption of cellular materials (Kozempel, Annous, Cook, Scullen, & Whiting, 1998).

1.5.2. Microwave-enhanced photocatalysis of TiO2 for inactivating microorganisms

Titanium (Ⅳ) oxide can be considered as an effective and widely photocatalyst due to its capability to effectively transform the light energy into the chemical energy. A commonly accepted explanation of the antimicrobial effect associated with TiO2 photocatalysis is that light of wavelengths below a threshold of 390 nm can generate electron hole pairs of strong redox capability on the TiO2 surface, converting water and oxygen into hydroxyl radicals (•OH), and superoxide ions (O•), and hydrogen peroxide

•− (O2 ) that are main reactive agents in killing microbes (Markowska-Szczupak, Ulfig, &

Morawski, 2011). Conventionally, photocatalysis of TiO2 can only be driven by UV range light sources, which restricts its application in many settings and often necessitates modification of TiO2 by doping of noble metals to enable activation by lights of other wavelengths such as visible light (Kowalska, Wei, Karabiyik, Herissan, Janczarek, Endo, et al., 2015). However, recent studies have demonstrated that microwave irradiation was also capable of activating TiO2 photocatalysis reaction. When used in combination with

UV irradiation, microwave irradiation can significantly enhance the photocatalysis reactions possibly owed to synergetic effect of the two irradiations (Církva & Relich, 2011;

Horikoshi, Hidaka, & Serpone, 2003). A promising system setting combining the two is the application of electrodeless discharge lamp (EDL) as a novel light source which generates efficiently UV light when placed into a microwave field. In the recent years, microwave enhanced photocatalysis using EDL has been widely investigated as an advanced coupling technology to treat organic wastewater and very promising results have been reported in terms of complete and efficient degradation of various organic compounds such as plasticizers, dyes, and pesticides (Horikoshi, Kajitani, & Serpone, 2007; Z. Zhang,

Jiatieli, Liu, Yu, Xue, Gao, et al., 2013; Zhong, Shaogui, Yongming, & Cheng, 2009).

These results suggested the great potential of microwave enhanced photocatalysis to inactivate microbes.

1.5.3. The use of microwave in food processing

Microwave is a conventional heating treatment that has been used to cook or sterilize foods. Microwaves are electromagnetic waves with frequencies ranging from 300

MHz to 300 GHz. 915 and 2450 MHz of microwave are most widely used in the worldwide.

The domestic microwave oven of frequency 2450 MHz is a relatively inexpensive apparatus compared with other classical heating sources. Microwave has been tested on a number of liquid and solid foods. A 2.45 GHz pilot plant microwave system was used to disinfect three common species of stored-grain (Vadivambal, Jayas, & White, 2007). Four different power levels 250, 300, 400, and 500 W were used for infested grains for two exposure times of 28 and 56 s. The data indicated that no significant difference was observed in the quality aspects of grain protein, flour protein, flour yield, flour ash, and loaf volume after microwave treatments. Disinfection of in-shell egg by microwaves was studied (Dev, Raghavan, & Gariepy, 2008). Microwave pasteurization of shell eggs could be achieved without losing the shell integrity of eggs (Dev, Raghavan, & Gariepy, 2008).

Similarly, microwave pasteurization was employed to kill Salmonella typhimurium in the yolk of shell eggs (Shenga, Singh, & Yadav, 2010). A 22% reduction of microbes was achieved by microwave irradiation for 15 s. While 36% of reduction was obtained after 15 minute- moist heat treatment (Shenga, Singh, & Yadav, 2010). More evidences indicated that microwave heating could provide high temperature and short time food processing with minor quality change. For example, Lau and Tang inactivate pickled asparagus in glass jars using 915 MHz microwaves (Lau & Tang, 2002). The process offered uniform heating while shorted one and a half processing time compared to water-bath heating.

Furthermore, the microwave pasteurization significantly reduced thermal degradation of asparagus.

1.6. Objective

The overall goal of this research was to develop and study non-thermal technologies for pasteurization of particulate foods. The specific objectives are to:

(1) To develop an experimental IPL system, and assess the effects of IPL treatments on microbial inactivation and physicochemical property of non-fat dry milk (NFDM).

(2) To optimize IPL processing parameters for different particulate foods and evaluate the impacts of IPL on these particulate foods.

(3) To improve and modify the experimental IPL system and processes based on the findings on microbial inactivation and physicochemical properties. A prototype system will be subsequently designed.

(4) To develop and assemble a prototype IPL system and evaluate performance of the system on particulate foods.

(5) To evaluate nonthermal hurdle technologies including CAP, PAW, microwave systems for processing of particulate foods.

Chapter 2. Develop and evaluate an experimental intense pulsed light system for processing of particulate foods

2.1. Introduction

There are a variety of potential processing issues need to be addressed before industrial application of IPL on particulate foods. For some sensitive powdered foods, moisture absorption from ambient environment and elevated temperature caused by IPL treatment usually resulted in undesirable agglomeration during IPL treatment (Peleg &

Bagley, 1983). The agglomeration therefore limits the IPL disinfection on microbes.

Temperature and humidity controllers potentially prevent the samples from overheating and caking during IPL treatment. In addition, IPL treatment is a sort of surface treatment, and has limited penetration which prevent it from utilizing in solid foods (N Elmnasser,

Guillou, Leroi, Orange, Bakhrouf, & Federighi, 2007). Penetration limitation of IPL occurred in most cases and cannot reach areas shaded by seed hull (Wallen, May, Rieger,

Holloway, & Cover, 2001). Partial decontamination could be induced if no homogenous methods were used. On the other hand, a previous study reported that a 1.5-log reduction of C. sakazakii in infant milk powder laid on a petri dish was achieved with IPL treatment

(Choi, et al., 2009). Therefore, a three-dimensional exposure of food samples to IPL needs to be carried out to maximize IPL decontamination. To resolve the issue, a vibratory treatment chamber will be developed to help rotate or tumble particles in order to achieve better exposure of entire samples to IPL.

2.2. Materials and methods

2.2.1. Bacteria inoculum preparation and enumeration

NFDM was received from Land O’Lakes, Inc. (Arden Hills, MN). The NFDM was made from pasteurized milk, and all samples were double vacuum-sealed and conveyed to the laboratory at the University of Minnesota until use. The methods to inoculate C. sakazakii ATCC 29544 on NFDM, water activity level adjustment, and bacterial enumeration were according to our previous study (Chen, Wiertzema, Peng, Cheng, Liu,

Mao, et al., 2018).

2.2.2. Intense pulsed light treatment

The experimental IPL system is composed of an IPL lamp, a vibratory feeder, a humidifier, and an air conditioner. The IPL source was a lab scale Z-1000 steripulse- XL system (Xenon Corporation, Woburn, MA) with the wavelength in the range of 190 nm–

1100 nm. A lamp cooling system was used to prevent the lamp from overheating during the process (Figure 2.1). High intense pulses were generated at a rate of 3 pulses per second with pulse width of 360 μs. Each pulse delivered 1.27 J/cm2 at an input of 3800 voltage.

Different residence time was controlled by adjusting the vibratory frequency. There was a

1-min interval among treatments to cool down the IPL system. Each treatment was carried out in triplicate.

Figure 2.1. A schematic diagram of the IPL system

2.2.3. Measurements of the sample physicochemical properties

Five grams aliquot of untreated and treated samples were used to determine particle size, color, amino acid composition, and sample temperature (Chen, et al., 2018).

2.3. Results and discussion

2.3.1. Inactivation kinetics of IPL treatment of C. sakazakii

Before treating a complex food matrix with IPL, one needs to evaluate C. sakazakii inactivation models with current IPL system. IPL dose-response curve for dispersed C. sakazakii in a filter paper can be fitted by first-order kinetics (EPA, 2003; G. Liu, 2005).

UV-light or pulsed light inactivation curve is sigmoidal (CFSAN-FDA, 2000) with shoulder and tail. Shoulder or tailing effect occurs because bacteria is shielded by external matrix (CFSAN-FDA, 2000; EPA, 2003). Therefore, pulsed light may not cause shoulder

16 and tailing effects for bacteria inactivation in a clear filter paper for a relatively short period of time. The first order inactivation equation can be expressed as (EPA, 2003)

퐷 −( ) (−푘퐷) 퐷 푁 = 푁0e = 10 10 (Equation 1)

Where N is the population of microorganisms after IPL treatment (CFU/mL), N0 is the population of microorganisms before IPL treatment, k represents the first order inactivation

2 2 coefficient (cm /J), D is the total dose delivered or fluence (J/cm ). D10 is IPL dose required to achieve 90% reduction in microbial population. Therefore,

푁 퐷 Log ( 0) = (Equation 2) 푁 퐷10

D10 value of a microorganism is to show the extent of the resistance of this microorganism to IPL light. Low D10 value means low resistance to IPL exposure, while high D10 value indicates high resistance to IPL.

Equation 1 can also be converted to the equation as follow,

푁 1 푘 Log ( 0) = kD ∗ = ( ) ∗ 퐷 (Equation 3) 푁 In(10) 2.303

푘 푁0 Where ( ) is the slope of the fitted straight line, and Log ( ) is the log10 reduction of 2.303 푁 the microorganism. Figure 2.2 shows the correlation between the IPL dose and log10

2 reduction of C. sakazakii. Based on the equation above, the D10 = 6.2 J/cm , and the k =0.37 cm2/J, R2 = 0.95. The samples were located at 8 cm from IPL lamp quartz window and was treated up to 30 s at 2.5 kW and 1 kV, the broadband energy intensity available at this location was measured at 0.5 J/cm2/pulse using a Vega laser power meter., It took 16.2 ms of total pulsed duration (360 µs per pulse) to achieve ~5 log reductions of C. sakazakii. In contrast, a previous study indicated 4.6 ms of total pulsed duration was required to achieve

~5 log reductions of C. sakazakii at 5.5 kW and 10kV(Choi, et al., 2009). The shorter pulsed duration might be as a result of significantly higher power and voltage were used.

10 9 y = 0.161x + 1.4169 R² = 0.9533 8

7 CFU/mL)

10 6 5 4

3 reduction (log

10 2

Log 1 0 0 10 20 30 40 50 2 IPL dose (J/cm )

Figure 2.2. Inactivation of C. sakazakii using IPL treatment

2.3.2. IPL energy distribution on the surface of the vibratory feeder

The Figure 2.3 shows the distribution of energy received on the vibratory feeder along the length and width. The width of the blue area indicates the highly effective IPL region. These data are helpful for the design of vibratory feeder for IPL system. The area outside of the blue area is less effective. The most effective width in vibratory feeder is ~5 cm at 8 cm using this Z-1000 IPL system. And the energy is diluted at both sides of IPL lamp. Energy density at central point is much higher than both sides. This non-uniform distribution of fluence is caused by the fact of close distance between xenon lamp and quartz window (Artíguez & de Marañón, 2014). And the study further revealed a homogeneous light distribution was found if increasing the distance. As evidenced by our

18 recent data, relatively small fluctuation was found for X-1100 IPL system, the range of IPL intensity was from 0.707 to 0.953 J/ cm2/pulse as length was increased from 0- 76 cm. The data of energy distribution plays a critical role in guiding IPL development: i.e. the width of vibratory feeder can be reduced to decrease unnecessary energy consumption; the IPL lamp with homogenous light distribution can be employed to increase the IPL efficiency, as well as to make the environmental conditions (humidity and temperature) in chamber easier to control.

(a) (b)

Figure 2.3. Energy distribution and fluence received during the original IPL illumination.

*(a) Width vs intensity; (b) Length vs intensity.

2.3.3. Study of IPL efficiency as a function of IPL spectrum range

The disinfection of IPL was due to its broad-spectrum, UV part of spectrum mainly took photochemical effect, and infrared regions mainly took photothermal effect (N

Elmnasser, Guillou, Leroi, Orange, Bakhrouf, & Federighi, 2007). Therefore, the effect of segmental IPL spectrum range on C. sakazakii in NFDM was then evaluated. The effect of these three ranges of IPL spectra on C. sakazakii in NFDM yielded varied results. The IPL

19 sterilization caused by different lamps was in the order of IPL lamp A< lamp B< lamp C after 28 s. Initial water activity level of the sample was set at 0.25; the treatment temperature was ~57 ºC (Table 2.1). The lamp A only containing visible and infrared red spectrum shows limited extent of C. sakazakii inactivation. Although few studies investigated C. sakazakii inactivation in the range of spectrum, a study reported 5 log10 reduction of S. aureus was achieved at the wavelengths over 400 nm using 630 J/cm2 in 30 min (Papageorgiou, Katsambas, & Chu, 2000). Cell death was resulted from the generation of reactive species and predominantly singlet delta through stimulation of intracellular porphyrins. The lamp B contains UV-A other than the spectrum existing in lamp A. The result showed the lamp B had significantly higher C. sakazakii inactivation than lamp A.

UV-A was used for destroying the membrane function during microorganism inactivation

(Bosshard, Bucheli, Meur, & Egli, 2010). The Lamp C including UV-C part (200-280 nm) caused the highest log10 reduction of microbe among them. The results are consistent with many other studies (Slieman & Nicholson, 2000; T. Wang, MacGregor, Anderson, &

Woolsey, 2005), which indicated that UV-C part plays the most important role in microbial inactivation. The sterilization effect of UV-C light on bacteria is mainly due to the formation of thymine dimers, which inhibits the process of cell replication for the new

DNA chain (Giese & Darby, 2000). Therefore, in comparison among different range of

IPL spectrum, the broad spectrum in the range of 190-1100 nm has the highest C. sakazakii reduction in NFDM.

Table 2.1. C. sakazakii inactivation as a function of different spectrum range

Lamp type Log reduction of C. sakazakii (log CFU/g) ± SD

Lamp A 0.61± 0.08A

Lamp B 1.41± 0.07B

Lamp C 2.96± 0.09C

*The residence time is 28 s at the distance of 8 cm for all lamps. The spectrum range of the lamp A is 380-1100 nm, the spectrum range of the lamp B is 280-1100 nm, and the spectrum range of the lamp C is 190-1100 nm. Means with the same uppercase letter within a column are not significantly different (p >0.05). All experiments were performed in triplicate.

2.3.4. Study of IPL efficiency as a function of sample thickness and lamp height.

The distance between the IPL lamp quartz window and top surface of the vibratory feeder is related to light intensity and coverage by the lamp. And layer thickness of the sample is another important paramount parameter. The surface response model was developed to predict the log10 reduction of C. sakazakii in NFDM with IPL. Variables including distance between samples and IPL lamp, layer thickness of NFDM, and log10 reduction of microbe were used to describe the model. Similar surface response models on alfalfa seed have been investigated (Sharma & Demirci, 2003) to estimate the regression coefficients. The general equation for the model was shown as equation (4):

2 2 y = 푎0 + 푎1푥1 + 푎2푥2 + 푎3푥1 + 푎4푥2 + 푎5푥1푥2 (4)

Where y is the log10 reduction of C. sakazakii in NFDM under IPL, ao is the constant term, a1 to a5 are the coefficients, x1 and x2 is the distance and layer thickness of the powder particle, respectively. Therefore, the surface response model (Figure 2.4) developed to predict the log10 reduction is shown as equation (5):

2 2 y = −16.9 + 5.325푥1 − 0.9666푥2 − 0.3319푥1 + 0.4475푥2 − 0.1178푥1푥2 (5) 21

2 With an R of 0.9718, which indicates that the model fits data with 90% of sample variation.

Based on the data of C. sakazakii inactivation in NFDM from 6-10 cm. The results showed the C. sakazakii inactivation at a distance of 10 cm is lower than that of 8 cm. This might be due to the increased energy dissipation as the IPL pulses at 10 cm travel a longer path from source to the powder than those at 8 cm. A research reported a reduction of 4.93 log10 CFU/g after 100 s pulsed light treatment at the distance of 3 cm, while only reaching

2.95 log10 CFU/g reduction at the distance of 13 cm using the same amount of treatment time (Jun, Irudayaraj, Demirci, & Geiser, 2003). On the other hand, our results showed that the microbial inactivation at a distance of 8 cm was higher than that of 6 cm; because the

IPL effective coverage area at 6 cm is smaller than the coverage of powdered sample layer, resulted in only partial decontamination on the samples. Subjecting samples to shorter distance was shown to cause undesirable agglomeration due to excessive heating and hence was not investigated. Regarding the thickness of the powder layer, our result revealed that the inactivation decreased with increasing average thickness of sample from 1.2 to 2.0 mm.

The result was in agreement with some previous studies, which determine the E. coli O157:

H7 inactivation as a function of distance in alfafa seeds, the result indicated inactivation decreased with increasing thickness of the seed layer (Sharma & Demirci, 2003). A reduction was achieved of 4.8 log10 CFU/g for the thickness of 1.02 mm. The log10 reduction decreased to 0.53 log10 CFU/g at the thickness of 6.25 mm. It is well known that the pulsed light is limited by its relatively low degree of penetration of solid foods (N

Elmnasser, Guillou, Leroi, Orange, Bakhrouf, & Federighi, 2007). Therefore, the feeding rate was set as 4200 g/h to maintain the particle layer thickness at ~1.2 mm, and the distance

22 between the IPL lamp quartz window and top surface of the vibratory feeder at 8 cm for all treatments.

Figure 2.4. Response surface diagram of IPL on log10 reduction of C. sakazakii

2.3.5. Study of IPL efficiency as a function of different water activity levels and sample temperature

The synergic effect between treatment temperature and UV was verified in a previous study (Gayan, Serrano, Raso, Alvarez, & Condon, 2012). However, few studies determined whether the combination of IPL and temperature were effective for bacteria inactivation in milk powder. On the other hand, a study reported that there was an decontamination effect as a function of the water activity level on C. sakazakii in PIF. C. sakazakii in PIF was more resistant at a water activity level of 0.20 than in PIF at water activity levels from 0.25 to 0.30 at both 21 and 31 °C (Beuchat, Kim, Gurtler, Lin, Ryu, &

Richards, 2009). Therefore, it is necessary to evaluate the effect of IPL on NFDM under different variables. In the current study, water activity levels from 0.20 to 0.35; treatment

23 time from 18 to 28 s; initial temperature from 25-35 ºC (final temperature is ~57 ºC) to assess the bactericidal effect on C. sakazakii in NFDM (Figure 2.5). For the residence time at different water activity level ranging from 0.20 to 0.35, there was a significant time- dependent pattern for C. sakazakii inactivation in NFDM from 18 to 28 s (p < 0.05 using two-way ANOVA), which caused a 0.27–3.18 log10 CFU/g reduction of C. sakazakii in

NFDM. Higher IPL dose of pulsed light or long treatment time was required to achieve higher C. sakazakii inactivation at each water activity level. Comparing between water activity levels at 0.20 and 0.25, it was observed that microbial inactivation at the water activity level of 0.25 was significantly higher than the water activity level of 0.20 (p < 0.05 using two-way ANOVA). This result indicated that C. sakazakii exhibited less resistance in relatively high moisture conditions than in a dry environment. Similar results were found in other studies, microbial inactivation decreased with increasing the water activity levels of NFDM from 0.25 to 0.35 (p< 0.05 using two-way ANOVA). Undesirable agglomeration was observed at high water activity level significantly limited the C. sakazakii inactivation using IPL (Figures 2.5 & 2.6). This agglomeration might provide a shield for the inner bacteria against IPL treatment. Due to the limitation of IPL penetration depth, the IPL fluence decreased as thickness of the particles increased. A research clarified that the penetration of pulsed light through sausage slices was a function of thickness and the IPL fluence at a thickness 0 mm was 1.10 J/cm2, while the fluence at 2.5 mm was 0.10 J/cm2

(Uesugi & Moraru, 2009). The result indicated that IPL treatment is more effective for bacteria inactivation on particle exterior surfaces than bacteria hid inside particles.

Overall, for the operation of IPL in the processing of NFDM, a balance among a treatment is required with sufficient residence time/fluence, appropriate water activity, and

24 temperature to eliminate C. sakazakii in NFDM. Nevertheless, the process needs to be mild enough to ensure that adverse quality effects of NFDM are not encountered. In addition, it also needs to be economically and commercially viable for the processor (i.e. minimal residence times/energy consumption). Therefore, the research was designed to acquire IPL inactivation data over a range of process variables that are most applicable to the related powdered food industry at a water activity level of 0.25, an initial temperature of 25 °C, and a residence time of 28 s for the current original IPL system (Chen, et al., 2018).

4 T=18 s, Tem= 35 ºC d cd T=25 s, Tem=30 ºC 3 abc b b T=28 s, Tem= 25 ºC a

2 f f e i

1 h Log reduction/(CFU/g) Log g

0 0.20 0.25 0.30 0.35 Water activity

Figure 2.5. Log reduction (CFU/g) of NFDM treated with IPL at different water activity levels

110 e Control 105 e e 100 T=18 s, Tem= 35 ºC

m m 95 μ T=25 s, Tem= 30 ºC 90 T=28 s, Tem= 25 ºC 85 d c 80 c c 75

NFDM diameter/ diameter/ NFDM ab ab b 70 ab a ab b b ab 65 60 0.20 0.25 0.30 0.35 Water activity level Figure 2.6. The NFDM mean particle size as a function of water activity,

*Initial temperature (Tem), and treatment time (T).

2.3.6. Analysis of surface appearance and amino acid composition

The surface appearance of NFDM particles was observed by SEM. In addition to a few craters, no obvious changes in spherical form and surface wrinkles were found in

NFDM particles after the IPL treatments (figure not shown). The possible reasons were 1) nonfat dry milk contains no more than 0.6% of milk fat. No or limited lipid globule, thus, can be formed; 2) the final temperature of samples was controlled under 57 °C, the temperature is below the glass temperature of NFDM, no obvious agglomeration can be generated at the temperature. In the current study, initial temperature of NFDM was controlled at 25 °C; initial aw was maintained at ~0.25, the IPL had no significant effects on amino acid composition of NFDM when the doses below a certain level (Table 2.2).

The results were consistent with a previous study, which reported no significant difference in amino acid composition of protein and lipid oxidation occurred after IPL treatment

(Noura Elmnasser, et al., 2008). The author further investigated condition of protein structure, the data of SDS-PAGE showed the formation of dimers after treatment of β- lactoglobulin beyond 5 pulses. The phenomenon was mainly due to the changes in the polarity of the tryptophanyl residue microenvironment of β-lactoglobulin solutions or variation in the tryptophan indole structure and some protein aggregation. Consequently, although there are no changes occurred in amino acid composition after IPL treatment, the protein structures (secondary, tertiary, and quaternary structures) were likely to be affected by IPL.

Table 2.2. Composition of major amino acids (% w/w) in control and IPL-treated

NFDM.

Amino acid types Control 28s-IPL treatment

Alanine 2.88±0.22 2.77±0.15

Arginine 0.83±0.10 0.93±0.04

Aspartic Acid 2.65±0.29 2.32±0.11

Glutamic acid 31.36±2.48 30.08±1.39

Glycine 1.04±0.15 0.97±0.09

Histidine 0.67±0.27 0.70±0.28

Isoleucine+Leucine 9.01±0.63 9.33±0.35

Lysine 25.87±4.32 24.92±5.03

Phenylalanine 2.12±0.14 2.05±0.22

Proline 13.17±1.81 15.15±4.87

Serine 2.16±0.26 2.31±0.20

Threonine 1.78±0.25 1.77±0.16

Tyrosine 1.23±0.16 1.41±0.50

Valine 5.23±0.17 5.29+0.05

*Values were expressed as the mean ± standard deviation of measurements made in triplicate.

2.3.7. Evaluation of IPL effect on different powdered foods after multiple passes

To achieve the enhanced microorganism inactivation (>4 log10 CFU/g reduction), the effects of IPL on microorganism inactivation in NFDM, wheat flour, and egg white

27 powder with multiple IPL treatments under the conditions of water activity level of 0.25, an initial temperature of 25 °C, and a residence time of 28 s for each IPL pass. The sample conditions (water activity level and temperature) were adjusted to the initial condition after each pass. The results showed that C. sakazakii on NFDM, wheat flour, and egg white powder were significantly inactivated by 5.27, 4.92, and 5.30 log10 CFU/g, respectively, after 3 or 4 passes of IPL treatments. In contrast, for decontamination of E. faecium, 3 or 4 passes of IPL treatments reduced the E. faecium on NFDM, wheat flour, and egg white by

3.67, 2.79, 2.74 log10 CFU/g, respectively (Figure 2.7). It generally agreed that higher resistance of E. faecium than C. sakazakii was found in all three of powdered samples.

