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Kinetic Behavior of a Pectinase Cocktail at High Hydrostatic Pressure Conditions

Permanent Link: http://ufdc.ufl.edu/UFE0043783/00001

Material Information

Title: Kinetic Behavior of a Pectinase Cocktail at High Hydrostatic Pressure Conditions
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Tomlin, Brittany D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: juice -- pectinase -- pressure
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, M.E.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Apple juice is one of the most popular fruit juices consumed in the United States. Pectinase cocktails, containing pectinases, hemicellulases, and cellulases are used in the production of commercial apple juice to reduce juice viscosity, increase yield, and to clarify the final product (Jayani and others 2005). High hydrostatic pressure (HHP) at moderate levels has stabilized and activated some enzymes. This project focused on testing a high hydrostatic pressure process to stabilize pectinase enzymes at raised temperatures above levels customarily used for clarification for possible economic benefits to traditional thermal processing. In the first set of experiments, the kinetics of inactivation of a commercial pectinase formulation was studied at 0.1 to 400 MPa and 55-85 °C. High pressure slowed the rate of inactivation of the pectinase cocktail treated at inactivating temperature conditions. Samples treated at temperatures of 77 °C or 85 °C and high pressures (200 - 400 MPa) retained activity for at least 8 times longer than samples treated at atmospheric pressure. In the second set of experiments, the stabilization of a commercial pectinase formulation at high hydrostatic pressures (200 and 300 MPa) at moderate temperatures (42.4 to 62.4 °C) was studied. The rate of viscosity reduction increased with temperature with a maximum of 0.0960 Pa-1.s-2 occurring at 62.4 °C and 300 MPa. Negative apparent activation volumes of -0.22 to -5.21 cm3.mol-1 demonstrated that pressure favored the increase in the rate of viscosity reduction with pressure having the greatest effect at 57.1 °C. Apparent activation energies suggest that temperature had greater effects at high pressure (200-300 MPa) than at atmospheric pressure. In the third set of experiments, the effects of high hydrostatic pressure on the shift in pH of pH indicator solutions were explored. Absorbance spectra for color acid/base indicators (pH 1.2 to 6.8) in the visible light range were used to quantify the change in pH induced by high pressures up to 600 MPa. Increases in pressure produced an apparent acidic pH shift in acid/base color indicators.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brittany D Tomlin.
Thesis: Thesis (M.E.)--University of Florida, 2011.
Local: Adviser: Correll, Melanie J.
Local: Co-adviser: Reyes De Corcuera, Jose Ignacio.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043783:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043783/00001

Material Information

Title: Kinetic Behavior of a Pectinase Cocktail at High Hydrostatic Pressure Conditions
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Tomlin, Brittany D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: juice -- pectinase -- pressure
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, M.E.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Apple juice is one of the most popular fruit juices consumed in the United States. Pectinase cocktails, containing pectinases, hemicellulases, and cellulases are used in the production of commercial apple juice to reduce juice viscosity, increase yield, and to clarify the final product (Jayani and others 2005). High hydrostatic pressure (HHP) at moderate levels has stabilized and activated some enzymes. This project focused on testing a high hydrostatic pressure process to stabilize pectinase enzymes at raised temperatures above levels customarily used for clarification for possible economic benefits to traditional thermal processing. In the first set of experiments, the kinetics of inactivation of a commercial pectinase formulation was studied at 0.1 to 400 MPa and 55-85 °C. High pressure slowed the rate of inactivation of the pectinase cocktail treated at inactivating temperature conditions. Samples treated at temperatures of 77 °C or 85 °C and high pressures (200 - 400 MPa) retained activity for at least 8 times longer than samples treated at atmospheric pressure. In the second set of experiments, the stabilization of a commercial pectinase formulation at high hydrostatic pressures (200 and 300 MPa) at moderate temperatures (42.4 to 62.4 °C) was studied. The rate of viscosity reduction increased with temperature with a maximum of 0.0960 Pa-1.s-2 occurring at 62.4 °C and 300 MPa. Negative apparent activation volumes of -0.22 to -5.21 cm3.mol-1 demonstrated that pressure favored the increase in the rate of viscosity reduction with pressure having the greatest effect at 57.1 °C. Apparent activation energies suggest that temperature had greater effects at high pressure (200-300 MPa) than at atmospheric pressure. In the third set of experiments, the effects of high hydrostatic pressure on the shift in pH of pH indicator solutions were explored. Absorbance spectra for color acid/base indicators (pH 1.2 to 6.8) in the visible light range were used to quantify the change in pH induced by high pressures up to 600 MPa. Increases in pressure produced an apparent acidic pH shift in acid/base color indicators.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brittany D Tomlin.
Thesis: Thesis (M.E.)--University of Florida, 2011.
Local: Adviser: Correll, Melanie J.
Local: Co-adviser: Reyes De Corcuera, Jose Ignacio.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043783:00001


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1 KINETIC BEHAVIOR OF A PECTINASE COCKTAIL AT HIGH HYDROSTATIC PRESSURE CONDITIONS By BRITTANY DANIELLE TOMLIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQU IREMENTS FOR THE DEGREE OF MASTER OF ENGINEERING UNIVERSITY OF FLORIDA 20 11

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2 2011 Brittany Tomlin

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3 To my family

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4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Reyes De Corcuera for his support, knowledge and guidance throughout my project. Dr. Reyes has a great love for his work with high pressure processing and in so encourages his students to be thorough, honest researchers while producing quality results. For providing funding for my research and conferences, I would like to th ank Dr. Reyes, the Agricultural and Biological Engineering departmen t, and the USDA NIFA AFRI grant. My committee members Dr. Teixeira, Dr. Correll, and Dr. Danyluk also provided a gre at deal of support throughout my research Thanks to my whole committee for reading countless papers and poster drafts. Thanks to Dr. Correll and Dr. Danyluk for being generous enough to provide laboratory space for different portions of my research. Thanks to Dr. Teixeira for instilling an excitement for food processing. I would also like to thank the laboratory members for their support. To Shelley and Veronica, the lab mangers, for being able to fix and handle almost any problem that occurred in the lab. To Juan Manuel, Rosie, Elyse, Sabrina, and Juan Fernando for the supp ort of peers during research. Finally I want to thank my family and friends who cheered me on during this final venture through school. My parents followed every step of my progress offering all their help and guidance. My younger brother provided an ear to listen and some comic relief along this rigorous process. My best friend, Jeslyn, who always believed in me and provides inspiration to live each day passionately. I could not have made it through this degree program without everyone mentioned and the blessing.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 BACKGROUND INFORMATION AND LITERATURE REVIEW ............................. 14 Apple Juice Production ................................ ................................ ........................... 14 Apple Juice Production Overview ................................ ................................ ..... 14 Processing ................................ ................................ ................................ ........ 15 Haze molecules ................................ ................................ ......................... 18 Clarification ................................ ................................ ................................ 20 Clarification enzymes ................................ ................................ ................. 21 Clarification processing conditions ................................ ............................. 26 Pasteurization ................................ ................................ ............................ 27 Microbes pertinent to apple juice ................................ ............................... 27 Pasteurization standards ................................ ................................ ........... 29 Juice nutr ients ................................ ................................ ............................ 30 Current Innovations ................................ ................................ .......................... 31 Innovation in Clarification enzymes ................................ ............................ 32 Juice pasteurization ................................ ................................ ................... 35 HHP alternatives ................................ ................................ ........................ 37 High Hydrostatic Pressure Processing ................................ ................................ .... 37 General History and Economics ................................ ................................ ....... 37 Technological Challenges and Opportunities ................................ ................... 39 Enzymes in HHP ................................ ................................ .............................. 41 Comparison HHP with thermal processing ................................ ................ 47 HHP and microorganisms ................................ ................................ .......... 48 Comparison with thermal pasteurization ................................ .................... 50 Effects of High Hydrostatic Pressure on pH ................................ ............................ 51 General History ................................ ................................ ................................ 51 Current Research ................................ ................................ ............................. 53 Gap of Knowledge ................................ ................................ ................................ .. 54 Juice Processing ................................ ................................ .............................. 54 High Hydrostatic Pressure Processing ................................ ............................. 55 Shift in pH with HHP ................................ ................................ ......................... 55 Summary ................................ ................................ ................................ ................ 56

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6 Objectives ................................ ................................ ................................ ............... 57 Specific Objective 1 ................................ ................................ .......................... 57 Specific Objective 2 ................................ ................................ .......................... 58 Specific Objective 3 ................................ ................................ .......................... 58 2 HIGH HYDROSTATIC PRESSURE DECREASED THE RATE OF THERMAL INACTIVATION OF A PECTINASE COCKTAIL ................................ ..................... 63 Materials and Methods ................................ ................................ ............................ 63 Materials ................................ ................................ ................................ ........... 6 3 Equipment ................................ ................................ ................................ ........ 63 Methods ................................ ................................ ................................ ............ 64 Sample preparation and HHP processing ................................ .................. 64 Processing conditions ................................ ................................ ................ 65 Activity measurements ................................ ................................ ............... 65 Rate of enzyme inactivation ................................ ................................ ....... 66 Results and Discussion ................................ ................................ ........................... 67 Activity Measurements ................................ ................................ ..................... 67 Residual Enzymatic Activity ................................ ................................ .............. 68 Rate of Enzy me Inactivation ................................ ................................ ............. 72 Effect of Pressure on the Rate of Inactivation ................................ .................. 74 Effect of Temperature on the Rate of Inactivation ................................ ............ 75 3 INCREASED RATE OF VISCOSITY REDU CTION OF A PECTINASE COCKTAIL AT HIGH HYDROSTATIC PRESURE ................................ ................. 84 Materials and Methods ................................ ................................ ............................ 84 Materials ................................ ................................ ................................ ........... 84 Equipment ................................ ................................ ................................ ........ 84 Methods ................................ ................................ ................................ ............ 84 Sample preparation and HHP processing ................................ .................. 84 Processing conditions ................................ ................................ ................ 85 Activity measurements ................................ ................................ ............... 86 Results and Discussion ................................ ................................ ........................... 87 Viscosity Reduction ................................ ................................ .......................... 87 Rate of Viscosity Reduction ................................ ................................ .............. 88 Activation Volume ................................ ................................ ............................. 93 Activation Energy ................................ ................................ ............................. 94 Preliminary Microbia l Inactivation ................................ ................................ ..... 95 4 EFFECT OF HIGH PRESSURE ON PH COLOR INDICATORS IN SOLUTION .. 101 Materials and Methods ................................ ................................ .......................... 101 Materials ................................ ................................ ................................ ......... 101 Equipment ................................ ................................ ................................ ...... 101 Methods ................................ ................................ ................................ .......... 102 Sample preparation ................................ ................................ .................. 102

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7 Processing conditions ................................ ................................ .............. 102 Peak area measurements ................................ ................................ ........ 103 Calibration curves and compression compensation ................................ 103 Results and Discussion ................................ ................................ ......................... 104 Peak Area ................................ ................................ ................................ ....... 104 Calibration Curves ................................ ................................ .......................... 105 Effect of Pressure on pH ................................ ................................ ................ 105 5 FINAL COMMEN TS ................................ ................................ .............................. 111 Overview ................................ ................................ ................................ ............... 111 Future Work ................................ ................................ ................................ .......... 111 APPENDIX: INACTIVATION OF E. COLI K12 WITH HHP TREATMENT .................... 113 Materials and Methods ................................ ................................ .......................... 113 Results and Discussion ................................ ................................ ......................... 114 Conclusions ................................ ................................ ................................ .......... 114 LIST OF REFERENCES ................................ ................................ ............................. 116 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 123

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8 LIST OF T ABLES Table page 1 1 Turbidity values for processed juices ................................ ................................ .. 60 1 2 Enzymes in juice clarification research ................................ ............................... 62 2 1 Rate cons tant of enzyme inactivation ................................ ................................ 81 3 1 Average viscosity reduction (%) of a 30 min process time ................................ 97 3 2 Rate of viscosity reduction ................................ ................................ ................ 98 4 1 Acid/base color indicators ................................ ................................ ................. 107 4 2 Parameters for lin ear relationships correlating pH vs. peak area ..................... 109

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9 LIST OF FIGURES Figure page 1 1 Overview of apple juice production to obtain either cloudy or cl ear juice. ........... 59 1 2 Basic summary of pectinase behavior ................................ ................................ 61 2 1 High pressure laboratory equipment set up. ................................ ....................... 78 2 2 treated at 69.3 C, 250 MPa, 15 min. ................................ ................................ 78 2 3 Maximum rate of viscosity reduction with a pseudo second order rate of rea ction plot for pectinase treated at 69.3 C, 250 MPa, 30 min ........................ 79 2 4 Observed and predicted viscosity for sample treated at 69.3 C, 250 MPa, 30 min. ................................ ................................ ................................ ..................... 79 2 5 Average pectinase residual activity ................................ ................................ ... 80 2 6 ................................ ................................ ... 80 2 7 Rate of pect inase inactivation with lines representing linear regression behavior ................................ ................................ ................................ .............. 81 2 8 Eyring plot for enzyme inactivation ................................ ................................ ..... 82 2 9 Activati on volumes for pectinase samples treate d at 0.1 MPa and 200 to 400 MPa ................................ ................................ ................................ .................... 82 2 10 ................................ ................................ .. 83 2 11 Ac tivation energies for pectinase samples treated at high hydrostatic pressure. ................................ ................................ ................................ ............ 83 3 1 treated at 62.4 C, 2 50 MPa, 15 min. ................................ ................................ 96 3 2 Viscosity reduction for samples ................................ ................................ .......... 96 3 3 Rate of viscosity reduction with a pseudo second order rate of reaction plot for pectinase ................................ ................................ ................................ ....... 97 3 4 Eyrin g plot for viscosity reduction ................................ ................................ ...... 99 3 5 Effect of pressure on the activation volume of the rate of viscosity reduction of pectin solutions. ................................ ................................ .............................. 99

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10 3 6 Effect of temperature on the activation energy of the rate of viscosity reduction of pectin solutions. ................................ ................................ ............ 100 4 1 Adjusted absorbance spectra for acidic pH points. ................................ ........... 107 4 2 Adjusted absorbance spectra for low acid pH points ................................ ........ 108 4 3 Adjusted absorbance spectra for Bromophenol Blue ................................ ........ 108 4 4 The pH calibration plot ................................ ................................ ...................... 109 4 5 Apparent effect of pressure on pH ................................ ................................ .... 110 4 6 Apparent pH shift with pressure ................................ ................................ ........ 110 A 1 E. coli K12 colonies gr owing on a MacConkey agar plate ................................ 115

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11 LIST OF ABBREVIATION S HHP High Hydrostatic P ressure PG Polygalacturonase PL Pectin L yas e PME Pectin Methyl E sterase

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Engineering KINETIC BEHAVIOR OF A PECTINASE COCK TAIL AT HIGH HYDROSTATIC PRESSURE CONDITIONS By Brittany Danielle Tomlin December 2011 Chair: Melanie J. Correll Cochair: Jos I. Reyes De Corcuera Major: Agricultural and Biological Engineering Apple juice is one of the most popular fruit juices cons umed in the United States. Pectinase cocktails, containing pectinases, hemicellulases, and cellulases are used in the production of commercial apple juice to reduce juice viscosity, increase yield, and to clarify the final product (Jayani and others 2005) High hydro static pressure (HHP) at moderate levels has stabilized and activated some enzymes. This project focused on testing a high hydrostatic pressure process to stabilize pectinase enzymes at raised temperatures above levels customarily used for clarification fo r possible economic benefits to traditional thermal processing. In the first set of experiment s the kinetics of inactivation of a commercial pectinase formulation was studied at 0.1 to 400 MPa and 55 85 C. High pressure slowed the rate of inactivation o f the pectinase cocktail treated at inactivating temperature conditions. Samples treated at temperatures of 77 C or 85 C and high pressures (200 400 MPa) retained activity for at least 8 times longer than samples treated at atmospheric pressure.

