Effects of Pulsed Ultraviolet Light on Microflora and Temperature of Propylene Glycol Cooling Medium

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Title:
Effects of Pulsed Ultraviolet Light on Microflora and Temperature of Propylene Glycol Cooling Medium
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1 online resource (80 p.)
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english
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Ozturk, Samet
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University of Florida
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Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Yang, Weihua Wade
Committee Members:
Correll, Melanie J
Wright, Anita Christine

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Subjects / Keywords:
glycol -- pulsed -- temperature -- thermocouple
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, M.S.
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theses   ( marcgt )
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born-digital   ( sobekcm )
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Abstract:
As a nonthermal inactivation technology, PL has gained significant interest in the food industry. Due to its decontamination effect on different food products, PL may offer a potential disinfection tool for the microfloracontrol of PG, a refrigeration medium widely used in the food industry. Theobjective of this study was to examine the effect of PL on inactivating themicroflora present in PG and determine the sample temperature profile during PLillumination. Propylene Glycol samples of 5, 10, and 15 ml in aluminum disheswere treated in a Xenon PL processor (Model RC847) at a distance of 6 cm fromthe quartz window for 5, 10, 15, 20, and 25 s. Thickness of the sample was 2.5,5.0 and 7.6 mm for 5, 10, and 15 ml of PG, respectively. The initial total andlactic acid bacteria were in the concentration of 6.48-log10 and3.88-log10, respectively. PL illumination for 15 s for 5 ml PG and20 s for 10 ml glycol resulted in complete sterilization, whereas 20 s illumination for 15 ml glycol resulted in 5.30-log10 reduction inAPC. Complete sterilization, 5.70-log10 and 4.11-log10reduction in APC was obtained for 5, 10, and 15 ml samples, respectively, with15 s illuminations. Lactic acid bacteria were totally inactivated.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Samet Ozturk.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Yang, Weihua Wade.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-02-28

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1 EF FECTS OF PULSED ULTRAVIOLET LIGHT ON MICROFLORA AND TEMPERATURE OF PROPYLENE GLYCOL COOLING MEDIUM By SAMET OZTURK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Samet Ozturk

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3 To my family and friends

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4 ACKNOWLEDGEMENTS I would like to gratefully acknowledge my major advisor Dr. Wade Yang for providing me with this opportunity to be a part of UF FSHN family, his mentorship with patience and advice in my masters degree. Im also thankful to Dr. Anita Wrig ht and Dr. Melanie J. Correll for serving on my m aster s d egree committee and taking their precious time to advise me when I met with them I would like to say that a great advantage of being at U niversity of Florida is to have met my great lab group members, Akshay K. Anugu, Bhaskar Janve, Braulio Macias, Dr. Cheryl R. Rock, Kelsey Guo, Senem Guner, Tara Faidhalla and Xingyu Zhao. I thank you all for your splendid friendship and mental support. I also would like to thank Dr. Hale Z. Toklu for her contribution in the statistical analysis of my data. Last but not least, I would like to express my sincere appreciation to my family for their love, encouragement, and support. They always believed in me. I would also like to thank my great fr iend Busra Koksal, Caglar Doguer and my uncles Ahmet Kada and Hakan Kada for their mental s upport in my good or bad times.

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5 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................... 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 10 CHAPTER 1 BACKGROUND ...................................................................................................... 12 1.1 Introduction ....................................................................................................... 12 1.2 Justification of Study ......................................................................................... 14 1.3 Overall Objective............................................................................................... 15 1.4 Specific Objectives ............................................................................................ 15 2 REVIEW OF LITERATURE .................................................................................... 16 2.1 Propylene Glycol (PG) ...................................................................................... 16 2.1.1 Production of Propylene Glycol ............................................................. 16 2.1.2 Applications of Propylene Glycol ........................................................... 17 2.1.3 Propylene Glycol as a Coolant .............................................................. 17 2.1.4 Toxicology of Propylene Glycol ............................................................. 17 2.1.5 Biodegradation of Glycols Under Oxic Conditions ................................. 18 2.1.6 Propylene Glycol Degradation by Pure Cultures of Anaerobic Bacteria ................................................................................................. 19 2.1.7 Anaerobic Degradation of EG and PG by Mixed Microbial Populations ............................................................................................ 19 2.2 Pulsed UV Light ................................................................................................ 20 2.2.1 PhotoChemical Mechanism .................................................................. 21 2.2.2 PhotoThermal Mechanism .................................................................... 22 2.2.3 PhotoPhysical Mechanism ................................................................... 23 2.2.4 Microbial Inactiv ation by Pulsed UV Light .............................................. 24 2.2.5 Applications of Pulsed UV Light ............................................................. 26 2.2.6 Effects of Pulsed UV Light on Food Components and Quality ............... 27 2.2.7 Inactivation Studies by Continuous and Pulsed UV Light ...................... 28 2.2.8 Economical Impact of UV Light Disinfection System ............................. 31 2.3 Lactic Acid Bacteria (LAB) ................................................................................ 32 2.4 Aerobic Plate Count (APC) ............................................................................... 34

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6 3 EFFECT OF PULSED LIGHT ON THE TEMPERATURE PROFILE OF PROPYLENE GLYCOL .......................................................................................... 37 3.1 Material and Methods ....................................................................................... 38 3.1.1 Sample Preparation ............................................................................... 38 3.1.2 Pulsed UV Light Treatment .................................................................... 38 3.1.3 Temperature Measurements ................................................................. 39 3.1.4 Experimental Desi gn .............................................................................. 39 3.1.5 Statistical Analysis ................................................................................. 40 3.2 Results and Discussion ..................................................................................... 40 4 EFFECT OF PULSED ULTRAVIOLET LIGHT ON AEROBIC PLATE COUNT IN MICROFLORA OF PROPYLENE GLYCOL ............................................................ 51 4.1 Material and Methods ....................................................................................... 51 4.1.1 Pulsed Ultraviolet Light Treatment ......................................................... 51 4.1.2 Aerobic Plate Count (APC) .................................................................... 51 4.1.3 Colony Forming Unit (CFU) ................................................................... 52 4.1.4 Evaluation of P L Efficiency .................................................................... 53 4.1.5 Sample Preparation ............................................................................... 53 4.1.6 Statistical Analysis ................................................................................. 53 4.2 Results and Discussion ..................................................................................... 53 5 EFFICACY OF PULSED UV LIGHT ON INACTIVATION OF THERMAL RESISTANCE BACTERIA IN MICROFLORA OF PROPYLENE GLYCOL ............ 61 5.1 Material and Methods ....................................................................................... 61 5.1.1 Pu lsed Ultraviolet Light Treatment ......................................................... 62 5.1.2 Colony Forming Unit .............................................................................. 62 5.1.3 Evaluation of PL Effi cacy ....................................................................... 62 5.1.4 Statistical Analysis ................................................................................. 63 5.2 Results and Discussion ..................................................................................... 63 6 CONCLUSIONS ..................................................................................................... 69 7 RECOMMENDATION ............................................................................................. 71 LIST OF REFERENCES ............................................................................................... 73 BIOGRAPHICAL SKETCH ............................................................................................ 80

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7 LIST OF TABLES Table Page 2 1 Properties of Propylene Glycol ........................................................................... 35 4 1 Reduction of Aerobic Plate Count in propylene glycol after Pulsed UV Light Treatment with in increase in temperature for 6 cm distance from quartz window ............................................................................................................... 58 4 2 Reduction of Aerobic Plate Count in propylene glycol after Pulsed UV Light Treatment with in increase in temperature for 9 cm distance from quartz window ............................................................................................................... 59 5 1 Log reduction of lactic acid bacteria in propylene glycol during Pulsed UV light treatment with in increase in temperature for 6 cm distance from quartz window ............................................................................................................... 66 5 2 Log reduction of lactic acid bacteria in propylene g lycol during Pulsed UV light treatment with in increase in temperature for 9 cm distance from quartz window ............................................................................................................... 67

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8 LIST OF FIGURES Figure Page 2 1 Scheme of organic products formed from anaerobic degradation of ethylene and propylene (R = H or CH3) ............................................................................ 36 3 1 A pilot scale PL system at the Food Science Pilot Plant, University of Florida, Gainesville, FL, U.S.A. ....................................................................................... 43 3 2 Schematics of the pulsed ultraviolet system with TC 08 thermocouple data logger ................................................................................................................. 44 3 3 Lab scale TC 08 thermocouple data logger temperature recording system ....... 45 3 4 A view of K type thermocouples immersed in propylene glycol .......................... 46 3 5 Change in initial temperature of proyplene glycol samples (5,10 and 15 mL) during PL treatment at 6 cm for 25 s .................................................................. 47 3 6 Change in initial temperature of proyplene glycol samples (5,10 and 15 mL) during PL treatment at 9 cm for 25 s. ................................................................ 48 3 7 Increase in the initial temperature of propylene glycol sample during PL heating for 50 s at 6 cm. .................................................................................... 49 3 8 Increase in the initial temperature of propylene glycol sample during PL heating for 50 s at 9 cm. .................................................................................... 50 4 1 Aerobic Plate Count on the 10 mL sample volume plates after 20 s exposure at 6 and 9 cm distance ....................................................................................... 60 5 1 The formation of lactic acid bacteria colonies in the untreated and treated samples for 25s of PL ......................................................................................... 68

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9 L IST O F ABBREVIATIONS AB Aerobic Bacteria ADP Adenosine Di phosphate AOAC Association of Official Analytical Chemists APC Aerobic Plate Count APHA American Public Health Association ATP Adenosine Tri phosphate CFU Colony Forming Unit DOW The Dow Chemical Company DPG Dipropylene Glycol FDA Food Drug Administration GC Guanine and Cytosine LAB Lactic Acid Bacteria MRS De Man, Rogosa and Sharpe MPG Monopropylene Glycol PG Propylene Glycol PO Propylene Oxide PL Pulsed Ultraviolet Light RNA Ribonucleic Acid TPG Tri propylene Glycol UV Ultraviolet

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EF FECTS OF PULSED ULTRAVIOLET LIGHT ON MICROFLORA AND TEMPERATURE OF PROPYLENE GLYCOL COOLING MEDIUM By Samet Ozturk August 2013 Chair: Wade Yang Major: Food Science and Human Nutrition As a nonthermal inactivation technology, PL has gained significant interest in the food industry. Due to its decontamination effect on different food products, PL may offer a potential disinfection tool for the microflora control of PG, a refrigeration medium widely used in the food industry. The objective of this study was to examine the effect of PL on inactivating the microflora present in PG and determine the sample temperature profile during PL illumination. Propylene Gl ycol samples of 5, 10, and 15 mL in aluminum dishes were treated in a Xenon PL processor (Model RC847) at a distance of 6 cm from the quartz window for 5, 10, 15, 20, and 25 s. Thickness of the sample was 2.5 5.0 and 7.6 mm for 5, 10, and 15 mL of PG, respectively. The initial total and lactic acid bacteria were in the concentration of 6.48log10 and 3.88log10, respectively. PL illumination for 15 s for 5 mL PG and 20 s for 10 mL glycol resulted in complete sterilization, whereas 20 s illuminat ion for 15 mL glycol resulted in 5.30log10 reduction in APC. Complete sterilization, 5.70log10 and 4.11 log10 reduction in APC w as obtained for 5, 10, and 15 mL samples, respectively, with 15 s illuminations Lactic acid bacteria were totally inactiv ated (detection limit <10 CFU/mL) for all the samples treated for 10 s. The initial temperature was 30.6C and an increase of 22.3, 16.5, and 14. 8 C was