Because E. faecium as a gram- positive bacteria possess a thicker (20-80 nm) cell wall and increased amounts of peptidoglycan than gram-negative bacteria (1.5-10 nm), as well as the gram-negative bacteria contain an outer membrane. These features of gram-positive bacteria impede the UV efficiency on microbial inactivation (Williams, Eichstadt, Kokjohn,

& Martin, 2007). Comparing among these three products, microorganism inactivation in white egg is higher than those in wheat flour and NFDM. Protein and lipid in powdered foods are known to absorb UV wavelength at 190 and 280 nm, some fatty acids especially saturated fatty acids also absorb visible and UV light (Noura Elmnasser, et al., 2008). Egg white typical is consist of lower protein (10%) and fat (0%) than the other two samples.

Therefore, the microbes can be rapidly inactivated in egg white.

Overall, the log-reduction curves exhibited significant time-dependent patterns for both types of bacteria in these three different powdered foods (p< 0.05 using one-way

ANOVA). All processing and environmental conditions were strictly controlled to reach a high IPL disinfection. 1) The water activity levels of the samples were recovered to the

28 initial level after each pass (Table 2.3). Microbes were proved to be inactivated more easily at relatively high-water activity level than low water activity level (Beuchat, Kim, Gurtler,

Lin, Ryu, & Richards, 2009). However, the data in section 2.3.5. indicated undesirable agglomeration of NFDM might be formed when water activity level excesses 0.3. 2) After each pass, temperature of the samples was cooled down to ~25 ºC, the approach not only can prevent samples from overheating (>58 °C), but also meet the requirement of nonthermal claim. Thus, significantly less nutrient loss was brought (Mancebo‐Campos,

Fregapane, & Desamparados Salvador, 2008). On the other hand, vibratory feeder employed help entire surface of samples expose to IPL, it contributes to higher microbial inactivation, in the meantime, IPL fluence was distributed to samples uniformly. Treatment time was extended without proper homogenous methods. The population of E. coli in liquid egg white was only reduced by 4.3 log10 CFU/g after 160 s- UV exposure (Geveke, 2008).

As a result, Table 2.4 shows no obvious agglomeration was observed after multiple IPL treatments in these three types of powdered foods under the appropriate environmental conditions.

NFDM 6.0 Wheat flour d 6.0 d 5.0 c 5.0 b C 4.0 a c 4.0 b 3.0 B B 3.0 B B a 2.0 A 2.0 A

Log Log reduction (CFU/g) A C. sakazakii 1.0 Log reduction (CFU/g) E. faecium 1.0 C. sakazakii 0.0 E. faecium 1 pass 2 passes 3 passes 4 passes 0.0 1 pass 2 passes 3 passes 4 passes

(a) (b)

Egg white 6.0 c 5.0

4.0 b C 3.0

2.0 a B Log Log reduction (CFU/g) 1.0 A C. sakazakii E. faecium 0.0 1 pass 2 passes 3 passes

(c)

Figure 2.7. Effect of multiple passes of IPL on the inactivation of C. sakazakii and E. faecium.

*Each fluence (energy flux) of pass is 29.36 J/cm2 for 28 s. (a) NFDM, (b) Wheat flour,

(c) Egg white. Data were expressed as the mean ± standard deviation of measurements made in triplicate. Different letters represent statistically significance (p <0.05).

Table 2.3. Processing parameters of each IPL treatment (28s)

Initial temperature± Final temperature± Initial water Final water

SD (ºC) SD (ºC) activity level± SD activity level± SD

NFDM

The 1st pass 24.6±0.6A 57.0±0.8B 0.26±0.01C 0.20±0.01E

The 2nd pass 25.6±0.5A 56.4±0.6B 0.26±0.02C 0.21±0.01E

The 3rd pass 25.2±0.3A 56.5±0.9B 0.25±0.01C 0.20±0.01E

The 4th pass 25.9±0.6A 56.2±0.6B 0.26±0.01C 0.20±0.01E

Wheat flour

The 1st pass 25.1±0.6A 56.9±0.6B 0.40±0.02D 0.31±0.01F

The 2nd pass 25.2±0.9A 56.4±0.8B 0.39±0.01D 0.32±0.01F

The 3rd pass 25.3±0.3A 56.7±0.9B 0.40±0.02D 0.32±0.01F

The 4th pass 25.9±0.8A 56.3±0.5B 0.39±0.02D 0.31±0.01F

Egg white powder

The 1st pass 24.6±0.4A 56.7±0.7B 0.25±0.01C 0.19±0.02E

The 2nd pass 25.7±0.6A 56.4±0.8B 0.25±0.02C 0.19±0.02E

The 3rd pass 25.5±1.0A 56.1±0.8B 0.24±0.02C 0.19±0.02E

*Means with the same uppercase letter within a column are not significantly different

(p >0.05)

Table 2.4. The product mean particle size as a function of different number of passes.

Samples type IPL treatment conditions Particle diameter (µm)

NFDM Control 31.67±1.04A

1 pass 31.88±1.07A

2 passes 31.95±0.87A

3 passes 32.24±1.60A

4 passes 33.57±1.52A

Wheat flour Control 44.71±0.74B

1 pass 44.74±0.56B

2 passes 44.95±1.01B

3 passes 45.01±0.74B

4 passes 44.81±0.98B

Egg white Control 55.68±0.48C

1 pass 55.66±0.28C

2 passes 55.65±0.68C

3 passes 55.69±0.43C

* Each fluence (energy flux) of pass is 29.36 J/cm2 for 28 s. Control is the sample without

IPL treatment. Data are represented as means ± standard deviations (n = 3) (p <0.05).

Means with the same uppercase letter within a column are not significantly different

(p >0.05)

Chapter 3. Develop and evaluate the prototype intense pulsed light on particulate foods

3.1. Introduction

Chapter 2 indicated high microbial inactivation (>5 log10 CFU/g) could be achieved after 3-4 pass-IPL treatment (Chen, Peng, et al., 2019). Our original IPL system was proved as an effective disinfection on C. sakazakii, E. faecium in egg white powder, wheat flour, and NFDM. However, operators need to adjust sample conditions after each pass, the overall process to achieve sufficient disinfection was an intermittent treatment. On the other hand, although a variety of environmental and sample conditions such as humidity, water activity level, residence time, and temperature had been evaluated above (Chen, et al., 2018), the impacts of other attributes including pulsed duration, intensity, and frequency on microbes need further investigation.

The disinfection capacity of UV combined with titanium dioxide (TiO2) had been investigated extensively. However, no study reported catalytic intense pulse light (CIPL) on powdered foods. The synergistic effects of TiO2 and UV is significantly higher than individual UV (Tsuang, Sun, Huang, Lu, Chang, & Wang, 2008). Antimicrobial behavior was enhanced under the condition of UV emission was due to the large band gap (3.2e V) of TiO2, and thus TiO2 could only be activated under UV emission (wavelength <390 nm).

The charge carriers on the surface of TiO2 can react with microorganisms (Figure 3.1). The positive holes (h+) in the valance band are highly oxidative, which can directly react with organic molecules that are adhesive to the TiO2 but also oxidize the pathogens or water indirectly through the production of •OH radicals. The chemical compounds •OH radicals are highly reactive and thereby responsible for redox chemistry reactions of the oxidation

33 process of pathogens during photocatalysis (Fujishima, Zhang, & Tryk, 2008). In the economic and food safety aspects, the TiO2 powder has been approved by the American

Food and Drug Administration (FDA, 2002). On the other hand, TiO2 powder is also considered as a cheap semiconducting material and widely used in many commercial applications (Ahmed et al., 2010).

Figure 3.1. Schematic diagram illustrating the principle of TiO2 photocatalysis

The objectives of the chapter 3 are to (1) fabricate and assemble a continuous IPL system incorporating TiO2 photocatalysis for powdered food pasteurization; (2) to explore

IPL disinfection on Cronobacter sakazakii, Enterococcus faecium (NRRL B-2354), and

B.cereus spore inactivation in NFDM and wheat flour as a function of voltage, frequency, pulsed duration, and feed rate; (3) to evaluate and compare the synergistic effects of CIPL on these microorganisms.

3.2. Materials and methods

3.2.1. The intense pulsed light system design and development

During the past four years, we have developed three generations of the IPL system.

The first-generation IPL system had no capacity to maintain a stable sample temperature and water activity during the process. Samples were exposed to atmosphere during IPL without environmental controllers. Therefore, temperature or moisture loss/gain during

IPL treatment (Figure 3.2(a)). To enhance IPL decontamination levels (4-5 log10CFU/g reduction of microbes), researchers need to pause treatment when the sample temperature reaches 60 °C and equilibrate the sample to room temperature and desirable water activity level before next pass of IPL treatment to avoid powder agglomeration, water activity loss, and maintain a “non-thermal” pasteurization claim to reduce unnecessary nutrient or quality degradation. Namely, in order to achieve 4-5 log10CFU/g reduction, researchers need to carry out 3-4 passes of 28-s IPL treatments (Chen, Cheng, Peng, Liu, Wang, Ma, et al., 2019). Although each pass could be considered as a continuous process, the overall treatment to reach 5 log10CFU/g reduction then became an intermittent process. In addition, the pulse frequency and intensity of pulsed light might also be too low to deliver the sufficient energy required for a higher inactivation and throughput.

In the next phase, adjustable climate controllers (temperature or humidity) were mounted and sufficient to maintain sample temperature and water activity levels at desirable levels during the IPL treatment (Figure 3.2(b)). However, the width of vibratory feeder is significantly wider than the IPL coverage causing ununiform treatment; the peak intensity, pulsed duration, and voltage are still in need of adjustment. Therefore, the energy consumption is relatively high. These findings and experience mentioned above have been

35 used to successfully develop a new pilot scale demonstration system that have following features: pre-condition of food products, exact control of temperature, relative humidity, continuous process, potential higher pulse frequency and intensity IPL source, higher throughput, and enhanced nutrition and quality preservation. Therefore, the system can ensure no significant change in particle diameter for all treated samples since the temperature and water activity levels were maintained during the entire IPL treatment.

Extended length of IPL lamp with higher pulse intensity and lower pulse duration was also used, and thereby, much higher bactericidal effects have been achieved in one pass (Figure

3.2 (c)). All of these characters contributed to a potential low-cost prototype IPL system.

Taking the C. sakazakii inactivation in NFDM as an example, it consumed 101.9 kJ/kg to achieve 1 log10 CFU/g reduction using the original IPL system, while it consumed only

41.9 kJ/kg to reach 1 log10 CFU/g reduction with the prototype IPL system. This prototype system has proven effective in inactivating pathogens in a variety of powdered food samples including non-fat dry milk, wheat flour, white egg powder, mesquite flour, barley seeds, wheat kernel, sunflower seeds, etc. The proposed technology could potentially be fitted into various current food processing plants such as milling and seed granulation.

(a) (b)

(c)

(d)

Figure 3.2. (a) Schematic diagram of original IPL system; (b) the modified IPL system;

3.2.2. Bacteria inoculum preparation, water activity adjustment, and enumeration

The inoculation method was followed by procedures of a previous study with minor modifications (Wiertzema, Borchardt, Beckstrom, Dev, Chen, Chen, et al., 2019). NFDM and wheat flour were homogenized with a stainless-steel whisk to enable an even distribution of inoculum on samples. Inoculated powders were then transferred to desiccators (Thermo Fisher Scientific, Waltham, MA). The inoculated NFDM powder samples were subsequently adjusted to a water activity (aw) level of 0.25 ± 0.2 by equilibrating in 25 °C. Lithium chloride (Sigma-Aldrich) for 7-10 days before the IPL treatment. For the water activity adjustment, the inoculated wheat flour samples were adjusted to an aw of 0.43 ± 0.2 by equilibrating in 25 °C potassium carbonate for 7-10 days

(Greenspan, 1977). A PawKit (Decagon Devices, Inc., Pullman, WA) was used to ensure the aw of each powder had equilibrated prior to testing. The obtained Cronobacter sakazakii

(ATCC 29544), Enterococcus faecium (NRRL B-2354), and Bacillus cereus (ATCC 14579) on these samples were around 7.8 log10 CFU/g, 7.5 log10 CFU/g, and 7.4 log10 CFU/g, respectively.

One gram of samples was serially diluted in 9 ml of 0.1% peptone and plated in duplicate onto Trypticase Soy Agar with 0.6% Yeast Extract (TSAYE) for C. sakazakii and E. faecium. TSAYE plates were incubated at 37°C for 48-72 hours before enumeration.

B. cereus samples were through heat shocked (80°C for 12 min) prior to serially diluting and plating onto Standard Methods Agar (SMA). SMA plates were incubated at 30°C for

24-48 hours. Each plate was counted manually, and the number of colonies were then expressed as colony-forming units per gram (CFU/g).

3.2.3. Coating TiO2 on substrates

To enable sufficient contacting surfaces and material recycle, coating substrates such as glass beads were used for coating TiO2, the coating procedure was stated as follows.

2 mm diameter-glass beads (Sigma‐Aldrich, St. Louis, MO) were etched with hydrofluoric acid (5%) for 24 h to create the porous surface (Daneshvar, Salari, Niaei, Rasoulifard, &

Khataee, 2005). 200 mL of TiO2 slurry was made from 1.5 g TiO2 powder (99.5% Sigma‐

Aldrich, St. Louis, MO). The etched glass beads were subsequently soaked in TiO2 slurry for 20 min. A magnetic flea was used to stir TiO2 slurry with etched glass beads. Then the coated glass beads were transferred to a porcelain crucible and placed in an oven for 1 h at

150 ºC. Coated beads were subsequently transported to a 500 ºC furnace for 2 h. Distilled water was then used to remove the free TiO2 particles of the surface of coated TiO2 beads.

3.2.4. Measurements of the IPL fluence, temperature, and flow rate

Fluence received from IPL was measured by a Vega laser power meter (Ophir

Optronics Inc., Wilmington, MA) equipped with a PE-50C pyroelectric energy sensor

(Ophir Optronics Inc., Wilmington, MA). The detailed procedures were described as followed, the pyroelectric sensor was placed in the central point of the vibratory feeder.

Noteworthy, the height of the energy sensor is 2 cm and to account for this, the IPL lamp was raised from 8 to 10 cm. The interval time is 30 s among each treatment. The temperature curves of the powdered samples were monitored a non-contact infrared thermometer with laser targeting (Cen-Tech, Montessori, NV). Air flow rates in the chamber and subsurface were measured using a traceable hot wire anemometer (Control company, Friendswood, TX). All measurements were carried out in triplicate.

3.2.5. Particle size measurements

Particle size of samples before and after IPL treatment were determined by a LS 13

320 laser diffraction particle size analyzer (Beckman Coulter, Inc, Brea, CA). Five grams of samples were randomly collected from each treatment. All experiments were conducted in triplicate. The particle size of powdered samples was recorded with the unit of diameter

(μm).

3.2.6. Gamma radiation and IPL treatment of mesquite flour

The mesquite flour was induced from Casa de Mesquite LLC and stored at ambient condition until use. ~5 logs of Bacillus cereus spores were naturally present in mesquite floor. The initial water activity level of mesquite flour sample was 0.42.

Gamma radiation. The treatment was conducted by researchers in U.S.

Department of Agriculture (Eastern Regional Research Center, Wyndmoor, PA), and based on a previous study (Fan, Felker, & Sokorai, 2015).

IPL treatment. Inoculated powdered foods such as NFDM and wheat flour were conveyed through the vibratory feeder under the different parameters of IPL with a treatment time up to 28 s. The 2 mm photo-catalyst TiO2 (Anatase) were employed to take synergistic decontamination effect with IPL. For the IPL+TiO2 mixture, the weight ratio of powdered sample and TiO2 coated glass bead is around 1:1 in the current study.

Vibratory feeder was adjusted to enable samples to pass the IPL for 28 s. Environmental humidity was set at 35-40 %. The processing temperatures of mesquite flour were maintained at 56±1°C during IPL treatment by adjusting the temperature of thermostatic circulating water bath incorporated with fin-fan coolers (LabX, Midland, ON, Canada).

3.2.7. Physicochemical property analysis of treated mesquite flour

The control and treated mesquite flour powder were extracted by methanol at a ratio of 1:10 (w:v). After vortexing and 5-min ultrasonic mixing, the samples were centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new 1.5 mL Eppendorf tube and stored at -20 °C. Triphenylphosphine (TPP), and 2-hydrazinoquinoline (HQ) were obtained from Alfa Aesar (Ward Hill, MA); 2,2’-dipyridyl disulfide (DPDS) from MP

Biomedicals (Santa Ana, CA); LC-MS-grade water and acetonitrile from Fisher Scientific

(Houston, TX).

Chemical derivatization. Prior to the liquid chromatography-mass spectrometry

(LC-MS) analysis, the methanol extract was first derivatized by HQ using a method (Lu,

Yao, & Chen, 2013).

3.2.8. Cytotoxicity analysis

Cytotoxicity analysis was conducted by determining the viability of human colorectal adenocarcinoma cell line (Caco-2) in contact with solutions containing untreated and treated samples. Caco-2 cell line is widely used for in vitro prediction of human intestinal absorption. A total concentration of 1.0 × 105 Caco-2 was purchased from ATCC,

USA, were uniformly distributed in a 96-well flat-bottom plate. After 37 °C for 24 h of cell growth, the supernatant medium of each well was discarded and a fetal bovine serum

(FBS)-free MEM solution containing a series of concentrations (20, 40,100 mg/mL in 0.1%

DMSO) of mesquite flour under different treatments. FBS-free MEM with 0.1% DMSO was added to cells as the vehicle control (VC) wells. FBS-free MEM solution containing different concentration of untreated mesquite flour was used as positive control (PC) wells.

After incubation for 24h, cell viability was evaluated using 3-(4.5-Dimethylthiazol-2-yl)-

2.5-diphenyltetrazolium bromide (MTT) assay as previously described (Bu, Narayanan,

Dalrymple, Cheng, & Serajuddin, 2016). The absorbance value was measured at 570 nm using a microplate reader and the cell viability (%) was calculated as a percentage of VC using GraphPad Prism 6.0.

3.3. Results and discussion

3.3.1. Optimizing processing parameters for pathogen disinfection

To determine capacity of the prototype IPL system on powdered foods, a variety of

IPL parameters and environmental factors such as treatment temperature, humidity, sample temperature, residence time, lamp height, layer thickness were already evaluated. The optimizing conditions to treat particulate foods were obtained. More critical parameters including voltage, frequency, intensity, feed rate would be discussed in this segment.

Powder foods such as NFDM and wheat flour inoculated with different microbes were loaded in the volumetric feeder. Samples were subsequently transported through a vibratory feeder under the IPL lamp. Prototype IPL disinfection effects were shown in

Figure 3.3 (a-d). Frequency from 1-14 Hz was investigated first. At the same volt and feed rate levels, the results show that lower IPL frequency causes significantly higher disinfection (i.e., inactivation at the condition of 1Hz, 3000 V, and 4200 g/h was significantly higher than that of 3Hz, 3000V, and 4200 g/h) because lower frequency resulted in higher energy density. For example, the energy density was ~0.291 J/cm2/pulse at 1 Hz, which is higher than the energy density at 14 Hz (0.021 J/cm2/pulse). Therefore, microbe inactivation can be significantly increased by reducing the frequency. Based on

42 the law of Lambert-Beer, the IPL disinfection increases with increasing of the IPL density or intensity on foods is due to the penetration enhancement of IPL. The equation to determine the total energy received by samples as the equation (6) (Luksiene, Gudelis,

Buchovec, & Raudeliuniene, 2007):

퐷 = 퐸푝 ∗ 푡 ∗ 푓 (6)

2 2 Where D, Ep, t, and f stand for the total energy (J/cm ), pulsed density (J/cm /pulse), treatment time (s), and frequency (Hz), respectively. According to the equation, under the conditions of certain treatment time and total energy consumption (J/cm2), higher energy density could be facilitated by decreasing frequency. It revealed 1 Hz-IPL contributed to the highest microorganism inactivation in the current study. The results are in agreement with a previous study, which reported that the pulsed light disinfection on virus inactivation increased with increasing energy density from 0.25-2.0 J/cm2/pulse (Roberts & Hope,

2003).

On the other hand, high voltage induced higher inactivation than that of low voltage in most of cases. It is well known that higher voltage can contribute to higher peak intensity, which led to the fact that the same amount of fluence was produced in a shorter pulse duration (µs). The pulsed duration was 342 µs at 3000 V and frequency of 1 Hz, while the pulsed duration was 773 µs at 2200 V and frequency of 1 Hz. Thus, the IPL sterilization is attributed to the pulse duration. Furthermore, high inactivation effect of IPL treatment might be due to the rich UV component (Oms-Oliu, Martin-Belloso, & Soliva-Fortuny,

2010). The model X-1100 has substantially richer UV content than model Z-1000 as shown in their lamp spectrum. A high UV component has been found more effective for eliminating bacteria. A study was carried out to investigate the high-intensity pulsed light

43 emission of high at low UV composition on E. coli inactivation (Rowan, MacGregor,

Anderson, Fouracre, McIlvaney, & Farish, 1999). The data revealed that low-UV PL resulted in a very poor inactivation effect (1 to 2 log10 CFU/g), while microorganisms were reduced by 5 to 6 log10CFU/g using high-UV PL. Similarly, our previous study also indicated UV-C wavelength in IPL played a most important role in microbicidal effects

(Chen, Cheng, et al., 2019). Therefore, high UV portion as a result of high voltage may be another factor causes higher microbial inactivation.

Moreover, IPL disinfection can be affected by feed rate, the microbial inactivation decreased with increasing the feed rate from 4200 to 8100 g/h and 3600 to 7200 g/h for

NFDM and wheat flour, respectively. The inactivation difference between these feed rates might be due the limited depth of the IPL penetration. A research investigated the E. coli

O157: H7 inactivation as a function of thickness of alfafa seeds, the study uncovered that microbial inactivation decreased with increasing thickness of the seed layer. A reduction of 4.8 log10 CFU/g was reached at 1.02 mm, the reduction was only 0.53 log10 CFU/g at

6.25 mm (Sharma & Demirci, 2003).

IPL treatment on C.sakazakii in NFDM (28 s) IPL treatment on C.sakazakii in Wheat flour (28 s) 4.0 3.0 Volt=3000 V, feed h Volt=3000 V, feed 3.5 i rate=4200 g/h rate=3600 g/h 2.5 Volt=3000 V, feed h Volt=3000 V, feed rate=7200 g/h 3.0 rate=8100 g/h g efg f 2.0 Volt=2200 V, feed Volt=2200 V, feed e e 2.5 g d d rate=3600 g/h rate=4200 g/h Volt=2200 V, feed 2.0 Volt=2200 V, feed 1.5 rate=7200 g/h f rate=8100 g/h c 1.5 f e de 1.0 b

d a a Log reduction (CFU/g) reduction Log Log reduction (CFU/g) reduction Log 1.0 ab a c bc 0.5 0.5

0.0 0.0 1 Hz 3 Hz 14 Hz 1 Hz 3 Hz 14 Hz Frequency of IPL Frequency of IPL

(a) (b)

IPL treatment on E.faecium in NFDM (28 s) IPL treatment on E.faecium in Wheat flour (28 s) 1.6 2.5 e Volt=3000 V, feed f Volt=3000 V, feed cde e 1.4 rate=4200 g/h rate=3600 g/h d cd Volt=3000 V, feed ef Volt=3000 V, feed ef 2.0 cd rate=7200 g/h 1.2 rate=8100 g/h c c Volt=2200 V, feed e Volt=2200 V, feed rate=3600 g/h 1.0 rate=4200 g/h Volt=2200 V, feed e 1.5 rate=7200 g/h d Volt=2200 V, feed 0.8 b b rate=8100 g/h ab c 1.0 a 0.6 c bc

ab Log reduction (CFU/g) reduction Log Log reduction (CFU/g) reduction Log 0.4 a a 0.5 0.2

0.0 0.0 1 Hz 3 Hz 14 Hz 1 Hz 3 Hz 14 Hz Frequency of IPL Frequency of IPL

Figure 3.3. Inactivation of C. sakazakii and E. faecium in wheat flour and NFDM at different peak voltage and feed rate

*The total fluence (energy flux) of each treatment is 7.13 J/cm2 (28 s). The feed rate of

NFDM at 4200 and 8100 g/h were associated with layer thickness of ~1.2 and 2.0 mm, respectively. The feed rate of wheat flour at 3600 and 7200 g/h were associated with layer thickness of ~1.2 and 2.0 mm, respectively. Different letters represent statistical significance (p <0.05).

