COMBINATION OF ULTRA-HIGH PRESSURE AND -DERIVATIVES TO INACTIVATE FOOD-BORNE SPOILAGE AND PATHOGENIC BACTERIA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Joy Gail Waite, M.S.

* * * * *

The Ohio State University 2007

Dissertation Committee: Approved by Professor Ahmed E. Yousef, Adviser

Professor Steven J. Schwartz ______Professor MacDonald Wick Adviser Food Science and Nutrition Graduate Program Professor Jeff Culbertson

ABSTRACT

Food processing methods can lead to the development of processing-resistant spoilage and pathogenic microorganisms that can cause further cost to processors and increased health risks to consumers. Thus, inactivation of these potentially problematic bacterial strains by combinations of physical and/or chemical treatments needs to be investigated. Previous studies have shown combination treatments of ultra-high pressure

(UHP) with antimicrobial peptides, oxidants, and antioxidants to be efficacious at inactivating bacteria. The current study investigates the use of UHP in combination with hydroxyxanthenes, including FD&C Red No. 3, to inactivate pressure-resistant strains of

Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli O157:H7.

Combination treatments of UHP (400 MPa, 3 minutes) and FD&C Red No. 3 (3 to 10 ppm) resulted in significant enhancement of inactivation of several pressure-resistant

Gram-positive and Gram-negative strains. FD&C Red No. 3 is a known photosensitizer and was capable of inactivating Gram-positive species with short exposure to ambient light (15-30 minutes); however this compound had no effect on Gram-negative species without UHP treatment. UHP was found to cause an irreversible change in the barrier properties of the outer membrane with pressure treatments above 250 MPa leading to accumulation of FD&C Red No. 3 correlating with cell inactivation. Inactivation of

Gram-positive and Gram-negative species by combination treatment was light-dependent

ii

with low UHP treatments (<400 MPa), indicating a role of photooxidation. However,

with increasing pressures (>400 MPa) a light-independent mechanism was identified for

all species tested. Efficacy of light-dependent and light-independent inactivation was

determined in the food systems: carrot juice and turkey meat . Combination

treatment was effective against L. monocytogenes and E. coli O157:H7 in carrot juice

with FD&C Red No. 3 at concentrations of 10 to 100 ppm. These strains were resistant

to inactivation by combination treatment in turkey meat product, indicating that specific

food components or composition may decrease the efficacy of these treatments. Further

studies were completed to determine the impact of superoxide production on the

inactivation of E. coli K12 wild-type and sod mutants. Mutant strains were not

significantly different in sensitivity to any of the treatments tested, however, all E. coli

K12 strains were significantly more sensitive to UHP and UHP-FD&C Red No. 3

combination treatments when treated and recovered under aerobic conditions compared

to anaerobic conditions. Pressure-resistant strains were treated with UHP alone and in

combination with FD&C Red No. 3 under aerobic and anaerobic conditions to determine

the impact of type I and type II photooxidation on inactivation. The majority of

inactivation by combination treatment with light exposure is due to type I photooxidation

based on little difference between aerobic and anaerobic treatments. Interestingly,

inactivation without light exposure was oxygen dependent for all microorganisms tested.

Further study is needed to determine if oxygen dependent effect is due to oxygen presence during treatment or during recovery. Inactivation of UHP processing-resistant microorganism can be achieved using combination treatment of UHP and FD&C Red No.

3 in food systems. iii

ACKNOWLEDGMENTS

I would like to take this opportunity to thank the people that have made this

research and my education at The Ohio State University possible. First and foremost, I

would like to thank Dr. Ahmed Yousef for sharing his unbelievable wisdom and passion

for research and his unwavering trust in my abilities. Your support has allowed me to

grow into a better researcher and teacher; I only hope to possess a small fraction of your

mentoring abilities and compassion for others. Secondly, I would like to thank the members of the Yousef lab for their friendship, encouragement, and wisdom: Yoon-

Kyung Chung, Zengguo (France) He, Luis Rodriguez-Romo, Mohammed Khadre, Aaron

Malone, Erin Horton, Mustafa Vurma, Shara Johnson, Xueying Zhang, Yuan Yan,

Jennifer Perry, Amrish Chawla, and Joe Jones. I would like to specifically thank Joe

Jones for being a wonderful person and friend and having an extraordinary ability to learn from someone as disorganized as myself. Thank you, Joe, for making dilution blanks and liters upon liters of media for me; but above all for all of the hard work and time spent on the salad dressing project, you are a life-saver, or more appropriately a project-saver.

I would like to thank my committee members, Dr. Steven Schwartz, Dr.

MacDonald Wick, and Dr. Jeff Culbertson for their patience through the latter stages of

iv

this process. Thank you Dr. Schwartz and Dr. Wick for teaching graduate courses that

were thought-provoking and educational. Thank you to Dr. Culbertson for listening to

my crazy rants about teaching problems.

A special thanks to James Douglass, Richard Nist, Dan Aruscavage, and Michele

Manuzon for all of the help with FST/MICRO 636.02, putting up with my rantings, and laughing together over “maximum penetration number” and “Asperilla”. Extra special

thanks to Matt Mezydlo for making the term projects work; they would have failed

without you.

I would especially like to thank the extremely close friends I have made over the

last few years: Mary Kay Folk, Jennifer Ahn-Jarvis, April Wax, Julie Jenkins, Nurdan

Kocaoglu-Vurma and Robert King. You have made the time go by quickly and have

helped distract me when necessary for me to retain my sanity.

Special thanks to my mother Sharon, my father Joe, and my sisters Laurie and

Mindy for believing in me and supporting my crazy ideas. Most importantly, I would

like to thank my son Allan. You are the best kid in the world, thank you for being a

wonderful, caring, and unique individual; I love you more than anything.

v

VITA

June 20, 1980………………………………………. Born, Florence, Oregon, U.S.A.

2002……………………...... B.S. Food Science and Technology, Oregon State University.

2004………………………………………………... M.S. Microbiology, Oregon State University

2004-present……………………………………….. Graduate Research and Teaching Associate, The Ohio State University

PUBLICATIONS

Research Publication

1. Waite, J.G., M.A. Daeschel. 2007. Contribution of wine components to inactivation of food-borne pathogens. J. Food Sci. 72(7):M286-M291.

FIELDS OF STUDY

Major Field: Food Science and Nutrition

vi

TABLE OF CONTENTS

Page Abstract...... ii Acknowledgements...... iv Vita...... vi List of Tables ...... ix List of Figures...... xi

Chapters:

1. Literature Review...... 1

1.1. Color and food ...... 1 1.2. Xanthene ...... 23 1.3. Photosensitizers and photooxidation ...... 36 1.4. Ultra-high pressure ...... 61 1.5. “Hurdle” Technologies ...... 75

2. Xanthene-derived food colorants sensitize processing-resistant foodborne pathogenic and spoilage bacteria to ultra-high pressure...... 96

3. Destabilization of the outer membrane of Escherichia coli by physical and chemical methods leads to inactivation by FD&C Red No. 3 ...... 119

4. Contribution of photooxidation to inactivation of Gram-positive and Gram-negative bacteria by ultra-high pressure and xanthene-derivatives...... 145

5. Inactivation of bacteria by ultra-high pressure and xanthene-derivatives: a secondary light-independent mechanisms...... 166

6. Efficacy of FD&C Red No. 3 and ultra-high pressure combination treatment against foodborne pathogens in food systems...... 168

7. Impact of aerobic and anaerobic treatment and recovery on inactivation of foodborne spoilage by FD&C Red No. 3 and ultra-high pressure combination treatment...... 205

8. Conclusions...... 232

vii

Appendix A: Partition coefficient determination of xanthene-derivatives...... 235

Appendix B: Preliminary results with FD&C colors...... 238

Appendix C: Selection and identification of a Listeria monocytogenes surrogate for optimization of ultra-high pressure and pulsed electric field process validation...... 240

Appendix D: Variability in barotolerance of Lactobacillus plantarum and Lactobacillus fermentum strains: impact of pressure dose, holding time and suspension medium ...... 268

Bibliography ...... 298

viii

LIST OF TABLES

Table Page

1.1 Selected food products colored with toxic compounds and adverse outcomes associated with ingestion of these colorants ...... 3

1.2 Original certified synthetic food colorants approved by the Food, Drug, and Cosmetic Act of 1906 ...... 5

1.3 Certified synthetic food colorants approved between 1916 and 1929...... 6

1.4 Certified synthetic food colorants approved since 1939...... 8

1.5 Major categories of processed food that use certified colors in their manufacture and corresponding color concentration levels...... 9

1.6 FDA specifications for certification of FD&C Red No. 3 and FD&C Blue No. 1...... 12

1.7 Currently certified FD&C colorants ...... 17

1.8 Chemical and physical properties of currently approved certified FD&C colorants...... 18

1.9 Characteristics of selected hydroxyxanthene compounds ...... 25

1.10 Variability in partition coefficients of hydroxyxanthenes reported by different researchers...... 30

1.11 Fluorescence and phosphorescence characteristics of hydroxyxanthenes...... 33

1.12 Quantum yields of fluorescence, triplet formation, and for selected hydroxyxanthenes ...... 54

3.1 Final molar concentrations of citrate-phosphate buffer components at various pH levels ...... 126

ix

6.1 Final molar concentrations of citrate-phosphate buffer components at various pH levels ...... 187

x

LIST OF FIGURES

Figure Page

1.1 Chemical structure of aniline...... 4

1.2 Basic structures of classes of xanthene dyes ...... 24

1.3 Chemical synthesis of and numbering of fluorescein molecule ...... 27

1.4 Chemical structures of fluorescein and phenolphthalein...... 31

1.5 Illustrated interpretation of photooxidation of a generic photosensitizer...... 38

1.6 Superoxide production pathways involving NADH as a result of -mediated photooxidation as proposed by Martin and Logsdon (1987)...... 41

1.7 Fenton reaction...... 42

1.8 Basic structures of synthetic and natural photosensitizers...... 49

2.1 Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli O157:H7 strains by ultra-high pressure treatment...... 103

2.2 Chemical structures of xanthene-derivatives used in this study ...... 104

2.3 Inactivation of Listeria monocytogenes OSY-328 by xanthene-derivatives (10 ppm) and/or ultra-high pressure treatment...... 105

2.4 Inactivation of Escherichia coli EDL 933 by xanthene derivatives (10 ppm) and/or ultra-high pressure treatment ...... 106

2.5 Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli O157:H7 strains by FD&C Red No. 3 ...... 108

2.6 Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli O157:H7 strains by combination FD&C Red No. 3 and ultra-high pressure treatment ...... 110

xi

2.7 Inactivation of Salmonella Typhimurium, , and Pediococcus acidilactici strains by Red No. 3 (10 ppm) with or without ultra-high pressure treatment ...... 112

3.1 Effect of ultra-high pressure treatment on inactivation of Escherichia coli EDL 933 by FD&C Red No. 3 added before or after pressure treatment ...... 130

3.2 Fluorescent microscopy images of Escherichia coli EDL 933 exposed to 10 ppm FD&C Red No. 3 during or after high pressure treatment...... 131

3.3 Illustration of density-dependent membrane isolation from Escherichia coli K12 treated with FD&C Red No. 3 (0-100 ppm) and with or without ultra-high pressure (500 MPa, 1 min)...... 133

3.4 Effect of pH on inactivation of Escherichia coli EDL 933 by FD&C Red No. 3. .136

3.5 Fluorescent microscopy images of Escherichia coli EDL 933 exposed to 10 ppm FD&C Red No. 3 at pH 7.0 or 3.0...... 137

4.1 Effect of FD&C Red No. 3 concentration and light exposure on Listeria monocytogenes ScottA, Lactobacillus plantarum OSY-104, and Escherichia coli EDL 933...... 152

4.2 Effect of FD&C Red No. 3 concentration, light exposure, and ultra-high pressure on Listeria monocytogenes ScottA, Lactobacillus plantarum OSY-104, and Escherichia coli EDL 933...... 154

4.3 Effect of light exposure on inactivation of Escherichia coli EDL 933 by FD&C Red No. 3 concentration and ultra-high pressure treatment ...... 156

4.4 Effect of light exposure on inactivation of Listeria monocytogenes OSY-328 by combination treatment of xanthene-derivatives and ultra-high pressure...... 158

4.5 Effect of light exposure on inactivation of Escherichia coli EDL 933 by combination treatment of xanthene-derivatives and ultra-high pressure...... 160

5.1 Effect of pressure level on inactivation of Listeria monocytogenes OSY-328 by FD&C Red No. 3 treatment with and without light exposure ...... 174

xii

5.2 Effect of pressure level on inactivation of Escherichia coli EDL 933 by FD&C Red No. 3 treatment with and without light exposure ...... 175

5.3 Inactivation of Listeria monocytogenes OSY-328 and Escherichia coli EDL 933 by combination treatment of xanthene-derivatives and ultra-high pressure without light exposure...... 178

6.1 Inactivation of Escherichia coli EDL 933 by combination pH, FD&C Red No. 3 and ultra-high pressure treatment...... 191

6.2 Inactivation of Listeria monocytogenes OSY-328 in carrot juice by combination FD&C Red No.3, light exposure, and ultra-high pressure treatment...... 192

6.3 Inactivation of Escherchia coli EDL 933 in carrot juice by combination FD&C Red No. 3, light exposure, and ultra-high pressure treatment ...... 194

6.4 Inactivation of Escherichia coli EDL 933 in turkey product by combination FD&C Red No. 3, light exposure, and ultra-high pressure treatment...... 195

7.1 Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure alone (500 MPa, 1 min) ...... 214

7.2 Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure (500 MPa, 1 min) and FD&C Red No. 3 (10 ppm) with no light exposure...... 215

7.3 Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure (500 MPa, 1 min), FD&C Red No. 3 (10 ppm) and 30 minutes light exposure following pressure treatment...... 216

7.4 Comparison of efficacy of ultra-high pressure (500 MPa, 1 min) combination treatments and aerobic versus anaerobic treatment and recovery by compiling inactivation of Escherichia coli wild-type and sod mutants...... 217

7.5 Aerobic versus anaerobic treatment and recovery of Lactobacillus plantarum MDOS-32 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min)...... 219

xiii

7.6 Aerobic versus anaerobic treatment and recovery of Listeria monocytogenes OSY-328 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min)...... 221

7.7 Aerobic versus anaerobic treatment and recovery of Escherichia coli EDL 933 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min)...... 223

7.8 Comparison of efficacy of ultra-high pressure (500 MPa, 1 min) combination treatments and aerobic versus anaerobic treatment and recovery by compiling inactivation of Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli inactivation...... 224

xiv

CHAPTER 1

LITERATURE REVIEW

In the process of performing an unrelated ultra-high pressure (UHP) experiment,

red food coloring was used as a tool to verify the homogenization of a lactic acid

bacterium inoculum into a ranch-style salad dressing. Following UHP treatment of these samples, inactivation of the inoculum was significantly higher than expected. Based on this exceptionally poor recovery, further experiments were performed to determine if food colorants alone, or in combination with UHP, could inactivate important food-borne spoilage and pathogenic bacteria.

1.1 Color and Food

Color is ingrained in people’s minds as an extremely important part of everything in the world. The colors of food products are one of the first sensual perceptions that a consumer has with a food product (125). Traditionally, people have used the natural color of a fruits and vegetables to determine ripeness and thus palatability of these items.

As the developed world has changed from a primarily agrarian society to an industrialized society, the majority of food products are processed to maintain their quality during processing, transport and, ultimately, consumption. Due to the elongated time between the farm and the table, several of these factors contribute to lower color 1

quality in food products. Therefore, food coloration is necessary for the following reasons: products have weak coloration that is unacceptable by the consumer, the color of the product is not uniform due to changes in raw materials, the color of the product is degraded during processing and/or storage conditions, or the inherent color of the product is colorless or unappealing (125). As more “fun” foods have been developed, color has been increasingly associated with particular flavors (50).

History of Food Coloration and Regulations

Humans have been fascinated with colors throughout history. There is evidence of drug colorants in ancient Egyptian writings (123). The use of the first food colorants began around 1500 B.C. Original food colorants were derived from substances found in nature (i.e., tumeric, paprika, and saffron) (123). Since the 1300s, butter has been colored yellow (62).

As knowledge of chemistry and color increased, synthetic colorants were developed in the laboratory. By analyzing different colored materials, it was discovered that certain atomic groups (chromophores) favor the absorption of certain wavelengths of light, thus imparting color. Specificity of absorption of certain wavelengths is caused by the presence of non-localized electrons (π-electrons) in the molecule. These electrons can be easily energized to higher energy levels by the absorption of visible light.

Common chromophores are azo groups, nitro groups linked to benzene rings, and systems of numerous conjugated bonds (125). In addition to the chromophore, the presence of an electron dispatching group (auxochrome) and an electron attracting group

(antiauxochrome) to the end of the molecule, increases the color effect of the molecule.

Examples of auxochrome groups are hydroxyl groups or amino groups. Examples of 2

antiauxochrome groups are nitro groups or carbonyl groups. Other functional groups,

like sulfa-acid groups enhance the water solubility of the color compound (125).

The driving forces for the change from natural food colorants to synthetic were

simple: ease of production, cost, superior coloring properties, superior blending

properties, amount needed, and the absence of undesirable flavors. In the 1800s, food

manufacturers colored products with potentially poisonous mineral- or metal-based

compounds. Table 1.1 displays some of the instances of foods that had been colored with

toxic compounds. Beginning in the late 1800s, the bulk of synthetic colors were derived from a toxic petroleum product, aniline (Figure 1.1). These aniline-derived dyes were

Food Product Colorant Outcome of Consumption Pickles Copper sulfate Illness and death Cheese Vermillion (HgS) and Red Lead Illness (Pb3O4) Used tea leaves for Copper arsenite, Lead chromate No report of outcome resale and Indigo Candies Lead chromate, Red Lead, No report of outcome Vermillion, and White Lead (basic lead carbonate) “Green” pudding Copper arsenite Death

Table 1.1. Selected food products colored with toxic compounds and adverse

outcomes associated with ingestion of these colorants (Adapted from National

Research Council 1971 (118)).

3

Figure 1.1. Chemical structure of aniline (1).

originally named coal-tar colors because the starting material was bituminous coal (123).

In 1900, approximately 80 synthetic color compounds were used in food products. These

compounds were not regulated and toxicology data and purity standards did not exist

(62).

The United States Food and Drug Act of 1906 allowed for the use of seven synthetic colors (Amaranth, Ponceau 3R, Erythrosine, Orange I, Naphthol Yellow S,

Light Green SF Yellowish and Indigo Carmine) for food use and established a voluntary certification program (65, 118). More information on these colorants is shown in Table

1.2. Between 1916 and 1929, ten additional dyes were approved for use in food products:

Yellow AB, Yellow OB, Tartrazine, Sunset Yellow FCF, Guinea Green B, Fast Green

FCF, Brilliant Blue FCF, Ponceau SX, Butter Yellow and Sudan I (65, 118). The Federal

Food, Drug and Cosmetic Act of 1938 made certification through the FDA mandatory for the fifteen approved dyes from the previous acts (except Butter Yellow and Sudan I, which had been delisted in 1918) (62, 118). More information on these dyes is shown in

Table 1.3. This Act also broadened the coverage of certification to three categories of colorants with appropriate abbreviations for ease of classification: colors used in foods, drugs, and cosmetics (FD&C); colors used in drugs and cosmetics (D&C); and colors used in externally applied drugs and cosmetics (Ext. D&C). This Act also

4

Structure Approved for external drugs and Japan in cosmetics Not approved elsewhere Approval Status Approval Elsewhere Approved for in use cosmetics most countries Approved for external drugs and Japan in cosmetics Not approved elsewhere Approved forfood use in mostother countries Approved forfood use in mostother countries Approved forfood use in mostother countries Approved for external drugs and Japan in cosmetics Not approved elsewhere and Cosmetics Act of 1906. g, the Food Dru y Approval in U.S. Status Approval Delisted forfood use in 1956. Delisted for exterior drugs and cosmetics use in 1968 Delisted forfood use in 1959 Permittedfor use in externally applied drugs and cosmetics Provisional listingterminated in 1966 – to due lackeconomicof importance Food, Drugs, Cosmetics and for dye Listing Permanent Provisional Listingfor lake Permanentlisting for indye food dye for listing Provisional applied externally in terminated drugscosmetics and Provisional listingfor Lake 1990 in terminated Provisional listingterminated in 1976 Petition forpermanent listing denied in1980 Provisional listingin food, drugs, andcosmetics terminated 1961 in roved b pp Synonyms FD&C Orange No. 1, Ext. D&C OrangeNo. 3, C.I. Acid Orange 20, JapanOrange #20, E111 FD&C Yellow No. 1, 1, No. FD&C Yellow Ext. D&C Yellow No. 1, Yellow Acid C.I. 7, Japan Yellow #403 FD&C Green No. 2, C.I. Food Green2, C.I. Acid Green 5 FD&C Blue No. 2, 2, No. Blue FD&C Indigotin, C.I.Food 1,Blue C.I. Acid Blue 74, JapanBlue 2, E132 FD&C Red No. 3, C.I. C.I. 3, No. Red FD&C Food Red 14, C.I. Acid Red 51, Japan E127 #3, Red FD&C Red No. 2, Red Food C.I E110, C.I6, Acid Red 27, Japan Red #2 FD&C Red No. 1, Ext. D&C Red No. 15, C.I. Acid Dye, Japan #502 Red 16155 Color Index Number 14600 10316 42095 73015 45430 16185 nthetic food colorants a y Chemical Group Chemical Abstracts Chemical (CAS) Service Number Trimethylaniline 3564-09-8 Azo 915-67-3 Xanthene 16423-68-0 Indigoid 860-22-0 Triarylmethane 5141-20-8 Nitro 846-70-8 Azo 523-44-4 inal certified s inal certified g Colorant (All listed in 1906) Orange I Naphthol Yellow S Light Green SF Yellowish Indigo Carmine Erythrosine Amaranth Ponceau 3R Table 1.2. Ori

5

Structure Approved in most other countries Unknown Approved for external drugs andcosmetics in Japan Not approved elsewhere Unknown Unknown Approved for external drugs andcosmetics in Japan Not approved elsewhere Approved in most other countries Approved in most other countries Approved for most in cosmetics other countries Approved forfood in most othercountries Approval Status Status Approval Elsewhere Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for dye Provisional Listing for lake Delisted 1959 in use food for Delisted for exterior drugs and cosmetics use in 1960 Delisted 1959 in use food for Delisted for exterior drugs and cosmetics use in 1960 Delisted within 6 months of approval Delisted within 6 months of approval Provisional listingterminated in 1966 – to due lackeconomic of importance Cosmetics and Drugs, Food, Permanent Listing for dye Provisional Listing for lake Cosmetics and Drugs, Food, Permanent Listing for dye Provisional Listing for lake Provisional listingterminated in 1976 Cosmetics and Drugs, Food, Permanent Listing for dye Provisional Listing for lake Approval StatusApproval in U.S. roved between 1916 and 1929. pp Synonyms FD&C Yellow No. 5, C.I. Food Yellow 4, C.I. Acid Yellow 23, Japan Yellow E102 #4, FD&C Yellow No. 3, Ext. D&C Yellow No. 9, Solvent Yellow 5 FD&C Yellow No. 4, Ext. D&C Yellow No. 10, C.I. Solvent Yellow6, Japan Yellow #405 Solvent Yellow14 Solvent Yellow2 FD&C Green No. 1, C.I. Acid Green 3, C.I. Food GreenJapan 1,Green #402 FD&C Green No. 3, C.I. Food Green3, Japan Green #3, E143 FD&C Blue No. 1, C.I. Food Blue 2, C.I. Acid Blue 9, Japan Blue #1, E133 FD&C Red No. 4, C.I. Food Red 1, Japan#504 Red FD&C Yellow No. 6, C.I. Food Yellow 3, Japan Yellow #5, E110 Color Index Number 19140 11380 11390 12055 11020 42085 42053 42090 14700 15985 nthetic food colorants a y Chemical Group Chemical Chemical Service Abstracts Number Azo 1934-21-0 Azo 85-84-7 Azo 131-79-3 Azo 842-07-9 Azo 60-11-7 Triarylmethane 4680-78-8 Triarylmethane 2353-45-9 Triarylmethane 3844-45-9 Azo 4548-53-2 Azo 2783-94-0 Table 1.3. Certified s Colorant Tartrazine Approved in 1916 Yellow AB Approved in 1918 Yellow OB Approved in 1918 Sudan I Approved in 1918 Butter Yellow Approved in 1918 Yellow Sunset FCF Approved in 1929 Guinea Green B Approved in 1922 FCFFast Green Approved in 1927 Brilliant Blue FCF Approved in 1929 Ponceau SX Approved in 1929

6

designated the colorants with specific numbers for ease of identification (64, 91).

However, the use of letter and number designations for the certified colorants was not mandatory. Four additional food colors were added to the permitted list between 1939 and 1950: Naphthol Yellow S (potassium salt), Oil Red XO, Orange SS and Benzyl

Violet 4B (64, 118). Additional information on these dyes is shown in Table 1.4.

Safety of food colorants was reconsidered in the early 1950s, when two occurrences of human toxicity arose. Black and orange Halloween taffy was implicated in a diarrheal outbreak and Orange I (FD&C Orange No. 1) was determined as the causative agent. Soon after the Halloween candy incident, a similar complaint was sent to the FDA about popcorn that had been colored with Oil Red XO (FD&C Red No. 32).

Due to these instances, interest peaked about the potential harmful effects of food colorants and extensive experiments into the toxicity of these compounds were initiated

(137).

The Color Additive Amendment of 1960 listed food colorants on a “provisional” list due to the lack of toxicology data on these compounds. This was particularly important because of the existence of the Delaney Clause, which prohibited the addition of known animal carcinogens to food products (62). This amendment enforced using the simplified nomenclature system for certified synthetic colorants. Each colorant would have an abbreviated number with the abbreviation of FD&C (i.e., FD&C Red No. 3).

This amendment also required food manufacturers to label their products with the term

“color added” if any colorants were included in the product (173). In 1971, it was estimated that the concentration of certified food colorants in the food supply was 9 parts per million 7

Structure Sameas No. Yellow salt 1, potassium Approved for Approved drugs and external cosmetics Japan in Not approved elsewhere for Approved in cosmetics use other most countries for Approved drugs and external cosmetics Japan in Not approved elsewhere for Approved in cosmetics use Poland and Thailand Not approved elsewhere in Not approved other countries Not approved in in Not approved other most countries in for use Approved other most countries Approval Status Elsewhere food to hot dogs hot food to Approval Status in U.S. Approval Delisted forfood use in 1956 Delisted and drugs for exterior cosmetics use in 1963 Delisted forfood use in 1959 Delisted and drugs exterior from cosmetics use in 1962 Delisted forfood use in 1955 Delisted and drugs for exterior cosmetics in 1963 Provisional listing terminated in 1973 Use as a denaturant in animal 1976 in terminated carcasses Restricted use in and sausagecasings Restricted use in foodorange to peels Food, Drugs, Cosmetics and Permanent Listing for and dye lake roved since 1939. pp FD&C Red No. 32, Ext. D&C Red No. Solvent C.I. 14, OrangeJapan 7, Red #505, Sudan II No. FD&C Yellow 2, Ext. D&C Yellow No. 8 No. Orange FD&C 2, Ext. D&C Orange No. 4, C.I. 2, Orange Solvent Orange Japan #403 FD&C Violet No. 6B 1, Violet C.I. Acid Orange 137 C.I. Solvent Red 80 Allura Red AC, C.I.Red 17, Food E129 Synonyms 12140 Color Color Index Number 10316 12100 42640 19235 12156 16035 nthetic food colorants a y Azo 3118-97-6 Azo 846-70-8 Azo 2646-17-5 Triphenylmethane 1694-09-3 Azo 6358-53-8 Azo 15139-76-1 Azo 25956-17-6 Chemical Group Group Chemical Chemical Abstracts Number Service Oil Red XO Red Oil 1939 in Approved Naphthol Yellow S, salt potassium 1939 in Approved SS Orange 1939 in Approved 4B Violet Benzyl 1950 in Approved B Orange 1966 in Approved FD&C Red No. 40 Red FD&C 1971 in Approved Citrus Red 2 1959 in Approved Colorant Table 1.4. Certified s

8

with the daily consumption estimated at 53.5 mg (118). Due to changes in lifestyle and

availability of more novelty food products, this concentration is likely an underestimate.

Example concentrations of food colorants used in various food products is shown in

Table 1.5. Good Manufacturing Practices (GMPs) limit the amount of food colorants that

may be added to a particular product (62, 102). Approximate usage levels of colorants in

a majority of food products in the European Union are 50-200 mg/kg (153).

The Nutrition Labeling Regulations of 1973 required the specific labeling of

FD&C Yellow No. 5 on the label (173). The Nutrition Labeling and Education Act of

1990 required that all food colorants must be listed separately in the ingredient statement by 1994 (62).

Color Concentration Category Range (ppm) Average (ppm) Candy and confections 10-400 100 Beverages 5-200 75 Dessert powders 5-600 140 Cereals 200-500 350 Maraschino cherries 100-400 200 Pet foods 100-400 200 Bakery goods 10-500 50 Ice cream 10-200 30 Sausage surfaces 40-250 125

Table 1.5. Major categories of processed food that use certified colors in their

manufacture and corresponding color concentration levels (Adapted from National

Research Council (1971) (118)).

9

Currently, the possibilities for food coloring are numerous: food with coloring

properties (red beets), natural colorants that do not exist in food (cochineal-carmine),

water-soluble synthetic colorants (certified artificial dyes) and their insoluble

counterparts (certified artificial lakes), and inorganic pigments (iron oxide) (125).

Certified food colorants

The FDA is responsible for regulating all color additives and ensuring that foods

containing color additives are safe to eat, contain only approved ingredients and are

accurately labeled. The permitted colors are classified as subject to certification or

exempt from certification. The synthetic food colorants are subject to certification. Food

colorants that are exempt from certification are derived from natural sources such as

plants, animals, minerals or man-made equivalents (62). The exempt colorants tend to

impart color in a less effective manner than the synthetic certified colorants and therefore

need to be used at higher concentrations (50). Regardless of this classification, the colors

are subject to rigorous safety standards prior to their approval and listing for food use

(62).

The Code of Federal Regulations, Title 21, parts 70 to 82 contains the approved

colorants for use in foods, drugs and cosmetics (169). For each colorant, the CFR

contains: purity specifications, application restriction, labeling requirements, obligatory

warning information and certification status (125). Each batch of a synthetic colorant has

to be certified through a process of official chemical determination to assure the safety,

quality, and consistency prior to food use (62, 154). Preparation and purification of safe certified food colorants is dependent on the purity of initial and intermediate compounds

used in its manufacture (8). Maximum limits of harmful contaminants, such as arsenic 10

and lead, were established and enforced in the certification process. Specifications for

certain components (volatile matter, water insoluble matter, ether extract, etc) have been

established for each individual dye. Additionally, certified dyes must contain an

established minimum pure dye percentage. Pure dye percentage is the percentage of the colorant molecule in the final product, by weight. Required pure dye percentages range from 82 to 89 percent for water-soluble colors, however most certified dyes contain 90 to

92 percent pure dye. Oil-soluble colors are required to have higher pure dye percentages

(95 to 98 percent) (64). As an example, the FDA specifications for FD&C Red No. 3 and

FD&C Blue No. 1 are shown in Table 1.6. The FDA certified almost 19 million pounds of color additives in 2006 (168).

Certification of dyes for use in foods, drugs, or cosmetics does not include microbiological testing. Historically, there have been recalls of cosmetic products by the

FDA due to the presence of pathogenic Gram-negative bacteria including Pseudomonas and members of Enterobacteriaceae. Sanitizing or sterilizing treatments of color additives are required to not affect the chemical, physical, or toxicological properties of the colorant. Dry heat results in decomposition of many colorants. Ethylene oxide is an effective antimicrobial treatment; however safety concerns have prevented widespread use of this technique. Jasnow and Smith (1975) found microwave treatment to be an effective means to reduce the microbiological population present in several certified colorants (66).

11

Specification FD&C Red No. 3 FD&C Blue No. 1 Pure dye 87.0% 85.0% Permitted range of organically 56.8-58.5% Not applicable combined iodine in pure dye (free from water of crystallization) Moisture Not more than 15.0% Not more than 10.0% Volatile matter (at 135°C) Not more than 12.0% Not more than 10.0% Subsidiary dyes Not applicable 6.0% Sodium acetate Not applicable Not more than 3.0% Sodium chlorides and sulfates Not more than 2.0% Not more than 4.0% Mixed oxides Not more than 1.0% Not more than 1.0% Sodium carbonate Not more than 0.5% Not applicable Sodium iodide 0.4% Not applicable Calcium Not more than 0.3% Not more than 0.3% Magnesium Not more than 0.3% Not more than 0.3% Water insoluble matter 0.2% 0.2% Ether extracts Not more than 0.1% Not more than 0.4% Aluminum Not more than 0.1% Not more than 0.1% Iron Not more than 0.1% Not more than 0.3% Arsenic 3 ppm Lead 10 ppm Mercury 0.0001 Heavy metals Trace

Table 1.6. FDA specifications for certification of FD&C Red No. 3 and FD&C Blue

No. 1 (8, 40, 64, 102).

12

Dyes

Dyes are soluble in water and produced as powders, granules, liquids or other

special-purpose forms. Currently, the dyes are manufactured as a byproduct of the

petroleum industry (50). The dye content of the certified dyes is between 86 and 96

percent (173). For most products, there are no specific colorant concentration limits.

GMPs suggest that dyes should be used at less than 300 ppm unless regulations stipulate

otherwise (50).

The certified food dyes are utilized due to their desirable coloring attributes and

stability in food products. However, manufacturers must observe specific precautions to obtain consistency in coloration of products. Exposure of certain food colorants to metals in processing equipment, including iron and aluminum, or metal from hard water, such as calcium and magnesium, will result in oxidation or reduction of food colorants causing decoloration and/or precipitation of dye (65, 153).

Lakes

Lakes are water-insoluble pigments made from the certified dyes by adsorption on an approved base or . The only approved substrate for FD&C lakes is composed of alumina (102, 133). The substrate is made by adding a sodium carbonate or sodium hydroxide solution to an aluminum sulfate solution. A solution of the certified dye is added followed by aluminum chloride to produce an aluminum salt which will adsorb to the alumina. This product is filtered and the cake is washed, dried and ground to the

desired size (102). Lakes are insoluble in water and are more stable than dyes (62).

Lakes may be used in foods or on materials that may come in contact with foods and be ingested (i.e., printing inks). The dye content of lakes is often between 1 and 40 percent 13

(133, 173). Lakes are also commonly used in products containing fats or oils (i.e., mixes,

hard candies, chewing gum) which lack sufficient moisture to dissolve the dye form (62).

Due to the acidic nature of the stomach contents, it is believed that that alumina

substrate of the lake is dissolved and results in the presence of the original dye. Based on

this information, no additional toxicity studies are required (133). This assumption may

be highly flawed as some of the oil-soluble azo lakes have been determined to be the

most toxic of food colorants used in the history of the United States. Chemically, the

lakes that are known to be highly toxic (and thus are now banned) are completely

unsulfonated (137).

Provisional Listing

Dyes that are provisionally listed can be legally added to food products until a

final decision is made based on completion of all scientific toxicological studies. The final decision will be to discontinue the provisional listing or allow a permanent listing

(173).

Permanent Listing

When all required toxicology tests are completed and the FDA deems the information satisfactory and the dye is considered “safe,” it can then be transferred to a permanent listing status (50).

Process for Approval of a New Food Colorant

The process to achieve approval of a new food colorant is quite extensive. To

start, the food colorant manufacturer must petition the FDA for approval. The petition

must show the efficacy of the colorant and submit results from various consumption

studies (50): 14

1. One subchronic 90-day feeding study in a non-rodent species

2. Acute toxicity feeding study in rats

3. Chronic 24-30 months feeding studies in two animal species, with at least one

performed with in utero exposure

4. One teratology study

5. One multigeneration reproduction mouse study

6. One mutagenicity test

The FDA considers numerous factors, including the physical properties of the compound, likely consumption, and safety factors. Once approved, the FDA determines regulations for use of the colorant, such as types of foods that can be colored, maximum amounts to be used, and labeling considerations. After approval, the FDA continues to monitor any adverse affects that may be identified after release via the Adverse Reaction

Monitoring System (ARMS) (62).

Currently Approved Synthetic Colorants in the United States

Currently, the United States Federal Food and Drug Administration (FDA) approves the use of nine synthetic food coloring agents through the Food Drug and

Cosmetic Act (FD&C): FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3,

FD&C Red No. 3 (dye only), FD&C Red No. 40, FD&C Yellow No. 5, FD&C Yellow

No. 6, Orange B and Citrus Red No. 2 (169). All of the dyes and the FD&C Red No. 40 lake currently have a permanent listing status with the remaining lakes possessing provisional listing status, other than FD&C Red No. 3 (50, 173). Additional information

15

about each dye is presented in Table 1.7, including quantity of each colorant certified by the FDA in 2006. Chemical and physical properties of the currently certified food

colorants are displayed in Table 1.8.

Colorant Classification

Four classes of colorants are currently approved for food use in the United States: azo, triphenylmethane, indigoid, and xanthene. Briefly, the characteristics of each class

will be discussed along with metabolism and toxicity information. Xanthene-derivatives will be discussed in further detail.

Azo Dyes

Azo dyes are the most widely used of dyes in all industries and constitute the

largest group of certified dyes in the United States. More than 2000 different azo dyes

are used in textiles, rubber, plastics and printing (138). Azo dyes are manufactured by

coupling a diazotized primary aromatic amine to another molecule, usually a naphthol

(50). The azo dyes may be mono-, di-, or tri- sulfonated compounds containing a

naphthalene or a pyrazolone ring linked via an azo-bond to another ring (133).

Azo bonds can be reduced in food products causing a cleavage of the molecule

forming colorless amine products. The fading of azo compounds is pronounced when the

food contains ascorbic acid, however packaging in opaque containers can limit the color

loss (133).

Metabolism and Toxicity of Azo Dyes

The intact sulfonated azo dyes are poorly absorbed in the intestinal tract; however

the reductive cleavage products are efficiently absorbed. Anaerobic intestinal microflora

are capable of reducing azo dyes extracellularly. Extracellular cleavage requires a 16

Structure Dye: 208,430.45 Dye: Dye: 527,028.53 Dye: Lake: 221,550.49 Dye: 3,337,658.86 Lake: 1,249,115.84 Dye: 16,436.18 Dye: Dye: 593,380.93 Dye: Lake: 448,712.28 Dye: 2,645.53 Dye: Information not available Dye: 3,079,142.14 Lake: 1,187,998.23 Dye: 5,620,508.88 Lake: 1,634,387.77 Quantity Certified Certified Quantity 2006 in FDA by (pounds) r dye terminated in in terminated dye r Permanent listing for in dye Food Provisionalfo listing externally applied drugsand cosmetics Provisional listing for Lake terminated in 1990 Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for Provisionaldye Listing lake for Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for Provisionaldye Listing lake for Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for Provisionaldye Listing lake for Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for Provisionaldye Listing lake for Restricteduse in foodorangeto peels Restricteduse in food dogshot to and sausage casings Food, Drugs, and Cosmetics and Drugs, Food, Permanent Listing for Provisionaldye Listing lake for Cosmetics and Drugs, Food, Permanent Listing for anddye lake Approval Status in U.S. in Status Approval Erythrosine, Food Red 14,E127, 3 Red Japan Indigotine, E132, E132, Indigotine, 2 Blue Japan Tartrazine, E102, E102, Tartrazine, 4 Yellow Japan Fast Green FCF, FCF, Green Fast 3, Green Japan E143 Brilliant Blue FCF, E133 2, Blue Japan Solvent Red 80 CI Acid Orange137 Sunset YellowFCF, E110, Japan Yellow 5 Allura Red AC, E129 Synonyms 45430 73015 19140 42053 42090 12156 19235 15985 16035 Color Color Index Number certified FD&C colorants. colorants. certified FD&C y Xanthene 16423-68-0 Indigoid 860-22-0 Azo 1934-21-0 Triphenylmethane 2353-45-9 Triphenylmethane 3844-45-9 Azo 6358-53-8 Azo 15139-76-1 Azo 25956-17-6 Azo 2783-94-0 Chemical Group Chemical Chemical Abstracts Number Service Red No. 3 Listed in 1906 Blue No. 2 No. Blue Listed in 1906 Yellow No. 5 in Approved 1916 Green No. 3 in Approved 1927 Blue No. 1 No. Blue in Approved 1929 Orange B in Approved 1966 Citrus Red 2 in Approved 1959 Red No. 40 in Approved 1971 Yellow No. 6 in Approved 1929 FD&C Certified Colorant Table 1.7. Currentl

17

Insoluble Insoluble Insoluble Insoluble Insoluble Vegetable Oil Insoluble Insoluble 20 0.1 7 20 20 2.2 1.5 Propylene Propylene Glycol Glycerin 20 1 18 20 20 20 3 Solubility (g/100Solubility ml) 25% Ethanol 8 0.5 12 20 20 10 9.5 currently approved certified FD&C colorants. colorants. approved certified FD&C currently

9 1.6 20 20 20 19 25 Water Poor Poor Good Good Good (unstable alkali) in Good Good pH pH Change search Council (118). Fair Poor Fair Poor Poor Fair Fair Oxidation Stability to… Stability Fair Poor Very Good Fair Fair Moderate Very Good Light Red No. 3 Red No. (Erythrosine) Blue No. 2 Blue No. (Indigotine) Yellow No. No. 5 Yellow (Tartrazine) Green 3 No. (Fast Green FCF) Blue No. 1 Blue No. (Brilliant Blue FCF) Yellow No. No. 6 Yellow (Sunset Yellow FCF) 40 Red No. (Allura Red) FD&C FD&C Certified Colorant (Common Name) Adapted from National Re Table 1.8. Chemical and physical properties of Table 1.8. Chemical and

18

complex system of intracellular and extracellular flavins. This enzymatic

function is very efficient, leaving only trace amounts of the intact compounds in fecal

material (133). Dyes that are sulfonated on both sides (i.e., FD&C Yellow No. 6) of the azo bond will yield only sulfonated breakdown products that are poorly absorbed. Azo dyes that are sulfonated on only one side (i.e., FD&C Orange No. 1) of the azo bond will produce some products that are not sulfonated and may be more easily absorbed (137).

After absorption, breakdown products are further modified by the liver and excretion products can be found in both the bile and the urine (133).

Triphenylmethane Dyes

The triphenylmethane dyes, FD&C Blue No. 1 and FD&C Green No. 3 are not widely used as singular dyes, but are very important as blending agents to achieve purple and green hues (133). Triphenylmethane dyes are the result of condensing a form of benzaldehyde with a benzylethylaniline, followed with oxidation and conversion to result in a disodium salt (64). The triphenylmethane dyes all contain sulfonic acid groups that allow for their high degree of water solubility (137). Triphenylmethane dyes are easily reduced in food systems to colorless compounds. Contact with metal surfaces or reducing agents such as ascorbic acid potentiate fading of these compounds (133).

Metabolism and Toxicity of Triphenylmethane Dyes

Intestinal absorption of intact triphenylmethane dyes has been quantitated at less than ten percent of consumed quantity (133). The small degree of absorption is thought to be related to the compounds low pKa values (137). What little of these compounds

19

that is absorbed, are rapidly excreted in the bile. The remainder of the fed dye is excreted

intact in the fecal material. These dyes exhibit a low degree of toxicity during chronic

feeding experiments, likely due to the low absorption of the dyes (133).

Indigoid Dyes

FD&C Blue No. 2 is the only indigoid dye permitted as a food additive in the

United States. FD&C Blue No. 2 was one of the first seven color additives allowed by

the U.S. Food and Drug Act of 1906 (133). FD&C Blue No. 2 is prepared by a series of chemical reactions beginning with naphthalene and ending with indigo. Indigo is sulfonated using sulfuric acid to produce this colorant (64). FD&C Blue No. 2 is unstable in aqueous solutions and becomes oxidized to isatin-5-sulfonic acid and 5- sulfoanthranilic acid, both of which are colorless. The presence of nonionic surfactants and sugars increase oxidation and thus, fading (133).

Metabolism and Toxicity of FD&C Blue No. 2

Based on experiments involving radioactively labeled FD&C Blue No. 2, less than three percent of the oral dose is absorbed and excreted in the urine. The remainder can be detected in the feces as intact, unabsorbed dye or oxidation products.

Approximately 24 percent of the ingested oxidation product, 5-sulfoanthranilic acid, is excreted in the urine within two days of ingestion (133).

Xanthene Dyes

The xanthene dyes are among the oldest and most commonly used of all synthetic dyestuffs. In 1938, 32 xanthene dyes were approved for certification by the FDA in one of the three categories; as of 1995 only 10 remain. Some were removed from certification due to safety concerns, but most were delisted due to lack of commercial 20

impact. Currently certifiable xanthene derivatives are FD&C Red No. 3, D&C Yellow

Nos. 7, 8, D&C Orange Nos. 5, 10, 11, D&C Red Nos. 21, 22, 27, and 28 (91).

Extensive information on the chemistry of xanthene dyes will be discussed in the

following section titled “Xanthene Dyes”.

FD&C Red No. 3 was one of the first seven color additives allowed by the U.S.

Food and Drug Act of 1906 and is the only xanthene derivative to be certified for food use in the United States (133). The dye form of FD&C Red No. 3 received permanent listing status for ingested drugs and foods in June of 1969 (15). FD&C Red No. 3 is prepared by iodinating fluorescein (64). This colorant is a very close match for primary red, is often used for blending, and does not bleed (56). FD&C Red No. 3 is extremely sensitive to light which has limited the applications of this food colorant to confections, powders and baked products (133). Under processing and storage conditions, specifically with heat treatment or storage in the presence of metal ions, FD&C Red No. 3 may release iodine (35, 133).

Metabolism and Toxicity of Red No. 3

FD&C Red No. 3 is poorly absorbed in the intestine and what is absorbed is excreted in the bile as intact and deiodinated forms. The fluorescein nucleus of the molecule is not degraded during digestion (60, 133, 180). FD&C Red No. 3 may serve as a source of dietary iodide due to iodide release when fed to rats, however studies suggest that iodide availability is too low to impact thyroid function (68, 174). Passage of the compound intact in the feces has been known to cause alarm to individuals and doctors.