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13 In the second set of experiments the stabiliz ation of a commercial pectinase formulation at high hydrostatic pressure s ( 200 and 300 MPa ) at moderate temperatures (42.4 to 62.4 C ) was studied The rate of viscosity reduction increased with temperature with a ma ximum of 0.0960 Pa 1 s 2 occurring at 62.4 C and 300 MPa. Negative apparent activation volumes of 0.22 to 5.21 cm 3 mol 1 demonstrated that pressure favored the increase in the rate of viscosity reduction with pressure having the greatest effect at 57.1 C. Apparent activation energies suggest that temperature had greater effects at high pressure (200 300 MPa) than at atmospheric pressure. In the third set of experiments the effect s of high hydrostatic pressure on the shift in pH of pH indicator solutio ns w ere explored A bsorbance spectra for color acid/base indicators (pH 1.2 to 6.8) in the visible light range were used to quantify the change in pH induced by high pressures up to 600 MPa Increases in pressure produced an apparent acidic pH shift in aci d/base color indicators.

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14 CHAPTER 1 BACKGROUND INFORMATI ON AND LITERATURE RE VIEW A pple juice is the second most popular fruit juice consumed in the United S tates only behind orange juice In 2007 2008 over 535,000 metric tons of concentrated apple juice were consumed (USFAS 2008) American s drank an average of 8.34 L of apple juice (single strength equivalent) pe r capita in 200 6/07, while consuming only 3.79 L of all other non citrus juices such as grape, pineapple, cranberry and prune juice (Pollack and Perez 2008) Improved processing for fruit juices can lead to higher quality products. Consistent high quality p roduct s are more likely to see incr eased popularity among juice drinkers and greater economic pro fits over competitors The objective of this chapter was to give an overview of clarified fruit juice production focusing specifically on clarification an d pas teurization. The effects of high hydrostatic pressure (HHP) on the stabilization of enzymes and inactivation of microorganisms will be reviewed in detail Also i t is known that HHP induces shift s in pH which in turn may be responsible for some of the effec ts of HHP on juice processing. A review on the thermodyn amics and methods of assessing effects of pH in food processing are provided. This chapter describes the current methods of juice clarification processing the behavior of enzymes with HHP treatments and identifies relevant gaps of knowledge in industrial juice processing. Finally, the specific objectives of this research are presented. Apple Juice Production Apple Juice Production Overview Apple juice consumption has increase d with the rise of orang e juice prices. Because most apple juice in the U.S. is imported from other countries such as China,

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15 Chile, and Argentina, apple juice has a large economic impact on the U.S. In 2007, th e U.S. spent a half b illion dollars importing over 90% of its consume d apple juice (USFAS 2008) The U.S. produces apples for fresh consumption and mostly cull apples are sent to juice processing. While the amount of fruit for fresh consumption has increased over the past thirty years, apples for processing (juice and cider, canned, frozen, and fresh apple slices) have fluctuated In 1980 the amount of apples processed was 3,866 million tons, and t his amount increased to over 4,000 million tons in the 1990s Howeve r, since the 1990s the amount of apples processed decreased and in 2007 was down to 2,979 the first l has not shown a clear trend over the past thirty years. From the period of 2000 2007, four $0.093/ kg for juice and cider processing and four seasons produced over $0.110/ kg Growers received more for a season when lower quantities of apples were produced A pple juice consumption increased slightly for each season. In the early 1980s consumption per person was under 5.68 Ly r 1 and has increased to 8.33 Lyr 1 for th e 2006/07 year (Pollack and Perez 2008) Processing Lea (1990) and Lozano and SpringerLink (2006) have reviewed in detail the processing of apple juice. A summary is provided here with a visual flow diagram of apple juice processing in Figure 1 1 R aw apples typically culls, are processed into juice products that range from clear single streng th juice to cloudy or natural juice, cider or juice concentrates The juice concentrates will be made into juice blends or diluted back to single streng th after shipping Apple juice can be made into several different

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16 final products, but all the juices have the same initial process. The fro nt end operation is the portion o f the processing that involves the collection and classification of the fruit. The a pples are sorted, washed and then milled before pressing. The fruit is washed with rotatory brushes that remove unwanted contaminants such as rot, soil, pesticides, and microorganisms A variety of mills can be used, fruit grinding mills, rasp or grater mi lls, or fixed blade hammer mills to reduce fruit size. During pressing, the juice is extracted and the pomace is removed. The pressing process varies slightly depending on equipment; popular equipment includes the rack and cloth press, horizontal pack pres ses, screw presses and continuous belt presses. After pressing, the production process normally depends on the type of juice to be produced: cloudy single strength juice, clear single strength juice, or clear juice concentrate. Concentrated products have the aromas stripped off befor e the clarification process. M et hanol created from the depectini zation with pectin methylesterases (PME) in the clarification process can damage the aroma essences and decr ease the recovered aroma volume C loudy juice only goe s through a rough filtration or centrifugation to remove large solid particles and defects before it is pas teurized and packaged The clear juice products go through a clarification step, in which polysaccharides, starch, and cellulose molecules that caus e the juice haze are broken down with enzymes and allowed to settle over a period of time. Clarification can include fining, centrifugation, and several filtration steps before the juice is pasteurized and pa ckaged Fining agents, such as gelatin and bento nite, are use d in the fi ning process to remove haze ca using particles from the juice The fining agents cause particles to sink out of solution by either promoting attractive charge s or increasing the particles

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17 weight Next p asteurization can be performed with different metho ds; two popular methods include high temperat ure short time pasteurization ( 15 s at 72 C) or ultrahigh temperature (0.1 s at or greater than 121 C). Pasteurization is covered in g reater detail in a future section of this review For concentrate d products centrifuged and filtered juice is evapor ated to remove the water and increase the concentration of soluble solids to around 70 Brix The concentrate can be shipped then reconstituted back to single strength and/or blended with other juices and filtered before the product is pas teurized and packaged To maintain quality and conf idence of apple juice customers, producers must have strict quality control The Code of Federal R egulations (CFR) states 100% single strength apple juice mu st have a minimum soluble solids content (SSC) of 11.5 Brix (21CFR101.30). The Food and Drug Administration ( FDA ) has guidelines in the form of a Hazard Analysis Critical Control Point (HACCP) plan with r equirements for juice manuf acturers to ensure consu mer safety (USFDA 2004) The hazards that concern the FDA in apple juice production are patulin, microorganisms, and metal fragments. Patulin is a toxin produced by molds such as Penicillium Aspergillus and Byssochylamys that can grow on apples and appears in higher quantities on damaged fruit (rotting or bruised fruit caused by falling, bi rd s insects, etc) The FDA recommends removing damaged fruit or fruit pieces before processing and having the fruit supplier guarantee the quality of fruit harvests (not from damaged fruit). The pertinent microorganisms for apple juice are Escherichia col i O157:H7 and Cryptosporidium parvum and a standard 5 log reduction of these organisms during pasteurization is re quired Pasteurization conditions including temperatures and holding times will vary from company to company

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18 due to the difference in process equipment production size and pasteurization temperature. F inally the FDA re quires using screens during processing to ensure the metal fragments from processing equipment are not found in the final juice product (USFDA 2004) A ju ice company must guarantee consumer safety for a profitable product Haze m olecules The molecules that cause h aze formation and increase juice viscosity come mainly from the fruit cell wall. These molecules include p olysaccharides (pectins, starc hes, and gums), proteins, polyphen ols, polyvalent cations, and lipids Starch is mainly found in young fruit and therefo re is not as prominent as pectin in the juice haze from the production of mature apples. Pectin however is the leadin g cause of the juice haze and it is specific to the middle lamella of fruit, t he section between cells walls (Jayani and others 2005) Pectin and othe r cell wall components are responsible for providing su pport and texture to the fruit (Alkorta and others 1998) Pectin s negative charge repels one molecule from the other (Binning and Possmann 1993) Pectin amounts vary in fruit and vegetables. Pectin amounts can be as high at 10 30 % dry matter in sugar beet pulp Fresh apple tis sue can have between 0.5 to 1.6 % pectin, which is similar t o bananas but more than strawberries and che rries (Jayani and others 2005) Pectin molecular weights can vary in apples and lemons (200 360 kDa), while in other fruits such as pears and p runes (25 35 kDa) or oranges and sugar beet pulp (40 50 kDa) the molecular weig ht has little variation Apple pectin molecule s are large multifaceted molecules that are highly methoxylated (Lea 1990) Though pectin is not a uniform substance, the core chain of molecules is made up of anhydrogala cturonic acid (Jayani and others 2005) The linear

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19 portion of pectin chai ns is termed the smooth region (Pedrolli and others 2009) The galacturonate of the core chain are connected by alpha 1 4 linkages. The methyl groups common in apple pectin are attached to the c arboxyl group of the galacturon ic acid. Besides the esterification or of the carboxyl group or acetylation of the hydroxyl groups of other carbons (C 2 or C 3 ), sodium, potassium and ammonium ions can neutralize the carboxyl groups by removing the final hydrogen and replacing it with the ion Pectin is highly variable and can have side chains of arabinan, galactan, arabin og a lalactin, xylose, and f r u ct ose (Jayani and o thers 2005) Besides the long smooth linear portion of pectin, there are portions o f the pectin molecule with side chains made up of Rhamnogalacturonan I and II. These portions of pectin are termed the hairy region. Sections of rhamnogal a cturonan I (RG I) have disaccharide rhamnose in between the galacturonic units in the pectin chain and can contain the previously mentioned side chain. The other portion of the hairy region of pectin contains rhamnogalacturonan II (RG II) have the linear galacturo nan chain with the side chains (Pedrolli and others 2009) Pectin has been separated into four main groups by the American Chemical Society: protopectin, pectic acid, pectinic acids, and pectin (Jayani and others 2005) Protopectins are the insoluble for m in the fruit. T he remaining three groups differ in the amount of methyl groups on the esterified carboxyl groups: Pectic acid has very little esterification, pectinic aci d esterification is below 75 %, and pectin chains contain carboxyl groups t hat at least 75 % have been est eri fied with methyl groups (Jayani and others 2005) Often pectin molecules can interact with proteins to create proto pectin

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20 (Kashyap and others 2001) A pple juice contains highly methylated pectin which must be taken into account when selecting a suitable blend of enzymes for clarification. Clarification During juice extraction, particles from the cell wall form a cloudy haze in the juice making it viscous and difficult to filter (Binning and Possmann 1993) These substances also decrease the jui For clear juice products the clarification step, i.e. the removal of haze causing particles, is performed after the apples are pressed and before filtra tion centrifugation, fining, and evaporation. The haze causing particles are removed by addition of enzymes that break down pectin and suspended insoluble molecules allowing the particles to settle Enzymes are used during fruit mashing and clarification to decrease viscosity and improve ju ice yield (Binning an d Possmann 1993) In white grape juice clarification, the commercial product Pectinex (Novo Industries, Denmark) had the highest degrading activit y for pectin and starch of commercial products and removed 98 99% of turbidity T he other commercial enzyme preparation Celluclast (Novo Industries, Denmark) and a laboratory A, niger pectinase only removed 50 60% These results illustrate the difference in activities of selected commercial product s (Sreenath and Santhanam 1992) A fter enzymatic treatment, small insoluble particles remain in the fruit juice and cause a turbid haze (Gutirrez Lpez and others 2008) Therefore other p rocessing st eps including decanting, centrifugation filtration, and fining are often necessary Clarification can be monitored by measuring the turbidity of the juice and has been studied in various dark and clarified juices. Turbidity is measured in different units depending on the observation methods; two notable units are Fo rmazinain Nephelometric u nits ( F N U) or Nephelometric Turbidity u nits (NTU). Formazinain

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21 Nephelometric unit measurements are more common in Europe and made with infrared light while NTU measure ments are made with white light. Stable clarified juices are those having low turbidity of less than 2 NTU (Araya Farias and others 2008) As seen with the turbidity values from several clarified f ruit juices in Table 1 1, e nzymatic treatments play a critical role in clarification Clarification e nzymes There are several classes of enzymes used in the apple juice clarification process, including pectinases, l y a ses or trans elminases protopectinas es and proteases among others Pectinase s have been described elsewhere ( Alkorta and others (1998) Jayani and others (2005) and Pedrolli and others (2009) ) therefore, this section only briefly summarizes the findings of enzymes specific to the pectin m olecules The first main enzyme pectin methyl esterase (PME E.C, 3.1.1.11) is a hydrolase that demethyloxylate s the galacturonic acid in apple pectin shown in Figure 1 2(a) (Lea 1990) Figure 1 2 was adapted from (Pedrolli and others 2009) P ectin methyl esterase belongs to the group of e nzymes that de esterify pectin This enzyme goes by many names: pectinase, pect in methoxylase, pectin demethoxylase, and pectolipase The molecular weight of PMEs range from 35 60 kDa and have an optimum temperature range of 40 50 C Though the average pH range for pectin PMEs is from 4 to 8, fungal PMEs are mor e active at an acid ic pH and bacterial PMEs are more active at a basic pH. These enzymes are carboxylic acid esterase s that hydroly ze me thyl groups of methylated pectin The final product of the de esterification is pectic acid seen in Figure 1 2(a) .The side product of the de esterification is methanol If the pectin is esterified with an acetyl group, there are similar enzymes called pectin acetyl esterases that