3.3.1.1. Temperature profile and particle size

Glass transition temperature (Tg) is defined as the temperature at which the transition between the glassy and rubbery state for amorphous materials (Chuy & Labuza,

1994). Undesirable agglomerated form of amorphous powdered foods can be induced when the sample temperature exceeds Tg (Aguilera, del Valle, & Karel, 1995). Undesirable agglomeration of powdered foods inhibits the micrbial inactivation during the IPL treatments. Glass transition temperature of NFDM is ~ 58 ºC (Chen, Wiertzema, et al.,

2018). Treatment temperature beyond 60 ºC might potentially cause nutrient loss and organoleptic change in food products (Mancebo‐Campos, Fregapane, & Desamparados

Salvador, 2008). Therefore, controlling temperature underneath 58 ºC (Tables 3.1 (a) &

(b)) in the current study. Mean particle size of powdered foods was a direct indicator to determine agglomeration for powdered foods. Particle size of IPL and UVC treated NFDM and wheat flour under various attributes after 28 s was measured. The results indicated no significant change in particle size was detected for all treated samples (Tables 3.2 (a) &

(b)). This might be due to the adequate control of temperature and humidity used in the sealed vibratory chamber. In terms of wheat flour, although wheat flour has high Tg (>100

ºC), the final temperature was controlled under the 58 ºC in order to minimize the physical and chemical changes in wheat flour (G. H. Ryu & Ng, 2001; Thanatuksorn, Kawai,

Kajiwara, & Suzuki, 2009) and maintain the nonthermal claim.

Table 3.1. Temperature and water activity profiles of different treatments.

(a) (b)

Initial Final Initial water Final water Initial Final Initial water Final water

temperature± temperature± activity activity temperature± temperature± activity activity

SD (ºC) SD (ºC) level± SD level± SD SD (ºC) SD (ºC) level± SD level± SD

NFDM Wheat

120s- 55.2±1.0A 56.3±0.8B 0.25±0.01C 0.24±0.01E flour

UVC 120s-UVC 54.2±0.8A 56.9±1.1B 0.40±0.01D 0.35±0.01F

1 55.9±0.6A 56.2±0.6B 0.26±0.01C 0.25±0.01E 13 54.9±0.6A 56.8±0.6B 0.40±0.01D 0.36±0.01F

2 55.2±0.3A 56.7±0.6B 0.25±0.01C 0.25±0.01E 14 54.2±0.6A 56.9±0.9B 0.40±0.01D 0.37±0.01F

3 55.9±0.6A 56.2±0.6B 0.26±0.01C 0.26±0.01E 15 54.9±0.6A 56.2±1.1B 0.39±0.01D 0.35±0.01F

4 56.1±0.6A 56.9±0.6B 0.27±0.02C 0.26±0.01E 16 54.1±0.6A 56.9±0.6B 0.41±0.02D 0.35±0.01F

5 55.2±0.9A 56.4±0.8B 0.24±0.01C 0.24±0.01E 17 54.2±0.9A 56.6±0.8B 0.39±0.01D 0.36±0.01F

6 54.9±0.9A 55.7±1.2B 0.25±0.02C 0.25±0.01E 18 54.4±0.8A 56.7±0.9B 0.40±0.01D 0.36±0.01F

7 55.9±0.8A 56.3±0.5B 0.26±0.02C 0.25±0.01E 19 54.5±0.8A 56.3±0.5B 0.40±0.01D 0.35±0.01F

8 55.2±0.3A 56.5±0.9B 0.24±0.01C 0.24±0.01E 20 54.5±0.3A 56.5±0.8B 0.39±0.01D 0.37±0.01F

9 55.9±0.6A 56.1±0.8B 0.25±0.01C 0.25±0.01E 21 53.9±0.9A 56.5±1.3B 0.41±0.01D 0.35±0.01F

10 55.4±0.3A 56.5±0.5B 0.24±0.02C 0.24±0.01E 22 54.6±0.9A 56.6±0.9B 0.41±0.01D 0.36±0.01F

11 55.5±0.3A 56.5±0.9B 0.24±0.01C 0.24±0.01E 23 54.7±0.6A 56.3±0.8B 0.41±0.01D 0.36±0.01F

12 55.9±0.6A 57.2±0.5B 0.25±0.01C 0.24±0.01E 24 54.7±0.6A 56.1±0.5B 0.41±0.02D 0.35±0.01F

*(a) NFDM, the relative humidity (RH) in IPL chamber was maintained at 25-30% during the IPL treatment, (b) Wheat flour, the RH in IPL chamber was maintained at 35-40% during the IPL treatment.

Table 3.2. Particle diameters of treated and untreated samples.

(a) (b)

NFDM Wheat flour

Particle Particle

Voltag Feed rate Frequen diameter Voltag Feed rate Frequen diameter

e (V) (g/h) cy (Hz) (µm) e (V) (g/h) cy (Hz) (µm)

Control NA NA NA 50.13±1.76a Control NA NA NA 54.19±0.99a

120s- 120s-

UVC NA NA NA 52.41±0.68a UVC NA NA NA 55.34±0.89a

1 3000 4200 1 52.08±0.85a 13 3000 3600 1 54.94±1.32a

2 3000 4200 3 51.41±1.92a 14 3000 3600 3 52.95±0.71a

3 3000 4200 14 49.58±0.67a 15 3000 3600 14 54.70±0.35a

4 3000 8100 1 48.52±2.03a 16 3000 7200 1 53.53±1.51a

5 3000 8100 3 53.38±2.02a 17 3000 7200 3 55.39±1.20a

6 3000 8100 14 51.27±0.98a 18 3000 7200 14 55.46±1.50a

7 2200 4200 1 51.52±1.70a 19 2200 3600 1 51.83±3.20a

8 2200 4200 3 51.79±0.59a 20 2200 3600 3 52.41±1.53a

9 2200 4200 14 51.71±1.32a 21 2200 3600 14 53.18±2.20a

10 2200 8100 1 51.63±0.73a 22 2200 7200 1 55.47±1.21a

11 2200 8100 3 51.15±0.89a 23 2200 7200 3 52.88±1.89a

12 2200 8100 14 50.30±1.75a 24 2200 7200 14 53.62±1.78a

Data in the same column followed by the same uppercase letter Data in the same column followed by the same uppercase letter

are not significantly different (P > 0.05). are not significantly different (P > 0.05).

*(a) The mean particle size of NFDM as a function of variable attributes. (b) The mean particle size of Wheat flour as a function of variable attributes. The untreated powdered samples are taken as control.

3.3.1.2. Color change

Color change of powdered foods is an important quality indicator. Color difference

(∆E) of NFDM and wheat flour after IPL and 120s-UVC treatments were then investigated

(Table 3.3). Compared different IPL treatment conditions, parameters such as the voltage, feed rate, and frequency affected ∆E to some degree. Frequency and feed rate are two major factors that affected the ∆E. In contrast, the voltage had no obvious effect on the ∆E (Three- way ANOVA within-group comparison: feed rate: p= 0.04< 0.05, Frequency: p=0.008<

0.01, Voltage: p=0.102> 0.05). For wheat flour, no significant color difference was observed on wheat flour after IPL treatment (∆E <0.5). While UVC affected color of

NFDM and wheat flour significantly (Table 3.3 & Figure 3.3). 120s-UVC treatments resulted in 2.14±0.12 and 1.94±0.59 log reductions for C. sakazakii and E. faecium in

NFDM, respectively. And 120s-UVC treatments caused 1.79±0.11and 2.94±0.04 log reductions for C. sakazakii and E. faecium in wheat flour, respectively. To reach a similar bactericidal effect, significantly higher ∆E was caused by UVC than IPL (i.e., treatments

NO. 7 & NO. 19). Comparing with our previous system (Z-1000), significantly less ∆E was caused with this prototype IPL system than the original IPL system(Chen, et al., 2018).

Probable reasons might owe to lower IPL fluence, appropriate temperature control, humidity control, and nitrogen gas usage. Gas barrier was provided by nitrogen gas which could prevent samples from contacting with air. Resulting in less photo-oxidation to

NFDM during IPL. Many sensitive food processing has already utilized nitrogen gas as an antioxidation technology (G. H. Ryu & Ng, 2001; Sharma & Demirci, 2003). Moreover, potential economic benefits will be brought in under the use of enclosed gas circulation system.

Table 3.3. Color difference (ΔE) of IPL treated samples

(a) (b)

NFDM Wheat flour

Voltag Feed rate Frequenc Feed rate Frequency

e (V) (g/h) y (Hz) ∆E Voltage (V) (g/h) (Hz) ∆E

120s- 120s-

UVC NA NA NA 3.36±0.28a UVC NA NA NA 0.66±0.22a

1 3000 4200 1 2.49±0.12b 13 3000 3600 1 0.23±0.15b

2 3000 4200 3 2.17±0.16c 14 3000 3600 3 0.20±0.11b

3 3000 4200 14 1.94±0.03d 15 3000 3600 14 0.22±0.14b

4 3000 8100 1 2.12±0.08c 16 3000 7200 1 0.11±0.10b

5 3000 8100 3 1.99±0.22cd 17 3000 7200 3 0.10±0.06b

6 3000 8100 14 1.79±0.29cd 18 3000 7200 14 0.16±0.14b

7 2200 4200 1 2.27±0.23bc 19 2200 3600 1 0.19±0.06b

8 2200 4200 3 2.02±0.29cd 20 2200 3600 3 0.16±0.06b

9 2200 4200 14 1.82±0.09d 21 2200 3600 14 0.10±0.08b

10 2200 8100 1 2.09±0.16cd 22 2200 7200 1 0.21±0.10b

11 2200 8100 3 1.75±0.15e 23 2200 7200 3 0.21±0.05b

12 2200 8100 14 1.58±0.07e 24 2200 7200 14 0.11±0.06b

Data in the same column followed by the same uppercase letter are not Data in the same column followed by the same uppercase letter are not

significantly different (P > 0.05). The data were expressed as the mean significantly different (P > 0.05). The data were expressed as the mean ±

± standard deviation of measurements made in triplicate. standard deviation of measurements made in triplicate.

*(a) NFDM and (b) wheat flour subjected to UVC and IPL treatments at various attributes

3.3.2. Effect of photocatalyst

The optimized parameters of prototype IPL system for pathogen decontamination are the voltage of 3000 V, frequency of 1 Hz. The IPL treatment time was extended to 60 s after securing the optimized treatment conditions. Bringing the total log reduction to

3.97±0.08 (C. sakazakii), 3.00±0.04 (E. faecium), and 1.68±0.38 (B. cereus) in non-fat dry milk, and 4.15±0.07 (C. sakazakii), 3.31±0.06 (E. faecium), 1.53±0.21 (B. cereus) in wheat flour (Table 3.4). Further increasing the IPL treatment time did not result in higher microbial inactivation (data not shown). Then the CIPL was investigated, additional one log10 CFU/g reduction was induced in both powdered foods with TiO2 and IPL than individual IPL for all types of bacteria or spores. However, further extending IPL treatment time did not foster microbial inactivation. The possible reasons may be due to the presence of cracks and pores in all sorts of powder particles. Microbes hidden in open pores are more difficult to be inactivated than those located on the surface of powdered food. Another explanation is related to the shadow effects. The bacteria of the upper layers are inactivated by IPL, cover and prevent the rest of the bacteria from exposing to IPL. Inhibiting the rest of the bacteria exposure to IPL. As evidenced by a study which used scanning electron microscopy to observe the B. subtilis cells on spice after IPL (Nicorescu, Nguyen, Moreau-

Ferret, Agoulon, Chevalier, & Orange, 2013).

Although extensive studies have been reported that enhanced bactericidal effect of

TiO2 photocatalysis was observed under the UV or solar light, no study ever used TiO2 photocatalysis with IPL in powder foods. A previous research investigated Heterosigma akashiwo inactivation in ballast water using CIPL. The study indicated CIPL could bring higher inactivation than UV+TiO2 because IPL had higher intensity and deeper penetration

50 than UV (Feng, Xu, & Liu, 2015). On the other hand, less energy of CIPL was consumed than IPL alone, which was consistent with our current results. Noteworthy, the results showed one more log10 CFU/g reduction was achieved with the use of TiO2 for B. cereus spore. The thickened cell wall of bacterial endospores contributes to a high resistance of spore than the vegetative forms. Reactive species (i.e. hydroxyl radicals and hydrogen peroxide) generated during photocatalysis process are able to disrupt the cell wall and cytoplasmic membrane (Foster, Ditta, Varghese, Steele, & biotechnology, 2011).

Thus, bacterial endospores would be directly inactivated. Furthermore, the study also demonstrated that the disinfection effect can be maximum at the condition of close contact between organisms and TiO2 catalysts. To fulfill the requirement, the tiny TiO2 coated beads were used in our study. IPL might be a more feasible technology to eliminate pathogens in NFDM and wheat flour than other non-thermal technologies in terms of quality maintenance and microbial inactivation for powder foods. The current study indicated that significantly less color change was induced by IPL light than UVC. This can be attributed to short pulse durations (300ns- 1ms) and the half-life of Π-bonds (10-9 to 10-

4 s), which could prevent samples from coupling with dissolved or free oxygen (Fine &

Gervais, 2004). Moreover, IPL induced more serious DNA damage on microorganism cellular structures than UVC light (Cheigh, Park, Chung, Shin, & Park, 2012).

Table 3.4. Disinfections of 60s-IPL and 60s-CIPL

Type of Log reduction of microorganism (log

Sample type microorganism CFU/g)

60s-IPL 60s-CIPL

C. sakazakii 3.97±0.08a 4.71±0.07b

NFDM E. faecium 3.00±0.04a 3.49±0.01b

B. cereus 1.68±0.38a 2.52±0.10b

C. sakazakii 4.15±0.07a 5.42±0.10b

Wheat flour E. faecium 3.31±0.06a 4.95±0.24b

B. cereus spore 1.53±0.21a 2.80±0.23b

*Effects of 60s-IPL and 60s-CIPL on inactivating C. sakazakii, E. faecium, and B. cereus spore inoculated in NFDM and wheat flour. The total fluence (energy flux) of 60s-IPL treatment is 17.46 J/cm2. Data represent mean of at least triplicates ±one standard deviation.

Data in the same row of the same table followed by the same lowercase letter are not significantly different (P > 0.05).

3.3.3. Comparison of the intense pulsed light and gamma radiation on Bacillus cereus spores in mesquite pod flour

The section 3.3.1. clarified that the highest IPL disinfection could be obtained by controlling voltage at 3000 V, frequency at 1 Hz, pulsed duration of 342 µs, etc. Based on these parameters, the impact of IPL on mesquite flour was then investigated. We maintained water activity levels of both IPL and gamma treated samples at ~0.40 during the whole process through humidity input since relatively high water activity levels contributed high microbial inactivation (Sanchez-Maldonado, Lee, & Farber, 2018). Log reduction (CFU/g) of naturally contaminated B. cereus in mesquite as a function of gamma dose (kGy) and IPL residence time (s) (Figure 3.4). The data was fitted for dose/residence time dependent patterns (one-way ANOVA within-group comparison, IPL: p< 0.0001;

52 gamma radiation: p< 0.0001). Around 3.5 log10CFU/g reductions were observed with 8 kGy gamma radiation. While approximate 1.5 log10CFU/g reductions were obtained after

28 s-IPL treatment. Furthermore, additional 0.2 log10CFU/g reductions of B. cereus spore were inactivated with a photocatalyst TiO2. However, prolonging IPL treatment time did not cause significantly higher log reduction. The probable reasons might be due to the low transparency and dark color of the mesquite flour. As described by (J. Dunn, 1996), the energy of light transmitted below the surface decreased with increasing of depth. As shown in the Lambert-Beer law equation (7):

−[훼]푥 E(x) = (1 − r)E0e (7)

In this equation, E0 stands for the initial energy reaches on the surface of products.

The energy E(x) of light transmitted below the surface of material decreases with increasing depth (x) below the surface. The α means extinction coefficient indicating the transparency of the products. And r represents the reflection coefficient of sample. The low decontamination effects may be attributed to high extinction coefficient of mesquite flour.

In contrast, ionizing radiation type such as gamma ray had high penetration capacity

(Tuncbilek, Ercan, & Canpolat, 2012). A study reported 6-Mev gamma rays can penetrate as deep as 160 cm in water (Roys, Shure, & Taylor, 1954). 104 spores of Aspergillus ochraceus spores in coffee beans was inactivated using 1-kGy gamma radiation (Kumar,

Kunwar, Gautam, & Sharma, 2012).

With respect to enhancement of IPL treatment under the photocatalysis. Some studies reported the photocatalyst TiO2 could be excited by the certain wavelength (< 390 nm). Superoxide radicals, hydroxyl radicals, and hydrogen peroxide were formed when free electrons were reacted with surrounding oxygen and water. (Maness, Smolinski, Blake,

Huang, Wolfrum, & Jacoby, 1999; Pichat, 2016). Our IPL system consists of ~54% ultraviolet light (wavelength<390 nm) in the whole wavelength. Therefore, catalysis effects can be fully utilized. Extensive researches have been carried out to investigate on pathogen inactivation with photocatalysis because TiO2 nanoparticle capable of increasing disinfection effect significantly. However, one challenging task of the process is difficult to recycle the TiO2 photocatalyst (Espino-Estevez, Fernandez-Rodriguez, Gonzalez-Diaz,

Navio, Fernandez-Hevia, & Dona-Rodriguez, 2015). In the current study, immobilizing

TiO2 onto solid substrate (i.e. quartz, stainless, or polymer beads) could potentially be used to separate and recycle the photocatalysts.

Color difference as an important quality indicator was also investigated since some of studies indicated both gamma radiation and pulsed light treatments caused undesirable color change for wheat flour to some degree (Bhat, Wani, Hamdani, Gani, & Masoodi,

2016; Fine & Gervais, 2004). 28s-IPL and 2kGy gamma treatments achieve the similar microbiocidal effects on mesquite flour. However, 28s-IPL caused significantly less color change than that of 2kGy (Table 3.5). This can be mainly attributed to uniform exposure to IPL with help of vibratory feeder; nitrogen gas protection; and environmental controllers.

The results were in agreement with our previous study (chapter 3.3.1.), which illustrated prototype IPL system caused no noticeable color change on wheat flour after 28 s.

4.0 2.0 3.5 3.0 1.5 2.5

2.0 1.0 1.5

1.0 0.5 Log Log reduction (CFU/g) 0.5 Log reduction (CFU/g) 0.0 0.0 0 2 4 6 8 10 0s 7s 12s 21s 28s 28s+TiO2 Gamma irradiation dose (kGy) IPL treatment conditions

(a) (b)

Figure 3.4. Microbial inactivation of mesquite flour under different treatments

*(a) Effects of the gamma radiation on the inactivation of naturally contaminated B. cereus in mesquite flour. (b) Effects of the new prototype IPL/ IPL+TiO2 on the inactivation of naturally contaminated B. cereus in mesquite flour. The total fluence (energy flux) of 28s-

IPL treatment is 7.13 J/cm2

Table 3.5. The L*, a*, b* values, and color difference (ΔE) of mesquite flour subjected to IPL treatment of 28 s and different dose of gamma radiation

L* a* b* ∆E

Control 70.51±0.33 7.24±0.25 25.89±0.06

2kGy 69.88±0.33 6.98±0.04 26.67±0.05 1.03±0.15

4kGy 69.54±0.16 7.09±0.12 26.37±0.42 1.09±0.11

6kGy 69.16±0.26 7.14±0.02 25.96±0.41 1.35±0.27

8kGy 68.93±0.22 7.16±0.08 26.11±0.17 1.59±0.23

IPL-28s 69.89±0.08 7.13±0.10 26.09±0.12 0.65±0.11

IPL-56s 69.58±0.46 7.36±0.02 26.69±0.24 1.22±0.49

L*: lightness (ranging from 0 to 100), a*: greenness to redness (ranging from −60 to +60) and the b*: blueness to yellowness (ranging from −60 to +60), ΔE: calculated color differences. All experiments were performed in triplicate. Data followed by the same lowercase letter are not significantly different (P > 0.05).

To investigate chemical changes caused by 28s-IPL and -radiation treatments in mesquite flour, untargeted chemometric analysis was used. A PCA model based on the LC-

MS analysis of methanol extracts of the mesquite flour samples from three treatments

(untreated control, 28s-IPL, and -radiation treatments) showed the separation of γ- irradiated and IPL-treated samples from the untreated control in a dose-dependent pattern

(Fig. 3.5A). The markers increased by IPL and -iradiation treatments (I-IX) were identified in the S-plot of an OPLS-DA model, and further characterized by database search and structure confirmation using chemical standards if available (Fig. 3.5B and Table 3.6).

Oxidized fatty acids, including 9-hydroxy-10,12-octadecadienoic acid (9-HODE), octadecanedioic acid, and trihydroxy-octadecenoic acid, were identified among these markers (Table 3.6). Quantitative analysis of 9-HODE showed that only IPL treatment significantly increased the concentration of 9-HODE in mesquite flour (Fig. 3.5C).

Although this observation indicated that IPL treatment caused an increase of 9-HODE, this increase of 9-HODE (~5 µg/g) after IPL treatment may not be biologically significant, lipid oxidation was, thereby, very limited. Linoleic acid, the parent compound of 9-HODE, is the most abundant fatty acid present in mesquite flour which contains 1.87-2.64 g total fat/100 g powder.(Cruz-Gracida, Siles-Alvarado, Méndez-Lagunas, Sandoval-Torres,

Rodríguez-Ramírez, & Barriada-Bernal, 2019) In addition, α-ketoisovaleric acid was

56 significantly increased by IPL and 2-, 6-, and 8-kGy treatments. Because α-ketoisovaleric acid has been previously identified as a deamination product of valine in γ-irradiation- treated protein samples (Hatano, 1960). Therefore, these two markers indicate that two non-thermal disinfection methods could affect fatty acids and amino acids in mesquite flour to different degree. Reaching a similar level of decontamination, IPL treatment generated higher chemical changes than -radiation in mesquite flour (Figs. 3.4 & 3.5A). The results were in agreement with previous studies, although some fatty acids such as unsaturated fatty acids were not able to directly absorb visible light or ultraviolet light, lipid photooxidation could occurred through coloring matters such as riboflavin in samples.(Chan, 1977) Moreover, some study indicated low dose (<10 kGy) of gamma radiation contribute to little lipid breakdown in foods.(Hampson, Fox, Lakritz, & Thayer,

1996) Further investigation into the sensory and nutritional value that these changes may cause is a potential future direction of this research.

Acetic acid and propionic acid were identified as pungent odor contributors in mesquite flour, the thresholds of acetic acid and propionic acid were 22 and 2.2 ppm.(Takeoka, Felker, Prokopiuk, & Dao, 2008) Propionic acid and acetic acid content of treated mesquite flour, after 28s-IPL and 2-8 kGy gamma treated mesquite flour were shown in Figure 3.6. The results showed no significant increase of acetic acid concentration was caused after IPL or γ-irradiation treatment. On the contrary, both acetic acid and propionic acid concentration was decreased by both IPL and γ-irradiation (Fig 3.6B).

Therefore, these pungent flavors are not expected to be affected by the treatments. In our separate study of evaluating effect of 28s-IPL treatment on odor, flavor, texture of wheat flour was conducted. The results showed no significant difference in these sensory

57 properties following 28s IPL treatment (data not shown). The reason might be owing to the high intensity (700 J/pulse), low frequency (1 Hz), high voltage (3000 V), and short pulsed duration (342 µs) IPL setting used in the current study, as well as nitrogen circulation system applied. Short pulse durations and the half-life of Π-bonds (10-9 to 10-4 s) enable to prevent mesquite flour from linking with dissolved or free oxygen (Fine &

Gervais, 2004). On the other hand, the data of this study are in agreement with a previous study (Marathe, Machaiah, Rao, Pednekar, & Sudha Rao, 2002) on wheat flour sensory evaluation after gamma radiation. The author evaluated various sensory attributes such as mouth-feel, appearance, taste, aroma, etc., after 1 kGy-gamma treatments. The sensory attributes were highly acceptable after treatments. Furthermore, it was found that relatively low gamma dose might maintain or modify the sensory after 6 months storage.