FD&C Red No. 3 is known for its ability to raise protein-bound iodine measurements and it is thought that consumption of this dye contributes to dietary iodine intake (17, 137). 21

On January 29, 1990, the FDA revoked the provisional listing of the lake form of

FD&C Red No. 3 by applying the Delaney Clause based on increases in thyroid tumors in male rats (15, 56). A study completed in 1982 by the International Research

Development Corporation for the Certified Color Manufacturer’s Association and published by Borzelleca et al (1987) found FD&C Red No. 3 to have adverse effect in utero and an increased incidence of thyroid follicular adenomas in male rats that were fed a diet containing FD&C Red No. 3 at a level of 4 percent (15, 18). Subsequent subchronic feeding studies have helped to elucidate the mechanism of these adverse effects. FD&C Red No. 3 inhibits conversion of certain hormones resulting in an increase in thyrotropin secretion by the pituitary gland. Thyrotropin overstimulates the thyroid resulting in tumor formation (50). Additional studies have investigated the impact of FD&C Red No. 3 on thyroid-stimulating hormone (TSH) (68, 72). No correlation between thyroid symptoms in rats and humans has been found (50). The Joint

FAO/WHO Expert Committee on Food Additives (JECFA) has evaluated toxicology studies of FD&C Red No. 3 on mice, rats, gerbils, guinea pigs, rabbits, dogs and pigs. In chronic feeding studies of rats, dogs, mice and gerbils no toxic effects were noted (137).

FD&C Red No. 3 was not mutagenic using the Ames Salmonella/microsome test, at high concentrations, it increased the mutation rate of a streptomycin-dependent Escherichia coli strain (133, 137). A review of the results in mutagenesis studies was compiled by

Lin and Brusick in 1969 (89).

Currently, the dye form (FD&C Red No. 3) can be used in food products and oral medications but the FDA has announced plans to reevaluate the risk of these uses (56,

123). FD&C Red No. 3 is widely used in the food industry and would be difficult to 22

replace if the use of this dye is banned (123). Xanthene dyes are known to form reactive oxygen species, including singlet oxygen, as a result of photodynamic activity. The potential risk of these oxidation products is considered to be minimal due to deactivation of reactive oxygen species by food components, particularly components with antioxidant qualities (153). Photooxidation of xanthene dyes will be discussed in further detail in the

“Photooxidation and Photosensitizers” section.

1.2. Xanthene Dyes

Xanthene dyes are diphenylenemethane derivatives and include fluorenes

(aminoxanthenes), rhodols (aminohydroxyxanthenes), and fluorones (hydroxyxanthenes); these are among the oldest and most commonly used dyestuffs (120). Basic structures of xanthene classes are shown in Figure 1.2. Fluorones will be referred to as hydroxyxanthenes or xanthene-derivatives throughout this chapter

This review will emphasize the hydroxyxanthenes: fluorescein, Eosin, Erythrosin,

Phloxine and . General characteristics and regulatory status of these compounds for use in food, drugs and cosmetics are shown in Table 1.9.

Synthesis and purity

Hydroxyxanthenes have been utilized as dyestuffs for many years. Von Baeyer discovered fluorescein in 1871 and shared the compound with Caro who brominated fluorescein to produce what would be marketed as Eosine in 1874. Bindschedler and

Busch first produced Erythrosine B (under the name Eosine bleuatre) in 1876. Gnehm synthesized Rose Bengal between 1880 and 1884 and Monnet produced in 1887

(57, 119).

23

Xanthene

Fluorenes/Aminoxanthenes Pyonins Rhodamines

Fluorones/Hydroxanthenes

Rhodols/Aminohydroxyxanthenes

Figure 1.2. Basic structures of classes of xanthene dyes.

24

Structure Approval Status Elsewhere Approved for most in cosmetics countries other Approved for most in cosmetics countries other Approved for most in cosmetics countries other Approved for defined food products in Japan Approved forfood other many in use countries Approved for select in cosmetics countries Approved for defined food products in Japan ounds. p Approved for in drugs use and cosmetics, not allowed for area eye to the application Approved for in drugs use and cosmetics, not allowed for area eye to the application Approved for in drugs use and cosmetics, not allowed for application areato eye Permanent listingfor in dye Food Provisional listing for dye terminated in externallyapplied drugscosmetics and Provisional listing for Lake terminated in 1990 Not approved forfood,drugs, or cosmetics Approval Status in U.S. Approval xanthene com y drox y D&C Yellow No. 7, 7, No. D&C Yellow Japan203 Yellow 8, No. D&C Yellow Japan202 Yellow 22, No. Red D&C Japan Red 230 21, No. Red D&C Japan Red 223 21 No. Red D&C Lake 28, No. Red D&C Japan Red Food 104 27, No. Red D&C Japan Red 218 27 No. Red D&C Lake FD&C Red No. 3, Japan Red Food 3, E127 Japan Red Food 105 Synonyms 45350:1 45350 45380 45380:1 45380:2 45410 45410:1 45410:2 45430 45440 Color Index Number 332.30 376.28 691.88 829.7 785.7 879.84 1017.64 Molecular Weight Table 1.9. Characteristics of selected h Fluorescein 2321-07-5 Uranine (Na salt) 518-47-8 Eosin Y 17372-87-1 Erythrosin B 16423-68-0 Rose Bengal 11121-48-5 632-69-9 Phloxine B 18472-87-2 13473-26-2 Xanthene- Derivative Chemical Abstracts Number Service

25

Fluorescein is commercially synthesized by condensation of and

using heat or a condensing agent (Figure 1.3). This condensation may

be catalyzed by zinc-chloride (Baeyer synthesis) or sulfuric acid (8, 57, 64, 120). After

several hours of heating, a solid product will remain. Impurities can be removed by washing the product with alcohol. The alcohol will dissolve some of the fluorescein, but the majority will remain as a red powder. The powder can be further processed as described by Hewitt (1922) depending on desired purity and desired formula weight

(anhydride or hydrate) (57). Marshall (1976) analyzed commercially available fluorescein for contaminants using thin-layer chromatorgraphy and found traces of fourteen different unidentified components. These are likely minor products resulting

from the synthesis of fluorescein (103).

Halogenation of the fluorescein molecule is performed to produce the halogenated hydroxyxanthenes Eosin and Erythrosin using elemental and iodine, respectively

(42). Substituting agents (i.e., bromine or iodine) will first attack positions 4’ and 5’, followed by positions 2’ and 7’ (See Figure 1.3 for positions) (57). For the tetrachlorinated species, Phloxine and Rose Bengal, the tetrachlorinated fluorescein must be produced prior to bromination or iodination. Production of 4,5,6,7- tetrachlorofluorescein is accomplished by the acid condensation of resorcinol and tetrachlorophthalic acid (or anhydride). Alkaline hydrolysis is used to produce the disodium salts following halogenation (42).

26

2 +

Resorcinol Phthalic Anhydride

+ ZnCl2 or H2SO4

4’ 5’ 3’ 6’

2’ 7’ 1’ 8’ 4

5 7 6

Fluorescein

Figure 1.3. Chemical synthesis of fluorescein and numbering of fluorescein molecule.

27

Hydroxyxanthenes are difficult to purify and commercially available materials are

not significantly purified following synthesis and may contain inorganic salts (120).

FD&C Red No. 3 may contain up to 9.0% lower iodinated , including 4’-,2’-,

4’,5’-, 2’,5’-, 2’,7’-, 2’,4’,5’-, and 2’,4’,7’-iodofluoresceins. These lower iodinated compounds can be separated and identified by HPLC, paper-chromatographic, and column-chromatographic procedures (102). Marshall (1976) reported the composition and purity of various hydroxyxanthenes as determined using thin-layer chromatography.

The bromination of fluorescein to give Eosin results in significant quantities of 4’-, 4’,5’-,

2’,4’,5’- and 2’,4’,5’,7’-bromofluoresceins with the last being the desired end product.

Erythrosin B contained 4’-, 2’,4’,5’-, and the desired 2’,4’,5’,7’-iodofluoresceins.

Interestingly, several batches of Erythrosin B also contained 2’,4’,5’-triiodo-4,5,6,7- tetrachlorofluorescein and 2’,4’,5’,7’-tetraiodo-4,5,6,7-tetrachlorofluorescein (Rose

Bengal). Methanol-insoluble residues were obtained from Erythrosin B samples and contained carbonate, chloride and iodide salts. Phloxine analysis revealed intermediate quantities of 4’-, 4’,5’-, and 2’,4’,5’-bromo-4,5,6,7-tetrachlorofluorescein. Rose Bengal was contaminated with considerable amounts of 4’-, 4’,5’-, and 2’,4’,5’-iodo-4,5,6,7- tetrachlorofluoresceins. Phloxine contained contaminating residues of carbonate and chloride salts, whereas Rose Bengal only contained chloride salts (103).

Purification of hydroxyxanthenes has been reported using chromatographic techniques. Most effective purification is achieved by conversion of the disodium salts to the lactone or lactone diacetate forms followed by precipitation using hydrochloric acid.

The precipitate is recrystallized and reconverted to the disodium salt (119, 120).

28

Tinctoral strength

Fluorescein, or Uranine (fluorescein, disodium salt) is a yellow-colored dye with

low tinctorial power (120). Fluorescein will dye wool and silk under acidic conditions,

however it does not stick (not fast), and thus is termed a highly fugitive (119).

Fluorescein is thought to be the most fluorescent compound, per unit weight, known. It exhibits strong fluorescence at high dilutions, detectable to the naked eye at 1 part per 40 million and was used to trace the course of several rivers and underground streams in the

early 1900s (57, 119).

Solubility

Hydroxyxanthenes are water soluble and are poorly soluble to insoluble in non- polar solvents (119). Non-crystallized fluorescein is easily soluble in alcohol and ether; however the crystallized form requires considerable heat to dissolve in acetic acid. The crystalline form is insoluble in chloroform and benzene (57). Erythrosin is considered insoluble in acids, with low solubility at pH 4.0 and below, and stable to alkali (35, 173).

Hydroxyxanthenes are amphiphilic. Several authors have reported partition coefficients of various hydroxyxanthenes using water and octanol as solvents to estimate the degree of lipophilicity of these compounds. Partition coefficients for hydroxyxanthenes reported by different sources were variable (Table 1.10). Oros et al

(2003) found hydrophobicity to correlate with antimicrobial activity of a wide variety of colorants, including hydroxyxanthenes (124).

29

Partition coefficient in different studies Wang et al Levitan Oros et al Appendix A4 Hydroxyxanthene (2006)1 (178) (1977)2 (88) (2003) (124)3 Fluorescein -0.28 -4.77 -0.49 Eosin Y -0.25 -1.27 6.49 -0.41 Erythrosin B -0.24 -0.15 -0.38 Phloxine 8.98 -0.20 Rose Bengal -0.21 3.14 9.91 0.35 1 Reported log P value of partition coefficients between 1-octanol and deionized water 2 Reported log P value of partition coefficients octanol/water 3 Reported as calculated hydrophobicity log P using computer software by Advanced Chemistry Development Inc. 4 Reported as partition coefficient between n-octanol and water (log P)

Table 1.10. Variability in partition coefficients of hydroxyxanthenes reported by

different researchers.

Chemistry

Several studies involving the chemistry of hydroxyxanthenes have emphasized

photooxidation processes. The photochemistry of hydroxyxanthenes is discussed later.

Xanthene-derivatives structurally differ from triphenylmethanes only in the

bridging oxygen of the xanthene ring. Fluorescein and phenolphthalein are good

examples of this relationship (see Figure 1.4). Terminology of the classification of these

dyes is based on the substrates used for synthesis of these compounds. Xanthene- derivatives are made from resorcinol, whereas triphenylmethanes are derived from phenol. Both classes of chemical compounds have been used as acid-base indicators

(119).

30

Fluorescein Phenolphthalein

Figure 1.4. Chemical structures of fluorescein and phenolphthalein.

Electron and energy transfer to and from hydroxyxanthenes has been exploited for industrial and scientific applications. Fluorescein may act as either an energy acceptor or donor depending on the chemical nature of other compounds in the system, thus it has been used in singlet-singlet energy transfer studies. Eosin, in the excited state, may act as either an oxidizing or reducing agent (120). Reduction potentials of hydroxyxanthenes have been reported for fluorescein (E1/2 = -0.523 volts), Eosin Y (-0.463 volts),

Erythrosin (-0.290 volts), and Rose Bengal (-0.533 volts) (24, 53).

The halogenated hydroxyxanthenes may release free halogens under certain conditions. Erythrosin can be degraded by heat or with light exposure releasing free iodine, this is enhanced in the presence of iron, tin and organic acids (35). Erythrosin can be bleached in the presence of electron donors (120).

Eosin and Erythrosin adsorb strongly to bovine serum albumin in a 1:1 molar ratio (49, 190). Likewise, Rose Bengal and Eosin have been shown to bind to the dextransucrase isolated from Streptococcus sanguis (20). 31

Gutierrez and Garcia (1998) investigated interactions and complex formation

between hydroxyxanthenes and quinones. Association of Erythrosin and Rose Bengal with quinones leads to a red shift in the absorbance spectra (53). Katsuki et al (1994) found electron transfer reactions occured between xanthene dyes and p-quinones with light exposure as measured by electron paramagnetic resonance (EPR) spectroscopy.

Quinone anion radicals were the product of photooxidation using Eosin Y, dibromofluorescein, and Erythrosin B, but not fluorescein (75).

Fluorescence and phosphorescence

Hydroxyxanthene compounds are known for their fluorescence capabilities and fluorescein is commonly used for microscopic and flow cytometric fluorescence techniques. Fluorescence and phosphorescence information on the hydroxyxanthenes is displayed in Table 1.11. Fluorescein is one of the most fluorescent organic compounds known and the yellow-green fluorescence it produces is detectable at very high dilutions, especially with excitation using ultra-violet light. At higher concentrations (> 10-4 M), the absorbance of hydroxyxanthenes does not follow Beer’s Law due to aggregation of the dye molecules (dimeric dye) (119). Fluorescein is susceptible to photobleaching and its fluorescence properties are sensitive to pH. Numerous derivatives of fluorescein are available for use as fluorescent probes to target a variety of cellular components, including lipophilic derivatives for binding to membranes. Eosin and Erythrosin do not possess particularly good fluorescence characteristics for biological studies, with Eosin exhibiting only 10-20% the fluorescence quantum yield of fluorescein, and Erythrosin even less (114). Erythrosin has been used as a viability stain for microscopic studies, however this compound is quite sensitive to photobleaching (74). The fluorescence 32

Hydroxyxanthene Fluorescein Eosin Phloxine Erythrosin Rose Properties Bengal Quantum yield 0.93 0.63 0.08 0.08 fluorescence (фf)1 Absorbance2/Emission3 494/518 524/543 538/5704 530/555 548/571 maximum (nm) Phosphorescence 686 691 maximum (nm)5 1 Reported by Neckers and Valdes-Aguilera 1993 (120). 2 Reported by Webb et al 1962 (180). 3 Reported by Du et al (1998) (37). 4 Reported by de Carvalho and Taboga (1996) (33). 5 Reported by Lettinga et al (2000) (87).

Table 1.11. Fluorescence and phosphorescence characteristics of hydroxyxanthenes.

emission wavelength maximum of Rose Bengal shifts to longer wavelengths in non- hydrogen-bonding solvents compared to water. This shift in absorbance maximum may be exploited to determine localization of dye molecules, however quantification is difficult due to the enhanced emission quantum yield of Rose Bengal in organic solvents compared to water (32).

Isothiocyanates of Eosin and Erythrosin have been used as phosphorescence probes to study rotational properties and immunolocalization of proteins (34, 114).

Protein bound Eosin and Erythrosin leads to a “red shift” of 10 nm in the absorbtion spectra (49).

Medical and scientific uses of hydroxyxanthenes

Fluorescein is commonly used for ophthalmalic examinations and applied topically to determine the extent of corneal damage. Fluorescein is also intravenously injected for a procedure termed retinal angiography (42). Erythrosin has been used for

33

many years as a plaque disclosing agent in dental evaluations and antimicrobial activity towards dental microflora has been noted (19, 185). Eosin has been used as a histological stain since the 1800s to observe leukocytes (eosinophiles) (120). Rose Bengal has been used to test liver function in humans (119). Halogenated hydroxyxanthenes have been described as effective tools for spectrophotometric determination of various metals (172).

Eosin and Rose Bengal are well known as selective inhibitors in microbiological media to favor the growth of Gram-negative bacteria and fungi, respectively (13). Rose

Bengal had been used to preferentially isolate fungi from soil samples for at least 60 years (126).

Toxicity of hydroxyxanthenes

Toxicological studies have been performed on a variety of hydroxyxanthenes.

Erythrosin is the most commonly studied due to its use in food products and discussion of these studies was presented earlier under “Metabolism and Toxicity of FD&C Red No. 3.

Sasaki et al (2002) completed a large study to determine DNA damage via the comet assay (single-cell gel electrophoresis) as caused by 39 currently approved food additives in Japan. Erythrosin, Phloxine, and Rose Bengal were administered to mice orally. DNA damage to the glandular stomach, colon, and urinary bladder was apparent

3 hours post feeding of 10 mg/kg Rose Bengal and 100 mg/kg Phloxine and Erythrosin.

However, DNA damage was not evident at 24 hours. Rose Bengal and Phloxine at >10 mg/kg induced DNA damage in the urinary bladder, whereas >100 mg/kg of Erythrosin induced the same effect (150).

There have been reports of Erythrosin causing inhibition of specific enzyme activity including choline esterase and 17α-ethinylestradiol (82). Erythrosin was found to 34

inhibit liver detoxification enzymes UGT1A6 and UGT2B7 in vitro at concentrations of

0.9 mM and 0.18 mM, respectively (82).

Adams et al (1982) suggested a potential use for Erythrosin as a Salmonella control agent for chickens. Birds fed Erythrosin (0.5 g/kg) exhibited of the gut and gut contents and lead to distended ceca two to three times the size of untreated birds.

It was postulated that this distention was due to the impaired function of the ceca to absorb water and other nutrients from the fecal material. No Erythrosin could be detected in the meat from treated birds. (2).

Fluorescein and its halogenated derivatives are phototoxic under specific conditions. Specifics to phototoxicity will be discussed here, while other facets of photooxidation and photochemistry will be discussed in a later section. Anecdotal evidence of intravenous injection of fluorescein has resulted in phototoxicity of the eye and skin. Experiments were performed to determine if intravenous injection or topical application of Eosin could lead to skin photosensitivity. Rabbits injected with hydroxyxanthenes showed phototoxic reactions, while topical exposure did not produce toxicity (42, 117). Cutaneous exposure of Eosin and Rose Bengal has resulted in phototoxicity of scarred skin, but not intact healthy skin (42, 69). Tissue culture experiments have found Erythrosine and Phloxine B to be phototoxic to human skin fibroblasts, including DNA damage, when exposed to visible light (16, 42, 181, 182).

Due to these reports, the FDA requested further investigation into the toxicity, specifically phototoxicity and photocarcinogenicity of D&C Red Nos. 27 and 28

(2’,4’,5’,7’-tetrabromo-4,5,6,7-tetrachlorofluorescein, conjugate acid, base pairs) in 2000

(42). 35

1.3. Photosensitizers and Photooxidation

Photooxidation

Photooxidation is a process where a photosensitizer is excited by light and reacts

with other compounds to produce oxidized endproducts (44). This process is also

referred to as photosensitized oxidation or photodynamic effect. This transfer of energy

from light to a chemical (photosensitizer) to be used to inactivate cells or proteins has

been studied since the 1880s (54).

Depending on the system, photooxidation may result in desirable or undesirable

endproducts. In food products, photooxidation is often an undesirable process due to the production of off-flavors from oxidation of lipids, proteins, and carbohydrates (9).

Products, such as surimi, are often prepared with food colorants that may act as

photosensitizers and mishandling or packaging may result in unfavorable lipid oxidation

(132). Photooxidation in food systems is often due to the presence of naturally occurring photosensitizers in the food products (i.e., riboflavin, chlorophyll) and a lack of protection of the food product from light exposure. Photooxidation has also been used to inactivate undesirable cells in various systems, including pathogenic microorganisms or cancer cells (178). However, the toxicity of photooxidation products are non-specific and are capable of damaging many cell types (44). Selectivity of photooxidation is determined by the photosensitizer and its chemical and physical properties.

Photooxidation of a generic photosensitizer is shown in Figure 1.5. The singlet

(ground state) photosensitizer contains two electrons with opposite spins in a low energy orbital. Absorption of a photon of light results in the movement of one of the electrons to a high energy orbital while maintaining its spin direction; this is referred to as first 36

excited singlet state. At this stage, the first excited singlet state can lose energy by

emitting light (fluorescence) or by giving off heat (internal conversion) to return to

ground state. Alternatively, the spin direction of the excited electron may invert to become parallel with its partner to form an excited triplet state of the photosensitizer that is relatively long-lived (microseconds). The excited triplet state may lose energy in three different pathways: reaction with cellular or soluble components (type I reaction), reaction with triplet oxygen (type II reaction) or light emission (phosphorescence). These reactions are not mutually exclusive, the ratio of each result is dependent on the photosensitizer and concentrations of reactants (i.e., substrates and oxygen) (23).

Type I reaction

In a type I reaction, the excited triplet state of the photosensitizer reacts with a reducing substrate in the system by transferring an electron or hydrogen atom to result in the formation of radical species. These radicals can react further with oxygen, creating

reactive oxygen species. Due to electron or hydrogen atom transfer that occurs in a type I

reaction, recycling of the ground state photosensitizer does not occur (9, 43, 106). Type I reactions are favored in aqueous systems, under conditions of low oxygen concentrations

(0.20-0.25 mM), or in the presence of strong reducing agents (106). Flavins are examples of photosensitizers that favor the type I reaction. Flavins are easily reduced by NADH, followed by a spontaneous autooxidation resulting in superoxide and hydrogen peroxide production (105).

37

2 ox 2 PS O 3 Compounds other than O (Adapted from Castano et al 2004 - 2 Type I Type * O 2 2 O O Photooxidation 3 1 Type II Type Radicals Photooxidation Light PS * 3 Triplet PS Triplet Heat ooxidation of a generic photosensitizer. ooxidation of a generic photosensitizer. PS 1 Light Internal Internal Ground state conversion photosensitizer Phosphorescence crossing Intersystem Intersystem Fluorescence Light transition Electronic PS * 1 photosensitizer Excited state singlet singlet state Excited

(23). Figure 1.5. Illustrated interpretation of phot

38

Type II reaction

In a type II reaction, the excited triplet photosensitizer transfers energy to triplet

oxygen to form singlet photosensitizer and singlet oxygen (23, 43). Due to the recycling

of photosensitizer in a type II reaction, the process of energy transfer from

photosensitizer to triplet oxygen will occur repeatedly (9). Singlet oxygen can react with

many cellular targets, including lipids, amino acids, and nucleotides (106, 140, 158).

Quinones are a frequent byproduct of type II photooxidation of aromatic hydrocarbons

and their derivatives (53).

Type II reactions are favored under conditions of high oxygen concentrations, in

the presence of some organic solvents, or in the absence of strong reducing agents (106).

Singlet oxygen generated extracellularly is unlikely to reach essential targets, such as

DNA; reaching these targets could lead to death. Singlet oxygen would likely be

quenched by unsaturated fatty acids or membrane proteins prior to accessing the

cytoplasm (30).

Synthetic dyes, including methylene blue and Rose Bengal, are thought to favor

the type II reaction pathway of photooxidation due to the high reaction rates that have

been determined between triplet state dyes and triplet oxygen (41, 105).

Superoxide production

There are discrepancies in the literature as to classification of superoxide

production as a type I or a type II reaction. Reaction of the triplet photosensitizer with

triplet oxygen via electron transfer will yield superoxide (43). Superoxide, while not

particularly damaging itself, leads to the production of highly reactive oxygen species

including hydroxyl radicals. The highly reactive radicals can oxidize other substrates in a 39

chain reaction (23). This discrepancy is dependent on the definitions of type I and type II

reactions. Some authors identify the difference between the two reactions as type I

resulting in transfer of electrons and/or protons and type II resulting in transfer of energy

(23). Others identify the difference as type I reacting with substrates other than triplet

oxygen and type II reacting with triplet oxygen (44).

Superoxide production is likely part of the general cycle of photooxidation occurring with repeated oxidation and reduction of synthetic dyes. Superoxide production is thought to be a rare occurrence with photooxidation, happening in less than

1 in 100 collisions with oxygen for most photosensitizers (29). Martin and Logsdon

(1987) proposed two mechanisms for superoxide production involving NADH, shown in

Figure 1.6. Thiazine, acridine, and xanthene dye triplets will likely favor type I reactions

in biological systems due to the high levels of reductants available, including NADH,

resulting in reduced dye species. These reduced products may be oxidized by molecular

oxygen resulting in the production of the superoxide anion as shown in Pathway B in

Figure 1.6. It is likely that other cellular reductants, e.g., glutathione, could replace

NADH in the scenario proposed in Figure 1.6 (12, 107). Superoxide is not particularly

reactive on its own and is incapable of damaging DNA directly; however superoxide can

lead to more serious oxidative damage via the Fenton reaction (167).

40

Pathway A

3 1 Dye + O2 Æ O2 + Dye 1 - + NADH + O2 Æ NAD + O2 + H + - NAD + O2 Æ NAD + O2

Pathway B Dye3 + NADH Æ Dye - + NAD + H+

+ - NAD + O2 Æ NAD + O2 - - Dye + O2 Æ Dye + O2 + - 2H + 2O2 Æ H2O2 + O2

●- Figure 1.6. Superoxide (O2 ) production pathways involving NADH as a result of dye-mediated photooxidation as proposed by Martin and Logsdon (1987) (107).

41

Fenton chemistry

Cells contain low and controlled levels of iron, copper and other transition metals

within the cytoplasm for essential functions. These ions exist in reduced forms due to the reducing conditions within the cell (i.e., Fe2+). Reduced metal ions catalyze the

conversion of hydrogen peroxide to hydroxyl radical and hydroxide (23). Production

of hydroxyl radicals in the presence of reduced transition metals is referred to as Fenton-

type chemistry or the Fenton reaction as is depicted in Figure 1.7 (9). Hydroxyl radicals

are capable to removing a hydrogen atom from various organic compounds, leading to

the production of another free radical eventually causing damage to numerous biological

macromolecules (9, 167).

The Fenton reaction requires the presence of free ferrous iron, which is likely not

present in the cell due to the reducing conditions and presence of strong reductants

(including glutathione and NADPH). Therefore, under normal unstressful conditions,

there would be no free iron available for damage-inducing Fenton reaction. Under stress

conditions, an excess of superoxide may lead to release of free iron from biomolecules,

specifically Fe-S cluster containing proteins (167). Superoxide has been shown to

inactivate several 4Fe-4S cluster containing proteins, including aconitase, and other

(de)hydratases. However, sensitivity of Fe-S clusters to superoxide may vary by enzyme

with some being resistant to inactivation by this mechanism (48).

H O + Fe2+ Æ Fe3+ + OH + OH- 2 2

Figure 1.7. Fenton Reaction (167). 42

Photooxidation to inactivate microorganisms

Photooxidation as a means to inactivate microorganisms has been studied since

the 1880s (54). Martin and Burch (1988) proposed that inactivation of cells was more

likely the result of type I reactions due to the reducing environment within the cell that

would favor the electron transfer pathway over reaction with triplet oxygen (105).

Regardless of the type of photooxidation that takes place in the studied system, damage to cellular components including lipids, enzymes, and DNA may lead to cell death (95,

106, 178).

Localization and target sites

Localization of the photosensitizer has a significant impact on the efficacy of photooxidation on inactivating cells (120). Localization of the photosensitizer is crucial because of the high reactivity and short-half life of reactive species produced by photooxidation. As an example, the half-life of singlet oxygen is estimated to be less than 40 nanoseconds in biological systems and thus the radius of action is approximately

20 nanometers. Localization is dependent on the chemical properties of the photosensitizer including molecular weight, lipophilicity, amphiphilicity, ionic charge, and binding characteristics. In addition, photosensitizer concentration, incubation time and light exposure impact efficacy of photooxidation on inactivation of target cells (23).

Despite the short half-lives of these reactive oxygen species, studies have found that localization of the photosensitizer is not always a prerequisite for inactivation by photooxidation. Investigating the potential application of photooxidation for waste water treatment, Schafer et al (2000) found that physical separation of the photosensitizer from

bacterial cells did not prevent inactivation. Photosensitizer molecules bound to surfaces 43

could be used as sanitizers for mobile-phase cells without being bound to the cell (151).

Multiple target sites within the cell have been identified as important for inactivation of microorganisms by photooxidation.

Membranes

The cytoplasmic membrane contains unsaturated fatty acids that are susceptible to lipid peroxidation via photooxidation processes (52, 120). Phospholipids in the membrane bilayer can be oxidized to hydroperoxides. This is likely a primary cause of damage if the photosensitizer is amphiphilic and localized within the membrane (52).

Extensive oxidation of the phospholipid bilayer can lead to a loss of membrane integrity and/or changes in membrane environment affecting membrane protein functionality (135,

158). Loss of membrane integrity has been measured by a leakage of UV-absorbing material from the cell or accumulation of exogenously added chemicals (54). Oxidation of the cytoplasmic membrane also leads to changes in functionality of ion transport channels and depolarization of the membrane (151, 158). Oxidation of membrane components by singlet oxygen may lead to downstream oxidative DNA damage via intermediate reactive oxygen species (127). Pooler and Valenzeno (1979) demonstrated that various photosensitizers, including Rose Bengal and Eosin, were efficacious at inactivating sodium channels of giant axon membranes from lobster, in vitro (136).

Proteins and enzymes

Proteins and enzymes may be inactivated by direct binding of the photosensitizer oxidation of key amino acid residues within the protein. Protein binding is of particular importance when the photosensitizer is anionic in nature. In vitro studies have indicated

44

the ability of Rose Bengal to bind and inhibit DNA and RNA polymerase (120).

Likewise, lysozyme activity is sensitive to photooxidation by Eosin (14, 76).

Tryptophan, cysteine, methionine, tyrosine, and histidine are known to be important targets of photooxidation within protein molecules (23, 120). Proteins, particularly histidine and tryptophan residues, are susceptible to oxidation via singlet

oxygen (23, 39). Metalloproteins, particularly heme containing proteins, are believed to

resist redox reactions associated with singlet oxygen. The resistance of the metal core to

redox reactions is thought to have evolved due to the essential biological functions of

these metalloproteins. Estevam et al (2004) determined that oxidative damage of heme

containing cytochrome c from singlet oxygen is due to the oxidation of amino acid side

chains and does not target the heme prosthetic group (39).

DNA and RNA

Several photosensitizers are known to cause damage to DNA, usually with

evidence of mutation (54). In vitro studies have found certain photosensitizers,

specifically Rose Bengal, to cause double-stranded breaks in DNA as a result of

photooxidation. These reactions are 20 times more efficient in the absence of oxygen,

indicating the likelihood of a type I mechanism (27, 120). However, others have reported

lower efficiency under anaerobic conditions, thus identifying singlet oxygen as an

intermediate (121).

DNA can be oxidatively damaged in the nucleic acid and sugar portion of the

molecule. Additionally, DNA could be oxidatively cross-linked to proteins which are

particularly difficult for cells to repair (23). It has been proposed that photooxidation

could lead to mutagenesis (135). 45

Other targets

Other studies have suggested numerous other damaged sites within the cell as a result of photooxidation. Changes in DNA, RNA, cell wall, and membrane synthesis were proposed by Malik et al (1992) (95). Oxidation of important redox molecules within the cell may be affected by photooxidation, including NADH, glutathione, and cytochrome c (105). Photooxidation of biologically important quinones or hydroxyquinones may be important biological targets for cellular inactivation (53).

Protection from photooxidation

Cellular response to photooxidation

Reactive oxygen species, including superoxide and hydrogen peroxide, directly or indirectly, regulate gene expression of oxidative stress defense genes including several classes of peroxidases (41). Catalase has been identified as an inhibitor of hydroxyl radical production via photooxidation (105). Superoxide dismutase expression, specifically manganese superoxide dismutase, is induced by exposure to specific photosensitizers (106, 107). Martin and Logsdon (1987) reported that photooxidation in

E. coli resulted in an increase in cyanide-insensitive respiration indicating potential damage to the electron transport chain (107).

Outer membrane

Gram-negative bacteria are substantially more resistant to inactivation by photooxidation than Gram-positive bacteria, indicating a protective effect of the outer membrane (13, 67, 67, 73, 81, 151). The barrier properties of the outer membrane may prevent localization of the photosensitizer and/or block the penetration of photooxidation 46

products (i.e., singlet oxygen, hydroxyl radical) (67, 73). Deep rough mutants of

Salmonella and E. coli are sensitive to photodynamic inactivation, including singlet oxygen damage, compared to wild-type strains indicating a protective barrier effect of the of the outer membrane (30, 32).

Expression of antioxidants

Microorganisms that produce high levels of pigments (carotenoids), such as

Corynebacterium poinsettiae, Sarcina lutea, Micrococcus spp., and Halobacterium

salinarium, have been demonstrated to be more resistant to photooxidation than pigment- negative mutants (54). Maxwell et al (1966) demonstrated the protective ability of

carotenoids produced by Rhodotorula glutis protect cells from inactivation by photooxidation of toluidine blue compared to non-pigmented mutants (112). Kreitner et al (2003) found pigmented yeast, including Rhodoturula mucilaginosa, to be more resistant to photooxidation by specific photosensitizers than non-pigmented yeast (81).

Likewise, Tatsuzawa et al (1997) and Sano et al (1999) found E. coli cells transformed to express lycopene were more resistant to singlet oxygen than wild-type cells, indicating a protective affect of lycopene against singlet oxygen damage (148, 163).

Dahl et al (1987) found that E. coli mutants that overexpress histidine are protected from singlet oxygen toxicity. The protective effect of histidine is likely due to its extreme reactivity with singlet oxygen, thus quenching singlet oxygen prior to it reaching a more lethal target (30).

Exogenously added chemicals

Studies have found that the addition of certain chemicals to reaction media may prevent the production of harmful photooxidation products. Thiourea, EDTA, DMSO, 47

urea, sodium borate, and sodium benzoate have been found to inhibit hydroxyl radical

production or act as hydroxyl radical scavengers and could protect cells from photooxidation damage (105, 106). Sodium azide has also been effective at protecting or preventing damaging photooxidative effects on membranes (136). Known antioxidants, including α-tocopherol and β-carotene are capable of quenching singlet oxygen or quenching the excited photosensitizer (120, 131, 136, 188). Protective ability of carotenoids may also occur by competing for light absorption with the photosensitizer if their absorption spectra overlap (112). External application of antioxidants, such as β- carotene, to cells may afford less protection from photooxidation than antioxidants produced by the cells. Aditionally, antioxidants may offer more or less protection depending on the photosensitizer (136).

Photosensitizers

Numerous photosensitizers have been discovered from a variety of sources, including natural and synthetic, and vary significantly in solubility, structure, and molecular weight. Photosensitizers are defined as compounds which are excited by light and can pass this energy to other molecules within the system (178). Chemical structures of some of the well-studied synthetic and natural photosensitizers are shown in Figure

1.8. These photosensitizers vary in structure, size and elemental composition; however they all possess the high degree of conjugation necessary for excitation with light exposure. The focus of this review will be on the hydroxyxanthene group of photosensitizers.

48

Synthetic Natural Photosensitizers Photosensitizers

Phenothiazine Porphyrin (Methylene Blue, Toluidine Blue) (Protoporphyrin, Chlorophyll)

Acridine (Acridine Red, Quinicrine)

Flavin (Riboflavin)

Naphthalimide (Lucifer Yellow)

Polyacetylene (Thiopene)

Hydroxyxanthene (Rose Bengal, Phloxine)

49

Figure 1.8. Basic structures of synthetic and natural photosensitizers.

Hydroxyxanthenes

Halogenated hydroxyxanthenes are known as potent photosensitizers and numerous applications for their use have been proposed. Xanthene-derivatives have been developed for use as phototoxic pesticides (178). Phloxine B is a useful pesticide and bactericidal agent against Agrobacterium tumefaciens (141). Rose Bengal has been suggested as a useful photosensitizer for the inactivation of fecal microorganisms, including E. coli, in wastewater in developing countries (67, 142). Bezman et al (1978) suggested using Rose Bengal immobilized on polystyrene beads for wastewater treatment

(14). Sterilization of seawater using hydroxyxanthenes was proposed by Martin and

Burch (1987) (104). Erythrosin has been used for numerous years as a dental plaque disclosing agent and has been suggested as a photosensitizer for photodynamic therapy against oral plaque biofilms (19, 185). Eosin and Erythrosin were used to inactivate snake venom in 1906 (54). Erythrosin was suggested as a Salmonella control agent for chickens (2). Rose Bengal is effective at inactivating Trypanosoma cruzi with light exposure (29). Electron transfer processes involving hydroxyxanthenes have been used for photoinitiation of polymerization of chemical compounds (120, 134).

Antimicrobial efficacy of hydroxyxanthenes

Multiple studies have determined the relative efficacy of hydroxyxanthenes as photosensitizers against microorganisms. Fluorescein does not exhibit antimicrobial properties against various Gram-positive, Gram-negative bacteria, or yeast at concentrations of 500 µM (108, 178). Fluorescein is consistently ineffective or the least effective hydroxyxanthene, followed with increasing efficacy by Eosin, Erythrosin, and 50

Rose Bengal (54, 73). Iwamoto et al (1989) found 100 µM Rose Bengal and Erythrosin to be the most effective of the hydroxyxanthenes tested at inactivation of Saccharomyces cerevisiae followed by Phloxine and Eosin. Rose Bengal and Erythrosin were also effective at a concentration of 10µM (63).

The antibacterial activity of hydroxyxanthenes via photooxidation was first reported in 1904 by Jadlbauer and von Tappeiner and in 1908 by Reitz (54, 73).

Differences in microbial sensitivity to Eosin were reported in the 1930s (54, 162).

Streptococcus hemolyticus, Cornyebacterium diphtheriae, and Neisseria intracellularis were particularly sensitive to Eosin, while Staphylococcus albus, S. paratyphi, S. paradysenteriae Flexner, and Brucella abortus were resistant to all conditions tested

(162).

Photooxidation of Eosin has been shown to inactivate viruses and yeasts and damage photosystem II in leaf tissues (120). Erythrosin at a concentration of 22 µM was effective as a photosensitizer for inactivaton of Streptococcus mutans biofilms with light exposure as low as 15 minutes (185). Rose Bengal is used as an ingredient in agar media to inhibit the growth of bacteria in favor of fungal growth. Exposure of these media to daylight can cause suppression of the growth of both fungal and bacterial isolates at concentrations of 350 and 670 mg/L (126). Thakur and Fung (1995) found Eosin to be ineffective at inhibiting the growth of 43 food-related food molds when incorporated into growth media (166).

Most studies have reported minimal, if any, effect of hydroxyxanthenes on Gram- negative bacteria. Ineffectiveness of these compounds against Gram-negative bacteria has been attributed to the barrier properties of the outer membrane (32). Wang et al 51

(2006) found the yeast Saccharomyces cerevisiae to be more sensitive to photooxidation

by hydroxyxanthenes than the Gram-positive Staphylococcus aureus. The later was more

sensitive than Gram-negative E. coli (178). Erythrosin, at concentrations of 0.5 and

1.0%, effectively inhibits the growth of numerous organisms found in dental plaque, including Streptococcus spp. and some yeast, but ineffective against Gram-negative bacteria (19). Several studies using Rose Bengal as a photosensiziter for water treatment have reported efficacy against fecal coliforms, including E. coli (67, 104, 142). This effect is enhanced by increasing Rose Bengal concentration (1 µM to 10 µM) and light exposure time (0 to 240 minutes) (67). Studies have also found that immobilized Eosin and Rose Bengal can be an effective method to inactivate E. coli in drinking water (14,

120). Oros et al (2003) performed an extensive study on the growth inhibition of

numerous colorants, including Rose Bengal, Eosin, and Phloxine B, on a large number of

bacterial isolates. The three hydroxyxanthenes (1 µM impregnated discs) were effective against all Gram-positive species tested (Cornyebacterium spp., Staphylococcus aureus,

Micrococcus luteus, and Bacillus spp.). All Gram-negative species were inhibited by

Rose Bengal including Agrobacterium spp., Bradyrhizobium sp., E. coli, Erwinia spp.,

Pseudomonas spp. and Xanthomonas spp. Eosin and Phloxine B were ineffective against

E. coli, Erwinia spp., and Pseudomonas spp. Lighting conditions were not described in

this study; however incubation was at 21°C and is presumed to be benchtop incubation

with ambient light exposure. Spectral mapping techniques were applied to all microbial

results to determine a relative increasing effectiveness of Eosin, Phloxine B, and Rose

Bengal (124). Dahl et al (1988) suggested that penetration of Rose Bengal through the

52

outer membrane may occur slowly, which may explain differences in efficacy of hydroxyxanthenes on Gram-negative bacteria due to differences in exposure times of various studies (31).

Photochemistry of hydroxyxanthenes

Relative antimicrobial activity of hydroxyxanthenes has been correlated with increasing halogen substitutions on the fluorescein base. Increasing halogen substitution correlates with increased singlet oxygen yield upon illumination (178). Heavy atoms, such as bromine and iodine, are postulated to enhance the intersystem crossing of the excited singlet state of the photosensitizer molecule by spin perturbation to yield high levels of the reactive triplet molecule and also high phosphorescence quantum yields (80,

146, 178). Table 1.12 displays quantum yields of fluorescence, triplet formation, and singlet oxygen for selected hydroxyxanthenes as reported by Neckers and Valdes-

Aguilera (1993) (120). Increasing halogen substitution results in an increase in intersystem crossing from singlet to triplet state; thus, increasing the quantum yield of phosphorescence at the expense of the quantum yield of fluorescence. Higher quantum yields of triplet formation for hydroxyxanthenes correlate with enhanced antimicrobial activity (49).

Irradiation of halogenated hydroxyxanthenes may lead to dehalogenation via type

I photooxidation. Debromination of Eosin can occur during photooxidation if triplet reacts with amines and phenols (120). Radiation with sunlight results in the

53

Quantum yield of… Fluorescence Triplet Formation Singlet Oxygen

(фf) (фt) (ф 2) Hydroxyxanthene O Fluorescein 0.92-0.93 0.03 0.03-0.09 Eosin 0.20-0.63 0.28-0.32 0.39-0.57 Phloxine B 0.40 0.59-0.65 Erythrosin 0.02-0.08 0.62-0.69 0.62-0.63 Rose Bengal 0.018-0.08 0.76-0.86 0.75-0.79

Table 1.12. Quantum yields of fluorescence, triplet formation, and singlet oxygen for selected hydroxyxanthenes (45, 61, 67, 120, 125).

debromonation of Phloxine B likely resulting in the formation of free radicals (42).

Photolysis of Phloxine B results in debromination yielding 2’,4’,5’-tribromo-4,5,6,7- tetrachlorofluorescein and 4’,5’-dibromo-4,5,6,7-tetrachlorofluorescein (179).

Extensive light exposure of hydroxyxanthenes leads to photobleaching and formation of stable photolysis products (151). Halogenated hydroxyxanthenes bleach when irradiated in the presence of oxygen. The bleaching occurs due to the removal of the chromophore, most likely via peroxide formation across the quinoid (A-ring), however bleaching may be reversible if reducing agents are added (119)..

Photodegradation of fluorescein and Phloxine have been estimated to have a half-life of one hour in sunlight. Jemli et al (2002) reported a half-life of Rose Bengal at approximately 20 minutes in sunlight (67). The antimicrobial activity of these photolysis products has only been minimally studied (151). Photolysis products of Erythrosin were found to be non-mutagenic in the Salmonella/Ames assay (128).

54

Eosin, Erythrosin, Phloxine and Rose Bengal are capable of generating singlet oxygen with irradiation with visible light (Type II). Lipid oxidation via photooxidation of halogenated hydroxyxanthenes can be effectively inhibited by carotenoids (131).

Eosin is also capable of electron transfer to tryptophan and other amino acids (type I).

Triplet Eosin can serve as both an oxidizing and reducing agent (120). Photooxidation of

NADH by hydroxyxanthenes varies significantly. Martin and Burch (1988) found Rose

Bengal to be a potent photooxidizer of NADH, whereas Erythrosin was virtually incapable of causing NADH oxidation (105).

Proposed mechanism of inactivation by hydroxyxanthenes

Rasooly et al (2005) studied the efficacy of photooxidation of Phloxine B on the inactivation of E. coli (140). Previous studies indicated that Gram-positive bacteria were quite sensitive to inactivation using Phloxine B as a photosensitizer (141). E. coli is resistant to inactivation by Phloxine B treatment; however incubation of cells in the presence of EDTA led to its susceptibility. The author proposed a mechanism for inactivation of bacteria by Phloxine B photooxidation. When Phloxine B is ionized, the anion has affinity for positively charged cellular components and interacts with Gram- positive cells. Illumination of the dye will lead to debromination of Phloxine B and formation of free radicals, via type I photooxidation. Another possible outcome is the production of singlet oxygen from type II photooxidation of Phloxine B which could also be responsible for antimicrobial acitivity. This mechanism is proposed to be applicable to Gram-negative bacteria if the outer membrane is compromised using EDTA (140).

This mechanism could be applied to photooxidation using other halogenated hydroxyxanthenes. 55

Due to the reducing environment of the cell, it is likely that inactivation of microorganisms via photooxidation occurs via a type I mechanism. Hydroxyxanthenes are known to oxidize a variety of substrates potentially producing radicals with light exposure at low intensity (108).

Schafer et al (2000) studied the efficacy of photooxidation of E. coli and

Deinococcus radiodurans using Rose Bengal. There appears to be a threshold level for inactivation of bacteria by photooxidation, likely indicating a multitarget mechanism of inactivation. Rose Bengal is an effective producer of singlet oxygen upon illumination with visible light. Cellular membrane could be compromised by photooxidation even when the photosensitizer was physically separated from the cells, thus indicating the presence of an intermediate reaction product that diffused through the liquid to the cell

(151).