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22 remove acetyl group from the pectin t o make pectic acid and acetate De esterifying enzymes are found in fungi, y east and bacteria and they are also involved in the aging and ripening process in fruit s and vegetables Pectin methyl ester ases from fungi randomly remove the methyl groups, while plant PMEs have a single chain mechanism in which the enzyme reacts with n on reducing end or next to a free carboxyl group In fruits and vegetables, PMEs are part of the changes that occur in the cell wall during growth A gel diffusion assay is common for PME activity determinations: as PME removes the methyl groups, more ruth en ium red (used to stain for spectroscopy) can bind to the pectin thus greater ability to measure pectin in solution (Downi e and others 1998) The pH changes are another way to determine PME activity due to the release of the hydrogen atom from water during the ionization of the carboxyl group (Whitaker 1984) effect on viscosity is minimal but allows the pectin molecule to be broken down into smaller chains by enzymes such as p olygalacturonase Polygalacturonase (PG) and pectin lyase (PL) are depoly merizing enzymes .The depolymerizing enzymes break down pectin in two way elimination Polygalacturonase s endo PG (E.C. 3.2.1 .15 and exo PG (E.C. 3.2.1.67) use hydrolysis by using water to break the galacturonic 1 4 glycosidic bo nd ( oxygen bridge) shown in Figure 1 2(b) The enzymes that use hydrolysis to break down pectin molecules are varied; polygalacturonases are used with pectate while polymethylgalacturonases break down pectin (highly esterified) and endo enzymes attack ran domly while exo enzymes focus on the term inal ends of the molecule. E xo p olygalacturonases are more common in fruit and vegetables, and endo galacturonases

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23 are found in other higher plants as well as fungi, b acteria, yeasts, and nematodes Microbial endo P G s have been c loned and studied for research Fungal and bacterial exo galacturonases prod uce different products; monogala cturonic acid and di galacturonic acid respectively In apple juice clarification PG can only break down galacturonic acid units, but not methylated galacturonic acid. P olygalacturonase causes the proto pectin complexes to settle out by partially removing the negative pectin charge, but the PG enzyme cannot deme thoxylate the pectin (Lea 1990) T he method used to measure PG activity involves 3,5 dinitrosalicylate reagent method or arsenomolybdate copper reagent method to determine the rate of increas e in reducing mL 1 min 1 galacturonic acid (Somogyi 1952; Miller 1959) Another m ethod to measure PG activity uses the flow rates of water, a test solution, and test solution with enzyme to determine the reduction in viscosity with the activity being described as amount of enzyme required to produce a specific decrease in viscosity per unit of time (Roboz and others 1952) Polygalacturonases vary greatly from one another; molecular weights range from 38 496 kDa (most range from 38 79 kDa), optimum pH range from 3 to 11, and opt imum temperatures range from 30 to 69 C. Common microbial PGs are most active in the acidic range (pH 3.5 5.5) and warm temperatures (30 50 C ) Aspergillus Bacillus Fu sarium Penicil lium and Termoascus species are some of the producers of PG Ly a ses or trans el i minases are the second major group of enzym es classified as depolymerizing enzymes. Lyases endo PL (E.C. 4.2.2.2) and exo PL (E.C.4.2.2.9) use trans elimination instead of water to break 1 4 glycosidic bond as shown in Figure 1 2(c) When ly ases cleave the 1 4 glycosidic bond hydrogen from the C 5 and

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24 oxygen are moved to create a hydroxyl group on one product and double bond o n the second galacturonic acid Similar to the polyga lacturonase enzymes, lyases can break down highly est er ified pectin or pectate Polymethylgal a cturonate lyases (PMGL) do not remove the methyl group from the esterified galacturonic acid While the enzymes that are specific for pectate (polygalactu ro nate l yases or PGLs ) can be cleave random (endo) or at terminal ends (exo), the enzymes specific to pectin ( PMGLs or PLs ) are mainly endo enzymes. For PGLs the endo form of the enzyme is more common than the exo form. Another difference between PGL and PMGL are that PGLs require calcium ions, while PGMLs do not Calcium and other ions can increase PGMLs behavior and chelating agents can inh ibit PGL Lyases can be made by bacteria and pathogenic fungi For apple jui ce clarification, PL degrades polygalacturonate chains of methylated pectin (Lea 1990) There are several ways to determine the activity of lyases. A common way i s to observe the production on the double bonds in the product with the increase of absorbance (235 saturated product per minute Other methods for measuring PL activity are through reducing group me thods, r eduction in viscosity and HPLC or GC analysis Pectin lyases are mainly found in microorganisms such as bacteria and fungi, but rarely in higher plants Lyases from bacteria are often associated with plant pathogenicity to break into fruit and veg etable s Lyases have an average molecular weight of 30 40 kDa, but some PLs have large molecular weights greater than 50 kDa. Also, this enzyme tend s to be active at higher temperatures than other pectinase enzymes as the optimum ranges is between 40 and 7 0 C and the optimum pH range is 7 to 10 Although, a few PMGLs from species

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25 Aureobasidium pullulans LV 10 and Pichia pinus have lower optimum pH of 5 and 4.5 respectively Aspergillious Bacillus Debaryomyces and Penicillium species are some of the mai n producers of PL Th e final group of enzymes is specific to protopectins and is t herefore named protopectinases The enzymes can also be called pectinosinases and these enzymes use water while co nverting protopectin to pectin The enzymes are cl assified by the site they cleave to break insolubl e protopectin into pectin; type A enzymes are specific to the polygalactur onic acid region and type B enzymes are specific to the polysa ccharide chains of protopectin Protopectinases are usually produced by yea sts and fungi similar to yeast O ther enzymes are used to break down the remaining chains found in the pectin molecule. There are enzymes that are specific to the rhamnogalacturonan chains and chains containing xylose molecules. The rhamnogalacturonan enzymes that hydrolyze the rhamnogalacturonan chain are rhamnogal a cturonan rhamnohydrolases, galacturonohydrolases, and hydrolases that produce rh a mnose, monogalacturonate, and oligogalacturonates respectively. Other enzymes include rhamnogalacturonan lyases, rha mnogalacturonan acetylesterases, a nd xylogalacturonan hydrolases F or apple juice clarification pectin is the main target molecule because apple pectin molecules are highly est er ified. As the particles are removed from solution by PME PG and PL the jui ce becomes less viscous and yield increases W ithout the pectin the juice appears clear and after fining is appealing to the customers. Though clarification is an established technology, new research continues to focus on different enzymes in respect to t heir sources, production methods, and be havior under different

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26 conditions in order to improve juice processing and clarification Research focusing on clarification enzymes will be covered more in depth within the innovations portion of this review. Clar ification processing c onditions After pressing, apple juice and pulp are stirred at least 1 to 20 min for fruit oxidases to oxidize the polyphenol s, which are enzyme inhibitors Sometimes the juice is centrifuged to remove large particles before clarifica tion (Kashyap and others 2001) The clarification process is carried out between 15 and 55 C (Lea 1990) As mentioned, p olysaccharides (pectins, starc hes and gums), proteins, polyphen ols, polyvalent cations, and lipids ar e some of the particles that ca n create the juice cloud, and enzymes such as pectinases, amylases, and proteases are used to tre at these particles (Binning and Possmann 1993) I ndustry uses specific tests to ensure their process has removed the necessary amount of pectin from the apple juice. One of the standa rd methods is the alcohol precipitation test, in which one part juice and two parts ethanol are combined If flocculation does not occur in about 15 min, the process was successful (Lea 1990) However this test provides results that are subjective and hard to quantify. As researchers, we have more conclusive ways to analyze the clarification than industry. For clarifi cation, it is desirable to determine the effect of temperature on enzyme cocktail activity to optimize th e process for each juice One way to observe the activity of a pectinase cocktail is to measure its effect on the viscosity of a pectin solution and t his method will be used for this research As enzyme activity increases, then a greater decrease in a treated pectin s olution viscosity will be observed The rate constants of enzyme inactivation or viscosity reduction can be used with the Arrhenius equat ion to

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27 calculate the activation energy (Ceci and Lozano 1998; Ortega and others 2004; Eisenmenger and Reyes De Corcuera 2009b) The activatio n energy value establishes the effect of temperature on enzyme activity with low values having neg ligible temperature effects on enzymes; activation energies are important in characterizing enzyme behavior at different conditions. The extent of clarificati on can also be monitored by the transmission of light through a centrifuged sample of juice. Clarified juices will absorb less light than cloudy juices achieving a higher transmission value. Pasteurization A pple juice consumers desire a safe product with organoleptic and nutritional properties that are comparable to fresh juice. Pasteurization is important to inactivate microbial populations that would either cause product spoilage or consumer illness A low pH in many juices (orange, a pple, and grapefrui t) due to naturally present organic acids helps prevent a large variety of microorganisms from growing in fruit juices; yet, some microorgan isms can survive and some may become acid adapted (Mosqueda Melgar and others 2008) Pathogenic microorganisms that can survive and grow in apple juice ar e the focus of producers and the FDA to create a safe product for consumers. Microbes p ertinent to a pple j uice The acidic pH of apple juice (typically between pH 3. 0 to 4.5) decreases the types of microorganisms that can survive in the juice and many huma n pathogens do not actively grow in fruit juice. But some pathogens such as E. coli O157:H7 and some Salmonella speci es can survive. The microorganisms that have adapted to acidic environments are termed acid adapted (Mazzotta 2001) The FDA recognizes that pathogens occurring in nature and from animal manure c an contaminate acidic juices

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28 (pH below 4.6) and low acid juices (pH above 4.6) and cau se foodborne illness in humans For acidic juices, E. coli O 157:H7, Listeria mon o cytogenes Cryptosporidium parvum and a variety of Salmonella species can taint juice; a nd low acid juice, like carrot juice, can host Clostridium botulinum (USFDA 2004) One of the earliest noted outbreaks of apple products was apple cider containing Salmonella T yphi i n 1922 (Paquet 1923) Escherichia coli O 15 7:H7 was noted later with cases in fresh apple cider in Canada (19 80) and the USA (1991) (Steele and others 1982; Besser and others 1993) Cases continued to appear in the 1990s, and Cryptosporidium parvum caused an outbreak in New York in fresh apple cider i n 1996 (USCDC 1997) These outbreaks were mainly illnesses with limited deaths. Illness associated E. coli O 157:H7 have symptoms of bloody diarrhea and hemolytic uremic syndrome (HUS) (Luedtke and Powell 2000) Many of these cases were due to inadequate sanitation or fecal conta mination of apples. The possibility of contamination of pathogenic microorganisms du ring apple productio n and juice processing presents the reason for which proper pasteurization i s a necessity for safe human consumption. For each juice, the FDA has requirements for the treatment of the pertinent microorganism or microorganism associated with any of the spec ific juice outbreaks. The FDA considers Cryptosporidium parvum and E. coli O157:H7 to be the pertinent microorganisms for apple juic e (USFDA 2004) However, j uice processors must be mindful of all pathogens that can contaminate juice including Salmonella E e nteritidis which has also been found in grapefruit juice and lemonade (Mosqueda Melgar and others 2008) and the commonly occu rring Listeria monocytogenes (USFDA 2004)

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29 In addition to pathogenic strains, p roducer s mus t also be concerned about spoilage microorganisms that deplete shelf life and cause economic losses with destroyed products. S poilage microorganism s cause unwanted sensory characteristics and resist thermal processing A major juice spoilage microorga nism of concern for apple juice producers is the spore former Alicyclobacillus acidoterrestris that is a thermo acidophilic bacterium Alicyclobacillus acidoterrestris f ound in soil thrives in optimal conditions of pH 4.5 to 5 at 36 to 53 C, and can surv ive in the pH range of 2.5 to 5. 8 at 20 to 70C. Currently no strains of A. acidoterrestris are considered pathogenic. Spoilage is noted as off flavors, odors, as well as possible sedimentation or change in color or c loudiness Guaiacol, and halopheno ls 2 ,6 dichlorophenol and 2,6 di bromophenol are responsible for the off flavors and odors creating medicinal, antiseptic and sm oky notes Alicyclobacillus acidoterrestris spores are resistant to thermal treatments, but vegetative cells are responsible for spoi lage (Smit and others 2011) Paste urization s tandards The FDA has mandated a 5 log reduction of the pathogen s pertinent or relevant to each juice for each pasteurization process A study cited by the FDA recommends treating j uice for 3 s at 71.1 C for a 5 log reduction of E. coli O157:H7 Salmonella and L. monocytogenes ( Mazzotta 2001 ) The pasteurization conditions in apple juice are often recommended for the pathogen E. coli O157:H7, because of the lack of information on the pathogen C. parvum The FDA makes recommendations for a 5 log reduction of C. parvum which is more heat resistant th an other microbes by thermal processes ranging from 7 1.1 C for 6 s to 82.2 C for 0.3 s (USFDA 2004)

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30 Juice n utrients A major focus of juice production is creating safe shelf stable products while maintaining vital juice nutrients. A section is included on juice research focused on optimizing juice processing to retain important nutritiona l characteristics of juice such as antioxidants and vitamins. This section provides examples of juices nota bly high in antioxidants: blueberry and pomegranate juices Anthocyanins, beneficial nutrients commonly found in plants such as blueberries eggplant s and grapes are part of juice antioxidants and contribute to the color of the fruit and its juice (Buckow and others 2010) Thermal treatments of blueberries and its juice have to be higher than 80 C to deactivate polyphenol oxidases (PPO) which can breakdown anthyocyanins in blueberry juice; however thermal degradations of nutrients and f lavors are an unwelcome side effect. The anthocyanin concentrations in juices and reactions to processing vary due to different types and amounts of anthocyanins, pH, presence and concentration of degrading enzymes, free radicals, and ascorbic acid. With t his complicated matrix, experimental degradati on of blueberry anthocyanins gave a thermal degradation that acted as a 1.4 th order reaction. Though high pressure and high temperature slow ed the degradation of blueberry anthocyanins in storage when compared to untreated juice; thermal processing had the slowest degradation and high est concentration of anthocyanins after storage. The total anthocyanins half life w as highest for a storage temperature of 4 C with a half life of 251.7 d for the thermal treatment of 90 C. For storage at 4 C, the juice processed at 600 MPa at 90 C, had a half life of 245.7 days. Half lives decreased with higher storage temperature blocks (25 and 40 C) and thermal processing had higher half lives than pressure/thermal treatment s with warmer storage. Pressure/thermal and thermal processing both