This cell viability test is an essential analysis prior to looking into the potential nutraceutical and/or therapeutical properties of the flour. Gamma dose below 1 kGy on fruits and vegetables are allowed by the United States Food and Drug Administration.(FDA,

2004a) The total accumulated fluence of IPL below 12.0 J/cm2 can be used to treat food products.(FDA, 1996) However, in depth cytotoxicity or oral toxicity investigations of higher dose of gamma radiation or IPL on foods are lacking. Figure 5 shows the data obtained in basal conditions after direct treatment of Caco-2 cells with different treatments under different concentration of mesquite flour. The cell viability of Caco-2 cell was relatively low at mesquite flour concentrations at 40 and 100 mg/mL. At 20 mg/mL mesquite flour level, both IPL and gamma radiation had no significant impact on cell viability, which showed no significant difference with PC treatment. A research indicated ionizing radiation caused a loss of intestinal epithelial integrity.(Kang, Kim, Yi, & Son,

2009) which was linked to the gastrointestinal syndrome such as abdominal pain, diarrhea, and bacterial infection. Overall, the results show that the 20 mg/mL treated flour did not affect the Caco-2 cells very much, suggesting that these two processes within the dosages tested in this study did not induce obvious cytotoxicity.

190218S_WM_MesquiteFlourPowder_MeOH_extraction_neg.M4 (OPLS/O2PLS-DA), Ctl vs Trt w[Comp. 1]/p(corr)[Comp. 1] 190218S_WM_MesquiteFlourPowder_MeOH_extraction_neg.M6 (PCA-X) A t[Comp. 1]/t[Comp. 2] B 1.0 I IV V 40 0.8 VII VII I 30 0.6 II III 20 -irradiation 0.4 IX VI 0.2 10 -0.0

0 t[2]

IPL p(corr)[1] -0.2 -10 -0.4 -20 Control -0.6 -30 -0.8 -40 -1.0 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 -0.15 -0.10 -0.05 -0.00 0.05 0.10 0.15 t[1] w[1] R2X[1] = 0.505703 R2X[2] = 0.169128 Ellipse: Hotelling T2 (0.95) SIMCA-P+ 12 - 2019-12-03 14:10:44 (UTC-6) R2X[1] = 0.155678 SIMCA-P+ 12 - 2019-10-03 14:01:42 (UTC-6) α-Ketoisovaleric Acid C HODE D 30 *

25 *

e c

20 n

r d

e 20

n d

15 u

g 10

v i

g 10

 l

5 e R

0 0 Ctl IPL 2 4 6 8 Ctl IPL 2 4 6 8

-irradiated (kGy) -irradiated (kGy)

Figure 3.5. Chemometric analysis of potential chemical changes in mesquite flour after

IPL- and -irradiation treatments

*A. Score plot of a PCA model on all treatment groups. B. S-plot of an OPLS-DA model on the comparison between untreated control and treatments. The prominent markers increased by either IPL or -irradiation annotated were identified and labeled (Table 2).

C. Concentration of 9-HODE in samples. D. Concentration of α-ketoisovaleric acid in samples.

A B Acetic Acid Proprionic Acid 250 40

200

r a r

e 30

e d

150 d

w w

o b o p b b 20

p b

g 100 /

g b

g  50  10

0 0 Ctl IPL 2 4 6 8 Ctl IPL 2 4 6 8

-irradiated (kGy) -irradiated (kGy)

Figure 3.6. Concentration of short-chain fatty acids in mesquite flour after IPL- and - irradiation treatments.

*A. Acetic acid concentrations. B. Proprionic acid concentrations.

Figure 3.7. Direct effect of flour with different treatments on Caco-2 cell viability

*Results expressed as mean ± standard deviation (n = 6). Different letters within the same flour concentration denote significant differences (p < 0.05).

Table 3.6. Chemical markers separating IPL and γ-irradiation treatments from Control

ID Chemical Identity m/z (-) Chemical Formula

* I α-ketoisovaleric acid 115.0391 C5H8O3

* II Phenyllactic acid 165.0546 C9H10O3

* III 2-Isopropylmalic acid 175.0601 C7H12O5

# IV Caffeic Acid 179.0335 C9H8O4

* V Nonate 187.0960 C9H16O4

# VI HODE 295.2264 C18H32O3

* VII Octadecanedioic acid 313.2372 C18H34O4

* VIII Trihydroxy-octadecenoic acid 329.2319 C18H34O5

* IX Phosphoethanolamine (18:2) 476.2774 C23H44NO7P

#: markers confirmed by chemical standards. *: markers identified by database search.

3.3.4. Effects of the intense pulsed light on microbes in seeds

All seed samples were treated with IPL up to 120 s (Table 3.6). Comparing between

IPL+TiO2 and IPL treatments, synergistic effects of IPL and TiO2 contributed to higher C. sakazakii inactivation than that of individual IPL after 120 s. And IPL disinfection prone to lower on B. cereus spore than C. sakazakii or E. faecium. Based on the energy consumption, less energy was consumed by almond, sunflower seed, and peanut than the rest seed samples. The probable reason was owed to the presence of hull for rice grain and wheat kernel, the hull or crevice kept the microbes hidden inside from exposing to IPL.

Therefore, significantly lower microbial inactivation was achieved. The microbial inactivation obtained from our study was significantly higher when comparing with other studies, the maximum IPL disinfection on Salmonella Enteritidis in almond was less than

2 logs after 160s (Harguindeguy, 2016). A study reported the highest Aspergillus flavus inactivation was 1.7 logs in malting barley. Overall, the data indicated enhanced microbial inactivation could be brought using the prototype IPL system (Zenklusen, Coronel, Castro,

Alzamora, & Gonzalez, 2018). For the germination rate, some previous studies investigated

IPL treatment on radish, pak choi, and wheat kernels. The results indicated germination rate of seeds was not affected by IPL significantly. 97-100 % and 95-100 % of germination rate was maintained after up to 37.80 J/cm2 for pak choi seeds and radish seeds respectively

(S.-M. Kim, Hwang, Cheigh, & Chung, 2019); percentage of germination was only reduced

14-15 % after receiving 32.0 J/cm2 of IPL fluence (Aron Maftei, Ramos‐Villarroel,

Nicolau, Martín‐Belloso, & Soliva‐Fortuny, 2014).

Table 0.1. Energy consumption and microbial inactivation for different seeds under

IPL, the data is mean values of duplicated experiments.

Bacteria and treatment Wheat Sunflower Almon Rice Peanut

condition seed d grain

C. sakazakii (IPL) Energy consumption 37.3 J/g 27.6 J/g 9.9 J/g 77.2 17.3 J/g

(per log reduction) J/g

Total log reduction 4.21 logs 3.78 logs 2.69 0.71 1.18

and treatment time (120s) (120s) logs logs logs

(40s) (40s) (40s)

C. sakazakii Energy consumption 31.4 J/g 22.1 J/g 9.2 J/g

(IPL+TiO2) Total log reduction 5.00 logs 4.71 logs 2.90

and treatment time (120s) (120s) logs

(40s)

E. faecium Energy consumption 37.7 J/g 36 J/g 12.6 J/g 41.8 25.5 J/g

J/g

Total log reduction 4.16 logs 3.86 logs 1.42 1.31 0.70

and treatment time (120s) (120s) logs logs logs

(40s) (40s) (40s)

B. cereus spore Energy consumption 96.8 J/g 66.2 J/g 18.4 J/g 114.2 32.4 J/g

J/g

Total log reduction 1.62 logs 2.10 logs 0.97 0.48 0.55

and treatment time (120s) (120s) logs logs logs

(40s) (40s) (40s)

3.3.5. C. sakazakii inactivation kinetics in NFDM

3.3.5.1. Kinetic modeling of inactivation curves

To analyze the kinetic modeling of inactivation curves, the Matlab software

(Mathworks, Natick, MA) was used to test both non-linear and linear survival curves including Weibull, Weibull plus tails, log linear, and biphasic.

The Weibull model is presented as equation (8) (Mafart, Couvert, Gaillard, & Leguerinel,

2002):

푡 푝 푙표푔 (푁) = 푙표푔 (푁 ) − ( ) (8) 10 10 0 훿

The Weibull plus tail model uses the equation (9) (Albert & Mafart, 2005):

푡 푝 푙표푔푁0 푙표푔푁푟푒푠 (− ) 푙표푔푁푟푒푠 푙표푔10(푁) = 푙표푔10(10 − 10 ) ∗ 10 훿 + 10 (9)

Where δ is used to indicate the time for the first decimal reduction, and p (dimensionless) represents the shape parameter of the curve. The curve shows convexity if p>1, while the curve is concave when the p<1. N0(CFU/g) is the initial concentration of microorganisms,

Nres is the population of resistant cells, N is the number of surviving microbes, and t is the treatment time (s).

The log linear model described as equation (10) (Bigelow, 1921):

푙표푔10(푁푓) = 푙표푔10(푁0) − 푘 ∗ 푡 (10)

The biphasic model used to express microbial survival curves is in equation (11) (Cerf,

1977):

−푘1∗푡 −푘2∗푡 푙표푔10(푁) = 푙표푔10(푁0) + 푙표푔10(푓 ∗ 푒 ) + (1 − 푓)푙표푔10(푒 ) (11)

Where k, k1, and k2 are inactivation rates, f is the resistant capacity of microorganisms.

3.3.5.2. Statistical analysis

Data were expressed by means of Log10 values of mean± standard deviation from triplicated experiments. The fitting curve functions of Matlab software were employed to determine the mean square error (MSE), root mean square error (RMSE), correlation coefficient (R2) adjusted correlation coefficient. The model with smallest RMSE can be considered the best fit.

3.3.5.3. Results and discussions

The fitness parameters of these four kinetic inactivation models are in Table 3.7.

Data indicated the C. sakazakii inactivation kinetics fitted Weibull plus tail and Biphasic fit better than the rest models because of low RMSE. Weibull model is the most frequently used to describe the microbial inactivation by IPL in many studies (Ferrario, Alzamora, &

Guerrero, 2013; Hsu & Moraru, 2011; B. Miller, Sauer, & Moraru, 2012). Tail plus Weibull model was observed in the current study, two possible theories could be used to explain the conditions. The overlapped microorganisms could potentially cover the other microbes due to the limited penetration of IPL, the energy fluence of IPL decreased significantly with increasing depth (Bialka, Demirci, & Puri, 2008). Tail could be also result of rough or porous surface of milk matrix, which might prevent microorganisms from exposing to

IPL (Ringus & Moraru, 2013). Biphasic model was usually to describe two microbes with different resistances. However, only one type of microbe presented in the current experiment. On the other hand, after many literature reviews, Log-linear model was not identified as a microbial inactivation curve in food matrices (Bradley, McNeil, Laffey, &

Rowan, 2012; Hayes, Laffey, McNeil, & Rowan, 2012; Said & Otaki, 2013). Therefore, the Weibull plus tail model was chosen as the model with best fit to plot the curve of C. sakazakii inactivation in NFDM with prototype IPL (Figure 3.5).

Table 0.2. Chemical Statistical parameters of four models for kinetic inactivation of

C. sakazakii in NFDM

Log linear Biphasic Weibull Weibull

plus tail

MSE 0.2833 0.0509 0.1992 0.0457

RMSE 0.5323 0.2256 0.4463 0.2137

R-Square 0.8817 0.9820 0.9232 0.9839

R-Square adjusted 0.8726 0.9771 0.9104 0.9795

The residence time (s) to reach 4D reduction 64.4 46.2 46.2 45.3

7.00

6.00

5.00

4.00

3.00 Log10(N)

2.00

1.00

0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Treatment time (s)

Measured Identified

Figure 0.1. Mean surviving population of C. sakazakii during prototype IPL

*Black dots are measured points. Red curve is plotted based on identified curve of C. sakazakii inactivation using Weibull plus tail model. The treatment conditions are 1 Hz and 3000 V. All samples were maintained at ~56.8 °C and relative humidity of 30-40%.

3.4. Conclusion

This prototype IPL system can be considered as a more environmentally friendly, cost-effective, and efficient system than our previous IPL system (Chen, Wiertzema, et al.,

2018). In the aspect of bactericidal effects, to achieve a similar C. sakazakii inactivation

2 (~3 log10 CFU/g), the current prototype system (7.13 J/cm ) consumed significantly less energy/fluence than the original IPL system (29.36 J/cm2). Factors other than high energy intensity contributed to the higher energy efficiency were shown as follows. The width of the vibratory feeder was decreased from 30 to 18 cm and a pathway was reduced and

68 parallel with IPL lamp to avoid energy waste. On the other hand, higher voltages (3000 V) were employed in this prototype IPL system when compared with the previous IPL system

(1000V). Our prototype IPL system used for pathogens inactivation in powdered foods showed a better performance when comparing with other IPL systems. The maximum B. cereus spore inactivation in ground black pepper and red pepper were only 0.8 and 1 log10

CFU/g reduction, respectively, with 10 J/cm2 of IPL (Nicorescu, Nguyen, Moreau-Ferret,

Agoulon, Chevalier, & Orange, 2013). The possible reasons could be attributed to insufficient homogenization, low IPL intensity, lack of environment control, nonuniform distribution of IPL energy. Therefore, our prototype IPL system under the controlled environment, appropriate IPL parameters, and conveying process results in a greater inactivation effect in powdered foods.

In terms of quality aspect, to achieve a ~ 3 log10 CFU/g reduction of C. sakazakii, the current innovative prototype IPL system caused significantly less ∆E than the original

IPL system (Chen, et al., 2018). This may be due to the application of low IPL fluence, more optimizing temperature, humidity control, and the circulation of nitrogen gas.

Nitrogen gas could serve as a gas barrier to prevent the samples from contacting with air

(G. H. Ryu & Ng, 2001; Sharma & Demirci, 2003). Therefore, reducing photo-oxidation of NFDM during IPL treatment. And an economic feature of this enclosed gas circulation system enables most of the feed gas to be recycled.

Chapter 4. Effects of cold atmospheric plasma on Cronobacter sakazakii inactivation and physicochemical property changes of non-fat dry milk powder

4.1. Introduction

Much research has reported that CAP jet only took effect in surface treatment.

Therefore, this gas-phase disinfection technology was not able to eliminate the excessive microbes located at entire surface of samples without using sufficient exposure under CAP

(Fernandez, Shearer, Wilson, & Thompson, 2012). To overcome the drawback, a proper fluidized plasma reaction chamber needs to be designed with features of allowing free-flow gas and restricting sample flow at the same time. The samples can be thereby tumbled and fully exposed to CAP. In this chapter, the main objectives are to (1) develop a fluidized plasma reaction system for C. sakazakii inactivation in NFDM; (2) explore the C. sakazakii inactivation by CAP in NFDM under different treatment conditions; (3) analyze the impacts of CAP treatments on some physicochemical properties of NFDM; (4) evaluate the effects of PAW treatments on the DON degradation in barley; (5) investigate germination rate after PAW treatments, and compare with IPL treatments.

4.2. Materials and methods

4.2.1. Sample inoculation and enumeration

NFDM (Land O’Lakes, Inc., Arden Hills, MN) was received from local diary company. The NFDM composition was relied on the United States Standards (USDA,

2001). The C. sakazakii (ATCC 29544) inoculation and enumeration was conducted using the previous steps (Chen, Peng, et al., 2019). Microbial concentration was express as

CFU/g.

4.2.2. CAP system development and treatment

An DYNE-A-MITETM VCP 3D surface treater (Enercon Industries Corp,

Menomonee Falls, WI) was used to generate plasma. The output power of the sytsem is

480 W, the primary voltage was stepped up to 4.4 KV. The gas flow fluidized bed was designed to tumble the NFDM during treatment. The dimention of CAP chamber is length

3.5 cm × width 1 cm × height 2 cm, and made of polycarbonate to reduce the potential arcing and resistant to chemicals and thermal effects during treatment. In addition, polyetheretherketone meshs (0.011 mm × 0.064 mm) were located on both upper and lower parts of chamber to enable plasma go through while the powder was kept inside (Figure

4.1).

One hundred milligrams of NFDM was loaded in the CAP treatment chamber, these samples were substantially treated with CAP from 0-120 s at flow rate from 8-20 Liter/min, the intial water activity level of NFDM was maintained at 0.25, and environmental humdity was controlled at ~35% during treatments.

Figure 4.1. Schematic diagram of the CAP system

4.2.3. Physicochemical property evaluation after CAP

Various physicochemical properties including color, crystallinity, amino acid composition, phenolic contents of NFDM were determined according to our previous study

(Chen, Peng, et al., 2019).

4.3. Results and discussions

4.3.1. Effects of CAP on C. sakazakii inoculated in NFDM

C. sakazakii inactivation in NFDM increased with increasing treatment time and flow rates from 20-120 s, contributed to 1.17- 3.27 log10 CFU/g reductions (Figure 4.2).

High C.sakazakii inactivation could be owed to the fluidized plasma reaction system combined with approximate environmental control. NFDM particles were exposed to CAP

72 fully by tumbling the particles in the air. This new strategy increased exposure of microbes to CAP in this study. This CAP disinfection was significantly higher than that of other studies. For example, a research indicated only 0.910 log10 CFU/g C. sakazakii inactivation was observed using cold plasma treatment. Insufficient contact between CAP and the milk particles resulted in only partial decontamination (Oh, Lee, Kim, Lee, Cho, & Min, 2015).

The CAP disinfection was less when comparing with the plasma on inorganic surfaces. For example, 5-30 s-CAP on C. sakazakii in polytetrafluoroethylene beeds led to the reductions of 0.12-6.94 log10 for C. sakazakii at a flow rate of 6L/min. This result might be contributed to the fact that the organic materials in NFDM such as protein, lipid, and photosensitizers could absorb or react reactive species (RS) and UV energy. Therefore,

CAP effects on orgnic samples were inhibited (Thirumdas, Sarangapani, & Annapure,

2015).

With respect to gas flow rate, C. sakazakii inactivation increased significantly with increasing gas flow rate from 8-20 L/min (p< 0.05 using Two-way ANOVA). The first explanation is owed to higher volumn of plasma was induced with higher gas flow rate.

Another possible explanation is higher velocity of the flow rate leads to the rise of the plasma flame. The extended flame can serve as an extended electrode in reducing the effective distance between electrodes (Cha, Lee, Kim, & Chung, 2005). The results were consistent with a former study, which indicated that E.coli O157:H7 and Salmonellas

Stanley inactivation on golden delicious apples were more effective at higher flow rate

(Niemira & Sites, 2008).

The temperature curve of NFDM surface fits a time-dependent pattern during CAP treatments from 0-120 s (Figure 4.3, p< 0.05 using one-way Anova). Synergistic effects of

CAP and heating contributed to higher micribial inactivation during plasma treatment

(Laroussi & Leipold, 2004). However, overheating could potentially cause undesirable agglomeration/caking for powdered foods such as milk powder during cold plasma treatment (Chen, et al., 2018). Therefore, the sample conditions including temperature or treatment time needs to be controlled to enable the final temperature was sufficient to optimize the C. sakazakii inactivation. However, adverse quality issues such as caking needs to be avioded. The highest temperatures achieved was 58.73 ºC, which did not excess the threshold of the thermal treatment (Osaili, Shaker, Al‐Haddaq, Al‐Nabulsi, & Holley,

2009). Overall, combined effects of heat, UV, and reactive species (RS) generated in CAP probably played main role in microbial inactivtion.

Diverse non-thermal technologies were employed to inactivate C. sakazakii in PIF, these technologies are not always sufficient, rapid, or practical in application. For example, it inactivated 3.28 log10 CFU/g Crononbacter in PIF after 120 min of ozonation at 5.3 mg/L

(Torlak & Sert, 2013). 1.22 log10 CFU/g reduction of C. sakazakii was observed in PIF with pulsed electric field treatments (Brendan A. Niemira, 2012).In addition, it took 20 min to eliminate approximate 3 log10 CFU/g C. sakazakii in PIF using a combination of

UV radiation and 60 ºC water bath treatments (Q. Liu, Lu, Swanson, Rasco, & Kang, 2012).

Hydrostatic pressure processing (HPP) disinfection caused as much as 3.11 log10 CFU/g reduction on C. sakazakii. However, this technology could only be used on the application of reconstituted PIF pasteurization (Gonzalez, Flick, Arritt, Holliman, & Meadows, 2006).

These approaches have failed to address the PIF sterilization prior to packaging. The findings in the present study could potentially meet the need.

4 8 L/min

12 L/min

16 L/min G FG 3 20 L/min FG F F EF EF EF DE E D D

2 D Log reduction (CFU/g) reductionLog D D C D B C AB BC A B A 1 20 40 60 80 100 120 Treatment time (s)

Figure 4.2. Log reduction (CFU/g) of NFDM treated with CAP at different flow rates and treatment times

*Data in the figure followed by the same uppercase letters are not significant difference

(p>0.05). The data were expressed as the mean ± standard deviation of measurements made in triplicate.

65.00 8 L/min 12 L/min 60.00 O 16 L/min N O MN O 20 L/min L 55.00 KL MN N J KL LM

) IJ JK

℃ IJ 50.00 HI HI I GH FG FG G 45.00 F DE E Temperature ( Temperature D CD 40.00 BC C BC B A A B 35.00 A A

30.00 0 5 10 15 20 25 30 35 40 Treatment time (s)

Figure 4.3. Surface temperature of NFDM samples during atmospheric cold plasma treatment from 0-40 s

*Data in the figure followed by the same uppercase letters are not significant difference

(p>0.05). Data are represented as means± standard deviations of measurements made in triplicate.

4.3.3. Effects of CAP on the color of NFDM

Color properties were important parameters for sample quality. Color properties including L*, a*, b*, and color difference (∆E) of CAP-treated NFDM were investigated up to 120 s-CAP treatment (Table 4.1). The data revealed that ∆E of NFDM did not show significant changes (∆E <0.5) from 8-20 L/min following 80 s-CAP treatment and100 s-

CAP at flow rates of 8 and 12 L/min. The short period-CAP treatment did not induce noticeable color change to NFDM. A slightly color change (0.5-1.5) only was observed after 100 s-CAP treatment. The association of a slight decrease in the L* value, a slight increase in a* value, and a slight decrease in b* value stands for slight brown pigment formation in NFDM after CAP. However, the largest ∆E for CAP treated NFDM was 1.14, which was still underneath the standard of slightly noticeable (∆E <1.5). The results denoted that CAP treatment had slightly noticeable effects on appearance of NFDM generally. The results are in agreement with previous studies. For example, a study related to the effects of CAP treatment on liquid milk indicated only slightly color change (∆E=

0.52) occurred after 20 min treatment (Gurol, Ekinci, Aslan, & Korachi, 2012). Compared with the results of other powdered foods, which reported distinct color changes in powdered food such as black pepper seeds, crushed oregano, and red paprika powder after

5-min CAP treatments, the probable reasons were owed to CAP-induced oxidation

(Hertwig, Reineke, Ehlbeck, Erdoğdu, Rauh, & Schlüter, 2015). Overall, limited quality loss of NFDM during the CAP treatments in this study was likely as a result of short treatment time and protection of inert nitrogen gas.

Table 4.1. The L*, a*, b* values, and color difference (∆E) of NFDM subjected to CAP treatment at gas flow rate from 8-20 L/min up to 120 s.

Time (s) Flow rate (L/min) L⁎ a⁎ b⁎ ∆E

untreated NFDM 95.34±0.18FGH -2.34±0.09CDE 13.94±0.22ABC

20 8 95.29±0.10GH -2.55±0.04F 13.83±0.09ABC 0.24±0.04EF

12 95.09±0.17DEFG -2.37±0.01E 13.81±0.02C 0.28±0.14EFG

16 95.14±0.15DEFGH -2.37±0.15CDEF 13.95±0.06B 0.19±0.07FG

20 94.96±0.24CDEFG -2.33±0.17BCDEF 13.82±0.11ABC 0.40±0.17BCDEF

40 8 95.14±0.03EF -2.35±0.05DE 13.83±0.14ABC 0.27±0.02F

12 95.09±0.07E -2.29±0.12BCDE 13.67±0.17AC 0.35±0.14CDEF

16 95.20±0.02FG -2.26±0.04CD 13.82±0.10ABC 0.20±0.08FG

20 95.03±0.09DE -2.24±0.03C 13.88±0.15ABC 0.33±0.11DEF

60 8 95.10±0.22DEFGH -2.33±0.13CDE 13.83±0.30ABC 0.26±0.11EFG

12 95.35±0.06H -2.25±0.09BCDE 13.87±0.21ABC 0.11±0.07G

16 95.07±0.05E -2.31±0.09CDE 13.83±0.13ABC 0.29±0.07EF

20 94.90±0.15CDE -2.24±0.07BCD 13.78±0.20ABC 0.48±0.06BCD

80 8 95.15±0.04EFG -2.21±0.09BCD 13.83±0.15ABC 0.26±0.05EF

12 95.10±0.21DEFGH -2.25±0.06BCD 13.83±0.09ABC 0.28±0.11EFG

16 94.90±0.18CDE -2.34±0.12CD 14.00±0.12BC 0.47±0.18BCDE

20 94.82±0.17CD -2.31±0.16BCDE 14.00±0.25ABC 0.45±0.17BCDE

100 8 95.12±0.06EF -2.31±0.21BCDE 13.78±0.06ABC 0.27±0.10EFG

12 95.17±0.12EFGH -2.30±0.17BCDE 13.80±0.07ABC 0.22±0.06FG

16 94.88±0.07CD -2.26±0.11BCDE 13.69±0.33ABC 0.53±0.21BCDE

20 94.85±0.32CDEFG -2.12±0.06B 13.73±0.26ABC 0.58±0.10BC

120 8 94.67±0.25BC -2.28±0.10BCDE 13.92±0.09BC 0.67±0.25BCD

12 94.43±0.34ABC -2.15±0.15ABCE 13.90±0.13ABC 0.93±0.36AB

16 94.36±0.13AB -2.13±0.06B 13.69±0.36ABC 1.03±0.08A

20 94.28±0.07A -1.99±0.04A 13.71±0.07A 1.14±0.08A

*Data were expressed as the mean ± standard deviation of measurements made in triplicate.