Transforming DNA (200 µg) was damaged by 100 µM Erythrosin, Eosin,

Phloxine B, and Rose Bengal with 5 hours of light exposure. The extent of DNA damage was not significantly enhanced at pH 5.6 compared to pH 7.2. Likewise, these compounds were also effective at inducing DNA damage in Bacillus subtilis in the recombination repair assay (rec-assay) with light exposure (189).

Hydroxyxanthenes have been shown to adsorb to proteins. Eosin and Erythrosin adsorb to bovine serum albumin approximating a 1:1 ratio (49). Rose Bengal binds with

RNA and DNA polymerase. Inhibition of DNA polymerase is irreversible with light exposure and reversible in the dark (120). 56

Mutagenesis has been reported to occur in certain studies of hydroxyxanthene photooxidation. Increased mutation frequencies were reported in Neurospora crassa spores, Serratia marcensens, E. coli, Bacillus subtilis, Haemophilus influenzae and

Penicillium notatum following photooxidation with Eosin or Erythrosin (54, 67, 85).

However, FD&C Red No. 3 (Erythrosin) has repeatedly been negative for causing mutagenicity in Salmonella/Ames reversion assays (67, 85).

Light-independent toxicity of hydroxyxanthenes

Most studies on inactivation of microorganisms by photooxidation of hydroxyxanthenes have found little to no antimicrobial activity of these compounds without light exposure. Martin and Logsdon (1987) found Rose Bengal and Erythrosin incapable of reducing oxygen levels without light exposure (107). Erythrosin B,

Phloxine B and Eosin B had no effect on entomopathogenic fungi without light exposure

(80). Wang et al (2006) found that high concentrations (>62.5 µM) of halogenated caused growth inhibition of E. coli, S. aureus, and S. cerevisiae (178).

Despite the lack of effect of hydroxyxanthenes in the dark in most studies, these compounds have been used historically for bacterial inhibition in growth media. Rose

Bengal has been used for suppression of bacterial growth in agar media. This suppression occurs under normal incubation conditions (dark) at concentrations of 350 mg/L. Suppression of Streptomyces spp. by Rose Bengal-containing media was also reported at concentrations of 670 mg/L under normal incubation conditions (126). T’ung and Zia (1937) reported an inhibitory effect of Eosin (2%) in saline against Streptococcus hemolyticus and Neisserria intracellularis under dark conditions (162). 57

Adams et al (1982) found that feeding chickens with Erythrosin (0.5 g/kg) led to

significant reduction in Salmonella (> 4 log cfu/g). Any reactions of Erythrosin with

Salmonella cells occurred within the digestive tract and was therefore under dark

conditions. Erythrosin treatment had no effect on native caecal or fecal microbiota, with

the exception of a reduction in the total aerobic count of the cecum (2). Martin and

Burch (1987) found exposure of E. coli to high Rose Bengal concentrations (25 µM) in

the dark to modestly increase expression of superoxide dismutase (107).

Localization of hydroxyxanthenes

Localization of hydroxyxanthenes on or within the bacterial cell is likely an

important factor contributing to antimicrobial efficacy of these compounds. There is

some discrepancy in the literature as to where these compounds localize and cause

destructive damage. Hydroxyxanthenes are anionic molecules that are incapable of

passing through the outer membrane of Gram-negative bacteria (106). Studies using

immobilized photosensitizers, such as Eosin and Rose Bengal, are reported to be

effective, suggesting that localization may not be a prerequisite for inactivation via

photooxidation (14, 120).

Cell membranes

Wang et al (2006) states that xanthene-derivatives typically localize in the cellular

membrane and photooxidation leads to destructive damage of lipid and protein

components (178). The lipophilic tendency of hydroxyxanthenes as measured by

partition coefficients suggests some partition of these compounds into the .

However, the transfer from aqueous solution to the cellular membrane may be impeded

58

by the negative surface charge of the cellular membrane and the negative charge of hydroxyxanthenes (32).

Dahl et al (1989) exploited the shift in absorbance maximum wavelength of Rose

Bengal to determine localization within wild-type and deep rough mutants of Salmonella.

Wild-type strains exhibited typical fluorescence patterns, whereas deep rough mutants

displayed a shift in absorbance indicating that some Rose Bengal was present in a more

lipid environment, likely a membrane (32).

Cell wall - peptidoglycan

Molnar et al (1992) treated Gram-positive and Gram-negative cells with

chlorpromazine followed by treatment with Rose Bengal and determine birefringence

changes using polarization microscopy. These authors concluded that the

hydroxyxanthene is localized within the cell wall (115). Chlorpromazine complexes with

various xanthene dyes and the resulting complex can be studied via biological and

spectrophotometric methods due to a bathochromic shift of 30-40 nm in the absorbance

spectrum of the free and bound dyes (83, 116). Dual treatment with chlorpromazine and

Rose Bengal resulted in an intense birefringence of the bacterial cell wall due to the

formation of charge-transfer (CT) complexes. Birefringence was negligible if cells were

pretreated with lysozyme, indicating localization of the CT complex within the cell wall.

Birefringence remained in cells treated with chloroform and methanol indicating that

localization was not within the membrane components of the cell (115). While these data

appear convincing, localization of the hydroxyxanthene dyes is likely altered due to the

treatment of the cells with chlorpromazine.

59

Proteins

Several authors have suggested association of hydroxyxanthenes with

proteinaceous components of the cell. Additional studies have focused on the activity of hydroxyxanthenes toward inactivation of specific bacterial proteins in vitro.

Conway and Adams (1989) grew Lactobacillus fermentum cells in the presence of

0.1% Erythrosin, stained cells with cotton blue and analyzed staining using conventional and fluorescent microscopy. Cells with associated Erythrosin were not associated with cotton blue and vice versa. Cells were disrupted and fractions were collected by ethanol and protease treatments. Ethanol precipitates without protease treatment contained

Erythrosin, while ethanol precipitates with protease treatment did not contain Erythrosin.

These findings indicate that Erythrosin is likely associated with protein components of the cell wall (28).

In vitro studies have found Rose Bengal, Eosin and Erythrosin to be effective inhibitors of E. coli DNA polymerase I both with (irreversible) and without (reversible) light exposure. Rose Bengal has also been shown to inhibit RNA polymerase and several

NAD+ and NADP+ -dependent dehydrogenases in vitro (159).

DNA

Hydroxyxanthenes likely do not interact directly with DNA, inducing cross-

linking, due to size, charge restrictions, and accessibility. Lakdawalla and Netrawali

(1988) suggest that the potential planarity of the hydroxyxanthene molecules could lead

to intercalation of the dye in the DNA resulting in frameshift mutations (85). In vitro

60

studies have identified oxidative damage due to photooxidation via hydroxyxanthenes

(21, 27). However, few studies have shown mutagenicity associated with

hydroxyxanthenes, in vivo (133, 137).

1.4. Ultra-High Pressure

Consumers desire processed food products that are safe and nutritionally equivalent to fresh products. Traditional food processing has relied on thermal inactivation of microorganisms to provide shelf-stability and safety. Thermal treatment, however, causes significant changes in nutritional and sensory quality of food products.

Nonthermal processing technologies are being developed and studied for their ability to

provide the desired safety, shelf-stability and quality demanded by consumers. These

nonthermal technologies include ultra-high pressure (UHP) treatment, otherwise called

high pressure processing (HPP) or high hydrostatic pressure (HHP). UHP was first

suggested as a means for preservation of fluid milk in 1899 (161). UHP treatment has

minimal impact on the perceived natural qualities of most foods, however some changes

are notable, particularly qualities relating to protein functionality (161). A number of

food products that are processed using UHP technology are available commercially,

including juices, meats, seafood, and guacamole (130). Impact of UHP on food-borne

microorganisms will be addressed in this review.

UHP involves treating a food product with isostatic pressure above 100 MPa and

up to 1000 MPa. The basic procedure for UHP treatment is to load the pressure chamber

with vacuum-sealed food in an appropriate container, usually a plastic pouch but may be

any container with at least one flexible side. The packages are surrounded by a food-

grade pressure transducing fluid, usually water or a water-glycol mixture. The chamber 61

is closed so that no gas is present. Once closed, pressure is applied for a specified

amount of time, pressure is released, and samples are removed from the chamber.

Treatment time is usually on an order of magnitude of minutes. Temperature of the

sample is controlled by the temperature of the transducing fluid, temperature of the water

jacket surrounding the chamber, and the pressure applied (11).

Efficacy of UHP technology is based on Le Chatelier’s principle and the isostatic

rule resulting in a treatment that is uniform and instantaneous. Therefore, the food

product will change in volume under pressure and reactions resulting in decreases in

volume will be favored (59, 84). The compression that occurs during UHP treatment results in an increase in temperature during processing (compression heating). Degree of compression heating is dependent on nature of compression fluid and sample properties.

Water is compressed 15% with UHP treatment of 600 MPa, resulting in an increase in temperature by 3°C per 100 MPa, whereas lipid increases 7°C per 100 MPa (51, 130). In order for the process to be considered nonthermal, holding temperature must be at or below ambient temperature (≤ 25°C).

Acidic or basic groups of molecules with negative reaction volumes (∆V°) will dissociate under pressure, resulting in a reversible change in pH; this includes side chains of many biomolecules, particularly proteins (51, 78, 130, 160). Under UHP treatment conditions, water will self-ionize due to its net volume reduction upon dissociation (∆V º

= -22.2 cm3/mol), resulting in a pH drop of approximately 0.3 pH units per 100 MPa (79,

130).

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Inactivation of microorganisms

Numerous studies have been performed to determine the efficacy of UHP on the

inactivation of various microorganisms. General trends in sensitivity and resistance of

microorganisms to UHP treatment have been reported in the literature. Gram-positive

bacteria generally show higher resistance to UHP treatment than Gram-negative bacteria

(7, 10). The resistance of Gram-positive cells is thought to be due to the high degree of

physical strength of the thick peptidoglycan layer. Coccoid-shaped bacteria are generally

more resistant than rod-shaped bacteria to UHP. The reduced surface area of cocci may

limit cell leakage that contributes to inactivation by UHP (10, 84). Spores are generally

the cell-type with the most resistance to UHP processing (101). Cells in stationary phase

are significantly more resistant to UHP treatment than exponential phase cells (93, 101).

The resistance of stationary-phase cells may be attributable to changes in membrane

lipids, thickening of the peptidoglycan, increases in cross-linking between membrane

proteins and cross-linking between peptides and lipoproteins (22). Differences in resistance are generalized and large variations in sensitivity to UHP processing have been reported for various species and strains (7, 97, 99, 100, 155, 161, 164, 176, 177).

Conditions effecting microbial resistance to UHP

UHP processing parameters including pressure level, holding time, and holding temperature have been extensively studied for their impact on microbial inactivation.

Conditions beyond the growth of microorganisms and processing parameters of UHP have also been found to contribute to the lethality of UHP treatments.

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Dose response

Efficacy of UHP treatment is dependent on the level of pressure applied to the sample. Inactivation of microbial cells begins with pressure treatments of 180 MPa with the rate of inactivation increasing as pressure increases (84).

Kinetics

Inactivation of microorganisms begins during the pressurization process. The pressure come-up time is defined as the time that is needed to raise the pressure within the treatment vessel from atmospheric pressure to the treatment pressure. The length of the come-up time is dependent on the UHP equipment and the lethality that occurs during come-up time should be distinguished from that occurring during pressure holding time

(84).

First-order inactivation kinetics have been reported for Lactobacillus plantarum and Lactobacillus brevis (96). However, survival plots are often sigmoidal in shape, thus deviating from first-order kinetics. These plots often exhibit shouldering, where a certain holding time is relatively ineffective, followed by a relatively rapid inactivation rate, and finally a tailing stage, where the inactivation rate diminishes progressively (144, 145).

Log-logistic and Weibull models are often used to predict inactivation by UHP treatment.

The sigmoidal shape of survival plots has been found to be independent of suspension medium, however the slopes may differ significantly from one medium to another (25).

Detailed description of UHP inactivation kinetics is presented by Palou et al (2007)

(130).

64

Holding temperature

Temperature is an important parameter to control during UHP processing. ter

Steeg et al (1999) studied the impact of holding temperature (8°-40°C) on the inactivation of Lactobacillus plantarum, Escherichia coli, and Saccharomyces cerevisiae by UHP

(150-200 MPa). UHP was the least effective at near the optimum growth temperature of these microorganisms (25°-37°C) (165). UHP holding temperatures above or below

optimum growth temperatures have been shown to be more effective at inactivating microorganisms (59). Enzymes are often resistant to UHP inactivation at temperatures between 20 and 40°C, but sensitive at higher or lower holding temperatures (90).

Pre-UHP stress

Cells that have been exposed to stresses other than pressure have been shown to be more resistant to subsequent UHP treatment. Sub-lethal heat treatment or cold shock has been shown to increase the pressure resistance of microorganisms. Heat stress and

cold shock may lead to changes in membrane fluidity that may result in increases in

barotolerance (59). Increase in membrane fluidity in bacteria is accomplished by a

variety of alterations in the membrane lipid structures. Listeria monocytogenes responds to a decrease in temperature by altering the acyl chain length in the membrane, an increase in 15:0 and a reduction in 17:0 (147). Alternatively, L. monocytogenes may also alter the degree of branching on the methyl end of the fatty acid chains (184).

Lactobacillus plantarum responds to cold temperatures by increasing unsaturation of the

membrane lipids. Russell et al (1995) found a correlation between an increased level

diphosphatidylglycerol (DGP) in Lactobacillus plantarum and sensitivity to UHP due to

the membrane-stiffening properties of these molecules (147). Casadei et al (2002) found 65

that membrane fluidity contributed significantly to the UHP resistance of exponential- phase E. coli cells but not stationary-phase cells (22). Cross-protection of cold shock may also be due to the heightened expression of cold shock proteins (CSPs).

Wemekamp-Kamphuis et al (2002) found that L. monocytogenes expressing higher levels of CSPs due to cold pre-treatment resulted in enhanced survival upon exposure to UHP.

These authors also suggested that cold pre-treatment may stabilize ribosomes and protect them from the potentially damaging effects of UHP (184). Aertsen et al (2004) found heat-shocked E. coli cells to be significantly more resistant to inactivation by UHP (250

MPa) than non-heat-shocked cells. Induction of pressure-resistance by heat treatment was only significant in the presence of nutrients, indicating the induced expression of heat shock proteins DnaK, GroEL, GroES, GrpE, ClpB, and HtpG was necessary to prevent damage due to pressure and aid in cell recovery (5).

Suspending Media

The barotolerance of microorganisms is greatly affected by the suspending medium in which they are pressure processed. Inactivation achieved by UHP with treatment in buffer systems may poorly correlate with results obtained in complex food systems. Complex food matrices, such as meat, juice, or milk, protect cells from inactivation by UHP (58, 59, 101, 113, 161). Protection in these systems may be due to the reduction of osmotic pressure across the cytoplasmic membrane (157). Little research has been performed to determine the impact of specific food components on protecting microorganisms from UHP. Simpson and Gilmour (1997) investigated the impact of protein (bovine serum albumin), carbohydrate (glucose), and lipid (olive oil), independently, on the inactivation of Listeria monocytogenes strains by UHP. Each of 66

these components provided protection to L. monocytogenes from inactivation by UHP

(155). Mackey et al (1995) found xylitol (15%) to offer near complete protection to

Listeria monocytogenes treatment with UHP at 300 MPa for 10 minutes (93). Other

factors of the food or medium matrix, including water activity and pH may alter UHP

efficacy (130).

UHP treatment results in a temporary shift in pH thought to be approximately 0.3 pH units per 100 MPa in water. Differences in pH-shifting depend on complexity and buffering capacity of the system (130). Thus, the pH and buffering capacity of the

suspending medium impact the efficacy of UHP treatment. Microbial inactivation during

UHP treatment increases with decreasing medium pH (59, 93, 93, 96, 144, 165, 177).

Acids may dissociate under UHP conditions if this dissociation results in a net volume

reduction, thus leading to a drop in pH of the suspension medium under pressure. A net

reduction in volume for the dissociated acid is expected for phosphate and citrate and

thus these acids are considered to be pressure-sensitive. For other buffers (i.e., HEPES,

MES), the dissociation of acidic groups would result in a net increase in volume and

therefore the undissociated form would be favored under pressure and a pH decrease

would be unfavorable. Thus, these acids are considered pressure-insensitive (51).

Chemical nature of the specific acids (weak vs. strong acids) present in the suspension

medium may affect UHP efficacy. Weak acids (phosphoric, malic, citric) have been

found to produce greater inactivation of Lactobacillus spp. treated with UHP compared to

strong acids (hydrochloric, sulfuric) (149). The efficacy of weak acids in the suspension

media may be attributed to the antimicrobial activity of the undissociated form of these

weak acids (51). 67

Modeling microbial inactivation by UHP

Numerous studies have been performed to model inactivation of various

microorganisms with UHP treatment (36, 59, 77, 144). Due to the contribution of the

aforementioned parameters to pressure efficacy, care must be taken when attempting to

apply these models to a new food system.

Mechanisms of inactivation by UHP

UHP treatment is uniform throughout the sample and is independent of sample mass. Application of UHP is known to compress molecules and favor reactions resulting in reduced volume (59, 90). Multiple target sites and/or cumulative effects of pressure are thought to determine inactivation by UHP treatment (59, 156, 164). UHP treatment causes morphological and biochemical changes in microorganisms (90). Membrane damage and protein denaturation are the mechanisms most commonly discussed for inactivation by UHP.

Membrane damage

Inactivation of microorganisms by UHP has been primarily attributed to membrane damage, however UHP does not usually result in cell lysis (100). UHP reduces the volume of the treated cell; this results in an increase in the packing density of the membranes. Generically, biological membranes consist primarily of phospholipids and membrane proteins which differ in their degree of compressibility. UHP leads to a tight packing of the phospholipids in the membrane causing membrane lipid gelation (22,

171). Depending on the nature of the acyl chains of the phospholipids, UHP treatment may lead to a phase change within the cytoplasmic membrane. Membranes that contain higher levels of unsaturated fatty acids, thus higher membrane fluidity, provide the cell 68

with additional resistance to UHP treatment (22, 84, 129, 170). Membrane fluidity

appears to be an important parameter for predicting UHP resistance in exponential-phase

cells, but seems less important for stationary-phase cells (22, 101). The difference in

compressibility of phospholipids and proteins in the membrane may lead to a loss of

membrane integrity due to UHP allowing the movement of components in or out of the

cell (3, 22, 59, 90). The loss of crucial intracellular components, including magnesium,

sodium, and calcium ions as well as RNA, proteins and other solutes could contribute to

inactivation of bacterial cells by UHP due to a loss of membrane integrity (46, 58, 100,

101, 170).

The loss of membrane integrity due to UHP treatment may be reversible or

irreversible, depending on level of pressure used (90, 129). Reversible damage to

biological membranes is difficult to determine experimentally and thus some researchers

have found little to no change in membrane morphology with and without UHP treatment

using transmission electron microscopy (186). Additional studies using fluorescent

probes that are impermeable to the cytoplasmic membrane showed that membrane

damage occurs with UHP treatment, but loss of membrane integrity does not directly

correlate with cell death in Lactobacillus plantarum TMW 1.460 (170). In studies using

exponential-phase E. coli, a good relationship between a loss of membrane integrity and inactivation was found (129). Further investigations using exponential-phase cells and a fluorescent lipophilic probe (FM 4-64) identified the formation of vesicles and other changes in membrane structure with UHP treatment. These changes were not apparent with stationary-phase cells; no apparent irreversible change in membrane structure or integrity with pressure treatments up to 600 MPa for both viable and dead cells (101). 69

Manas and Mackey (2004) postulated that the differences in vesicle formation between exponential and stationary phase cells were due to differences in the compressibility of membranes at different growth phases (101).

Protein denaturation

Hydrophobic and electrostatic interactions within and between proteins are affected by pressure, thus UHP treatments can alter the tertiary and quaternary structure of proteins. Proteins are highly variable in their tertiary and quaternary structure, thus extent of denaturation due to UHP depends on the type of protein (3). UHP compresses proteins and may lead to ionization of amino acid side chains as well as exposing hydrophobic residues. Dissociation of intermolecular protein bonding is thought to occur at relatively low pressures (200 MPa) (171). Irreversible protein denaturation

(intramolecular) is believed to occur with pressures above 300 MPa which also corresponds to the UHP range that results in microbial inactivation (55, 84), however more recent studies have argued that microbial inactivation does not correlate with protein denaturation. Manas and Mackey (2004) investigated changes in proteins due to

UHP treatment by visualizing protein aggregation using fluorescent-labeled (FITC) proteins. Protein aggregation occurred in most cells with pressure treatments of 200 MPa and no appreciable change was noted with higher pressure treatments. These findings suggest that inactivation of microorganisms by UHP may not be directly due to protein aggregation (101).

Other researchers attributed microbial inactivation by UHP to enzyme denaturation (90, 170, 187). Wouters et al (1998) indirectly investigated the impact of

UHP treatment on F0F1 ATPase activity in Lactobacillus plantarum LA 10-11 by 70

measuring the ability of UHP treated cells to maintain a pH gradient across the

membrane. Intracellular pH was found to decrease, however this did not correlate with a

loss of viability due to UHP treatment indicating that inactivation of microorganisms by

UHP is a complex process (187). Ulmer et al (2000) found inactivation of Lactobacillus

platnarum TMW 1.460 by UHP to correlate with a loss of metabolic activity as measured

by tetrazolium salt reduction potentially indicative of enzyme inactivation (170). Ulmer

et al (2002) discovered inactivation of Lactobacillus plantarum multridrug resistance

transporter HorA due to UHP treatment correlated with sublethal injury (171). Malone et

al (2006) suggested that UHP treatment could lead to destabilization of Fe-S clusters

within metalloproteins. This destabilization could lead to the release of free iron within

the cell resulting in oxidative stress via the Fenton reaction (97).

Additional changes associated with microbial inactivation by UHP

While membrane damage and protein denaturation are considered to be the

primary mechanisms of inactivation by UHP, additional research has focused on other

cellular components that may directly or indirectly impact cellular inactivation. These

changes may be caused directly by UHP or indirectly due to changes in membrane

integrity and/or protein denaturation.

Ribosome dissociation

UHP treatment caused dissociation of ribosomes in vitro and in vivo and therefore these organelles may be targeted by UHP treatment (122, 170). Protein synthesis is known to be an extremely pressure-sensitive process; inhibition of protein synthesis occurring with pressures as low as 68 MPa (86, 152). Niven et al (1999) used differential scanning calorimetry to detect changes in ribosome conformation before and after UHP 71

treatments. The number of native and intact ribosomes decreased with UHP treatments.

At the highest pressure tested (250 MPa), no intact native ribosomes were identified.

Death of the cell may be attributed to a critical loss in the number of functional ribosomes beyond which the cell could not recover (122). Previous studies by Mackey et al (1994) determined ribosome loss due to UHP treatments of 250 MPa with a holding time of 10 minutes for Salmonella Thompson using transmission electron microscopy (94). The destabilization of ribosomes due to UHP may be directly due to the UHP treatment or indirectly due to leakage of ribosome-stabilizing magnesium ions from the cell (122).

Inability to deal with oxidative stress

Bacterial cells treated with UHP have been found to be sensitive to oxidative stress. Enhanced recovery of sublethally-treated cells has been achieved by the inclusion of reactive oxygen species scavengers in treatment media and/or recovery in anaerobic conditions (3, 98). Aertsen et al (2005) developed a mechanism to determine oxidative stress within the cytoplasm of pressure-treated cells. E. coli mutants were created to express a cytoplasmic alkaline phosphatase (CAP) in which activation of the enzyme can only occur with oxidative stress in the cytoplasm. Activation of CAP occurred in wild- type E. coli as a function of pressure level indicating that cells experienced oxidative stress during UHP. However, pressure-resistant strains showed a significantly less inactivation of CAP with identical pressure treatments, suggesting ability to deal with oxidative stress is a large contributor to UHP-resistance (3). Aertsen et al (2005) found that anaerobic recovery of E. coli treated with UHP at 400 MPa for 15 minutes resulted in

100-fold higher survival than aerobic recovery. These findings indicate the UHP

72

treatment leads to an impaired ability for the cell to deal with oxidative stress. Mutants

defective in oxidative stress management (catalase and superoxide dismutase mutants)

were more sensitive to UHP treatment (3).

Changes in the outer membrane of Gram-negative bacteria

Ritz et al (2000) investigated changes in outer membrane proteins of Salmonella exposed to UHP. UHP treated cells were more resistant to EDTA-lysozyme treatments for spheroplast formation compared to control cells and the outer membrane protein profile (as measured by SDS-PAGE) was significantly altered following treatment. The authors postulated that UHP treatment caused a reorganization of the outer membrane, likely accompanied by a decrease in the number of porins present. Major and minor proteins present in the outer membrane of control cells were no longer present in the membrane following UHP treatment (143).

Ganzle and Vogel (2001) used 1-N-phenylnaphthylamine (NPN) as a fluorescent probe to determine changes in the outer membrane of E. coli during UHP treatment.

NPN was incorporated into UHP treated samples either during or after treatment to determine reversible and irreversible effects on the outer membrane. Changes in the outer membrane was dependent on pressure level, however outer membrane damage was not predicative of cell death. The authors proposed that increased permeability of the outer membrane due to UHP treatment was the result of a loss of from the outer membrane (46).

73

Nucleoid conformation

Manas and Mackey (2004) utilized fluorescent microscopy to determine changes

in nucleoid conformation in E. coli cells treated with UHP. Gross conformation changes

in the nucleoid were caused by UHP in both exponential- and stationary-phase cells.

Nucleoid conformation was apparent in exponential-phase cells with treatments greater

than 100 MPa, while stationary-phase cells displayed changes as pressures exceeding 200

MPa. Conformational changes only correlated with cell inactivation for exponential-

phase cells (101). Changes in nucleoid conformation have also been observed with UHP treatment of Lactobacillus plantarum, Listeria monocytogenes, and Salmonella

Thompson (94, 187).

Gene regulation

UHP treatment leads to a change in gene expression in E. coli. Sublethal treatment with UHP (100 MPa, 15 minutes) resulted in significant down-regulation of some of the genes associated with stress response (i.e., uspA, dps) and Fe-S cluster status

(sufS). The UHP treatment also resulted in the significant up-regulation of other stress response genes, including hsIV, ibpA, pphA, and cspA. Thiol-disulfide redox system genes (grxA, nrdI) and Fe-S cluster status genes (fnr and iscR) were significantly up- regulated in response to UHP stress (97). Further studies have found an induction of heat shock, cold shock, and SOS response in E. coli treated with UHP (4). Changes in gene expression profile may be due to pressure-induced changes in promoters or on promoter/effector interactions (38).

74

Levels of cold shock proteins (CSPs) and heat shock proteins (HSPs) increase in

response to UHP treatment in L. monocytogenes and E. coli (183, 184). CSPs are found

in most, if not all, bacteria and they have been associated with a variety of functions within the cell, including RNA chaperones, transcription activators or antiterminators

(184).

Additional cellular impacts of UHP have been noted; these include damage to the cell wall, plasmid loss, and delayed lysis (38, 90, 100). Repeated exposure to UHP may lead to the development of pressure-resistant mutants to protect cellular targets or cellular processes. This has been demonstrated for E. coli K12 and E. coli O157:H7 strains (4,

55, 99).

1.5. “Hurdle” Technologies

Due to the resistance, native or adapted, of microorganisms to various inactivation methods, food processors may need to combine physical and chemical treatments to achieve targeted inactivation of microorganisms in food products. Several studies have investigated combining UHP treatment with other physical or chemical treatments to achieve additive or synergistic microbial inactivation.

Pressure-assisted thermal processing (PATP)

UHP treatment at ambient temperatures is relatively ineffective at reducing spore populations in buffer systems and food products. UHP treatment in combination with high temperatures (45°-105°C) has been shown to be effective at reducing spore populations (84, 139). This combination of high heat and high pressure has been referred to as pressure-assisted thermal processing (PATP).

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High pressure-low temperature

Luscher et al (2004) investigated the efficacy of low temperature (below 0°C) in

combination with UHP treatment against Listeria innocua. Treatments with these

combinations of temperature (-25°C to -45°C) and pressure (200 MPa to 400 MPa) result

in phase transitions of water from ice I to ice III during compression. Inactivation of L.

innocua under liquid conditions resulted in linear inactivation with increasing holding times. Treatments that included ice phase transitions resulted in greater inactivation of

bacterial cells; however increasing the holding time had little to no effect on inactivation.

This finding suggests that the actual phase transition is the source of stress to the cells

and repeated pressure cycling for cells to experience multiple phase transitions did

enhance inactivation (92).

Organic Acids and UHP

UHP inactivation of microorganisms can be enhanced by reducing the pH of the

treatment medium (6). Alpas et al (2000) found enhanced inactivation of various

bacterial species with the inclusion of lactic acid in the treatment medium (6). Studies by

Giron and Shellhammer (2005) and Sarangapani and Shellhammer (2007) have demonstrated that suspension of Lactobacillus plantarum in media containing organic acids (citric, phosphoric, or malic) resulted in significantly higher inactivation compared to mineral acids at the same pH (51, 149). Changes in dissociation of organic acids under pressure may lead to enhanced inactivation due to the antimicrobial properties of the undissociated form of these organic acids.

76

Antimicrobial peptides and proteins and UHP

Several researchers have investigated the efficacy of combinations of UHP and various antimicrobial peptides against Gram-positive and Gram-negative bacteria. Gram- negative bacteria are commonly resistant to antimicrobial peptides and proteins due to the permeability barrier provided by the outer membrane. In combination with UHP treatment, Gram-negative bacteria become sensitive to lysozyme, lysozyme-derived peptides, the lactoperoxidase system, nisin, lactoferrin, lactoferricin, and pediocin AcH,

likely due to a loss of integrity of the outer membrane by UHP (47, 59, 70, 71, 109, 165).

Efficacy of lysozyme in combination with UHP was found to only be effective if present

during UHP treatment, indicating a transient change in outer membrane permeability at

low pressures (<300 MPa) (111). Synergistic inactivation by UHP and antimicrobial

proteins and peptides has been observed for both Gram-negative and Gram-positive

bacteria, usually with lower pressures displaying synergy with Gram-positive bacteria

(110, 111, 165). UHP likely facilitates the access of these antimicrobial agents to target

sites within the cytoplasmic membrane for both groups of bacteria (165).

UHP and oxidants

Researchers have used a combination of oxidants and UHP treatment to inactivate

microorganisms. Aertsen et al (2005) found that sublethal levels of tert-butyl

hydroperoxide (tBHP) or plumbagin (superoxide generator) with UHP resulted in

enhanced inactivation of E. coli compared to UHP alone. UHP treatment likely leads to

an inability of the cell to cope with the oxidative stress produced by these compounds (3).

Other compounds or enzyme systems may enhance inactivation of bacteria by oxidative

stress including the lactoperoxidase enzyme system (47). 77

UHP and antioxidants/preservatives

Mackey et al (1995) investigated the impact of chemical preservatives on the inactivation of Listeria monocytogenes by UHP. Potassium sorbate (1 mM) was

minimally effective and butylated hydroxyanisole (BHA) (1.55 mM) was substantially effective at enhancing the inactivation of L. monocytogenes with UHP treatments of 300

MPa for 10 minutes. These compounds do not show any lethal effect on this bacterium without UHP treatment and exhibited little to no effect if added after UHP treatment.

Sodium ascorbate (20 mM), butylated hydroxytoluene (BHT) (2.7 x 10-5 M) and parabens

(1 mM) were reported as ineffective in combination with UHP (93).

Chung et al (2005) and Vurma et al (2006) found the combination treatment of

UHP (400 MPa, 5 minutes) and tert-butyl hydroquinone (TBHQ) (100 ppm) to be

significantly more lethal to L. monocytogenes compared to UHP alone (26, 175).

Combination treatment of TBHQ (300 ppm) with UHP (600 MPa, 5 min) was also

effective in sausage and eliminated low numbers of survivors associated with UHP

treatment alone (26). The presence of reducing agents (cystine and glutathione) offer

protection to E. coli O157:H7 from inactivation by UHP-TBHQ combination treatment

(98).

Based on the current literature and preliminary observations, further studies were

performed to determine the efficacy of hydroxyxanthenes, particularly FD&C Red No. 3,

against important food-borne bacteria, including strains recognized as pressure-resistant.

Further investigation was needed to determine the mechanism of microbial inactivation

by Red No. 3 and the synergy between the dye and UHP. The contribution of outer 78

membrane to the resistance of Gram-negative bacteria to Red No. 3, and the use of UHP to destabilize the membrane and sensitize these bacteria to the dye were investigated. The antimicrobial properties resulting from dye photosensitization, in combination with UHP treatments, were determined for Gram-positive and Gram-negative bacteria, and particular attention was given to the light-independent inactivation. Contributions of type

I and type II photooxidation to bacterial inactivation were studied. Finally, combination treatments were also evaluated for efficacy in two food systems.

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CHAPTER 2

XANTHENE-DERIVED FOOD COLORANTS SENSITIZE PROCESSING-

RESISTANT FOODBORNE PATHOGENIC AND SPOILAGE BACTERIA TO

ULTRA-HIGH PRESSURE

ABSTRACT

Variability in barotolerance among foodborne microorganisms has been

demonstrated at the genus, species and strain levels. Identification of conditions and

additives that enhance the efficacy of ultra-high pressure (UHP) against foodborne pathogenic and spoilage microorganism is crucial for maximizing product safety and stability. Based on preliminary work in the laboratory, food colorants, specifically xanthene-derivatives may be used in combination with UHP treatment to enhance the

inactivation of bacteria. FD&C Red No. 3 was the only U.S. certified food colorant

found to be bactericidal and to act synergistically with UHP against Lactobacillus

plantarum strains. In addition to Red No. 3, other halogenated xanthene-derivatives

(Eosin Y, Phloxine B, and Rose Bengal) were effective against L. monocytogenes OSY-

328 and the dye-UHP combination inactivated large populations of E. coli EDL 933.

Inactivation of Listeria monocytogenes and Escherichia coli O157:H7 strains by UHP

(400 MPa, 3 min) resulted in synergistic lethality with the presence of Red No.3 at 3 ppm 96

and above. Treatments with combinations of 10 ppm Red No. 3 and UHP resulted in

maximum inactivation of barotolerant L. monocytogenes OSY-328. Treatment of this

resistant strain with UHP inactivated 0.3 log cfu/ml and the dye inactivated 1.8 log

cfu/ml; however the combination treatment inactivated 8.1 log cfu/ml. Staphylococcus aureus, Pediococcus acidilactici, and Salmonella Typhimurium varied in sensitivity to the dye and dye-UHP combinations. Gram-positive strains were inhibited by halogenated

xanthene-derivatives alone, while Gram-negative strains were only affected when treated

with both the colorant and UHP, suggesting a role of the outer membrane as a protective

layer that can be disrupted with UHP processing. This is the first study to determine

strain variability to inactivation by Red No. 3 alone and in combination with UHP.

INTRODUCTION

Ultra-high pressure (UHP) processing is receiving increased attention due to the ability of this process to inactivate microbial contaminants without impairing the quality attributes of food. Lethality of foodborne microorganisms by UHP has been widely investigated and considerable variability in barotolerance has been demonstrated at the genus, species and strain levels (3). Barotolerant strains of Listeria monocytogenes (17),

Lactobacillus plantarum, Lactobacillus fermentum (19), and Escherichia coli O157:H7

(5) have been identified; these pose potential risk in foods processed using pressure.

Consequently, it is important to identify conditions and additives that enhance the

efficacy of pressure against foodborne pathogenic and spoilage microorganisms.

Recently, investigators screened a large number of additives, particularly antimicrobial peptides, for synergy with high pressure against foodborne pathogens.

Antimicrobial peptides (e.g., nisin) are usually effective against Gram-positive bacteria, 97

but efficacy of these compounds against Gram-negative bacteria is limited.

Ineffectiveness of these compounds against Gram-negative bacteria is thought to be due to the shielding effect of the outer membrane. Channels, or porins, within the outer membrane allow hydrophilic compounds less than 700 daltons (depending on osmotic conditions) to pass to the periplasmic space and access the cytoplasmic membrane.

Hydrophobic compounds are entirely excluded due to the polar nature of the outer leaflet; however specific molecules may have separate permeation mechanisms to cross the outer membrane (9).

Enhanced inactivation of both Gram-positive and Gram-negative bacteria with

UHP has been determined with nisin, lysozyme, pediocin, lactoferrin, and lactoferricin

(7, 8). The sensitivity of Gram-negative bacteria to these combination treatments is thought to be caused by the permeabilization of the outer membrane due to the high pressure treatment (7). None of the previously tested compounds, coupled with UHP, were effective against all organisms; however each organism tested was sensitive to at least one additive-pressure combination (8). Chung et al (1) and Vurma et al (18) found that phenolic antioxidants, particularly TBHQ, sensitize L. monocytogenes to UHP.

Food colorants are traditionally used to impart a desired color to a food product and are thought to have limited effects on the food product beyond tincture strength.

Limited studies have addressed the effect of food colorants on microorganisms beyond traditional toxicity screenings for mutagenicity (4, 12, 13). Owen (1930) investigated the efficacy of synthetic dyes against various bacteria (11). Additional inactivation studies using impregnated disc assays have been performed to determine antimicrobial activity of various synthetic food colorants (10). 98

Seven synthetic food color additives were tested for antimicrobial activity against

Lactobacillus spp. alone and in combination with UHP. Preliminary studies showed the

xanthene-derivative, FD&C Red No. 3 (Color Index number 45460, Erythrosin B), to be

the only certified food colorant to possess antimicrobial activity against Lactobacillus

spp. when the dye was tested alone or in combination with ultra-high pressure (data not

shown). Based on these observations, the antimicrobial activity of FD&C Red No. 3 and

other xanthene-derived colorants and the potential synergy of these compounds with

UHP processing were investigated.

MATERIALS AND METHODS

Bacterial strains.

Listeria monocytogenes (Scott A and OSY-328), Lactobacillus plantarum (OSY-

104 and MDOS-32), Escherichia coli O157:H7 (EDL 933, OSY-MBM, and OSY-ASM),

Staphylococcus aureus (ATCC 6538), Salmonella enterica serovar Typhimurium (ATCC

2637), and Pediococcus acidilactici (PXL) were obtained from the culture collection of the Food Safety Laboratory at The Ohio State University (Columbus) and tested in this study. Stock cultures were suspended in appropriate broth media, containing 40%

(vol/vol) glycerol, and stored at -80ºC. Immediately before experiments, L. plantarum strains was transferred from the frozen stock-culture to MRS broth and incubated at 30°C for 48-72 hours. Similarly, L. monocytogenes, E. coli, S. aureus, and Salmonella were

transferred from frozen stock-culture to tryptic soy agar (TSA) (Criterion, Santa Maria,

CA) and incubated at 37ºC for 48 to 72 hours. P. acidilactici strains were transferred to

De Man, Rogosa and Sharpe (MRS) agar (Criterion, Santa Maria, CA) and incubated at

99

37ºC for 48 to 72 hours. Three isolated colonies of each strain were transferred to the

appropriate broth (MRS or TSA) and incubated overnight at 30°C (for L. plantarum) or

37°C (for all other bacteria).

Xanthene-derivatives.

FD&C Red No. 3 [Color Index (CI) number 45430, Japan Food Red No. 3] was

obtained from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Fluorescein (CI

number 45350), Erythrosin B (CI number 45430, Japan Food Red No. 3), Phloxine B (CI number 45410, Japan Food Red No. 104), Eosin Y (CI number 45380), and Rose Bengal

(CI number 45440, Japan Food Red No. 105) were purchased from Sigma-Aldrich (St.

Louis, MO). Stock solutions (10-1000 ppm) were prepared by dissolving the colorant in

sterile distilled water. All solutions were used within 30 minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, WA) containing 1:1 (vol/vol) glycol/water

pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International Inc., Valley

Forge, Pa). The press consists of a jacketed vessel with end closures, has a 2-liter

capacity, and is designed to operate at pressures up to 900 MPa. To reduce temperature

effect on ultra-high pressure inactivation, initial glycol:water processing fluid was 5-10ºC

to achieve a holding temperature of 20-25ºC. All experiments were performed at 400

MPa with a 3-min holding time.

Bacteria treatment in buffer.

Overnight cultures were centrifuged at 10,000 rpm (12,400 x g) for 10 minutes

(Sorvall RC-5B refrigerated superspeed centrifuge equipped with SM-24 rotor) and 100

suspended in sterile citrate-phosphate buffer (0.087 M phosphate, 0.0065 M citrate, pH

7.0). Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into sterile polyethylene bags (Fisher Scientific Co.,

Pittsburgh, Pa.) containing 0.1 ml of food colorant stock solution to achieve the desired

final concentration of these additives, and the bags were heat-sealed.

Sample bags were then placed into a larger polyethylene bag and sealed using a

vacuum sealer (Vacmaster, Kansas City, Mo.). Samples were then placed on ice for

approximately 30 minutes prior to high pressure treatment. After pressurization, bags

were immediately placed on ice until plated. Non-pressure-treated bags were held on ice

throughout the duration of the experiment. Bags were opened aseptically and the

contents were decimally diluted in 0.1% peptone water and plated on the appropriate agar

medium. L. plantarum strains were plated on MRS agar and incubated at 30°C for 72

hours prior to enumeration. L. monocytogenes, E. coli, Salmonella, and S. aureus strains

were plated on TSA and incubated at 37ºC for 48 hours. P. acidilactici strains were

plated on MRS agar and incubated at 37ºC for 48 hours. Experiments were performed in

triplicate, unless otherwise indicated.

Statistical analysis.

Data analysis of variance (ANOVA) was performed using the General Linear

Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the mean log

reductions of treatments were made using the Tukey’s Studentized Range test. Synergy

was defined as a significant difference between (log reduction dye alone + log reduction

UHP alone) and (log reduction dye + UHP combination) with significance determined

using a two-sampled paired t-test. 101

RESULTS

Species and strain variability to UHP lethality.

Inactivation of various species by UHP alone is shown in Figure 2.1. A pressure treatment of 400 MPa with a holding time of three minutes resulted in significant differences in inactivation amongst the strains tested. Average log inactivation ranged from 0.1 log for L. monocytogenes OSY-328 to 3.9 for L. plantarum OSY-104. This is the first study to compare these strains side-by-side with uniform treatment conditions.

L. plantarum MDOS-32 and L. monocytogenes OSY-328 strains were the most pressure resistant whereas L. plantarum OSY-104 and L. monocytogenes Scott A were the most sensitive to this pressure treatment. E. coli strains did not vary significantly in their pressure resistance under the tested conditions.

Screening xanthene-derivatives for synergy with ultra-high pressure.

FD&C Red No. 3 belongs to a class of compounds called xanthene-derivatives.

These share a common xanthene ring and may have halogen substitutions on the xanthene ring or the single ring structure (Figure 2.2). Inactivation of L. monocytogenes

OSY-328 and E. coli EDL 933 by xanthene-derivatives alone and in combination with pressure treatment is shown in Figures 2.3 and 2.4.

102

9.00

8.00

7.00

6.00

5.00 a 4.00 b 3.00

Log Reduction (cfu/ml) b,c 2.00 c,d b,c,d 1.00 c,d d 0.00 Lactobacillus Lactobacillus Listeria Listeria Escherichia Escherichia Escherichia plantarum plantarum monocytogenes monocytogenes coli coli coli MDOS-32 OSY-104 Scott A OSY-328 O157:H7 O157:H7 O157:H7 EDL 933 OSY-MBM OSY-ASM 933 ASM MBM OSY-104 Scott A Listeria Listeria MDOS-32 OSY-328 plantarum plantarum Lactobacillus Lactobacillus O157:H7 EDL O157:H7 O157:H7 OSY- O157:H7 OSY- O157:H7 monocytogenes monocytogenes Escherichia coli Escherichia coli Escherichia coli Species and Strains

Figure 2.1. Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and

Escherichia coli O157:H7 strains by ultra-high pressure treatment. Pressure treatments were 400 MPa with a holding time of three minutes. Temperature during holding time was between 20 and 25ºC. Error bars indicate standard error, n = 3.

Different letter designations indicate significant differences (p-value <0.05) in barotolerance amongst all strains shown.

103

Erythrosin B Rose Bengal

Fluorescein

Eosin Y Phloxine B

Figure 2.2. Chemical structures of xanthene-derivatives used in this study.

Erythrosin B is a synonym for FD&C Red No. 3.

104

9 * Dye alone 0.048 0.162 8 UHP + dye * 0.014 7

6

5 0.260

4

3 Log Reduction (cfu/ml) 2 * 0.002 1 0.723 0 Red 3 Red No dye Eosin Y Phloxine B Fluorescein Rose Bengal Rose Erythrosin B Xanthene-derivatives (10 ppm)

Figure 2.3. Inactivation of Listeria monocytogenes OSY-328 by xanthene-derivatives

(10 ppm) and/or ultra-high pressure treatment. Pressure treatments were 400 MPa with a holding time of three minutes. Temperature during holding time was between 20 and 25ºC. Error bars indicate standard error, n = 2. * indicates statistically synergistic effect of colorant and ultra-high pressure combination (p-value reported for synergy).

105

9 Dye Alone 8 UHP + dye

7

6 * * * * 0.022 0.004 0.005 0.002

5 * 0.036 4

3 Log Reduction (cfu/ml) Reduction Log 2 0.813 1

0 Red 3 Red No dye Eosin Y Eosin Phloxine B Fluorescein Rose Bengal Rose Erythrosin B Xanthene-derivative (10 ppm)

Figure 2.4. Inactivation of Escherichia coli EDL 933 by xanthene-derivatives (10 ppm) and/or ultra-high pressure treatment. Pressure treatments were 400 MPa with a holding time of three minutes. Temperature during holding time was between 20 and

25ºC. Error bars indicate standard error, n = 3. * indicates synergistic effect of colorant and ultra-high pressure combination (p-value reported for synergy).