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11 observed respectively for 5, 10, and 15 mL of PG sample treated for 20 s at 6 cm distance from quartz window Sample temperature and reduction in microflora decreased with increased sample thickness, as the penetration depth of the sample was a limiting factor for PL illumination. This study confirmed that the efficacy of PL in inactivating microflora in PG He nce, PL c ould be used as a novice technology for the food industry in inactivating the microflora present in PG

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12 CHAPTER 1 BACKGROUND 1.1 Introduction G round source heat pump system s, which can utilize the energy of the earth s shallow geothermal sources for heating and cooling, has been significantly used in industry along with different types of organic anti freeze substances such as ethylene glycol (EG), propylene glycol (PG) and betaine in the system as a coolant (Klotzbucher and others 2007). Additionally PG (C3H8O2) is increasingly used for therapeutic, cosmetic purposes and food applications in the industry. Propylene glycol may exist in the following forms : mono(MPG), di (DPG), and tri propylene glycols (TPG) (DOW 2005). The glycols are obtained from the hydrolysis of propylene oxide (PO)(C3H6O ), in which MPG is the most popular with the common commercial name of 1,2 propanediol. Furthermore, PG is a colorless, nearly odorless and tasteless viscous liquid ( Shigeno a nd Nakahara 1991). It may be used as an effective humectant, preservative and stabilizer in diverse food and cosmetic products such as pet food, bakery goods, food flavorings, salad dressing and shaving cream (DOW 2012). The F ood and D rug A dministration (FDA) allows PG to be used in foods in the following percentages : 2.5% in frozen dairy products, 5% in alcoholic beverages, 5% nuts and nut products, 24% in confections and frostings, 97% in seasonings and flavorings ( FDA 2012). Moreover, United States Pharmacopeia (USP) recomm ended the use of PG in chiller systems and also approved it for food applications and for high quality food processing. T here are several problems related to different sources f or heating and cooling systems. The most major one is groundwater, which is a potential contaminant for

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13 PG.As previously alluded to, addition of other solvents such as water to PG during circulation in the chilling a nd freezing systems may cause contamination problems ( Dentinger and others 199 5 ). Moreover, improper handling and management practices could also result in the contamination of PG. C onsequently these practices may reduce the microbial safety, overall quality and also cause biodegradation of PG (Aas and others 1993). Although PG poss esses antibacterial and antifungal properties (Kinnunen and other 1991) it may lose these properties because of oxidation due to chemical and microbial action. That is why food preservation techniques can play a critical role for modern mass food product i on and distribution besides handling systems. As such, different preservation technologies have been developed and adopted successfully in food industry. There are several technologies such as, power ultrasound (PU), irradiation, microwave, pulsed electric field (PEF) magnetic field (MF), high pressure processing (HPP) and ohmic heating (OH) treatments available for using in industry to inactivate microorganism s (pathogenic or spoilage) ( FDA 2012c ). However, there are many limitations affiliated with the aforementioned technologies and consequently, there is continual need to develop novel preservation techniques to improve efficiency, minimize cost, and achieve minimal quality changes to the product Such limitations may included the following: Attenuation of ultrasonic intensity based on the medium, inactivation of thermo resistant microorganisms in HPP (cold sterilization) and contamination of the medium with metal deposits from the degradation and corrosion of electrodes used in OH systems.

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14 By the beginning of nineteen century ultraviolet (UV) light has been used as a novel technology for bactericidal inactivation ( Demirci and other s 2008). However, pulsed UV light has been proven to be more effici ent than other commercial techniques such as steam, microwave on the inactivation of pathogens and decontamination of food surfaces and packing material. The use of PL has achieved high levels of microbial inactivation on relatively simple surfaces, while resulting in only 13 log10 reductions on complex surfaces such as meats (Demirci and other s 2008) O ther applications of PL include the pasteurization of milk and mitigation of food allergens (Demirci and other s 2008). The mechanism of PL light produces t hree major effects, which are the following: P hotochemical, photo thermal and photophysical ( Krishnamurthy and others2010). The main mechanism of the PL treatment on microbial inactivation is attributed to the photochemical effect, in which the structural changes in DNA of most of microorganism s are observed. DNAs are converted into thymine dimers ( Krishnamurthy and others 2008) ,thus inhibit ing the microorganism from DNA transcription and replication and le ading to cell death (Miller and others 1999). The degree of microbial inactivation exerted by PL depends also on food composition attributed to macromolecules such as: carbohydrates, water, protein and fats. Krishnamurthy and others (2004) reported that PL is more suitable for solids surfaces than liquids (e.g., wine, fresh juices) in which the process differs as affected by solids concentration closely related to absorbance. 1.2 Justification of Study As previously mentioned, PL is a nonthermal technology gai ning commercial interest. It has been proven to be effective in inactivating microorganisms in several food products and liquids, and hence is expected to exhibit similar effect on microflora present in PG. The significance and applications of PG in the fo od industry provides

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15 premise to evaluate the effects of PL on inactivation of microorganism. Additionally, t here is no published data regarding the effect of PL on the microflora in PG as well as its effects on quality. It is hypothesized in this study tha t PL exposure may have an equally synergistic effect on inactivation of bacteria and microflora of PG. 1.3 Overall Objective The overall objective of this study is to investigate the efficacy of PL on the microflora (lactic acid bacteria and aerobic plate count ) of PG. 1.4 Specific Objectives Listed below are the specific objectives of this study which will subsequently appear in their respective chapters. The specific objectives of this research are to: 1. Determine the temperature profile of the PG during PL illumination at 6 and 9 cm distance from quartz window 2. Determine thermal and/or nonthermal effect of PL on inactivation of APC in PG at the same sample thickness with different distances from quartz window 3. Determine the efficiency of PL treatment to inact ivate LA B in microflora of PG

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16 CHAPTER 2 REVIEW OF LITERATURE 2.1 Propylene Glycol (PG) Propylene gl ycol (PG) is an organic compound with formula C3H8O2, which is present in numerous forms including mono(MPG), di (DPG), and tripropylene glycols (TPG) (Charles and J ohn 2002). The most popular PG is refer r ed to as 1,2propanediol and also known as 1,2 propylene glycol, 1,2dihydroxypropane, methylene glycol, and methyl glycol The chemical structure of PG is simple, in whi ch two hydroxyl groups characterize it as a glycol (Harris 1992). With both a primary and a secondary hydroxyl, the 1,2propanediol (MPG) is a difunct ional alcohol (Parker and Issaacs 1959). Therefore, the solubility characteristics and other properties of glycols tend to be bet ween simple alcohols and glycerin ( Ruddick 1972) T able 2 1 shows some physical properties of PG. 2.1.1 Production of Propylene Glycol In the industry, PG is synthesized from the hydrolysis of PO (C3H6O, PO). There are two different hydrolysis methods for PG. First occurs in the presence of ion exchange resin, and the other occurs in the presence of a small amount of sulfuric acid. Both reactions are noncatalytic under high pressure in the range of 1200 psi to 1600 psi and high temperature at 200 or 220C or w ith a catalytic reaction at lower temperature suc h as 150 to 180C (DOW 2012). After the production process, PG consists of 20 % 1,2 propanediol, 1,5 DPG and a minute amounts of other PG. Moreover, PG can be obtained from hydrolyses of glycerol and biodies el byproduct (Shigeno and Nakahara 1991). Additionally the subsequent formation of dipropylene

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17 (DPG) and tripropylene (TPG) glycol is obtained by the same process involving the production of PG. 2.1.2 Applications of Propylene Glycol Propylene glycol can be use d as an effective humectant, preservative and stabilizer especially in or food products (ice cream, wine, pet foods) or personal care products such as shampoo, conditioner and soap (DOW 2012). Additionally, PG is liste d as a direct additive for particular foods in the regulation, and sorted as generally recognized as safe (GRAS) by Food and Drug Administration (FDA). The PG can be used for direct and indirect food additive applications such as antioxidant and emulsifier to give food some physical and t echn ical attributes (DOW 2012). 2.1.3 Propylene Glycol as a Coolant A coola nt is a fluid, which prevents overheating and provides the transfer of heat between product and heating source. Coolants can be found in different forms such as liquid, gas or solids ( Martin and Murphy 1994 ). Propylene glycol also acts as a coolant that keeps products from melting in heat and/or freezing when it is cold to enhance penetration, as previously introduced in C hapter 1 (DOW 2005 ). Because of some reasons such as consequently cooling of liquids, there may be either crystal or ice formation caus ing the fluid to become viscous and decreasing the flow rate. Propylene glycol does not have any sharp freezing points. Because of that it can be mixed with other liquids foods such as w ine and beer t o prevent super cooling effects, which may initiate the formation of ice and crystals (DOW 2012). 2.1.4 Toxicology of Propylene Glycol All glycols have a lower degree of toxicity for human health and desirable formulation properties; thus, it has been a significant ingredient for different application

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18 areas such as food industry and cosmetics (DOW 2012). Additionally, it is considered to be biodegradable, and hence it will not remain as a chemical remnant, and it can be used for aerobic and anaerobi c conditions as a source of carbon (DOW 2012). Moreover, an animal study showed that a mixture of PG and stearic acid enhanced the in vitro permea bility of nimodipine through rat skin. Nimodipine is a calcium channel blocker with vasodilati ng properties, which may be used as an anti hypertensive drug (FDA 2012). Also, it has been shown that a mixture containing PG enhanced the absorption of verapa mil, which is another calcium channel blocker (Breslin and others 1996) 2.1.5 Biodegradation of Glycols Under Oxic C onditions The PG and EG are widely used as a carbon and energy source among aerobic microorganisms. Some bacteria groups have ability of degrading PG and EG under oxic conditions ( Klotzbucher and others 2007). Willetts (1979) reported that the degradation of PG is based on metabolic pathways and proceeds via lactaldehyde and pyruvate. In the next steps, pyruvate is metabolized to acetyl CoA, which is oxidized to CO2 in the tricarboxylic acid cycle. As regards to this information, degradation of PG may occur without any accumulation of toxic and persistent organic intermediates under oxic conditions (Klotzbucher and others 2007). There is not sufficient information about the kinetics of PG for aerobic degradation in groundwater. However, rapid aerobic biodegr adation of PG was investigated in studies conducted with sewagesludge and soil samples. For instance, Klecka (1993) investigated that degradation of PG in soil at concentration of 6000 ppm was changed at different conditions like an average rate of 2 ppm/day at 2C ppm/day at 8C and 93 ppm/day at 25C. The rate of degradation of PG and EG in