And Data in the same column followed by the same uppercase letter are not significantly different (p > 0.05).

4.3.4. Effects of CAP on NFDM crystallinity

Crystallization caused various deteriorative alterations in milk powders, the adverse effects such as undesirable agglomeration, loss of lysine, lipid oxidation, and discoloration are usually induced. Crystallization of amorphous lactose was presented in crystal forms of α-lactose monohydrate, anhydrous β-lactose, stable and unstable anhydrous α-lactose, and an anhydrous mixture of α- and β-lactose in a molar ratio of 5:3 and 4:1. XRD analysis can be used to evaluate and identify these forms (Jouppila, Kansikas, & Roos, 1998). The

XRD pattern of CAP treated NFDM is used to measure the degree of lactose crystals after

CAP treatment. Figure 4.4 shows NFDM samples that diffraction patterns of all treated and untreated NFDM milk powder posed no significant under different flow rates. The lactose crystallization was monitored based on the peaks with XRD at different angles, for example, the characteristic peaks at 20º stands for formation of α-lactose monohydrate and anhydrous crystals with anhydrous crystals with α-and β-lactose in a molar ratio of 5:3

(Jouppila, Kansikas, & Roos, 1998). No significant change in the peak levels at diffraction angle of 20° indicated no obvious lactose crystallization was formed after CAP treatment.

Therefore, the physiochemical properties of NFDM were maintained well after CAP treatments. Similar to our former research under intense pulsed light (Chen, et al., 2018), the data of this study confirmed that relatively short treatment time, appropriate water activity, mild temperature, and humidity could avoid or minimize adverse effects on the quality of NFDM.

1800 Control 1600 8L/min 12L/min 1400 16L/min 20L/min 1200

1000

800

600

Intensity (arbitaray counts) Intensity 400

200

0 5 7 9 11 13 15 17 19 21 23 25 27 28 30 32 34 36 38 Two-theta (degrees)

Figure 4.4. XRD results for the untreated samples and the CAP treated NFDM samples from flow rate of 8-20 L/min for 120 s

4.3.5. Effects of CAP on the amino acid composition of NFDM

The impacts of CAP on amino acid composition of NFDM were analyzed at a flow rate of 20L/min after 0-120s-CAP treatments (Table 4.2). The CAP treatments did not bring significant changes to the amino acid composition of NFDM (p>0.05 using one-way

ANOVA). Amino acids are sensitive to oxidation induced from ROS and RNS. It is well known that the atomic oxygen (i.e. 309 nm) and hydroxyl radicals (i.e. 777 nm) can cause severe oxidation among all reactive species formed by plasma (Ragu, Faye, Iraqui,

Masurel-Heneman, Kolodner, & Huang, 2007). However, no noticeable signals of these

ROS were observed according to the optical emission spectra (Figure 4.5). The reasons can

80 be attributed to relatively low humidity (~35%) controlled during the CAP treatments and limited amount of atomic oxygen was generated when using N2 as feed gas. As evidenced by a previous study (Soloshenko, Tsiolko, Pogulay, Kalyuzhnaya, Bazhenov, & Shchedrin,

2009), high humidity level (80%) led to almost three-fold increase of OH radicals as low humidity level (20%). On the other hand, relatively short duration of CAP treatment (120 s) was adopted to reduce the oxidation either. CAP caused slight oxidation of the proteins for whey protein isolate below 15 min (Segat, Misra, Cullen, & Innocente, 2015). However, noticeable protein change was detected when expanding CAP treatment time from 30-60 min. This CAP treatment of current study induced significantly less changes in amino acids when comparing with some conventional treatments such as heating treatment. A study clarified that lysine in UHT milk decreased significantly after heating treatment because

Maillard reaction occurred at relatively high temperatures (at 120 ºC or greater) (Van

Boekel, 1998).

Table 4.2. Composition of major amino acids (% w/w) as a function of CAP treatment time

Amino acid types CAP treatment time (s)

0 40 80 120

Alanine 2.88±0.22A 2.91±0.14A 2.94±0.06A 3.10±0.18A

Arginine 0.83±0.10A 0.85±0.09A 0.92±0.11A 0.91±0.08A

Aspartic Acid 2.65±0.29A 2.71±0.34A 2.60±0.29A 2.75±0.20A

Glutamic acid 31.36±2.48A 31.39±1.55A 32.25±0.90A 33.23±0.81A

Glycine 1.04±0.15A 1.06±0.07A 1.09±0.10A 1.19±0.13A

Histidine 0.67±0.27A 0.6±0.25A 0.58±0.15A 0.69±0.48A

Isoleucine+Leucine 9.01±0.63A 9.13±0.75A 8.66±0.46A 8.62±0.44A

Lysine 25.87±4.32A 22.29±2.15A 22.61±2.00A 21.51±0.50A

Phenylalanine 2.12±0.14A 1.99±0.08A 1.83±0.16A 1.94±0.16A

Proline 13.17±1.81A 16.51±1.81A 16.05±2.61A 15.38±0.75A

Serine 2.16±0.26A 2.16±0.20A 2.21±0.26A 2.41±0.12A

Threonine 1.78±0.25A 2.01±0.18A 1.96±0.23A 1.96±0.07A

Tyrosine 1.23±0.16A 1.04±0.13A 1.02±0.44A 1.10±0.32A

Valine 5.23±0.17A 5.34±0.18A 5.29±0.27A 5.21±0.20A

*Data were expressed as the mean ± standard deviation of measurements made in triplicate.

And Data in the same row followed by the same uppercase letter are not significantly different (p > 0.05).

4.3.6. Effects of CAP on total phenolic content

Total phenolics can reflect antioxidant condition of milk. Phenolics took important effects on the functional properties of milk such as foamability, microbiological stability, oxidative stability, heat stability, and sensory properties of milk (O’connell & Fox, 2001).

Therefore, the impacts of CAP treatment on the phenolic content in NFDM were investigated. Table 4.3 shows the relationship between total phenolic compounds of NFDM and flow rate from 0-120s at 20 L/min. The data indicated no significant change of phenolic content from 0-120s CAP treated samples (p>0.05 using One-way ANOVA). CAP tends to be a promising technology to remain phenolic content in comparison with the high temperature short time (HTST) pasteurization process (72 ºC for 15 s) (Calligaris,

Manzocco, Anese, & Nicoli, 2004). The HTST pasteurization caused the decline of the antioxidant properties in milk because of the development of the Maillard reaction. Higher

82 temperature (>85 °C) treatment decreases the total phenolic contents and antioxidants of milk samples probably because of increased polyphenol-protein interactions. In contrast, the impact of heating at lower temperature (30-40 °C) on total phenolic contents was minimal (Chávez-Servín, Andrade-Montemayor, Vázquez, Barreyro, García-Gasca,

Martínez, et al., 2018; Kılıç Bayraktar, Harbourne, & Fagan, 2019). Overall, the data implied the oxidative stress did not induced obvious changes to milk’s antioxidant property with CAP.

Table 4.3. Total phenolic content as a function of CAP treatment time

CAP treatment time (s) Total phenolic content in NFDM (mg GAE/g)

0 6.73±0.39A

40 6.51±0.18A

80 6.85±0.19A

120 7.00±0.39A

*Data were expressed as the mean ± standard deviation of measurements made in triplicate.

And Data in the same column followed by the same uppercase letter are not significantly different (p > 0.05).

4.3.7. Characterization of CAP through optical emission spectroscopy

It is well known that UV photons and reactive species generated from CAP.

Nevertheless, UV photons are easily absorbed by gas atoms and molecules when applying ambient pressure cold plasma treatment (Brendan A. Niemira, 2012). Therefore, UV probably does not play a critical role in microbicidal effects during this type of plasma treatment, whereas ions, radicals, and reactive gas species were identified as main biocidal agents. The discharge of CAP caused the breakdown of gas and development of plasma.

The major electron induced reactions in the active zone of N2 plasma are shown as follows

(Equations 12 & 13) (Deng, Nikiforov, Vanraes, & Leys, 2013).

+ N2 + e → N2 + 2e (12)

3 3 3 N2 + e → N2(A Σ, B Π, C Π) + e (13)

On the other hand, the generation of atomic N in the states 4S, 2D, and 2P as a result of electron disassociation also took a main part in the sustainability of plasma in nitrogen

(Itikawa, Hayashi, Ichimura, Onda, Sakimoto, Takayanagi, et al., 1986). Relatively weak

NO lines were observed in the range of UV region between 210- 300 nm since ~0.1% of oxygen involved in the industrial N2 used in the study (Figure 4.5). The emission from

281.4 nm-380 nm were dominated by the presence of the second positive system of the

3 3 nitrogen molecule (N2 (C Πµ - B Πg), e.g. 337 and 354 nm) and the first negative system

+ 2 2 of the nitrogen molecular ion (N2 (B Σµ -X Σg), 390-426 nm, e.g. 391 nm), and first

3 3 positive system of the nitrogen molecule (N2 (B Πg-A Πµ), 500-900 nm, e.g. 580 and 654 nm) were the primary contributions to the spectra. All signals were disappeared once removing the CAP.

(a)

(b)

Figure 4.5. Optical emission spectra of CAP discharge (6.5 cm above the nozzle) at the flow rate of 20 L/min

*(a) spectrum from 190-650 nm was monitored by STS spectrometer, (b) spectrum from

450-1000 nm was monitored by USB 4000 spectrometer.

4.4. Conclusions

With respect to CAP on NFDM, this chapter concluded that (1) fluidized CAP can be considered as an alternative non-thermal technology for C. sakazakii inactivation in

NFDM. This fluidized reaction system was particularly useful when the feeding gas of the

CAP was at a relatively high flow rate; (2) CAP had no or minor adverse effects on physicochemical properties of NFDM including color, amino acid composition, crystallinity, and phenolic content following 120-s CAP treatment. However, additional experiment needs to be carried out to scale-up this continuous treatment: the larger sample loading region and power up discharge heads before applying in the powdered food industry.

Chapter 5. Decontamination of Deoxynivalenol in Raw and Germinating Barley using Plasma-Activated Water and Intense Pulsed Light

5.1. Introduction

Although direct cold atmospheric plasma is considered as an effective decontamination method to inactivate microorganisms or toxins in food samples, some sensitive or fragile food samples such as barley seeds are not suitable to be exposed under plasma directly. Therefore, indirect exposure under CAP may also be a potential method. i.e. plasma activated water is a way to “save” the microbicidal effect in a medium and treat samples with reactive species, ions, and radicals only. The UV irradiation emitted from plasma jet is not included (Scholtz, Pazlarova, Souskova, Khun, & Julak, 2015). We will investigate the effect of plasma-activated water on Deoxynivalenol in raw and germinating barely in this chapter.

5.2. Methods and materials.

5.2.1. Sample preparation

Raw barley samples were obtained from Rahr Malting Co., Minnesota, US. The initial DON level reached around 1.45 ppm in this study. The samples were then vacuum sealed at 4 °C until use. The protocol of germinating barley making was followed our previous research (Chen, Chen, Cheng, Peng, Liu, Ma, et al., 2019).

5.2.2. PAW generation and treatment

A nonthermal atmospheric-pressure plasma jet (The DYNE-A-MITETM VCP 3D surface treater was used to generate PAW. The detailed process was based on a previous

87 method (Chen, Chen, et al., 2019). 50 g of raw and germinating barely were merged in a

100 mL of PAW up to 20 min. A ultra-thin magnetic stirrer was empolyed to mix PAW during treatment.

5.2.3. ORP, conductivity, and pH value measurements

A multimeter pH/ORP/Con (Cole-Parmer P100 pH/ORP/Con Meter, Vernon Hills,

IL) were used to measured these three physicochemical parameters in PAW during treatment.

5.2.4. Laboratory germination test

50 g treated or untreated raw and germinating barley samples were steepped in water at 18 ºC for 72 hours and 48 hours, respectively, with regular turning of the grain.

The number of kernels with rootlet over one kernel length was recorded as germinated.

Seed germination was expressed as a percentage of the total number of tested seeds

(germination rate). Germination score was given from 1-5 points for samples from 0-100 % germination rate (Table 5.1).

Table 5.1. Germination score based on the number of kernels with rootlet over one kernel length

Number of grains with rootlet Germination Score

over one kernel length (%)

0-20 1

21-40 2

41-60 3

61-80 4

81-100 5

5.3. Results and discussions

5.3.1. Effect of PAW on DON Reduction and Germination Rate

5.3.1.1. Physicochemical Properties of PAW

The physicochemical properties including ORP, electrical conductivity, and pH values of PAW were measured to determine how PAW works. ORP represents the global oxidative capacity of the disinfectant, as well as the concentration level or strength of oxidizers. ORP took main part in the PAW based disinfection process (Liao, Chen, & Xiao,

2007; Tian, et al., 2015; Q. Zhang, et al., 2013). The ORP of PAW increased significantly from 224.7 mV to 463.8 mV after 20 min plasma generation (p < 0.05, Table 5.2). Electrical conductivity revels the level of active ions in PAW. the electrical conductivity of water rose up to 451.5 μS/cm after plasma formation (p< 0.05, Table 5.2). The data was consistent with other studies (Tian, et al., 2015; Q. Zhang, et al., 2013), these authors reported many active ions or electrons were formed in PAW. On the other hand, pH value in water decreased substantially from 7.6 to 2.8 during this period (p< 0.05). One study demonstrated that DON was unstable in acidic aqueous condition (pH=1-3) due to an unknown degraded product formed (Mishra, Dixit, Dwivedi, Pandey, & Das, 2014).

Overall, DON degradation in barley was also as a result of low pH value. In addition, air was used as feed gas to generate PAW, pH values dropped also hint the generation of acidic reactive species such as nitrites, nitrates and H2O2 (Oehmigen, Hähnel, Brandenburg,

Wilke, Weltmann, & Von Woedtke, 2010). These reactive species contribute to the

89 sterilization capacity of plasma active water. Some study suggested the efficacy of nitrite and H2O2 at acidic pH enhanced antimicrobial effects of PAW over neutral state (Traylor,

Pavlovich, Karim, Hait, Sakiyama, Clark, et al., 2011).

Table 5.2. Physicochemical properties (ORP, conductivity, and pH) of control (DI water) and PAW after 20 min treatment without samples

Treatment ORP (mV) Conductivity pH Temperature

(μS/cm) (℃)

Control 224.7± 8.5a 8.2± 0.1a 7.6± 0.2a 24.9± 0.9a

PAW 463.8± 3.3b 451.5± 3.0b 2.8± 0.02b 43.0± 1.4b

*Data are represented as means± standard deviations (n=3) and the same column followed by the different lowercase letter are significantly different (p< 0.05).

The physiochemical properties of PAW during treatments were also monitored.

The conductivity in PAW treated raw and germinating barely were reduced by 131.7 and

248.3 μS/cm, respectively (Table 5.3). ORP levels were significantly reduced by 136.4 mV and 141.0 mV in PAW treated germinating barley and raw barley, respectively, after first

5 min. Then decreasing rate became slower in the following 15 min. The data showed loss of accumulated ions and ROS which indicated that these antimicrobial agents had reacted with DON. Our results were in accordance with a previous study (Zahoranova et al. 2016).

On the other hand, pH values of PAW for both germinating barley and raw barley increased by 1.5 after 5 min. These data pointed out that the oxidative ability of PAW was the more efficient in the first 5 min of PAW treatment, then its oxidative ability declined during

90 period of 5- 20 min. Comparing these physiochemical properties in both barley samples, less radicals/ions can be reacted with raw barley because of the presence of the hull, which reduces the absorption of radicals and ions. The similar phenomenon was also observed in pH change.

Table 5.3. The values of (a) ORP, (b) electrical conductivity, and (c) pH value of control (PAW without samples), raw barley, and germinating barley, respectively, from 0-20 min plasma activation time.

ORP (mV)

Germinating

Treatment Control Raw barley barley

PAW- 0 462.0± 2.6aA 462.3± 4.5aA 465.7± 5.5aA

PAW- 5 461.5± 2.2aA 321.3± 5.0bB 329.3± 4.7bB

PAW- 10 461.0± 1.7aA 293.3± 5.5cB 295.7± 8.7cB

PAW- 15 459.8± 1.6aA 253.3± 4.2dB 262.0± 6.2dB

PAW- 20 459.0± 2.6aA 238.7± 4.2eB 243.3± 2.1eB

(a)

Conductivity (μS/cm)

Germinating

Treatment Control Raw barley barley

PAW- 0 453.0± 2.6aA 451.0± 3.1aA 453.3± 3.1aA

PAW- 5 452.8± 1.0aA 359.0± 2.7bB 236.0± 5.6bC

PAW- 10 452.7± 0.8aA 353.0± 2.7bB 215.3± 3.8cC

PAW- 15 451.8± 1.4aA 325.0± 4.6cB 208.3± 2.1dC

PAW- 20 450.3± 1.2aA 319.3± 2.5cB 205.0± 1.7dC

(b)

pH Value

Germinating

Treatment Control Raw barley barley

PAW- 0 2.8± 0.03aA 2.9± 0.03aA 2.8± 0.03aA

PAW- 5 2.9± 0.02aA 4.4± 0.07bB 4.3± 0.1bB

PAW- 10 2.9± 0.01aA 5.7± 0.05cB 4.8± 0.07cC

PAW- 15 2.9± 0.04aA 6.0± 0.04dB 4.9± 0.04dC

PAW- 20 2.9± 0.03aA 6.5± 0.04eB 5.0± 0.05eC

(c)

*Data are represented as means± standard deviations (n=3). Data in the same column followed by the different lowercase letter are significantly different (p < 0.05). Data in the same row followed by the different uppercase letter are significantly different (p < 0.05).

5.3.1.2. Effect of PAW Treatment Evaluation on DON Level

Table 5.4 shows DON levels as a function of treatment time from 0- 20 min with

PAW in raw and germinating barley. However, the DON level in both samples did not rely on time-dependent decline from 0-20 min (p> 0.05 by One-way Anova). DON level in raw barley first decreased to 77.5% after 5 min, the DON concentration had no change (74.2%)

92 from 5-20 min PAW treatments. DON degradation in germinating barley was consistently higher than raw barley (p< 0.05). For germinating barley, PAW treatment caused 34.6%-

38.3% DON degradation from 5- 20 min. The pairwise correlations of DON and electrical conductivity of PAW in raw and germinating barley samples were 0.9728 and 0.9996 (p<

0.05, Tables 5.3 & 5.4), respectively, from 0- 20 min. The data suggested that the close correlation between behavior of the electrical conductivity and the PAW disinfection. In addition, high correlation value was also observed between DON level and ORP value (pair correlations of raw barley and germinating barely are 0.9640 and 0.9556, respectively, p<

0.05). In terms of pH value, high correlation value was obtained between DON level and pH (raw barley and germinating barley are 0.9063 and 0.9725, respectively, p<0.05) hint the high correlations between them.

As stated in previous studies, PAW contains effective agents for microbial inactivation. PAW disinfection is more effective at first several minutes (Filatova,

Azharonok, Lushkevich, Zhukovsky, Gadzhieva, Spasic, et al., 2013; Traylor, et al., 2011).

The PAW treatment yielded a drop of 5.6 logs CFU/g on E. coli in saline solutions in the first 15 min, then the microbial concentration decreased by 2.4 logs CFU/g in the following

15 min. However, the sterilization efficacy was significantly declined after 30 min (Traylor, et al., 2011). PAW treatment on S. aureus was also investigated, approximate 6 logs CFU/g reduction was observed after 10 min PAW treatment. However, no significant reduction of microbe was detected in the next 30 min (Filatova, et al., 2013). The present study indicated that PAW could degrade DON in barley samples effectively. The advisory level of DON was set below 1 ppm for commodities intended for use in human food and animal feed

(FDA, 2010). Don in germinating barley was underneath 1 ppm after 5 min-PAW treatment

93 in the current study (0.89-0.95 ppm DON residue), the technology thereby can be used to reduce the potential risk of illness in barley samples.

Plasma jet generates both long and short-live RS. Long-live RS such as hydrogen

- peroxide (H2O2), ozone (O3), and nitrate ion (NO3 ) can be remained in PAW up to several hours, and thus, probably play a major role in the degradation process during PAW

- - treatment. Short-live RS such as hydroxyl radical (OH ), superoxide (O2 ), and singlet oxygen will disappear when removing nonthermal plasma (Arjunan, Friedman, Fridman,

& Clyne, 2011). Half-life time of OH- and singlet oxygen are as short as 10-9 and 10-6 s respectively (Pryor, 1986). Therefore, short-live RS took limited effects during PAW treatment on DON detoxication in barley samples. More exposure of DON in germinating barley under the RS in PAW, resulting in higher DON degradation. In contrast, lower DON degradation achieved for raw barley because of barley hull, which prevents DON hidden inside from being exposed to RS.

Table 5.4. Effect of PAW treatment on the DON level (%) in raw and germinating barley samples.

DON concentration

Treatment Raw barley Germinating barley

PAW- 5 77.5%± 3.4%aA 65.4%± 2.7%aB

PAW- 10 76.1%± 4.2%aA 63.2%± 3.5%aB

PAW- 15 75.4%± 2.4%aA 62.3%± 4.1%aB

PAW- 20 74.2%± 3.1%aA 61.7%± 3.6%aB

5.3.1.3. Evaluation of Germination Rate after PAW Treatment

Germination was an important indicator of seed quality and biological structure during PAW treatment. Operating parameters including stage of the barley samples and treatment time were investigated on seed germination. The germination scores of the barley sample after the PAW treatment were in comparison with untreated samples (PAW-0). The data showed that the germination rate of the raw and germinating barely samples was remained unchangeable when treatment time was below 10 and 15 min, respectively (Table

5.5). Then the germination rate of both barley samples was reduced significantly as a function of PAW treatment time. Although no study investigated PAW on the germination rate of barley kernel, some research evaluated the impact of direct plasma treatment on the germination rate of wheat seeds. The study indicated short plasma treatment time has no effects on the germination rate of wheat seeds. Plasma treatment did not cause the change of germination of wheat seeds within 5 min (Los, Ziuzina, Akkermans, Boehm, Cullen,

Van Impe, et al., 2018). The findings suggested that barley samples could tolerate certain oxidative stress because of antioxidant enzymes in their roots. However, prolonged plasma treatment inhibited the seeds germination significantly (Maksimovic, Zhang, Zeng,

Zivanovic, Shabala, Zhou, et al., 2013). Similarly, germination rate of wheat seeds decreased significantly after 20 min plasma treatment (Filatova, et al., 2013), which was owed to the fact that RS entered the seed through porous seed hull, then reacted with

95 cellular components, and thereby affecting metabolism of the seed (Sera, Spatenka, Sery,

Vrchotova, & Hruskova, 2010).

Table 5.5. Raw and germinating barley samples germination rate score as a function of PAW treatment time.

PAW treatment Germination score

Raw barley Germinating barley

PAW- 0 5 5

PAW- 5 5 5

PAW- 10 5 5

PAW- 15 4 5

PAW- 20 4 3

*Values are presented as means (n=2).