106

Rose Bengal, Phloxine B, Erythrosin B and FD&C Red No. 3 were effective at

inactivating L. monocytogenes OSY-328 without pressure treatment. Rose Bengal was the most effective, leading to 5.0 log reduction. Phloxine B inactivated 3.6 log of L.

monocytogenes OSY-328, followed by FD&C Red No. 3 and Erythrosin B inactivating

2.2 log and 2.0 log, respectively. Eosin Y and fluorescein did not significantly inactivate

L. monocytogenes. In combination with UHP, Red No. 3 and Erythrosin B resulted in

synergistic inactivation of L. monocytogenes OSY-328, reducing the population by 7.0

log and 7.4 log, respectively. Combination treatment of L. monocytogenes with UHP and

Eosin Y also resulted in synergistic lethality, leading to an inactivation of 1.0 log

compared to 0.3 log by UHP alone. Efficacy of Rose Bengal and Phloxine B was

enhanced by combining with ultra-high pressure; however the effect was not significantly

greater than additive effect of pressure and dye lethalities.

None of the xanthene-derivatives were effective against E. coli EDL 933 without

UHP treatment (Figure 2.4). Rose Bengal, Phloxine B, FD&C Red No.3, Erythrosin B,

and Eosin Y in combination with UHP caused synergistic inactivation of E. coli EDL

933. Eosin Y was less effective than the other xanthene-derivatives mentioned, leading

to an average inactivation of only 3.8 log, whereas the other combinations resulted in

approximately 5 log inactivation. Fluorescein was ineffective against E. coli alone and in

combination with UHP. These findings indicate the importance of halogenation of the

xanthene-derivative with regard to antimicrobial properties and sensitization of bacteria

to UHP treatment.

107

9 1 ppm 8 3 ppm a a 5 ppm a 7 7 ppm 6 10 ppm a a,b 5 a,b 4

3 b,c Log Reduction (cfu/ml) 2 a c b b 1 b,c c cb,c 0 Lactobacillus Lactobacillus Listeria Listeria Escherichia Escherichia Escherichia plantarum plantarum monocytogenes monocytogenes coli coli coli MDOS-32 OSY-104 Scott A OSY-328 O157:H7 O157:H7 O157:H7 EDL 933 OSY-MBM OSY-ASM 933 ASM MBM OSY-104 Scott A Scott Listeria Listeria MDOS-32 OSY-328 plantarum plantarum Lactobacillus Lactobacillus O157:H7 EDL O157:H7 O157:H7 OSY- O157:H7 OSY- monocytogenes monocytogenes Escherichia coli Escherichia coli Escherichia coli Escherichia Species and Strain

Figure 2.5. Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and

Escherichia coli O157:H7 strains by FD&C Red No. 3. Error bars indicate standard error, n = 3. Different letter designations indicate significant differences in dye efficacy for each strain tested (p-value <0.05).

108

Dose-dependent inactivation of bacteria by Red No. 3 treatment.

Species varied in their sensitivity to Red No. 3 treatment with results shown in

Figure 2.5. L. plantarum strains were the most sensitive to Red No. 3 treatment followed by L. monocytogenes strains. E. coli strains were unaffected by Red No. 3 at the concentrations listed. Increasing Red No. 3 concentration to 300 ppm had no significant effect on any of the E. coli strains tested (data not shown). The outer membrane of

Gram-negative bacteria serves as a protective shield preventing deleterious compounds from accessing potential cellular targets (9, 16). The selective permeability of the outer membrane may protect E. coli from inactivation by Red No. 3.

For the Gram-positive organisms, inactivation correlated with Red No. 3 concentration. Strains of L. monocytogenes were significantly more resistant to Red No.

3 treatment than were L. plantarum strains. L. plantarum strains and L. monocytogenes

OSY-328 showed significant differences in inactivation with increasing dye concentration. L. monocytogenes Scott A showed a similar trend in dose-dependence; however these differences were not significant (p-value 0.060). Strain variability was noticeable among Gram-positive species. Lactobacillus plantarum MDOS-32 was extremely sensitive to small concentrations of Red No. 3, whereas L. plantarum OSY-

104 was more resistant. A treatment with 3 ppm colorant resulted in an average reduction 3.4 log of L. plantarum MDOS-32, whereas the same treatment resulted in a

0.7 log reduction in L. plantarum OSY-104. Higher treatment levels of Red No. 3 (10 ppm) resulted in an average log reduction of 1.7 for L. monocytogenes OSY-328 and 0.5 for L. monocytogenes Scott A. This is the first study to determine strain variability in resistance to xanthene-derivatives. 109

9 1 ppm * * * * 3 ppm 8 * * * 5 ppm * 7 7 ppm * * 10 ppm 6 * * * * 5 * * * * 4 * * 3

Log Reduction (cfu/ml) * 2

1

0 Lactobacillus Lactobacillus Listeria Listeria Escherichia Escherichia Escherichia plantarum plantarum monocytogenes monocytogenes coli coli coli MDOS-32 OSY-104 Scott A OSY-328 O157:H7 O157:H7 O157:H7 EDL 933 OSY-MBM OSY-ASM 933 ASM MBM OSY-104 Scott A Listeria Listeria MDOS-32 OSY-328 plantarum plantarum Lactobacillus Lactobacillus O157:H7 EDL O157:H7 O157:H7 OSY- O157:H7 OSY- O157:H7 monocytogenes monocytogenes Escherichia coli Escherichia coli Escherichia coli Species and Strain

Figure 2.6. Inactivation of Lactobacillus plantarum, Listeria monocytogenes, and

Escherichia coli O157:H7 strains by combination FD&C Red No.3 and ultra-high pressure treatment. Pressure treatments were 400 MPa with a holding time of three minutes. Temperature during holding time was between 20 and 25ºC. Error bars indicate standard error, n = 3. * indicates synergistic inactivation of FD&C Red No. 3 and ultra- high pressure (p-value <0.05).

110

Synergy between UHP and Red No. 3 against food-borne bacterial strains

Inactivation of tested strains by UHP and 1-10 ppm concentrations of Red No. 3

is shown in Figure 2.6. Efficacy of these treatments was dependent on the dose of Red

No. 3, with increasing colorant concentration resulting in increased inactivation. For the

purposes of this study, synergistic inactivation by the combination treatment is defined as

a significant difference between the additive effect of the two treatments (sum of the

inactivation by UHP and Red No. 3, measured separately and the inactivation by the

combination treatment. For both L. plantarum strains the combination was extremely

effective, but synergy was often not statistically significant due to the excessive

sensitivity of these strains to individual treatments. L. plantarum MDOS-32 did not show

significant synergistic inactivation by the combination treatment at any concentration due to the high degree of inactivation by Red No. 3 alone. Sensitivity of L. plantarum OSY-

104 to UHP masked the synergy of the combined treatment; synergy was apparent only when 3 ppm Red No. 3 was combined with pressure.

Synergistic inactivation of the combination treatment was apparent for L. monocytogenes and E. coli strains at Red No. 3 concentrations of 3 ppm and higher.

Interestingly, E. coli strains were sensitive to the combination treatment, whereas these strains were resistant to Red No. 3 alone. E. coli OSY-MBM and OSY-ASM tended to be more resistant to the combination treatments compared to E. coli EDL 933; however for most colorant concentrations this result may be explained by relative differences in resistance of these strains to the ultra-high pressure treatment. Additional bacterial species of importance to the food industry were tested for their relative sensitivity to Red

No.3 with or without UHP treatment (Figure 2.7). Similar to E. coli, Salmonella 111

9.00

8.00 <0.0001 <0.0001 7.00

6.00

5.00 Dye Alone UHP alone 4.00 Dye+UHP

3.00 Log Reduction (cfu/ml)

2.00

1.00

0.00 SalmonellaSalmonella Typhimurium StaphylococcusStaphylococcus aureusaureus PediococcusPediococcus acidilactici acidilactici TyphimuriumATCC 2637 ATCCATCC 6538 6538 PXLPXL ATCC 2637 Strain

Figure 2.7. Inactivation of Salmonella Typhimurium, Staphylococcus aureus, and

Pediococcus acidilactici by Red No. 3 (10 ppm) with or without ultra-high pressure treatment. Pressure treatments were 400 MPa with a holding time of three minutes.

Temperature during holding time was between 20 and 25ºC. Error bars indicate standard error, n = 3. Numbers above bars are p-values associated with the synergistic effect between Red No. 3 and UHP.

112

Typhimurium was resistant to 10 ppm Red No. 3. This finding supports the previous hypothesis that the outer membrane protects Gram-negative bacteria against treatment with Red No. 3. Efficacy of combination treatment against Gram-negative bacteria may be due to disruption of the outer membrane by UHP treatment. S. aureus and P. acidilactici were also tested to verify trends seen previously with Gram-positive organisms. S. aureus was comparable to L. monocytogenes OSY-328 in resistance to colorant, pressure, and combination treatment. P. acidilactici was extremely sensitive to

Red No. 3 treatment, resulting in greater than 7.0 log inactivation. P. acidilactici was extremely resistant to UHP treatment with less than 0.1 log inactivation with 400 MPa for

3 minutes. Synergistic inactivation by combination treatment was measurable for

Salmonella Typhimurium and S. aureus.

DISCUSSION

Previous studies have reported variability among strains of various species to inactivation by UHP treatment. In the present study, strains of L. plantarum, L. monocytogenes, and E. coli O157:H7 were selected from previous studies noting

significant differences in barotolerance at the strain level (6, 18, 20). This is believed to

be the first study comparing these strains side-by-side. Strains behaved as expected, with

the previously reported processing-resistant strains being less affected by UHP treatment

than the processing-sensitive strains. L. plantarum MDOS-32 was significantly more

resistant to UHP treatment than L. plantarum OSY-104. Likewise, L. monocytogenes

OSY-328 was significantly more resistant than L. monocytogenes Scott A. E. coli

O157:H7 strains did not vary significantly in their pressure resistance under the tested

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conditions, however the trend remained consistent with previous reports of strain OSY-

MBM and OSY-ASM being more pressure resistant than EDL 933 (6).

Previous studies have investigated the impact of xanthene-derivatives and their chemical structures on inactivation of bacteria. Wang et al (2006) found that the inherent toxicity of xanthene-derivatives was significantly dependent on parent molecule structure

(fluorescein skeleton) and that halogen substitutions contributed slightly to activity against E. coli, S. aureus, and S. cerevisiae. Phloxine and Rose Bengal were the most effective xanthene-derivatives tested (21); this agrees with the results of the current study that shows the bactericidal action of xanthene-derivatives against L. monocytogenes

OSY-328 (Figure 2.6). Rasooly and Weinz (2002) tested numerous xanthene-derivatives

(100 ppm) for their ability to inhibit the growth of S. aureus in broth. Halogen substitutions on the hydroxyxanthene moiety and tetrachlorination in the benzoic moiety were found to be important structural requirements to inhibit growth of S. aureus (15).

Several xanthene-derivatives, especially Rose Bengal, are known for their photosensitizing ability and this has been hypothesized to be a mechanism for inactivation of bacteria by these compounds (16). Impact of photooxidation was not measured nor optimized in this study, further investigations on the impact of photooxidation will be completed in future studies.

Dose-dependence of inactivation of bacteria by xanthene-derivatives has been reported in previous studies. Schafer et al (2000) found increasing concentrations of

Rose Bengal (0-5 ppm), with light treatment, to be increasingly effective at inactivating

E. coli and Deinococcus radiodurans. At the lowest concentration (0.5 ppm) tested by

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these researchers, there was not significant inactivation. Rose Bengal at concentrations

equal or greater than 1 ppm produced detectable lethality to E. coli and D. radiodurans

(16).

In the present study, Gram-negative bacteria were resistant to xanthene-

derivatives; for example, Red No. 3 at concentrations up to 300 ppm was not bactericidal

to E. coli strains. Previous investigators have reported conflicting data on inactivation of

Gram-negative bacteria by xanthene-derivatives. Rasooly (2005) tested the effect of

Phloxine B on inactivation of various Gram-positive and Gram-negative bacteria. Gram-

positive bacteria, including S. aureus, , B. mycoides, B. thuringienisis, and

B. subtilis, were inactivated by concentrations ranging from 25 to 100 µg/ml Phloxine B.

Gram-negative bacteria, including Salmonella, E. coli, and E. coli O157:H7 strains were

resistant to 100 µg/ml Phloxine B (14). These reports are in agreement with the findings

reported in this study. However, Wang et al (2006) found 125 µM Erythrosin B (~100

ppm) to be effective against E. coli with light exposure, and 500 µM (~400 ppm) were effective without light exposure (21). Differences in sensitivity of Gram-negative bacteria may be due to strain variability, light intensity and dye concentration. These

variabilities are likely due to major differences in technique and methodology and

whether the goal of the study was to inactivate or just inhibit growth of bacteria.

Regardless, researchers agree Gram-negative bacteria are substantially more resistant to

xanthene-derivatives than Gram-positive bacteria, signifying a role for the outer

membrane. Penetration of a biocide through the outer membrane of Gram-negative

bacteria may often be determined by the compound’s physical character (charge,

hydrophobicity, or amphipathicity) rather than chemical structure (2). 115

In the current study, halogenated xanthene-derivatives were bactericidal against

Gram-positive bacteria and these compounds in combination with UHP treatment lead to synergistic lethality against Gram-positive and Gram-negative bacteria. Red No.3 is approved for food use in the United States and could be used as an additive to promote food safety. This investigation shows the feasibility of using the Red No. 3 to enhance the shelf-life and safety of pressure-processed food products. Further investigations are needed to understand the mechanism of inactivation of the combination treatment as well as understanding effectiveness of the colorant within food systems.

ACKNOWLEDGEMENTS

The authors thank Dr. Mark Daeschel (Oregon State University) for providing L. plantarum MDOS-32. The authors thank Aaron Malone (The Ohio State University) for providing the wild-type and pressure resistant mutants of E. coli O157:H7 EDL 933.

REFERENCES

1. Chung Y-K, Vurma M, Turek EJ, Chism GW, and Yousef AE. 2005. Inactivation of barotolerant Listeria monocytogenes in sausage by combination of high-pressure processing and food-grade additives. Journal of Food Protection 68:744-750.

2. Denyer S P and Maillard J-Y. 2002. Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria. Journal of Applied Microbiology 92:35S-45S.

3. Lado BH and Yousef AE. 2002. Alternative food-preservation technologies: efficacy and mechanisms. Microbes and Infection 4:433-440.

4. Lakdawalla A A and Netrawali M S. 1988. Mutagenicity, comutagenicity, and antimutagenicity of erythrosine (FD and C Red 3), a food dye, in the Ames/Salmonella assay. Mutation Research 204:131-139.

5. Malone AS, Chung Y-K, and Yousef AE. 2006. Genes of Escherichia coli O157:H7 that are involved in high pressure resistance. Applied and Environmental Microbiology 72:2661-2671.

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6. Malone AS, Rodriguez-Romo L A, Baldauf N A, Rodriguez-Saona L E, and Yousef AE. Differentiation of Escherichia coli O157:H7 Processing-resistant Isogenic Mutants Recovered From High-pressure Processed Apple Juice by Fourier-transform Infrared Spectroscopy. International Association for Food Protection. 2006. Calgary, Alberta, Canada.

7. Masschalck B, Deckers D, and Michiels CW. 2003. Sensitization of outer- membrane mutants of Salmonella Typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under high pressure. Journal of Food Protection 66:1360- 1367.

8. Masschalck B, Van Houdt R, and Michiels CW. 2001. High pressure increases bactericial activity and spectrum of lactoferrin, lactoferricin and nisin. International Journal of Food Microbiology 64:325-332.

9. Niedhardt F C, Ingraham J L, and Schaechter M. 1990. Physiology of the bacterial cell: a molecular approach, p. -506. Sinauer Associates, Inc., Sunderland, Massachusetts.

10. Oros G, Cserhati T, and Forgacs E. 2003. Separation of the strength and selectivity of the microbiological effect of synthetic dyes by spectral mapping technique. Chemosphere 52:185-193.

11. Owen S E. 1930. The relation of media pH to the bacteriostatic action of dyes. American Journal of Physiology 102:154-158.

12. Ozaki A, Kitano M, Itoh N, Kuroda K, Furusawa N, Masuda T, and Yamaguchi H. 1998. Mutagenicity and DNA-damaging activity of decomposed products of food colors under UV irradiation. Food and Chemical Toxicology 36:811-817.

13. Parkinson T M and Brown J P. 1981. Metabolic fate of food colorants. Annual Reviews in Nutrition 1:175-205.

14. Rasooly R. 2005. Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria. FEMS Immunology and Medical Microbiology 45:239-244.

15. Rasooly R and Weisz A. 2002. In vitro antibacterial activities of phloxine B and other halogenated fluoresceins against methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 46:3650-3653.

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16. Schafer M, Schmitz C, Facius R, Horneck G, Milow B, Funken K-H, and Ortner J. 2000. Systematic study of parameters influencing the action of rose bengal with visible light on bacterial cells: Comparison between the biological effect and singlet-oxygen production. Photochemistry and Photobiology 71:514- 523.

17. Tay A, Shellhammer TH, Yousef AE, and Chism GW. 2003. Pressure death and tailing behavior of Listeria monocytogenes strains having different barotolerances. Journal of Food Protection 66:2057-2061.

18. Vurma M, Chung Y-K, Shellhammer TH, Turek EJ, and Yousef AE. 2006. Use of phenolic compounds for sensitizing Listeria monocytogenes to high- pressure processing. International Journal of Food Microbiology 106:263-269.

19. Waite J G, Horton E M, and Yousef AE. Identification of pressure-resistant Lactobacillus spp. for optimizing non-thermal processing of perishable foods. 2005. Atlanta, Ga, American Society for Microbiologists Annual Meeting.

20. Waite J G and Yousef AE. Comparison of barosensitive and baroresistant strains of Lactobacillus plantarum and Lactobacillus fermentum by investigating the impact of dose response and kinetic parameters, buffer composition and buffer pH. 2006. Calgary, Canada, International Association for Food Protection Annual Meeting.

21. Wang H, Lu L, Zhu S, Li Y, and Cai W. 2006. The phototoxicity of xanthene derivatives against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Current Microbiology 52:1-5.

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CHAPTER 3

DESTABILIZATION OF THE OUTER MEMBRANE OF Escherichia coli BY

PHYSICAL AND CHEMICAL METHODS LEADS TO INACTIVATION BY

FD&C RED NO. 3

ABSTRACT

The outer membrane of Gram-negative bacteria provides excellent barrier properties thus protecting these organisms from harmful chemicals such as bacteriocins and organic acids. Previous studies have shown that Gram-negative bacteria are inherently resistant to inactivation by xanthene-derivatives, but that pre-treatment with membrane-modulating chemicals, such as EDTA, alters the barrier properties of the outer

membrane leading to sensitivity to these compounds. The objective of this study was to

compare the impact of various physical and chemical treatments on the efficacy of the

xanthene-derivative, FD&C Red No. 3 against Escherichia coli and correlate these

findings with outer membrane properties. Inactivation by these treatments was

determined by plate counting procedures and outer membrane permeability was assessed

using fluorescence microscopy. Treatments with ultra-high pressure (UHP) produced

reversible (>250 MPa) or irreversible (>300 MPa) outer membrane damage as

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determined by fluorescence microscopy. Treatment of E. coli with UHP or acid (pH 3.0)

increased dye accumulation as judged by microscopy and enhanced the bactericidal

properties of Red No. 3. Treatment with EDTA did not lead to cellular uptake of Red No.

3 or inactivation of E. coli by the dye. Bacterial cells were treated with dye or dye-UHP

combination and inner and outer membranes were isolated by ultra-centrifugation on a

sucrose density gradient to determine localization of the dye. Dye accumulation was

associated with the outer membrane following UHP treatment. Interestingly, UHP

treatment resulted in a loss of density of the outer membrane. Further investigations are

needed to identify which membrane components are released during UHP treatment.

INTRODUCTION

Bacteriologists have known, since the late 1960s, that Gram-negative bacteria are

protected from antimicrobial agents, such as antibiotics, dyes, and bile salts due to the

presence of an outer membrane (17). The outer membrane possesses unique barrier

properties, protecting the cell from both polar and nonpolar substances due to its

asymmetrical structure (5, 24). The outer membrane is a bilayer with the inner leaflet

composed of phospholipids and the outer leaflet composed of lipopolysaccharide (LPS).

Like the inner membrane, proteins are imbedded in the outer membrane (2). A majority of the proteins imbedded in the outer membrane are porins that regulate the movement of small hydrophilic compounds (<600 Daltons) and their expression may be regulated by solute concentrations and temperature (2, 17, 22, 24). The outer membrane is stabilized by the presence of divalent cations associated with the LPS and covalent linkage of outer membrane proteins with the peptidoglycan (2, 17). The LPS is primarily responsible for

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protecting the cell from hydrophobic agents and is composed of three distinct regions that are covalently linked: lipid A, core polysaccharide, and O-polysaccharide chain. Rough mutants (deficient in O-polysaccharide chain) and deep rough mutants (deficient in O- polysaccharide chain and core polysaccharide) are susceptible to hydrophobic antimicrobial agents (2).

Due to the barrier properties of the outer membrane, combinations of treatments may be required for antimicrobial compounds to access lethal target sites within the

Gram-negative cell. This is especially true for compounds with inherent activity against

Gram-positive organisms that exhibit minimal or no effect against Gram-negative bacteria. Destabilization of the outer membrane may be accomplished by chemical and physical treatments. Chemical treatments may effect the integrity of the outer membrane by intercalating into membrane structure or releasing membrane components (5).

Chemical treatment with chelators (i.e., EDTA) is known to destabilize LPS and can be used to alter the permeability of the outer membrane to antimicrobial agents (2, 10).

Short treatments (two minutes) of Gram-negative cells with EDTA caused the loss of approximately 50% of the LPS, rendering these cells sensitive to antibiotics (16, 17).

Selectivity of porins is known to be affected by pH, ionic strength and polycations (15).

Physical treatments, including ultra-high pressure (UHP), have also been proposed to permeabilize the outer membrane. The hypothesized change in permeability of was based on the observation that Gram-negative bacteria are resistant to nisin, lysozyme, pediocin, lactoferrin, and lactoferricin, but sensitive to these compounds when treatment is combined with UHP (4, 8, 9, 13, 14). Massachalck et al (2003) investigated the

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efficacy of antimicrobial peptides and pressure on inactivation of outer membrane-

defective mutants of Salmonella and Pseudomonas aeruginosa. Lipolysaccharide length

and hydrophobicity of the outer membrane likely play a role in the inactivation of Gram-

negative bacteria by combinations of antimicrobial peptides and high pressure (13).

Previous studies have found halogenated xanthene-derivatives to be effective antimicrobial agents against Gram-positive bacteria, but less effective or ineffective against Gram-negatives (19, 23). Hydroxyxanthenes, including fluorescein, do not penetrate the outer membrane of Gram-negative bacteria (12, 23). Halogenated xanthene-derivatives are known to be potent photosensitizers and inactivate bacterial cells via the production of reactive oxygen species (ROS) and subsequent reactions with crucial target sites within the cell (11, 18). The outer membrane could protect the cell from ROS produced by photooxidation of xanthene-derivatives, or other photosensitizers, by serving as a permeabilization barrier or as a non-lethal target for ROS (11). Due to the short half-life of ROS, association of the xanthene-derivative with a specific cellular target may be necessary for effective inactivation by photooxidation or other mechanisms.

Phoenix et al (2003) investigated the photooxidative lethality of various phenothiazolium dyes against E. coli and S. aureus with a light dose of 3.15 J/cm2. These dyes were generally more efficacious in inherent toxicity and phototoxicity towards S. aureus compared to E. coli. This is consistent with other studies that suggest that the outer membrane provides an additional layer of protection to Gram-negative bacteria and hinders the uptake of photosensitizing molecules or intercepting photo-generated reactive species (18). Sensitization of the outer membrane by divalent cations, such as calcium

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chloride, has been found to be necessary to enable inactivation of E. coli by photodynamic therapy in a physically separated surface/sensitizer system (21). Rasooly

(2005) suggested that permeabilization of the outer membrane by EDTA treatment could lead to inactivation of Gram-negative cells by Phloxine B with extended light exposure

(19).

The objective of this study was to determine whether altering the barrier properties of the Gram-negative outer membrane (by chemical and physical treatments) enhances lethality caused by halogenated xanthene-derivatives, specifically FD&C Red

No. 3.

MATERIALS AND METHODS

Bacterial strains.

Escherichia coli O157:H7 EDL 933 and K12 were obtained from the culture collection of the Food Safety Laboratory at The Ohio State University (Columbus) and tested in this study. Stock cultures were suspended in appropriate tryptic soy broth (TSB), containing 40% (vol/vol) glycerol, and stored at -80ºC. Immediately before experiments,

E. coli was transferred from frozen stock-culture to tryptic soy agar (TSA) and incubated at 37ºC for 48 to 72 hours. Three isolated colonies of each strain were transferred to TSB and incubated overnight at 37°C.

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FD&C Red No. 3 preparation.

FD&C Red No. 3 (Color Index number 45430, Japan Food Red No. 3) was

obtained from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Stock solutions

(50-1000 ppm) were prepared by dissolving the food colorant in sterile distilled water.

All solutions were used within 30 minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol)

glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

2-liter capacity, and is designed to operate at pressures up to 900 MPa. To reduce

temperature effect on ultra-high pressure inactivation, initial glycol:water processing

fluid was adjusted to achieve a holding temperature of 20-25ºC.

Bacteria treatment in buffer with high pressure.

Overnight cultures were centrifuged at 10,000 rpm (9,000 x g) for 10 minutes

(International Equipment Company IEC Centra MP4R) and suspended in sterile citrate- phosphate buffer, pH 7.0 (as described in Table 3.1). Cell suspensions contained

approximately 109 CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into

sterile polyethylene bags (Fisher Scientific Co., Pittsburgh, Pa.) containing 0.1 ml of food

colorant stock solution to achieve the desired final concentration of these additives (5-10

ppm), and the bags were heat-sealed. Samples were then placed on ice for approximately

30 minutes prior to high pressure treatment. UHP treatments ranged from 200 MPa to

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300 MPa with a holding time of 1 minute. After pressurization, bags were immediately

placed on ice until plated. Control bags were held on ice throughout the duration of the

experiment. Alternatively, food colorant was added to samples after pressure treatment.

In this case, the bags were aseptically opened, the colorant was added to achieve the

desired final concentration and the bags were resealed and placed on ice until plating.

Bags were opened aseptically and the contents were decimally diluted in 0.1% peptone

water, plated on TSA and incubated at 37ºC for 48 hours. Experiments were performed

in triplicate, unless otherwise indicated.

Treatment of cells at different pH values.

Overnight cultures were centrifuged at 10,000 rpm (12,400 x g) for 10 minutes

(Sorvall RC-5B Refrigerated Superspeed Centrifuge, equipped with SM-24 rotor) and

suspended in sterile citrate-phosphate buffers at pH ranging between 3.0 and 7.0. Buffers

varied in molar concentrations depending on desired pH, as shown in Table 3.1.

Suspensions of E. coli in buffers with pH 3.0-7.0 contained approximately 109

CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into sterile polyethylene bags containing 0.1 ml of food colorant stock solution to achieve the desired final concentration of these additives (10 ppm), and the bags were heat-sealed. Sample bags were placed on the bench top for 30 minutes of ambient light exposure. Incident luminescence 485 lux as measured using a light meter (Sekonic Flashmeter L-358,

Elmsford, New York). Bags were opened aseptically and the contents were serially diluted in 0.1% peptone water, plated on TSA and incubated at 37ºC for 48 hours.

Experiments were performed in triplicate.

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pH M Na2HPO4 M citric acid 3.0 0.020 0.040 4.0 0.039 0.031 5.0 0.051 0.024 6.0 0.064 0.018 7.0 0.087 0.007

Table 3.1. Final molar concentration of citrate-phosphate buffer components at

various pH levels.

Treatment of E. coli with EDTA.

Overnight cultures were centrifuged at 10,000 rpm (12,400 x g) for 10 minutes

(Sorvall RC-5B Refrigerated Superspeed Centrifuge, equipped with SM-24 rotor) and

suspended in sterile citrate-phosphate buffer, pH 7.0 containing 0, 10, 15, or 25 mM

EDTA. Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell

suspensions were transferred into sterile polyethylene bags containing 0.1 ml of food

colorant stock solution to achieve the desired final concentration of these additives (10

ppm), and the bags were heat-sealed. Sample bags were placed under ambient light at

25ºC for 30 minutes. Incident luminescence 485 lux as measured using a light meter

(Sekonic Flashmeter L-358, Elmsford, New York). Bags were opened aseptically and the

contents were serially diluted in 0.1% peptone water and plated on TSA incubated at

37ºC for 48 hours. Experiments were performed in triplicate.

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Microscopic evaluation.

Treated bacterial suspensions were also analyzed by fluorescent microscopy

(Olympus BX 61 DSU, Olympus America, Melville, NY) using the GFP and RFP filter

cubes. A volume of 0.5 ml was removed from the sample bag and counterstained with

0.5 µl SYTO9 (BacLight Live/Dead Kit, Molecular Probes, Eugene, OR) to allow

visualization of all cells. Cells were incubated at 25ºC under dark conditions for 15 minutes prior to viewing. Images were captured using commercial software (SlideBook,

Olympus America, Melville, NY) using both green and red channels. Multiple images were captured for each treatment and representative images are displayed in results section.

Membrane isolation.

Overnight cultures of E. coli K12 in TSB (2 L per sample) were centrifuged at

10,000 rpm (16,300 x g) for 10 minutes (Sorvall RC-5B Refrigerated Superspeed

Centrifuge, equipped with GSA-1 rotor) and concentrated to 120 mL in sterile citrate- phosphate buffer, pH 7.0. Suspended cells were transferred to sterile polyethylene bags containing sterile water or FD&C Red No. 3 stock solution to achieve a final dye concentration of 10 ppm or 100 ppm and bags were heat-sealed. Samples were then placed on ice for approximately 30 minutes prior to high pressure treatment of 500 MPa with a holding time of 1 minute. After pressurization, bags were immediately placed on ice. Control bags were held on ice throughout the duration of the experiment. Membrane isolation of E. coli K12 was performed using a density-dependent ultracentrifugation method adapted from Ishidate et al (1986) (7). Briefly, samples were centrifuged and

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washed with 10 mL citrate-phosphate buffer, pH 7.0 and suspended in 5 mL 20% ultracentrifugation grade sucrose in 10 mM HEPES buffer. Cell concentration was approximately 1011 cfu/ml. Samples were frozen at -20ºC until further processed. Frozen treated cells were melted and lysed using a French pressure cell (Aminco, SLM

Instrument, Inc., Urbana, Illinois) was used for cell lysis. EDTA was added to lysate to achieve a final concentration of 5 mM. Samples were centrifuged at 5,000 rpm (3,100 x g) for 20 minutes (Sorvall RC-5B Refrigerated Superspeed Centrifuge, equipped with

SM-24 rotor) to pellet cell debris. Cell lysate supernatant (1 ml) was applied to the following sucrose gradient in 5 mL ultracentrifuge tubes: 1 mL each 45%, 40%, 35%, and 30% ultracentrifugation grade sucrose. Samples were spun at 44,800 rpm (241,000 x g) for 16 hours (Beckman L8-55 Ultracentrifuge, equipped with SW 50.1 swinging bucket rotor). Following ultracentrifugation, tubes were removed, membrane bands were visually identified and distance traveled was measured using a ruler.

Statistical analysis.

Data analysis of variance (ANOVA) was performed using the General Linear

Models Procedure of SAS (SAS Institute, Cary, NC). Average log reductions of treatments were compared using the Tukey’s Studentized Range test.

RESULTS

Impact of order of treatment of FD&C Red No. 3 and ultra-high pressure on inactivation of E. coli EDL 933.

E. coli EDL 933 was subjected to ultra-high pressure treatment ranging from 200

MPa to 300 MPa with a holding time of one minute and with Red No. 3 (10 ppm) added

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before or after pressure treatment. Inactivation results using UHP alone and combination treatments are shown in Figure 3.1. As previously reported in Chapter 2, E. coli is resistant to Red No. 3 treatment alone. E. coli was not significantly inactivated by these

UHP treatments, resulting in less than 0.5 log reduction with 300 MPa treatment. Dye concentration at 10 ppm showed an increase in inactivation with both 250 MPa (1.0 log) and 300 MPa (2.6 log) treatments. If dye addition was completed after pressure treatment,efficacy remained dependent on pressure treatment. Addition of 10 ppm following UHP treatment with (300 MPa) resulted in significant reduction of E. coli cells

(0.8 log); however the resulting inactivation was significantly less than with the dye present during the UHP treatment.

Uptake of Red No. 3 correlates with enhanced inactivation of E. coli by UHP

Cell suspensions, treated with different sequences of pressure and Red No. 3, were analyzed by fluorescent microscopy. Red No. 3 possesses fluorescent properties, emitting red fluorescence, when excited with 530 nm wavelength, with with intensity corresponding to its concentration. E. coli cells were counterstained with SYTO9, a cell- permeant DNA probe, to allow visualization of all cells in samples. Fluorescent images of E. coli treated with the dye during pressure or after pressure treatment are shown in

Figure 3.2. Preliminary image analyses showed that Gram-positive bacteria immediately uptook Red No. 3; these organisms are known to be inherently sensitive to dye treatment with light exposure (data not shown). As discussed earlier, Gram-negative cells do not accumulate the dye and are not sensitive to the dye treatment unless treated with ultra- high pressure. By comparison of the images in Figure 3.2 and the quantitative data

129

4.00 200 MPa, 1 min 250 MPa, 1 min 3.50 300 MPa, 1 min * 3.00

2.50

2.00

1.50 * Log Reduction (cfu/ml) * 1.00

0.50

0.00 No Red 3 Red 3 added before UHP Red 3 added after UHP FD&C Red No. 3 conditions

Figure 3.1. Effect of ultra-high pressure treatment on inactivation of Escherichia coli EDL 933 by FD&C Red No. 3 added before or after pressure treatment. FD&C

Red No. 3 concentration was 10 ppm. Ultra-high pressure treatment varied between 200

MPa and 300 MPa with a holding time of 1 minute. Light exposure was 30 minutes.

Error bars indicate standard error, n = 3. * indicates significant inactivation (p-value

<0.05).

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Dye present during HPP

Dye added after HPP

200 MPa, 1 min 250 MPa, 1 min 300 MPa, 1 min

Figure 3.2. Fluorescent microscopy images of Escherichia coli EDL 933 exposed to

10 ppm FD&C Red No. 3 during or after high pressure treatment. Cells were counterstained with SYTO9 after pressure treatment. Images are an overlay of individual images captured using the GFP and RFP filter sets. Magnification 1000X.

131

shown in Figure 3.1, the following conclusions can be drawn. Low pressure treatments

(i.e., 200 MPa) do not cause significant disruption of the outer membrane to allow for dye uptake by E. coli, the majority of cells in these images are green. The majority of cells pressure treated with 250 MPa or 300 MPa with dye present during pressure treatment are

red, indicating that higher pressure treatments (≥ 250 MPa) cause substantial damage to

the outer membrane to allow uptake of the dye by the cells. Dye uptake, as determined

by microscopy, corresponded to treatments resulting in inactivation of E. coli, as shown in Figure 3.1. Likewise, if the dye is added after the pressure treatment, differences in dye accumulation are apparent between the 250 MPa and 300 MPa treatments. For the

250 MPa treatments, the majority of cells were green if Red No. 3 was added after UHP

whereas cells were mostly red if the colorant was present during pressure treatment.

These findings indicate that the outer membrane damage with pressure treatments at 250

MPa is reversible. Reversible outer membrane damage is also explained by the lack of

inactivation of E. coli with dye added after the 250 MPa treatment compared to the dye

present during UHP. Treatments of cells with 300 MPa, regardless of when the dye was

added, produced a considerable number of red cells, indicating that the outer membrane

damage caused by 300 MPa is irreversible. Dye uptake correlated positively with cell

lethality (Figure 3.1).

UHP decreased density of outer membrane and increased uptake of Red No. 3.

Isolation and resolution of the outer membrane of E. coli K12 with and without

UHP treatment were accomplished (Figure 3.3). The denser fraction is composed of the

outer membrane while the less dense fraction is primarily composed of the inner

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1.8 cm 3.1 cm No UHP

1.8 cm 2.4 cm UHP

0 ppm 10 ppm 100 ppm

Figure 3.3. Illustration of density-dependent membrane isolation from Escherichia coli K12 treated with FD&C Red No. 3 (0-100 ppm) and with or without ultra-high pressure (500 MPa, 1 min). Isolation was performed via ultracentrifugation (44,800 rpm x 16 hours) on a sucrose gradient ranging between 30 and 45 percent sucrose.

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membrane (7). The outer membrane was visible as a dense white layer in the sucrose

column. The location of the outer membrane within the sucrose gradient changed with

UHP treatment. Samples with no UHP treatment resulted in the outer membrane

traveling 3.1 cm with ultra-centrifugation. With UHP treatment the outer membrane

traveled 2.4 cm. This change indicates a decrease in the density of the outer membrane

due to UHP treatment. This change occurred regardless of Red No. 3 concentration.

Dye accumulation associated with the outer membrane was weakly noticeable in

control samples (no UHP treatment) and dose-dependent with the 100 ppm samples appearing slightly more pink in color than the 10 ppm samples. With UHP treatment, a substantial quantity of Red No. 3 was associated with the outer membrane bands, again more apparent with the 100 ppm than the 10 ppm treatments. With dye and UHP treatment, the outer membrane band maintained a white opacity and was surrounded in the column by a pink or red defined area. In the case of the 100 ppm UHP treatment, the red color was close to overwhelming the visualization of the outer membrane band.

With all treatments, the inner membrane was resolved, but poorly visualized using this procedure but could be faintly detected as a less dense brownish colored band.

Attempts to improve inner membrane isolation and visualization using increased cell density and changes in sucrose gradient were unsuccessful. Despite this drawback, accumulation of Red No. 3 with the inner membrane was not observed for any of the treatments, suggesting that the inner membrane may not be the target for inactivation of

Gram-negative bacteria using this combination treatment.

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Following UHP treatment and during the washing step, there was a noticeable

difference in the Red No.3 concentration in the supernatants of pressure treated and non-

treated cells. Spectrophotometric measurements showed an increased association of Red

No. 3 with the UHP treated, compared to non-UHP treated cells. Concentration of Red

No. 3 with cell supernatant decreased by 60% for cells treated with UHP, compared to

non-treated cells, regardless of colorant concentration (data not shown).

Low pH leads to sensitivity of E. coli to Red No. 3.

Outer membrane stability is known to be effected by extrinsic conditions such as

the pH of suspension medium (15). E. coli cells were suspended in citrate-phosphate

buffer with varying pH levels and exposed to Red No. 3. Efficacy of Red No. 3 in

combination with pH against E. coli EDL 933 is shown in Figure 3.4. E. coli was not significantly inactivated by suspension in various buffers. Inclusion of 10 ppm Red No. 3 caused significant inactivation of E. coli cells only at pH 3.0 (2.7 log reduction).

Additionally, samples were analyzed using fluorescence microscopy to determine if accumulation of dye correlated with inactivation. Microscopic images of pH treatments

7.0 and 3.0 are shown in Figure 3.5. Little to no association of Red No. 3 was detectable in samples suspended in pH 7.0 buffer. Association of the dye with E. coli cells was apparent at pH 3.0, with a majority of the cells being red in color. Uptake of dye by cells correlates with inactivation patterns shown in Figure 3.4

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1.50 0 ppm dye 10 ppm dye

*

1.00

0.50 Log Reduction (cfu/ml) Reduction Log

0.00 7.06.05.04.03.0 pH

Figure 3.4. Effect of pH on inactivation of Escherichia coli EDL 933 by FD&C Red

No. 3. FD&C Red No. 3 concentrations were 10 ppm. Light exposure was 30 minutes.

Error bars indicate standard error, n = 3. * indicates significant inactivation of E. coli by a particular treatment (p-value <0.05).

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pH 7.0 pH 3.0

Figure 3.5. Fluorescent microscopy images of Escherichia coli EDL 933 exposed to

10 ppm FD&C Red No. 3 at pH 7.0 or 3.0. Cells were counterstained with SYTO9 after light exposure. Images are an overlay of individual images captured using the GFP and RFP filter sets. Magnification 1000X.

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DISCUSSION

Depending on UHP treatment dose, the permeability of the outer membrane is reversibly (250 MPa) or irreversibly (300 MPa) compromised, thus leading to uptake of

Red No. 3 and inactivation. The reversible nature of outer membrane disruption by UHP has been previously noted by Hauben et al (1996) (4). E. coli cells were treated with combinations of UHP and nisin or lysozyme added before or after UHP treatment.

Inclusion of lysozyme or nisin during UHP at 270 MPa increased inactivation compared to UHP alone, indicating a change in permeability of the outer membrane. Lysozyme or nisin treatment after UHP treatment of 270 MPa displayed to increased inactivation, compared to UHP alone, indicating that the change in permeability of the outer membrane was reversible (4).

Membrane isolation experiments suggest that the decrease in density of the outer membrane due to UHP also correlates with accumulation of Red No. 3 with the remaining portions of the outer membrane. These lost components may provide the barrier properties to the outer membrane that protect E. coli from inactivation by Red No.

3 treatments alone. LPS is usually associated with the barrier properties to the outer membrane. Ganzle and Vogel (2001) used 1-N-phenylnaphthylamine (NPN) as a fluorescent probe to determine changes in the outer membrane of E. coli during UHP treatment. NPN was incorporated into UHP treated samples either during or after treatment to determine reversible and irreversible effects on the outer membrane.

Changes in the outer membrane were dependent on pressure level, however outer membrane damage was not predicative of cell death. The authors proposed that increased

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permeability of the outer membrane due to UHP treatment was the result of a loss of lipopolysaccharide component (3). Ritz et al (2000) investigated changes in outer membrane proteins of Salmonella exposed to UHP. UHP treated cells were more resistant to EDTA-lysozyme treatments for spheroplast formation compared to control cells and protein profile of the outer membrane was altered following treatment. The authors postulated that UHP treatment caused a reorganization of the outer membrane, likely accompanied by a decrease in the number of porins present. Major and minor proteins normally present in the outer membrane of control cells were absent following

UHP treatment (20). Further studies are needed to identify which components of the outer membrane are lost or modified by UHP treatment, leading to dye uptake.

Hydroxyxanthenes theoretically should localize in the cellular membrane (lipid environment) based on the lipophilic nature of these compounds based on measured partition coefficients. However, the transfer from aqueous solution to the cellular membrane may be impeded compared to transfer from water to octanol due to the negative charges on both cellular membrane surface and hydroxyxanthenes (1). Wang et al (2006) states that xanthene-derivatives typically localize in the cellular membrane and photooxidation leads to destructive damage of lipid and protein components (23). Dahl et al (1989) exploited the shift in absorbance maximum wavelength of Rose Bengal to determine localization within wild-type and deep rough mutants of Salmonella. Wild- type strains exhibited typical fluorescence patterns, whereas deep rough mutants displayed a shift in absorbance indicating that some Rose Bengal was present in a more lipid environment, likely a membrane (1). Using confocal fluorescence microscopy,

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Inbaraj et al (2005) found Phloxine B to localize within the cytoplasmic membrane of keratinocytes. However, increases in incubation time (10 minutes) resulted in diffusion of the colorant in the cytoplasm indicating that the membrane may not be the lethal target site (6).

Inactivation of bacteria by xanthene-derivatives, including FD&C Red No. 3, is thought to be caused by the photooxidation products of these dyes when exposed to light.

With light exposure, xanthene-derivatives can undergo both type I and type II photooxidation, producing damaging free radicals and singlet oxygen, respectively.

Schafer et al (2000) directly measured the production of singlet oxygen by photooxidation of 2 ppm Rose Bengal, a xanthene-derivative, for different pH indices

(pH 4.5, 7.0, and 9.6). The patterns of singlet oxygen production were identical regardless of pH indicating no effect on singlet oxygen production by pH. However, singlet oxygen could damage parts of the membrane or penetrate the cell wall more easily if the pH of the medium is altered. Inactivation of E. coli by Rose Bengal was enhanced at pH 4.5 and 9.6 compared to pH 7.0 (21).

EDTA treatments (10, 15, and 25 mM) were ineffective at reducing populations of E. coli (data not shown). Fluorescence microscopy confirmed that EDTA treatment did not result in any accumulation of the dye with E. coli cells (images not shown).

Rasooly (2005) used EDTA and magnesium chloride treatments to alter the outer membrane permeability of E. coli in an attempt to sensitize the cells to Phloxine B.

These experiments were reported as successful; however bactericidal inactivation was only apparent with light exposures at or exceeding five hours (19).

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The outer membrane plays an important role in protecting E. coli from

inactivation by xanthene-derivatives, including Red No. 3. UHP is capable of changing

the outer membrane, resulting in a decrease in density and significant uptake of Red No.

3 within or surrounding the outer membrane. These results taken together suggest that localization of a threshold level Red No. 3 within the outer membrane is essential for inactivation of Gram-negative bacteria and that UHP is an effective mechanism for providing the colorant access to the outer membrane.

ACKNOWLEDGEMENTS

This research was supported by a grant from the Center for Advanced Processing and Packaging Studies (CAPPS). The author would like to thank Jeremy Somerville for supplying the light meter for illuminance measurements. The author would like to thank

Dr. Steve Schwartz and Rachel Kopec for use of and assistance with the Beckman ultracentrifuge. The author would also like to thank the Department of Microbiology and

Richard Nist at The Ohio State University for use of and assistance with the French

Pressure Cell. Final thanks to Dr. Monica Giusti and Dante Vargas for UV-vis spectrophotometric measurements of colorant samples.

REFERENCES

1. Dahl T A, Valdes-Aguilera O M, Midden W R, and Neckers D C. 1989. Partition of rose bengal anion from aqueous medium into a lipophilic environment in the cell envelope of Salmonella typhimurium: implications for cell-type targeting in photodynamic therapy. Journal of Photochemistry and Photobiology: B 4:171-184.

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2. Denyer S P and Maillard J-Y. 2002. Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria. Journal of Applied Microbiology 92:35S-45S.

3. Ganzle MG and Vogel RF. 2001. On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli. Systematic and Applied Microbiology 24:477-485.

4. Hauben K J A, Wuytack EY, Soontjens C C F, and Michiels CW. 1996. High- pressure transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability. Journal of Food Protection 59:350- 355.