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19 aquifers are lower than soils owing to microbial population densities like in oligotrophic systems. Further studies still need to be quantified such degrada tion rates. 2.1.6 Propylene Glycol Degradation by Pure Cultures of Anaerobic B acteria Diverse pure bacterial strains and enrichment cultures are capable of degrading PG anaerobically. These types of microorganism were isolated from various habitats, high nutrient capacity of sewage sludge, wastewater (Dwyer and Tiedje 1983; Obradors 1988), also sediments of an oligotrophic lake (Sass and Cypionka2004). Anaerobic biodegradation of PG takes plac e in most anoxic environments. Gaston and Stadtman (1963); and Toraya (1979) reported that fermenting bacteria were able to degrade EG and PG to acetate and propionate, and ethanol and propanol (Figure 2 1 ). Eichler and Schink (1985) investigated that EG and PG were degraded to their acids like acetate as sole organic produc ts via production of CH4 or H2. Thus evidence of the corresponding aldehydes such as acetaldehyde and propionaldehyde were investigated as degradation products (Toraya 1979; Eichler and Schink 1985). In addition to fermenting, EG and PG were degraded by s ulfatereducing bacteria (SRB) (Klotzbucher and others 2007). There is not known data about the complete oxidation of the glycols to CO2 by SBR. Sass and Cypionka (2004) reported that most SBR have capability to use other electron acceptors thus, anaerobi c degradation of glycols may involve reduction of nitrate, iron (III) or manganese (IV) as potential electron acceptors. 2.1.7 Anaerobic Degradation of EG and PG by Mixed Microbial P opulations Klotzbucher and others (2007) demonstrated that under anoxic conditions in sewage sludge and in soil, PG may be degraded by mixed microbial populations Dwyer and Tiedje (1983), and Veltman (1998) reported that EG and PG were initially degraded by fermentation in sludge to the same amount of acids and alcohols. In further

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20 degradation methane and CO2 were obtained. During the microbial degradation of PG in soil columns, Fe3+ and Mn4+ oxides were poorly used (Jaesche and others 2006). The reduction of iron and manganese is based on two sequential processes, which are directly o xidation of PG and oxidation of fermentation product ( propanol and propionate) of PG. Therefore, it is likely PG degradation can depend on aerobic microorganism reactions. 2.2 Pulsed UV Light Pulsed UV light (PL) has been used as an emerging processing technology in many different areas of the food industry most commonly to decontaminate food products and surfaces. Pulsed UV light treatment involves the use of radiation, which comes from the ultraviolet region of the electromagnetic spectrum, which aids in product disin fection. Additionally, PL, which is generated by lamps, has a wide spectrum ranging from UV ( 10 nm 390 nm ) to the infrared (750nm 1mm) (Demirci and others 2008). During the generation of P L light, electrical energy is stored in a capacit or, and the energy is released as short pulses in several times per second with pulse lasting between 100 ns and 2 ms (Demirci and others 2008). Thus, the P L light has higher energy than continuous ultraviolet light (UV) system (Xenon 2003). Pulsed UV light has intensity 20,000 times more than the sunlight and the germicidal effect of P L seems below 400 nm (Dunn and others 1995). There are three effects exhibited by P L light. These are photochemical, photothermal and photophysical, and contribute to the inactivation mechanisms of P L light and are described in more detail in the sections below.

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21 2.2.1 PhotoC hemical Mechanism Any chemical reaction is caused by absorption of light (including visible, ultraviolet, and infrared). The light excites atoms and molecules (shifts some of their electrons to a higher energy level) and thus makes them more reactive. In comparison to ordinary reactions using thermal energy alone, photochemical reactions can follow different routes and are more likely to produce free radicals, which can trigger and sustain chain reactions. The germicidal effect of P L light includes photochemical damage to deoxyribonucleic acid ( DNA) and ribonucleic acid (RNA) of microorganisms. In the wavelength of 240 to 280 nm, the most important absorbers of light are known microorganism nucleic acids ( Krishnamurthy and others2010). Any damage to either of these substances (DNA and RNA) can sterilize the organisms because they carry necessary genetic information for reproduction. Pulsed ultraviolet (P L ) does not inactivate microorganisms by chemical reaction but radiation of light can cause a photochemical reaction in the organism s DNA and RNA (Murov 1973). The mechanism is based on changing the structure of DNA and performing chemical modifications. The photochemical transformation of pyrimidine, which is based on DNA of bacteria, viruses, and other pathogens, underlies the germicidal effec ts of UV light (Giese and Darby 2000). Such modifications to DNA bonds cause unzipping implicated in replication, and henc e the organisms are unable to proliferate. This is as a result of mutations; abnormal gene transcriptions and impaired replications occur and then cause death ( Krishnamurthy and others2010). Some experiments showed that after PL treatment there is no t any enzymatic repair of DNA. It may be considered that the same effect can inactivate the DNA repair system (Dunn and others 1995; McDonald and others 2002; Smith and others 2002).

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22 Although the UV light shows germicidal properties between 100280 nm, according to some previous studies the shorter wavelengths are more efficient on the inactivation of microorganism than longer wavelengths because of their higher energy levels (Rowan and others 1999). UV light part of the lamp can provide almost 69 log10 re duction effects on the microorganism, and UV C is considerable for 50% of all effects. Furthermore, using a flash lamp for UV C fluxes can provide a sufficient inactivation for all microorganisms (Wekhof 2000). When light pulses combine with UV C light the re is a synergistic inactivation of conidia of Botrytis cinerea and Monilia frugtigena fungi (Marquenie and others 2003b ). Pulsed ultraviolet (P L ) l ight has a germicidal effect between 230300 nm on Escherichia coli Although P L shows a maximum effect at 270 nm there is no inactivation, which can be observed above 300 nm ( Krishnamurthy and others2010). After PL treatment there are some DNA damage such as the formation of single strand breaks and pyrimidine dimers, which have been induced in, yeast cells. B ut, the inactivation effect of continuous UV light at 254 nm is slightly greater than P L does although killing level of treated yeast cells is almost the same in both cas es (Takeshita and others 2003). 2.2.2 PhotoT hermal Mechanism Photothermal effect is a phen omenon associated with electromagnetic radiation. It is prod uced by photo excitation resulting in the production of thermal energy (heat). This mechanism is based on the differences in the heating rates of bacteria and the surrounding media. It can cause damage to bacterial cells because of absorption of energy of light by bacteria cells. The intensity in destruction of microbes via pulsed could be partly due to the photothermal effect. It was proposed that energy exceeding

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23 0.5J cm2 could enhance disinfection by bacterial disruption during short time overheating resulting from absorption of all UV light from a flash lamp (Wekhof 2000). In t his process, the water content of bacteria vaporizes and lead s to bacterial disruption. Thus ultimate inactivation occurs (Takeshita and others 2003). With Aspergillus niger spores, the overheating due to internal explosion resulted in evacuation of the cell contents during the light pulse. During the PL treatment, photothermal effects are enhanced via biocidal action. Since proteins are heat sensitive, it can be supposed that high doses would result in cell death (Cover and others 2001). 2.2.3 PhotoP hysical Mechanism Photophysical mechanism of P L is based on the structural damage to bacterial cells because of disturbances of intermittent highenergy pulses There is much research, which illustrates this mechanism. For example, damage to proteins and cell membranes during exposure to PL are definitely correlated with destruction of nucleic acid. In a study for in inactivation of Saccharomyces ceravisiae cells by pulsed light and classic UV was observ ed (Takeshita and others 2003) It was found that concentration of eluted protein from yeast cells after treatment with P L was higher than under UV treatment. This indicates that there was potential cell membrane damage. The electron micrograph of yeast cells revealed changes in cell s tructure including large vacuoles, cell membrane distortion, and change to circular shape (Wang and others 2005). On the contrary, the UV treated cells were almost similar to the nontreated cells (Takeshita and others 2003.) Electron micrograph of treated A. niger spores showed ruptured in the top surface of the spore. In the treatment of light pulse, there were collapsed and deformed spores with deep craters observed (Wekhof and others 2001). In UV

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24 treatment of most Bacillus subtilis spores had become disintegrated or lost its shape (Wekhof and others 2001) 2.2.4 Microbial Inactivation by Pulsed UV Light Although PL obtains energy from UV, the visible and infrared regions contribute to the inactivation mechanism with UV being the predomina nt cause However, UV light of the wide spectrum is known as the predominate cause of inactivation. Moreover, PL may cause different damage to cells such as cell wall breakage, cellular membrane structure, and also induce leakage of the cell content in S aureus ( Krishnamurthy and others 2010). On the other hand, a small increase of 23 C in the temperature was seen as negligible during the treatment, and hence P L can induce some shocking effect on the cell wall of bacteria ( Krishnamurthy 2006). In additio n to shocking effect there is a thermal stress on the bact eria cell because of exposure to PL, especially at higher density of light (0.5 J/cm2), and it might lead to cell rupture. Absorption of energy from light depends on the characteristic of the bacter ia and the surrounding medium; therefore, bacteria may also be overheated in different amounts (Fine and Gervais 200 4). There might be some changes, which may lead to membrane destruction in the cells such as steam flow and vaporization. There are a lot of researchers, who have proven these approaches. For instance, according to Gomez Lopez (2005) different microorganism s were inoculated on agar media to represent high dec ontamination effects, ranging from 1.2 to 5.9 log10 of intense pulsed light with a pu lse duration of 30 s and pulse intensity of 7 J (Gomez Lopez and others 2005a ). Another study chose E. coli and S enter i tidis to study inactivation effects of pulsed light. 5 to 100 pulses with a range of 200530 nm w ere applied to bacterial suspension c ontained in petri dishes. The results show a 9log10 order reduction after treatment with 100 pulses of 9 J for E.

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25 coli, and 100 pulses of 4.5 J for each pulse produced a 7log10 reduction, and merely 0.5 log10 reduction were observed after 5 pulses. Resul ts indicate that high energy per pulse is heavily r esponsible for this mechanism. Low reduction rate in the beginning of the first few pulses is possibly due to high cel l population, that is approximately 1.3x 109CFUmL1, which causes the intensity to shift off through 3.28 mm depth of the sa mple (Ghasemi and others 2003). Results show a 6log10 reduction in Listeria monocytogenes, E. coli 0157:H7, S enteriditis P. aeruginosa, B cereus and S aureus These pathogens were inoculated on agar plates usi ng 200 pulses of highUV light. When low UV light was used, there was only 12 log10 reduction. Pulsed light source was also less effective than higher UV intensity, which makes the UV part of spectrum to be solely responsible for microbial deactiv ation (R owan and others 1999). Other results show that 64 light pulses (spectral range of 200530 nm) of 1 s duration and 3J intensity are required to inactivate and reduce E. coli O157:H7 and L. monocytogenes populations by 24 log10 reductions. This reduction i s increased to 7 and 6 by increasing the pulse rate of 512 (Macgregor and others 1998). 78 log10 reduction can be achieved for S aureus in both suspended cultures and agar seeded cells, which were treated for 5 s by pulsed UV light at 5.6 Jcm2 with a pulse duration of 360 s (Krishnamurthy and others 2004). Another recent study showed that a single light pulse at a dose of 1 J cm2 is adequate for reduction of bacterial population of P. aerouginosa, about 106 CFUmL1 was suspended in solution for injection (Feuilloley and others 2006). A different study showed that 20 pulses with 1 Jcm2 of 0.3 s duration can have a reduction of more than 6 logs for Bacillus pumilus spores in aqueous suspension in a polyethylene cont ainer (Dunn and others 1997).