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31 degrade ascorbic acids; however the increase d free radicals and oxygen concentration during pressurization could lead to the faster degradation rates than thermal processing (Buckow and others 2010) A ntioxidants and vitamins are a lso crucial to the popularity of pomegranate juice Hydrolysable tannins are the main antioxidant of pomegranate juice, but anthocyanins and phenols also contribute to the antioxidant composition. These molecules are affected by light, pH, temperature and oxygen content. High pressure is being consi dered an alternative to control microbial populations while limiting the loss of antioxidants and nutrients. Pomegranate juice with an initial microbial load of 10 4 cfu/mL was reduced to a population at or below 1 cfu/mL by high pressure processing for 400 600 MPa, 5 10 min, and 25 50 C. Temperature and process time had the greater effects on color and aroma of pomegranate juice than high pressure. Turbidity decreased with high pressure processing. Anthocyanins decreased with high pressure processing, exce pt when processed at 500 MPa for 5 10 min and 600 MPa at 10 min (both with the processing temperature of 50 C). Phenols increased in concentration for samples trea ted at 400 MPa and 50 C; the untre ated juice polyphenol content was 1.36 g/L of gall ic aci d and treated samples had a phenol content 1.48 g/L of gallic acid and 1.92 g/L of gallic acid for 5 and 10 minutes processes respectively (Ferrari and others 2010) Current Innovations J uice producers and researchers continue to search for and develop processe s that make juice production more economical and more consistent with fresh pressed juice. This section will highlight some of the recent research involving clarification

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32 enzymes juice production / pasteurization and new clarification techniques that will i mprove future production and that is relevant to this research project. Innovation in Clarificatio n e nzymes Juice clarification is a current area of study due to its importance in the juice processing. Juice clarification reduces viscosity and cloudy haze for ease of downstream processing and visual desirability; however, the clarification step is a time consuming phase with the added expense of heating and the addition of enzymes. Current research focu ses on improving clarification b y de creasing clarifica tion times and reducing expenses. E nzyme immobilization to retain and reuse enzymes during processes as well as new sources of pectinases and mixtures could decrease clar ification time and enzyme requirements Researchers have looked at immobilizing enzyme s for enzyme retention during apple juice clarification and have found immobilized pectinases performed better with pan (polyacrylonitrile) bea ds over glass and nylon beads Immobilized pectinases have higher optimum pH (pH 6.2) than free enzymes pH 5 .6 b ut do not perform as well as free enzymes at acidic co nditions. Diano and others (2008) found the optimum temperature range of 48 to 65 C fo r apple juice clarification for immobilized enzymes w as similar to that of free enzymes ( 43 to 65 C) Clarificatio n research compares new enzymes from different sources to concentrated commercial products traditionally used in industry. Table 1 2 highlights so me of the new enzymes in juice clarification research The fungi Aspergillus niger is popular in research due to its high pectinase activity and commercial safety for food products. Sandri and others, (2011) observed the effect of A. niger T0005007 2 (TE1) and A. oryzae IPT 301 (TE2) to compare commercial clarification enzymes

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33 Pectinex Clear (PC) and Pectinex BeCo lour (PB) for light and dark clarified juices. Viscosity reduction has been used for total pectinase activity with one unit of activity stated as the necessary amount of enzyme required to reduce the viscosity by 50% (Sandri and others 2011) Pecti nex Clear and PB had total pectinase activities of 919 U mL 1 and 1150 U mL 1 respectively and TE1 and TE2 had activities of 75 U mL 1 and 44 U mL 1 However, the new enzyme extract TE1 had a larger exo PG activity then PB and performed comparably in clar ification studies to commercial products. For apple juice clarification, based on turbidity reduction, TE1 and PC had similar results of about 60% clarification with treatments with PC for 60 min at 30 C, and TE1 treated samples at 50 C for periods of 30 and 60 min. A. niger T0005007 2 had compara ble or greater clarification results than PC in Butia palm juice and PB in blueberry and grape juice. However TE2 did not produce the largest turbidity reduction for any of the four juices. Though the new extrac t TE1 had lower total activity, its PG and PL activities were higher resulting in similar results as commercial products (Sandri and others 2011) The research shows that the ratio of individual enzymes ( PG, PME, and PL) in products is a large fact or in overall pectinase behavior. Another area in innovation in enzymes is the potential to clone plasmids with PGs specific to yeast such as Pichia pastoris One of the main purposes of these experiments is to produce enzymes that work best in acid ic pHs su ch as those found in juices. Polygalacturonase s from Penicillium sp. CGMCC 1699 and Bispora sp. MEY 1 have been successfully produced by Pichia pastoris Penicillium sp. CGMCC 1699 had optimal activity of pH 3.5 at 40 C and has been studied with commer cial pectinases and lyases to reduce intrinsic viscosity (Yuan and others 2011) The viscosity was

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34 measured with a glass capillary viscometer as a function of flow time, density and the same conditions observed with water; viscosity reductions were obtained from the difference in viscosity of treated and untreated juice divided by the viscosity of the control sample of untreated juice (Yang and others 2011; Yuan and others 2011) Optimum activity in apple juice was obtained when the PG (0.1% v/v) was mixed with an unnamed commer cial PL product (0.05% w/w); this mixture had higher viscosity reductions than a commercial pectinase product ( also unnamed in the study) and the c ommercial PL product alone The optimum mixture of endo PG and the commercial PL product obtained intrinsic viscosity reductions of 22.7% and 33.1% in apple juice for 180s and 240s at 40 C (Yuan an d others 2011) Viscosity reduction was also assessed for endo PG from Biospora sp. MEY 1; with an amount of 10 U/mL, the viscosity of apple juice was reduced by 7.7% and transmittance increase d more than 84% (Yang and others 2011) Viscosity r eduction is an additional factor in determining pectinase releva nce in juice clarification for viscosity is an important par ameter in processing and filtering juice Viscosity reduction can be used as a measurement of total enzyme activity of enzyme mixtures These studies demonstrated that a solution of multiple enzymes achieved higher clarifi cation and viscosity reduction than a solution with an individual product Shelf life is another factor important and should be characterized for enzymes used in juice clarification The ability to maintain enzyme activity for long storage periods is bene ficial for researchers and industry alike in that enzymes are often expensive and are often not used immediately after their production For example, p olygalacturonase from A. niger ( ATCC9642 ) maintained it s initial activity when stored

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35 for 40 d in low tem peratures of 4, 10, and 80 C (Gomes and others 2010) Therefore, the identification of new, highly active, stable enzymes at juice processing condition could lower costs for juice processing. Juice p asteurization P asteurization is a critical step to insure safety of consumers and increase shelf lives of products. Pasteurization research is at present focused on techniques that minimize thermal degradations o f nutrients while inactivating pathog enic and spoilage microorganisms. study pasteurization processes that do not include high temperatures and are termed non thermal processing. Researchers are also combining other proce ssing techniques quality while inactivating pathogenic an d spoilage microorganisms. Non thermal processes such as UV irradiation and pulsed light t reatments, must be appro ved by the FDA. Unlike other novel non thermal processing techniques, h igh pressure processing (and other processes not using radiation or chemical agents) d oes not ne ed FDA approval for validation because it has already been proven effective and safe as a pasteurization technique (USFDA 2004) High pressure, pulse electric fields, ultraviol et light and several other non thermal pasteurization techniques are being investigated to enhance fruit juice processing. High intensity pulsed el ectric fields (HIPEF) have been proven to be successful at pasteurization and is more applicable with the ad dition of organic acids, enzymes, bacteriocins, and spices. The technique has also been combined with generally recognized as safe products (GRAS) such as citric acid and cinnamon bark in apple, pear orange, and strawberry juices. N on linear microbial po pulation reductions were

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36 noted with m ost of the inactivation occurring at the beginning of treatment. Tailing the decrease in the rate of microbial reductions, can be caused by several possibilities such as heterogeneous sensit ivities among population s or shielding by juice mat rix and dead cells. Electric pulsed fields have had FDA approval and is starting to be used on a commercial scale (Mosqueda Melgar and others 2008) Another alte rnative to thermal processing, ultraviolet light (UV) can be applied at low temperatures, is not known to prod uce any toxic effect s on products, and requires very little energy One disadvantage of UV processing is its lack of penetration in solid or on opaque surfaces ; thus air, direct surfaces, and clear liquids have the greatest sensitivity to UV light treatmen t. For juices, the typical penetration depth is 1 mm with 90% of the light absorbed and the type of liquid, soluble solids and suspend matter can affect penetration. Turbulent flow is often used to address the stated issues to expose as much of the juice t o the UV light and prevent clustering of microorganisms. UV light is effective for inactivating microorganisms by causing cross linkages in DNA (between thymine and cysteine) which causes cell death. Radiant exposure dosages can be stated as energy per are a or volume. For example, c larified apple juice inoculated with E. coli K12 had 3.5 log reductions in aerobic plate counts while ye ast and molds were reduced by 3 log reductions after UV treatments (Keyser and others 2008) Lastly, supercritical gases are capable of pasteurizing fruit juices as an alternative to thermal processing. Supercritical gases require l ower temperatures and pressures than thermal and high pressure processing respectively. In apple juice with initial concentrations of natural microflora of 5 x 10 2 cfu mL 1 and a treatment of 36 C, 100 bar (CO 2 or N 2 O), 300 rpm (stirring), the microbial populations were reduced to 8.01%

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37 and 7.42% by CO 2 and N 2 O respectively. Both gases completely inactivated natural microflora populations after 10 minutes of the same treatment (Gasperi and others 2009) HHP alternatives As discusse d previously, the three important facto rs in clarified juice production are maintaining vitamins and nutrients, clarifying juice in a timely and an inexpen sive manner, and inactivating pertinent microorganisms. All three of these factors are critical to a successful juice product. HHP processing is being highlighted as a viable process to perform all three of these steps. The remaining portion of this review will explore HHP research in the clarifie d juice field to demonstrate its potential benefits to the food processing industry. High Hydrostatic Pressure Processing General History and Economics In recent years, consumer s have started to demand more fresh like products that have minimal processing and that have little to no artificial stabilizers in them (Deliza and others 2005) Fruit juice as a whole h as seen a decrease in consumption in the past several years (Pollack and Perez 2008) Improved processing techniques leading to a fresher tasting product could help provide a bo and high pressure processing is a current topic of research to improve fruit juice quality. High h ydrostatic p ressure processing as a non thermal alternative (below 40 C) or as a pressure assisted thermal tre atment are two techniques that offer improved benefits to thermal processing by retaining juice nutrients, antioxidants, coloration, and clarity after pasteurization.

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38 At the end of the nineteenth century, experimentation important for food processing bega n to occur. Demazeau and Rivalain (2011) have reviewed the history of high pressure processing, and the key research is summarized in the following section. One of the first major high pressure discoveries relevant to food processing was that 300 MPa did n ot inactivate Staphylococcus aureus however the same pressure could inactivate other Streptococcus species. Enzymatic studies involving high pressure began when Bchner induced ferm pressures of 40 to 50 MPa. I n 1899 Hit e investigated high pressure as an alternative to thermal processing, and established that a pressure over 463 MPa (for 1 h) could slow milk souring by 24 h. From this initial research, high pressure studies have been performed focusing on vaccines, protei n denaturation, enzyme activity and its effect on microorganisms for food preservation The Japanese were the first to commercially produce food products with HHP processing. These products were jams and were available in market in the early 1990s (Demazeau and Rivalain 2011) Other food s pasteurized by high pressure processing expanded to include squid and rice cakes in Japan, orange and apples juice i n Europe, and oysters and guacamole in the American market (Rastogi and others 2 007) P ressure treated juices available today include orange grapefruit and apple juice (Patterson and others 2007) The initial equipment and operational costs of HHP continue to decrease as research and popularity of this non thermal option grows. The HHP can be performed by either a batch process or a semi continuous process in which the food product and its package are treated or where the juices are processed with high pre ssure before being packaged For semi continuous, a pressure medium and piston are used to

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39 pressurize the fluid product in two separate vessels An additional pressure vessel can be added (three in total), so while one product vessel is being pressurized the other product vessel is being filled or discharged The typical system consists of a pressure vessel, seal closures, pumps, and systems to monitor control and transport product during processing (Balasubramaniam and others 2008) Commercial manufacturers of high pressure equip ment include Avure Technologies (Franklin, TN), NC Hyperbaric ( Burgos, Spain ), and Uhde (Dortmund, Germany) Food companies that have incorporated high pressure into their production line include Hormel (Austin, MN), Kraft (Northfield, IL), Perdue (Salisbu ry, MD), Foster Farms (Livingston, CA) and Wellshire Farms ( Swedesboro, NJ ). Around 11 % of commercial products processed by high pressure are fru it juices and beverages, and 80 % of the commercial equipment was put into processing in 2000 or later (Balasubramaniam and others 2008) High pressure equipment can range from $500,000 to $2.5 million and the vessels can typically hold from 30 to over 600 L of product (Balasubramaniam and others 2008) An estimate of cost of HHP processing ranges from $ 0.05 and $ 0.5 0 per liter or kilogram with t he lowest end of the range being near thermal processing costs (Rastogi and others 2007) With r e search and the acceptance by mainstream consumers, commercial HHP processing could bec ome a more feasible option for juice production. Technological Challenges and Opportunities High pressure treatments offer many opportunities for the food and fruit juice industr ies. Some HHP treatments can be performed at room temperature, thus it can be used as a pasteurization process that may eliminate the need for heat and preservatives (Rastogi and others 2007) Thermal treatments can result in temperature gradients that can be detrimental if products do not have the same exposure to the