5. Helander IM and Mattila-Sandholm T. 2000. Fluorometric assessment of Gram-negative bacterial permeabilization. Journal of Applied Microbiology 88:213-219.

6. Inbaraj J J, Kukielczak B M, and Chignell C F. 2005. Phloxine B phototoxicity: A mechanistic study using HaCaT keratinocytes. Photochemistry and Photobiology 81:81-88.

7. Ishidate K, Creeger E S, Zrike J, Deb S, Glauner B, MacAlister T J, and Rothfield L I. 1986. Isolation of differntiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murien skeleton of the cell envelope. The Journal of Biological Chemistry 261:428-443.

8. Kalchayanand N, Sikes A, Dunne CP, and Ray B. 1998. Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria. Journal of Food Protection 61:425-431.

9. Kalchayanand N, Sikes T, Dunne CP, and Ray B. 1994. Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Applied and Environmental Microbiology 60:4174-4177.

10. Leive L. 1965. Release of lipopolysaccharide by EDTA treatment of Escherichia coli. Biochemical and Biophysical Research Communications 21:290-296.

11. Malik Z, Ladan H, and Nitzan Y. 1992. Photodynamic inactivation of Gram- negative bacteria: problems and possible solutions. Journal of Photochemistry and Photobiology: B 14:262-266.

12. Martin J P Jr and Logsdon N. 1987. Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and lucifer yellow CH. Photochemistry and Photobiology 46:45-53.

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13. Masschalck B, Deckers D, and Michiels CW. 2003. Sensitization of outer- membrane mutants of Salmonella Typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under high pressure. Journal of Food Protection 66:1360- 1367.

14. Masschalck B, Van Houdt R, and Michiels CW. 2001. High pressure increases bactericial activity and spectrum of lactoferrin, lactoferricin and nisin. International Journal of Food Microbiology 64:325-332.

15. Muller D J and Engel A. 1999. Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. Journal of Molecular Biology 285:1347-1351.

16. Muschel L H and Gustafson L. 1968. Antibiotic, detergent, and complement sensitivity of Salmonella typhi after ethylenediaminetetraacetic acid treatment. Journal of Bacteriology 95:2010-2013.

17. Nakae T and Nikaido H. 1975. Outer membrane as a diffusion barrier in Salmonella typhimurium. The Journal of Biological Chemistry 250:7359-7365.

18. Phoenix DA, Sayed Z, Hussain S, Harris F, and Wainwright M. 2003. The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus. FEMS Immunology and Medical Microbiology 39:17-22.

19. Rasooly R. 2005. Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria. FEMS Immunology and Medical Microbiology 45:239-244.

20. Ritz M, Freulet M, Orange N, and Federighi M. 2000. Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium. International Journal of Food Microbiology 55:115-119.

21. Schafer M, Schmitz C, Facius R, Horneck G, Milow B, Funken K-H, and Ortner J. 2000. Systematic study of parameters influencing the action of rose bengal with visible light on bacterial cells: Comparison between the biological effect and singlet-oxygen production. Photochemistry and Photobiology 71:514- 523.

22. Scudamore R A, Beveridge T J, and Goldner M. 1979. Outer-membrane penetration barriers as components of intrinsic resistance to beta-lactam and other antibiotics in Escherichia coli K-12. Antimicrobial Agents and Chemotherapy 15:182-189.

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23. Wang H, Lu L, Zhu S, Li Y, and Cai W. 2006. The phototoxicity of xanthene derivatives against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Current Microbiology 52:1-5.

24. Wiese A, Brandenburg K, Ulmer A J, Seydel U, and Muller-Loennies S. 1999. The dual role of lipopolysaccharide as effector and target molecule. Biological Chemistry 380:767-784.

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CHAPTER 4

CONTRIBUTION OF PHOTOOXIDATION TO INACTIVATION OF GRAM-

POSITIVE AND GRAM-NEGATIVE BACTERIA BY ULTRA-HIGH PRESSURE

AND XANTHENE-DERIVATIVES

ABSTRACT

Xanthene-derivatives, including FD&C Red No. 3, are known to act as photosensitizers leading to inactivation of bacteria with intense light exposure. Previous studies have found the combination treatment of ultra-high pressure (UHP) and xanthene- derivatives to display synergistic inactivation against Gram-positive and Gram-negative bacteria. The contribution of photooxidation to the inactivation by this combination treatment needs to be determined to optimize treatments for maximum bacterial inactivation. In the present study, combination treatments of UHP and Red No. 3 were compared for efficacy with and without light treatment to quantify photooxidative inactivation of Lactobacillus plantarum, Listeria monocytogenes, and Escherichia coli.

Treatment of these species with dye treatment alone (no UHP) only resulted in inactivation of the Gram-positive organisms with light exposure. E. coli was resistant to the colorant treatment regardless of light exposure. Using the combination treatment

(with UHP, 300 MPa, 1 min), enhanced inactivation of all three organisms was realized

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only with light exposure, indicating a significant contribution of photooxidation. Order

of exposure of light and UHP treatment had a significant impact on inactivation of E. coli,

indicating that changes in localization of the colorant caused by UHP treatment leads to

sensitivity of the cells to inactivation by photooxidation. Similarly, L. monocytogenes is

more sensitive to photooxidation that occurs following UHP. Additional xanthene- derivatives were tested to determine impact of photooxidation on combination treatment.

FD&C Red No. 3, Erythrosin B, Phloxine B, and Rose Bengal exhibited photooxidative

capabilities in combination with UHP against both L. monocytogenes and E. coli.

Additionally, Eosin Y was effective against E. coli with light exposure. Interestingly, some of the xanthene-derivatives exhibited enhanced inactivation of bacteria with UHP

(500 MPa, 1 min) without light exposure indicating a potential light-independent effect of the combination treatment. Further investigations into this light-independent effect are warranted.

INTRODUCTION

Photooxidation is a process where a photosensitizer is excited by light and reacts with other compounds to produce oxidized endproducts (7). This process is also referred to as photosensitized oxidation or photodynamic effect. Photooxidation to inactivate cells or proteins has been studied since the 1880s (8). Briefly, photooxidation requires the transfer of energy to the photosensitizer resulting in an excited triplet state of the photosensitizer. The excited molecule may lose energy in three different pathways: reaction with cellular or soluble components (type I reaction), reaction with triplet oxygen (type II reaction) or light emission (phosphorescence). These reactions are not

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mutually exclusive, and the ratio of each result is dependent on the photosensitizer and

concentrations of reactants (substrates and oxygen) (4). The products of type I and type

II reactions possess antimicrobial activity.

The antibacterial activity of hydroxyxanthenes via photooxidation was first reported in 1904 by Jadlbauer and von Tappeiner and in 1908 by Reitz (8, 11).

Photooxidation of xanthene-derivatives has been shown to inactivate viruses, yeasts, damage photosystem II in leaf tissues, and inactivate bacterial biofilms (16, 24). Several studies have investigated utilizing photooxidation of Rose Bengal for water treatment (10,

13, 20, 2, 16).

Differences in microbial sensitivity to Eosin were reported in the 1930s (8, 22).

Streptococcus hemolyticus, Cornyebacterium diphtheriae, and Neisseria intracellularis were particularly sensitive to Eosin, while Staphylococcus albus, S. paratyphi, S. paradysenteriae Flexner, and Brucella abortus were resistant to all conditions tested (22).

Similar studies continue today. Wang et al (2006) found the yeast Saccharomyces cerevisiae to be more sensitive to photooxidation by hydroxyxanthenes than the Gram- positive Staphylococcus aureus and S. aureus more sensitive than Gram-negative E. coli

(23). Differences in sensitivity to hydroxyxanthenes have been exploited for selective growth of microorganisms in laboratory media. Rose Bengal is used as an ingredient in agar media to inhibit the growth of bacteria in favor of fungal growth, while Eosin is used to inhibit the growth of Gram-positive bacteria in favor of Gram-negative bacteria (1, 17).

Most studies have reported minimal, if any, effect of hydroxyxanthenes on Gram- negative bacteria. Ineffectiveness of these compounds against Gram-negative bacteria

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has been attributed to the barrier properties of the outer membrane (6). Rasooly (2005)

tested the effect of Phloxine B on inactivation of various Gram-positive and Gram-

negative organisms. Only Gram-positive organisms were inactivated including S. aureus,

Bacillus cereus, B. mycoides, B. thuringienisis, and B. subtilis. Concentrations of 50 ppm of Phloxine B inactivated 1 to 2.5 log cfu of various Gram-positive bacteria with 120 minutes of light exposure. Gram-negative organisms were resistant to inactivation at levels of 100 ppm including Salmonella, E. coli, and E. coli O157:H7 strains (19).

Erythrosin, at concentrations of 0.5 and 1.0%, is effective at inhibiting the growth of numerous organisms found in dental plaque, including Streptococcus spp. and some yeast, but ineffective against Gram-negative bacteria (3). Dahl et al (1988) suggested that penetration of Rose Bengal through the outer membrane may occur slowly, which may explain differences in efficacy of hydroxyxanthenes on Gram-negative bacteria due to differences in exposure times of various studies (5).

Combination treatment of ultra-high pressure (UHP) and xanthene-derivatives is effective against Gram-positive and Gram-negative bacteria. Previous studies were completed with minimal control over light exposure. The objective of this study was to determine the impact of photooxidation on inactivation of Gram-positive and Gram- negative bacteria by UHP and hydroxyxanthene combination treatment.

MATERIALS AND METHODS

Bacterial strains.

Listeria monocytogenes (OSY-328 and ScottA), Lactobacillus plantarum OSY-

104 and MDOS-32, Escherichia coli O157:H7 EDL 933 were obtained from the culture

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collection of the Food Safety Laboratory at The Ohio State University (Columbus) and

tested in this study. Stock cultures were suspended in appropriate broth media,

containing 40% (vol/vol) glycerol, and stored at -80ºC. Immediately before experiments,

L. plantarum strains was transferred from the frozen stock-culture to De Man, Rogosa

and Sharpe (MRS) agar and incubated at 37°C for 48-72 hours. Similarly, L.

monocytogenes and E. coli were transferred from frozen stock-culture to tryptic soy agar

(TSA) and incubated at 37ºC for 48 to 72 hours. Three isolated colonies of each strain

were transferred to the appropriate broth (MRS or TSB) and incubated overnight at 37°C

(for L. plantarum) or 37°C (for all other bacteria).

Xanthene-derivatives.

FD&C Red No. 3 (CI number 45430, Japan Food Red No. 3) was obtained from

Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Fluorescein (CI number 45350),

Erythrosin B (CI number 45430, Japan Food Red No. 3), Phloxine B (CI number 45410,

Japan Food Red No. 104), Eosin Y (CI number 45380), and Rose Bengal (CI number

45440, Japan Food Red No. 105) were purchased from Sigma-Aldrich (St. Louis, MO).

Stock solutions (50-1000 ppm) were prepared by dissolving the food colorant in sterile

distilled water. All solutions were used within 30 minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol)

glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

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2-liter capacity, and is designed to operate at pressures up to 900 MPa. To reduce

temperature effect on ultra-high pressure inactivation, initial glycol:water processing

fluid was adjusted to achieve a holding temperature of 20-25ºC.

Bacteria treatment in buffer.

Overnight cultures were centrifuged at 10,000 rpm (9,000 x g) for 10 minutes

(International Equipment Co., IEC Centra MP4R) and suspended in sterile citrate- phosphate buffer (0.087 M phosphate, 0.007 M citrate, pH 7.0). Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into clear sterile polyethylene bags (Fisher Scientific Co., Pittsburgh, Pa.) or

black sterile polyethylene bags (Nasco Whirl-Pak, Fort Atkinson, Wi.) containing 0.1 ml of colorant stock solution to achieve the desired final concentration of these additives, and the bags were heat-sealed. Sample bags were then placed into a larger polyethylene bag and sealed using a vacuum sealer (Vacmaster, Kansas City, Mo.). Samples were then placed on ice for approximately 30 minutes prior to high pressure treatment. UHP treatments ranged from 300 to 500 MPa with holding times of one minute. After pressurization, bags were immediately placed on ice until plated. Control bags were held

on ice throughout the duration of the experiment. Bags were opened aseptically and the

contents were decimally diluted in 0.1% peptone water and plated on the appropriate agar

medium. L. plantarum strains were plated on MRS agar and incubated at 37°C for 48

hours prior to enumeration. L. monocytogenes and E. coli were plated on TSA and incubated at 37ºC for 48 hours. Experiments were performed in triplicate, unless otherwise indicated.

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Light exposure of samples.

Light exposure consisted of samples in clear polyethylene bags being spread onto

laboratory bench under ambient lighting conditions for 15 or 30 minute increments before

and/or after pressure treatment, depending on experiment. Incident luminescence 485 lux

as measured using a light meter (Sekonic Flashmeter L-358, Elmsford, New York).

Following controlled light exposure, samples were protected from light by wrapping the

samples in aluminum foil until dilution and plating were performed.

Statistical analysis.

Data were analyzed by analysis of variance (ANOVA) by using the General

Linear Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the

mean log reductions of treatments were made using the Tukey’s Studentized Range test.

RESULTS

Light exposure and Red No. 3 concentration cause inactivation of Gram-positive

bacteria.

Inactivation of L. monocytogenes ScottA, L. plantarum OSY-104 and E. coli EDL

933 by Red No.3 with and without light exposure is shown in Figure 4.1. As expected, E.

coli EDL 933 was unaffected by dye concentration regardless of light exposure. Gram-

positive bacteria were sensitive to Red No. 3 with light exposure. Samples protected

from light were unaffected by the colorant. L. monocytogenes was more resistant to dye

compared to L. plantarum. The only effective treatment against L. monocytogenes ScottA was 10 ppm Red No. 3 with light exposure (1 hour total) (0.9 log reduction). L. plantarum OSY-104 was inactivated by Red No. 3 at 5 and 10 ppm, in combination with

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9 0 ppm, Light 8 5 ppm, Light 10 ppm, Light 7 0 ppm, Dark 5 ppm, Dark 6 10 ppm, Dark a 5

4 b

3 Log Reduction (cfu/ml)

2 a 1 b b b b b c c c c 0 ListeriaListeria monocytogenes monocytogenes ScottA LactobacillusLactobacillus plantarum plantarum OSY- Escherichia coli EDLO157:H7 933 Scott A OSY-104104 EDL 933 Species and Strain

Figure 4.1. Effect of FD&C Red No. 3 concentration and light exposure on Listeria monocytogenes ScottA, Lactobacillus plantarum OSY-104, and Escherichia coli EDL

933. FD&C Red No. 3 concentrations varied from 0 to 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for two 30 minute periods. Dark samples were protected from light by being stored in black polyethylene bags. Error bars indicate standard error, n = 3. Different letter designations indicate significant differences in dye treatments for each strain (p-value <0.05).

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light exposure. The 10 ppm treatment was significantly (p-value = 0.004) more effective

(5.0 log reduction) than the 5 ppm treatment (3.3 log reduction). The findings indicate that light exposure is essential for inactivation of Gram-positive organisms by Red No. 3.

Impact of light exposure on bacterial inactivation of various species with UHP-Red

No. 3 combination (300 MPa, 1 min).

Inactivation of various species by combination treatment of FD&C Red No. 3 and ultra-high pressure (300 MPa, 1 min) with and without light exposure (1 hour total) is shown in Figure 4.2. Pressure treatment was only mildly lethal, with the pressure- sensitive L. plantarum OSY-104 strain displaying the largest inactivation (1.2 log reduction). Inactivation of L. monocytogenes Scott A and E. coli EDL 933 by UHP alone was minimal with inactivation of 0.14 and 0.41 log reduction, respectively. L. monocytogenes, L. plantarum, and E. coli inactivation were significantly enhanced (p- values = 0.004, 0.001, and 0.001, respectively) by the combination of 10 ppm FD&C Red

No. 3, pressure treatment, and light exposure, compared to pressure treatment alone with log reductions of 2.8, 6.2, and 1.0 respectively. L. plantarum OSY-104 also displayed enhanced inactivation with 5 ppm colorant and light exposure (5.2 log reduction).

Samples protected from light were not significantly different (p-values greater than 0.10) from samples with UHP treatment alone, indicating enhancement of inactivation to be due to photooxidation.

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9.00 0 ppm, Light 8.00 5 ppm, Light 10 ppm, Light 7.00 0 ppm, Dark a 5 ppm, Dark 6.00 10 ppm, Dark a

5.00

4.00 a 3.00 Log Reduction (cfu/ml) b b 2.00 b b b a 1.00 a,b b b b b b b b b 0.00 Listeria monocytogenes Lactobacillus plantarum Escherichia coli Listeria monocytogenes Lactobacillus plantarum OSY- Escherichia coli O157:H7EDL 933 ScottScottA A OSY-104104 EDL 933 Species and Strain

Figure 4.2. Effect of FD&C Red No. 3 concentration, light exposure, and ultra-high pressure on Listeria monocytogenes ScottA, Lactobacillus plantarum OSY-104, and

Escherichia coli EDL 933. FD&C Red No. 3 concentrations varied from 0 to 10 ppm.

Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for two 30 minute periods. Dark samples were protected from light by being stored in black polyethylene bags. Ultra-high pressure treatment was 300 MPa for 1 minute. Error bars indicate standard error, n = 3. Different letter designations indicate significant differences in dye treatments for each strain (p-value <0.05).

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Order of light exposure and UHP impacts inactivation of E. coli EDL 933.

Impact of order of pressure treatment (400 MPa, 1 min) and light exposure was

investigated using E. coli as the target organism to determine whether light exposure is

more efficacious, before or after UHP treatment. Inactivation resulting from light

exposure prior to UHP treatment could be attributed to the production of stable

photooxidative products, whereas inactivation resulting from light exposure after UHP is

likely attributable to the localization of the dye and production of photooxidative products that may be less stable. Samples were exposed to light for 0 minutes, 15 minutes before pressure treatment, 15 minutes after pressure treatment or 15 minutes before and after pressure treatment (30 minutes total). Resulting inactivation is shown in

Figure 4.3. As expected, light treatment had no effect on inactivation of E. coli with

UHP alone (1.0 log inactivation without light and 1.1 log inactivation with 30 minutes light). Samples exposed to a total of 30 minutes of light exhibited the highest inactivation for 5 and 10 ppm, 4.0 and 4.6 log inactivation respectively. Order of light exposure was a significant factor in explaining inactivation by combination treatment with 5 and 10 ppm. Light exposure prior to ultra-high pressure treatment (400 MPa, 1 min) had a minimal effect on inactivation of E. coli. Inactivation was enhanced with 10 ppm (1.7 log reduction) when compared to 0 ppm (0.8 log reduction) with light exposure prior to UHP, however inactivation was less than 10 ppm Red No. 3 with no light exposure (2.1 log reduction). Inactivation by 5 ppm with 15 minutes of light exposure prior to UHP was significantly less effective than 5 ppm with no light exposure (p-value

= 0.046). Light exposure after pressure treatment caused significantly more lethality

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9.00 0 minutes light 15 minutes light before UHP 8.00 15 minutes light after UHP 30 minutes light total 7.00

6.00

5.00 a a a 4.00

b 3.00 Log Reduction (cfu/ml) b c b 2.00 a d a a a 1.00

0.00 0510 FD&C Red No. 3 (ppm)

Figure 4.3. Effect of light exposure on inactivation of Escherichia coli EDL 933 by

FD&C Red No. 3 concentration and ultra-high pressure treatment. FD&C Red No.

3 concentrations varied from 0 to 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for 15 minute periods before and/or after pressure treatment. Dark samples were protected from light by being stored in black polyethylene bags. Ultra-high pressure treatment was 400 MPa for 1 minute. Error bars indicate standard error, n ≥ 2. Different letter designations indicate significant differences in light treatments for each dye concentration (p-value <0.05).

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of E. coli with Red No. 3 concentrations of 5 and 10 ppm compared to samples with no

light exposure (p-values of 0.046 and 0.004, respectively). Inactivation with 10 ppm with

light exposure after UHP treatment (4.0 log inactivation) was similar to 10 ppm with 30

minute light exposure (4.6 log inactivation). These findings suggest that photooxidative

effects against Gram-negative bacteria are primarily attributable to photooxidation

following localization of the dye to the outer membrane or other compartment of the cell

(see Chapter 3).

It is worth mentioning that presence of the dye during UHP treatment and no light

exposure (1.9 and 2.1 log reduction for 5 and 10 ppm, respectively) did show enhanced

inactivation compared to UHP alone (1.0). This effect has not previously been observed

and further investigation into this phenomenon is reported in Chapter 5.

Effect of light exposure on inactivation by xanthene-derivatives-UHP combination.

Inactivation of L. monocytogenes by xanthene-derivative combination treatments

is shown in Figure 4.4. Light exposure for these studies was minimal with a total light

exposure time of only 15 minutes. Rose Bengal was the only xanthene-derivative to

display significant antimicrobial activity (p-value = 0.001) without UHP treatment under

this brief light exposure (4.2 log reduction), however longer light exposure (30 minutes)

would produce greater inactivation for Phloxine B and Erythrosin B (See Chapter 2,

Figure 2.6). Fluorescein and Eosin Y had no effect on microbial inactivation in

combination with UHP against L. monocytogenes under these treatment conditions. Red

No. 3, Erythrosin B, and Phloxine B enhanced inactivation (6.2, 5.7, 8.1 log reductions,

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9.00 15 minutes light, No UHP 0 minutes light, UHP * 8.00 15 minutes light, UHP * * 7.00 * * 6.00

5.00 * 4.00

3.00 Log Reduction (cfu/ml)

2.00

1.00

0.00 None FD&C Red Fluorescein Eosin Y Erythrosin B Phloxine B Rose Bengal No. 3 Xanthene-derivative (ppm)

Figure 4.4. Effect of light exposure on inactivation of Listeria monocytogenes OSY-

328 by combination treatment of xanthene-derivatives and ultra-high pressure.

Xanthene-derivatives concentrations were 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for 15 minutes after pressure treatment. Dark samples were protected from light by being stored in black polyethylene bags. Ultra-high pressure treatment was 500 MPa for 1 minute. Error bars indicate standard error, n = 3. * indicates significant inactivation beyond UHP alone (p- value <0.05).

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respectively) by combination treatment only with light exposure, indicating that

photooxidation following UHP treatment is responsible for the increased efficacy of these treatments.

Inactivation of E. coli by xanthene-derivatives combination treatments is shown

in Figure 4.5. Xanthene-derivatives without pressure treatment were ineffective at

reducing E. coli populations (data not shown). UHP treatments with Red No. 3, Eosin Y,

Erythrosin B, Phloxine B, and Rose Bengal and light exposure resulted in enhanced

inactivation of E. coli (4.7, 2.3, 5.1, 4.8, and 4.5 log inactivation, respectively). With

Gram-negative organisms, UHP leads to accumulation of hydroxyxanthenes (See Chapter

3) resulting in sensitivity to photooxidative processes. Fluorescein exhibited no effect on

the inactivation of E. coli with UHP treatment.

DISCUSSION

Schafer et al (2000) studied the efficacy of photooxidation of E. coli and

Deinococcus radiodurans using Rose Bengal. There appears to be a threshold level for

inactivation of bacteria by photooxidation, likely indicating a multitarget mechanism of

inactivation (21). The increase in lethality of photooxidation following UHP suggests

that UHP somehow sensitizes L. monocytogenes to photooxidation by these halogenated xanthene-derivatives. UHP may enable the halogenated xanthene-derivatives to localize to a more lethal target site within the cell. Alternatively, UHP may inhibit the ability of L.

monocytogenes to deal with photooxidation byproducts. Rose Bengal is reported to be

the strongest photosensitizer of the halogenated hydroxyxanthenes, thus this effect is not

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9.00 0 minutes light, UHP 15 minuts light, UHP 8.00

7.00

6.00 a a a 5.00 a

4.00 b b b,c 3.00

Log Reduction (cfu/ml) b,c b,c,d 2.00 c,d c,d c,d c,d 1.00 d

0.00 None FD&C Red Fluorescein Eosin Y Erythrosin B Phloxine B Rose Bengal No. 3 Xanthene-derivative (10 ppm)

Figure 4.5. Effect of light exposure on inactivation of Escherichia coli EDL 933 by combination treatment of xanthene-derivatives and ultra-high pressure. Xanthene- derivatives concentrations were 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for 15 minutes after pressure treatment. Dark samples were protected from light by being stored in black polyethylene bags. Ultra-high pressure treatment was 500 MPa for 1 minute. Error bars indicate standard error, n = 3. Different letter designations indicate significant differences between all treatments shown (p-value <0.05).

160

surprising (16). Previous studies have shown that the photodynamic action observed with

Rose Bengal depends on the availability and mobility of singlet oxygen instead of

localization of Rose Bengal. Inactivation by Rose Bengal has been shown where the dye

did not contact the cell membrane or penetrate the cytoplasm with experiments requiring

diffusion of singlet oxygen to interact and inactivate cells (21).

Studies on inactivation of microorganisms by photooxidation of hydroxyxanthenes have found little to no antimicrobial activity of these compounds without light exposure. Martin and Logsdon (1987) found hydroxyxanthenes Rose

Bengal and Erythrosin incapable of reducing oxygen levels without light exposure (14).

Hydroxyxanthenes (Erythrosin B, Phloxine B and Eosin B) had no effect on

entomopathogenic fungi without light exposure (12).

Previous studies have also found Gram-negative bacteria to be resistant to

inactivation by photooxidiation of halogenated hydroxyxanthenes, including species

tested in this study. Most studies have reported minimal, if any, effect of

hydroxyxanthenes on Gram-negative bacteria. Ineffectiveness of these compounds

against Gram-negative bacteria has been attributed to the barrier properties of the outer

membrane (6,23). Wang et al (2006) found the yeast Saccharomyces cerevisiae to be

more sensitive to photooxidation by hydroxyxanthenes than the Gram-positive

Staphylococcus aureus. S. aureus was more sensitive than Gram-negative E. coli (23).

Erythrosin, at concentrations of 0.5 and 1.0%, is effective at inhibiting the growth of

numerous organisms found in dental plaque, including Streptococcus spp. and some yeast,

but ineffective against Gram-negative bacteria (3). Several studies using Rose Bengal as

161

a photosensiziter for water treatment have reported efficacy against fecal coliforms,

including E. coli (10, 13, 20). This effect is enhanced by increasing Rose Bengal

concentration (1 µM to 10 µM) and light exposure time (0 to 240 minutes) (10).

Previous studies have shown fluorescein to be incapable of photooxidation and

therefore having no impact on inactivation of organisms with light exposure. Fluorescein

does not exhibit antimicrobial properties against various Gram-positive, Gram-negative

bacteria, or yeast at concentrations of 500 µM (15, 23).

Comparing efficacy of hydroxyxanthenes in combination with UHP against both

L. monocytogenes and E. coli, few differences were noticeable among the

hydroxyxanthenes. Eosin Y produced a significant enhancement of inactivation of E. coli

(p-value = 0.001), but not L. monocytogenes indicating that this compound has the

weakest effect of the halogenated compounds tested. Rose Bengal was the only

compound to have an effect on L. monocytogenes without UHP, suggesting that this is the most effective compound tested. With UHP treatment and light exposure, FD&C Red

No.3, Erythrosin B, Phloxine B, and Rose Bengal were not significantly different in their

lethal effect (p-value > 0.05). This finding is likely affected by the design of the study.

All compounds were tested at a level of 10 ppm, thus the concentrations were equal by

weight but not by molarity. By molar concentration, fluorescein was the highest (27 µM),

followed by Eosin Y (14 µM), Phloxine B (12 µM), FD&C Red No. 3 and Erythrosin B

(11 µM), and Rose Bengal (9.8 µM). Multiple studies have determined relative efficacy

of hydroxyxanthenes as photosensitizers against microorganisms. Fluorescein is consistently ineffective or the least effective hydroxyxanthene, followed with increasing

162

efficacy: Eosin, Erythrosin, and Rose Bengal (8, 11). Iwamoto et al (1989) reported 100

µM Rose Bengal and Erythrosin to be the most effective of the hydroxyxanthenes tested

at inactivating Saccharomyces cerevisiae followed by Phloxine and Eosin. Rose Bengal

and Erythrosin were also effective at a concentration of 10µM (9).

Wang et al (2006) found that the inherent toxicity of xanthene-derivatives was

dependent on parent molecule structure (fluorescein skeleton) and that halogen substitutions exhibited slight effects on activity against E. coli, S. aureus, and S. cerevisiae (23). Relative potencies of xanthene dyes with light exposure depend on the relative magnitudes of the phosphorescence quantum yields of particular compounds, which, in turn depend on the type and extent of halogenation (12). The presence of heavy atoms (such as bromine or iodine) may enhance the yields of intersystem crossing of these photosensitizers to create the reactive triplet state of the dyes, thereby relatively increasing photooxidative effects (23).

Inactivation of bacteria by photooxidation of Eosin, Erythrosin, Phloxine and

Rose Bengal may be due to the generation of singlet oxygen, superoxide anion, and/or radical production (16, 18). Due to the reducing environment of the cell, it is likely that inactivation of microorganisms via photooxidation occurs via a type I mechanism

(radicals). Hydroxyxanthenes are known to react with a variety of substrates potentially producing radicals with light exposure at low intensity, likely resulting in membrane

damage (15). Membranes are also primary targets for damage caused by UHP treatment.

Photooxidation and UHP likely lead to combined damage to the membrane which these

bacteria are incapable of recovering.

163

Of note, the combination treatment of Rose Bengal and UHP without light

exposure resulted in enhanced inactivation of L. monocytogenes compared to UHP alone.

Combination treatment of UHP and Phloxine B or Rose Bengal in the dark also resulted

in significantly enhanced inactivation of E. coli compared to UHP alone (p-value =

0.018). These results indicate a light-independent function of inactivation (not

photooxidation) by the combination treatment of certain halogenated xanthene-

derivatives in the dark. Further investigation of this mechanism is reported in Chapter 5.

Light-independent inactivation due to hydroxyxanthenes has not been previously reported

in the literature; however studies have reported some limited effects of growth inhibition

without light exposure at high concentration (17, 22, 23).

Photooxidation is responsible for the inactivation of Gram-positive bacteria by

halogenated hydroxyxanthenes. L. plantarum was significantly more sensitive to

photooxidation of FD&C Red No. 3 compared to L. monocytogenes (p-value <0.05).

Differences in sensitivity of these organisms may help to explain the cellular targets and/or resistance mechanisms of these treatments. Photooxidation also plays an important role in the inactivation of both Gram-positive and Gram-negative bacteria by

UHP-xanthene-derivative combination treatments. Further studies are needed to elucidate the contribution of type I and type II mechanisms of photooxidation relating to bacterial inactivation. However, a light-independent mechanism may play a role in the inactivation using this combination treatment. Further studies are needed to investigate the contribution of light-independent inactivation.

164

ACKNOWLEDGEMENTS

This research was supported by a grant from the Center for Advanced Processing and Packaging Studies (CAPPS).

REFERENCES

1. Begue W J, Bard R C, and Koehne G W. 1965. Microbial inhibition by erythrosin. Journal of Dental Research 45:1464-1467.

2. Bezman S A, Burtis P A, Izod T P J, and Thayer M A. 1978. Photodynamic inactivation of E. coli by rose bengal immobilized on polystyrene beads. Photochemistry and Photobiology 28:325-329.

3. Caldwell R C and Hunt D E. 1969. A comparison of the antimicrobial activity of disclosing agents. Journal of Dental Research 48:913-915.

4. Castano A P, Demidova T N, and Hamblin M R. 2004. Mechanisms in photodynamic therapy: part one - photosensitizers, photochemistry and cellular localization. Photodiagnosis and Photodynamic Therapy 1:279-293.

5. Dahl T A, Midden W R, and Neckers D C. 1988. Comparison of photodynamic action by rose bengal in Gram-positive and Gram-negative bacteria. Photochemistry and Photobiology 48:607-612.

6. Dahl T A, Valdes-Aguilera O M, Midden W R, and Neckers D C. 1989. Partition of rose bengal anion from aqueous medium into a lipophilic environment in the cell envelope of Salmonella typhimurium: implications for cell-type targeting in photodynamic therapy. Journal of Photochemistry and Photobiology: B 4:171-184.

7. Foote C S. 1991. Definition of type I and type II photosensitized oxidation. Photochemistry and Photobiology 54:659.

8. Harrison A P. 1967. Survival of bacteria: harmful effects of light, with some comparisons with other adverse physical agents. Annual Reviews in Microbiology 21:143-156.

9. Iwamoto Y, Tominaga C, and Yanagihara Y. 1989. Photodynamic activities of food additive dyes on the yeast Saccharomyces cerevisiae. Chemical Pharaceutical Bulletin 37:1632-1634.

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10. Jemli M, Alouini Z, Sabbahi S, and Gueddari M. 2002. Destruction of fecal bacteria in wastewater by three photosensitizers. Journal of Environmental Monitoring 4:511-516.

11. Karrer S, Szeimies R-M, Ernst S, Abels C, Baumler W, and Landthaler M. 1999. Photodynamic inactivation of staphylococci with 5-aminolaevulinic acid or photofrin. Lasers Medical Science 14:54-61.

12. Krasnoff S B, Faloon D, Williams J E, and Gibson D M. 1999. Toxicity of xanthene dyes to entomopathogenic fungi. Biocontrol Science and Technology 9:215-225.

13. Martin D F and Perez-Cruet M J. 1987. Preparation of sterile seawater through photodynamic action. Preliminary studies. Florida Scientist 50:168-176.

14. Martin J P Jr and Logsdon N. 1987. Oxygen radicals are generated by dye- mediated intracellular photooxidations: a role for superoxide in photodynamic effects. Archives of Biochemistry and Biophysics 256:39-49.

15. Martin J P Jr and Logsdon N. 1987. Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and lucifer yellow CH. Photochemistry and Photobiology 46:45-53.

16. Neckers D C and Valdes-Aguilera O M. 1993. Photochemistry of the xanthene dyes, p. 315-394. In Volman D, Hammond G S, and Neckers D C (eds.), Advances in Photochemistry. John Wiley & Sons, Inc.

17. Ottow J C G. 1972. Rose Bengal as a selective aid in the isolation of fungi and actinomycetes from natural sources. Mycologia 64:304-315.

18. Pan X, Ushio H, and Ohshima T. 2005. Effects of molecular configurations of food colorants on their efficacies as photosensitizers in lipid oxidation. Food Chemistry 92:37-44.

19. Rasooly R. 2005. Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria. FEMS Immunology and Medical Microbiology 45:239-244.

20. Rengifo-Herrera J A, Sanabria J, Machuca F, Dierfolf C F, Pulgarin C, and Orellana G. 2007. A comparison of solar photocatalytic inactivation of waterborne E. coli using Tris (2,2'-bipyridine)ruthenium(II), Rose Bengal and TiO2. Journal of Solar Energy Engineering 129:135-140.

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21. Schafer M, Schmitz C, Facius R, Horneck G, Milow B, Funken K-H, and Ortner J. 2000. Systematic study of parameters influencing the action of rose bengal with visible light on bacterial cells: Comparison between the biological effect and singlet-oxygen production. Photochemistry and Photobiology 71:514- 523.

22. T'ung T and Zia S H. 1937. Photodynamic action of various dyes on bacteria. Proceedings of the Society for Experimental Biology and Medicine 36:326-330.

23. Wang H, Lu L, Zhu S, Li Y, and Cai W. 2006. The phototoxicity of xanthene derivatives against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Current Microbiology 52:1-5.

24. Wood S, Metcalf D, Devine D, and Robinson C. 2006. Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms. Journal of Antimicrobial Chemotherapy 57:680-684.

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CHAPTER 5

INACTIVATION OF BACTERIA BY ULTRA-HIGH PRESSURE AND

XANTHENE-DERIVATIVES: A SECONDARY LIGHT-INDEPENDENT

MECHANISM

ABSTRACT

Numerous studies have identified xanthene-derivatives as photosensitizers and have attempted to identify optimal conditions for using this characteristic to inactivate various organisms. Inactivation of bacteria by xanthene-derivatives in combination with ultra-high pressure (UHP) is extremely effective at inactivating Gram-positive and Gram- negative bacteria with light exposure. While inactivation results were encouraging, photooxidation as a means of inactivation of bacteria in food products may lead to undesirable sensory characteristics of food products. Preliminary findings in an earlier study suggested that increases in UHP treatment may lead to enhanced inactivation without light exposure. The objective of this study was to identify treatment conditions that would maximize the inactivation of bacteria without light exposure therefore avoiding the production of off-flavors due to photooxidation. Escherichia coli and

Listeria monocytogenes were treated with various levels of UHP (400-600 MPa, 1 min),

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with and without FD&C Red No. 3 (10 ppm), with and without light exposure. Pressure

level had a significant effect on inactivation of both species with and without light and dye exposure. Results indicate a threshold pressure at which light-independent

inactivation occurs; this threshold pressure varies by species. L. monocytogenes appears

to have a threshold pressure for light-independent inactivation between 400 and 500 MPa,

whereas the threshold pressure for E. coli is between 300 and 400 MPa. Additional

xanthene-derivatives (Rose Bengal and Phloxine B) also displayed light-independent inactivation of L. monocytogenes and/or E. coli with UHP treatment of 500 MPa, 1 min.

Enhanced light-independent inactivation may occur with other xanthene-derivatives with

higher UHP treatments.

INTRODUCTION

Most studies on inactivation of microorganisms by photooxidation of

hydroxyxanthenes have found little to no antimicrobial activity of these compounds

without light exposure. Martin and Logsdon (1987) found hydroxyxanthenes Rose

Bengal and Erythrosin incapable of reducing oxygen levels without light exposure, thus

no photooxidation without the photons (7). Hydroxyxanthenes (Erythrosin B, Phloxine B

and Eosin B) had no effect on entomopathogenic fungi without light exposure (6).

Despite the lack of effect of hydroxyxanthenes in the dark in most studies, these

compounds have been used historically for bacterial inhibition in growth media. Rose

Bengal has been used for suppression of bacterial growth in agar media. This

suppression occurs under normal incubation conditions (dark) at concentrations of 350 mg/L. Suppression of Streptomyces spp. by Rose Bengal containing media was also

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reported at concentrations of 670 mg/L under normal incubation conditions (8). T’ung and Zia (1937) reported an inhibitory effect of Eosin (2%) in saline against Streptococcus hemolyticus and Neisserria intracellularis under dark conditions (9). Wang et al (2006) found that high concentrations of halogenated xanthenes caused growth inhibition of E. coli (500 µM), S. aureus (62.5-125 µM) and S. cerevisiae (125-250 µM) in an agar filter- disc diffusion assay (10). Adams et al (1982) found that chickens fed Erythrosin (0.5 g/kg) led to significant reduction in Salmonella (> 4 log cfu/g) in fecal material. Any reactions of Erythrosin with Salmonella cells occurred within the digestive tract and therefore under dark conditions. Erythrosin treatment had no effect on native cecal or fecal microbiota, with the exception of a reduction in the total aerobic count of the cecum

(1).

Previous hydroxyxanthene photooxidation studies in our laboratory have indicated a potential light-independent toxicity of these compounds when combined with

ultra-high pressure (UHP) treatment towards Listeria monocytogenes and Escherichia

coli (Chapter 4). The objective of this study was to confirm a light-independent effect of

this combination treatment on Gram-positive and Gram-negative bacteria. Other

xanthene-derivatives were used to determine if this effect is specific to Red No. 3 or

whether it occurs using other halogenated hydroxyxanthenes in combination with UHP.

MATERIALS AND METHODS

Bacterial strains.

Listeria monocytogenes (OSY-328 and ScottA), Lactobacillus plantarum

MDOS-32, and Escherichia coli O157:H7 EDL 933 were obtained from the culture

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collection of the Food Safety Laboratory at The Ohio State University (Columbus) and

tested in this study. Stock cultures were suspended in appropriate broth media,

containing 40% (vol/vol) glycerol, and stored at -80ºC. Immediately before experiments,

L. plantarum was transferred from the frozen stock-culture to MRS agar and incubated at

37°C for 48-72 hours. Similarly, L. monocytogenes and E. coli were transferred from

frozen stock-culture to tryptic soy agar (TSA) and incubated at 37ºC for 48 to 72 hours.

Three isolated colonies of each strain were transferred to the appropriate broth (MRS or

TSB) and incubated overnight at 37°C.

Xanthene-derivatives.

FD&C Red No. 3 (CI number 45430, Japan Food Red No. 3) was obtained from

Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Fluorescein (CI number 45350),

Erythrosin B (CI number 45430, Japan Food Red No. 3), Phloxine B (CI number 45410,

Japan Food Red No. 104), Eosin Y (CI number 45380), and Rose Bengal (CI number

45440, Japan Food Red No. 105) were purchased from Sigma-Aldrich (St. Louis, MO).

Stock solutions (100-1000 ppm) were prepared by dissolving the food colorant in sterile

distilled water. All solutions were used within 30 minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, WA) containing 1:1 (vol/vol) glycol/water

pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International Inc., Valley

Forge, Pa). The press consists of a jacketed vessel with end closures, has a 2-liter

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capacity, and is designed to operate at pressures up to 900 MPa. To reduce temperature

effect on ultra-high pressure inactivation, initial glycol:water processing fluid was

adjusted to achieve a holding temperature of 20-25ºC.

Bacteria treatment in buffer.

Overnight cultures were centrifuged at 10,000 rpm (9,000 x g) for 10 minutes

(International Equipment Co., IEC Centra MP4R) and suspended in sterile citrate- phosphate buffer (0.087 M phosphate, 0.007 M citrate, pH 7.0). Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into clear sterile polyethylene bags (Fisher Scientific Co., Pittsburgh, PA) or black sterile polyethylene bags (Nasco Whirl-Pak, Fort Atkinson, WI) containing 0.1 ml of colorant stock solution to achieve the desired final concentration of these additives, and the bags were heat-sealed. Sample bags were then placed into a larger polyethylene bag and sealed using a vacuum sealer (Vacmaster, Kansas City, MO). Samples were then placed on ice for approximately 30 minutes prior to high pressure treatment. Pressure treatments ranged from 400 to 600 MPa with holding times of one minute. After pressurization, bags were immediately placed on ice until plated. Control bags were held on ice throughout the duration of the experiment. Bags were opened aseptically and the contents were serially diluted in 0.1% peptone water and plated on the appropriate agar medium. L. plantarum strains were plated on MRS agar and incubated at 37°C for 48 hours prior to enumeration. L. monocytogenes and E. coli were plated on TSA and incubated at 37ºC for 48 hours. Experiments were performed in triplicate, unless otherwise indicated.

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Light exposure of samples.

Light exposure consisted of samples in clear polyethylene bags being spread onto

laboratory bench under ambient lighting conditions for 15 minutes after pressure

treatment, depending on experiment. Incident luminescence 485 lux as measured using a

light meter (Sekonic Flashmeter L-358, Elmsford, NY). Following controlled light

exposure, samples were protected from light by wrapping the samples in aluminum foil

until plating.

Statistical analysis.

Data were analyzed by analysis of variance (ANOVA) by using the General

Linear Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the

mean log reductions of treatments were made using the Tukey’s Studentized Range test.

RESULTS

Pressure dose determines efficacy of UHP-Red No. 3 combination with and without

light exposure.

Samples of L. monocytogenes OSY-328 and E. coli EDL 933 were treated with

and without Red No. 3 (10 ppm), with and without light (15 minutes after pressure

treatment) and various levels of ultra-high pressure (0, 400, 500, and 600 MPa, 1 minute).

Results for L. monocytogenes and E. coli are shown in Figures 5.1 and 5.2, respectively.

L. monocytogenes OSY-328 was weakly inactivated (0.3 log) by 10 ppm colorant and 15 minutes of light exposure with no pressure treatment. With increasing pressure treatments, there was an increase in the inactivation of L. monocytogenes with 0 ppm colorant, with and without light exposure. Inactivation by pressure alone increased from

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9.00 0 minutes light, 0 ppm dye 15 minutes light, 0 ppm dye 8.00 0 minutes light, 10 ppm dye 15 minutes light, 10 ppm dye a,b a 7.00 a a 6.00 a a,b 5.00 b 4.00

3.00 Log reduction (cfu/ml)

b 2.00 b 1.00 b a b a,b b b b 0.00 No UHP 400 MPa, 1 min 500 MPa, 1 min 600 MPa, 1 min Pressure treatment

Figure 5.1. Effect of pressure level on inactivation of Listeria monocytogenes OSY-

328 by FD&C Red No. 3 treatment with and without light exposure. FD&C Red No.

3 concentrations were 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for 15 minutes after pressure treatment. Dark samples were protected from light by being stored in black polyethylene bags. Error bars indicate standard error, n = 3. Different letter designations indicate significant differences in treatments for each pressure condition (p-value <0.05).

174

9.00 0 minutes light, 0 ppm dye 15 minutes light, 0 ppm dye 8.00 0 minutes light, 10 ppm dye a a 15 minutes light, 10 ppm dye 7.00

6.00

a b 5.00 b

b 4.00 c 3.00 Log Reduction (cfu/ml) c c 2.00 c c c 1.00

0.00 No UHP 400 MPa, 1 min 500 MPa, 1 min 600 MPa, 1 min Pressure treatment

Figure 5.2. Effect of pressure level on inactivation of Escherichia coli EDL 933 by

FD&C Red No. 3 treatment with and without light exposure. FD&C Red No. 3 concentrations were 10 ppm. Light exposure was achieved by placing clear polyethylene bag using ambient lighting conditions for 15 minutes after pressure treatment. Dark samples were protected from light by being stored in black polyethylene bags. Error bars indicate standard error, n ≥ 2. Different letter designations indicate significant differences in treatments for each pressure condition (p-value <0.05).

175

0.03 log at 400 MPa to 4.6 log at 600 MPa. With pressure treatments of 400 MPa, significant enhancement in inactivation of L. monocytogenes by combination treatment was only with light exposure, an increase from 0.03 log inactivation of pressure alone to

4.9 log inactivation with pressure, dye and light. With pressure treatments of 500 MPa, significant enhancement of inactivation with Red No. 3 occurred both with and without light exposure resulting in log reductions of 6.6 and 5.9, respectively (p-values <0.001).