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26 Similar study showed 68 log10 reductions for spores of Bacillus subtilis, Bacillus pumilus, and Bacillus stearothermophilus which are ut terly inactivated by 3 pulses. Roughly 3.7 and greater than 5.9log10 reduction was reported in Bacillus circulans and Bacillus cereus with approximately 50 pulses with each pulse having 7 J when this bacterium were treated on agar surface (Gomez Lopez and others 2005 a ). 2.2.5 Applications of Pulsed UV Light There are developing new applications areas, which use PL treatment to disinfect food products or packaging materials. Recently, some commercial compani es have started to use it on packaging materials in the final steps of disinfection in the lack of chemical preservatives and disinfectants. Moreover, PL is used on the where surface contamination is seen as a concern for microbial contamination such as food with smooth surfaces, for example, whole fruit, vegetables, meat and cheese. Add itionally, it can be used on eggs or mushrooms to enhance t he content of vitamin D. Due to the potential sunlight effect of PL, researchers have already changed 7 dehydoxy cholesterol to natural vitamin D3 (cholecalciferol) in the wavelength of 280310 nm. On the other hand, it is used to fortify edible mushrooms r ich in vitamin D2. Furthermore, while using the UV light combinations some researchers ranged the content of vitamin D2 from 22.92.7 to 184.0 5.7 for various mushrooms ( Krishnamurthy and others 2010). Another study showed that it has inactivation effect on the toxins, and there was a suf ficient reduction of 3 100 % afl atoxin M1 in milk when 260 min of PL treatment was applied ( Krishnamurthy and others 2010). Additionally, the authors noticed that it could be used to inactivate allergens in soybean and pe anuts. They reported that after PL treatment there was a remarkable reduction rate of two important peanut allergens,

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27 which are Ara h1 and h3, in the liquid peanut. Hereby, this study opens new avenues to reduce content of allergens in other food products. 2.2.6 Effects of Pulsed UV Light on Food Components and Quality There are some kind of effects of P L on the food quality and components although it can be used as a useful technology. One of them is that UV light can depolymerize starch in the presence of air, and metal oxides components (ZnO ) enhance this process (Tomasik 2004). Moreover UV light can increase other oxidation mechanism such as free radicals, and catalyze other steps of the process. Additionally, Kolakowska (2003) reported that UV light has an e ffect to form some components such as lipid radicals, superoxide radicals (S OR ), and H2O2. Some functions of components such as carbohydrate crosslinking, protein crosslinking, protein fragmentation, and peroxidation of unsaturated fatty acid can be induced by SOR under UV light. On the o ther hand, UV light can expose textural changes in milk because of denaturation of proteins, enzymes, and amino acids. Absorption of UV photons by water leads to production of OHand H+ radicals; therefore, high dose of UV light may affect product quali ty under hig h doses applications besides just changing chemistry of food components. But they seem m ostly useful changes because it is detrimental to microbial growth. Thus, for maintaining the quality of food products and to ensure their safety the optimal properties of disi nfections steps are necessary. Moreover, under UV light treatment the fat soluble vitamins like vitamin A and colored compounds like vitamin B2 can be affected by photodegradation and peroxides produced, and these changes in food components may cause changes in nutritional quality of food. Additionally, long time treatment with UV light and high doses of UV radiation may

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28 cause temperature increase of food products, which can lead to changes in food quality because of changes in flavor color and enzymatic browning. Under normal conditions the inactivation of microorganisms takes a few seconds to a minute but the time depends on the opacity of the food products, microorganism type, and doses of UV radiation. Furthermore, UV light may induce flavor of products, which is caused because of activation of riboflavin, which is responsible for the conversion of methionine from methanol which leads to a burnt protein like, burnt feathers like, or medicinal like flav or Generally, there is not any adverse effect after UV light treatment of food if it is applied under optimal conditions such as moderate amounts, which are needed to provide required inactivation of microorganism s or increase temperature. To have success ful implementation of the process in some foods, modification and optimization of the UV light treatment might be necessary. 2.2.7 Inactivation S tud ies by Continuous and P ulsed UV L ight There was a comparison between effectiveness of a continuous UV light source and a PL source to decontaminate surfaces. It showed almost the same level of inactivation of B. subtillis with 4x103J/cm2 of PL source and 8x103J/cm2 continuous UV light source (McDonald and others 2000). Also PL is used to inactivate E coli, S. Typhi Shigellasonnei, S faecalis, and S. aureus (Chang and others 1985). It was investigated that the bacteria were grown at 35C for 20 to 24 h in a nutrient broth. The prepared culture was filtered with a 0.45Re suspended and aggregated bacteria in sterile buffer water were removed while using a the filtering treatment with UV light, bacteria colonies were grown in nutrient agar and counted. Although E. coli, S

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29 aureus, S. s onnei and S. t y p hi displayed similar resistance to the UV light and approximately 7 x103J/cm2 energy caused 3log10 reductions, S. faecalis needed a 1.4 times higher dose to get 3log10 r eduction of inactivation. Rowan and others (1999) investigated the efficiency of UV light emission with low or high UV content on inactivation of bacteria such as L. monocytogenes, E. coli, S. e nteritidis Psudeomonas aeruginosa, B. cerus, and S. aureus. Seeded bacteria on the surf ace of Tryptone soyayeast extract agar were treated with a PL source with low and high UV content. After PL treatment with 200 pulsed while pulsed duration is 100 ns, 2 and 6 log10 reductions were obtained for low and high content, respectively. While usi ng diluted samples concentration of 1x109 (Sample A), 1x108 (Sample B), or 1x107 (Sample C) with sterile deionized water, the effect of highintensity UV light on inactivation of B. subtilis spores was investigated by Sonenshein (2003). The t hree types of positi ons were used and a 50 sample from each concentration were placed on them. The first position was on the lamp axis and at the midpoint of the lamp, second one was 1 cm above the lamp axis and at the midpoint of the lamp and last one was 1 cm above the lamp axis and 172 mm to the right of the midpoint of the lamp. When samples placed at the lamp axis and at the midpoint of the lamp, more than 6.5 log10 CFU/mL for sample B and 5.5log10 CFU/mL for sample C reduction was investigated after UV light tr eatment (three pulses for 1 s). Yaun and others (2004) investigated that efficiency of continuous ultraviolet energy for inhibition of the pathogens on fresh product. After inoculation of the surfaces of red delicious apples, leaf lettuce, and tomatoes wit h Salmonella spp. or E. coli O157:H7, the samples were treated with UV C light at a wavelength of 253.7 nm with

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30 range of 1.5 to 24x103 W/cm2. Different reduction amounts of E. coli O157:H7 were obtained for different samples like 3.3 log10 CFU/apple at 24x103 W/cm2 but 2.19 log10 CFU/ tomato slices at the same dose. Similar results were obtained for Salmonella spp. and E. coli O157:H7 on inoculated lettuce as 2.65 and 2.79log10 CFU/lettuce reductions, respectively. The mechanisms of damag e of yeast cells induced by PL and continuous UV light were investigated by Takeshita and others (2003). According to this research, the DNA damage induced by continuous UV light was slightly higher than that of PL. Moreover, efficiency of PL on protein el ution was higher than that of continuous UV light. The inactivation mechanism of PL was based on investigations of both the germicidal action of UV C light and rapture of microorganisms due to thermal st ress caused by the UV component (Wekhof and other 2000). Jun and others (2003) showed inactivation efficiency of PL on Aspergillus niger spores in corn meal. After 100 s treatment of PL when the sample distance from quartz window was at 8 cm, a 4.95log10 reduction of A. niger on inoculated corn meal was inv estigated (Jun and others 2003). Moreover, PL inactivated E. coli O157:H7 on alfalfa seeds with reduction of 0.09 to 4.89 log10 CFU/g for various thickness and treatment time (Sharma and Demirci 2003). As the thickness was adjusted at 1.02 mm, the completely inactivation (4.80log10 CFU/g) of E. coli O157:H7 was obtained after 30 s treatment of PL. An increase in treatment time caused higher reduction for all thickness. In another study, Hillegas and Demirci (2003) showed that PL is an effective treatment m ethod to inactivate Clostridium sporogenes in honey. In this study, almost 88 %of reduction was obtained for Clostridium sporogenes for 45 s treatment of PL ( initial inoculum level was 6.24-

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31 log10CFU/g) as a 2 mm depth of honey sample distance from quartz window was kept at 8 cm, although 180 s treatment s was required to reach 89.4 % reduction at 20 cm distance from quartz window. The bulk tank of bovine milk was exposed to PL created by pulsed laser excimer at 284 nm. After treated with PL (25 J/cm2 energy for 114 s exposure) there were not any obtained growing cultures of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella d ublin Yersinia enterocolitica, Staphylococcus arueus, Aeromonus hydrophillia, and Serratia marcescens in bovine milk. The reduction amount obtained was more than 2 log10 CFU/mL (Smith and others 2002). Furthermore, PL can also be effective to degrade toxins. 2 to 60 min treatment of PL achieved very important reduction range 3.6 to 100% of alfatoxin M1 in milk (Yousef and Marth 1985). These inactivation effects of PL are based on several factors such as, transmissi on of the food product, geometry of the waveguide, lamp power, and exposed wavelength range. 2.2.8 Economic al I mpact of UV Light Disinfection S ystem Pulsed UV light disinfect ion technology is cheaper than other available systems According to resear chers, the estimated treatment cost at 4 J/cm2 with the PureBright PL treatment system is 0.1/ft2 of treated area (Dunn 1997). The estimated cost comprises of conservative estimate of electricity, maintenance, and equipment amortization. Also with cost of investment in a hooded high intensity lamp and power unit the cost of treatment with the PureBright system is estimat ed as 0.1/ft2 by Lander (1996). For the apple cider industry, it is approximated to be cheaper to utilize PL pasteurization (Choi and other 2005), since its cost is approximately $15,000 (Higgins 2001). The cost of annual maintenance like power consumption and lamp replacement

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32 is based on lowest dosage of 30,000 MW.s/cm2 for multi lamp and single lamp disinfection source of continuous UV light as $2,465 and $3,060 for an 8,000 h run time (Anonymous 1989). Taghipour (2004) reported that the costs for 4 log1 0 reduction of E. coli in primary waste water by UV light, electron beam, and gamma irradiation were 0.4/m3, 1.25 /m3, and 25/m3, respectively. The treatment costs of UV light, electron beam, and gamma irradiation to inactivate E. coli ( 4 log10) in prima ry was tewater were reported as 0.4/m3, 1. 25/m3, and 25/m3, respectively (Taghi pour 2004). It is clear to indicate that the UV light treatment to inactivate pathogenic mic roorganisms is cost effective. 2.3 Lactic Acid Bacteria (LAB) L actic acid bacteria are gram positive rods, nonspore forming cocci or coccobacilli with a DNA base composition of h igh GC (Guanine and Cytosine content) They produce lactic acid as a major fermentation product or lactic acid, CO2 and ethanol simultaneously (Jay and others 1986). They are generally nonrespiratory and lack catalase, however, they have the superoxide dismutase and alternative means for detoxifying peroxide radicals. A ll members of this group obtain energy only from by substrate level phosphorylation because o f not carrying out electron transport phosphorylation (Hardie and Whiley 1995). T hey can grow in the presence of O2 as well as in its absence, and hence they are known as aerotolerant anaerobes (Holdeman and others 1975). Lactic A cid B acteria (LAB) has bee n classified according to their cell morphology, DNA composition, and also type of fermentative metabolism. Members of this group, which are Leuconostoc, Pediococcus, Lactococcus and Streptococcus, have almost familiar DNA composition, maybe there is a lit tle difference from strain to strain. Most of