5.3.2. Effects of IPL on DON Reduction and Germination Rate

5.3.2.1. Evaluation of DON Level after IPL Treatment

Surface temperature of both barley samples increased with an increasing number of pulses up to 360 pulses (Figure 5.1). In this study, the highest temperature was less than

70 °C for both germinating barley and raw barley samples. The temperature of raw barley samples was consistently higher than germinating barley samples. This was owed to higher moisture content of germinating barley than raw barley (Ellis & Roberts, 1980). On the other hand, since DON was highly resistant to heating treatment up to 120 °C, the photothermal effect was not a main factor for DON level degradation. (Kabak, 2009; Numanoglu,

Gokmen, Uygun, & Koksel, 2012). UV and visible light spectrum may play main roles in

96 the degradation of the DON level. A study stated 90.5%, 99.4%, and 95.3% of the DON levels could be detoxified by that short (254 nm), long (362 nm) UV waves, and fluorescent light, respectively, after 30 min exposure in wheat grains (Ataila, Hassanein, El-Beih, &

Youssef, 2004). Chemical mechanism from UV and visible light resulted in DON degradation. A fragmentation of the mycotoxin molecule was caused by IPL (Moreau,

Lescure, Agoulon, Svinareff, Orange, & Feuilloley, 2013).

The curves of DON in raw and germinating barley samples as a function of IPL pulses fit for pulse-dependent decline of DON level during IPL treatment (p< 0.05 by One- way Anova) (Table 5.6). DON in raw barley was substantially reduced to 69.1%-95.8% after 45-180 pulses of IPL treatment. DON degradation in Germinating barley was consistently higher than raw barley. 90-360 pulses-IPL treatment contributed to 26.1%-

71.7% DON degradation in germinating barley. DON in germinating barley after 180 pulses of IPL (<0.94 ppm DON residue) could be used in this study based on FDA standard

(USFDA 2000).

Similar as PAW on raw barley, low DON degradation rate may be due to the presence of barley hull for raw barley. The hull can serve as a shield to keep mycotoxin from the IPL beams. Resulting in only partial degradation. Pulsed light is a more effective technique to inactivate DON in a solution when comparing with food matrix. Degrading

92.7% using only 8 pulses- pulsed light treatment (1 J cm-2) (Moreau et al. 2013). Low

DON degradation is owed to the fact that DON could reside in crevices or in irregularities of the barley sample surface or may penetrate under the product epidermis (Moreau,

Lescure, Agoulon, Svinareff, Orange, & Feuilloley, 2013). The data suggested that the IPL

97 treatments on DON in barley samples were less efficient than media, significantly higher

IPL dose was thereby required in the current study.

) 60 ℃

40 Temperature ( Temperature

30 Raw barley Germinating barley 20 0 45 90 135 180 225 270 315 360 405 Number of pulses

Figure 5.1. Surface temperature of raw and germinating barley during IPL treatment

*Raw and germinating barley samples were illuminated with IPL directly on a petri-dish.

Data are expressed as means± standard deviations (n=3).

Table 5.6. DON concentration of raw and germinating barley during IPL treatment.

Treatment DON concentration

IPL-R-45 pulses 95.8%± 3.8%a

IPL-R-90 pulses 91.8%± 4.0%a

IPL-R-135 pulses 75.6%± 5.6%b

IPL-R-180 pulses 69.1%± 5.1%b

IPL-G-90 pulses 73.9%± 5.3%b

IPL-G-180 pulses 64.5%± 3.1%b

IPL-G-270 pulses 39.7%± 10.5%d

IPL-G-360 pulses 28.3%± 2.9%d

*The actual light intensity received on the surface of samples is 0.528 J/cm2/pulse at the distance of 8 cm. Data are represented as means± standard deviations (n=3). Values with different lowercase letters are significantly different (p< 0.05).

5.3.2.2. Evaluation of Germination Rate after IPL Treatment

The germination rates declined with increasing number of pulses for both germinating barley and raw barley. The barely germination of barley before and after IPL treatment are shown in Table 5.7. The results indicated that IPL had no significant or slight impacts on the germination rate of the raw and germinating barely within 45 and 90 pulses, respectively. The germination rate of both barley samples was affected heavily substantially. Close correlation was found between the surface temperature and germination for both barley samples. The surface temperatures beyond ~50 ℃ affected germination rate heavily (Figure 5.1 & Table 5.7) because of the overheating combined with photo-oxidation (Fine et al. 2004). Extended treatment time may potentially induce over oxidation to food samples, a phenomenon was also observed before, the germination of treated alfalfa seeds after 225 pulses was reduced to 34.1 % (Sharma & Demirci, 2003).

On the other hand, the temperature increased more slowly for germinating barley than raw barely because of higher moisture content in germinating barley (Ellis & Roberts, 1980).

Overall, the results showed that substantial impact on germination rate was observed for both barley samples after receiving high IPL doses.

Table 5.7. Raw and germinating barley samples germination rate score as a function of PAW treatment time.

Treatment Germination score

IPL-R-0 pulses 5

IPL-R-45 pulses 5

IPL-R-90 pulses 4

IPL-R-135 pulses 3

IPL-R-180 pulses 2

IPL-G-0 pulses 5

IPL-G-90 pulses 5

IPL-G-180 pulses 3

IPL-G-270 pulses 2

IPL-G-360 pulses 1

*Values are represented as means (n=2)

100

5.4. Conclusion

This study concluded that (1) barley germination could be affected by both PAW and IPL treatments to different extent. To achieve a same level of DON degradation

(36.5%), PAW induced significantly less germination change than that of IPL. High germination rate could be maintained (80-100%) in the first 15 min-PAW treatment; (2)

ORP, electrical conductivity, and pH in PAW had close correlation with DON degradation.

However, before commercial application, more quality measurements such as fatty acid value and the amino acid content of treated barley need to be investigated. Higher DON concentration in barely samples also needs to be evaluated. Finally, the economic benefits and safety of implementing IPL and PAW technology to remove DON in barley kernels need to be tested.

101

Chapter 6. Decontamination of wheat kernels and wheat flour using catalytic microwave system

6.1. Introduction

Microwave may cause sample overheating when using microwave heating for pasteurization of food products, care must be taken to make sure the homogeneity of the thermal process within the product and that the target suitable temperature (<60 °C) is maintained for a sufficient period of time to provide a safe product. A solution to these problems would be to agitate the particles to increase the uniform exposure to microwave irradiation, for example, using a cyclone or a mixing fluidized reactor, as well as adopting a temperature probe to monitor the temperature of samples during microwave treatments.

The objectives of this chapter are: (1) to evaluate the low and high temperature-microwave disinfection on different microbes in wheat kernel and wheat flour; (2) to investigate and compare the catalytic microwave treatment and microwave treatment.

6.2. Materials and methods

6.2.1. Sample preparation and inoculation

The bacterial strains utilized in this study were: Enterococcus faecium (The non- pathogenic surrogate of Salmonella spp.), Bacillus cereus, and Escherichia coli. Each microorganism will be revived from frozen culture (-80 °C) onto tryptic soy agar (Neogen,

Lansing, MI) supplemented with 0.6% (wt/vol) yeast extract (TSAYE; Sigma-Aldrich, St.

Louis, MO) and stored at 4 °C.

Seed samples were inoculated with known amount of Salmonella enterica subsp. enterica serovar Enterococcus faecium strain NRRL B-2354, Bacillus cereus strain ATCC

102

14579, or Escherichia coli. The inoculated samples were then homogenized and diluted in

10-fold serial dilution to acquire samples with bacterial concentration ranging from 0 to

108 CFU/g. Uninoculated samples were used as negative control. Each experiment will be performed in triplicate.

To enumerate wheat kernels, 5 g of samples were transferred to 750 ml (24 oz)

Whirl-Pak filtered blender bags (Nasco, Fort Atkinson, WI) with 45 ml of 0.1% peptone and stomached for 3 minutes at 260 rpm. One ml was transferred to 9 ml of 0.1% peptone, serially diluted and plated out to 10-8 in duplicate onto TSAYE. Plates will be incubated at

37°C for 48-72 h to allow for injured cells to resuscitate. The colony unit will be expressed in CFU/g concentrations.

6.2.2. Microwave and catalytic microwave treatments

A domestic household microwave system (2450 MHz) with output power of 1000

W was adopted. Inoculated wheat kernels were placed in porcelain crucible under the different parameters of microwave treatment with certain residence time (i.e. 30 or 60s).

The weight ratio of sample to TiO2 coated glass bead was ~1:1 (10g each of the samples and the TiO2 coated beads), then mixing samples and TiO2 coated beads uniformly in porcelain crucible. The detailed process after loading the samples in the microwave system are as follows, microwave was turned on for until samples reach the desirable temperature, turn off the microwave, cool down temperature of samples to around 53-55 °C, which took about 5-10 s. Then turn on the microwave again for another 5 s or so to reach about 60 °C sample temperature again and repeating the process until reaching desirable treatment time.

The temperature was monitored by thermocouple. After microwave treatment, the samples

103 were separated from TiO2 coated beads using a 2200 µm sieve. The schematic diagram of scale-up continuous catalytic microwave system is shown in Figure 6.1. The seed materials were fed in volumetric feeder, conveyed to cyclone reactor through air. Then seed samples were substantially treated under the microwave and photocatalysis. EDL was used to enhance the photocatalysis during the process.

Figure 6.1. A schematic diagram of catalytic microwave/microwave system

6.3. Results and discussions

6.3.1. Disinfection of wheat/wheat flour using microwave

Table 6.1 shows the microbial inactivation in C. sakazakii, E. faecium, and natural microorganism (mesophilic aerobic bacteria) in both wheat and wheat flour. For wheat kernel, higher log reduction could be achieved for both C. sakazakii and E. faecium than natural microorganism. The probable reasons might be due to ununiform distribution natural microorganisms. Microbes hidden inside kernel hull or crevice was more difficult to be eliminated (Villa-Rojas, Tang, Wang, Gao, Kang, Mah, et al., 2013). In terms of

104 wheat flour, lower temperature and relatively shorter treatment time microwave contributed to higher microbial inactivation. Extended treatment time resulted in obvious burning smell or color change (data not shown). Therefore, maintaining treatment temperature under the 60 °C could be considered as a better strategy to reach higher microwave disinfection while maintaining the sample quality. Comparing among different microorganisms, B. cereus spore inactivation was significantly higher than other bacteria.

Spore was equipped with thicker cell wall comparing with non-spore forming bacteria

(Foster, Ditta, Varghese, & Steele, 2011), thereby the spore was difficult to be inactivated.

Microwave disinfection on C. sakazakii was significantly higher than that of E. faecium.

The reasons were mainly due to the cell envelope and cell wall difference between gram- negative and gram-positive bacteria. Thicken cell walls and increased amounts of peptidoglycan were found in gram-positive bacteria than those in gram-negative bacteria(Williams, Eichstadt, Kokjohn, & Martin, 2007). On the other hand, the cell envelope of gram positive bacteria is more resistant in responses to antibiotics, heat, and

UV radiation (Mai-Prochnow, Clauson, Hong, & Murphy, 2016). Bacterial spores are typically 5-50 times more resistant than vegetative cells in the respects of UV radiation, heat, peroxides, etc (Setlow, 2001). Therefore, significantly lower microbial inactivation was achieved for bacterial spores in the study.

Table 6.1. Microwave disinfection as a function of different treatment conditions

acteria and treatment condition Log reduction (CFU/g)

Wheat kernel

105

C. sakazakii (60 °C microwave, 60 sec) 2.83 logs

E. faecium (60 °C microwave, 60 sec) 2.46 logs

Natural microorganism (60 °C microwave, 60 sec) 2.35 logs

Wheat flour

C. sakazakii (60 °C microwave, 30 sec) 1.84 logs

C. sakazakii (100 °C microwave, 15 sec) 1.13 logs

E. faecium (60 °C microwave, 30 sec) 1.32 logs

E. faecium (100 °C microwave, 15 sec) 0.75 logs

B. cereus spore (60 °C microwave, 30 sec) 1.27 logs

B. cereus spore (100 °C microwave, 15 sec) 0.88 logs

*All experiments were repeated in duplicate.

6.3.2. The synergistic effect of TiO2 and microwave on wheat kernel disinfection

Previous chapters indicated a prototype IPL technology that incorporates TiO2 as a photocatalyst into the process for fast and energy efficient disinfection was developed. A

4.21 log CFU/g reduction of C. sakazakii was achieved for wheat kernel under individual

IPL treatment for 120s. The C. sakazakii inactivation was increased to 5.00 log CFU/g when TiO2 photocatalyst was used. In the current study, catalytic microwave technique was evaluated for disinfection of wheat kernels. The data shows 2.2 log CFU/g reduction could be achieved after 60 s microwave treatment (Figure 6.2). When TiO2 photocatalysts combined with an electroless UV source (UV can be activated under the microwave) were used during microwave treatment, one additional log10 reduction was achieved. The temperature profile during the treatments of microwave combined with TiO2 was shown in

Figure 6.3. We believe that there is a great potential to enhance the microbicidal effect of the treatment by improving the TiO2 activity through physical and chemical modification and use of UV light and optimizing the microwave/ grain/TiO2 ratios. It is also important

106 to develop a continuous process that will not only provide a better way for us to conduct a more meaningful evaluation of the technology but also present the potential for practical application of the technology. The synergistic effect of UV and microwave during treatment might take an important effect on microbial inactivation (Law & Dowling, 2018).

2 population (CFU/g) population 1

C. sakazakii C.sakazakii Initial concentration 60 s Microwave 60 s Microwave+TiO2 Treatment conditions

Figure 6.2. Effect of C. sakazakii population inoculated in wheat kernels as a function of different microwave treatments

70 60

C) 50 ° 40 30

20 Temperature ( Temperature 10 0 0 10 20 30 40 50 60 70 treatment time (s)

107

Figure 6.3. Temperature of wheat kernel during microwave treatment combined with TiO2

6.3.3. Photocatalyst modification

Degussa (Evonik) P25, Aeroxide TiO2 P25 and S-doped TiO2 were developed to evaluate the enhanced photocatalysis on microbial inactivation in wheat kernels. Figure

6.4.(a) shows significantly higher log10 reduction was observed under IPL+P25 titanium dioxide than IPL+TiO2. It is known that P25 titanium dioxide photocatalyst that was widely used in many photocatalysis reactions because of its high purity (>99.5%), appropriate particle size (~20 nm), and appropriate ratio of anatase (~73%) and rutile (~18%) crystallities (Jensen, Joensen, Jørgensen, Pedersen, & Søgaard, 2004). It is well known that rutile, with a direct bandgap, can catch photons to generate photo-excited electrons and positive holes. While anatase, with an indirect bandgap, exhibits a longer lifetime of electrons and holes than direct band gap rutile. On the other hand, the lowest recombination rate of charge carriers in anatase TiO2 as a result of the lightest effective mass enable the fastest migration of electrons and holes from interior to surface of anatase TiO2 (J. Zhang,

Zhou, Liu, & Yu, 2014). S-doped TiO2 did not cause increased IPL disinfection when comparing with individual IPL treatment (data not shown). The probable reasons may be due to the unstable state of under the S-doped TiO2 IPL. In addition, the data indicated S- doped TiO2 induced significantly higher microbial inactivation under the microwave than

TiO2 alone. TiO2 has relatively high band-gap energy (3.2 eV). Two forms of titanium dioxide rutile and anatase only can be activated under UV light (<390 nm). Therefore, doping TiO2 with transition metals or anions such as Ru, Cr, Pt, S, N, and C could reduce band-gap energy of TiO2 photocatalysts (i.e. the energy to activate S-doped TiO2 is 1.7

108 eV). (Dong, Guo, Wang, Li, & Wu, 2011; Jaiswal, Bharambe, Patel, Dashora, Kothari, &

Miotello, 2015; Teruhisa Ohno, Miyako Akiyoshi, Tsutomu Umebayashi, Keisuke Asai,

Takahiro Mitsui, & Michio Matsumura, 2004). Moreover, dopant can contribute to higher quantum efficiency of TiO2 photocatalytic reactions, which prevent the generated electron- hole pairs from recombining (J. Liu, Yu, Liu, Liu, Shang, Zhang, et al., 2014).

109

3.5

3.0

2.5

2.0

1.5

1.0 Log Log reduction (CFU/g) 0.5

0.0 IPL+TiO2 IPL+P25 TiO2 (a)

4.0

3.5

3.0

2.5

2.0

1.5

Log Log reduction (CFU/g) 1.0

0.5

0.0 Microwave Microwave+TiO2 Microwave+P25 TiO2 Microwave+S-doped TiO2 (b)

Figure 6.4. Log10 reduction of C. sakazakii in wheat kernels as a function of different catalytic treatments

*(a) IPL treatment; (b) microwave treatment.

110

6.4. Conclusions

Feasible bacterial inactivation and high efficiency of microwave were observed based on our preliminary data presented above. This study aims to develop a continuous catalytic microwave system for sterilization of wheat grains, which has not been reported in the literature. Second, the use of photocatalysts and low temperature, which may present new killing mechanisms, and expected to significantly improve the treatment effectiveness and achieve higher inactivation. Third, coating photocatalysts onto substrates enable easy recovery and reuse of the catalysts. The proposed technology can be adapted to decontamination or treatment of other cereal grains and particulate foods. Therefore, a broader impact on the safety and production of foods and agricultural products expected.

For example, the proposed technology could be used to decontaminate other grains and seeds/powder production, reduce disease for crop reproduction, degrade mycotoxins in malts, prolong shelf life of grain and nuts, etc.

The treatment can be fitted into the step after wheat grain tempering since it is an important step in flour milling. The moistened surface may have positive effect on microwave treatment as spores may be turned to vegetative state and the added moisture on the surface improve absorption of microwave, which is expected to enhance inactivation of microbes on the surface and avoid excess internal heating. Therefore, in the future study, research will be carried out to investigate the microwave treatment of wheat grains without and with tempering.

111

Chapter 7. Summary and future work

7.1. Summary of the dissertation

Pathogen outbreaks in particulate foods such as infant formula, wheat flour, etc.

However, an effective and solid pasteurization method to eliminate the microbes is lacking.

This dissertation research mainly focused on system development, microbiological analysis, and quality analysis. The main goal of this dissertation research was to develop an effective and pilot scale IPL or other nonthermal technologies to inactivate pathogens in particulate foods while maintaining or reducing quality loss.

Chapters 2 & 3 introduced a continuous IPL as an alternative method to inactivate

C. sakazakii in particulate foods. Processing and environmental factors such as energy fluence, treatment time, humidity, temperature, particle size, IPL frequency, voltage, feed rate, sample quality change before and after IPL treatment were examined. This study revealed that an IPL inactivation effect of C. sakazakii on NFDM was significantly affected by these factors at some extent. Then optimized treatment conditions for different samples were obtained. IPL disinfections on non-spore forming bacteria and spore were substantial examined. Finally, IPL sterilization effects were significantly enhanced under the photocatalysis. The IPL system structure and facilities were modified for multiple generations to meet the requirements of energy save, high efficiency, and long duration.

This novel prototype system eliminates the disadvantages of partial decontamination of commonly used IPL treatment methods. The choice of this innovative prototype IPL system provides a feasible alternative to scale-up this technology.

Chapters 4 & 5 investigated two forms of nonthermal plasma technologies to eliminate pathogens in particulate foods: CAP and PAW. For the former technology, the

112 application of a fluidized reaction system was proved as a useful platform especially at high flow rate-feeding gas. Higher microbes could be thereby eliminated. This new strategy could potentially be used to treat the entire surface of samples during CAP treatments. No noticeable or slight changes occurred in color, amino acid composition, extent of crystallinity, and phenolic content after 120-s CAP treatment. As to the PAW method,

PAW exhibited a potential for reducing DON in germinating barley. To achieve a similar

DON degradation (i.e. 36.5%) for germinating barley, germination rate of barely was affected less by PAW than IPL treatment. RS and low pH took main effects on the antimicrobial activity of PAW.

In Chapter 6, the catalytic microwave treatment was used for pasteurization of wheat kernels and other seeds before they were milled. The process will combine low temperature microwave heating with titanium dioxide (TiO2)- a photocatalyst. The final goal of the catalytic microwave assisted decontamination process is expected to effectively reduce microbial counts by 3-5 logs in seeds.

7.2. Future work

More research needs to be conducted to understand the microbicidal effects of key process variables during catalytic microwave treatment. The critical processing parameters of catalytic microwave decontamination (cMAD) are comprise of (1) power supply; (2) average treatment time; (3) sample loading; (4) sample flow rate; (5) quantity of photocatalysts (i.e. TiO2); (6) sample preparations including sample tempering, moisture content, and water activity adjustments, etc.; (7) relative humidity of environment, and (8) treatment temperature.

113

In addition, the characters of TiO2 nanoparticles such as crystallinity, size, purity, etc., are other important factors that potentially affect the cMAD disinfection/energy consumption. Because samples are consistently moving in the fluidized chamber, the contacting surface and contacting time are in need of increasing during the whole processing to maximum utilize the synergistic effects between microwave and photocatalyst. Two choices to immobilize TiO2 will be evaluated and compared. The first choice is to immobilize TiO2 on the internal surface of cyclone reactor; the second option is to immobilize TiO2 on substrates (i.e. polymer beads or stainless beads), the medium selected needs to have similar flowing properties (density, size, and flow rate) as wheat kernel to ensure homogeneous movement and mixing during cMAD process.

On the other hand, TiO2 has relatively high band-gap energy. Two forms of titanium dioxide rutile and anatase only can be activated under UV light (<390 nm). Therefore, developing a photocatalysts that can be activated under both UV and visible light is highly sought after to utilize light energy more effectively in photocatalytic reactions. In this case, some study indicated doping TiO2 with transition metals or anions such as Ru, Cr, Pt, S,

N, and C could reduce band-gap energy of TiO2 photocatalysts (Anpo, 1997; Anpo &

Takeuchi, 2003). For example, Ohno et al. reported relatively high photocatalytic activity was observed under visible light (>500 nm) using S-doped TiO2 photocatalysts (Teruhisa

Ohno, Miyako Akiyoshi, Tsutomu Umebayashi, Keisuke Asai, Takahiro Mitsui, & Michio

Matsumura, 2004). After that, a study needs to be conducted to figure out the interactions among microwave, wavelength, and photocatalysts.

114

Bibliography

Abramson, D., House, J. D., & Nyachoti, C. M. (2005). Reduction of deoxynivalenol in barley by treatment with aqueous sodium carbonate and heat. J Mycopathologia, 160(4), 297-301.

Aguilera, J., del Valle, J., & Karel, M. (1995). Caking phenomena in amorphous food powders. J Trends in Food Science Technology, 6(5), 149-155.

Albert, I., & Mafart, P. (2005). A modified Weibull model for bacterial inactivation. J International journal of food microbiology, 100(1-3), 197-211.

Angulo, F. J., Cahill, S. M., Wachsmuth, I. K., Costarrica, M. d. L., & Embarek, P. K. B. (2008). Powdered infant formula as a source of Salmonella infection in infants. J Clinical infectious diseases, 46(2), 268-273.

Anpo, M. (1997). Photocatalysis on titanium oxide catalysts: approaches in achieving highly efficient reactions and realizing the use of visible light. Catalysis Surveys from Asia, 1(2), 169-179.

Anpo, M., & Takeuchi, M. (2003). The design and development of highly reactive titanium oxide photocatalysts operating under visible light irradiation. Journal of catalysis, 216(1-2), 505-516.

Archer, J., Jervis, E. T., Bird, J., & Gaze, J. E. (1998). Heat resistance of Salmonella weltevreden in low-moisture environments. J Journal of food protection, 61(8), 969-973.

Arjunan, K. P., Friedman, G., Fridman, A., & Clyne, A. M. (2011). Non-thermal dielectric barrier discharge plasma induces angiogenesis through reactive oxygen species. J Journal of the Royal Society Interface, 9(66), 147-157.

Aron Maftei, N., Ramos‐Villarroel, A. Y., Nicolau, A. I., Martín‐Belloso, O., & Soliva‐ Fortuny, R. (2014). Pulsed light inactivation of naturally occurring moulds on wheat grain. J Journal of the Science of Food Agriculture, 94(4), 721-726.

Artíguez, M. L., & de Marañón, I. M. (2014). Process parameters affecting Listeria innocua inactivation by pulsed light. J Food Bioprocess Technology, 7(9), 2759-2765.

Asakura, H., Morita‐Ishihara, T., Yamamoto, S., & Igimi, S. (2007). Genetic characterization of thermal tolerance in Enterobacter sakazakii. J Microbiology immunology, 51(7), 671-677.

115

Ataila, M., Hassanein, N., El-Beih, A., & Youssef, Y. (2004). Effect of fluorescent and UV light on mycotoxin production under different relative humidities in wheat grains. J ACTA Pharmaceutica Sciencia, 46(3).