Treatment of samples at 600 MPa resulted in an increase in inactivation with combination treatment with and without light; however these differences were only significant when comparing 0 ppm colorant and 10 ppm colorant with light exposure.

The same experiment was performed using E. coli as the target organism. As expected, the colorant was ineffective as an antimicrobial compound towards E. coli without pressure treatment regardless of light exposure. As pressure levels increased, inactivation of E. coli by pressure alone increased from 0.7 log at 400 MPa to 2.6 log at

600 MPa. Enhanced inactivation with dye present was significant at all pressure levels

(p-values < 0.05), regardless of light exposure, however there were also significant differences between dye-dark combinations and dye-light combinations with the light treatment being the most effective (p-values < 0.05).

Light-independent inactivation of bacteria by UHP-xanthene-derivatives combinations.

Figure 5.3 displays the inactivation of L. monocytogenes OSY-328 and E. coli

EDL 933 when treated with xanthene-derivatives and UHP without light exposure. At the concentration used in this study (10 ppm), the hydroxyxanthenes had no impact on

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survival of either organism without UHP treatment (data not shown). Inactivation by

UHP alone results in 0.5 log reduction and 1.1 log reduction for L. monocytogenes and E.

coli, respectively. For experiments with L. monocytogenes, Rose Bengal was the only

hydroxyxanthene to cause a significant increase in inactivation (4.5 log reduction) when

combined with UHP (p-value = 0.018). Previous experiments with Red No. 3 had shown

a light-independent effect when combined with UHP under similar treatment conditions

(See Figure 1). Differences in these results suggest that 500 MPa is close to the threshold

level for the light-independent effect of this combination treatment. It is likely that

increasing the pressure level would identify more of the colorants as effective in

combination with UHP without light exposure. Significant increases in inactivation of E.

coli were found for Red No. 3 (2.3 log reduction), Phloxine B (2.9 log reduction), and

Rose Bengal (3.1 log reduction) (p-values < 0.05).

DISCUSSION

The differences in impact of light exposure on inactivation by combination Red

No. 3 and UHP indicate the workings of two mechanisms of inactivation: light-dependent

and light-independent. The impact of the light-independent mechanism towards

inactivation of L. monocytogenes varies depending on level of pressure treatment with a

potential threshold pressure for light-independent activity. Pressure treatments of 400

MPa or below are significantly more effective than pressure alone if they are exposed to

light; inactivation due to a light-dependent mechanism only. Pressure treatments at 500

MPa, or above, displayed enhanced inactivation with colorant, both with and without

light exposure, thus indicating an impact of both light-dependent and light-independent

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9.00

8.00

7.00

6.00 *

5.00

4.00 * * 3.00 * Log Reduction(cfu/ml) 2.00

1.00

0.00 None None Eosin Eosin Red 3 Red 3 Red Phloxine Phloxine Erythrosin Erythrosin Fluorescein Fluorescein Rose Bengal Rose Bengal Rose

L. monocytogenes E. coli

Figure 5.3. Inactivation of Listeria monocytogenes OSY-328 and Escherichia coli

EDL 933 by combination treatment of xanthene-derivatives and ultra-high pressure without light exposure. Xanthene-derivatives concentrations were 10 ppm. Samples were protected from light by being stored in black polyethylene bags. Ultra-high pressure treatment was 500 MPa for 1 minute. Error bars indicate standard error, n = 3.

* indicates significant differences between combination dye plus UHP treatment and

UHP treatment without xanthene-derivative for a given organism (p-value <0.05).

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mechanisms. The impact of the light-independent mechanism on inactivation of E. coli

Red No. 3 and UHP combination treatment was apparent at all pressures tested (400

MPa-600 MPa). There may also be a threshold pressure for this mechanism to occur with

E. coli, likely between 300 and 400 MPa based on comparison with data from earlier

studies (See Chapter 3).

There are few previous reports of a light-independent effect of xanthene-

derivatives towards bacteria. Consistent with the findings from this study, fluorescein

has not been reported to be inhibitory to bacteria with or without light exposure even with concentrations up to 500 µM (~186 ppm). Halogenation of xanthene-derivatives increases the efficacy of these compounds towards microorganisms with light exposure

(10). Likewise, this report suggests that the same trend is true for inactivation of bacteria by UHP and hydroxyxanthene combination treatment without light exposure. Wang et al

(2006) found Rose Bengal to be more effective at inhibiting growth of S. aureus and S. cerevisiae growth with dark incubation than the less halogenated Eosin. Erythrosin was also more inhibitory than Eosin towards S. aureus (10). It is important to note that concentrations of dye used for growth inhibition by Wang et al (2006) were substantially higher than those presented in this study, indicating the significance of the dark inactivation with UHP combination treatment. A previous study by Cruz et al (1984) found that localization of Rose Bengal within Trypanosoma cruzi cells was independent of light exposure (4). Due to the effect of these compounds in combination with UHP, localization of the dye for both light and dark inactivation is believed to be essential for inactivation.

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This study identifies light-dependent and light-independent mechanisms of

inactivation of bacteria by combination treatments of xanthene-derivatives and UHP.

Light-independent inactivation was UHP dose-dependent. Previous studies, relating to

toxicity of xanthene-derivatives as pesticides, have identified light-independent effects, especially with Rose Bengal treatment, however mechanisms have not been identified (2,

3, 5).

ACKNOWLEDGEMENTS

This research was supported by a grant from the Center for Advanced Processing and Packaging Studies (CAPPS).

REFERENCES

1. Adams R F, Jones R L, and Conway P L. 1982. Reduction of Salmonella typhimurium in laboratory-inoculated chickens by the use of erythrosine. British Poultry Science 23:461-468.

2. Ballard J B, Vance A D, and Gold R E. 1988. Light-dependent and independent responses of populations of German and brownbanded cockroaches (Orthoptera: Blattellidae) to two photodynamic dyes. Journal of Economic Entomology 81:1641-1644.

3. Broome J R, Callaham M F, Lewis L A, Ladner C M, and Heitz J R. 1974. The effects of rose bengal on the imported fire ant, Solenopsis richteri (forel). Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 51:117-120.

4. Cruz F S, Lopes L A V, de Souza W, Moreno S N J, Mason R P, and Docampo R. 1984. The photodynamic action of rose bengal on Trypanosoma cruzi. Acta Tropica 41:99-108.

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5. Fondren J E Jr. and Heitz J R. 1978. Xanthene dye induced toxicity in the adult face fly, Musca autumnalis. Environmental Entomology 7:843-846.

6. Krasnoff S B, Faloon D, Williams J E, and Gibson D M. 1999. Toxicity of xanthene dyes to entomopathogenic fungi. Biocontrol Science and Technology 9:215-225.

7. Martin J P Jr and Logsdon N. 1987. Oxygen radicals are generated by dye- mediated intracellular photooxidations: a role for superoxide in photodynamic effects. Archives of Biochemistry and Biophysics 256:39-49.

8. Ottow J C G. 1972. Rose Bengal as a selective aid in the isolation of fungi and actinomycetes from natural sources. Mycologia 64:304-315.

9. T'ung T and Zia S H. 1937. Photodynamic action of various dyes on bacteria. Proceedings of the Society for Experimental Biology and Medicine 36:326-330.

10. Wang H, Lu L, Zhu S, Li Y, and Cai W. 2006. The phototoxicity of xanthene derivatives against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Current Microbiology 52:1-5.

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CHAPTER 6

EFFICACY OF FD&C RED NO. 3 AND ULTRA-HIGH PRESSURE

COMBINATION TREATMENT AGAINST FOODBORNE PATHOGENS IN

FOOD SYSTEMS

ABSTRACT

Combination treatments of FD&C Red No. 3 and ultra-high pressure (UHP) are effective at inactivating a variety of important processing-resistant strains of spoilage and pathogenic bacteria. Often, new combination treatments are effective against target organisms in simple buffer systems but ineffective against these organisms in complex systems including food. The objective of this study was to determine the impact of pH and two food systems on the inactivation of Listeria monocytogenes and Escherichia coli using Red No. 3 and UHP. Escherichia coli subjected to this combination in citrate- phosphate buffer systems ranging from 7.0 to 3.0. Combination treatment was effective at all pH levels tested, with pH 3.0 being the most effective treatment. Both L. monocytogenes and E. coli were inoculated into sterile carrot juice and canned turkey meat product to determine effect of food composition on inactivation by combination treatment. Red No. 3 concentrations of 10, 50, and 100 ppm in combination with UHP

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(500 MPa, 1 min) resulted in synergistic inactivation of L. monocytogenes with and without light exposure. Treatments of 100 ppm Red No. 3 in combination with UHP treatment resulted in synergistic inactivation of E. coli. UHP treatment was completely ineffective at reducing L. monocytogenes in turkey meat product; likewise combination treatment did not result in significant inactivation. UHP treatment reduced E. coli in turkey meat product by 3.9 log; however combination treatment did not result in a significant increase in inactivation, regardless of Red No. 3 concentration or light exposure. Food components have a significant impact on the efficacy of chemical and physical treatments with potential application in the food industry. Further studies are needed to identify specific components that protect bacteria from inactivation by both

UHP and Red No. 3 treatments.

INTRODUCTION

Efficacy of various food processing technologies, including thermal, microwave, pulsed-electric field, and ultrasonic treatments, against microorganisms is affected by suspension medium and food components (10, 12, 31, 32). Ultra-high pressure (UHP) treatment is an effective non-thermal treatment proven to inactivate various microorganisms in buffer systems and food systems. UHP treatment results in a temporary shift in pH, thought to approximately 0.3 pH units per 100 MPa in water, depending on complexity and buffering capacity of the system (18). Thus, the pH of the suspending medium has a significant impact on the efficacy of UHP treatment, with increasing inactivation with decreasing pH (11, 14, 20, 34). The chemical nature of the specific acids (weak vs. strong acids) present in the suspension medium may also affect

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UHP efficacy. Weak acids (phosphoric, malic, citric) have been found to produce greater inactivation of Lactobacillus spp. treated with UHP compared to strong acids

(hydrochloric, sulfuric) (21).

Inactivation of microorganisms treated with UHP in buffer systems compared to food systems is often poorly correlated. Media rich in ions, proteins, lipids and carbohydrates are significantly more protective than buffer systems (8, 15). Food systems may protect bacterial cells from inactivation by UHP by reducing the osmotic pressure across the cytoplasmic membrane, thus enhancing membrane integrity (26).

Chen and Hoover (2003) compared the inactivation of Yersinia enterocolitica by UHP in phosphate buffer and whole milk. Inactivation was significantly compromised when cells were suspended in whole milk due to the potential protection of the cells by fat and proteins (3). Solomon and Hoover (2004) assessed the ability of UHP to inactivate

Campylobacter jejuni in whole, skim, and soy milk and chicken puree. C. jejuni strains were more resistant to UHP treatment in food products than compared to buffer systems or spent broth (26). Food systems, however, are not always more protective. Styles et al

(1991) compared the inactivation of Listeria monocytogenes in buffer and milk and the inactivation of Vibrio parahaemolyticus in buffer and clam juice. Milk protected L. monocytogenes from inactivation by UHP, whereas inactivation of V. parahaemolyticus was enhanced in clam juice (27).

Likewise, efficacy of hydroxyxanthenes, including Red No. 3, to inactivate bacteria alone or in combination with UHP may be compromised by food components.

Very few studies have investigated the potential for xanthene-derivatives to serve as

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antimicrobial agents in food systems. Ray (2000) tested the ability of Phloxine B to inhibit growth of Listeria monocytogenes in cooked shrimp and hot dogs. Phloxine B had little to no effect on L. monocytogenes growth on these food products (19).

The objective of this study was to validate previous findings on synergy between

UHP and Red No. 3 on lethality of bacteria in food systems. Food systems are likely to cause a negative impact on the efficacy of this combination treatment and must be investigated for potential application of this treatment in the food industry. UHP-Red No.

3 combinations were tested in carrot juice and turkey meat product that were contaminated with selected pathogenic bacteria.

MATERIALS AND METHODS

Bacterial strains.

Listeria monocytogenes OSY-328 and Escherichia coli O157:H7 EDL 933 were obtained from the culture collection of the Food Safety Laboratory at The Ohio State

University (Columbus) and tested in this study. Stock cultures were suspended in appropriate broth media, containing 40% (vol/vol) glycerol, and stored at -80ºC.

Immediately before experiments, L. monocytogenes and E. coli were transferred from frozen stock-culture to Trypticase Soy Agar (TSA) and incubated at 37ºC for 48 to 72 hours. Three isolated colonies of each strain were transferred to Trypticase Soy Broth

(TSB) and incubated overnight at 37°C.

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FD&C Red No. 3 preparation.

FD&C Red No. 3 (CI number 45430, Japan Food Red No. 3) was obtained from

Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Stock solutions were prepared by

dissolving the food colorant in sterile distilled water. All solutions were used within 30

minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol)

glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

2-liter capacity, and is designed to operate at pressures up to 900 MPa. To reduce temperature effect on ultra-high pressure inactivation, initial glycol:water processing fluid was 5-10ºC to achieve a holding temperature of 20-25ºC.

Bacteria treatment in buffer.

Overnight cultures were centrifuged at 10,000 rpm (12,400 x g) for 10 minutes

(Sorvall RC-5B Refrigerated Superspeed Centrifuge, equipped with SM-24 rotor) and suspended in sterile citrate-phosphate buffer. Buffers varied in molar concentrations depending on desired pH, as shown in Table 6.1.

Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell

suspensions were transferred into sterile polyethylene bags (Fisher Scientific Co.,

Pittsburgh, Pa.) containing 0.1 ml of food colorant stock solution to achieve the desired

final concentration of these additives, and the bags were heat-sealed. Sample bags were

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pH M Na2HPO4 M citric acid 3.0 0.020 0.040 4.0 0.039 0.031 5.0 0.051 0.024 6.0 0.064 0.018 7.0 0.087 0.007

Table 6.1. Final molar concentration of citrate-phosphate buffer components at

various pH levels.

placed on the bench top for 30 minutes of ambient light exposure. Following light

exposure, sample bags were then placed into a larger polyethylene bag and sealed using a vacuum sealer (Vacmaster, Kansas City, Mo.). Samples were then placed on ice for

approximately 30 minutes prior to high pressure treatment (400 MPa, 3minutes, 25ºC

±3ºC). After pressurization, bags were immediately placed on ice. Sample bags were

removed from ice and placed on the bench top for an additional light exposure of 15

minutes. Incident luminescence 485 lux as measured using a light meter (Sekonic

Flashmeter L-358, Elmsford, New York). Following the second light exposure, samples

were protected from light until plated. Control bags were held on ice throughout the

duration of the experiment. Bags were opened aseptically and the contents were serially

diluted in 0.1% peptone water and plated on TSA and incubated at 37ºC for 48 hours.

Experiments were performed in triplicate, unless otherwise indicated.

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Bacterial treatment in carrot juice.

Overnight cultures were centrifuged at 10,000 rpm for 10 minutes (International

Equipment Company IEC Centra MP4R) and suspended in commercially sterile carrot

juice (Hollywood 100% pure carrot juice, The Hain Celestial Group, Inc., Melville, NY).

Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell

suspensions were transferred into clear sterile polyethylene bags (Fisher Scientific Co.,

Pittsburgh, Pa.) or black sterile polyethylene bags (Nasco Whirl-Pak, Fort Atkinson, Wi)

containing 0.1 ml of food colorant stock solution to achieve the desired final

concentration of these additives, and the bags were heat-sealed. Sample bags were then

placed into a larger polyethylene bag and sealed using a vacuum sealer (Vacmaster,

Kansas City, Mo.). Samples were then placed on ice for approximately 30 minutes prior

to high pressure treatment (500 MPa, 1 minute, 25ºC ±3ºC). After pressurization, bags

were immediately placed on ice. Bags were removed from ice and placed on the bench

top for light exposure of 30 minutes. Incident luminescence 485 lux as measured using a

light meter (Sekonic Flashmeter L-358, Elmsford, New York). Following controlled

light exposure, samples were protected from light until plated. Control bags were held on

ice throughout the duration of the experiment. Bags were opened aseptically and the

contents were serially diluted in 0.1% peptone water and plated on TSA and incubated at

37ºC for 48 hours. Experiments were performed in triplicate.

Bacterial treatment in turkey product.

Overnight cultures were centrifuged at 10,000 rpm for 10 minutes (International

Equipment Company IEC Centra MP4R) and suspended in citrate-phosphate buffer (pH

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7.0). Cell suspensions contained approximately 109 CFU/ml. Aliquots (1.0 ml) of cell

suspensions were transferred into clear sterile polyethylene bags (Fisher Scientific Co.,

Pittsburgh, Pa.) or black sterile polyethylene bags (Nasco Whirl-Pak, Fort Atkinson, Wi)

containing 10 g of turkey product (SPAM oven roasted turkey, Hormel Foods

Corporation, Austin, Wi), that had been homogenized in a food processor with FD&C

Red No. 3 dissolved in sterile distilled water to achieve the desired final concentration.

Bags were heat-sealed and samples were massaged by hand to incorporate inoculum

evenly into turkey. Sample bags were then placed into a larger polyethylene bag and

sealed using a vacuum sealer (Vacmaster, Kansas City, Mo.). Samples were then placed

on ice for approximately 30 minutes prior to high pressure treatment (500 MPa, 1 minute,

25ºC ±3ºC). After pressurization, bags were immediately placed on ice. Bags were

removed from ice and placed on the bench top for light exposure of 30 minutes. Incident

luminescence 485 lux as measured using a light meter (Sekonic Flashmeter L-358,

Elmsford, New York). Following controlled light exposure, samples were protected from

light until plated. Control bags were held on ice throughout the duration of the

experiment. Bags were opened aseptically and the contents were serially diluted in 0.1%

peptone water and plated on TSA and incubated at 37ºC for 48 hours. Experiments were

performed in triplicate.

Statistical analysis.

Data were analyzed by analysis of variance (ANOVA) by using the General

Linear Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the

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mean log reductions of treatments were made using the Tukey’s Studentized Range test.

Synergy was defined as a significant difference between (log reduction dye + log reduction UHP) and (log reduction dye+UHP combination).

RESULTS

Minimal impact of pH on efficacy of UHP-Red No. 3 combination treatments.

Inactivation due to UHP-Red No. 3 combinations in different pH buffers is shown in Figure 6.1. Suspension into various pH buffers did not result in significant inactivation of E. coli cells (p-value >0.05). E. coli is known to be unaffected by dye treatment alone at pH 7.0; there was no significant inactivation with dye treatment in solutions with pH greater than or equal to 4.0 (p-value >0.05). However, significant inactivation of E. coli by dye treatment alone was observed at pH 3.0 (2.7 log reduction) (p-value = 0.005).

UHP-Red No. 3 combination efficacy in carrot juice.

Efficacy of Red No. 3 at various concentrations and with and without light exposure against L. monocytogenes is shown in Figure 6.2. Colorant concentration in samples kept in the dark had no effect on the inoculated population. With a light exposure of 30 minutes, inactivation was observed in a dose-dependent manner. Average log reduction values for L. monocytogenes treated with 10, 50 and 100 ppm with light exposure were 0.1, 0.3, and 1.4, respectively. Inactivation of L. monocytogenes by dye alone was lower than expected when compared to citrate-phosphate buffer studies (see chapter 2); components of carrot juice may protect the cells from photooxidative effects.

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9.00 pH a a,b dye a,b 8.00 a,b UHP UHP + dye 7.00

a,b 6.00 b

5.00

4.00 c 3.00 Log Reduction (cfu/ml) c,d 2.00 c,d,e c,d,e c,d,e c,d,e 1.00 d,e d,e d,e d,e e d,e 0.00 76543 pH

Figure 6.1. Inactivation of Escherichia coli EDL 933 by combination pH, FD&C

Red No.3 (10 ppm) and ultra-high pressure treatment. Pressure treatments were 400

MPa with a holding time of three minutes. Light exposure was 30 minutes prior and 15 minutes after pressure treatment. Temperature during holding time was between 20 and

25ºC. Error bars indicate standard error, n = 2. Different letter designations indicate significant differences between all treatments shown (p-value <0.05).

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9 0 ppm <0.0001 8 10 ppm 50 ppm <0.0001 <0.0001 7 100 ppm <0.0001 <0.0001 6 0.0011 5

4

3 Log Reduction (cfu/ml)

2

1

0 0 minutes light, No 30 minutes light, No 0 minutes light, UHP 30 minutes light, UHP UHP UHP Light and UHP treatments

Figure 6.2. Inactivation of Listeria monocytogenes OSY-328 in carrot juice by combination FD&C Red No. 3, light exposure, and ultra-high pressure treatment.

Pressure treatments were 500 MPa with a holding time of 3 minutes. Holding temperature was 23ºC ± 2ºC. Light exposure was 30 minutes after pressure treatment.

Error bars indicate standard error, n = 3. Numbers above treatments indicate synergistic activity of combination treatment and p-value determined for the comparison.

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Pressure treated samples inoculated with L. monocytogenes both with and without light exposure showed a colorant dose-response. Synergistic activity of the colorant and pressure combination was apparent for all doses both with and without light exposure.

Efficacy of Red No. 3 at various concentrations with and without light exposure against E. coli in carrot juice is shown in Figure 6.3. As expected, E. coli was resistant to all colorant concentrations in the absence of pressure treatment. In combination with

UHP, inactivation of E. coli followed a colorant dose-dependent relationship regardless of light exposure. Synergy of inactivation by combination treatment was statistically significant with the highest concentration of colorant (100 ppm) both with and without light exposure, inactivating 6.2 and 4.8 log, respectively (p-values <0.001).

UHP-Red No. 3 combination treatment ineffective in turkey meat product.

Efficacy of UHP-Red No. 3 at various concentrations and with and without light exposure against E. coli EDL 933 in turkey meat product is shown in Figure 6.4. Again,

E. coli was resistant to Red No. 3 treatment without pressure treatment. UHP of 500

MPa for 1 minute produced significant inactivation of E. coli in the product. Presence of

Red No. 3 had no significant effect on E. coli inactivation, regardless of colorant concentration or light exposure.

Identical studies were completed with L. monocytogenes OSY-328. L. monocytogenes was completely resistant to all conditions in the study (data not shown).

L. monocytogenes OSY-328 in turkey meat product was resistant (<0.5 log inactivation) to FD&C Red No. 3, light and/or ultra-high pressure treatment. The L. monocytogenes strain used in this study (OSY-328) was isolated from a meat product and may be

193

9.00 0 ppm 8.00 10 ppm 50 ppm <0.0001 7.00 100 ppm

6.00 <0.0001 5.00

4.00

3.00 Log Reduction (cfu/ml)

2.00

1.00

0.00 0 minutes light, No 30 minutes light, No 0 minutes light, UHP 30 minutes light, UHP UHP UHP Light and UHP treatments

Figure 6.3. Inactivation of Escherichia coli EDL 933 in carrot juice by combination

FD&C Red No. 3, light exposure, and ultra-high pressure treatment. Pressure treatments were 500 MPa with a holding time of 3 minutes. Holding temperature was

23ºC ± 2ºC. Light exposure was 30 minutes after pressure treatment. Error bars indicate standard error, n = 3. Numbers above treatments indicate synergistic activity of combination treatment and p-value determined for the comparison.

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8.00 0 ppm 10 ppm 7.00 50 ppm 100 pm 6.00 * 5.00

4.00

3.00 Log Reduction (cfu/ml) 2.00

1.00

0.00 0 minutes light, No 30 minutes light, No 0 minutes light, UHP 30 minutes light, UHP UHP UHP Light and UHP treatments

Figure 6.4. Inactivation of Escherichia coli EDL 933 in turkey product by combination FD&C Red No. 3, light exposure, and ultra-high pressure treatment.

Pressure treatments were 500 MPa with a holding time of 3 minutes. Holding temperature was 23ºC ± 2ºC. Light exposure was 30 minutes after pressure treatment.

Error bars indicate standard error, n = 3. Different letter designations indicate significant differences between all treatments.

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specifically adapted for this food environment. Analysis of additional L. monocytogenes strains may provide further insight into the resistance of L. monocytogenes strains to UHP treatment in meat products.

DISCUSSION

Suspension of cells in acidic solutions is known to enhance inactivation by UHP; however a significant enhancement of inactivation of E. coli occurred only at pH 3.0.

Alpas et al (2000) found additional inactivation of E. coli 933 of 1.96 log in 100 mM

citric acid when pH was decreased from 6.5 to 4.5 with UHP treatment of 345 MPa for 5

minutes (holding temperature 35ºC) (1). Likewise, Sarangapani and Shellhammer (2007)

have reported that UHP is more effective against Lactobacillus spp. when suspension medium is composed of weak acids, including citrate, versus strong acids (21). Efficacy of these weak acid systems may be the result of antimicrobial activity associated with the undissociated form of these weak acids that may be favored due to the decrease in pH under pressure (9). Differences in pH effect may be attributable to buffering capacity of suspension medium; a citrate-phosphate buffer system is assumed to have more buffering capacity than citric acid solution. Previous experiments have shown Lactobacillus spp. to

be slightly more resistant to UHP treatment in citrate-phosphate buffer, pH 7.0 when

compared to phosphate-buffered saline and significantly more resistant when compared

to peptone water (34). Combination treatments of pH, colorant and pressure were

significantly effective compared to UHP alone. Combination treatments at pH 5 were

significantly more effective than at pH 6, however there was no obvious trend in efficacy

due to pH. Previous studies have indicated an enhancement of antimicrobial activities of

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xanthene-derivatives with lower pH. Ray (2000) found that inhibition of L.

monocytogenes growth by Phloxine B could be enhanced by reducing the pH of the growth medium (19). Red No. 3 has substantially decreased solubility in acidic solutions,

below pH 5.0; however there was no observable change in solubility with the concentration tested (10 ppm) (7, 33). Schafer et al (2000) directly measured the production of singlet oxygen by photooxidation of 2 ppm Rose Bengal for different pH

indices (pH 4.5, 7.0, and 9.6). The curves of singlet oxygen production were identical

regardless of pH, indicating no effect on singlet oxygen production due to pH (22).

Food systems are known to modulate the efficacy of food processing treatments,

including UHP. Therefore, two food systems, carrot juice and turkey product, were

chosen to determine the efficacy of combination treatments of Red No. 3 and UHP on

inactivation of L. monocytogenes and E. coli O157:H7. Studies have determined the

efficacy of UHP treatment on the inactivation of important foodborne microorganisms in

juice products. Carrot juice is often studied due to its relatively high pH (6.0) compared

to the other juice products. Van Opstal et al (2005) created extensive models to compare

inactivation of E. coli in buffer and carrot juice. Cells were consistently more resistant to

UHP treatment in carrot juice than buffer when treated in an identical manner (30). Teo

et al (2001) determined inactivation of E. coli O157:H7 strains and Salmonella serovars

in carrot juice using UHP treatment. A cocktail of E. coli O157:H7 strains was

inactivated 4.51 log cfu/ml and 6.40 log cfu/ml with pressure treatment of 615 MPa for 1

minute and 2 minutes, respectively (holding temperature 15ºC) (29). Dede et al (2007)

investigated the effect of UHP treatment on total aerobic count, antioxidant activity, and

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color of carrot juice (5). Initial microbial loads of carrot juice were 5.5 log cfu/ml and complete inactivation was achieved with UHP treatments of 250 MPa for 15 minutes

(holding temperature = 35ºC) with limited and acceptable changes in product quality (5).

The aforementioned studies (5, 29, 30) used freshly extracted carrot juice for all

experiments which may contain antimicrobial compounds. Beuchat and Brackett (1990)

identified an anti-Listeria effect in raw carrots and fresh carrot juice (2). The

antimicrobial effect was inactivated by cooking and is therefore presumed to be inactive

in the retorted juice used in this study. Carotenoids, naturally present in carrot juice, are

believed to be stable to UHP treatment and may protect cells from inactivation by

xanthene-derivatives (5). Carotenoids have been shown to protect cells and other

molecules from photooxidation, specifically protecting from damage induced by singlet

oxygen (6, 13, 17, 23). Several studies have investigated the protective affect of

carotenoids against lipid peroxidation of oil systems (6, 35). Yang et al (2002)

investigated the potential of β-carotene to quench singlet oxygen production by FD&C

Red No. 3 in soybean oil/acetone. β-carotene quenched both the singlet oxygen and the

excited triplet Red No. 3 (20 ppm) at or over 3.72 x 10-6 M (17, 35). Additional studies

have focused on the protective effect of carotenoid molecules as part of the membrane of

bacteria and yeast (23, 28).

Previous studies have found a protective effect of meat on the inactivation of

bacteria by UHP treatment, compared to inactivation in buffer systems. Multiple species have been tested for inactivation in phosphate buffer and model meat system when

treated at 500 MPa for 10 minutes (holding temperature 40ºC) (11). Some species and

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strains were more resistant to UHP treatment in the meat system, especially Lactobacillus sakei strains. However, E. coli CTC1018 was equally resistant to UHP treatment regardless of matrix tested (11). Solomon and Hoover (2004) found chicken puree to be significantly more protective to C. jejuni cells than buffer with 10 minute treatments at various pressure levels, however complete inactivation was achieved with 400 MPa treatments (26). Likewise, Shigehisa et al (1991) found that pork slurries offered protection to various bacteria treated with UHP at levels at or below 3000 atm, however higher pressure treatments were effective against tested organisms (24). Metrick et al

(1989) found Salmonella strains to be significantly more resistant to UHP treatment in chicken puree than phosphate-buffered saline (16). There is conflicting information in the literature as to the impact of specific food components on inactivation of bacteria by

UHP. Carbohydrates and proteins are thought to protect microorganisms from inactivation by UHP (11). Some authors have found fat to protect cells from UHP treatment, while others have found no effect (30).

Simpson and Gillmore (1997) investigated the efficacy of UHP treatment on the inactivation of two strains of L. monocytogenes: Lm1 and Lm2, in raw and cooked minced meats. Similar to OSY-328, strain Lm1 was resistant to UHP treatments (<0.5 log inactivation, 375 MPa, 5 minutes) in raw and cooked minced chicken. Strain Lm2 was significantly more sensitive to UHP treatment in minced chicken products (~3.5 log inactivation at 375 MPa, 5 minutes). Increases in holding times led to increased inactivation of Lm1 and Lm2 in minced chicken products (25), indicating that the resistance of OSY-328 in turkey product may be further characterized by increases in

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holding time. Chung et al (2005) reported efficacy of UHP and tert-butyl hydroquinone

(TBHQ) combination treatment on inactivation of surface-associated L. monocytogenes

ScottA, OSY-8578, and OSY-328 in Vienna sausages. Surface-associated bacteria and

surface treatment with TBHQ would likely lead to enhanced contact between bacterial

cells and TBHQ compared to the homogenous mixture reported in this study. Studies in

sausage were also treated with a higher dose of UHP (600 MPa, 5 minutes), indicating

that potentially elevating UHP treatment may lead to substantial inactivation (4).

Xanthene-derivatives may bind or interact preferentially with specific food

components and are thus unable to interact with microorganisms, especially in complex food systems similar to turkey meat product. Similarly, Ray (2000) found Phloxine B to be ineffective at inhibiting the growth of L. monocytogenes in cooked shrimp and hot dogs at concentrations up to 500 ppm (19). Proteins may bind with the negatively

charged groups of the xanthene-derivative molecule and thus be ineffective at targeting

bacterial cells in the food systems (19).

Food products impact the efficacy of Red No. 3 and UHP combination treatments.

Inactivation of L. monocytogenes and E. coli in carrot juice was enhanced by

combination treatment both with and without light exposure. UHP-Red No. 3

combination in ineffective against bacteria in the turkey meat product. Further studies

are needed to determine which food components or characteristics are protecting the cells

from the combination treatment.

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ACKNOWLEDGEMENTS

This research was supported by a grant from the Center for Advanced Processing

and Packaging Studies (CAPPS). The authors would like to thank Teresa Nguyen for

significant laboratory assistance during turkey meat product studies.

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35. Yang W T, Lee J H, and Min D B. 2002. Quenching mechanisms and kinetcis of α-tocopherol and β-carotene on the photosensitizing effect of synthetic food colorant FD&C Red No. 3. Journal of Food Science 67:507-510.

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CHAPTER 7

IMPACT OF AEROBIC AND ANAEROBIC TREATMENT AND RECOVERY

ON INACTIVATION OF FOODBORNE BACTERIA BY FD&C RED NO. 3 AND

ULTRA-HIGH PRESSURE COMBINATION TREATMENT

ABSTRACT

Ultra-high pressure (UHP) and FD&C Red No. 3 are thought to cause oxidative stress to bacterial cells. UHP may cause oxidation of various components of the cell or render the cells incapable of dealing with oxidative stress following UHP treatment.

Previous investigators have found significantly more recovery under anaerobic conditions of E. coli treated with UHP compared with aerobic recovery. Photooxidation contributes significantly to inactivation of bacteria by UHP and Red No. 3 combination treatments with light exposure. Differences in aerobic and anaerobic treatment and recovery will help to elucidate the mechanism of inactivation by UHP and Red No. 3 combination treatments. The objective of this study was to determine the impact of superoxide production via photooxidation on the inactivation of E. coli K12 using aerobic and anaerobic treatments and comparing inactivation patterns between wild-type and mutants deficient in superoxide dismutase (sodA, sodB, sodAB). The second objective of this study was to determine the impact of aerobic and anaerobic treatment and recovery of 205

processing-resistant strains of Listeria monocytogenes, Lactobacillus plantarum, and E.

coli O157:H7 using combination treatments to determine the impact of type I and type II

photooxidation on inactivation. Superoxide dismutase status had no impact on sensitivity

of E. coli K12 to inactivation by any of the combination treatments. However, treatment and recovery under anaerobic conditions consistently resulted in enhanced recovery. L. monocytogenes was significantly more sensitive to UHP treatment under anaerobic treatment and recovery conditions compared to aerobic treatment and recovery. E. coli

O157:H7 was the only strain to show significantly higher inactivation with aerobic verus

anaerobic conditions with UHP, dye, and light, indicating a contribution of type I

(majority) and type II (minority) photooxidation. Light exposure was limited for L.

monocytogenes and L. plantarum due to their higher degree of sensitivity to

photooxidation, however with lengthier light exposures contributions due to both type I

and type II photooxidation may become apparent. Interestingly, there was a significant

difference between aerobic and anaerobic treatment and recovery conditions with UHP

and dye in the dark for all strains with the exception of L. monocytogenes. Little is

known about the light-independent mechanism of inactivation by the combinationt

treatment, however from these findings this inactivation is oxygen dependent.

INTRODUCTION

Inactivation of microorganisms by hydroxyxanthenes, including Red No. 3, is

believed to be due to photooxidation leading to oxidation of cellular components,

ultimately resulting in cell death. Studies have focused on the efficacy of photooxidation

on the most inhibitory hydroxyxanthene, Rose Bengal, to inactivate microorganisms,

oxidize specific substrates, or cause breaks in DNA (13, 17, 19, 20). Studies have 206

suggested that Rose Bengal is the most effective because it has the highest quantum yield

for singlet oxygen (0.75-0.79) of the xanthene-derivatives due to its high degree of

halogenation (16). However, Rose Bengal is peculiar among the hydroxyxanthenes in

regard to certain characteristics, including partition coefficient, and has been reported to oxidize certain biological components that other xanthene-derivatives cannot (10, 13, 14).

Due to the high yield of singlet oxygen, Schafer et al (2000) speculated that inactivation of microorganisms by Rose Bengal is primarily due to type II photooxidation (17); however studies have not determined the impact of type I and type II photooxidation on microorganisms. Confusing these issues, photooxidation may lead to the production of superoxide anions, which is poorly defined by the type I and type II definitions in the literature. Superoxide production is the result of energy and electron transfer from the photosensitizer to triplet oxygen. Production of superoxide is a rare occurrence, less than

1 out of 100 collisions with oxygen, for most photosensitizers (4, 6). For the purposes of

this study, superoxide production will be classified as a type II photooxidation process,

due to the requirement for the presence of molecular oxygen. By this definition,

differences in the impact of type I and type II photooxidation on microorganisms can be determined by exposing cells to or protecting cells from molecular oxygen. Kuraoka et al

(1991) investigated the efficacy of several red dyes, including Erythrosin, Phloxine, and

Rose Bengal, on inactivating E. coli and several oxygen defense mutants via

photooxidation. Mutants deficient in catalase (katEG) were significantly more resistant

to photodynamic inactivation by Phloxine with anaerobic incubation, indicating a large

207

contribution of type II photooxidation (9). Photooxidation of proline by Rose Bengal was found to occur under both aerobic and anaerobic conditions, indicating the potential impact of the type I reactions on biological compounds (5).

E. coli possesses a repertoire of oxidative stress management components.

Included in this repertoire are the superoxide dismutase (SOD) enzymes. E. coli is known to constituently express Fe-SOD (sodB), induce expression of Mn-SOD (sodA) upon entry into stationary phase, and induce expression of low levels of periplasmic

CuZn-SOD (sodC) upon exposure to molecular oxygen (2). Exposure of E. coli cells to photooxidation by Toluidine Blue O resulted in induction of Mn-SOD. Induction of this enzyme occurred at a lower level with dye exposure in the dark. SOD induction by photosensitizers, including Rose Bengal, correlated with their ability to produce superoxide (15). Additionally, exogenous addition of superoxide dismutase or previous induction of superoxide dismutase has been shown to protect E. coli from inactivation by a variety of photosensitizers (14).

Ultra-high pressure (UHP) is believed to affect a number of cellular targets, ultimately resulting in cell death. Cellular membranes and essential proteins are believed to be the primary targets that lead to bacterial inactivation (1, 3, 12). However, several other hypotheses have been offered to explain differences in cellular resistance to UHP inactivation. Aertsen et al (2005) postulated that cells are exposed to oxidative stress during UHP treatment and that subsequent or combined oxidative stress may lead to enhanced inactivation. E. coli mutants defective in oxidative stress management, including superoxide dismutase (sodAB), were significantly more sensitive to UHP than the wild-type strain (1). Cells deficient in superoxide dismutase enzymes are likely more 208

sensitive to superoxide attack on Fe-S cluster-containing proteins leading to cellular damage via Fenton chemistry (18). Malone et al (2006) did not find a significant response in sod mRNA levels upon exposure of E. coli to sublethal UHP treatments (11).

The combination treatment of a photosensitizer, light, UHP treatment, and aerobic recovery may lead to extensive oxidative stress on the cell. Determination of the impact of each of these components on inactivation will help elucidate the primary mechanisms of inactivation by the UHP-Red No. 3 combination treatment.

The primary objective of this study was to determine the contribution of type I and type II photooxidation to the inactivation of selected processing-resistant bacteria by

UHP-Red No. 3 combination with aerobic or anaerobic treatment and recovery.

Additionally, superoxide dismutase mutants were compared to wild-type E. coli to identify the role of superoxide production on inactivation by combination treatment.

MATERIALS AND METHODS

Bacterial strains.

Escherichia coli K12 (wild-type), single knockout mutants lacking sodA, sodB and a double knockout mutant lacking sodAB were acquired by Aaron Malone and are maintained in the Food Safety Laboratory at The Ohio State University. Listeria monocytogenes OSY-328, Lactobacillus plantarum MDOS-32, and Escherichia coli

O157:H7 EDL 933 were obtained from the culture collection of the Food Safety

Laboratory at The Ohio State University (Columbus) and tested in this study. Stock cultures were suspended in appropriate broth media, containing 40% (vol/vol) glycerol, and stored at -80ºC. Immediately before experiments, L. plantarum was transferred from the frozen stock-culture to MRS agar and incubated at 37°C for 48-72 hours. Similarly, L. 209

monocytogenes and E. coli were transferred from frozen stock-culture to TSA agar and

incubated at 37ºC for 48 to 72 hours. Three isolated colonies of each strain were

transferred to the appropriate broth (MRS or TSB) and incubated overnight at 37°C.

FD&C Red No. 3.

FD&C Red No. 3 (CI number 45430, Japan Food Red No. 3) was obtained from

Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Stock solutions were prepared by

dissolving the food colorant in sterile distilled water. All solutions were used within 30

minutes of preparation.

High pressure equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol)

glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

2-liter capacity, and is designed to operate at pressures up to 900 MPa. To reduce temperature effect on ultra-high pressure inactivation, initial glycol:water processing fluid was 5-10ºC to achieve a holding temperature of 20-25ºC. All experiments were performed at 500 MPa with a 1-min holding time.

Bacteria treatment in buffer.

Overnight cultures were centrifuged at 10,000 rpm (9,000 x g) for 10 minutes

(International Equipment Co., IEC Centra MP4R) and suspended in sterile citrate- phosphate buffer (0.087 M phosphate, 0.007 M citrate, pH 7.0). Cell suspensions contained approximately 109 CFU/ml. Aliquots (0.9 ml) of cell suspensions were transferred into clear sterile polyethylene bags (Fisher Scientific Co., Pittsburgh, Pa.) or 210

black sterile polyethylene bags (Nasco Whirl-Pak, Fort Atkinson, Wi.) containing 0.1 ml of colorant stock solution to achieve the desired final concentration of these additives, and the bags were heat-sealed. Sample bags were then placed into a larger polyethylene bag and sealed using a vacuum sealer (Vacmaster, Kansas City, Mo.). Samples were then placed on ice for approximately 30 minutes prior to high pressure treatment. After pressurization, bags were immediately placed on ice until plated. Control bags were held

on ice throughout the duration of the experiment. Bags were opened aseptically and the

contents were serially diluted in 0.1% peptone water and plated on the appropriate agar

medium. L. plantarum strains were plated on MRS agar and incubated at 37°C for 48

hours prior to enumeration. L. monocytogenes and E. coli were plated on TSA and incubated at 37ºC for 48 hours. Experiments were performed in triplicate, unless otherwise indicated.

Light exposure of samples.

Light exposure consisted of samples in clear polyethylene bags being spread onto laboratory bench under ambient lighting conditions for 15 or 30 minute increments after pressure treatment, depending on experiment. Incident luminescence 485 lux as measured using a light meter (Sekonic Flashmeter L-358, Elmsford, New York).

Following controlled light exposure, samples were protected from light by wrapping the samples in aluminum foil until plating.

Anaerobic conditions.

Anaerobic samples were handled and manipulated within the anaerobe chamber

(Forma Scientific, Anaerobic System Model 1024, Marietta, Ohio) under anaerobic conditions. Necessary experimental supplies (i.e., buffer, peptone water, pipette tips) 211

were placed in an anaerobe chamber overnight prior to experiment. Aliquots of overnight cultures were centrifuged as described in bacteria treatment in buffer section. Following centrifugation, half of the samples were handled aerobically and half were handled anaerobically. Once polyethylene bags had been heat-sealed, they were removed from the anaerobe chamber, vacuum-sealed, held on ice, and pressure-processed. Following

UHP treatment, light exposure occurred and samples were returned to the anaerobe chamber for dilution and plating.

Statistical analysis.

Data were analyzed by analysis of variance (ANOVA) by using the General

Linear Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the mean log reductions of treatments were made using the Tukey’s Studentized Range test.

Synergy was defined as a significant difference between (log reduction dye + log reduction UHP) and (log reduction dye+UHP combination).

RESULTS

Wild-type and superoxide dismutase mutant strains of E. coli K12.

Superoxide dismutase status does not explain sensitivity of E. coli to UHP or

UHP-Red No. 3 combinations.

Inactivation of wild-type and superoxide dismutase mutant strains of E. coli K12 by UHP treatment (500 MPa, 1 min) is shown in Figure 7.1. Wild-type and mutant strains did not differ in their resistance to UHP treatment (p-value > 0.05). These results indicate that superoxide dismutase has little to no impact on inactivation of E. coli by

UHP treatment. Inactivation of the wild-type, sodB mutant and sodAB mutant strains

212

were significantly affected by the presence of oxygen (p-values <0.05). Anaerobic treatment and recovery of these strains resulted in significantly more resistance to UHP

treatment compared to aerobic treatment and recovery.

Efficacy of combination treatment without light exposure towards wild-type and

mutant strains is shown in Figure 7.2. Again, mutations in superoxide dismutase genes

had no affect on inactivation by these treatments, with all strains displaying similar

inactivation. Aerobic treatment and recovery resulted in significantly higher reduction of

all strains tested compared to anaerobic treatment and recovery.

Inactivation of wild-type and mutant strains by Red No. 3 and UHP with light

exposure is shown in Figure 7.3. Mutations did not have any effect on inactivation using

the combination treatment with light exposure. Aerobic recovery resulted in significantly

higher inactivation of sodB and sodAB strains compared to anaerobic treatment and

recovery.

Compilation of E. coli K12 strains and response to treatments.

Due to the absence of a strain effect on the inactivation by UHP and UHP-Red No.

3 combination treatments, log reduction data were compiled for all strains and all

treatments to determine overall reduction patterns (Figure 7.4). For comparable

treatments, aerobic treatment and recovery resulted in significantly higher inactivation

compared to anaerobic treatment and recovery. These strains are pressure-sensitive with

an average inactivation due to UHP alone (aerobic) of 4.8 log.

213

9.00 Anaerobic Aerobic 8.00

7.00 * 6.00 * *

5.00

4.00

3.00 Log Reduction (cfu/ml) Log Reduction

2.00

1.00

0.00 sod+ ∆sodA ∆sodB ∆sodAB Strain

Figure 7.1. Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure alone (500 MPa, 1 min).

* indicates significant difference (α = 0.05) between aerobic and anaerobic treatment and recovery (p-value < 0.05). Error bars represent standard error, n = 3.

214

9.00 Anaerobic Aerobic 8.00 * * * * 7.00

6.00

5.00

4.00

3.00 Log Reduction (cfu/ml) Log Reduction

2.00

1.00

0.00 sod+ ∆sodA ∆sodB ∆sodAB Strain

Figure 7.2. Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure (500 MPa, 1 min) and

FD&C Red No. 3 (10 ppm) with no light exposure. * indicates significant difference

(p-value < 0.05) between aerobic and anaerobic treatment and recovery. Error bars represent standard error, n = 3.