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33 LAB gain energy form metabolism of sugar; thus, they are usually restricted to environment s in which sugars are present. Lactic A cid B acteria (LAB) has only limited biosynthetic ability, thus they evolved in environments that are rich in amino acids, vitamins, purines and pyrimidines to provide nutritional need (Gilliland 1990). There is one important difference between subgroups of LAB, which is depended on sugar fermentation patterns. One subgroup produces only lactic acid as a major fermentation product, and is called homo fermentative. On the other hand, the other group produces ethanol and CO2 as well as lactic acid, and it is called hetero fermentative ( Facklam and Elliot 1995) They mostly live in beneficial or harmless associations with animal, al though some of them are pathogens. They may be found in different habitats such as milk and dairy products, and in decaying plant m aterials (Schillinger and Lucke 1987). There are some special places which LAB play a beneficial role and can be found naturally in the intestinal tract, oral and vaginal cavity of humans (Farrow and others 198 6 ). Moreover, LAB is one of the most common and important groups of microorganisms, which are used to ferment foods such as yogurt, cheese and beer. Besides they have an ability to contribute to the taste and texture of fermented products. Additionally, LAB can inhibit some undesirable microorganisms like food spoilage bacteria by producing growthinhi biting substances a nd large amounts of lactic acid (Essers 1982). There is an essential feature of LAB metabolism, which is productive carbohydrate fermentation coupled to substratelevel phosphorylation. Adenosine triphosphate (ATP) generated is subsequently used for biosynthesis. L actic A cid B acteria (LAB) has an effective capacity as a group to demote unique carbohydrates and relevant compounds such as lactic acid

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34 that is more than 50% of sugar carbon (Facklam and others 1995). On the other hand, LAB can adapt to different conditions and produce significantly various endproduct patterns because of the ability to chang e the metabolism. 2.4 Aerobic Plate Count (APC) There are some kinds of methods to enumerate the number of organism in food. One of them is the Aerobic Plate Count (APC), which indicates the level of microorganisms in a product and can sometimes be used to indicate the quality and spoilage level of the product. Detailed procedures for determining the A PC of foods have been developed by the Association of Off icial Analytical Chemists (AOAC 1990) and American Public Health Association (APHA 1984) Obtaining an estimate of the number of microorganisms in a food product will aid in evaluating sanitary pract ices during processing and handling, as well as determining potential sources of contamination (Jay 1986). Moreover, the APC may measure potential of bacteria flora, which can grow as visible colonies under random test conditions. Although APC is the best estimate, it is not available to measure the total bac teria population in food (AOAC 1995a). It can be effective to determine certain microorganisms such as thermophiles, mesophiles, psychrotrophiles, and proteolytic or lipolytic microorganisms (AOAC 1995 ) Additionally, a high APC may specify that a food product has been contaminated or has a poor quality ingredient. The suitable colony counting range was determined between 25 250 ( FDA 2012). Some conditions, which have already been altered such as temper ature of incubation or composition of the agar medium, may change display of the organism, which will grow in the medium. Thanks to these kinds of altering conditions, APC can be used as the best method to determine product q uality and microbial load (FDA 2001 )

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35 Table 2 1. Properties of Propylene Glycol (Source DOW 2012) Property Unit Molecular Weight 76.10 g/mole Formula C 3 H 8 O 2 Boiling Point at 101.3 kPa 187.4 C Melting Point 59.96 C Pour Point < 57 C Density at 25 C 1.036 gm/cm 3 Vapor Pressure at 25 C 0.017 kPa Viscosity at 25 C 48.6 mPa.s(=cp) Thermal Conductivity at 25 C 0.2061 W/(m.K) Heat of Formation 422 kJ/mol

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36 Figure 21. Scheme of organic products formed from anaerobic degradation of ethylene and propylene (R = H or CH3) permitted by T. Klotzbucher and others / Geothermics 36 (2007) 348 61

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37 CHAPTER 3 EFFECT OF PULSED LIGHT ON THE TEMPERATURE PROFILE OF PROPYLENE GLYCOL A temperatu re rise has been observed with pulsed light illumination for an extended duration due to the significant proportions of infrared spectra (Li and others 2011; Shriver and others 2011). Since pulsed light by Xenon lamp consists of around 20% infrared spectra besides approximately 54% UV spectra and around 22% visible spectra In the pulsed generation, the electrical energy is amplified and stored in capacitor over a short period of time like few milliseconds and released as very short period pulses (several nanoseconds). The stored electrical energy is passed through a lamp filled with inert gas (xenon or krypton), which causes ionization of gas and produces a broad spectrum of light in the wavelength from UV light to infrared region. The intensity of pulsed l ight has 20,000 times more powerful than that of sunlight (Dunn 1995). Generally the pulse rate is released as 1 to 20 pulses per second and the pulse width is 300 ns to 1 ms. Thus, light of puls es with high energy in several m egawatts are produced though the total energy is comparable to continuous UV light system. Use of pulsed light has been approved as a nonthermal technology as applied within short times (<10 s). In contrast, longer exposure time may cause increases in temperature when energy accumul ates in foods products Another possible ex planation to changes in temperature during PL treatment is based on the fluence threshold (Wekhof 2000). As mentioned in previously publications, fluence is evaluated as the energy delivered to the sample by the system during PL exposure. However, the fluency capacity of PL may be dependent on the characteristics of sample and the extent in which energy is

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38 absorbed. Given the limited absorption of energy in light food products, it is probable that samples did not reach a fluence threshold in any of the treatments. In this study, effect of PL on temperature variation of naturally contaminated ( FSHN Lake Water Gainesville, FL ) PG was investigated. The efficiency of PL was assessed based on the increase in temperature of PG. 3.1 Material and Methods 3.1.1 Sample Preparation Using an incubator the inoculated PG sample (Sara Lee Foods, Peoria, IL, U.S.A) was stored at a controlled temperature of 27C. The container containing the PG was tightly sealed in order to avoid oxidation before treatment. Each sample was loaded individually into the same weighing size dishes (Low Form Aluminum, Fluted, Fisher Scientific U.S.A ). 3.1.2 Pulsed UV Light Treatment T he PL treatments were performed at room temperature while using a pi lot scale continuous PL system designed by Xenon Corp. (LH840LMP HSG, Xenon Corp., Wilmington, MA, U S A) (Figure 3 1). The PL system consists of a controller unit, treatment chamber housing with two adjustable Xenon flash lamps, and a hydraulic conveyor belt. This unit generated a broadspectrum light (1001100 nm) at a pulse rate of three pulses/ 2) were adopted from measurements conducted by Krishnamurthy (2006) in a PL batch system of simila r characteristics. Exposure time and distance from the PL quartz window were established as the experimental variables. The PG samples were individually centered and treated for 5,

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39 10, 15, 20 and 25 s, at a distance of 6 and 9 cm from the light source. The thickness of th e PG samples was measured as 2.5, 5 and 7.6 mm for 5, 10 and 15 mL, respectively. 3.1.3 Temperature Measurements The temperature profile of PG samples during PL treatment was recorded by K type thermocouple using Pico eight channel thermocouple data logging interface (T C 08) attached to laptop runni ng Pico software (Figure 32 vs. Figure 33). The K type thermocouple was placed at the geometrical center of aluminum dish for every sample (Figure 3 4). The data recoding was started and stopped 30s before and after the actual experiment. The temperature of PG samples was continuously measured using K type thermocouples through a data acquisition system ( TC 08 Thermocouple Data Logger Pico Technology North America Inch, Tyler, TX). 3.1.4 Experimental Desi gn A full factorial design with three factors and 23 levels was utilized in this study. The factors were average temperature of the PG samples (69.9 and 93.5C), volu me of the PG sample ( 5, 10, and 15 mL), and treatment time (5, 10,15, 20 and 25 s) and tw o different distances (6 and 9 cm) from quartz window Three replications were performed for each condition. For the first set of experiments the program was used to control the temperature of the PG using K type thermocouple for 5,10,15,20 and 25 s at 6 cm distance from quartz window. In the second of experiments, the distance from quartz was adjusted as 9 cm for a 30.6Cinitial temperature for 5, 10, 15, 20 and 25 s PL treatment. In a third set of experiments, the treatment time was increased 50 s for vo lume of PG (5,10 and 15 mL) at two distances (6 and 9 cm) from quartz window. Also, the effect of short treatment times (5,10, 15, 20 and 25 s ) was investigated by treating the micr oorganisms for all volumes of PG samples.

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40 3.1.5 Statistical A nalysis Using Graph Pad Prism 5.0 Software compared the mean reduction rates. Oneway ANOVA test followed by Tukeys post hoc comparison was performed to evaluate the difference between the treatment groups. The difference p<0.05 was accepted as significant. 3.2 Results and Discussion The PL treatment is considered as a nonthermal process for short period of inactivation of microorganism s. Although it is considered as a nonthermal process, there may be an increase in temperature because of high energy content of pulses due to prolonged treatment resulting in the accumulation of energy in the sample (Krishnamurthy 2006) The sample temperature increased gradually (Figure 35 vs. 3 6 ) directly proportional with the exposure time. Also, as the thickness of the sample decreases, the temperature increase d. This may be due to an increase of energy absorption because of more penetration depth allowing more energy to be absorbed by th e sample. In accordance, i ncrease in temperature is also related to the viscosity and the surface of sam ple (Demirci and others 2003). In contrast an increase in sample volume resulted in a decrease in temperature rise which may be attributed to lower penetration depth of PL into the sample (Krishnamurthy 2006). Hence, higher temperatures could be expected on samples, which are located at a closer distance (from the quartz window) and have lower thickness as seen in Figure 35 According to the measurements undertaken with the K type thermocouple using Pico eight channel thermocouple data logging interface (PC 08) attached to the computer with Pico software (Figure 32 vs. Figure 33 ) the average initial temperature of samples was obtained as 3 0.6 C. At 5, 15 and 25 s, the increase in temperatur e of samples w as