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40 required temperature throughout the process In extremes this gradient can lead to incomp lete microbial reduction due to lack of uniformity in heating of the product and cause a decrease in quality of the product As pressure increases, the resulting compression can change the pH of juice with an acidic shift of 0.2 pH units per 100 MPa as see n with apple juice in 1995 (Rastogi and others 2007) Also with this compressio n, molecules shift to the most compact form. An increase of temperature is observed with an increase in pressure on the order of generally around 3 C per 100 MPa increase for liquid mediums (Rastogi and others 2007) Another challenge with HHP is its effects on enzymes and other proteins which can cause changes in activity or confi guration Luckily, these e ffects are normally reversi ble at pressures between 100 and 400 MPa and this reversibility is not found in most cases with thermal processing. One of the benefits of HHP is that the processing does not appear to affect desirable q ualities of food (flavor, nutrients, an color) as is often found with thermal processing (Rastogi and others 2007) High pressure homogenization (HPH) has also been linked with the ability to pasteurize juice while keeping fresh qualities For example, when fresh clear Annurca apple juice was pasteurized with HPH processing (20 C, 150 MPa, 3 passes), the juice maintained a longer storage life and color than untreated juice. The soluble solids content and color stayed constant in treated juice for 28 days while the Brix fluctuated in unprocessed apple juice throughout the 28 day st orage time. The HPH treated apple juice maintained a minimum shelf life of 28 days under refrigerated conditions that met European Commission regulations no. 2073/2007 of a microbial loa d under 100 cfu g 1 with no coliform contamination (Maresca and others 2011)

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41 Sometimes perceived as a challenge, the larger molecules with non covalent bonds such as proteins and polysaccharides are affected during high pressure processing (Rastogi and others 2007) The costs of equipment and unknown effects of high pressure on food components are the major limiting factors affecting the adoption of high pressure processing In addition, the p ressure vessel size is limited to around 25 L with intended pressures over 400 MPa, and the pre stressed wire wound vessels needed are expensive. For commercial use, p ressure vesse ls are limited to less than 680 MPa The vessel limitations make some processing (oyster shucking at 200 400 MPa) more economical to process with HHP than guacamole salsa (around 600 MPa). Equipment manufacturers are working on designing press ure vessels that can withstand and exceed these pressures and higher temperat ures (Torres and Velazquez 2005) The final technological hurtle is the unknown behaviors of many reactions (enzymatic and microbial) that have to be explored for the HHP technology to become widespread and effectively used in industry. Enzymes in HHP Most of the work on enzyme reaction rates in food processing has focused on inactivating a specific en zyme of concern. Conversely for some industrial enzyme processes, it is desirable to activate and stabilize the enzymes as in the cas e of fruit juice clarification for a combination of pectinases and cellulases. However, little is known about pressure act ivation of enzymes or the reasons for the increase in stability at higher pressures It has been hypothesized that activation and stabilization is a result of changes in the enzyme structure, reaction mechanism, or the substrate/solvent physical properties Even with advancement of mathematical modeling, actual data need to be acquired to accurately predict the outcome affected by pressure and

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42 temperature processing (Eisenmenger and Reyes De Corcuera 2009b) Recent studies that focused on enzyme stabilizatio n focused on individual enzymes More research needs to be conducted to characterize the effect of pressure on enzymes mixt ures in apple juice clarification. High pressure does not have the same effect on all enzymes. Some enzymes are inactivated with high pressure while others can be stabilized or activated with HHP at temperatures that would normally inactivate the enzymes (Hendrickx and others 1998; Eisenmenger and Reyes De Corcuera 2009b) Enzymes such as PME in cloudy juices like orange juice can break down pectin and other cloud forming molecules in the juice leading to undesirable cleare r product. Some PMEs are more resistant to heat and high pressure can be effectively used to decrease PME activity in orange juice (Nienaber and Shellhammer 2001) The condition of pulp, pasteurized or unpasteurized, affects the activity of PME for much of the PME is found within t he juice pulp Samples with 8.5 % of unpasteurized pulp had higher PME activity than samples with the same amount of pasteurized pulp treated at room temperature and high pressures (Nienaber and Shellhammer 2001) T he residual activities of PME in unprocessed samples for unpasteurized and pasteurized pulp wer e 100% and 64.8% respectively After t reatments using 500 MPa at 50 C for 5 min the P ME residual activity reduced to 19.9% in samples with unpasteurized pulp down to 11.3% in samples with pasteurized pulp. Higher pressures of 800 MPa at 25 C for 1 min s howed even further decrease s in PME activity with a drop to 6.4% and 3.9% in unpasteurized and pasteurized pulp, respectively (Nienaber and Shellhammer 2001)

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43 High pressure homogenization can be performed under a variety of different pressures, temperatures, and number of passes and is another non thermal alternative to inactivate PME in orange juice. For example, a linear inactivation of PME was seen for different temperature treatments for orange juice ( e.g., 22 C, 35 C, and 45 C) as the pressure increased for a single pass (Welti Chanes and others 2009) T he fastest inactivation was at 45 C with a rate of 0.262 % MPa 1 while at 22 C and 35 C there were similar inactivation rates of 0.1 92 % MPa 1 and 0.195 % MPa 1 respectively. The number of passes through the homogenizer did not have a significant effect on PME activity at 100 MPa, but led to a constant increase in inactivation for high pressure of 250 MPa. PME activity increased with storage time at 4 C, most likely due to isoenzymes that were split during processing and reacted with juice later. Higher pressures and increased number of passes slowed the PME activity rate in storage and all sa mples treated with high pressure retained the cloud during the 12 day storage while untreated juice lost the cloud after 6 h of storage. It was hypothesized that the resulting smaller haze molecules from sieving and homogenization contributed to the juice cloud Fruit juice smoothies are another area in which high pressure processing is being explored for its potential as a minimal process treatment The r esearch focused on this method includes smoothies containing a pples, apple juice concentrate bananas, strawberries, and oranges Specifically t hese studies were looking at the effects of HHP processing on antioxidants, phenols, and color compared to thermal and untreated juices For example, smoothies that were treated at 450 MPa at 20 C for 5 minutes or less were compared with thermal processin g that reached 70 C for at least 10 minutes

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44 (Keenan and others 2010) Interestingly, s moothies treated with a thermal process had higher antioxid ants than samples treated with HHP or untreated samples, while HHP had higher phenol concentrations than thermal ly processed sample s It is believed that thermal processing increases extractability of antioxidants while inactivating enzymes that would degr ade antioxidants (Keenan and others 2010) While HHP also has the capability to disrupt cell membranes and increase extractability of antioxidants and shorter industrial process times. Chilled storage conditions at 4 C also led to a decrease in antioxidant concentrations in all samples. Polyphenol oxidase (PPO) enzyme s from the bananas and apples also lead to browning of samples (Keenan and others 2010) While much of th e high pressure research focused on inact ivation of microorganisms and enzymes, some has focused on the stabilization and activation effects that high pressure can have on enzy mes. For example, t omato PME was activated at 300 MPa, and carrot PME was stabilized around 500 MPa (Ly Nguyen and others 2003; Hsu 2008) Reports on the stabilization of PMEs and PGs with HHP processing are prevalent in the literature d ue to the improved texture results seen in fruit and vegetables with this method The inactivation of PG and the stabilization of PME with or without calcium ions lead to higher quality in texture. Though most studies report that PME is pressure stable be low 400 to 500 MPa, PG has been reported as sensitive to pressure processing. Pressure and temperature behavior of two tomato polygalacturonases PG1 and PG2 have been reported. Polygalacturonase 1 was heat stable wh ile PG2 was heat labile, and both were

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45 inactivated at pressures of 300 500 MPa at room temperature for 15 min. The same process failed to inactivate tomato PME (Rodrigo and others 2006) Tomato PME was stable up to 700 MPa and still retained activity of 50% after treatment at 850 MPa for 15 min. Some researchers have focused on the behavior of tomato PME and PG in t he presence and absence of each other at high pressure. Polygalacturonase in the presence of highly methoxlyated pectin and PME at pH 4.4 had maximal activity at 50 C and 200 MPa. Samples treated at 100 and 200 MPa at 50 C had higher activity than sample s treated at atmospheric pressure or above 300 MPa. PME activity was optimal at 60 C, yet the presence of PG affected the activity at high pressures with 0.1 MPa having activity slightly higher tha n 200 MPa with PG present. A ll high pressures obtained hig her activity than 0.1 MPa with the absence of PG (Verlent and others 2007) High pressure has shown to decrease inactivation rates in t omato PME at ambient and inactivation temperatures, though citric acid buffer with a pH of 3.8 4 5 decreased the antagonistic effect pressure had against temperature. Finally PME activation has been achieved at different high pressures. Activation of tomato PME was found at temperatures of 59 60 C at 100 MPa (w ithout calcium ions) or 400 MPa (with calcium ions) (Hendrickx and others 1998) whereas others report tomato PME activation at 300 MPa (Hsu 2008) In addition to tomato, c arrot PME has been widely studied in HHP since this method can improve carrot texture quality. Carrot PME was stabilized around 500 MPa (Ly Nguyen and others 2003) Reported activation volumes at 30 to 55 C for purified carr ot PME were between 7.8 and 5.73 cm 3 1 and increased with temperatures greater than 40 C (Sila and others 2007) The small activation volumes confirmed the

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46 stability of t he enzyme to high pressure treatments. Activation energy (E a ) reported for carrot PME at thermally degrading temperatures showed no apparent trend with pressure 1 for 0.1 to 500 MPa treatments with no signi ficant decrease in E a with high pressure treatment at 200 to 500 MPa (31.6 1 ) than from 0.1 MPa treatments 1 ) (Sila and others 2007) Ortega and others, (2004) also investigated inactivation kinetics for the commercial blends of pectinase For temperatures of 40 60 C inactivation plots of residual polygalacturonase activity (natural log) versus time did not produce first order inactivation for commercial cocktails Pectinex 3XL (Novozyme) Pectinase CCM (Biocon) and Rapidase C80 (Gist Brocades) Therefore, a multi fraction first order model was fitted for the PG enzyme in the commercial products likely due to heat labile and heat stable forms in the mix tures. For Pectinex 3XL the same commercial product used in this research project, the heat labile form of PG was inactivated too quickly for inactivation rate calculation. The heat stable form of PG in Pectinex 3XL in thermal process studies had an act ivation energy of 160 kJmol 1 while the PG for heat labile and heat stable forms had energies of 92.4 and 145 kJmol 1 for Rapidase C80 and 166 and 76.6 kJmol 1 for Pectinase CCM (Ortega and othe rs 2004) Of the three commercial products studied, Rapidase C80 was the most heat tolerant due to the lowest activation energy for the heat stable form of PG. Taken together thermal treatments have varying effects on the activation energies of commerc ial pectinase products.

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47 Comparison HHP with thermal processing As described, HHP treatments can reduce the time or temperature of the thermal processing by stabilizing enzymes For microbial pectinases, treatments at 200 MPa and 45C and 300 MPa at 50 C h ave been proven optimal in A. aculeatus PME activity for rate of de esterifying apple pectin pH 4.5 At 0.1 MPa, the activity decreased with the increase in temperature above the optimal 45 C (Fraeye and others 2007) Also, researchers have found samples of A. aculeatus PME treated at 55 C and pH 4.5, at high pressures (400 and 700 MPa) for 30 min retained higher than 90% enzyme activity, while samples treated at the same parameters and atmospheric pressure retained less than 10% after 10 min trea tment At atmospheric conditions and pH 4.5, microbial Aspergillus aculeatus PME followed first order ki netics for inactivation at temperatures 46 56 C However, inactivation kinetics varied for enzyme source. Apple, tomato, and banana also followed first order, while strawberry and carrot demonstrated a biphasic thermal inactivation (Duvetter and others 2005) Maximum activity for commercial pectinase cocktails has been studied for thermal processing. The activity of PG in the commercial product was determined with the reducing sugar method. Pectinase 3X L (Novozyme) had the maximum activity at 50 C. Pectinex 3XL only retained 10% of initial activity after being thermally treated at 50 C for 1 hr; ot her commercial pectinases, Pectinase CMM (Biocon) and Rapidase C80 (Gist Brocades) retained 57 and 5 % activity (Ortega and others 2004) These results demonstrate that at moderate temperatures, p ectinases normally retain activity for an hour or less and activity can be stabilized with the application of HHP.

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48 HHP and m i croorganisms Un til recent years, a majority of the HHP research for all foods has centered on microbial inactivation. High pressure inactivates E. coli, Salmonella and Vibrio strains and is also an approved method for pasteurization by the USDA of L monocytogenes in pr ocessed meats (Balasubramaniam and others 2008) Current re search is still being conducted with pathogenic and spoilage microorganisms pertinent to fruit juices and the spores that are resistant to both thermal and pressure pasteurizations. Yeast organisms can be inactivated by pressures ranging from 300 600 MPa, but bacterial spores and pressure resistant bacteria are harder to destroy (Hendrickx and others 1998) Microbial spores are problematic due to their resistance to heat (moist and dry), radiation, a nd chemicals. One of the common mechanisms believed to inactivate spore concentrations is using high pressure and temperatures to cause germination and force spores to lose their resistance to processing (Black and others 2007) Injury to the cell membrane is considered to be the main mechanism to vegetative microbial inacti vation with pressure pasteurization. Cells that are actively growing are more sensitive to pressure than those in the stationary phase (Patterson 2005) Clostridium botul inum Clostridium perfringens and Bacillus cereus are all spore formers that can cause food borne illness i n humans Alicyclobacillus acidoterrrestris is a spoilage s pore former that can grow in acid ic foods like fru it juices (Black and others 2007) Many spore forming microorganisms cannot be treated with pressure alone, a nd thermal processing alone requires high degradation conditions for antioxidants and nutrients. Pressure assisted thermal sterilization is a focus for many researchers studying food preservation. Alicyclobacillus acidoterrestris spores can survive within a pH of 2.5 6 and temperatures o f 25 60 C These bacterial spores have D values (90%