Beuchat, L. R., Kim, H., Gurtler, J. B., Lin, L.-C., Ryu, J.-H., & Richards, G. M. (2009). Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation. J International journal of food microbiology, 136(2), 204-213.

Beuchat, L. R., Komitopoulou, E., Beckers, H., Betts, R. P., Bourdichon, F., Fanning, S., Joosten, H. M., & Ter Kuile, B. H. (2013). Low–water activity foods: increased concern as vehicles of foodborne pathogens. J Journal of food protection, 76(1), 150-172.

Bhat, N. A., Wani, I. A., Hamdani, A. M., Gani, A., & Masoodi, F. (2016). Physicochemical properties of whole wheat flour as affected by gamma irradiation. J LWT-Food Science Technology, 71, 175-183.

Bhavya, M., & Umesh Hebbar, H. (2017). Pulsed light processing of foods for microbial safety. J Food Quality Safety, 1(3), 187-202.

Bialka, K. L., Demirci, A., & Puri, V. M. (2008). Modeling the inactivation of Escherichia coli O157: H7 and Salmonella enterica on raspberries and strawberries resulting from exposure to ozone or pulsed UV-light. J Journal of Food Engineering, 85(3), 444-449.

Bigelow, W. (1921). The logarithmic nature of thermal death time curves. J The Journal of Infectious Diseases, 528-536.

Blazquez, E., Rodriguez, C., Rodenas, J., de Rozas, A. P., Segales, J., Pujols, J., & Polo, J. (2017). Ultraviolet (UV-C) inactivation of Enterococcus faecium, Salmonella choleraesuis and Salmonella typhimurium in porcine plasma. J PloS one, 12(4), e0175289.

Borge, G. I. A., Skeie, M., Sørhaug, T., Langsrud, T., & Granum, P. E. (2001). Growth and toxin profiles of Bacillus cereus isolated from different food sources. J International journal of food microbiology, 69(3), 237-246.

Bosshard, F., Bucheli, M., Meur, Y., & Egli, T. (2010). The respiratory chain is the cell's Achilles' heel during UVA inactivation in Escherichia coli. J Microbiology, 156(7), 2006-2015.

Bottone, E. J. (2010). Bacillus cereus, a volatile human pathogen. J Clinical microbiology reviews, 23(2), 382-398.

Bradley, D., McNeil, B., Laffey, J. G., & Rowan, N. J. (2012). Studies on the pathogenesis and survival of different culture forms of Listeria monocytogenes to pulsed UV-

116

light irradiation after exposure to mild-food processing stresses. J Food microbiology, 30(2), 330-339.

Bretz, M., Beyer, M., Cramer, B., Knecht, A., & Humpf, H.-U. (2006). Thermal degradation of the Fusarium mycotoxin deoxynivalenol. J Journal of agricultural food chemistry, 54(17), 6445-6451.

Bu, P., Narayanan, S., Dalrymple, D., Cheng, X., & Serajuddin, A. T. (2016). Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant. Eur J Pharm Sci, 91, 162-171.

Burlica, R., Grim, R., Shih, K. Y., Balkwill, D., & Locke, B. (2010). Bacteria inactivation using low power pulsed gliding arc discharges with water spray. J Plasma Processes Polymers, 7(8), 640-649.

Calligaris, S., Manzocco, L., Anese, M., & Nicoli, M. C. (2004). Effect of heat-treatment on the antioxidant and pro-oxidant activity of milk. J International Dairy Journal, 14(5), 421-427.

CDC. (2010). Salmonella Montevideo infections associated with salami products made with contaminated imported black and red pepper—United States. https://www.cdc.gov/mmwr/preview/mmwrhtml/mm5950a3.html.

CDC. (2016). Multistate Outbreak of Salmonella Infections Linked to Alfalfa Sprouts from One Contaminated Seed Lot (Final Update)https://www.cdc.gov/salmonella/muenchen-02-16/index.html.

Cerf, O. (1977). A review tailing of survival curves of bacterial spores. J Journal of Applied Bacteriology, 42(1), 1-19.

CFSAN-FDA. (2000). Ultraviolet light. In Kinetics of Microbial Inactivation for Alternative Food Processing Technologies. Atlanta, GA: Center for Food Safety and Applied Nutrition – Food and Drug Administration. Available at: http://www.cfsan.fda.gov/~comm/ift-uv.html. Accessed 19 April 2006.

Cha, M., Lee, S., Kim, K., & Chung, S. (2005). Soot suppression by nonthermal plasma in coflow jet diffusion flames using a dielectric barrier discharge. J Combustion flame, 141(4), 438-447.

Chan, H. W.-S. (1977). Photo-sensitized oxidation of unsaturated fatty acid methyl esters. The identification of different pathways. J Journal of the American Oil Chemists’ Society, 54(3), 100-104.

Chávez-Servín, J. L., Andrade-Montemayor, H. M., Vázquez, C. V., Barreyro, A. A., García-Gasca, T., Martínez, R. A. F., Ramírez, A. M. O., & de la Torre-Carbot, K. (2018). Effects of feeding system, heat treatment and season on phenolic

117

compounds and antioxidant capacity in goat milk, whey and cheese. J Small Ruminant Research, 160, 54-58.

Cheigh, C.-I., Park, M.-H., Chung, M.-S., Shin, J.-K., & Park, Y.-S. (2012). Comparison of intense pulsed light-and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157: H7. J Food Control, 25(2), 654-659.

Chen, D., Chen, P., Cheng, Y., Peng, P., Liu, J., Ma, Y., Liu, Y., & Ruan, R. (2019). Deoxynivalenol decontamination in raw and germinating barley treated by plasma- activated water and intense pulsed light. J Food Bioprocess Technology, 12(2), 246-254.

Chen, D., Cheng, Y., Peng, P., Liu, J., Wang, Y., Ma, Y., Anderson, E., Chen, C., Chen, P., & Ruan, R. (2019). Effects of intense pulsed light on Cronobacter sakazakii and Salmonella surrogate Enterococcus faecium inoculated in different powdered foods. J Food chemistry, 296, 23-28.

Chen, D., Peng, P., Zhou, N., Cheng, Y., Min, M., Ma, Y., Mao, Q., Chen, P., Chen, C., & Ruan, R. (2019). Evaluation of Cronobacter sakazakii inactivation and physicochemical property changes of non-fat dry milk powder by cold atmospheric plasma. J Food chemistry, 290, 270-276.

Chen, D., Wiertzema, J., Peng, P., Cheng, Y., Liu, J., Mao, Q., Ma, Y., Anderson, E., Chen, P., & Baumler, D. J. (2018). Effects of intense pulsed light on Cronobacter sakazakii inoculated in non-fat dry milk. J Journal of Food Engineering, 238, 178- 187.

Choi, M. S., Cheigh, C. I., Jeong, E. A., Shin, J. K., Park, J. Y., Song, K. B., Park, J. H., Kwon, K. S., & Chung, M. S. (2009). Inactivation of Enterobacter sakazakii inoculated on formulated infant foods by intense pulsed light treatment. J Food Science Biotechnology, 18(6), 1537-1540.

Církva, V., & Relich, S. (2011). Microwave photochemistry and photocatalysis. Part 1: Principles and overview. Current Organic Chemistry, 15(2), 248-264.

Cordier, J.-L. (2014). Methodological and sampling challenges to testing spices and low- water activity food for the presence of foodborne pathogens. In The Microbiological Safety of Low Water Activity Foods and Spices, (pp. 367-386): Springer.

Cruz-Gracida, M., Siles-Alvarado, S., Méndez-Lagunas, L., Sandoval-Torres, S., Rodríguez-Ramírez, J., & Barriada-Bernal, G. (2019). Quantitative analysis of fatty acids in Prosopis laevigata flour. J Grasas y Aceite, 70(3), 321.

Daneshvar, N., Salari, D., Niaei, A., Rasoulifard, M., & Khataee, A. (2005). Immobilization of TiO2 nanopowder on glass beads for the photocatalytic

118

decolorization of an azo dye CI Direct Red 23. J Journal of Environmental Science Health, Part A, 40(8), 1605-1617.

Deng, X., Nikiforov, A. Y., Vanraes, P., & Leys, C. (2013). Direct current plasma jet at atmospheric pressure operating in nitrogen and air. J Journal of Applied Physics, 113(2), 023305.

Dev, S., Raghavan, G., & Gariepy, Y. (2008). Dielectric properties of egg components and microwave heating for in-shell pasteurization of eggs. Journal of food engineering, 86(2), 207-214.

Dong, F., Guo, S., Wang, H., Li, X., & Wu, Z. (2011). Enhancement of the visible light photocatalytic activity of C-doped TiO2 nanomaterials prepared by a green synthetic approach. J The Journal of Physical Chemistry C, 115(27), 13285-13292.

Dunn, J. (1996). Pulsed light and pulsed electric field for foods and eggs. 75(9), 1133-1136.

Dunn, J. E., Clark, R. W., Asmus, J. F., Pearlman, J. S., Boyer, K., Painchaud, F., & Hofmann, G. A. (1989). Methods for preservation of foodstuffs. In): Google Patents.

EFSA. (2009). The community summary report on food-borne outbreaks in the European Union in 2007. EFSA Journal, 271, 1-102.

Ellis, R., & Roberts, E. (1980). The influence of temperature and moisture on seed viability period in barley (Hordeum distichum L.). J Annals of Botany, 45(1), 31-37.

Elmnasser, N., Dalgalarrondo, M., Orange, N., Bakhrouf, A., Haertle, T., Federighi, M., & Chobert, J.-M. (2008). Effect of pulsed-light treatment on milk proteins and lipids. J Journal of agricultural food chemistry, 56(6), 1984-1991.

Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., & Federighi, M. (2007). Pulsed-light system as a novel food decontamination technology: a review. J Canadian journal of microbiology, 53(7), 813-821.

EPA. (2003). UV disinfection guidance manual. EPA document no. 815-D-03-007. Washington, DC: Environmental Protection Agency.

Espino-Estevez, M. R., Fernandez-Rodriguez, C., Gonzalez-Diaz, O. M., Navio, J. A., Fernandez-Hevia, D., & Dona-Rodriguez, J. M. (2015). Enhancement of stability and photoactivity of TiO2 coatings on annular glass reactors to remove emerging pollutants from waters. J Chemical Engineering Journal, 279, 488-497.

Fan, X., Felker, P., & Sokorai, K. J. (2015). Decontamination of Mesquite Pod Flour Naturally Contaminated with Bacillus cereus and Formation of Furan by Ionizing Irradiation. J Food Prot, 78(5), 954-962.

119

FDA. (1996). Pulsed light for the treatment of food. In): In Available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=179. 41 Accessed 24 July 2019.

FDA. (2002). Listing of color additives exempt from certification. In Title 21-Food and Drugs. Food and Drug Administration. Code of Federal Regulations. 21 CFR 73.2575.

FDA. (2004a). Overview of Irradiation of Food and Packaging. In Available at https://www.fda.gov/Food/IngredientsPackagingLabeling/IrradiatedFoodPackagi ng/ucm081050.htm Accessed 13 Feb 2019).

FDA. (2004b). Overview of Radiation of Food and Packaging. In Available at https://www.fda.gov/Food/IngredientsPackagingLabeling/IrradiatedFoodPackagin g/ucm081050.htm Accessed 13 Feb 2019.

FDA. (2010). Guidance for Industry and FDA: Advisory Levels for Deoxynivalenol (DON) in Finished Wheat Products for Human Consumption and Grains and Grain By- Products used for Animal

Feed. Available at https://www.fda.gov/Food/GuidanceRegulation/G u i d a n c e D o c u m e n t s R e g u l a t o r y I n f o r m a t i o n /ChemicalContaminantsMetalsNaturalToxinsPesticides/ucm120184.htm. Accessed 7 Nov 2018.

Feeney, A., Kropp, K. A., O’Connor, R., & Sleator, R. D. (2014). Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen. J Gut microbes, 5(6), 711-718.

Feng, D., Xu, S., & Liu, G. (2015). Application of immobilized TiO2 photocatalysis to improve the inactivation of Heterosigma akashiwo in ballast water by intense pulsed light. J Chemosphere, 125, 102-107.

Fernandez, A., Shearer, N., Wilson, D., & Thompson, A. (2012). Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium. J International journal of food microbiology, 152(3), 175-180.

Ferrario, M., Alzamora, S. M., & Guerrero, S. (2013). Inactivation kinetics of some microorganisms in apple, melon, orange and strawberry juices by high intensity light pulses. J Journal of Food Engineering, 118(3), 302-311.

Filatova, I., Azharonok, V., Lushkevich, V., Zhukovsky, A., Gadzhieva, G., Spasic, K., Zivkovic, S., Puac, N., Lazovic, S., & Malovic, G. (2013). Plasma seeds treatment as a promising technique for seed germination improvement. In Proceeding of the 31st International Conference on Phenomena in Ionized Gases).

120

Fine, F., & Gervais, P. (2004). Efficiency of pulsed UV light for microbial decontamination of food powders. J Journal of food protection, 67(4), 787-792.

Foster, H. A., Ditta, I. B., Varghese, S., & Steele, A. (2011). Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity. J Applied microbiology biotechnology, 90(6), 1847-1868.

Foster, H. A., Ditta, I. B., Varghese, S., Steele, A. J. A. m., & biotechnology. (2011). Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity. 90(6), 1847-1868.

Fujishima, A., Zhang, X., & Tryk, D. A. (2008). TiO2 photocatalysis and related surface phenomena. J Surface science reports, 63(12), 515-582.

Garrett, K. A., Thomas-Sharma, S., Forbes, G. A., Nopsa, J. H., Ziska, L., & Dukes, J. (2014). Climate change and plant pathogen invasions. J Invasive species global climate change, 4, 22-44.

Gayan, E., Serrano, M., Raso, J., Alvarez, I., & Condon, S. (2012). Inactivation of Salmonella enterica by UV-C light alone and in combination with mild temperatures. J Appl. Environ. Microbiol., 78(23), 8353-8361.

Geveke, D. J. (2008). UV inactivation of E. coli in liquid egg white. J Food Bioprocess Technology, 1(2), 201-206.

Giese, N., & Darby, J. (2000). Sensitivity of microorganisms to different wavelengths of UV light: implications on modeling of medium pressure UV systems. J Water Research, 34(16), 4007-4013.

Gonzalez, S., Flick, G., Arritt, F., Holliman, D., & Meadows, B. (2006). Effect of high- pressure processing on strains of Enterobacter sakazakii. J Journal of food protection, 69(4), 935-937.

Greenspan, L. (1977). Humidity fixed points of binary saturated aqueous solutions. J Journal of research of the national bureau of standards, 81(1), 89-96.

Guerrero-Beltr· n, J., & Barbosa-C· novas, G. (2004). Advantages and limitations on processing foods by UV light. J Food science technology international, 10(3), 137- 147.

Gurol, C., Ekinci, F., Aslan, N., & Korachi, M. (2012). Low temperature plasma for decontamination of E. coli in milk. J International journal of food microbiology, 157(1), 1-5.

Hampson, J. W., Fox, J. B., Lakritz, L., & Thayer, D. W. (1996). Effect of low dose gamma radiation on lipids in five different meats. Meat Sci, 42(3), 271-276.

121

Harguindeguy, M. (2016). Efficacy of pulsed light technology for the inactivation of Salmonella Enteritidis PT 30 on almond kernel surface: Illinois Institute of Technology.

Hatano, H. (1960). Studies on Radiolysis of Amino Acids and Proteins III. On Radiolysis of Peptides and Proteins in Aqueous Solutions by Gamma Irradiation. J Journal of Radiation Research, 1(1), 38-45.

Hayes, J. C., Laffey, J. G., McNeil, B., & Rowan, N. J. (2012). Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐ intensity pulsed UV light irradiation. J International journal of food science technology, 47(9), 1925-1934.

Hertwig, C., Reineke, K., Ehlbeck, J., Erdoğdu, B., Rauh, C., & Schlüter, O. (2015). Impact of remote plasma treatment on natural microbial load and quality parameters of selected herbs and spices. J Journal of Food Engineering, 167, 12-17.

Hillegas, S. L., & Demirci, A. (2003). Inactivation of Clostridium sporogenes in clover honey by pulsed UV-light treatment. In 2003 ASAE Annual Meeting, (pp. 1): American Society of Agricultural and Biological Engineers.

Hoang, H. H., Sechet, J., Bailly, C., Leymarie, J., & Corbineau, F. (2014). Inhibition of germination of dormant barley (H ordeum vulgare L.) grains by blue light as related to oxygen and hormonal regulation. J Plant, cell environment, 37(6), 1393-1403.

Hollosy, F. (2002). Effects of ultraviolet radiation on plant cells. J Micron, 33(2), 179-197.

Horikoshi, S., Hidaka, H., & Serpone, N. (2003). Hydroxyl radicals in microwave photocatalysis. Enhanced formation of OH radicals probed by ESR techniques in microwave-assisted photocatalysis in aqueous TiO2 dispersions. Chemical Physics Letters, 376(3-4), 475-480.

Horikoshi, S., Kajitani, M., & Serpone, N. (2007). The microwave-/photo-assisted degradation of bisphenol-A in aqueous TiO2 dispersions revisited: Re-assessment of the microwave non-thermal effect. Journal of Photochemistry and Photobiology A: Chemistry, 188(1), 1-4.

Hsu, L., & Moraru, C. I. (2011). A numerical approach for predicting volumetric inactivation of food borne microorganisms in liquid substrates by pulsed light treatment. J Journal of Food Engineering, 105(3), 569-576.

Itikawa, Y., Hayashi, M., Ichimura, A., Onda, K., Sakimoto, K., Takayanagi, K., Nakamura, M., Nishimura, H., & Takayanagi, T. (1986). Cross sections for collisions of electrons and photons with nitrogen molecules. J Journal of physical chemical reference data, 15(3), 985-1010.

122

Jaiswal, R., Bharambe, J., Patel, N., Dashora, A., Kothari, D., & Miotello, A. (2015). Copper and Nitrogen co-doped TiO2 photocatalyst with enhanced optical absorption and catalytic activity. J Applied Catalysis B: Environmental, 168, 333- 341.

Jensen, H., Joensen, K. D., Jørgensen, J.-E., Pedersen, J. S., & Søgaard, G. (2004). Characterization of nanosized partly crystalline photocatalysts. J Journal of Nanoparticle Research, 6(5), 519-526.

Jouppila, K., Kansikas, J., & Roos, Y. H. (1998). Crystallization and X‐ray diffraction of crystals formed in water‐plasticized amorphous lactose. J Biotechnology progress, 14(2), 347-350.

Jun, S., Irudayaraj, J., Demirci, A., & Geiser, D. (2003). Pulsed UV‐light treatment of corn meal for inactivation of Aspergillus niger spores. J International journal of food science technology, 38(8), 883-888.

Kabak, B. (2009). The fate of mycotoxins during thermal food processing. J Journal of the Science of Food Agriculture, 89(4), 549-554.

Kang, M. H., Kim, D. Y., Yi, J. Y., & Son, Y. (2009). Substance P accelerates intestinal tissue regeneration after γ‐irradiation–induced damage. J Wound repair regeneration, 17(2), 216-223.

Keener, L., & Krishnamurthy, K. (2014). Shedding light on food safety: Applications of pulsed light processing. J Food Safety Magazine, 20(3), 28-33.

Khadre, M., Yousef, A., & Kim, J. G. (2001). Microbiological aspects of ozone applications in food: a review. J Journal of Food Science, 66(9), 1242-1252.

Kılıç Bayraktar, M., Harbourne, N. B., & Fagan, C. C. (2019). Impact of heat treatment and acid gelation on polyphenol enriched milk samples. J Lebensmittel- Wissenschaft+ Technologie.

Kim, S.-H., & Park, J.-H. (2007). Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula. J Journal of microbiology biotechnology, 17(2), 364-368.

Kim, S.-M., Hwang, H.-J., Cheigh, C.-I., & Chung, M.-S. (2019). Bactericidal effect of intense pulsed light on seeds without loss of viability. J Food Science Biotechnology, 28(1), 281-287.

Korachi, M., & Aslan, N. (2011). The effect of atmospheric pressure plasma corona discharge on pH, lipid content and DNA of bacterial cells. J Plasma Science Technology, 13(1), 99.

123

Kowalska, E., Wei, Z., Karabiyik, B., Herissan, A., Janczarek, M., Endo, M., Markowska- Szczupak, A., Remita, H., & Ohtani, B. (2015). Silver-modified titania with enhanced photocatalytic and antimicrobial properties under UV and visible light irradiation. Catalysis Today, 252, 136-142.

Kozempel, M. F., Annous, B. A., Cook, R. D., Scullen, O., & Whiting, R. C. (1998). Inactivation of Microorganisms with Microwaves at Reduced Temperaturas. Journal of food protection, 61(5), 582-585.

Kumar, S., Kunwar, A., Gautam, S., & Sharma, A. (2012). Inactivation of A. ochraceus spores and detoxification of ochratoxin A in coffee beans by gamma irradiation. J Food Sci, 77(2), T44-51.

Laroussi, M., & Leipold, F. (2004). Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. J International Journal of Mass Spectrometry, 233(1-3), 81-86.

Lasagabaster, A., Arboleya, J. C., & De Maranon, I. M. (2011). Pulsed light technology for surface decontamination of eggs: Impact on Salmonella inactivation and egg quality. J Innovative Food Science Emerging Technologies, 12(2), 124-128.

Lau, M., & Tang, J. (2002). Pasteurization of pickled asparagus using 915 MHz microwaves. Journal of food engineering, 51(4), 283-290.

Law, V. J., & Dowling, D. P. (2018). Converting a Microwave Oven into a Plasma Reactor: A Review. J International Journal of Chemical Engineering, 2018.

Lee, J., Sung, T., Lee, K., & Kim, M. (2004). Effect of gamma‐irradiation on color, pungency, and volatiles of Korean red pepper powder. J Journal of Food Science, 69(8), C585-C592.

Liao, L. B., Chen, W. M., & Xiao, X. M. (2007). The generation and inactivation mechanism of oxidation–reduction potential of electrolyzed oxidizing water. J Journal of Food Engineering, 78(4), 1326-1332.

Liu, G. (2005). An investigation of UV disinfection performance under the influence of turbidity & particulates for drinking water applications. University of Waterloo.

Liu, J., Yu, X., Liu, Q., Liu, R., Shang, X., Zhang, S., Li, W., Zheng, W., Zhang, G., & Cao, H. (2014). Surface-phase junctions of branched TiO2 nanorod arrays for efficient photoelectrochemical water splitting. J Applied Catalysis B: Environmental, 158, 296-300.

Liu, Q., Lu, X., Swanson, B. G., Rasco, B. A., & Kang, D. H. (2012). Monitoring ultraviolet (UV) radiation inactivation of Cronobacter sakazakii in dry infant formula using fourier transform infrared spectroscopy. J Journal of Food Science, 77(1), 86-93.

124

Los, A., Ziuzina, D., Akkermans, S., Boehm, D., Cullen, P. J., Van Impe, J., & Bourke, P. (2018). Improving microbiological safety and quality characteristics of wheat and barley by high voltage atmospheric cold plasma closed processing. J Food research international, 106, 509-521.

Lu, Y., Yao, D., & Chen, C. (2013). 2-Hydrazinoquinoline as a derivatization agent for LC-MS-based metabolomic investigation of diabetic ketoacidosis. J Metabolites, 3(4), 993-1010.

Luksiene, Z., Gudelis, V., Buchovec, I., & Raudeliuniene, J. (2007). Advanced high‐power pulsed light device to decontaminate food from pathogens: effects on Salmonella typhimurium viability in vitro. J Journal of applied microbiology, 103(5), 1545- 1552.

Ma, R., Wang, G., Tian, Y., Wang, K., Zhang, J., & Fang, J. (2015). Non-thermal plasma- activated water inactivation of food-borne pathogen on fresh produce. J Journal of hazardous materials, 300, 643-651.

MacGregor, S., Rowan, N., McIlvaney, L., Anderson, J., Fouracre, R., & Farish, O. (1998). Light inactivation of food‐related pathogenic bacteria using a pulsed power source. J Letters in Applied Microbiology, 27(2), 67-70.

Mafart, P., Couvert, O., Gaillard, S., & Leguerinel, I. (2002). On calculating sterility in thermal preservation methods: application of the Weibull frequency distribution model. J International journal of food microbiology, 72(1-2), 107-113.