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41 recorded as 1 4 3.1 30 .5 1.3 and 39.9 1.2 C, respectively; using a sample volume of 5 mL was kept at 6 cm distance from quartz window In this study, the temperature increase initiated by PL was moderately high, especially during the first seconds of exposure (~6C). For instance, at 10 s treatment at 6 cm distance from q uartz window resulted in a 20.4 2.5, 1 6.5 1.2 and 14.8 3.2 C increase in sample temperature for 5, 10 and 15 mL PG sample volumes, respectively (Figure 3 5) On th e other hand, there was a proportional increase for all PG sample volumes during longer exposure because of the increasing photothermal effects of P L on the temperature at both distances (Figure 3 5 vs. 3 6 ). However, when the distance from the lamp source increased and treatment time is concomitantly decreas ed, the food samples may not be exposed to a more intense treatment (Gmez Lpez and others 2005a). In the second part of the study, the distance to quartz window was i ncreased while sample volume (5, 10 and 15 mL) and treatment times were kept constant. As shown in the Figure 39, the temperature profiles of PG sample s were determined for 50 s As sh own in the Figure 36, for 10 mL sample, increase in t emperature w as observed as 12.8 0.6, 14.8 and 20.8 0.5C for 5, 15 and 25 s e xposure at 9 cm distance, respectively The critical temperatur e chang e was obtained as 7.6 1 3.2 and 1 6.1 C for 15 mL PG sample during 5,10 and 20 s PL treatments at 9 cm respectively Figure 38 show s relationship between temperature and distance when di stance increases from quartz window it cause s a decrease in temperature. Krishnamurty (2006) reported that lower temperatures in combination with shorter treatment times and lesser volume resulted in lower reductions. As expected, in third part of this study, the rat io of temperature increase was higher when less volume of PG sample was treated, as a result of more

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42 energy accumulation that is re adily available to heat the PG sample. The temperat ure was raised from 55.1 to 5 8.9 C within fifty seconds of PL treatment for 5 mL volume at 6 and 9 cm distances, respectively (Figure 37 vs. 3 8) Optimizing the temperature of PG samples during PL treatment could result in less detrimental quality changes. In this study we showed that t he effect of the sample th ickness did not have any significant effect on increase in temperature until 10 s for 5 mL sample (p<0.05) (Figure 3 5 vs. 3 6) at both distances The temperature of the sample increased as the treatment time increased after several seconds (Figure 35 vs 3 6 ); however, no significant temperature change was observed during the first 5 s for all volumes. But after 15 s there was a significan t increase for 5 mL sample. This may indicate that nonthermal effect of PL displays only in short treatment periods On the other hand, a minor increase in temperature within seconds may cause a considerable thermal effect for some bacteria species such as psychrophilic organism (Struvay and Feller 2012). Another disadvantage of a high increase in temperature is that specific features of the sample may be damaged. During a 20s treatment time, the temperature increase was about 2 3 C and 1 9 C for a 10 mL PG sample at 6 and 9 cm distance, respectively Our statistical analysis demonstrated that the treatment duration and the interaction (treatment time*depth) had significant (p<0.05) impact on temperature increase. .

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43 Figure 31 A pilot scale PL system at the Food Science Pilot Plant, University of Florida, Gainesville, FL, U.S.A.

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44 Figure 32 Schematics of the pulsed ultraviolet system with TC 08 thermocouple data logger

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45 Figure 33 Lab scale TC 08 thermocouple data logger temperature recording system ( Photo courtesy of Samet Ozturk)

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46 Figure 34 A view of K type thermocouples immersed in propylene glycol ( Photo courtesy of Samet Ozturk)

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47 Figure 35 Change in initial temperature of proyplene glycol samples (5,10 and 15 mL) during PL treatment at 6 cm for 25 s Data represent the mean SD (n=3). There is a significant delta temperature change in for all volumes after 10 seconds when compared with the initial temperature. p< 0.05 represents significance between sample volumes 0 10 30 40 50 60 0 10 30 40 50 60Increase in Temperature( C) Time (s) 5ml 10ml 15ml

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48 Figure 36 Change in initial temperature of proyplene glycol samples (5,10 and 15 mL ) during PL treatment at 9 cm for 25 s Data represent the mean SD (n=3). There is a significant delta temperature change in for all volumes after 10 seconds when compared with the initial temperature. p< 0.05 represents significance between sample volumes 0 10 30 40 50 60 0 10 30 40 50 60 Increase in Temperature( C) Time (s) 5ml 10ml 15ml

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49 Figure 37 Incr ease in the initial temperature of propylene glycol sample during PL heating for 50 s at 6 cm Data represent s the mea n (n=3). 0 10 30 40 50 60 0 40 60Increase in Temperature ( C ) Time (s) 5ml 10ml 15ml

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50 Figure 38 Increase in the initial temperature of propylene glycol sample during PL heating for 50 s at 9 cm Data represent the mean (n=3) 0 10 30 40 50 60 0 40 60Increase in Temperature ( C ) Time (s) 5ml 10ml 15ml

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51 CHAPTER 4 EFFECT OF PULSED ULTRAVIOLET LIGHT ON AEROBIC PLATE COUNT IN MICRO FLORA OF PROPYLENE GLYCOL Aerobic plate count ing (APC) is a method used as an indicator of the level of microorganisms in a sample of raw material, inprocess ma terial, or finished product which is not usually associated with food safety concerns since it is not related to pathogenesis or toxicity of bact eria (Neusely and others 2013). However, enumeration of these microorganisms plays an integral role in determining the quality, shelf life and post treatment processing contamination. The food industry uses this method to determine the sanitation levels betw een processing and the distri bution steps besides determining the sufficiency of sanitation. The higher APC there is a higher possibility to increase environmental and sanitation controls Closed water and chilling system s h ave a potential to encourage the growth of microorganism s and promote oxidation of propylene glycol in the chilling system. The level of bacterial growth is related to the rate of decomposition in PG The FDA recalls and initiates court actions towards infections in handling systems (FDA 2001). In this study, we determined the efficiency of PL on the disinfection of APC of the microflora in PG. 4.1 Material and Methods 4.1.1 Pulsed Ultraviolet Light Treatment Propylene glycol samples were treated wit h PL as described in Chapter 3. 4.1.2 Aerobic Plate Count (APC) The AOAC 966.23 C method was used as the reference procedure in determining the APC of PG From each P L treated PG sample, a serial dilution (1/10 mL ) was made up to 104. One mL from each diluted sample was put into sterile petri

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52 dishes using sterile pipette. Plate Count Agar (at 45C) (Fisher Scientific, Pittsburg, PA, U.S.A) was poured into each of petri dish containing 1 mL inoculum. The plates were allowed to solidify on a flat surface, inverted and incubated at 35C for 48 hours. After incubation, reducti on of bacteria in ten fold was calculated for different treated PG samples The data was analyzed using a statistical analysis system (Graph Pad Prism 5.0 ). O neway ANOVA followed by post hoc Tukey s multiple comparison test was performed. T he level of sig nificance was determined at P < 0.05 4.1.3 Colony Forming Unit (CFU) The colony forming of units is an estimate to use for determining the number of viable bacterial cells in a sample per mL or per gram. Hence, it tells the degree of contamination in samples of water, vegetables, soil or fruits or the magnitude of the infection in humans and animals (FDA 2012). To obtain APC, count duplicate or triplicate plating from the same dilution, whic h produces 25 to 300 colonies and take the average of the plate counts. I n short, 10fold dilutions were employed in the rinse solutions (1/10, 1/100, 1/1000) initially. The PL treated and untreated samples (1 mL) were vortexed and tran sferred to dilutio ns tubes (9 mL PW) a using sterile pipette tip for each transfer. Then, 1 mL portion of dilutions was transferred to empty plates and 910 mL plate count agar (Fisher Scientific, Pittsburg, PA, U.S.A) was poured. After this process, plates were incubated at 48 3 h at 35C1C. To c alculate the APC the total number of colonies counted is multiplied by the reciprocal of the dilution factor on the plate.

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53 4.1.4 Evaluation of PL Efficiency The level of microbial inactivation (Log [N/No]) was calculated by subtracting the survivor counts (N) resultant from PL treatment from the initial counts (No) represented by the control samples. Resul ts were expressed in log CFU/mL (PG) Additionally, survivor curves were built by creating scatter plots of log survivor counts (N) v ersus treatment time (t). 4.1.5 Sample Preparation The obtained PG (Sara Lee Foods, Peoria, IL, U.S.A) was contaminated with lake water to increase level of microorganism in natural flora according to chilling system uses (4 5 % Propylene Gl ycol to 5 5 % Lake Water). Lake water (FSHN Lake Gainesville, FL ) was obtained at 04/25/2013. Then it was incubated at 30C up to 5 days. 4.1.6 Statistical Analysis The data obtained was analyzed using a statistical analysis system (Graph Pad Prism 5.0 ). Analysis of variance (oneway ANOVA) was performed and the significant differences in the means were separated using the Tukey s studentized range test. The data was tabulated as an average of triplicates standard deviation, and the level of significa nce was determined at P < 0.05. 4.2 Results and Discussion Several treatment methods in industry are used to prevent and reduce microbial contamination in food processing equipment These methods range from chemical treatments to heat and other nonthermal methods. However, t o remove the source of microbial contamination effectively without chemicals, there is a need for novel

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54 technolog ies. One example of a novel technology is the use of P L as a treatment Use of PL has been approved to be more effective on inactivation of pathogens. It is also relatively effective on simple surfaces with 13 log10 on complex surfaces like meat. T he P L treatment was also used against P. aeruginosa biofilms growing on steel and helped to improve the efficiency of gentamicin against the same biofilms ( Huang and others 1988) Here, the effect of PL on the microflora of PG was studied by measuring the reduction of APC. Specifically, t h e effects of treatment time, sample volume and distance f rom quartz window were studied as to their effects on APC. The initial average APC obtained was approximately 6. 5 log10 CFU/mL for control groups grown at 30.6C T he complete inactivation time was found to be 20 s of PL for the 10 mL PG sample whereas 10 s of PL treatment was adequate for in activation for the 5 mL sample for samples treated 6 cm from the quartz window o f the UV lamp (Table 41) For the 15 mL sample, the maximal log reduction occurred at 25 s of PL but it may have been further reduced if t reatment times were extended. These results corresponded to temperature increase s of the sample of 33.4 2 0 .1 and 16.7 C at 20 s for the treatments in the 5,10 and 15 mL sample, r espectively suggesting temperature effects 21C ( Figure 3 5 ) W hen samples were placed at 9 cm distance from quartz window a slight drop in the log reduction occurred for the 10 mL and 15 mL samples at 20 s treatment time s when compared to samples that were placed 6 cm from the quartz window under the same treatment time (Table 4 1 v s.T able 42 ) For example, the 15 mL sample treated with 20 s of PL dropped from a 5.9log10 reduction at the 6 cm treatment to a 4.7 log10 reduction at the 9 cm treatment. The temperature increases were lower at the 9 cm distance compared to the 6cm treatment in this case ( 22.2 C for 6 cm vs. 19.6C for 9

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55 cm; Figure 35 vs. Figure 3 6 ) suggesting that temperature may in part be part of the cause of the lower reduction in bac teria. However, there was still microorganisms on p late of PL treated sample (10 mL) at 9 cm distance after 20 s although there was an approximately 19 6 C increase in temperature. In addition, the poor penetration capacity of the light at the farther dist ance from the quarts window is also likely involved in the lower reduction of bac teria at the farther distance. Previously, the effects of PL on the inactivation E. coli and S. enteritidis at 5 to 100 pulses with a range of 200530 nm at different energy l evels was studied in effects of pulse light on reduction in microflora of milk ( Ghasemi a nd others 2003). For E. coli a 9 log10 order reduction occurred after treatment with 100 pulses of 9 J but for a lower energy pulse of 4.5J at 100 pulses there was less of a log reduction (7 log10 reduction). In addition, a fewer number of pulses (5) even at higher energy (9J) resulted in a low log reduction (0.5 log10 reduction). For S. enteritidis 6 log CFU/mL was observed after 20 s treatment times. These results suggest that the high energy per pulse is heavily responsible for the reduction in bacteria, but requi re a minimum amount of pulses. In another study, when microflora of milk ( 3 mL) was treated for 1, 2, and 4 min of PL a 0.3, 3.4 and 8.4log10 CFU/mL redu ctions in bacteria were obtained, respectively (Krishnamurthy 2006). I nactivation of microorganisms in PL is not only related to the increase in temperature because many organism s have high heat resistance (D value Heat Resistance ; Sungur 1994) but ar e inactivated under PL. For t hese high heat resistance organisms, the complete inactivation will require a hi gh dose of UV treatment (Walker 1984).