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49 reduction times) of 15 23 min at 90 C or 2.4 2.8 min at 95 C. When tested with a pressure pasteurization process, the bacteria were reduced to less than 1 cfu/mL (5.5 log reduction) with a process of 414 or 621 MPa at 71 C for 10 mi n (Lee and others 2002) Pressure assisted thermal pasteurization of a 5 log reduction has been seen for C botulinum at conditions of 82 7 MPa, 5 min, 50 55 C (type E); 600 MPa, 70 min, 80 C (proteolytic type B); and 600 MPa, 6 min, 80 C (proteolytic type A) (Rendueles and others 2011). For less thermally resistant microorganisms, a pressure process of 400 MPa or higher and at 20 C or less can be used for fruit juice pasteurization and can inactivate E. coli O157:H7 a common pathogen in apple a nd orange juice (Patterson 2005) Researchers have tested pressure resistant E. coli and found that storage after the pressure pasteurization helped destroy the bacteria. After a 15 minute process at 20 C and 300 MPa, there was only a 1.1 log reduction of the pressure resistant bacteria E. coli LMM1010 H owever after the same treatment and a 2 d storage period, the bacter ia were reduced by almost 5 log The optimum process with storage afterwards, 15 min at 20 C and 500 MPa, in a juice of pH 4 had a 5 log reduction in the bacteri a count (Garcia Graells and others 1998) These results show that pressure can be used as an adequate pasteurization process. E. coli O157:H7 strains also show variation in their susceptibility to high p ressure treatment. Strain C9490 is more resistant to pressure than E. coli NCFB 1989, which was completely inactivat ed with pressure treatment of 350 MPa An addition al storage

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50 period of 24 h and high pressure treatments of 300 MPa, destroyed all survivors (Jordan and others 2001) Comparison with t hermal p asteurization Commercial thermal pasteurization processes are used to d estroy pathogenic and spoilage microorganism with high temperatures, but the thermal processing can damage antioxidants and vitamins. Spore forming microorganisms are the most difficult to treat with heat treatments. Alicyclobacillus acidoterrestris therma l resistance decreased without linearity with the increase in temperature. High temperatures also resistance. Resis tance to thermal processing varied between strains of A ac idoterrestris with D values for 90 C ranging from 7.38 to 20.80 min. The D value decreased with increasing temperature, with D values for 95 C ranging from 2.3 to 2.8 min (Smit and others 2011) Acid adapted E.coli O157:H7 showed higher resistance to thermal processing than stationary no n acid adapted microorganisms in apple, orange, and white grape juice. However as temperature increased, the D values for both acid adapted and stationary phase samples decreased; a low temperature of 56 C produced D values of 7.0 and 4.1 min for acid ada pted and stationary E. coli respectively an d a high temperature produced D values of 1.5 and 0.8 min res pectively in apple juice. The z value was slightly higher for acid adapted E. coli O157:H7 (z = 5.9 C) than stationary phase E. coli O157:H7 (z = 5.6 C). E scherichia coli O157:H7 had higher heat resistance than Salmonella and L. monocytogenes ; and a complete thermal pasteurization for all three microbes could be performed with a thermal process of 3 s at 71.1 C for a 5 log reduction (Mazzotta 2001)

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51 Pressure alone is not always effective in destroying resi stant endospores. However, a pressure assisted thermal pasteurization has the ability to lower the pasteurization temperature to reduce possible thermal degradation and/or heating expenses. Effects of High Hydrostatic Pressure on pH General History The f inal portion of this research involves the investigation of the shift in pH with HHP processing. During HHP treatment, pH should be monitored because it is a physical factor that affects the processing and food quality. The role of pH in high pressure proc essing is critical due to effects on proteins, microorganisms, as well as chemical reactions (Hayert and others 1999; Stippl and others 2004) Currently there are no commercial pH sensors for pressurized vessel s available C ommon pH sensors with glass and reference electrodes cannot withstand HHP processing Studies on the pH behavior of solutions at HHP will aid in producing accurate measurements for any future pH sensors designed for pressurized vessels. Acid thermodynamic principle demonstrates the relationship between the reaction volume, temperature and ionization constant (Bruins and others 2007) .The study of dissociation constants of weak electrolytes is important not only to fo od processing with high pressure, but the study of the science of high pressure. Researchers have conducted studies to determine the effects of HHP on dissociation constants with concentration cell emf (electromagnetic force or voltage ), optical density, a s well as density and fluorescent measurements (Hayert and others 1999) Method and models have been developed for prediction of pH with equilibrium constants that are often unknown for

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52 food systems. Yet pressure will affect pH with the volume change that directly impacts equilibrium constants (Stippl and others 2004) An increase in the ionization constant with pressure can lead to an acidic pH shift (El'yanov and Hamann 1975) Reports vary on the exact amount of pressure required to affect pH. The effect of 100 MPa at room temperature caused a shift in the pH of water from 0.16 pH units to .073 pH uni ts (Hayert and others 1999) Compressibility and densi ty are two physical factors that are associated with pH and their behaviors under pressure are more commonly noted than pH behavior (Min and others 2010) The NIST Chemistry WebBook provides the compressibility of water up to at least 900 MPa (USNIST 2008) Another fa ctor affected by the change in pH with pressure is a solution s absorbance levels. Absorbance levels are affected with an increase in pressure as HHP compresses and decreases the volume (Hayert and others 1999) Color indicators absorb light at a specific wavelength, and produce single or multiple peaks in th e visual light range of 400 700 nm. Acid/base color indicators have a pH range in which colors shift with the shift in pH; some examples are Bromocresol Purple that shifts from yellow at pH 5.2 to purple at pH 6.8 and Metanil Yellow that shifts from hot pi nk at pH 1.2 to orange at pH of 2.4. The combination of compression of water and the change in the absorbance of light by color acid/base indicators together can be used to crea te pH calibration charts for determining the change in pH with the application of pressure. Color acid/base indicators are solutions that have been used to observe the effect of pressure on pH with their change in the transmission of light (Stippl and others 2004)

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53 The b uffer s sensitivity to pressure and temperature and thus its potential shift in pH at high pressure are important consideration s when choosing a buffer for high pressure studies The need for use of barostable buffers in high pressure research is often overlooked. Some buffers are temperature sensitive yet resistant to pressure, such as ACES (N (2 acetamido) 2 aminoethane sulfonic acid) and TRIS (2 amino 2 hydroxymethyl 1,3 propanedio l). Yet other buffers are sensitive to pressure; phosphate buffer (initial pH of 7) drops to pH 5.4 at 600 MPa (Bruins and others 2007) Researchers have used a wavelength intensity ratio of Fluorescein to determine the change in pH with the increase in pressure for water and buffers (Hayert and others 1999) An acidic shift, or decrease in pH was seen for all solutions tested except for MES buffer; the largest shift was seen with orthophosphoric acid with a change of 0.92 pH units at 200 MPa. Distilled water and acetic acid started with initial pH of 5 .8 and 4.1 respectively. Shifts in pH of 0.30 and 0.22 pH units at 100 MPa and 0.31 and 0.40 respectively at 200 MPa were observed (Hayert and others 1999) These results were similar to values seen in previous literature (Owen and Brinkley 1941; Distche 1972) Current Research Thermodynamic models that considered molar volumes, equilibrium constants and activity coefficients have been used to predict change in pH in CO 2 systems as well as orange juice at high pressure The models accurately predicted a pH decrease of a water and CO 2 solution of 2.53 pH units when p ressure was increased from 0 to 5.516 MPa. However, the model did not accurately predict the behavior of orange ju ice, (Meyssami and others 1992) Color indicator systems with up to 16 indicators encompassing a pH ra nge of 1 10 have been used with chemometric models (principal component regression and partial least squares) to predict pH behavior at high

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54 pressures. With the large pH indicator system, pH at high pressures was predicted with the spectrometric prediction s at HHP minus the spectrometric prediction for atmospheric pressure and pH measured value at atmospheric pressure (Stip pl and others 2004) Other indicator systems to observe the effect of pressure on pH behavior have been developed. Dual wavelength indicators LysoSenser Yellow/Blue DND 160 as well as seminaphthofluorescein and seminapthorhodafluor utilize ratio of two wavelengths to determine pH at HHP (Salerno and others 2007; DePedro an d Urayama 2009) Though several systems and models have been produced to quantify the shift in pH with high pressure, variations in predictions show that research is still need ed to confirm actual shifts. Gap of Knowledge Juice Processing C larif ication at atmospheric pressure and thermal pasteuriz ation of fruit juice s are well established industrial technologies New techniques beyond thermal processing to retain nutritional value, organoleptic properties and aromas are being explored to improve apple and fruit juice quality. However, there are still unknown factors that should be investigated before novel research can move into commercial practice. Mechanisms for microbial and enzymatic inactivation and a ctivation need to be elucidated for HHP and other no n thermal processing. While knowledge has advanced on how to retain nutritional qualities of juice, f urther research is needed to improve antioxidant and vitamin retention during the complete juicing process while manufacturing a safe and quality product Finally the effects of high pressure therm al assisted processing on nutritional and flavor characteristics of fruit juice need to be further documented

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55 High Hydrostatic Pressure Processing There are several gaps in knowledge that remain for HHP juice processing. First the activity of pectinase enzymes varies greatly T he formula of the enzyme mixture, pressure applied, temperature, pH and matrix ( whether it is food or buffer ) are some of the important factors to consider when using HHP For industr ial processing, the behavior of commercial pectinases at moderate and high temperatures has yet to be explored in combination with high pressure processing. Secondly the need exists for an enzymatic activity quantification of the overall activity of a mixtu re of enzymes to determine the effec t of high pressure treatments on the enzyme mixture as a whole The rate of v iscosity reduction is a parameter that can be used for enzymatic activity. Also, the few studies using viscosity reduction all have variations in assay technique. F urther experimentation with viscosity reduction to ensure an accurate and consistent representation of enzyme activity for a commercial pectinase cocktail is needed Lastly assessing antioxidant and vitamin stability in a pressure ass isted thermal pasteurization process es is required to determine whether the cost benefit of a non thermal technology justifies the investment. Shift in pH with HHP Theoretical equations have been derived and experimentation has been performed to predict p H shift at high pressures. However, w ithout a high pressure vessel equipped with a pH sensor all pH shifts can only be estimated with simple solutions. Determination of pH shifts in situ is crucial in helping characterize this phenomenon. Colored pH indic ators have been studied to quantify this shift in pH

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56 however their respective dissociation constant behavior in HHP treatments has not been studied. Predictions of pH shift with any absorbance or reflectance values can only be applied to optically clear solutions which is rare in most food systems. Model s for pH shift can be limited when applied to food systems due to the complex nature of food matrixes that affect the pH. Additionally, the combined effects of pressure and temperature on food systems pH should be further evaluated for equilibrium constants of organic and weak acids which vary in concentration and could be affected differently with HHP processing Summary Over the past several decades a great deal of progress had been made to propel HHP processing from a novel technology into a commercially viable process used worldwide. Research has highlighted relevant effects of high pressure on microbial activity, enzymatic activity and pH behavior, all of which are important factors for commercial fo od processing. With advances in research, high pressure is a suitable treatment for apple juice that could allow the combination of juice clarification and pasteurization with a high pressure assisted thermal process. Without studies that optimize juice cl arification and pasteurization with pressure assisted thermal processing, a true comparison to thermal processing cannot be made for each specific juice product. Juice clarification experiments must be performed with commercial pectinase blend for valid HH P results, since industry does not process juice with only individual pectinases. Finally, without a constant quantitation of shift in pH with HHP processing, high pressure sensors will be difficult to manufacture without accurate calibration charts

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57 availa ble. T he use of high pressure should be further investigated to improve and enhance commercial food processing. Objectives This project focused on testing a HHP process to stabilize pectinase enzymes at raised temperatures above levels customarily used for clarification. Kinetic behavior of enzyme inactivation and viscosity reduction can be used to optimize HHP treatments for juice clarification. This high pressure, high temperature process could be used to clarify and pasteurize apple simultaneously. The d ata from this project c ould benefit the apple juice producers and consumers alike by testing the feasibility of combining the clarification and pasteurization processes. The project will focus on the clarification process with enzymes specific for the pol ysaccharide pectin molecules. The purpose of this study is to find a pressure, temperature, and processing time combination that will stabilize and activate a mixture of commercial pectinases to reduce the clarification time and required amount of enzyme u sed in apple juice processing. In virtue of the increased temperature, we also anticipate reduction of pathogenic microbial popu lations to a safe level. This research is expected to translate into operating costs savings and increased product quality. The final portion of the projec t is to observe and quantify the change in pH with the absorbance of color acid/base indicator water solutions. Specific Objective 1 To determine the effect of HHP on the stability of a commercial pectinase cocktail at selected h igh temperatures for pectinas es used for juice clarification.

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58 Specific Objective 2 To maximize the activity of a pectinase cocktail at HHP for optimal clarification processing that minimizes thermal degradation of pectinase enzymes. As preliminary results for future experimentation, inactivation of E. coli K12 will be performed at inactivating conditions to determine if complete pasteurization is obtainable with clarifying treatments. Specific Objective 3 To assess pH behavior under high pressure conditio ns with the visible spectrum produced by acid/base color indicators and monitored with a high resolution spectrometer.

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59 Figure 1 1. Overview of apple juice p roduction to obtain either cloudy or clear juice

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60 Table 1 1. Turbidity values for p roces sed j uices Juice Process Turbidity Reference Apple Juice Fresh centrifuged 259 436 NTU (Araya Farias and others 2008) Depectinized centrifuged 8.9 9.5 NTU (Araya Farias and others 2008) Depectinized centrifuged, and electrofloatation 7.9 10.2 NTU (Araya Farias and others 2008) Depectinized centrifuged, and electrofloatation wi th gelatin 3.4 NTU (Araya Farias and others 2008) Banana Enzyme treated and centrifuged 600 5000 NTU (Vaillant and others 2008) Blackberry Grinded/sieved and enzyme treated 9600 16200 NTU (Vaillant and ot hers 2008) Pressed and enzyme treated 350 750 NTU (Vaillant and others 2008) Cherry Non Clarified 55.6 F N U (Pinelo and others 2010) Clarified 11.0 F N U (Pinelo and others 2010) Clarified centrifuged 5.79 F N U (Pinelo and others 2010) Clarified, centrifuged, and filtered 2.75 F N U (Pinelo and others 2010) Pineapple Non treated 1500 4000 NTU (Vaillant and others 2008) Enzyme treated and centrifuged 150 600 NTU (Vaillant and others 2008) Notes: Standard clarification and processing vary slightly by juice and research team for specific processing conditions ; please note original research paper noted in the refere nce section.