215

9.00 Anaerobic Aerobic 8.00 * *

7.00

6.00

5.00

4.00

3.00 Log Reduction (cfu/ml) Log Reduction

2.00

1.00

0.00 sod+ ∆sodA ∆sodB ∆sodAB Strain

Figure 7.3. Aerobic versus anaerobic treatment and recovery of Escherichia coli wild-type and sod mutants treated with ultra-high pressure (500 MPa, 1 min),

FD&C Red No. 3 (10 ppm) and 30 minutes light exposure following pressure treatment. * indicates significant difference (p-value < 0.05) between aerobic and anaerobic treatment and recovery. Error bars represent standard error, n = 3.

216

9.00

8.00 a a 7.00 b 6.00 b 5.00 c c 4.00

3.00 Log Reduction (cfu/ml) Log Reduction

2.00

1.00

0.00 Anaerobic, UHP Aerobic, UHP Anaerobic, Aerobic, Anaerobic, Aerobic, UHP+Dye+Dark UHP+Dye+Dark UHP+Dye+Light UHP+Dye+Light Treatment

Figure 7.4. Comparison of efficacy of ultra-high pressure (500 MPa, 1 min) combination treatments and aerobic versus anaerobic recovery by compiling inactivation of E. coli wild-type and sod mutants. Treatments with different letter designations indicate significant difference (p-value < 0.05) in treatment efficacy. Error bars represent standard error, n = 12.

217

UHP resistant wild-type strains.

Contribution of aerobic and anaerobic treatment and recovery on

inactivation of Lactobacillus plantarum MDOS-32 by UHP-Red No. 3 combination.

Inactivation of L. plantarum MDOS-32 by various treatments is shown in Figure

7.5. Red No. 3 produced significant inactivation (p-value <0.05) of L. plantarum

MDOS-32 with 15 minutes of light exposure with a 1.3 log reduction under aerobic

conditions. The sensitivity of L. plantarum to photooxidation by Red No. 3 was expected

due to previous results (See Chapter 2). There was no significant difference (p-value

>0.05) in inactivation between samples treated aerobically or anaerobically with UHP,

light, and dye with inactivations of 5.9 and 5.0 log, respectively, thus indicating that the

presence of molecular oxygen is not necessary for inactivation. This suggests that

inactivation of L. plantarum by photooxidation of Red No. 3 occurs via a type I

mechanism (radical production).

UHP treatment (500 MPa, 1 min) resulted in an expected low level of inactivation

(0.7 log reduction) for this pressure-resistant strain. There was no difference (p-value >

0.05) in inactivation by UHP with or without light exposure or treated aerobically or

anaerobically. Inactivation of L. plantarum with UHP and Red No. 3 in the dark resulted in a significant increase in inactivation (p-value < 0.05) when treated aerobically compared to same treatment under anaerobic conditions. Treatment with the combination and light exposure did significantly enhance the inactivation of L. plantarum (p-values <

0.05) independent of aerobic or anaerobic treatment. No significant difference between

aerobic and anaerobic treatments with UHP, dye, and light suggests that photooxidative

damage following UHP treatment is the result of type I photooxidation. 218

9.00 Aerobic Anaerobic 8.00

7.00

6.00

5.00

4.00

3.00 Log Reduction (cfu/ml) Reduction Log *

2.00

1.00

0.00 Dark Dye, Dark Light Dye, Light Dark Dye, Dark Light Dye, Light

No UHP UHP

Figure 7.5. Aerobic versus anaerobic treatment and recovery of Lactobacillus plantarum MDOS-32 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min). Light exposure was 15 minutes after pressure treatment. * indicates significant difference (p- value < 0.05) between aerobic and anaerobic treatment and recovery. Error bars represent standard error, n = 3.

219

Contribution of aerobic and anaerobic treatment and recovery on

inactivation of Listeria monocytogenes OSY-328 by UHP-Red No. 3 combination.

Inactivation of L. monocytogenes OSY-328 by various combination treatments is shown

in Figure 7.6. Red No. 3 was ineffective at reducing L. monocytogenes without UHP

treatment, regardless of light exposure. Light exposure was kept to a minimum of 15 minutes for the Gram-positive organisms in an attempt to recover survivors from the most extreme treatment. Longer exposure times to light (30 minutes) would have resulted in inactivation without UHP based on previous studies in the laboratory (See

Chapters 2 and 4). Inactivation of L. monocytogenes OSY-328 by UHP alone resulted in the expected low inactivation with aerobic treatment and recovery; however inactivation was significantly higher with anaerobic treatment and recovery, regardless of light

exposure.

The combination treatment was only effective in the dark with aerobic treatment

and recovery. There was no significant difference between aerobic and anaerobic treatment; however aerobic recovery was significantly more lethal than UHP alone, whereas anaerobic recovery was similar to UHP alone. As expected, inactivation of L.

monocytogenes by combination treatments was enhanced with light exposure, but there

was no difference in the efficacy of these treatments treated aerobically or anaerobically.

220

9.00 Aerobic Anaerobic 8.00

7.00

6.00

5.00

4.00 * * 3.00 Log Reduction (cfu/ml)

2.00

1.00

0.00 Dark Dye, Dark Light Dye, Light Dark Dye, Dark Light Dye, Light

No UHP UHP

Figure 7.6. Aerobic versus anaerobic treatment and recovery of Listeria monocytogenes OSY-328 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min).

Light exposure for was 15 minutes after pressure treatment. * indicates significant difference (p-value < 0.05) between aerobic and anaerobic treatment and recovery. Error bars represent standard error, n = 3.

221

Contribution of aerobic and anaerobic treatment and recovery on

inactivation of Escherichia coli EDL 933 by UHP-Red No. 3 combination.

Inactivation of E. coli EDL 933 by combination treatments is shown in Figure 7.7.

As expected, Red No. 3 had no impact on E. coli without UHP treatment. Inactivation

due to UHP treatment alone resulted in 1.3 log reduction with no significant difference

when treated and recovered aerobically or anaerobically. Combination treatment of Red

No. 3 and UHP without light exposure resulted in significant enhancement of inactivation

with aerobic treatment and recovery. Anaerobic treatment and recovery under dark

conditions produced no increase in inactivation compared to UHP alone. Combination

treatments including light exposure were more effective than those protected from light, as expected. However, inactivation was significantly enhanced with treatment under aerobic conditions.

Compiled data for UHP-resistant strains

Figure 7.8 displays compiled data for UHP and UHP-combination treatments for the three UHP-resistant strains used in this study. Data was compiled to aid in understanding of consistent affects of these treatments. It must be noted that strains were compiled, despite the strain having a significant impact on efficacy of these treatments.

As a whole, UHP alone treatments were not significantly different from one another regardless of light exposure and aerobic versus anaerobic treatment (p-value > 0.05).

UHP-Red No. 3 combination without light exposure was only effective under aerobic conditions (p-value < 0.05). UHP-Red No. 3 combination with light exposure was not significantly enhanced in the presence of oxygen (p-value < 0.05).

222

9.00 Aerobic Anaerobic 8.00 *

7.00

6.00

5.00 *

4.00

3.00 Log Reduction (cfu/ml) Log Reduction

2.00

1.00

0.00 Dark Dye, Dark Light Dye, Light Dark Dye, Dark Light Dye, Light

No UHP UHP

Figure 7.7. Aerobic versus anaerobic treatment and recovery of Escherichia coli

EDL 933 treated with and without FD&C Red No. 3 (10 ppm), with and without light, and with and without ultra-high pressure (500 MPa, 1 min). Light exposure was 30 minutes after pressure treatment. * indicates significant difference (p-value <

0.05) between aerobic and anaerobic treatment and recovery. Error bars represent standard error, n = 3.

223

9.00

8.00

7.00

a 6.00 a 5.00

b 4.00

3.00

Log Reduction (cfu/ml) Log Reduction c 2.00 c c c c 1.00

0.00 Anaerobic, Aerobic, Anaerobic, Aerobic, Anaerobic, Aerobic, Anaerobic, Aerobic, UHP, Dark UHP, Dark UHP, Light UHP, Light UHP, Dye, UHP, Dye, UHP, Dye, UHP, Dye, Dark Dark Light Light Treatment

Figure 7.8. Comparison of efficacy of ultra-high pressure (500 MPa, 1 min) combination treatments and aerobic versus anaerobic recovery by compiling inactivation of L. plantarum, L. monocytogenes, and E. coli inactivation. Treatments with different letter designations indicate significant difference (p-vlaue < 0.05) in treatment efficacy. Error bars represent standard error, n = 9.

224

DISCUSSION

Differences in aerobic and anaerobic treatment and recovery of Red No. 3 and

UHP combination treatment may lead to further understanding of photooxidative mechanisms of inactivation. Type II photooxidation requires the presence of molecular oxygen to form singlet oxygen and/or superoxide, whereas type II photooxidation can occur in the absence of molecular oxygen. Experiments were designed to determine the extent of type I and type II photooxidation on the lethal affect of Red No. 3 and UHP-Red

No. 3 combination treatments by performing these experiments with and without

exposure to molecular oxygen. In addition, superoxide dismutase mutants of E. coli K12

were compared to a wild-type strain to identify any contribution of superoxide to the

lethal affect of these treatments.

Aertsen et al (2005) found aerobic recovery of E. coli MG1655 to produce 100-

fold higher inactivation compared to anaerobic recovery with UHP treatment of 400 MPa

for 15 minutes. These authors also attempted anaerobic recovery of several oxidative

stress mutants, including sodAB, following UHP treatment which resulted in increased

recovery, however numbers were not reported. These authors also reported that the

double mutant sodAB was significantly more sensitive (~4.8 log reduction) to UHP

inactivation than its wild-type (~2.3 log reduction) with a treatment of 350 MPa for 15

minutes (1). Superoxide dismutase deficient mutants are known to be defective in 4Fe-

4S cluster containing enzyme activity. This lack of enzyme activity is thought to be due

to superoxide leading to iron release from these clusters which can lead to the production

of additional reactive oxygen species via the Fenton reaction (18). UHP may exacerbate

this problem by causing destabilization of Fe-S clusters, thus increasing the presence of 225

iron to interact with superoxide (11). The current study found no significant effect of

superoxide dismutase production ability on resistance to UHP or UHP-Red No. 3 combination treatments. Differences in relative resistance to UHP of wild-type and mutant strains may be attributable to differing UHP treatment conditions and suspension media.

These UHP-resistant strains were less sensitive to changes in aerobic and anaerobic treatments compared to the more UHP-sensitive E. coli K12 strains. Thus, an ability to deal with oxidative stress during and following UHP treatment may be partially indicative of strain resistance to UHP treatment. Likewise, Malone et al (2006) found significant changes in gene expression profile of E. coli strains exposed to UHP to occur only in the UHP resistant strain, EC-88, suggesting that pressure-sensitive strains may not induce a significant response to UHP stress to recover from the treatment (11). Aertsen et al (2005) developed a mechanism to determine oxidative stress within the cytoplasm of pressure-treated cells. E. coli mutants were created to express a cytoplasmic alkaline phosphatase (CAP) in which activation of the enzyme can only occur with oxidative stress in the cytoplasm. Activation of CAP occurred in wild-type E. coli as a function of pressure level indicating that cells experienced oxidative stress during UHP. However,

pressure-resistant strains showed significantly lower inactivation of CAP with identical

ressure treatments, suggesting ability to deal with oxidative stress is a large contributor to

UHP-resistance. These strains were also more resistant to oxidative stress in the form of

superoxide generated by plumbagin without UHP treatment (1).

In vitro studies designed to identify photooxidation mechanisms and products

have found that the presence of superoxide dismutase may inhibit radical formation by 226

hydroxyxanthenes, suggesting that superoxide is an important intermediate product for

further oxidative damage to lipids and other cellular components (6, 7, 14). Other studies

have identified superoxide dismutase as an effective protector against cytochrome c

reduction in vitro (15). The application of these chemical studies to biological systems is

limiting due to the complexity of the cell and its environment. As shown in this study,

presence of superoxide dismutase has no impact on the survival of E. coli exposed to

photooxidation by Red No. 3. Previous studies conducted by Kuraoka et al (1991) found

E. coli mutants deficient in katEG were significantly more sensitive to photodynamic treatment with Erythrosin, Phloxine, and Rose Bengal. Other mutants, including recA,

and xth nfo, did not display any differences in sensitivity to photooxidation by these dyes.

Mutants lacking recA were significantly more resistant (~800-fold) to Phloxine when

exposed under aerobic conditions compared to anaerobic, indicating a large impact of

type II photooxidation (9). Studies of superoxide dismutase mutants of Gram-positive

bacteria would provide an ideal situation to determine impact of superoxide production

on inactivation.

Efficacy of combination treatments in the dark was dependent on treatment and

recovery conditions, with aerobic conditions resulting in significantly higher levels of

inactivation compared to anaerobic treatment which resulted in the same level of

inactivation compared with UHP alone. Very little is known about the mechanism of

inactivation due to combination treatment in the dark but these data indicate that

inactivation is oxygen-dependent. From the study design used, it is impossible to

distinguish if the inactivation caused by aerobic treatment is due to the presence of

oxygen during the actual treatment or if it is due to the presence of oxygen during 227

recovery and incubation. Further studies are needed to determine which of these conditions, or both, lead to the efficacy of the combination treatment in the dark.

Inactivation by the combination treatment with light exposure resulted in significantly higher levels of inactivation compared to UHP alone, again treatment and recovery conditions were important. Aerobic treatment and recovery again resulted in higher levels of inactivation compared to anaerobic conditions. This is suggestive of the impact of type I and type II photooxidation mechanisms; however the differences may be due to the differences caused by UHP alone using these strains. If type II photooxidation was a significant contributor to inactivation by combination treatment with light, superoxide dismutase mutants should have been substantially more sensitive to this treatment compared to the wild-type. This was not the case, suggesting that the differences in aerobic and anaerobic treatment and recovery of these strains is linked to their wild-type strain’s sensitivity to UHP and resulting oxidative stress (1).

Differences in efficacy and/or recovery of L. monocytogenes due to aerobic versus anaerobic UHP treatment have not been previously reported in the literature. Knabel et al

(1990) found improved recovery of thermally-treated L. monocytogenes with anaerobic recovery, the opposite of the results presented in this study. The authors speculated that the heat treatment resulted in inactivation of superoxide dismutase and catalase enzymes leading to their sensitivity under aerobic conditions (8). Further investigations are warranted to understand mechanism of sensitivity of L. monocytogenes to UHP via anaerobic treatment and recovery.

Inactivation of E. coli K12 and superoxide dismutase mutants via UHP is significantly effected by the presence of molecular oxygen during treatment and recovery. 228

Superoxide dismutase production ability was not explanatory of sensitivity of E. coli to

UHP or UHP-Red No. 3 combination treatments. Pressure-resistant wild-type strains of

E. coli and L. plantarum were equally resistant to UHP treatment under aerobic and anaerobic conditions, whereas L. monocytogenes OSY-328 was significantly more sensitive to UHP treatment under anaerobic conditions. Inactivation of all strains via

UHP-Red No. 3 combinations without light exposure is oxygen-dependent. Inactivation of E. coli by UHP-Red No. 3 combinations with light exposure indicates that type I photooxidation is the primary mechanism of inactivation of bacteria following UHP- Red

No. 3 combination treatments. Type II photooxidation plays a smaller role depending on extent of light exposure and the photosensitizer tested.

ACKNOWLEDGEMENTS

This research was supported by a grant from the Center for Advanced Processing and Packaging Studies (CAPPS). The author would like to thank Aaron Malone for collecting and organizing the sod mutant and wild-type strains. The author also thanks

Dr. Mark Daeschel, for supplying L. plantarum MDOS-32 for these studies. Special thanks to Dr. Yoon-Kyung Chung for assistance with the anaerobic treatment chamber.

REFERENCES

1. Aertsen A, De Spiegeleer P, Vanoirbeek K, Lavilla M, and Michiels CW. 2005. Induction of oxidative stress by high hydrostatic pressure in Escherichia coli . Applied and Environmental Microbiology 71:2226-2231.

2. Benov L T and Fridovich I. 1994. Escherichia coli expresses a copper- and zinc- containing superoxide dismutase. The Journal of Biological Chemistry 269:25310-25314.

3. Casadei MA, Manas P, Niven GW, Needs E, and Mackey B. 2002. Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164. Applied and Environmental Microbiology 68:5965-5972.

229

4. Cruz F S, Lopes L A V, de Souza W, Moreno S N J, Mason R P, and Docampo R. 1984. The photodynamic action of rose bengal on Trypanosoma cruzi. Acta Tropica 41:99-108.

5. Endo K, Hirayama K, Aota Y, Seya K, Asakura H, and Hisamichi K. 1998. Photooxidative decarboxylation of proline, a novel oxidative stress to natural amines. Heterocycles 47:865-870.

6. Foote C S. 1976. Photosensitized oxidation and singlet oxygen: consequences in biological systems, p. 85-133. In Pryor W A (ed.), Free Radicals in Biology. Academic Press, New York.

7. Inbaraj J J, Kukielczak B M, and Chignell C F. 2005. Phloxine B phototoxicity: A mechanistic study using HaCaT keratinocytes. Photochemistry and Photobiology 81:81-88.

8. Knabel S J, Walker H W, Hartman P A, and Mendonca A F. 1990. Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization. Applied and Environmental Microbiology 56:370-376.

9. Kuraoka I, Kawakami K, Hiratsu K, Ono T, Yamada K, Nunoshiba T, and Nishioka H. Sensitivity of Escherichia coli mutants lacking active oxygen defense systems to photodynamic action. Science and Engineering Review of Doshisha University 32[2], 210-216. 1991.

10. Levitan H. 1977. Food, drug, and cosmetic dyes: Biological effects related to lipid solubility. Proceedings of National Academy of Science USA 74:2914-2918.

11. Malone AS, Chung Y-K, and Yousef AE. 2006. Genes of Escherichia coli O157:H7 that are involved in high pressure resistance. Applied and Environmental Microbiology 72:2661-2671.

12. Manas P and Mackey B. 2004. Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death. Applied and Environmental Microbiology 70:1545-1554.

13. Martin J P Jr and Burch P. Oxygen radicals are generated by dye-mediated intracellular photooxidiations. Cerutti P A, Fridovich I, and McCord J M. 82 (Oxy-Radicals in Molecular Biology and Pathology), 393-404. 1988. New York, NY, Alan R. Liss, Inc. Proceedings of an Upjohn - UCLA Symposia on Molecular and Cellular Biology.

230

14. Martin J P Jr and Logsdon N. 1987. Oxygen radicals are generated by dye- mediated intracellular photooxidations: a role for superoxide in photodynamic effects. Archives of Biochemistry and Biophysics 256:39-49.

15. Martin J P Jr and Logsdon N. 1987. Oxygen radicals are generated by dye- mediated intracellular photooxidations: a role for superoxide in photodynamic effects. Archives of Biochemistry and Biophysics 256:39-49.

16. Neckers D C and Valdes-Aguilera O M. 1993. Photochemistry of the xanthene dyes, p. 315-394. In Volman D, Hammond G S, and Neckers D C (eds.), Advances in Photochemistry. John Wiley & Sons, Inc.

17. Schafer M, Schmitz C, Facius R, Horneck G, Milow B, Funken K-H, and Ortner J. 2000. Systematic study of parameters influencing the action of rose bengal with visible light on bacterial cells: Comparison between the biological effect and singlet-oxygen production. Photochemistry and Photobiology 71:514- 523.

18. Touati D. 2000. Iron and oxidative stress in bacteria. Archives of Biochemistry and Biophysics 373:1-6.

19. Wang H, Lu L, Zhu S, Li Y, and Cai W. 2006. The phototoxicity of xanthene derivatives against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Current Microbiology 52:1-5.

20. Yoshikawa K, Kurata H, Iwahara S, and Kada T. 1978. Photodynamic action of fluorescein dyes in DNA-damage and in vitro inactivation of transforming DNA in bacteria. Mutation Research 56:359-362.

231

CHAPTER 8

CONCLUSIONS

Halogenated xanthene-derivatives, including Red No. 3, are effective at

inactivating Gram-positive bacteria via a photooxidative mechanism at low concentration

(1-10 ppm) with limited light exposure (15-30 minutes, 485 lux). Lactic acid bacteria,

specifically Pediococcus acidilactici and Lactobacillus plantarum strains, were

extremely sensitive to colorant and light treatments. Other Gram-positive bacteria

(Staphylococcus aureus and Listeria monocytogenes) strains were sensitive to colorant

and light treatments, but significantly more resistant than lactic acid bacteria (see Chapter

2). Xanthene-derivatives were ineffective at inactivating Gram-negative bacteria

(Escherichia coli and Salmonella Typhimurium) under the conditions tested. E. coli was resistant to Red No. 3 at concentrations up to 300 ppm. The outer membrane likely plays a protective role in preventing inactivation of Gram-negative bacteria via photooxidation by halogenated xanthene-derivatives, via unknown mechanisms.

Dose-dependent inactivation of Gram-positive and Gram-negative bacteria using combination of halogenated xanthene-derivatives and ultra-high pressure (UHP) was achieved without light treatment and further enhanced with light treatment, indicating two mechanisms of inactivation: light-dependent (photooxidative) and light-independent

232

(non-photooxidative). Combination treatments showed that light-independent inactivation is an oxygen-dependent process. Light-dependent inactivation by

combination treatments is primarily via type I photooxidation processes due to

insignificant differences in inactivation with and without the presence of molecular

oxygen. Further studies are needed to elucidate if oxygen-dependence of light-

independent inactivation is a function of treatment conditions or recovery conditions.

Gram-negative bacteria are inherently resistant to inactivation by halogenated

xanthene-derivatives. These compounds accumulate with Gram-positive cells, but not

with Gram-negative bacteria, indicating that the outer membrane is preventing the

association of these colorants with these cells. Accumulation of Red No. 3 occurs in

Gram-negative bacteria following UHP treatment or suspension in pH 3.0 buffer.

Inactivation by these combinations was apparent with treatments resulting in dye uptake,

signifying that these chemical and physical treatments alter the barrier properties of the

outer membrane. Membrane isolation following UHP treatment revealed that the density

of the outer membrane is significantly decreased, possibly due to a release of outer

membrane proteins and/or lipopolysaccharide. Outer membranes isolated from pressure

treated cells showed a significant increase in colorant association compared to non-

pressure treated cells. This localization of halogenated hydroxyxanthenes may signify

the presence of a lethal target site within the outer membrane.

Combinations of Red No. 3 and UHP result in synergistic inactivation of

important foodborne spoilage and pathogenic bacterial species, including E. coli

O157:H7, L. monocytogenes, L. plantarum, P. acidilactici, Salmonella enterica, and S.

233

aureus. Inactivation of bacteria by the combination treatment was effective in citrate- phosphate buffer over a range of pH (3.0-7.0) and in carrot juice against UHP-resistant strains of L. monocytogenes and E. coli O157:H7. Combination treatment was ineffective in a homogenized turkey meat product, indicating that food composition may protect bacteria from inactivation. The inclusion of permissible levels (<300 ppm) of

Red No. 3 in select food systems could provide enhanced safety to commercially pressure treated products.

234

APPENDIX A

PARTITION COEFFICEINT DETERMINATION OF XANTHENE-

DERIVATIVES

235

Information supplied by Dante Vargas (Advisor: Monica Giusti)

Materials and Methods

To determine the partition coefficient of the various xanthenes, the Shake-Flask method was used as explained by El-Gendy (2004) with certain modifications to adjust to determined conditions and compounds.

• Standard curves: First, equal amounts of 1-octanol and water were placed in a 500mL flask. This was inverted manually several times. Then, the mixture was placed on a mechanical shaker (New Brunswick Scientific Classic C24 incubator shaker) at 50RPM for 8 hours. Mixture was then left overnight for proper solvent separation. Standard curves for each of the xanthenes were developed in water (previously shaken with octanol). These curves were created keeping in mind the maximum absorbances handled by the UV spectrometer. The maximum concentration of the standard curves was 20 ppm. Standard curves for 1-octanol were also developed but the concentration used did not give significant absorbance values for most colors. At the same time, 1-octanol color samples seemed to degrade at a faster rate, even in the dark.

• Shake-Flask Method: After the water/octanol mixture had rested, solutions of 20ppm of each colorant were prepared (in water previously shaken with octanol). These “reference” samples were measured for absorbance at their max absorbance wave lengths on a Shimadzu UV-2450 UV-Visible Spectrometer. Out of these “reference” samples, 5mL of each were mixed with 5mL of octanol. These solutions were manually mixed and then placed on a shaker at 180RPM for 0.5 hours. These samples were let to rest overnight. Then, the aqueous colored solvent was extracted and its absorbance was measured. The values of the concentration in octanol were obtained by the difference of the concentration of the reference sample and the final concentration in the aqueous solvent. (This would determine how much sample was transferred to the octanol). The partition coefficient is measured by the logarithm of the molar concentration of the sample in octanol divided by its molar concentration in water.

Results

The aqueous and octanolic colored solutions had different maximum absorbance wave lengths. These wave lengths will differ in as much as 21 nm. This can be observed in Table 1.

The partition coefficient is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two phase system considering two largely immiscible solvents. (European Chemicals Bureau, 2007) Positive values point out that the compound is more soluble in 1-octanol while the contrary points out that the compound is more soluble in water. The results obtained indicate that Rose Bengal is more hydrophobic that the 236

other colorants (dissolved better in octanol). At the same time, it can be observed that Fluorescein is the most hydrophilic color. The other xanthenes partition coefficient values fall in the negative side which also is a sign of their hydrophilicity. The partition coefficient values of the xanthenes can be observed in Table 2.

References: • European Chemicals Bureau, 2007. http://ecb.jrc.it/DOCUMENTS/Testing-Methods/ANNEXV/A08web1992.pdf • El-Gendy, A.; Adejare, A. Membrane permeability related physicochemical properties of a novel γ-secretase inhibitor

Table 1: Max Absorbance Wave Lenghts

Colorant Max Abs Wave Length Max Abs Wave Length in H20 in 1-Octanol (OcOH) Fluorescein 489.5 490.0 Eosin 517.0 538.0 Erythrosin 526.5 545.5 Phloxine 539.0 555.5 Rose Bengal 549.0 567.5

Table 2: Partition coefficient results for xanthenes

Reference Color concentration Concentration (after shaking) Partition

in H2Oa (ppm) Coefficientc (ppm) H2O 1-Octanolb Meand SDe Meand SDe Log P Fluorescein 19.65 0.0514 14.84 0.1283 4.81 -0.4894 Eosin 21.51 0.7521 15.51 0.2174 6.01 -0.4119 Erythrosin 19.54 0.6066 13.83 0.3350 5.71 -0.3844 Phloxine 20.02 0.2604 12.32 0.1471 7.70 -0.2042 Rose Bengal 19.33 0.2916 5.95 0.0768 13.38 0.3517 a: Solution was made to obtain a concentration of approximately 20ppm. b: Result obtained from the difference of the concentration of the reference and the concentration of the aqueous sample after shaking. c: Log P = Log ( C octanol / C water ) d: Number of repetitions = 3 e: Standard Deviation of 3 repetitions

237

APPENDIX B

PRELIMINARY RESULTS WITH FD&C COLORS

238

9.00

8.00

7.00 UHP alone

UHP + Blue 6.00 No. 1 UHP + Blue 5.00 No. 2 UHP + Green No. 3 4.00 UHP + Red No. 3

Log Reduction (cfu/ml) Log Reduction 3.00 UHP + Red No. 40 2.00 UHP + Yellow No. 5 UHP+ Yellow 1.00 No. 6

0.00 L. plantarum MDOS-32 L. plantarum OSY-104 Strains

Figure A.1. Inactivation of L. plantarum MDOS-32 and L. plantarum OSY-104 by ultra-high pressure (400 MPa, 3 min) and FD&C certified food colorants (300 ppm).

Temperature during holding time was 30ºC. Error bars indicate standard error, n = 3.

239

APPENDIX C

SELECTION AND IDENTIFICATION OF A Listeria monocytogenes

SURROGATE FOR ULTRA-HIGH PRESSURE AND PULSED ELECTRIC

FIELD PROCESS VALIDATION

240 Selection and identification of a Listeria monocytogenes surrogate for optimization

of ultra-high pressure and pulsed electric field process validation

J.G. Waite, B.H. Lado, and A.E. Yousef

ABSTRACT

Ultra-high pressure (UHP) and pulsed-electric field (PEF) are promising

alternative processing techniques that are being optimized to enhance safety and/or shelf- stability of treated food. It is crucial to recognize the pathogens of concern (targeted pathogens) for food treated with these technologies. Additionally, it is equally important to identify low-risk surrogate microorganisms that can be used, in lieu of the targeted pathogens, in food processing facilities. The objective of this study was to determine appropriate surrogate organisms for Listeria monocytogenes for UHP- and PEF-process

validation. Candidate surrogates tested include four Lactobacillus spp., a Pediococcus

sp. and a Listeria innocua strain. Candidate surrogates and nine L. monocytogenes strains

were treated with UHP and PEF and processing sensitivity of these microorganisms was

determined. For UHP, strains were suspended in citrate-phosphate buffer (pH 7.0 or pH

4.5), sweet whey, or acidified sweet whey and processed at 500 MPa with a holding time

of 1 minute. For PEF, each strain was suspended in 0.1% NaCl or 50% acid whey and

was individually processed with a continuous PEF apparatus at 25 kV/cm. Inactivation

by PEF and UHP was strain-dependent and varied with media composition and pH.

Pressure treatments of L. monocytogenes strains suspended in citrate-phosphate buffer

(pH 7.0) inactivated 0.3 to 2.0 log10 CFU/ml whereas suspension in citrate-phosphate

buffer (pH 4.5) inactivated 4.8 to 6.6 log10 CFU/ml. Similarly, inactivation of L.

241 monocytogenes strains was enhanced when processed in acidified sweet whey (pH 4.2)

compared to sweet whey (pH 6.0). When PEF-processed in 0.1% NaCl, L.

monocytogenes populations decreased 0.7 to 5.3 log10 CFU/ml. Listeria monocytogenes

V7 and OSY-8578 were among the most resistant strains to both UHP and PEF treatments and would be ideal target organisms. Lb. plantarum was similar or more

resistant to the target organisms in resistance to UHP and PEF treatments and would be a

good surrogate organism for both processes. In the case of UHP, a Pediococcus sp. was

the most resistant bacterium tested with a maximum of <0.3 log10 inactivation under all

conditions tested.

INTRODUCTION

Alternative food processing technologies have been developed to combat the

negative quality impacts on food due to traditional thermal processing. Ultra-high pressure (UHP) and pulsed electric field (PEF) are emerging technologies capable of inactivating spoilage and pathogenic microorganisms in food products with minimal impact on food quality. UHP and PEF can eliminate pathogens from food products. Low processing temperature (≤ 55°C) is desirable to retain the functionality of heat-labile nutrients and can still result in bacterial inactivation for both technologies (5, 8).

Satisfactory UHP and PEF processing results in the elimination of the most resistant pathogen potentially present in the food.

It has been suggested that the destruction of Listeria monocytogenes is a critical measure for UHP- and PEF-process validation (4, 9, 18). Previous studies have determined the variability in resistance of L. monocytogenes strains to UHP and PEF

treatments when suspended in spent broth and 0.1% NaCl, respectively (21, 32). L.

242 monocytogenes OSY-8578 was proposed to be the target strain for process validation,

however analysis of processing-resistance in food systems has not been completed.

Inactivation studies of target pathogenic strains are restricted to controlled

laboratory settings and cannot be introduced into the food processing environment for

safety reasons. Therefore, a surrogate microorganism for L. monocytogenes is urgently

required to validate pilot- and industrial-scale UHP and PEF processing parameters. As

defined by the U.S. Food and Drug Administration, a surrogate organism is “a non-

pathogenic species and strain responding to a particular treatment in a manner equivalent

to a pathogenic species and strain” (13). Several studies have been performed to identify

surrogates for specific pathogens and specific technologies. Geobacillus

stearothermophilus or Clostridium sporogenes have frequently replaced C. botulinum for the validation of commercial sterilization via retorting or temperature-assisted thermal processing (PATP) (1). Montville et al (2005) found several appropriate avirulent

Bacillus spores to serve as surrogates for virulent B. anthracis thermally processed in milk and orange juice systems (24). Eblem et al (2005) identified E. coli ATCC 25922 as a candidate surrogate for E. coli O157:H7 and Salmonella Poona on apple surfaces treated with hydrogen peroxide and in broth that was heat-treated (10).

Surrogate organisms are often screened based on close genetic relationships to the target pathogen, assuming that genetic similarities equate to similar resistance patterns.

Listeria innocua has been suggested as a thermal-resistance indicator of L. monocytogenes in laboratory-scale analyses (11-13), however detection of Listeria, regardless of the species, in a food product is a sufficient hazard in the US to command additional testing. Therefore, in this study, potential surrogates of L. monocytogenes for

243 UHP and PEF processing were primarily sought among bacterial species “generally

recognized as safe”, and which have frequently been isolated from food. Lactic acid bacteria such as lactobacilli and pediococci are common in food, phylogenetically close

to Listeria spp., and can co-exist with this pathogen in food (19, 29, 35). Sensitivity to

PEF varies with bacterial morphology and cell wall structure; like Listeria sp.,

lactobacilli are rod-shaped, Gram-positive bacteria with peptidoglycan cross-linkage

between position 3 and 4 of two peptide subunits (3). Pediococci are gram-positive cocci

of diameter size close to the length and width of Listeria sp., and have similar cell wall

structure (2). Whey was selected as the food model to select a suitable Lactobacillus or

Pediococcus surrogate of L. monocytogenes for UHP- and PEF- process validation.

Whey and, more particularly, whey protein concentrates (WPC) are frequently

used in food formulation (8, 20, 30). Excessive heating may reduce whey protein

functionality (8, 17). Whey may become contaminated with L. monocytogenes during

cheese manufacturing (26, 27). The heating steps in WPC production aims at eliminating

pathogens and non-pathogenic psychrotrophs. Failure to inactivate these microorganisms

would result in microbial concentration and multiplication during ultrafiltration, thereby

reducing WPC quality (17, 27). Thermal pasteurization (72°C for 15 s) efficiently

eliminates L. monocytogenes in sweet whey (17). Similarly, acid whey is held at ~ 52°C to reduce microbial load (17). However, sub-lethal injury of L. monocytogenes was observed in artificially contaminated acid whey (pH 4.6) that was cooked at 57.2°C for

30 min, and viable cells were recovered after several months’ storage at refrigeration temperature (26). WPC may be adversely affected by thermal treatments; therefore alternative processing conditions to control Listeria spp. and minimize product quality 244 deterioration have been investigated. Gallo et al (2007) found a combination of nisin, pH, and low temperature to control L. innocua in liquid cheese whey (15). This same group investigated the efficacy of nisin and PEF on inactivation of L. innocua in whey

(14). A satisfactory surrogate should be, in whey, at least equally resistant to UHP- and

PEF-processing as the target L. monocytogenes strain.

In this study, the UHP- and PEF-resistance of four Lactobacillus spp., one

Pediococcus sp. and one L. innocua strain were compared to the PEF-resistance of nine

L. monocytogenes strains. The most suitable surrogate microorganism was selected, based on its superior UHP- and PEF-resistance, when suspended in various media, including whey products.

MATERIALS AND METHODS

Microorganisms.

Listeria monocytogenes Scott A, California, V7, OSY-8517, OSY-8576, OSY-

8578, OSY-8579, OSY-8580, OSY-8732 were obtained from the culture collection of the

Food Safety laboratory at the Ohio State University. The following non-pathogenic microorganisms were obtained from the same culture collection: L. innocua ATCC

33090, Pediococcus acidilactici PXL, Lactobacillus plantarum ATCC 8014, Lb. leichmanii ATCC 4797, Lb. bulgaricus OSY 135 and Lb. acidophilus ATCC 19992.

Listeria strains and lactic acid bacteria were incubated overnight at 37ºC in tryptose broth

(TB) and MRS respectively. Stock cultures were stored at – 80ºC in TB containing 40%

(v/v) glycerol for Listeria, and in De Man, Rogosa, and Sharpe (MRS) broth containing

40% (v/v) glycerol for lactic acid bacteria.

245 Processing media for ultra-high pressure experiments.

Bacterial cells were suspended in citrate-phosphate buffers (pH 7.0 and 4.5),

reconstituted sweet dairy whey, and reconstituted acidified sweet whey for UHP process.

Powdered sweet dairy whey (Bob’s Red Mill, Milwaukee, Oregon) was suspended in

sterile distilled water to achieve a final concentration of 10% (w/v). Final pH of

reconstituted sweet whey was 6.0. Acidified sweet whey was prepared by lowering the

pH of the sweet whey to 4.2 with lactic acid. Both types of whey were sterilized by tindalization. Whey preparations were stored at +4ºC prior to the experiment.

Ultra-high pressure (UHP) equipment and processing conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol) glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

2-liter capacity, and is designed to operate at up to 900 MPa.

UHP processing.

Cultures of Listeria spp., Lactobacillus spp. and Pediococcus sp. were harvested by centrifugation at 10,000 rpm (12,400 x g) for 10 min (Sorvall RC-5B, DuPont,

Wilmington, DE) at 4°C, and suspended in selected processing media. The following four processing media were used: (i) citrate-phosphate buffer (pH 7.0), (ii) citrate-phosphate buffer (pH 4.5), (iii) sweet whey (10% w/v), and (iv) acidified sweet whey. Aliquots of 1 mL were transferred to sterile polyethylene bags and heat sealed. Samples were placed inside a larger polyethylene bag, vacuum-sealed and held on ice for 30 minutes prior to

UHP treatment to maximize resistance to UHP treatment. Samples were then pressure

246 treated at 500 MPa for 1 minute with a holding temperature of 23ºC ± 3ºC. All samples

were returned to ice until sampled and plated. Listeria spp. were plated onto tryptose agar for enumeration and lactic acid bacteria onto MRS agar. Three independent trials of each experiment were executed.

Processing media for pulsed electric field experiments.

Bacterial cells were suspended in 0.1% NaCl, 50% acid whey, 66% sweet whey and 50% acidified sweet whey for PEF process. The 0.1% (w/v) NaCl solution was at pH

7.0, with an electrical conductivity of 0.21 S/m. Acid whey (pH 4.2) was collected during

cottage cheese production (Arps Dairy, Inc., Defiance, OH). Sweet whey (pH 6.8) was

recovered from the manufacture of Parmesan-type cheese from skimmed milk in our

laboratory (Unpublished procedure from A. E. Yousef). Each type of whey was filter-

sterilized (0.4 µm pore size, Osmonics Inc., Westborough, MA) and diluted with

deionized water to standardize the conductivity at 0.46 S/m. Acidified whey was diluted

in an equal volume of deionized water, and were therefore referred to as “50% acidified

whey”. One volume of deionized water was added to two volumes of sweet whey, and

hence, the processing medium was referred as “66% sweet whey”. All whey preparations

were stored at +4ºC and degassed with a vacuum pump (Model 115V, Curtin Matheson

Scientific, Houston, TX) prior to the experiment to prevent arcins.

Pulsed electric field (PEF) equipment.

An OSU 4-C PEF unit (Department of Food Science & Technology, Ohio State

University, Columbus, OH) was used for pulsed electric field process (39). This unit

contained four co-field treatment chambers (0.23 cm-diameter, 0.29 cm-gap distance

between electrodes for each treatment chamber) connected in series. Before and after

247 passing through a pair of treatment chambers, each sample passed through cooling coils

submerged in a water-bath (23°C) to maintain processing temperature of 0.1% NaCl and whey samples below 56°C and 40°C, respectively. The flow rate was set at 1 ml/s for the single point inactivation study in 0.1% NaCl, and 2 ml/s in all other assays. An electric field of 25 kV/cm, with bipolar pulses of 3 µs, was applied at a frequency of 1,000 Hz for cells suspended in NaCl and 667 Hz for cells suspended in whey. An oscilloscope (TDS

340A; Tektronix, Beaverton, OR) monitored the square wave pulse, input voltage, and current. Processing time was calculated as follows:

Processing time (µs) = Number of chamber × gap distance between electrodes (cm) × (π

× electrode diameter2 (cm2) / 4) × pulse duration (µs / pulse) × pulse frequency (pulse / s)

/ flow rate (cm3 / s)

PEF processing.

Cultures of Listeria spp., Lactobacillus spp. and Pediococcus sp. were harvested

by centrifugation at 5,000 x g for 10 min (Sorvall RC-5B, DuPont, Wilmington, DE) at

4°C, and washed briefly by suspension in 0.1% NaCl. Washed cells were suspended to a

concentration ~ 108 CFU/ml in processing medium held at 23°C. The following three

processing media were used: (i) 0.1% NaCl, for treatment of all strains; (ii) 50% acid

whey, for Listeria spp. and Lb. plantarum;; and (iii) 66% sweet whey, for all L.

monocytogenes strains. Cell suspensions were then exposed to an electric field of 25

kV/cm. Processing time for species comparisons was 48 µs and 144 µs for suspensions in

248 whey and 0.1% NaCl, respectively. To establish survivor plots, suspensions of Listeria

sp. and Lb. plantarum were processed in 0.1% NaCl six times, by 72 µs-increments, so

that the total processing time varied from 0 to 432 µs. All treated and untreated samples

were rapidly cooled in water-ice mixture, and serially diluted in phosphate buffer (pH

7.2; Weber Sci., Hamilton, NJ). Appropriate dilutions of Listeria spp. were plated onto

tryptose agar for enumeration, and of lactic acid bacteria onto MRS agar. At least two

independent trials of each experiment were executed.

Data analysis.

Statistical analyses were performed using SAS statistical programs (Cary, N.C.).

Averages of the inactivation of the tested strains were compared by one-way analysis of variance and Tukey’s test.

RESULTS

Efficacy of UHP against surrogate organisms in citrate-phosphate buffer and impact of pH.

The UHP inactivation in citrate-phoshate buffer (pH 7.0) of potential surrogate strains varied from 0.0 to 1.4 log CFU/ml after processing 1 minute at 500 MPa (Figure

C.1). Inactivation levels of surrogates were compared to the inactivation of L. monocytogenes strains tested under identical conditions. L. monocytogenes V7 was consistently resistant to UHP treatments relative to other L. monocytogenes strains with an inactivation of 0.3 log CFU/ml in the pH 7.0 buffer. L. monocytogenes ScottA was consistently sensitive to UHP treatments relative to other strains with an inactivation of

249 7.0

6.0

5.0

4.0

3.0

ab a 2.0 UHP Inactivaton (log CFU/ml) (log UHP Inactivaton 1.0 bc bc c c bc c 0.0

L. innocua

Lb. plantarum Lb. leichmanii Lb. bulgaricus P. acidilactici Lb. acidophilus

Figure C.1. Decrease in count (UHP inactivation) of Listeria, Pediococcus, and

Lactobacillus strains caused by ultra-high pressure at 500 MPa and 23°C for 1

minute, when cells were suspended in citrate-phosphate buffer, pH 7.0. Cell

suspensions were ~109 CFU/ml before treatment. The dashed and the dotted lines

indicate the corresponding inactivation of Listeria monocytogenes Scott A and V7, respectively. Different letter designations indicate strains with statistically different (P <

0.05) UHP inactivation levels, based on one-way ANOVA and Tukey mean comparison, n = 3.

250 2.0 log CFU/ml under the same conditions. P. acidilactici, Lb. plantarum and Lb.

acidophilus were the most resistant to UHP under these conditions with average

inactivations of 0.0, 0.1, and 0.1 log CFU/ml, respectively. L. innocua was comparable in resistance to L. monocytogenes V7. None of the potential surrogate strains were significantly different than L. monocytogenes V7 when treated in pH 7.0 buffer (p-values

> 0.05).

The UHP inactivation in citrate-phosphate buffer (pH 4.5) of potential surrogate strains varied from 0.3 to 4.7 log CFU/ml after processing 1 minute at 500 MPa (Figure

C.2). Inactivation levels of surrogates were compared to the inactivation of L. monocytogenes strains tested under identical conditions. L. monocytogenes V7 was again resistant to UHP treatments relative to other L. monocytogenes strains with an inactivation of 4.8 log CFU/ml in the pH 4.5 buffer. L. monocytogenes ScottA was again

sensitive to UHP with an inactivation of 6.6 log CFU/ml under the same conditions.

However, there were no significant differences in resistance among L. moncytogenes

strains treated in pH 4.5 buffer (p-value > 0.05). L. innocua was comparable in resistance

to L. monocytogenes strains with an average inactivation of 4.7 log CFU/ml. P.

acidilactici, Lb. plantarum and Lb. acidophilus were the most resistant to UHP under

these conditions with average inactivations of 0.3, 3.7, and 3.3 log CFU/ml, respectively.

P. acidilactici was significantly more resistant than all strains tested at this pH. Lb.

plantarum and Lb. acidophilus were significantly more resistant than L. monocytogenes

ScottA, but not significantly different than L. monocytogenes V7.

251 7.0 a

6.0 ab

ab b ab 5.0 ab

4.0 b

3.0

2.0 UHP Inactivation (log CFU/ml) UHP 1.0 c

0.0

L. innocua P. acidilactici Lb. plantarum Lb. leichmanii Lb. bulgaricus

Lb. acidophilus

Figure C.2. Decrease in count (UHP inactivation) of Listeria, Pediococcus, and

Lactobacillus strains caused by ultra-high pressure at 500 MPa and 23°C for 1 minute, when cells were suspended in citrate-phosphate buffer, pH 4.5. Cell suspensions were ~109 CFU/ml before treatment. The dashed and the dotted lines

indicate the corresponding inactivation of Listeria monocytogenes Scott A and V7, respectively. Different letter designations indicate strains with statistically different (P <

0.05) UHP inactivation levels, based on one-way ANOVA and Tukey mean comparison, n = 3.

252 Efficacy of UHP treatment on L. monocytogenes strains and potential surrogates in whey products.

Resistance to 500 MPa process for 1 minute in sweet whey (pH 6.0) did not significantly vary among Listeria spp. strains. Average inactivation by UHP was between 2.5 and 5.3 log CFU/ml for Listeria spp. (Figure C.3). P. acidilactici and Lb. plantarum were significantly more resistant to UHP in sweet whey than Listeria spp. with average inactivations of 0.0 and 0.3 log CFU/ml, respectively.