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56 Additionally, a bsorption of energy from the light depends on the characteristic of the bacteria and the surrounding medium; therefore, bacteria may also be over heated in different amounts (Fine and Gervais 2004). There might be some changes, which may lead to membrane destruction in the cells such as steam flow and vaporization. In accordance with the literature, our results sugg ested that reduction of APC is likely based on photothermal and photophysical effect of PL besides the thermal effect Moreover, o v erheating of the bacteria is based on t he differences in UV light absorption by microorganism and surrounding medium; thus, microorganisms become a local vaporization center and may generate a small steam flow performing membra ne destruction (Takeshita 2003) Although the thermal effect plays a vital role at extended treatments with PL (>5 s treatment time), it does not have a significant effect on shorter treatments. In this study, use of PL resulted in reduction of APC for 5 s although no significant incre ase in temperature for all sample volumes (<10 s) at 6 and 9 cm distance from quartz window (Figure 3 5 and 3 6 ) was observed. However, it may not be only related to increase in temperature. Also, it was investigated that PL was effective on killing microorganisms, which was a result of PL damage on DNA structure of microorganisms and also to bacteria cells because of absorption of energy of light by bacteria cells (Giese and others 2000; Wekhof 2000). T he present study clear ly demonstrates the potential of PL for APC inactivation in PG and that c omplete reduction can be achieved with PL while increase in temperature (within seconds) Thus, PL treatment can be used as an alternative to thermal and chemical sterilization for mi croflora of PG cooling medium.

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57 It was observed that increasing treatment time or decreasing sample volume the log10 reduction rate of APC increased regardless of distance from the quartz window (Table 41 v s. Table 42 ) However, decreasing the distance of the sample from the quartz window increased the effect of PL on the inactivation of APC.

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58 Table 41. Reduction of Aerobic Plate Count in propylene glycol after Pulsed UV Light Treatment with in increase in temperature for 6 cm distance from quartz window Treatment Time (s) Reduction (Log CFU/mL ) 5 m L 10 m L 15 m L 5 3.7 0.0 1.8 0.0 10 5.5 0.9** 3.0 0.0*** 15 6.5 0.00*** 5.6 0.8*** 4.3 0.0 *** 20 6.5 0.00*** 6.5 0.0*** 5.9 1.1 *** 25 6.5 0.00*** 6.5 0.0*** 6. 5 0.0*** One Way ANOVA followed by Tukeys test. P <0.05, ** P <0.01, *** P .001 vs 5 mL: Data are expressed as mean standard deviation (SD). N=3. CFU: Colony forming unit

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59 Table 42 Reduction of Aerobic Plate Count in propylene glycol after Pulsed UV Light Treatment with in increase in temperature for 9 cm distance from quartz window Treatment Time (s) Reduction (Log CFU/mL ) 5 m L 10 m L 15 m L 5 3.3 0.1 1.4 0.1 10 4.7 0.0** 3.0 0.0* 2.6 0.0*** 15 5.7 0.7*** 4.7 0.0*** 3.6 0.0*** 20 6.5 0.0*** 5.6 0.8*** 4.7 0.0*** 25 6.5 0.0*** 6.5 0.0*** 6.1 0.1*** One Way ANOVA followed by Tukeys test.* P <0.05, ** P <0.01, *** P <0.001 vs mL: Data are expressed as mean standard deviation (SD). N=3. CFU: Colony forming unit Increase in temperature presents average for all sample volumes.

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60 Figure 41 Aerobic P lat e C ount on the 10 mL sample volum e plates after 20 s exposure at 6 and 9 cm distance ( Photo courtesy of Samet Ozturk)

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61 CHAPTER 5 EFFIC ACY OF PULSED UV LIGHT ON INACTIVATION OF THERMAL RESISTANCE BACTERIA IN MICROFLORA OF PROPYLENE GLYCOL The thermal resistance of bacteria has been expressed with well known concepts of D and z values, which are the required time (in seconds) to reduce bacteria almost 90% (D Value) and the increase in temperature (in kelvin) to provide a 90% reduction in D value (z Value) respectively (Hansen and other 1963). Also the values of D and z can express inactivation of bacteria. Previous research has examined the resistance of pathogenic bacteria to heat such as E. coli O157:H7 (Ahmed and others 1995; Kotrola and others 1997), Salmonella strains (Roberts and others 1996), and Listeria monocytogenes (Farber and other 1990; Roberts and others 1996). Most of these studies have been performed using homogenized foods or liquid media. The heat resistance of lactic acid bacteria (LAB) has been widely examined due to its im portance to the food industry Franz and other (1996) investigated that heat resistance of meat spoilage for Lactobacillus in vitro with D values at 57, 60 and 63C for 52.9, 39.3 and 32.5 s treatments, respectively In this study, we investigated the efficiency of PL on inactivati on of LAB in propylene glycol. 5.1 Material and Methods T he 5, 10 and 15 mL of treated PG samples were sequentially diluted and plated on the de man, rogosa and sharpe ( MRS ) agar at pH 4.8 (Fisher Scientific, Pittsburg, PA, U.S.A ). After the diluting and plating process the samples were autoclaved, and then LAB strain isolation was carried out with adjusted pH to 4.8 with 10% citric acid. Plates which contained 100 mg/L cycloheximide (Fisher Scientific, Pittsburg, PA, U.S.A) to i nhibit growing of microorganism such as yeasts and other fungi w ere then incubated anaerobically at 30C for 3 5 days.

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62 5.1.1 Pulsed Ultraviolet Light Treatment Propylene Glycol (PG) samples were treated wit h PL as described in Chapter 3. 5.1.2 Colony Forming Unit The colony forming of units is an estimate to use for determining the number of viable bacterial cells in a sample per mL or per gram. Hence, it tells the degree of contamination in samples of water, vegetables, soil or fruits or the magnitude of the infection in humans and animals (FDA 2012). To ob tain the LAB count, duplicate or triplicate samples from the same dilution, which produces 25 to 300 colonies were plated. I n short, 10 fold dilutions were employed in the rinse solutions (1/10, 1/100, 1/1000) initi ally. The PL treated and untreated samples (1 mL) were vortexed and tran sferred to dilutions tubes (9 mL PW) a using sterile pipette tip for each transfer. Then, 1 mL portion of dilutions was transferred to empty plates and 910 mL plate count agar was poured. After this process, plates were incubated at 48 3 h at 35C1C. To calculate the LAB the total numbers of colonies counted were multiplied by the reciprocal of the dilution factor on the plate. 5.1.3 Evaluation of PL Effi cacy The level of microbial inacti vation (Log [N/No]) was calculated by subtracting the survivor counts (N) resultant from PL treatment from the initial counts (No) represented by the control samples. Results were expressed in log CFU/mL Additionally survivor curves were built by creating scatter plots of log survivor counts (N) versus treatment time (t).

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63 5.1.4 Statistical Analysis The data obtained was analyzed using a statistical analysis system (Graph Pad Prism 5.0 ). Analysis of variance (oneway ANOVA) was performed and the significant differences in the means were separated using the Tukey s studentized range test. The data was tabulated as an average of triplicates standard deviation, and the level of significa nce was determined at P < 0.05. 5.2 Results and Discussion In order to demonstrate efficiency of PL on inactivation of heat resistance bacteria in liquid medium, PL had been applied to inactivate LAB cells in PG samples up to 25 s treatment times at distance of 6 and 9 cm from the quartz window of the lamp The initial LAB count obtained was 3.9log10 CFU/mL fo r control groups at 30.6C The efficiency of PL on inactivation of LAB in PG samples is reported based on exposure time, sample thickness and distance from the quartz window (Table 51 and 5 2). In accordance with the literature, when distance from the light source decreased and exposure time increased concomitantly, the samples receive more intense treatment ( Gmez Lpez and others 2005a) Hence, as it can be seen in Figure 37 and 3 8, higher increase in sample temperature could be expected with extended exposure period at shorter distance fro m the quartz window. The temperature rise during PL treatment is also strongly dependent on propert ies of samples such as viscosity and color ( Oms Oliu and others 2010) but these were not factors studied here. As shown in Table 51, the complete inactivation for 5 mL sample was observed after 10 s PL treatment at 6 cm distance from the quartz window and this corresponded to an increase in temperature in the sample of 17C ( Figure 35 ). After 10 s treatment of PL there was still countable LAB colonies in 15 mL PG sample likely due to the

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64 increased sample thickness and poor penetration of pulse light into the sample (Figure 3 5 vs. 3 6) T he sample depth and distance from the quartz window both had significant impact on survival (p<0.05). No significant temperature increase was observed for the first 10 s between all sample volumes (Figure 35) ; however, when the treatment duration continued after several seconds there was a non linear increase in temperature until 20 s at 6 cm distance ( Fig ure 35). In contrast, a linear increase was observed for all sample volumes for the longer treatments of 50 s (Figure 37 ). In the second study we observed that while increase in distance of sample surface from lamp (9 cm) had a significant (p<0.05) effect on inactivation of LAB (Table 5 2), there was no significant increase in temperature within shorter time treatment between sample volumes at 9 cm distance (Figure 37) As mentioned in C hapter 3 the inactivation of LAB was not likely relevant to thermal effect because of no significant increase in (Figure 3 6 ). Also these increases in temperature is likely significant on reduction of some microorganism which are not resistance to heat treatment. Krishnamurthy (2006) investigated effectiveness on inactivation of S. aureus. The maximum log reduction o f S. aureus (8. 6 log10 CFU/mL) was observed in 30 mL of milk for 180 s at 8 cm sample distance from quartz window (Krishnamurthy 2006). The water content of bacteria vaporized while leading to bacterial disruption and ultimately inactivation (Takeshita and others 2003). In Aspergillus niger spores, the overheating due to internal explosion and resulted in evacuation of the cell contents during the light pulse. In this study, we did not observ e any significant evacuation for PG samples because of its density and also high boiling point ( 187.4C). However, while thickness of samples was kept shallow, the effectiveness of PL on inactivation of heat resistance

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65 LAB in microflora of PG was investigated with short time PL treatment at different distances (6 a nd 9 cm) and also different volumes of PG samples (5,10 and 15 mL ) As mentioned Chapter 1, PL is known as novel technology, which is limited only for short time treatments. The absorption of energy for longer treatments cause increase in temperature. Puls ed UV light treatment is considered non thermal, but holds only for short time treatments. Temperature increases as absorbed energy accumulates during longer treatments. For 20 C increase while 5 s treatment time inc C in all sample volumes at 6 and 9 cm distance from quartz window, respectively Finally, our results demonstrated that the effect of treatment duration and the interaction (treatment time*depth) have significant (p<0.05) effect on i nactivation of LAB in PG samples A complete inactivation was obtained for samples treated with 25 s for all sample sizes. The average corresponding log10 reduction was 3.92log10 CFU/mL (Figure 5 1).