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61 Figure 1 2. Basic summary of pectinase behavior: (a) R = COOCH3 and X = COOH, (b) X = COOH, (c) R = COOCH3 or COOH. Enzymes cut pectin molecule at position highlighted with arrow. PME, pectin methyesterase; PG, polygalacturonase; PL, pect in lyase. 1

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62 Table 1 2 E nzymes in juice clarification research a Viscosity Reduction b Reducing Sugars Method c Titration of Carboxylic Groups d Absorbance Readings at 235 nm and e Dinitrosalicylic acid absorption method Origin Enzyme Assay Assay Conditions ( C) Activity Optimum Conditions Comments Reference A. niger T0005007 2 VR a 30 75 U mL 1 40 C (Sandri and others 2011) Exo PG RS b 35 14 2 U mL 1 PME T c 30 0.03 U mL 1 PL A d 4 0 432 U mL 1 A. oryzae IPT 301 Total Pectinase Activity VR a 30 44 U mL 1 40 C (Sandri and others 2011) Exo PG RS b 35 40 U mL 1 PME T c 30 0.29 U mL 1 PL A d 4 0 180 U mL 1 A. nig er ATCC9642 PG DNS e 40 51.82 U mL 1 pH 5.5 and 37 C (Gomes and others 2010) Penicillium sp. CGMCC 1699 Endo PG DNS e 40 815.5 U mg 1 pH 3.5 and 40 C Expressed in the yeast Pichia pastoris (Yuan and others 2011) Bispora sp. MEY 1 Endo PG DNS e 55 1520 U mg 1 pH 3.5 and 55 C Expressed in the yeast Pichia pastoris (Yang and others 2011)

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63 CHAPTER 2 HIGH HYDROSTATIC PRE SSURE DECREASED THE RATE OF THERMAL INACTIVATION OF A PE CTINASE COCKTAIL To the best of our knowledge, the effect of HHP on the stability of commercial microb ial pectinases has not been studied. We hypothesize that using a HHP stabilized pectinase cocktail for fruit juice clarification in which temperature can be increased above the levels currently possible at atmospheric pressure could allow several processin g benefits. These possible benefits include shortening production time s ; reducing required amounts of enzyme needed for clarification; and if com bined with juice pasteurization, could lead to economically viable higher quality products. The objective of th is study was to determine the effect of HHP on the stability of a commercial pectinase cocktail at selected high temperatures. Materials and Methods Materials Pectinases from Aspergillus niger in an aqueous solution (Pectinex 3XL Product No P2736 Novoz ymes, Napa, CA ) was purchased from Sigma Aldrich (St. Louis, MO, USA). The commercial cocktail of enzymes contains pectintranseliminase, polygalacturonase, pectinesterase, and smaller portions of cellulases and hemicellulases. Sodium Citrate and Citric Ac id were both obtained from Fisher Scientific (Pittsburg, PA, USA). Pectin (Product No. P 9135) from citrus fruit was obtained from Sigma Aldrich (St. Louis, MO USA) and dissolved to make a 1.5% (w/v) solution in ultra filtered water. Equipment All high p ressure equipment was from Unipress Equipment (Warsaw, Poland). Samples were submerged and treated in a high hydrostatic pressure cell Model U111

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64 with silicone oil serving as the pressure medium. The high pressure cell was pressurized with a micropump mode l MP5 and controller. The high pressure chamber was jacketed to control temperature with alternating water baths model Isotemp 3016D from Fisher Scientific (Pittsburg, PA, USA) and pinch valves described previously in (Eisenmenger and Reyes De Corcuera 2009a) One water bath was set at 10 C and the other at desired process temperature. A computer program written in LabVIEW v 8.5 and data acquisition board model DAQ Card 6062E from National Instrumen ts (Austin, TX USA) were used to control and record pressure, temperatures and processing time. A temperature controlled cone and plate viscometer rheometer with a Wells Brookfield Cone and Plate and CP 40 cone spindle, model LVDV II+Pro and Rheocalc sof tware from Brookfield Engineering Laboratories, Inc. (Middleboro, MA USA) were used to record the viscosity reducti on of pectin solutions at 45 C. The jacketed cup was temperature controlled with a water bath also from Fisher Scientific A photo of the la boratory setup is provided in Figure 2 1. Methods Sample prepa ration and HHP processing The pectinase solution was d iluted in citrate buffer to 0.5 % w/v (15 unit/mL) and aliquots were place in 1 mL plastic pouches, heat sealed, and placed on ice unti l trea te d. Enzyme solutions were made with 0.1 M citrate buffer pH 3.5 B uffers and the enzyme cocktail were held at separately 4 C before use, and placed on ice when enzymes were added to citrate buffer. For treatment, a pectinase aliquot was placed in the hig h pressure cell, held at 10 C, and the pressure cell was closed. Pressure was raised to the process set point. Then, temperature was raised to the incubation set point and when 90% of the change in temperature was reached, processing time started.

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65 After p rocessing, the pressure cell was cooled back to 13 C, depressurized, and then the sample was placed on ice. Residual enzyme activity was assayed on the same day as the HHP treatment. Processing conditions Samples were treated at 0.1 MPa (control) or 200 t o 400 MPa at 50 MPa increments, 55 to 85 C at 7 to 8 C increments, and for a processing time of 0 to 45 min. The processing temperatures were 55 C, 62 C, 69.3 C, 77 C, and 85 C; temperature increments were chosen for even distribution of T 1 for the calculation of activation energy with the Arrhe nius equation. Ramp up and ramp down times wer e accounted for as follows. Ramp up time included pressurizing and heating the cell to 90% of set point temperature. Ramp down time included cooling the pressur e cell to 13 C followed by depressurization to atmospheric pressure. To determine 100% residual activity with an incubation time of 0 min, samples were cooled and depressurized immediately after reaching the set point pressure and 90% of the set point tem perature. Figure 2 2 shows the pressure and temperature profile for a sample treated at 69.3 C, 250 MPa, and 15 min. Samples of this time series data were processed in duplicate The study was performed in a randomized block design with temperature treate d as blocks. Pressure and process times were randomly selected. Activity measurements To assess the activity of the enzyme cocktail, 132 L of the treated enzyme sample was added to 1,865 L of pectin solution and stirred for 40 s with a miniature magneti c bar and stirrer. The mixture was then immediately withdrawn with a syringe and injected into a three way valve attached to a viscometer cup port, in order to record the viscosity reduction as soon as the mixing period had been completed. The viscosity

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66 of pectin was recorded every 1.2 s for 10 min with a maximal viscometer rotational speed of 20 rpm as the treated enzyme cleaved pectin molecules reducing the viscosity. Samples that contained pectin only or pectin with only citrate buffer were run for 5 min to check pectin consistency. The rate of viscosity reduction was used as the method to determine the treated enzymes residual activity after high pressure processing. The initial rate of viscosity reduction was not used as the parameter to measure enzym e activity because of the inconsistencies that occurred during the mixing process. Therefore, a pseudo second order rate of reaction was used to determine the rate of viscosity reduction. To determine the maximum rate of vi scosity reduction with a pseudo s econd order reaction, the inverse viscosity was plotted against assay time as seen in Figure 2 3 The maximum slope for a period of 60 s was taken as the maximum rate of viscosity reduction (the rate constant for viscosity reduction). The maximum rate cons tant of viscosity reduction (Pa 1 1 ) for each sample was used as measurement of the Figure 2 4 shows a pred icted viscosity from the pseudo second order rate of viscosity reduction plotted with the observed viscosity reductio n. The percent residual activity was calculated with respect to process time of 0 min (k 0 ). Rate of enzyme inactivation The rate of enzyme inactivation was determined from the relationship between residual activity and HHP treatment time. Orders of reacti ons 0, 1 st and 2 nd were analyzed for pectinase inactivation. N o order was a definitive fit for all temperature blocks and pressures. However, a pseudo first order reaction was the best fit for most treatments or sometimes second best fit for all pressure levels. Therefore, pseudo first order was used to calculate the rate constant of inactivation as discussed in the results

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67 section. This selection of order of inactivation was also pertinent for the purpose of comparing our results with those in the litera ture. Indeed, most enzyme inactivation studies are reported as first or pseudo first order reactions. 2 1) was used to estimate the activation volume where k o [min 1 ] the activation volume [cm 3 mol 1 ] R the universal gas constant (8.3145 J mol 1 K 1 ) T the absolute temperature [K] P the pressure [MPa] and P 0 the reference pressure of 0.1 MPa Arrhenius equation (Equation 2 2) was used to estimate the activation energy: where k [min 1 ] is the rate constant of enzyme inactivation E a the activation energy [ kJ mol 1 ] and T 0 is the reference temperature Results and Discussion Activity Measurements With the addition of pectinase cocktail, the viscosity of the pectin solution decr eased as seen with Figure 2 4 Viscosity reduction was selected as a measurement o f the overall activity of the cocktail because in practice, in the clarified juice process, it is the desired effect. Also, the activity of individual enzymes contained in the cocktail was not determined because the intent of this research was to assess th e impact of HHP on a commercially available cocktail. The maximum rate of viscosity reduction was

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68 chosen as the parameter to quant ify residual activity. O rders of reactions 1, 1.7, 2, 2.3 ff and Powell methods were used to determine the best fit (Smith 1981; Masel 2001) None of the proposed viscosity reduction is complex reaction. However, a pseudo second order reaction fitted best the maximum rate of viscosity reduction with high correlation coefficients (r 2 > 0.762) for samples that maintained activity and best predicted the initial viscosity better than previously tested orders. The predicted viscosity reduction using this rate constant is shown by the continuous line in Figure 2 4 The prediction from the pseudo second order only accounts for the viscosity reduction, not the initial mixing perio d. A linear relationship between t he changes in viscosity of pectic acid and the logarithm of the amount of enzyme, pectin depolymerase has been reported (Roboz and others 1952) Viscosity red uction had also been fitted to exponential models (Ceci and Lozano 1998) However ou r results did not fit an exponential trend. Despite their practical relevance these methods are not often used in current research methods, because a correlation has not been found between the reductio n in viscosity and hydrolysis of glycosidic bonds by po lygalacturonases (Jayani and others 2005) Yet, the quantification of the hydrolysis of glycosidic bonds is not a useful parameter for industry as it does not directly relate to the characteristics of fruit juices or pastes such as viscosity or turbidity. Residual En zymatic Activity For this study, enzyme activity was report ed as residual activity after treatment or as the rate of enzyme inactivation in order to compare results to previo usly published literature. T emperature treatments produced some irreversible inact ivation of the pectinase cocktail in that no pressure/treatment combination maintain ed 100% activity

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69 through the longest process time for each temperature block. At lower temperatures of 55 C and 62 C, activity over 22.5% was maintained for all sa mples t reated at atmospheric or high pressure. For temperatures of 69.3 C to 85 C, samples treated at high pressure maintain ed activity for longer process times than samples treated at atmospheric pressure as seen in Figure 2 5 At 69.3 C, samples treated at atmospheric pressure for 4.5 min only retained an average activity of 33.2%, while high pressure samples re tained at least 11.2 % activity for 45 min. High temperatures of 77 C and 85 C samples treated at 0.1 M Pa maintained only an average of 2.5% and 10 % residual activity for 1.5 mi n and 0.75 min respectively, in contrast to samples treated at 200 MPa that had residual activity of at least 2.2% and 7.4% for 15 and 6 min respectively. As process time increased for each temperature block, residual activi ty tended to decrease. Variation from this general decrease was seen for lower temperatures of 55 C and 62 C. At 55 C, activation of enzymes can be seen for 200, 250 and 300 MPa at the 15 min process times as seen in Figure 2 6 Residual activity reache d 106.4%, 119% and 102 % for 200, 250 and 300 MPa respectively for 15 min. However, after longer incubation times up to 45 min, an average of 49% residual activity was observed for samples treated at 0.1 MPa, and 57.8% for high pressure samp les. Stabilizat ion also occurred at lower temperatures, such as 62 C 400 MPa and 45 min, in which activity was higher than the previous 30 min time point This is an indication that the enzymes in the cocktail could be affected by different pr essure and time conditions. The difference between high and lower temperatures of the study was illustrated in Figure 2 4 for temperature blocks 55 C and 85 C Residual activity did not change greatly with the increase of pressure. From 200 to 400 MPa, resi dual activity fluctuated with a 0.61 to

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70 50.28% difference between samples with no defined tre nd; this could be explained by the large experimental error in processing and being located at edge of the elliptical pressure temperature diagram in which enzymes begin to denature. The general pressure temperature diagram for enzymes h as been previously summarized (Eisenmenger and Reyes De Corcuera 2009b) For 55 C and 62 C, samples treated at atmospheric pressure maintained steady activity after the initial decrease of 49.0% to 56% and 22.6% to 32.0% respectively for the entire process time from 15 to 45 min. Atmospheric pressure samples however had less residual activity than samp les treated at high pressure. For s amples treated at higher temperatures (69.3 C and greater), residual activity decreased faster for atmospheric pressure samp les. Samples treated with high pressure maintain ed activity 10 time s longer than atmospheric pressure samples for 69.3 C and 77 C and 7.9 times longer for 85 C High pressure has a stabilization effect on the pectinase cocktail; the effect is more pronounced for higher temperatures. Moderate temperatures and high pre ssure combinations, that do n ot produce thermal degradation, provide the best results for industrial processing. Pressure processing resulted not only in a stabilization effect but in an activation effect for short process times, 15 min in the case of this research. Most pectinase pressure and temperature stability studies focus on PME and PG (Hendrickx and others 1998; Duvetter and others 2009 ; Eisenmenger and Reyes De Corcuera 2009b) Tomato polygalac turonases was inactivated at 300 to 500 MPa, while tomato PME was stable up to 700 MPa (Rodrigo and others 2006) The activity of orange juice PME decreased with increased pressure, temperatures and processing

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71 times. Samples treated at atmospheric pressure for 15 min at 37.5 C had a residual activity of 89.9%, while samples t reated for 12 min at 50 C only retained 65%. Samples treated at 400 MPa, 50 C for 12 min had a residual activity of 50.1%. Orange PME inactivation increased with the increase in temperature as well as the increase in pressure (Nienaber and Shellhammer 2001) The two tomato enzymes, PME and PG also affect the behavior of each other. Verlent and others, (2007) reported that tomato polygalacturonase in the presence of highly esterified pectin and PME at pH 4.4 had peak activity at 50 C and 200 MPa, after which activity declined. This research confirms tomato PGs can be inactivated at pressures greater than 300 MPa. Tomato PME a lso affected the activity of PG Maximum PME activity was see n at 60 C and ambient pressure or at 50 C and 200 MPa with the presence of PG; however, with the absence of PG, PME peak activity was seen at 60 C and 400 MPa. Researchers have hypothesized the that shorter pectin chains created by tomato PG, may make better substrates for PME and increase its activity (Verlent and others 2007) These results differ from our inactivation study results in which high pressure treatments had greater activity than samples treated atmospheric pressure above 55 C. There are multiple enzymes within the pectinase mixture; t he behavior of each enzyme could be affecting mixture as a whole creating a n overall stabilization effect on total activity. Studies have also focused on the activity of commercial enzymes and microbial PME. For microbial PME, similar conditions of 200 M Pa at 45C or 300 MPa at 50 C were reported as optimal in A. aculeatus PME activity for de esterifying apple pectin. At 0.1 MPa, the activity decreased with the increase in temperature above the optimal 45 C (Fraeye and others 2007) Maximum activity for commercial pectinase cocktails has