Magan, N., Hope, R., Cairns, V., & Aldred, D. (2003). Post-harvest fungal ecology: impact of fungal growth and mycotoxin accumulation in stored grain. In Epidemiology of Mycotoxin Producing Fungi, (pp. 723-730): Springer.

Mai-Prochnow, A., Clauson, M., Hong, J., & Murphy, A. B. (2016). Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. J Scientific reports, 6, 38610.

Maksimovic, J. D., Zhang, J., Zeng, F., Zivanovic, B. D., Shabala, L., Zhou, M., & Shabala, S. (2013). Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance? J Plant Soil, 365(1-2), 141-155.

Mancebo‐Campos, V., Fregapane, G., & Desamparados Salvador, M. (2008). Kinetic study for the development of an accelerated oxidative stability test to estimate virgin olive oil potential shelf life. J European journal of lipid science technology, 110(10), 969-976.

125

Maness, P. C., Smolinski, S., Blake, D. M., Huang, Z., Wolfrum, E. J., & Jacoby, W. A. (1999). Bactericidal activity of photocatalytic TiO(2) reaction: toward an understanding of its killing mechanism. Appl Environ Microbiol, 65(9), 4094-4098.

Marathe, S., Machaiah, J., Rao, B., Pednekar, M., & Sudha Rao, V. (2002). Extension of shelf‐life of whole‐wheat flour by gamma radiation. J International journal of food science technology, 37(2), 163-168.

Markowska-Szczupak, A., Ulfig, K., & Morawski, A. (2011). The application of titanium dioxide for deactivation of bioparticulates: an overview. Catalysis Today, 169(1), 249-257.

Miller, B., Sauer, A., & Moraru, C. (2012). Inactivation of Escherichia coli in milk and concentrated milk using pulsed-light treatment. J Journal of dairy science, 95(10), 5597-5603.

Miller, D., Goepfert, J., & Amundson, C. (1972). Survival of salmonellae and Escherichia coli during the spray drying of various food products. J Journal of Food Science, 37(6), 828-831.

Mishra, S., Dixit, S., Dwivedi, P. D., Pandey, H. P., & Das, M. (2014). Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product. J Food Additives Contaminants: Part A, 31(1), 121-131.

Moeller, R., Raguse, M., Reitz, G., Okayasu, R., Li, Z., Klein, S., Setlow, P., & Nicholson, W. L. (2014). Resistance of Bacillus subtilis spore DNA to lethal ionizing radiation damage relies primarily on spore core components and DNA repair, with minor effects of oxygen radical detoxification. J Appl. Environ. Microbiol., 80(1), 104- 109.

Moisan, M., Barbeau, J., Crevier, M.-C., Pelletier, J., Philip, N., & Saoudi, B. (2002). Plasma sterilization. Methods and mechanisms. J Pure applied chemistry, 74(3), 349-358.

Moreau, M., Lescure, G., Agoulon, A., Svinareff, P., Orange, N., & Feuilloley, M. (2013). Application of the pulsed light technology to mycotoxin degradation and inactivation. J Journal of applied toxicology, 33(5), 357-363.

Mukisa, I. M., Muyanja, C. M., Byaruhanga, Y. B., Schüller, R. B., Langsrud, T., & Narvhus, J. A. (2012). Gamma irradiation of sorghum flour: Effects on microbial inactivation, amylase activity, fermentability, viscosity and starch granule structure. J Radiation Physics Chemistry, 81(3), 345-351.

Nazarowec-White, M., & Farber, J. (1997). Enterobacter sakazakii: a review. J International journal of food microbiology, 34(2), 103-113.

126

Nicorescu, I., Nguyen, B., Moreau-Ferret, M., Agoulon, A., Chevalier, S., & Orange, N. (2013). Pulsed light inactivation of Bacillus subtilis vegetative cells in suspensions and spices. J Food Control, 31(1), 151-157.

Niemira, B. A. (2012). Cold plasma decontamination of foods. Annual review of food science technology, 3, 125-142.

Niemira, B. A. (2012). Cold plasma reduction of Salmonella and Escherichia coli O157: H7 on almonds using ambient pressure gases. J Journal of Food Science, 77(3), 171-175.

Niemira, B. A., & Sites, J. (2008). Cold plasma inactivates Salmonella Stanley and Escherichia coli O157: H7 inoculated on golden delicious apples. J Journal of food protection, 71(7), 1357-1365.

Numanoglu, E., Gokmen, V., Uygun, U., & Koksel. (2012). Thermal degradation of deoxynivalenol during maize bread baking. Food Additives Contaminants, 29(3), 423-430.

O’connell, J., & Fox, P. (2001). Significance and applications of phenolic compounds in the production and quality of milk and dairy products: a review. J International Dairy Journal, 11(3), 103-120.

Oehmigen, K., Hähnel, M., Brandenburg, R., Wilke, C., Weltmann, K., & Von Woedtke, T. (2010). The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids. J Plasma Processes Polymers, 7(3‐4), 250-257.

Oh, Y. J., Lee, H., Kim, J. E., Lee, S. H., Cho, H. Y., & Min, S. C. (2015). Cold plasma treatment application to improve microbiological safety of infant milk powder and onion powder. J Korean Journal of Food Science Technology, 47(4), 486-491.

Ohno, T., Akiyoshi, M., Umebayashi, T., Asai, K., Mitsui, T., & Matsumura, M. (2004). Preparation of S-doped TiO2 photocatalysts and their photocatalytic activities under visible light. Applied Catalysis A: General, 265(1), 115-121.

Oms-Oliu, G., Martin-Belloso, O., & Soliva-Fortuny, R. (2010). Pulsed light treatments for food preservation. A review. J Food Bioprocess Technology, 3(1), 13.

Osaili, T. M., Shaker, R. R., Al‐Haddaq, M., Al‐Nabulsi, A., & Holley, R. (2009). Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula. J Journal of applied microbiology, 107(3), 928-935.

127

Papageorgiou, P., Katsambas, A., & Chu, A. (2000). Phototherapy with blue (415 nm) and red (660 nm) light in the treatment of acne vulgaris. J British journal of Dermatology, 142(5), 973-978.

Peleg, M., & Bagley, E. B. (1983). Physical properties of foods. In IFT basic symposium series (USA)): AVI Pub. Co.

Pereyra, S., & Dill-Macky, R. (2008). Colonization of the residues of diverse plant species by Gibberella zeae and their contribution to Fusarium head blight inoculum. J Plant Disease, 92(5), 800-807.

Pichat, P. (2016). Fundamentals of TiO 2 Photocatalysis. Consequences for some environmental applications. In Heterogeneous photocatalysis, (pp. 321-359): Springer.

Pryor, W. A. (1986). Oxy-radicals and related species: their formation, lifetimes, and reactions. J Annual review of Physiology, 48(1), 657-667.

Ragu, S., Faye, G., Iraqui, I., Masurel-Heneman, A., Kolodner, R. D., & Huang, M.-E. (2007). Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death. J Proceedings of the National Academy of Sciences, 104(23), 9747-9752.

Ramirez, C., Patel, M., & Blok, K. (2006). From fluid milk to milk powder: Energy use and energy efficiency in the European dairy industry. J Energy, 31(12), 1984-2004.

Richards, G., Gurtler, J., & Beuchat, L. (2005). Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice. J Journal of applied microbiology, 99(4), 844-850.

Ringus, D. L., & Moraru, C. I. (2013). Pulsed Ligh inactivation of Listeria innocua on food packaging materials of different surface roughness and reflectivity. J Journal of Food Engineering, 114(3), 331-337.

Roberts, P., & Hope, A. (2003). Virus inactivation by high intensity broad spectrum pulsed light. J Journal of virological methods, 110(1), 61-65.

Rosset, P., Noel, V., & Morelli, E. (2007). Time–temperature profiles of infant milk formula in hospitals and analysis of Enterobacter sakazakii growth. J Food Control, 18(11), 1412-1418.

Rowan, N., MacGregor, S., Anderson, J., Fouracre, R., McIlvaney, L., & Farish, O. (1999). Pulsed-light inactivation of food-related microorganisms. J Appl. Environ. Microbiol., 65(3), 1312-1315.

128

Roys, P., Shure, K., & Taylor, J. (1954). Penetration of 6-Mev Gamma Rays in Water. J Physical Review, 95(4), 911.

Ryu, G. H., & Ng, P. (2001). Effects of selected process parameters on expansion and mechanical properties of wheat flour and whole cornmeal extrudates. J Starch‐ Stärke, 53(3‐4), 147-154.

Ryu, J.-H., & Beuchat, L. R. (2005). Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid–based sanitizer. J Journal of food protection, 68(12), 2614-2622.

Said, M. B., & Otaki, M. (2013). Development of a DNA-dosimeter system for monitoring the effects of pulsed ultraviolet radiation. J Annals of Microbiology, 63(3), 1057- 1063.

Sanchez-Maldonado, A. F., Lee, A., & Farber, J. M. (2018). Methods for the Control of Foodborne Pathogens in Low-Moisture Foods. Annu Rev Food Sci Technol, 9, 177- 208.

Scholtz, V., Pazlarova, J., Souskova, H., Khun, J., & Julak, J. (2015). Nonthermal plasma—a tool for decontamination and disinfection. J Biotechnology advances, 33(6), 1108-1119.

Segat, A., Misra, N., Cullen, P., & Innocente, N. (2015). Atmospheric pressure cold plasma (ACP) treatment of whey protein isolate model solution. J Innovative Food Science Emerging Technologies, 29, 247-254.

Sera, B., Spatenka, P., Sery, M., Vrchotova, N., & Hruskova, I. (2010). Influence of plasma treatment on wheat and oat germination and early growth. J IEEE Transactions on Plasma Science, 38(10), 2963-2968.

Setlow, P. (2001). Resistance of spores of Bacillus species to ultraviolet light. J Environmental molecular mutagenesis, 38(2‐3), 97-104.

Sharma, R., & Demirci, A. (2003). Inactivation of Escherichia coli O157: H7 on inoculated alfalfa seeds with pulsed ultraviolet light and response surface modeling. J Journal of Food Science, 68(4), 1448-1453.

Shenga, E., Singh, R., & Yadav, A. (2010). Effect of pasteurization of shell egg on its quality characteristics under ambient storage. Journal of food science and technology, 47(4), 420-425.

Slieman, T. A., & Nicholson, W. L. (2000). Artificial and solar UV radiation induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis spore DNA. J Appl. Environ. Microbiol., 66(1), 199-205.

129

Soloshenko, I., Tsiolko, V., Pogulay, S., Kalyuzhnaya, A., Bazhenov, V. Y., & Shchedrin, A. (2009). Effect of water adding on kinetics of barrier discharge in air. J Plasma Sources Science Technology, 18(4), 045019.

Stenfors Arnesen, L. P., Fagerlund, A., & Granum, P. E. (2008). From soil to gut: Bacillus cereus and its food poisoning toxins. J FEMS microbiology reviews, 32(4), 579- 606.

Takeoka, G., Felker, P., Prokopiuk, D., & Dao, L. (2008). Volatile constituents of mesquite (Prosopis) pods. American Chemical Society Symposium Series, 9, 98-108.

Thanatuksorn, P., Kawai, K., Kajiwara, K., & Suzuki, T. (2009). Effects of ball‐milling on the glass transition of wheat flour constituents. J Journal of the Science of Food Agriculture, 89(3), 430-435.

Thirumdas, R., Sarangapani, C., & Annapure, U. (2015). Cold plasma: a novel non-thermal technology for food processing. J Food biophysics, 10(1), 1-11.

Tian, Y., Ma, R., Zhang, Q., Feng, H., Liang, Y., Zhang, J., & Fang, J. (2015). Assessment of the physicochemical properties and biological effects of water activated by non‐ thermal plasma above and beneath the water surface. J Plasma Processes Polymers, 12(5), 439-449.

Torlak, E., & Sert, D. (2013). Inactivation of Cronobacter by gaseous ozone in milk powders with different fat contents. J International Dairy Journal, 32(2), 121-125.

Trail, F. (2009). For blighted waves of grain: Fusarium graminearum in the postgenomics era. J Plant physiology, 149(1), 103-110.

Traylor, M. J., Pavlovich, M. J., Karim, S., Hait, P., Sakiyama, Y., Clark, D. S., & Graves, D. B. (2011). Long-term antibacterial efficacy of air plasma-activated water. J Journal of Physics D: Applied Physics, 44(47), 472001.

Tsuang, Y. H., Sun, J. S., Huang, Y. C., Lu, C. H., Chang, W. H. S., & Wang, C. C. (2008). Studies of photokilling of bacteria using titanium dioxide nanoparticles. J Artificial Organs, 32(2), 167-174.

Tuncbilek, A. S., Ercan, F. S., & Canpolat, U. (2012). Effect of ionizing (gamma) and non- ionizing (UV) radiation on the development of Trichogramma euproctidis (Hymenoptera: Trichogrammatidae). J Archives of Biological Sciences, 64(1), 287- 295.

Uesugi, A. R., & Moraru, C. I. (2009). Reduction of Listeria on ready-to-eat sausages after exposure to a combination of pulsed light and nisin. J Journal of food protection, 72(2), 347-353.

130

USDA. (2001). United States Standards for Grades of Nonfat Dry Milk (Spray Process) Available at: https://www.ams.usda.gov/grades-standards/nonfat-dry-milk- sprayprocess-grades-and-standards. Accessed 24 September 2019.

Vadivambal, R., Jayas, D., & White, N. (2007). Wheat disinfestation using microwave energy. Journal of stored products research, 43(4), 508-514.

Van Boekel, M. (1998). Effect of heating on Maillard reactions in milk. J Food chemistry, 62(4), 403-414.

Varga, L., & Szigeti, J. (2016). Use of ozone in the dairy industry: A review. J International Journal of Dairy Technology, 69(2), 157-168.

Villa-Rojas, R., Tang, J., Wang, S., Gao, M., Kang, D.-H., Mah, J.-H., Gray, P., Sosa- Morales, M. E., & Lopez-Malo, A. (2013). Thermal inactivation of Salmonella Enteritidis PT 30 in almond kernels as influenced by water activity. J Journal of food protection, 76(1), 26-32.

Wallen, R. D., May, R., Rieger, K., Holloway, J. M., & Cover, W. H. (2001). Sterilization of a new medical device using broad-spectrum pulsed light. J Biomedical instrumentation technology, 35(5), 323-330.

Wan, D., Huang, L., Pan, Y., Wu, Q., Chen, D., Tao, Y., Wang, X., Liu, Z., Li, J., & Wang, L. (2013). Metabolism, distribution, and excretion of deoxynivalenol with combined techniques of radiotracing, high-performance liquid chromatography ion trap time-of-flight mass spectrometry, and online radiometric detection. J Journal of agricultural food chemistry, 62(1), 288-296.

Wang, L., Shao, H., Luo, X., Wang, R., Li, Y., Li, Y., Luo, Y., & Chen, Z. (2016). Effect of ozone treatment on deoxynivalenol and wheat quality. J PloS one, 11(1), e0147613.

Wang, T., MacGregor, S., Anderson, J., & Woolsey, G. (2005). Pulsed ultra-violet inactivation spectrum of Escherichia coli. J Water Research, 39(13), 2921-2925.

Wiertzema, J. R., Borchardt, C., Beckstrom, A. K., Dev, K., Chen, P., Chen, C., Vickers, Z., Feirtag, J., Lee, L., & Ruan, R. (2019). Evaluation of Methods for Inoculating Dry Powder Foods with Salmonella enterica, Enterococcus faecium, or Cronobacter sakazakii. J Journal of food protection, 82(6), 1082-1088.

Williams, P. D., Eichstadt, S. L., Kokjohn, T. A., & Martin, E. L. (2007). Effects of ultraviolet radiation on the gram-positive marine bacterium Microbacterium maritypicum. J Current microbiology, 55(1), 1-7.

Zenklusen, M. H., Coronel, M. B., Castro, M. A., Alzamora, S. M., & Gonzalez, H. H. L. (2018). Inactivation of Aspergillus carbonarius and Aspergillus flavus in malting

131

barley by pulsed light and impact on germination capacity and microstructure. J Innovative Food Science Emerging Technologies, 45, 161-168.

Zhang, H. Q., Barbosa-Canovas, G. V., Balasubramaniam, V. B., Dunne, C. P., Farkas, D. F., & Yuan, J. T. (2011). Nonthermal processing technologies for food (Vol. 45): John Wiley & Sons.

Zhang, J., Zhou, P., Liu, J., & Yu, J. (2014). New understanding of the difference of photocatalytic activity among anatase, rutile and brookite TiO2. J Physical Chemistry Chemical Physics, 16(38), 20382-20386.

Zhang, Q., Liang, Y., Feng, H., Ma, R., Tian, Y., Zhang, J., & Fang, J. (2013). A study of oxidative stress induced by non-thermal plasma-activated water for bacterial damage. J Applied physics letters, 102(20), 203701.

Zhang, Z., Jiatieli, J., Liu, D., Yu, F., Xue, S., Gao, W., Li, Y., & Dionysiou, D. D. (2013). Microwave induced degradation of parathion in the presence of supported anatase- and rutile-TiO2/AC and comparison of their catalytic activity. Chemical engineering journal, 231, 84-93.

Zhong, H., Shaogui, Y., Yongming, J., & Cheng, S. (2009). Microwave photocatalytic degradation of Rhodamine B using TiO2 supported on activated carbon: Mechanism implication. Journal of Environmental Sciences, 21(2), 268-272.

132

Appendix A: Effects of IPL on different microbes in filter papers

Introduction

Organic matters in food matrix was able to prevent microorganisms from absorbing

IPL. Lipid and protein may inhibit certain UV spectrum absorption. Therefore, it is crucial to evaluate the IPL disinfection on different microbes including Cronobacter sakazakii,

Enterococcus faecium, and Bacillus cereus in non-contact food matrices such as filter paper.

Methods and materials

C. sakazakii strain ATCC 29544, E. faecium strain NRRL B-2354, and Bacillus cereus strain ATCC 14579 were investigated in this chapter. C. sakazakii was received from the American Type Culture Collection (ATCC) as a lyophilized culture. All three microorganisms were revived from frozen culture (-80 °C) and maintained on tryptic soy agar (Sigma-Aldrich, St. Louis, MO) supplemented with 0.6% (w/v) yeast extract (Sigma-

Aldrich) (TSAYE). Plates were stored at 4± 2 °C. 1000 mL of each bacterium was then plated onto filter paper, allowed them to dry in a biosafety hood in petri dishes. After drying, all samples were subjected at 0, 10, 20, and 30 s Z-1000 IPL treatments (Xenon Corporation,

Woburn, MA) at distances of 8 cm. The frequency of the system is 3 Hz with pulse width of 360 µs. Ten second intervals among each treatment was applied. After IPL treatment, microorganisms were plated into Triptone Soya Agar (Biolife), both C. sakazakii and E. faecium were serially diluted and plated in duplicate onto TSAYE. Then samples were incubated at 37°C for 48-72 hours before enumeration. B. cereus samples were heat shocked (80°C for 12 min) before serially diluting and plating onto standard methods agar

(SMA). SMA plates were then incubated at 30°C for 24-48 hours prior to enumeration. All

133 plates were counted manually and the concentration of colonies were described as colony- forming units per gram (CFU/g).

Results and Discussions

The figure A1 shows Cronobacter sakazakii, E. faecium, and B. cereus spore were inactivated by 8.11, 7.40, and 6.72 log10 reductions after 30s- IPL treatments. Higher resistance of E. faecium than C. sakazakii was observed because of Gram-positive pathogen is more resistant to irradiation and heating treatments (Blazquez, Rodriguez,

Rodenas, de Rozas, Segales, Pujols, et al., 2017). On the other hand, bacterial endospores were proved more resistant than vegetative cells, resulting in significantly less logs reductions of B. cereus spore than the other microorganisms (J.-H. Ryu & Beuchat, 2005).

Microbial inactivation in paper filters was significant less than that in food matrices such as wheat flour, NFDM, and egg white powder after the similar IPL treatment time. 28s IPL treatments caused 2-3 log10 reductions of C. sakazakii or E. faecium in these three food matrices (Chen, Cheng, et al., 2019). High concentration of protein in powdered foods can absorb UV wavelengths at 190 nm, 280 nm, and UV-B region, lipids also can absorb visible and UV light under the help of photosensitizers (Noura Elmnasser, et al., 2008; Hollosy,

2002).

134

9 8 7 6 5 4 3

2 Population (Log CFU/mL) 1 0 0 5 10 15 20 25 30 Treatment time (s)

C. sakazakii E. faecium B.cereus

Figure A1. Populations of Cronobacter sakazakii, Enterococcus faecium, and Bacillus cereus in filter papers as a function of IPL treatment time from 0-30s

135

Appendix B: Sensory evaluation of IPL treated wheat flour

Introduction

To ensure the commercial applicability, it is necessary to evaluate IPL attributes towards to sensory of IPL treated samples. In this chapter, the effects of IPL frequency, voltage, feed rate of the intense pulsed light apparatus on sensory attributes of all-purpose wheat flour were evaluated.

Materials and methods

Sensory evaluation was conducted to evaluate the acceptance of the IPL treated products and obtained optimized conditions that caused slight or no adverse impacts of the process on product quality. Therefore, the correlation between IPL parameters such as voltage, frequency, and feed rate were investigated to optimize the light source for maximum germicidal effects with minimum quality loss.

IPL treatments:

Wheat flour (General Mills Operations, Inc., Golden Valley, MN) were treated under IPL at different treatment conditions. The weight of each sample treatment is around

1000 grams. The detailed factorial design information of 14 treatments was shown in Table

B1. After IPL treatment, wheat flour was packaged in glass jars covered with foil and stored at -20 °C in a freezer until use.

Training:

Nine panelists participated in an initial five training sessions during which they will learn to use calibrated flavor/taste intensity and aroma intensity scales. During the first session, they developed a standardized flavor and aroma analysis procedure to evaluate wheat flour. During the first three sessions they individually described several pairs of a

136 specific powder using descriptive terms (lexicon) related to the aroma and flavor of the samples. At session four they practiced scaling the intensity of the sensory attributes with calibrated scales on our computerized data collection software. At session five, panelists learned to clarify the meanings of any poorly understood descriptors and gave panelists feedback on their performance.

Testing:

Nine panelists participated in all five test sessions. They evaluated a complete set of the wheat flour samples in the first two sessions. During the testing sessions, each panelist will evaluate each sample by rating the intensity of the aroma and flavor on 20 point-line scales labeled ‘none’ at the left end and ‘intense’ at the right end. Intensity ratings of flavor and aroma were made on the standard citric acid scale. Panelists will wear nose clips when evaluating the flavor.

Table B1. The IPL factorial design for sensory study

Code Voltage (V) Feed rate (g/h) Frequency (Hz)

1 3000 4200 1.0

2 3000 4200 3.0

3 3000 4200 14.0

4 3000 8100 1.0

5 3000 8100 3.0

6 3000 8100 14.0

7 2200 4200 1.0

8 2200 4200 3.0

137

9 2200 4200 14.0

10 2200 8100 1.0

11 2200 8100 3.0

12 2200 8100 14.0

13 (120 s-UV treatment) NA NA NA

14 (No treatment) NA NA NA

Results and discussions

The code 1 treatment resulted in the highest bactericidal effects based on data in chapter 4, the data clearly show that the overall aroma and flavor of code 1 treatment has no significant difference with untreated samples (Table B2). Moreover, the aroma and flavor intensity of all IPL treated wheat flour had no significant change. The results indicated these three parameters had no effects on flavor or aroma of wheat flour. In contrast, to achieve a same level of bacterial inactivation, 120s- UV treatment resulted in significant flavor and aroma change when comparing with untreated samples. Therefore, the results revealed this prototype IPL system was able to inactivate microbes in wheat flour and maintained the wheat flour sensory at the same time.

Table B2. Overall aroma and flavor ratings of IPL treated wheat flour at different processing conditions

Code Aroma Flavor

1 4.2 b 3.9 bc

2 4.7 b 3.9 bc

138

3 3.8 b 3.8 bc

4 3.8 b 3.4 c

5 4.9 b 4.0 bc

6 4.7 b 3.1 c

7 4.5 b 5.0 bc

8 5.1 b 4.3 abc

9 4.4 b 3.8 bc

10 4.9 b 4.1 bc

11 4.8 b 3.7 bc

12 3.5 b 3.2 c

13 (120 s-UV treatment) 6.8 a 5.5 a

14 (No treatment) 3.6 b 3.2 c

*Mean ratings within a row of lower-case letters do not differ significantly (SNK test, p>

0.05). The data was mean values of 9 panelists.

139