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66 Table 51. Log reduction of lactic acid bacteria in propylene glycol during P ulsed UV light treatment with in increase in temperature for 6 cm distance from quartz window Treatment Time (s) Reduction (Log CFU/mL ) 5 m L 10 m L 15 m L 5 3.4 0.8 2.4 0.0 1. 6 0.0 10 3.9 0.0 3.5 0.8* 2.8 1.0 15 3.9 0.0 3.9 0.0** 3.5 0.8* 20 3.9 0.0 3.9 0.0** 3.9 0.0** 25 3.9 0.0 3.9 0.0** 3.9 0 .0** One Way AN OVA followed by Tukeys test. *P<0.0 5, ** P P <0.05 vs 5 mL. Data are expressed as mean standard deviation (SD). CFU: Colony forming unit

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67 Table 52. Log reduction of lactic acid bacteria in propylene glycol during P ulsed UV light treatment with in increase in temperature for 9 cm distance from quartz window Treat ment Time (s) Reduction (Log CFU/mL ) 5 m L 10 m L 15 m L 5 2. 8 1. 1 2.0 0. 1 1 0 .0 10 3.9 0.0 3 0 0.8* 2. 1 0.0*** 15 3.9 0.0 3. 5 0.8*** 3.5 0. 8 *** 20 3.9 0.0 3.9 0.0*** 3.9 0.0*** 25 3.9 0.0 3.9 0.0*** 3.9 0.0*** One Way ANOVA followed by Tukeys test. *P<0.05, ** P <0.01, *** P <0.001 vs 5 seconds and P P Data are expressed as mean standard deviation (SD). CFU: Colony forming unit

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68 Figure 51 The formation of lactic acid b acteria colonies in the untreated and treated samples for 25s of PL (all samples volumes were similar) ( Photo courtesy of Samet Ozturk)

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69 CHAPTER 6 CONCLUSIONS T he main focus of this study was to investigate the potential effic acy of PL on temperature profile and microflora of PG cooling medium A ll these findings demonstrated that PL treatment resulted in reduct ion in microflora and increase in temperature of PG C onsistent with previous studies ( Krishnamurthy 2006), this effect d epended on sample thickness, which affects the penetration depth of PL, distance from the quartz window and exposure time. As the sample volume increases, the inactivation ratio decreases because of poor penetration of the PL into the sample. Although t her e was no significant temperature rise for all volumes after 10 s treatment times the inactivation of APC and LAB in PG microflora was achieved, indicating a nonthermal effect of PL. Thus it may be concluded that the inactivating effect of PL on microorga nisms was via its photochemical and photothermal properties in shorter time course (<10 s). On the other hand, at the shorter times (<10 s) the temperature increase, although not significant, may have a considerable effect on some species like p sychrophi lic bacteria since they and their enzymes are sensitive to lower temperature increases The effect of PL is also related to the microorganism type, i.e. Boeger (1999) demonstrated the Cryptosporidium parvum, a protozoa of major concern in water which is less resistant than Bacillus subtilis spores, was more susceptible to PL. T here was a significant increase in the temperature in the 5 mL PG sample at 15 s indicating that extended exposure time caused this high increase in temperature. Others also have reported an increase in temperature when treatment time increased (Demirci and others 2003). There was a greater increase in temperature as the sample was located closer to light source (6 vs. 9 cm). Based on these studies, it recommended that at 6 cm

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70 dis tance, 25 s treatment times showed complete reduction for APC and LAB in PG microflora. H owever, at 9 cm distance from the quartz window 25 s treatment times was not sufficient in complete reduction of APC bacteria. It is likely due to the poor penetratio n or lower thermal effect. For LAB, the treatment time of 25 s for either 6 or 9 cm distance from the light source was sufficient for complete reduction of bacterial population in PG microflora but reduction levels of both distance had differences Therefo re, further studies are required to determine exact treatment times based on type of contamination, characteristic of microorganism, location of sample from the light source, sample thicknesses and also thermal properties of sample.

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71 CHAPTER 7 RECOMMENDATION The se results may encourage future studies, which may be conducted with initial microbial load, different microorganism target s, repair mechanism s of microorganism s and their impact on quality and safety of food and devices. The initial population of APC and LAB in PG may not be representative of real life situations since the degree of contamination on food, surface or equipment tends to be lower. T he efficiency of PL treatment may be associated level of contamination on food samples, and also inappropriate environmental conditions which may contribute to light attenuation. Moreover, under inconvenient environmental conditions and handling methods microorganisms may accumulate and attach to surfaces forming biofilms making their inactivation more and more diff icult. A more detailed research needs to be done to find an optimum condition for the evaluation of inactivati ng effects of PL for continuous flow conditions to represent commercial cases. While setting up a light source of PL around the tubes, which PG is inside, to treat used PG after circulation in the system. Based on this system, the dimer of tubes, permeability of tubes according to light and location of light source are needed to be determining before to apply in industry. Furthermore, the efficacy o f the PL can be compared to equiv alent energy continuous UV light and infrared heating to conventional heating in order to demonstrate potenti al effects of PL on PG microflora. Also, future studies need to evaluate t emperature profiling during PL illuminat ions using a fiber optic sensor or infrared thermal imaging technique fo r static or continuous systems.

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72 In conclusion this study indicates that PL treatment may be an alternative technology to prevent contamination and also remove formed biofilms in or on cl osed system for future studies.

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73 LIST OF REFERENCES Aas P, Randall E, Hicks. 1993. Biodegradation of Organic Particles by surface and Benthic Nepheloid Layer Microbes from Lake Superior. Journal of Great Lakes Research 19:31021. Anonymous. 1989. Back to basics: The use of ultraviolet light for microbial control. Ultrapure water 4:62 8. AOAC. 1990. Total Plate Count. Official Methods of Analysis of AOAC International 15th ed. Gaithersburg, MD: AOAC International. p 105. AOAC. 199 5a. Aerobic plate count in foods: Dry rehydratable film. Sec. 17.02.07, Method 990.12. In Official Methods of Analysis of AOAC International 16th ed. Gaithersburg, MD: AOAC International. p 101. APHA. 1984. Compendium of Methods for the Microbiological E xamination of Foods 2nd ed. Washington, DC: American Public Health Association. p 663 81. Boeger JM, Cover WM, McDonald CJ. 1999. PureBright sterilization system: Advanced technology for rapid sterilization of pharmaceutical products, medical devices, pac kaging and water. Proceedings of Hypro99 Congress; 1999 November 2326; Wiesbaden, Germany. MessagoMesse GmbH, 1999. p 36574. Breslin WJ, Cieszlak FS, Zablotny CL, Corley RA, Verschuuren HG, Yano BL. 1996. Evaluation of the developmental toxicity of inhal ed dipropylene glycol monomethyl ether in rabbits and rats. Occup Hyg 2:16170. Chang JC, Ossoff SF, Lobe DC, Dorfman MH, Dumais CM, Qualls RG, Johnson, JD.1985. UV inactivation of pathogenic and indicator microorganisms. Appl Environ Microbiol 49: 1361 65. Charles AS, John WD. 2002. An examination of the physical properties, fate, ecotoxicity and potential environmental risks for a series of propylene glycol ethers. Chemosphere 49:6173. Choi LH, Nielsen SS. 2005. The effects of thermal and nonthermal processing methods on apple cider quality and consumer acceptability. Journal of Food Quality 28:13 29. Cover WH, Holloway, JM, Xue H, Busby TF editors. PPB 2001. Inactivation of lipid enveloped and nonenveloped viruses in human plasma proteins with broad spec trum pulsed light. The 2nd Plasma Product Biotechnology Meeting. 2001 May 1418; island of Malta. Plasma Sci; 2001. Demirci A, L Panico. 2008. Pulsed ultraviolet light. Food Science and Technology International 14:4436.

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74 Dentinger B, Faucher KJ, Ostrom SM, Schmidl MK. 1995. Controlling bacterial contamination of an enteral formula through the use of a unique closed system. Nutrition 11:74750. Dow Chem i cal Company Report (DOW). 2005. Propylene Glycol. Available from: http://www.dow.com/propyleneglycol. [Acc essed 2012 April 15] Dow Chemical Company Report (DOW). 2012. Propylene Glycol. Available from: http://www.dow.com. [Accessed 2012 March 21] Dunn J, Bushnell A, Ott T, Clark, W.1997. Pulsed white light food processing. Cereal Foods World 42:51015. Dunn J, Ott, T,Clark W. 1995. Pulsed light treatment of food and packaging. Food Technol 49: 95 8. Dwyer DF, Tiedje JM.1983. Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia. Appl Environ Microbiol 46:185 90. Eichler B, Schink B.19 85. Fermentation of primary alcohols and diols and pure culture of syntrophically alcohol oxidizing anaerobes. Arch Microbiol 143:60 6. Essers L. 1982. Simple identification of anaerobic bacteria to genus level using typical antibiotic susceptibility patterns. J Appl Bacteriol 52:31923. Facklam R, Elliot JA. 1995. Identification, Classification, and Clinical Relevance of CatalaseNegative, Gram Positive Cocci, Excluding the Streptococci and Enterococci, Clinical Microbiol. Reviews 8:479 95. Farrow JAE Phillips BA, Collins MD. 1986. Nucleic Acid studies on some heterofermentative lactobacilli; Description of Lactobacillus malefermentatans sp.nov. and Lactobacillus parabuchneri sp.nov., FEMS Microbiol Letters 55:1638. Feuilloley MGJ, Bourdet G, Orange N. 2006. Effect of white pulsed light on Pseudomonas aeruginosa culturability and its endotoxin when present in ampoules for injection. Eur J Parenteral Pharm Sci 11: 37 43. Fine F, Gervais P. 2004. Efficiency of pulsed UV light for microbial decontaminatio n of food powders. J Food Prot 67: 787 92. Food and Drug Administration. 2001. Bacteriological Analytical Manual Chapter 3 Aerobic Plate Count. Available from: http://www.fda.gov. [Accessed 2012 April 4] Food and Drug Administration. 2012 .Propylene glycol; SCOGS Report Number: 27. Available from: http://www.fda.gov. [Accessed 2012 March 17]

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80 BIOGRAPHICAL SKETCH Samet Ozturk was originally from Sivas, Turkey. He received his bachelors degree in food engineering from Gaziosmanpasa University in 2009. After a oneyear of industry experience, h e received a scholarship from the Ministry of Education of Turkey in 2010, which gave him the opportunity to study abroad, in the U.S. He attended to UC Davis Intensive Engl ish Course before he entered a Food S cience Master of Science program at the University of Florida under the supervision of Dr. Wade Yang. During his matriculation in his Masters program Samet presented his research at the i nstitut e of Food Technologists meeting, LA, Nevada in 2012. Samet plans to continue his academic journey by pursuing his Ph .D. degree upon his completion of his masters program