Inactivation of Listeria spp. strains did not vary significantly when processed in acidified sweet whey (pH 4.2) (Figure C.4). Average inactivation by UHP in acidified sweet whey was between 4.8 and 6.9 log CFU/ml for Listeria spp. Lb. plantarum was inactivated 3.2 log CFU/ml when treated with UHP in acidified sweet whey. This inactivation was statistically similar to the more resistant strains of L. monocytogenes. P. acidilactici was significantly more resistant to UHP in acidified sweet whey than all other strains tested with an average inactivation of 0.2 log CFU/ml.

Efficacy of PEF against surrogate organisms in 0.1% NaCl.

The PEF inactivation in 0.1% NaCl of the Listeria, Pediococcus, and

Lactobacillus strains ranged from 0.7 to 5.3 log CFU/ml after 144 µs-processing at 25 kV/cm (Figure C.5). These inactivation levels were compared to the range of inactivation

(0.7 – 3.7 log CFU/ml) of L. monocytogenes strains; inactivation of L. monocytogenes strains had been determined in a previous study, under similar processing conditions (21).

Lactobacillus plantarum ATCC 8014 and Pediococcus acidilactici PXL were the least sensitive non-pathogenic strains to the PEF processing, and had inactivation levels

253 7.0

6.0 a a 5.0 a a a a a a 4.0 a a

3.0

2.0 UHP InactivationUHP (log CFU/ml) 1.0 b b 0.0 a V7

ScottA Californi L. innocua OSY-8517 OSY-8576 OSY-8578 OSY-8579 OSY-8580 OSY-8732

P. acidilactici Lb. plantarum L. monocytogenes

Figure C.3. Decrease in count (UHP inactivation) of Listeria spp., Lactobacillus plantarum, and P. acidilactici processed for 1 minute with ultra-high pressure at 500

MPa in sweet whey (pH 6.0) at 23°C. Different letter designations indicate strains with statistically different (P < 0.05) UHP inactivation levels, based on one-way ANOVA and

Tukey mean comparison, n = 3.

254 a 7.0 a a ab ab ab ab ab 6.0 ab

ab 5.0

4.0 b

3.0

2.0 UHP Inactivation (log CFU/ml) UHP Inactivation (log 1.0 c

0.0 a V7

ScottA Californi L. innocua OSY-8732 OSY-8517 OSY-8576 OSY-8578 OSY-8579 OSY-8580

P. acidilactici Lb. plantarum

L. monocytogenes

Figure C.4. Decrease in count (UHP inactivation) of Listeria spp., Lactobacillus plantarum, and P. acidilactici processed for 1 minute with ultra-high pressure at 500

MPa in acidified sweet whey (pH 4.2) at 23°C. Different letter designations indicate strains with statistically different (P < 0.05) UHP inactivation levels, based on one-way

ANOVA and Tukey mean comparison, n = 3.

255 7.0

6.0 a

5.0 ab

bc 4.0 b

3.0

d 2.0 de PEF InactivationPEF (log CFU/ml) 1.0 e e

0.0

L. innocua Lb. plantarum Lb. leichmanii Lb. bulgaricus P. acidilactici

Lb. acidophilus

Figure C.5. Decrease in count (PEF inactivation) of Listeria, Pediococcus, and

Lactobacillus strains caused by pulsed electric field treatment at 25 kV/cm and 23°C for 144 µs, when cells were suspended in 0.1% NaCl. Cell suspensions were adjusted to ~108 CFU/ml before treatment. The dashed and the dotted lines indicate the corresponding inactivation of Listeria monocytogenes Scott A and OSY-8578, respectively, which were reported in an earlier study (21). Different letter designations

indicate strains with statistically different (P < 0.05) PEF inactivation levels, based on

one-way ANOVA and Tukey mean comparison, n = 2.

256 similar (P > 0.05) to the process-resistant L. monocytogenes OSY-8578. Inactivation of

L. innocua ATCC 33090 was not significantly different to that of P. acidilactici (P >

0.05), but was significantly intermediate between that of L. monocytogenes OSY-8578 and L. monocytogenes Scott A (P < 0.05). Lb. leichmanii ATCC 4797, Lb. bulgaricus

OSY 135 and Lb. acidophilus ATCC 19992 had mean PEF-inactivation levels higher than 3.0 log CFU/ml; these strains were either equally or more sensitive to PEF than L. monocytogenes Scott A, and more sensitive (P < 0.05) than all other tested strains.

Efficacy of PEF treatment on L. monocytogenes strains and potential surrogates in whey products.

Resistance to 25 kV/cm-PEF process for 48 µs in 50% acid whey (pH 4.2) varied greatly (P < 0.05) among nine L. monocytogenes strains, L. innocua ATCC 33090 and

Lb. plantarum ATCC 8014 (Figure C.6). Listeria monocytogenes populations decreased

1.8 to 3.6 log CFU/ml. No statistical difference in PEF-inactivation (P > 0.05) was observed among L. innocua and the most resistant L. monocytogenes strains (OSY-8579,

OSY-8578, V7, California, and OSY-8580). Lactobacillus plantarum was significantly

(P < 0.05) the most resistant to this PEF process, with a corresponding inactivation of 0.4 log CFU/ml. Inactivation of the nine L. monocytogenes strains was less than 1 log

CFU/ml, when processed under similar conditions in 66% sweet whey (pH 6.8; data not shown).

DISCUSSION

Inactivation of L. monocytogenes and potential surrogates by UHP.

The resistance of bacteria to inactivation by UHP was highly dependent on the suspension medium. Bacteria tested were most resistant to UHP when suspended in

257 7.0

6.0

5.0

4.0 a a ab ab 3.0 ab bc bc bc 2.0 c c

1.0 d

0.0 a

V7

ScottA Californi L. innocua OSY-8732 OSY-8517 OSY-8576 OSY-8578 OSY-8579 OSY-8580

Lb. plantarum L. monocytogenes

Figure C.6. Decrease in count (PEF inactivation) of Listeria spp. and Lactobacillus plantarum processed for 48 µs with pulsed electric field at 25 kV/cm in 50% acid whey (pH 4.2) at 23°C. The acid whey was diluted with deionized water to standardize conductivity to ~0.46 S/m. Different letter designations indicate strains with statistically different (P < 0.05) PEF inactivation levels, based on one-way ANOVA and Tukey mean comparison, n = 2.

258 citrate-phosphate buffer, pH 7.0 compared to other media tested (P <0.05). Average

inactivations ranged from 0.0 to 2.0 log CFU/ml for all strains tested. Previous studies

have shown various bacteria to be very resistant to UHP when treated in this buffer

system (34). Inactivation was significantly affected by changes in pH of suspension

medium. This was true for both the citrate-phosphate buffer systems and the whey

systems, with the lower pH significantly contributing to increased inactivation. Previous

studies have found pH to be a significant contributing factor to inactivation by UHP

treatment (34). Tay et al (32) determined the barotolerance of these same L.

monocytogenes strains in spent broth when treated with UHP at 500 MPa for 1 minute.

Most strains were completely inactivated by UHP treatment at 500 MPa in spent broth,

indicating the sensitivity of L. monocytogenes is affected by suspension medium.

Identification of a target strain of L. monocytogenes for inactivation studies was

determined by UHP treatment in various suspension media. The present study found L.

monocytogenes V7 to be consistently among the most resistant strains when processed in

citrate-phosphate buffer systems and L. monocytogenes ScottA to be consistently among

the most sensitive strains. L. monocytogenes OSY-8578 was previously identified as the most resistant strain to UHP when processed in spent broth; however L. monocytogenes

V7 was also one of the more resistant strains in the referenced study (32). L. monocytogenes ScottA was the most sensitive strain to UHP when treated in spent broth

(32). There were no significant differences among L. monocytogenes strains UHP treated in whey products. Therefore, targeting inactivation of L. monocytogenes V7 or OSY-

8578 would be suitable for process design and validation.

259 Lb. plantarum and P. acidilactici were consistently the most resistant strains to

UHP in various suspension media among the potential surrogates. Lb. plantarum was

comparable in resistance to UHP to the more resistant L. monocytogenes strains and

could serve as a useful surrogate for estimation of inactivation for a given UHP process, i.e., for every 1 log of inactivation of Lb. plantarum ATCC 8014 would be equal to or greater than 1 log of inactivation of L. monocytogenes OSY-V7. P. acidilactici was extremely resistant to UHP treatments under all conditions tested. Further studies need to be completed to determine efficacy of various UHP treatments on inactivation of P. acidilactici as none of the treatments in this study significantly inactivated 1 log CFU/ml of this strain. Sohn and Lee (31) found P. cerevisiae to be more resistant to UHP treatment in distilled water compared to other lactic acid bacteria. Panagou et al (25) identified and characterized a pressure-resistant strain of P. damnosus. This P. damnosus strain was inactivated less than 1 log CFU/ml by 500 MPa with a 2 minute holding time in gilt-head seabream. To date, P. acidilactici PXL is the most resistant to UHP processing that has been tested in our laboratory based on these data and other ongoing studies. Further studies are planned to characterize the pressure-resistance of this strain.

It should be noted that comparison of strains for UHP-resistance was performed with a single pressure condition of 500 MPa for 1 minute at room temperature. Relative sensitivity to UHP may vary among strains under different treatment conditions or in a different suspension medium (34).

Inactivation of L. monocytogenes and potential surrogates by PEF.

The screening for L. monocytogenes OSY-8578 surrogate showed important variations in PEF-sensitivity among and between genera, but causes were unclear. Cell

260 size was first hypothesized the leading factor of variations in inactivation (4, 37).

Lactobacillus spp. are frequently long rods with 0.5-1.2 µm diameter and 1.5-9.0 µm

length, compared to the size of Listeria sp. (0.4-0.5 x 0.5-2.0 µm) and Pediococcus sp.

(0.5-1.0 µm diameter) (2, 3). The identification of P. acidilactici PXL among potential

surrogates of L. monocytogenes OSY-8578 for PEF-process validation was therefore

expected. Similarly, three of the four tested lactobacilli were particularly sensitive to the

PEF process. Increased sensitivity to PEF as cell size increases, however, would not

explain why Lb. plantarum was the most resistant of all tested microorganisms despite its

large cell size (0.9-1.2 x 3.0-8.0 µm). Wouters et al (37) observed that the contribution of

cell size and shape to PEF sensitivity decreased as processing time increased. Therefore,

a more likely hypothesis was that differences in cellular structure and membrane

composition accounted for most of the strains variability.

Lb. plantarum ATCC 8014 was identified as potential surrogate to L.

monocytogenes OSY-8578 for short-time PEF-processing in 0.1% NaCl or 50% acid

whey. This surrogate was screened to validate inactivation rates during PEF processing

(data not shown). Lactobacilli can grow at pH as low as 4.2, which may have favored

survival of Lb. plantarum in PEF-treated acid whey. The increasing inactivation of Lb.

plantarum in 0.1% NaCl compared to that of the target L. monocytogenes strain, as PEF

processing time increased, showed the importance of confirming single-point

experiments with survivor plots; a more resistant surrogate microorganism would be needed, for instance, to ascertain eradication of a large L. monocytogenes population in

saline solution.

261 Inactivation levels of each strain and species varied with medium composition.

High conductivity, high pH, calcium ions, and low water activity enhanced bacterial

survival to PEF (7, 36, 38). Hypotonic media, such as 0.1% NaCl favored the bursting of

leaky bacterial cells (28, 33); such media was necessary to facilitate the identification of a

PEF-resistant surrogate, as the lab-scale PEF-equipment used in this study was limited to

mild electric field settings (≤ 30 kV/cm). Acid and sweet whey had to be diluted to fit the

equipment requirements and prevent arcins at 25 kV/cm. Dilutions reduced and

standardized the conductivity of processing media. These dilutions would be unnecessary

in larger-scale equipment, where higher electric field and flow rates can be achieved (23).

Listeria spp. were extremely resistant to PEF processing in sweet whey with less than 1

log CFU/ml inactivation under tested conditions. Gallo et al (14) found L. innocua to be

similarly resistant (~1 log CFU/ml inactivation) when treated with PEF (12 kV/cm, 3 µF,

60 pulses) in whey. Listeria spp. were killed faster in acid whey than in 0.1% NaCl (23),

which could be due to the low pH of acid whey (4.2) compared to that of the saline

solution (~ 7.0). Similarly, Geveke and Kozempel (16) observed an enhanced inactivation

of L. innocua at pH 3.8. Synergistic bactericidal effect between PEF and low pH (sorbic

and benzoic acids) was also reported at pH 3.4, but not at pH 6.4 (22). The contribution of conductivity to PEF inactivation appeared minor in the comparison of the two media, as the conductivity of 50% acid whey was approximately twice that of 0.1% NaCl.

Surrogate selection of L. monocytogenes for UHP- or PEF-process validation.

Listeria innocua has been frequently used as non-pathogenic surrogate of L. monocytogenes, because the two species are phylogenetically very closely related (6, 9,

38). This study demonstrated that, for UHP and PEF studies, L. innocua should be used

262 as surrogate cautiously. In UHP studies, L. innocua inactivation was comparable to L. monocytogenes strains, but L. innocua was never significantly more resistant to UHP treatments compared to L. monocytogenes strains. Listeria innocua was a potential surrogate to L. monocytogenes OSY-8578 for PEF-processing of acid whey, but not when processed in 0.1% NaCl. Processing conditions based on L. innocua inactivation by UHP or PEF could eliminate some but not all L. monocytogenes contaminants; this underestimation of the pathogen’s range of processing-resistance could result in sporadic isolation of L. monocytogenes in processed products.

Overall, Lb. plantarum ATCC 8014 was selected as the most suitable potential surrogate because it was the less affected by nature of the suspension medium and consistent inactivation equal to or greater than the most resistant target strains for each process. This study also showed the potential of UHP and PEF as substitutes for heat pasteurization for acid whey processing. The substantial resistance of Lb. plantarum to

UHP- and PEF-processing under acidic conditions supported its suitability as surrogate of

L. monocytogenes V7 or OSY-8578 for UHP- or PEF-process validation, especially in the case of acidic food products.

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267

APPENDIX D

VARIABILITY IN BAROTOLERANCE OF Lactobacillus plantarum AND

Lactobacillus fermentum STRAINS: IMPACT OF PRESSURE DOSE, HOLDING

TIME AND SUSPENSION MEDIUM

268

Variability in barotolerance of Lactobacillus plantarum AND Lactobacillus

fermentum strains: impact of pressure dose, holding time and suspension medium

Joy G. Waite and Ahmed E. Yousef

ABSTRACT

Previous work has identified Lactobacillus plantarum as a potential surrogate organism for Listeria monocytogenes in products treated with UHP. Studies on L. plantarum inactivation by UHP have been completed; however strain variability in barotolerance has not been previously determined. The objective of this study was to identify pressure-sensitive and pressure-resistant strains of L. plantarum and

Lactobacillus fermentum and analyze their inactivation behavior due to UHP treatment under a variety of circumstances to identify potential surrogate organisms for L. monocytogenes. Sixteen strains of L. plantarum and six strains of L. fermentum were screened for relative pressure-resistance at 500 and 600 MPa with a holding time of one minute. Four strains were chosen for further dose-response and kinetic analyses: pressure-resistant L. plantarum MDOS-32 and L. fermentum NKV-NF85, and pressure- sensitive L. plantarum OSY-104 and L. fermentum NKV-NB4. Relative sensitivity of L. plantarum strains as determined from screening methods was predicative of relative inactivation under additional UHP treatments. L. fermentum strains were often similar in their response to additional UHP treatments, likely due to their increased pressure- resistance compared to L. plantarum strains. Suspension medium was found to

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significantly effect inactivation of these strains, with higher inactivation by UHP when

suspended in peptone water compared to buffer systems. Also, pH contributed

significantly to inactivation by UHP (300 and 500 MPa) in buffer systems for all strains

tested. L. fermentum NKV-NF85 was determined to be an ideal target organism for

inactivation by UHP in low acid foods, while L. fermentum NKV-NB4 would be ideal for

UHP in high acid foods.

INTRODUCTION

Significant research on the efficacy of ultra-high pressure (UHP) in food

preservation has been completed in the recent years. Treatment of food products with

UHP compared to traditional thermal treatments results in products of “fresh-like” quality to the consumer while providing additional safety compared to fresh products (5). Several

key questions still need to be answered to ensure the safety and shelf stability of food

processed by UHP. Surrogate microorganisms are needed to evaluate the efficacy of UHP

treatments in food products at commercial scale operations. These surrogates have yet to

be identified for most barotolerant pathogens. It is no coincidence that microorganisms

proposed as suitable surrogates also cause spoilage of pressure-processed foods.

Therefore, successful recognition of these surrogates should be coupled with research to

control these problematic microorganisms.

Listeria monocytogenes is one of the most pressure-resistant pathogens known

and strains of this bacterium differ considerably in barotolerance (Tay et al 2003).

Species from the genus Lactobacillus may serve as an ideal surrogate for Listeria due to

their relatively close phylogenetic relationship. It is crucial to assess the efficacy of HPP

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against surrogate organisms so that processing conditions will be adequate to control food

safety concerns and minimize product spoilage. Preliminary studies in our lab have

determined L. plantarum ATCC 8014 to be a suitable surrogate for L. monocytogenes V7

(18). L. plantarum displayed comparable or higher resistance to UHP treatment than the

most barotolerant L. monocytogenes strain in the study (V7) in various suspension

systems. However, all UHP treatments were identical in dose and holding time. Further

investigation is needed to determine the impacts of dose and holding time on the

inactivation of additional strains of Lactobacillus spp. to identify additional surrogate

strains that may be useful for specific treatments. Few studies on UHP processing of L.

plantarum have been completed. Ulmer et al (2000) investigated the kinetics of

inactivation of L. plantarum TMW1.460 at various pressures (16). In another study by

this research group, Lb. plantarum strain TMW1.460 was treated with HPP in a model

beer system at the relatively low pressure of 300 MPa (3). This same group studied the

inactivation of HorA, a multidrug resistance transporter, by UHP at 200 MPa (17).

Wouters et al (1998) investigated the impact of HPP on Lb. plantarum LA10-11

combining pH effects and treatment at 250 MPa (19). Mallidis et al (2003) also investigated Lb. plantarum kinetics using a strain that was isolated from Greek salad (6).

However, published literature is void of studies that compare the barotolerance of strains of the same Lactobacillus species.

The main objective of this study was to identify barotolerant strains of L. plantarum and L. fermentum that could be used as potential surrogate organisms for L. monocytogenes. Therefore, numerous strains of Lactobacillus plantarum and

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Lactobacillus fermentum were obtained from various sources and screened for resistance to UHP. Additionally, strains differing significantly in barotolerance were analyzed for their resistance to UHP with different levels of pressure dose and holding time. The impact of suspension medium and pH was also investigated.

MATERIALS AND METHODS

Bacterial strains.

Sixteen strains of L. plantarum and five strains of L. fermentum were obtained from a variety of sources (Table D.1). Strains were stored in MRS broth (Difco, Becton

Dickinson, Sparks, Md.) containing 40% (vol/vol) glycerol at -80ºC. Each strain was transferred from frozen stock culture to MRS agar and incubated at 30°C for 48-72 hours.

Three isolated colonies of each strain were transferred to MRS broth and incubated overnight at 30°C for experimental use.

Species identification - Sequencing the spacer region between 16S rRNA – 23S rRNA genes.

Species confirmation for all strains was determined by sequencing the spacer region between the 16S rRNA and 23S rRNA genes using a method modified from

Tannock et al (1999) (14). The gel extracted PCR product was sequenced by The Ohio

State University Plant and Microbe Sequencing Facility. Sequence data was compared to published sequences in the NCBI database using the BLAST function

(www.ncbi.nlm.nih.gov). Species identification was considered to be confirmed if similarity was 97 percent or greater.

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Species Strain Source L. plantarum ATCC 8014 OSU Food Safety Laboratory ATCC10241 OSU Department of Microbiology OSU 140 OSU Department of Microbiology LB75 VanOpstal – Belgium (4) LA 10-11 Unilever – the Netherlands (19) TMW 1.460 Vogel – Germany hop resistant (16) MDOS-11 Daeschel – Oregon State University– pickle MDOS-12 Daeschel – Oregon State University – pickle MDOS-32 Daeschel – Oregon State University – NCFB strain MDOS-337 Daeschel – Oregon State University – negative polymer producer B2 Mallidis – NAGREF – Greece – Greek salad isolate (6) OSY-101 OSU Food Safety Lab stock OSY-102 OSU Food Safety Lab stock OSY-104 OSU Food Safety Lab stock OSY-106 OSU Food Safety Lab stock OSY-109 OSU Food Safety Lab stock L. fermentum OSY-103 OSU Food Safety Lab stock OSY-105 OSU Food Safety Lab stock OSY-107 OSU Food Safety Lab stock OSY-108 OSU Food Safety Lab stock NKV-NF85 OSU N. Vurma – NF85 – Swiss cheese NKV-NB4 OSU N. Vurma – NF85 – Swiss cheese

Table D.1. Lactobacillus plantarum and Lactobacillus fermentum strains obtained

from various sources.

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Screening for pressure resistance.

Overnight cultures were transferred to sterile centrifuge tubes and centrifuged at

10,000 rpm (12,400 x g) for 10 minutes using the Sorvall RC 5C Plus refrigerated

centrifuge equipped with the SS-34 rotor. The supernatant was removed and the pellet was suspended in phosphate-buffered saline (PBS) at pH 7.4. One mL aliquots were placed into sterile polyethylene bags and heat sealed minimizing the volume of air within the bag. Multiple bags to be treated at the same pressure were placed into a larger polyethylene bag containing bleach and vacuum sealed. Samples were then placed on ice for approximately 30 minutes prior to UHP treatment. All L. plantarum and L. fermentum strains were UHP treated at 500 MPa and 600 MPa with a holding time of 1 minute.

After pressurization samples were returned to ice and immediately decimally diluted in peptone water and plated on MRS agar. Plates were incubated at 30ºC for 72 hours prior to enumeration. Pressure resistance experiments were performed in triplicate.

Dose-response of Lactobacillus spp.

Based on findings from screening experiments, L. plantarum MDOS-32, L. plantarum OSY-104, L. fermentum NKV-NF85, and L. fermentum NKV-NB4 were used for dose-response studies. Samples were handled as described in the screening procedure. Strains were exposed to UHP at 350, 450, 550 and 650 MPa with a holding time of 0.1 second (come-up time), 1 minute, and 3 minutes. Experiments were performed in triplicate.

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Impact of holding time on inactivation of Lactobacillus spp.

Strains used for dose-response were also used for the kinetics experiments.

Samples were handled as described in the screening procedure. Strains were exposed to

UHP at 450 and 550 MPa with a holding time of 0.1 second (come-up time), 1 minute, 3 minutes, 5 minutes, 7 minutes, and 10 minutes. Experiments were performed in triplicate.

Suspension media.

Strains used for dose-response were also used for suspension media experiments.

Samples were treated as described in the screening procedure, except that pellets were

suspended in PBS (pH 7.4), 1% peptone water (pH 7.0), or citrate-phosphate buffer (CP)

(pH 7.0). Strains were exposed to UHP at 300 or 500 MPa with a holding time 3

minutes. Experiments were performed in quadruplicate.

Impact of pH.

Strains used for dose-response were also used for buffer composition

experiments. Samples were handled as described in the screening procedure, except that

pellets were suspended in CP with pH values of 3.0, 4.0, 5.0, 6.0 or 7.0. Buffer

composition is shown in Table D.2. Strains were exposed to UHP at 300 or 500 MPa

with a holding time 3 minutes. Experiments were performed in quadruplicate.

UHP equipment and conditions.

All pressure treatments were performed using a hydrostatic food processor

(Quintus QFP6, Flow Pressure Systems, Kent, Wash.) containing 1:1 (vol/vol)

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pH M Na2HPO4 M citric acid 3.0 0.020 0.040 4.0 0.039 0.031 5.0 0.051 0.024 6.0 0.064 0.018 7.0 0.087 0.007

Table D.2. Final molar concentration of citrate-phosphate buffer components at

various pH levels.

glycol/water pressure transmitting fluid (Houghto-Safe 620 TY, Houghton International

Inc., Valley Forge, Pa). The press consists of a jacketed vessel with end closures, has a

2-liter capacity, and is designed to operate at up to 900 MPa. Initial temperatures for the samples and pressure-transmitting fluid were between 2-6ºC to minimize the holding temperature during the pressure treatment.

Statistical analysis.

Data were analyzed by analysis of variance (ANOVA) by using the General

Linear Models Procedure of SAS (SAS Institute, Cary, NC). Comparisons between the mean log reductions of treatments were made using the Tukey’s Studentized Range test.

RESULTS AND DISCUSSION

Species identification.

All strains used in this study were confirmed to be either L. plantarum or L.

fermentum, as shown in Table D.1, based on the DNA sequence of the intergenic spacer

region between the 16S rRNA and 23S rRNA genes.

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Screening Lactobacillus spp. strains for pressure resistance.

Lactobacillus plantarum strains suspended in PBS (pH 7.4) were treated with

UHP at 500 MPa and 600 MPa with a 1-minute holding time. Inactivation of L.

plantarum strains using the 500 MPa treatment and the 600 MPa treatment are shown in

Figure D.1. and Figure D.2, respectively. L. plantarum strains varied significantly in their resistance to both treatments. Inactivation of L. plantarum strains treated with 500

MPa varied between 1.1 and 7.7 log reductions with strain MDOS-32 being the most resistant and OSY-104 being the most sensitive. Inactivation using 600 MPa varied between 3.6 and 8.8 log reduction with the same strains being the most resistant and sensitive at both pressures. Therefore, strain MDOS-32 will be referred to as the most barotolerance strain of L. plantarum. Strain OSY-104 will be referred to as the most barosensitive strain of L. plantarum. Further studies on UHP processing will be performed using these two strains. It is noteworthy that day to day variability was statistically significant for L. plantarum UHP treatments; this has been a common result for UHP processing experiments.

Lactobacillus fermentum strains were treated with UHP at 500 MPa and 600 MPa with a 1-minute holding time. Inactivation of L. fermentum strains was insignificant with a pressure treatment of 500 MPa with all strains exhibiting less than 1 log inactivation

(data not shown). Inactivation of L. fermentum strains with a treatment of 600 MPa for 1 minute is shown in Figure D.3. Inactivation of L. fermentum varied between 1.4 and 4.8 log reductions with the 600 MPa treatment. Strain NKV-NF85 was the most resistant L. fermentum strain and NKV-NB4 was the most sensitive. These strains will be used for

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10.0

9.0 a a 8.0 a,b a,b,c a,b 7.0

6.0 b,c,d b,c,d 5.0 b,c,d b,c,d b,c,d b,c,d b,c,d 4.0 d d

Log reduction (cfu/ml) 3.0 d d 2.0

1.0

0.0 B2 LB75 OSU 140 OSU OSY-101 OSY-102 OSY-104 OSY-106 OSY-109 LA 10-11 MDOS-11 MDOS-12 MDOS-32 MDOS-337 ATCC 8014 TMW 1.460 TMW ATCC 10241 ATCC L. plantarum strains

Figure D.1. Average log reduction of L. plantarum strains treated with UHP at 500

MPa with a 1-minute holding time. Initial cell counts were ~109 cfu/ml; n = 3; error

bars represent ± 1 standard error. Means with the same superscript are not different (p-

value <0.05).

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10.0 a,b a a,b a a a,b a 9.0

8.0

7.0 b,c 6.0

5.0 c 4.0

Log reduction (cfu/ml) reduction Log 3.0

2.0

1.0

0.0 B2 LB75 OSU 140 OSU OSY-109 OSY-101 OSY-102 OSY-104 OSY-106 LA 10-11 LA MDOS-11 MDOS-12 MDOS-32 MDOS-337 ATCC 8014 TMW 1.460 TMW ATCC 10241 ATCC L. plantarum strains

Figure D.2. Average log reduction of L. plantarum strains treated with UHP at 600

MPa with a 1-minute holding time. Initial cell counts were ~109 cfu/ml; n = 3; error

bars represent ± 1 standard error. Means with the same superscript are not different (p-

value <0.05).

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10.0

9.0

8.0 * 7.0

6.0

5.0

4.0 Log reduction (cfu/ml) reduction Log 3.0

2.0

1.0

0.0 OSY-103 OSY-105 OSY-107 OSY-108 NKV-NF85 NKV-NB4 L. fermentum strains

Figure D.3. Average log reduction of L. fermentum strains treated with UHP at 600

MPa with a 1-minute holding time. Initial cell counts were ~109 cfu/ml; n = 3; error

bars represent ± 1 standard error. * indicates significant differences in inactivation by

UHP (p-value < 0.05)..

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further UHP analysis along with at least one intermediate strain, likely OSY-105 and

NKV-B72. L. fermentum strains were more uniform in their pressure resistance than L. plantarum strains. Previous studies have shown significant variability to UHP inactivation for various species and strains (1, 7-9, 12, 13, 15, 18). This is the first investigation into the strain variability of L. plantarum and L. fermentum strains.

Dose-response of Lactobacillus spp. to UHP

Four strains were chosen for further UHP analysis based on relative pressure- resistance: two strains of L. plantarum [a pressure-sensitive strain (OSY-104) and a pressure-resistant strain (MDOS-32)] and two strains of L. fermentum [a pressure- sensitive strain (NKV-NB4) and a pressure-resistant strain (NKV-NF85)]. Strains were analyzed for their dose-response by exposure to UHP at 350, 450, 550 and 650 MPa with a holding time of 0.1 second (come-up time), 1 minute, and 3 minutes.

Inactivation of the pressure-sensitive L. plantarum OSY-104 at various pressures with holding times of 0.1 second, 1 minute, and 3 minutes are shown in Figure D.4. As expected with UHP treatment, increases in pressure level and increases in holding time resulted in increased inactivation of this strain. Strain OSY-104 was inactivated during the come-up time at pressure equal or greater than 450 MPa. The come-up time for 450

MPa resulted in 1.5 log inactivation while the come-up time for 650 MPa resulted in 6.1 log inactivation. This strain is extremely pressure-sensitive with significant inactivations of 1.7 and 4.1 log resulting from pressure treatments of 350 MPa with a holding time of 1 and 3 minutes, respectively.

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10.0

9.0 8.0 7.0 6.0 Log Reduction 5.0 (cfu/ml) 4.0

3.0 650 2.0 550 1.0 0.0 450 Pressure (MPa)

0.1 350 1 Time (min) 3

Figure D.4. Dose response of L. plantarum OSY-104 to high pressure processing at various pressures with holding times of 0.1 second, 1 minute and 3 minutes. Initial cell counts were ~109 cfu/ml; n = 4.

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Inactivation of the pressure-resistant L. plantarum MDOS-32 at various pressures

with holding times of 0.1 second, 1 minute, and 3 minutes are shown in Figure D.5. L.

plantarum MDOS-32 was resistant to inactivation by the less extreme pressure treatments

in this study. No significant inactivation for this strain was accomplished by any of the

350 MPa or 450 MPa treatments, as well as the come-up time treatment for 550 MPa.

Treatments of L. plantarum MDOS-32 with 550 or 650 MPa were more effective with

increased holding times. Inactivation using 550 MPa ranged from 0.3 to 5.1 log

reduction with holding times of 0.1 second and 3 minutes, respectively. Similarly,

inactivation at 650 MPa ranged from 2.7 to 8.1 log reductions for 0.1 second and 3

minutes, respectively.

The L. fermentum strains NKV-NF85 and NKV-NB4 displayed intermediate

inactivation compared to both L. plantarum strains during dose experiments (data not

shown). The L. fermentum strains did not separate as predicted at all pressures (i.e., the

“pressure-resistant” strain NKV-NF85 was not always more resistant than the “pressure-

sensitive” NKV-NB4). At higher pressures and longer holding times, the “pressure-

resistant” strain NKV-NF85 was more resistant than the “pressure-sensitive” NKV-NB4.

However, the opposite was true at lower pressures and shorter holding times.

Lactobacillus plantarum OSY-104 displayed an increasing inactivation with

increasing pressure, indicating that come-up time is an important component of inactivation for this particular strain. Come-up time had little effect on Lb. plantarum

MDOS-32 except at the highest pressure tested (650 MPa). Both Lb. fermentum strains displayed similar results for all pressures, with a 1-2 log reduction during come-up time

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10.0 9.0 8.0 7.0 6.0 Log Reduction 5.0 (cfu/ml) 4.0 650 3.0 2.0 550 1.0 0.0 450 Pressure (MPa)

0.1 350 1 Time (min) 3

Figure D.5. Dose response of L. plantarum MDOS-32 to high pressure processing at various pressures with holding times of 0.1 second, 1 minute and 3 minutes. Initial cell counts were ~109 cfu/ml; n = 4.

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regardless of pressure. With 1 minute or 3 minutes of holding time, all strains displayed

the same pattern of increasing inactivation with increasing pressure.

Inactivation due to pressure is a function of treatment time, although the relationship between the reduction and time is not linear. Inactivation curves do not follow first-order kinetics but tend to be exponential, with a rapid initial decrease in cell numbers during the first 15 min of treatment followed by a ‘tail’. Ritz et al (2000) indicate that treatment time is a significant variable, however gain in efficiency between

3 and 10 min was not significant(10, 15). For treatments at 200 and 600 MPa, the holding time had no significance on inactivation of L. monocytogenes (3 to 20 min). The greatest interaction between pressure and holding time was seen at 300 MPa where there was a gain of efficiency of 4 logs kill between 3 min and 20 min (10).

Kinetics of Lactobacillus spp. with UHP treatment.

Four strains were chosen for further UHP inactivation studies: two strains of Lb. plantarum [a pressure-sensitive strain (OSY-104) and a pressure-resistant strain (MDOS-

32)] and two strains of Lb. fermentum [a pressure-sensitive strain (NKV-NB4) and a pressure-resistant strain (NKV-NF85)]. Kinetics of these strains were analyzed by exposure to UHP at 450 and 550 MPa with a holding time of 0.1 second (come-up time),

1 minute, and 3 minutes, 5 minutes 7 minutes, and 10 minutes.

Kinetics of the four strains at 450 MPa is shown in Figure D.6. At 450 MPa, L. plantarum MDOS-32 was the most resistant stain throughout all time points. L. plantarum OSY-104 was the most sensitive strain throughout all time points. The L.

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Time (sec) -200 -100 0 100 200 300 400 500 600 700 0.0 b c -1.0 c c a a c b a -2.0 b b b -3.0 b b c b -4.0 b b,c -5.0 a

Log Ratio Log b -6.0

-7.0 a

-8.0 L. plantarum MDOS-32 a a L. plantarum OSY-104 a -9.0 L. fermentum NKV-NF85 L. fermentum NKV-NB4 -10.0

Figure D.6. Kinetics of four strains of Lactobacillus spp.: L. plantarum OSY-104

(■), L. plantarum MDOS-32 (x), L. fermentum NKV-NF85 (▲), L. fermentum NKV-

NB4 (●) to high pressure processing at 450 MPa with holding times of 0.1 second

(come-up time), 1 minute, 3 minutes, 5 minutes, 7 minutes, and 10 minutes. Initial cell counts were ~109 cfu/ml; n = 4. Means with the same superscript are not different

for strains compared at each time point (p-value < 0.05).

286

fermentum strains switched in their relative sensitivity depending on time of treatment,

however the only significant differences between these two strains existed at time points

1 minute and 3 minutes.

Kinetics of the four strains at 550 MPa is shown in Figure D.7. At 550 MPa, L.

plantarum OSY-104 was again the most sensitive of the strains tested at all time points.

The remaining three strains grouped together and were not significantly different at any

time points, with the exception of L. fermentum NKV-NF85 relative sensitivity during the

come-up time.

Inactivation by UHP is initiated during the pressure come-up time, i.e., the time needed to raise the pressure to the targeted level. The pressure come-up time depends on

the equipment and the headspace in the package (5). Although high-hydrostatic pressure

treatments are considered to be isostatic, Ritz et al (2001) showed that cellular damage is

not equally withstood by all the cells, suggesting that more resistant or less damaged cells

are present in the pressurized cell population (11).

Inactivation by high pressure does not always follow simple first-order kinetics.

In nonlinear survivor curves, cell inactivation is thought to be the result of multiple

events or cumulative damage to the cell. This is consistent with the generally accepted

belief that high pressure affects a combination of microbial processes and does not inhibit

or destroy just one specific cell site or function (12, 15). Most of the authors propose a

first-order or non-linear inactivation kinetics under a set of limited data points, usually less than 20 data points (2, 15).

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Time (sec) -200 -100 0 100 200 300 400 500 600 700 0.0 c -1.0 c

b -2.0 b

b -3.0 b a -4.0 b -5.0 b b Log Ratio Log -6.0 b b a,b b -7.0 a,b a,b a a a a -8.0 L. plantarum MDOS-32 a L. plantarum OSY-104 -9.0 L. fermentum NKV-NF85 a a a L. fermentum NKV-NB4 -10.0

Figure D.7. Kinetics of four strains of Lactobacillus spp.: L. plantarum OSY-104

(■), L. plantarum MDOS-32 (x), L. fermentum NKV-NF85 (▲), L. fermentum NKV-

NB4 (●) to high pressure processing at 550 MPa with holding times of 0.1 second

(come-up time), 1 minute, 3 minutes, 5 minutes, 7 minutes, and 10 minutes. Initial cell counts were ~109 cfu/ml; n = 4. Means with the same superscript are not different

for strains compared at each time point (p-value).

288

Chen and Hoover (2003) attempted to model the survival curves of Yersinia enterlcolitica exposed to high pressure. Inspection of all of the inactivation curves indicated a curvature and tailing. As pressure magnitudes increased, the degree of destruction also increased. The shapes of the survival curves at 300, 350, and 400 MPa were very similar in buffer. However, the survival curve at 450 MPa in buffer was very different from other curves, with a rapid drop at the beginning and a long tail (2).

Impact of suspension medium on inactivation of Lactobacillus spp. by UHP.

Suspension media (PBS, 1% peptone, and CP) were analyzed for their contribution to UHP inactivation of barotolerant and barosensitive strains of L. plantarum and L. fermentum. Strains were analyzed by exposure to UHP at 300 and 500 MPa with a holding time of 3 minutes. With UHP treatments at 300 MPa, there was no significant impact of suspension media on inactivation of any of the four strains tested (data not shown).

Impact of suspension media on inactivation of L. plantarum and L. fermentum strains by UHP at 500 MPa is shown in Figure D.8. At 500 MPa, L. plantarum MDOS-

32, L. plantarum OSY-104, and L. fermentum NKV-NB4 were significantly mores sensitive to inactivation by UHP when suspended in 1% peptone water than when suspended in either buffer tested. L. plantarum MDOS-32 showed the most drastic differences in sensitivity to UHP due to suspension medium at 500 MPa with inactivation in peptone water achieving 6.6 log reduction and CP achieving only 1.1 log reduction.

289

10.0 * Peptone water, 1%, pH 7.0 PBS, pH 7.4 9.0 Phos-Cit Buffer, pH 7.0 8.0 * 7.0

6.0

5.0 *

4.0 Log Reduction (cfu/ml) Log Reduction 3.0

2.0

1.0

0.0 L. plantarum MDOS-32 L. plantarum OSY-104 L. fermentum NKV-NF85 L. fermentum NKV-NB4 Strains

Figure D.8. Log reduction of four strains of Lactobacillus spp. resuspended in various suspension media to ultra-high pressure processing at 500 MPa with a holding time of 3 minutes. Initial cell counts were ~109 cfu/ml; n = 4; error bars

represent standard error. * indicates significantly higher inactivation due to suspension

medium compared to other media for the same strain (p-value < 0.05).

290

As expected, there were no significant differences in inactivation of the L. fermentum strains with treatments at 500 MPa. Also as expected, L. plantarum MDOS-32 was significantly more resistant in all media than L. plantarum OSY-104.

Impact of pH on inactivation of Lactobacillus spp. by UHP.

Buffer pH values (3.0, 4.0, 5.0, 6.0, and 7.0) were analyzed for their contribution to UHP inactivation of L. plantarum and L. fermentum strains. Strains were analyzed by exposure to UHP at 300 and 500 MPa with a holding time of 3 minutes.

Inactivation of L. plantarum and L. fermentum strains by pH and 300 MPa is shown in Figure D.9. Inactivation of all four strains was enhanced by decreases in pH.

Inactivation of L. plantarum MDOS-32 was significantly enhanced with UHP treatment in pH 3.0 (5.1 log reduction) and 4.0 (4.4 log reduction) buffer compared to pH 7.0 buffer

(0.2 log reduction). The pressure-sensitive L. plantarum OSY-104 exhibited enhanced inactivation with pH 3.0, 4.0, 5.0, and 6.0 compared to pH 7.0. L. fermentum strains were more resistant to changes in pH with 300 MPa treatment. Inactivation of L. fermentum

NKV-NF85 by UHP was significantly enhanced by pH 3.0 and 4.0 buffers compared to pH 7.0 buffer. Enhanced inactivation of L. fermentum NKV-NB4 was only achieved with suspension in the pH 3.0 buffer.

Inactivation of L. plantarum and L. fermentum strains by pH and 500 MPa is shown in Figure D.10. Again, inactivation of all four strains was enhanced by decreases in pH. L. plantarum MDOS-32 inactivation was significantly enhanced by pH 3.0, 4.0,

5.0, and 6.0 compared to pH 7.0. L. plantarum OSY-104 was very sensitive to UHP

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10.0 pH 7.0 pH 6.0 9.0 pH 5.0 8.0 pH 4.0 pH 3.0 * 7.0

* 6.0 * * 5.0 * * 4.0 *

Log Reduction (cfu/ml) Log Reduction 3.0 * * 2.0

1.0

0.0 L. plantarum MDOS-32 L. plantarum OSY-104 L. fermentum NKV-NF85 L. fermentum NKV-NB4 Strains

Figure D.9. Log reduction of four strains of Lactobacillus spp. resuspended in citrate-phosphate buffer with various pH values to high pressure processing at 300

MPa with a holding time of 3 minutes. Initial cell counts were ~109 cfu/ml; n = 4; error

bars represent ± 1 standard error. * indicates UHP treatment in a given pH was

significantly more effective than UHP treatment at pH 7.0 for a given strain (p-value <

0.05).

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pH 7.0 10.0 * pH 6.0 * 9.0 * pH 5.0 pH 4.0 * 8.0 * pH 3.0 * * 7.0

* * 6.0 * 5.0

4.0 Log Reduction (cfu/ml) Reduction Log 3.0

2.0

1.0

0.0 L. plantarum MDOS-32 L. plantarum OSY-104 L. fermentum NKV-NF85 L. fermentum NKV-NB4 Strains

Figure D.10. Log reduction of four strains of Lactobacillus spp. resuspended in citrate-phosphate buffer with various pH values to high pressure processing at 500

MPa (B) with a holding time of 3 minutes. Initial cell counts were ~109 cfu/ml; n = 4;

error bars represent ± 1 standard error. * indicates UHP treatment in a given pH was

significantly more effective than UHP treatment at pH 7.0 for a given strain (p-value <

0.05).

293

treatment at 500 MPa (7.9 log reduction). Significant enhancement was only achieved

with treatment at pH 3.0 (9.4 log reduction). L. fermentum NKV-NF85 inactivation was

enhanced by treatment in pH 3.0 and 4.0 buffers. L. fermentum NKV-NB4 was more

sensitive to UHP in buffer systems at or below pH 5.0. Overall, L. fermentum NKV-NB4

was the most resistant to the most extreme pH-UHP combinations.

With UHP treatment at 300 MPa and a holding time of 3 minutes, there were

overall differences in the buffers tested. The most inhibitory buffers had pH values of 3.0

and 4.0 which were produced significantly more reduction than buffers with pH values of

5.0 and 6.0. CP buffer at pH 7.0 was significantly more protective than pH 5.0, but not

pH 6.0. Similar results were found with pressure treatment at 500 MPa. The most

inhibitory buffers had pH values of 3.0, 4.0 and 5.0. These were significantly more

inhibitory than CP pH 6.0, which was significantly more inhibitory than CP pH 7.0. L.

plantarum OSY-104 was significantly more sensitive to UHP at all treatments than the

other three strains tested. It should be noted that L. plantarum MDOS-32 displayed an

additional 1.5 log reduction due to suspension in CP pH 3.0. There was no reduction

detected for any of the other buffers or strains tested. In general, these strains showed an

increase in inactivation with a decrease in buffer pH. L. fermentum strains appear to be

more resistant to the acid-HPP combination than L. plantarum strains.

Ritz et al (2000) completed a fractional study to compare four treatment variables:

pressure, time, temperature and pH and there impact on inactivation of L. monocytogenes.

All four variables were shown to be statistically significant and should be considered when designing an experiment. Pressure (1) and pH (2) of the suspending medium were

294

the most significant variables related to L. monocytogenes inactivation. Temperature (3)

and treatment time (4) were also significant. Pressure was more efficient at inactivating

L. monocytogenes at pH 5.6 compared to pH 7, except at 200 MPa and 600 MPa. At 400

MPa, there was a difference in of up to5.5 log (2.34 was mean) of inactivation of L. monocytogenes between pH 7 (phosphate buffer) and pH 5.6 (citrate buffer) (10).

This study identified pressure-resistant and pressure-sensitive strains of L. plantarum and L. fermentum that will be useful in developing surrogate microorganisms for pathogens such as L. monocytogenes. Relative pressure-resistance of L. plantarum strains as determined by screening procedure was predicative of the resistant and sensitive strain inactivation by UHP under different conditions. L. fermentum strains were more uniform in their pressure-resistance and relative barotolerance as determined by screening was often not predicative of their inactivation in other systems. L. fermentum NKV-NF85 would serve as an ideal target organism for inactivation in low acid food products, while L. fermentum NKV-NB4 would serve as an ideal target for

UHP inactivation in high acid food products.

ACKNOWLEDGEMENTS

This project was funded by the United States Department of Agriculture. The authors thank Erin Horton and Shara Johnson for their assistance with benchwork on this project.

The authors would also like to thank the following individuals for providing

Lactobacillus spp. strains for this study: Dr. Mark Daeschel (Oregon State University),

Bill Swoager and Nurdan Kocaoglu-Vurma (The Ohio State University), Dr. Isabelle Van

295

Opstal (Katholieke Universiteit Leuven, Belguim) , Dr. C. Mallidis (NAGREF, Greece),

Dr. Patrick Wouters (Unilever, The Netherlands), and Dr. Rudi Vogel (Technische

Universitat Munchen, Germany).

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