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72 been studied for thermal processing at atmospheric conditions ; Pectinase 3XL the same cocktail used for this study had the maximum activity at 50 C (Ortega and others 2004) Pectinex 3XL only retained 10% of initial activity when treated at 50 C for 1 h; other commercial pectinase Pectinase CMM and Rapidase C80 retained 57 and 5% activity (Ortega and others 2004) .These studies agree with our research that high pressure is stabilizing pectinase enzymes that are sensitive to high pressures and temperatures. Rate of Enzyme Inactivation The rate of pectinase inactivation was fitted to a pseudo first order rate of reaction. Zero, first and second orders were compared with first order rate providing the best fit for most experi ments or second best fit for all temperature and pressure combinations. First order kinetics for enzyme inactivation have been used to describe orange PME as well as A. niger PME in apple and cloudberry juices (Nienaber and Shellhammer 2001; Wilinska and others 2008) Ortega an d others, (2004) also investigated inactivation kinetics for the commercial blends; for temperatures of 40 60 C inactivation plots of the natural logarithm of residual polygalacturonase activity versus time did not produce linear results but rather the co mbination of two separate linear regions due to different forms of PG present. A multi fraction first order model was not suitable for the PG of Pectinex 3XL inactivation; a rate constant could not be calculated for heat labile form due to the sudden inac tivation, but the heat stable form of PG was fitted with a first order model. Rapidase C80 and Pectinase CCM were fitted with two fractional first order models for polygalacturonase activity (Ortega and others 2004) With viscosity reduction as an indirect form of the total pectinase activ ity, our experimental data showed a linear firs t order model could be used to fit inactivation rates for the pectinase

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73 cocktail with temperatures as low at 55 C. Research pertaining to specific enzymes, such as PG from Pectinex 3XL showed that only one form of PG may be contributing to the overall p ectinase activity. Rate constants of enzyme inactivation were shown in Table 2 1. At atmospheric pressure, the rate of enzyme inactivation increased as temperature increased. The same ge neral trend was noted for high pressures. Rates of inactivation at 55 and 62 C were under 0.03 min 1 at all pressures and rates of inactivation increased with the increase of temperature for 69.3, 77, and 85 C. Figure 2 7 show s the increase in inactivation rate for samples treated at 250 MPa. The larger inactivation rates at higher temperatures showed the effect of temperature inactivation increases for temperatures from 69.3 to 85 C for all pressures. The coefficient of correlation for pressures treated at 69.3 C and 77 C were equal or greater than 0.80, while R 2 values for 85 C were equal or greater than 0.90. For atmospheric pressure, the rate of enzyme inactivation was faster than all s amples treated at high pressure. This result indicates that high pressure stabilizes the pectinase cocktail at all temperatures stud ied in the results presented. Figure 2 7 graphs natural logarithm of residual activity versus process time to obtain rates of inactivation shown in Table 2 1. High pressure slowed the rate of inactivation b y 40.4%, 81.9%, 43.9%, 20.0% or 37.7% at 200, 250 300, 350 or 400 MPa respectively compared to samples treated at atmospheric pressure at 55 C. The rate of inactivation was slowed by over 83% for all high pressure samples treated at 69.3 to 85 C. The rate of inactivation for orange PME was reported t o increase with temperature and pressure. Samples treated a 400 MPa and 25 C were inactivated at a rate of 0.0197

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74 min 1 while an increase in pressure to 600 MPa increased the rate to .3308 min 1 The largest rate of inactivation was seen with the highest pressure temperature combination of 600 MPa and 50 C, with a rate of 0.50 min 1 (Nienaber and Shellhammer 2001) Although these results appear to contradict ours, the pressure levels used by (Nienaber and Shellhammer, (2001) were higher which explains the pressure induced enzyme inactivation. The rate of inactivation of PME from A. niger increased with the increase in temperature from 52 66 C following first order kinetics in apple juice and cloudberry juices. The rate of inactivation in 0.1 M sodium acetate buffer with a pH 3.6 was 0.010 to 0 .870 min 1 from the previously stated temperatures while in fresh and industrially produced apple juice the inactivation rate constants were 0.022 to 1.143 min 1 and 0.012 to 0.654 min 1 for temperature range of 54 to 66 C (Wilinska and others 2008) Orange PME at 400 MPa and PME from A. niger in apple juice had similar rates of inactivation for temperatures of those tested in this research study (55 t o 85 C) The pectinase cocktail also had smaller i nactivation rates at 62 and 69.3 C than those observed for A. niger at 66 C, which showed that the pectinase cocktail was more stable at atmospheric and high pressure than PME alone. Effect of Pressure on the Rate of Inactivation The Eyring plot (Figur e 2 8 ) shows the natural logarithm of the rate constant of enzyme inactivation (K inact ) plotted against pressure to obtain an apparent activation volume for each temperature. The trend is not linear throughout the pressure range. At pressure above 250 MP a, the curve levels off suggesting that beyond that pressure, pressure no longer has an effect on the overall stability of the enzym e cocktail in slowing thermal inactivation. This is in agreement with the earlier studies at 400 MPa and above on PME from d ifferent sources in which the rate of inactivat ion increased

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75 with pressure. A pressure induced inactivation could be anticipated from the trend shown in Figure 2 8 in higher pressures (> 400 MPa) Therefore, the activation volume of stabilization can only be calculated for up to 250 MPa. The calculated apparent ) had a small decrease from 55 C to 62 C, and increased with temperature reaching a maximum at 77 C; pressure had a greater effect on the enzyme as the temperature increases to 77 C with volume of 33.72 cm 3 mol 1 indicated in Figure 2 9 With positive activation volumes, pressures up to 250 MPa slow thermal inactivation, with the greater effect seen at 77 C. Also, the pressure range s tudied (200 400 MPa) was narrow, thereby inducing only small changes in activity between individual high pressure treatments. Future research should include lower pressures to better cor relate the effect of pressure on the stability of the pectinase cocktail. Reported activation volumes at 30 to 55 C for purified carrot PME were between 7.8 and 5.73 cm 3 mol 1 and increased with temperatures greater than 40 C (Sila and others 2007) Inactivation volumes for orange P ME were 30.9 to 35 cm 3 mol 1 with an increase in volume with the increase in temperature 25 50 C (Nienaber and Shellhammer 2001) The order of magnitude of these results wa s similar to this research. Even though we did not observe negative activation volumes, apparent activation volumes inc reased with temperature for carrot PME, orange PME and the results presented here for temperatures up to 77 C. Wi th a positive activation volume pressure slows thermal inactivation of the pectinase cocktail; the opposite trend was seen for carrot and ora nge PME. Effect of Temperature on the Rate of Inactivation The effect of temperature on the rate of inactivation of the pectinase cocktail was studied using Arrheni us approach as shown in Figure 2 10 in which K inact stands for the

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76 rate constant for enzyme inactivation. At all pressures, apparent Arrhenius behavior was observed as confirmed by the linear relationship between the logarithm of the rate constant and the reciprocal of temperature. The apparent activation energy of inactivation at atmospheric pr essure was 195.6 kJmol 1 Temperature had less effect at high pressure with apparent activation energies of 107.3 154.4 kJmol 1 than at atmospheric pressure, in particular at 300 to 400 MPa where the lowest activation energies were calculated Activati on energies at sel ected temperatures are shown in Figure 2 11 Error was reported using the standard error of the slope from the linear regression of Arrhenius plots. Therefore, in the ranges of temperature and pressure studied here, these two variables ha ve antagonistic effects on the rate of inactivation of the pectinase cocktail. Activation energy reported for carrot PME showed no apparent trend with values 1 for 0.1 to 500 MPa with no significant decrease in E a with high pressure treatment at 200 to 500 MPa (31.6 1 ) than from 0.1 1 ) (Sila and others 2007) Activation energies of orange PME, 13.5 30.1 kJmol 1 decrease with the increase in pressure, 400 to 600 MPa (Nienaber and Shellhammer 2001) Carrot and orange PME activation energies were generally lower for those obtained for this pectinase study (107.3 1 ) which can be explained by the use of a cocktail with multiple enzymes. The hea t stable form of polygalacturonase in Pectinex 3XL in thermal process studies had an activation energy of 160 kJmol 1 while the PG for heat labile and heat stable forms had energies of 92.4 and 145 kJmol 1 for Rapidase C80 and 166 and 76.6 kJmol 1 for Pectinase CCM (Ortega and others 2004) Apparent activation energy for Pecti nex 3XL calculated by

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77 Ortega and others (2004), is in the same order of magnitude as seen with the apparent activation energy for 0.1 MPa samples with this research. This result was expected ; though activity assay s were differen t for each study, the react ion wa s accelerated in the same way. Apparent activation energies for individual enzymes are smaller than those seen for commercial products indicating that the collective or one specific enzyme of the pectinase was more affected by temperature. For the pe ctinase product, an increase in temperature can affect the activity of one enzyme, which in turn affects the overall viscosity reduction and activation energy. The activity and stab ilization of lipase in hexane was recently studied in our laboratory using the same HHP system and similar conditions (Eisenmenger and Reyes De Corcuera 2009a) At low pressure (below 10 MPa) an E a of 42.7 kJmol 1 was reported, while at 400 MPa E a of 42.36 kJmol 1 was ob tained showing no significant difference from low pressure. Activation energies for lipase were lower than apparent activation energies found in this study for pectinases by an order of 10. Observing variation between apparent activation energies from atm ospheric pressure and high pressure, but little variation in lipase in hexane at different pressures indicates the high pressure conditions is dependent not only on temperatu re but dependent on the specific enzyme, solvent, and mobilization conditions as well.

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78 Fi gure 2 1. High pressure laboratory equipment set up. The high pressure piston pump is located on the bottom shelf of the cart, the HHP controller is located on th e middle shelf, and the water baths are on the top of the cart. The two HHP cells are located of the top of the cart with the smaller optic al cell circle d on the left an d the larger blind cell circled on the right. The HHP setup is controlled and monitored with the computer on the right Figure 2 2 treated at 69.3 C, 250 MPa, 15 min.

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79 Figure 2 3. Maximum rate of viscosity reduction with a pseudo second o rder rate of reacti on plot for p ectinase treated at 69.3 C, 250 MPa, 30 min with a rate consta nt for viscosity reduction of 0.127 Pa 1 s 2 and R 2 = 0.993. Figure 2 4. Observed and predicted viscosity for sample treated at 69.3 C, 250 MPa, samples, ( pectin + citrate b uffer 137 138 139 140 141 142 143 144 145 146 490 500 510 520 530 540 550 560 570 (Viscosity) 1 (Pas) 1 Time (s)

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80 Figure 2 5. Average pectinase residual activity for samples treated at 55 C and 0.1 300 MPa (X). Figu re 2 6. 0.1 MPa ( ), 200 MPa ( ), 250 MPa ( ), 300 MPa (X), 350 MPa ( ), and 400

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81 Table 2 1. Rate constant of enzyme i nactivation ( min 1 ) standard e rror (n = 2) for six pre ssure and five temperature treatments. Pressure (MPa) Temperature 55 C 62 C 69.3 C 77 C 85 C 0.1 0.015 0.006 0.029 0.011 0.281 0.100 2.331 0.743 3.067 0.644 200 0.009 0.003 0.008 0.004 0.028 0.007 0.244 0.066 0.433 0.091 250 0.003 0.011 0.010 0.003 0.028 0.008 0.125 0.021 0.282 0.020 300 0.008 0.003 0.007 0.006 0.024 0.006 0.120 0.030 0.201 0.032 350 0.012 0.004 0.011 0.004 0.047 0.011 0.194 0.018 0.205 0.049 400 0.009 0.004 0.012 0.009 0. 026 0.002 0.080 0.018 0.221 0.064 Figure 2 7. Rate of pectinase inactivation with lines representing linear regression behavior for samples treated at 250 MPa and 55 C ( ), 62 C ( ), 69.3 C (

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82 Figure 2 8. Eyri ng plot for enzyme inactivation for samples treated at 55 C ( ), 62 C ( ), 69.3 C ( Figure 2 9. Activation volumes for p ectinase samples treated at 0.1 MPa and 200 to 400 MPa. Error bars represent standard error of linear regression (n=2)

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83 Figure 2 10. 0.1 MPa ( ), 200 MPa ( ), 250 MPa ( ), 300 MPa (X), 350 MPa ( Figure 2 11. Activation energies for pectinase samples treated at high hydrostatic p ressure. E rror bars represent standard error of linear regression (n=2) -8 -6 -4 -2 0 2 0.00275 0.0028 0.00285 0.0029 0.00295 0.003 0.00305 0.0031 Ln(k inact ) Temperature 1 (K 1 )

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84 CHAPTER 3 I NCREASED RATE OF VIS COSITY REDUCTION OF A PECTINASE COCKT AIL AT HIGH HYDROSTATIC PRE SURE Chapter 2 reported the effects of HHP and high temperature on the kinetics of inactivation of a pectina se cocktail. Based on the results f rom the inactivation study this chapter describes the treatment of the cocktail moderate temperatures and high pressure The objective of this study is to maximize the activity of a pectinase cocktail at HHP for optimal clarification processing that minimizes thermal degradation of pectinase enzymes Only a summary of the preliminary microbial inactivation results is provided in this chapter; for greater detail, see Appendix. Materials and Methods Materials The pect i nase cocktail, citrate buffer and pectin products were the same as those used in Chapter 2 Sodium a cetate, g lacial a cetic a cid, and s odium c hloride were all purchased from Fisher Scientific (Pittsburg, PA, USA) Equipment The equipment used for thi s experimental study was the same as detailed in Chapter 2 with the addition of a third Isotemp 3016D water bath from Fisher. The three wat er baths were set for cooling at 4 C, the processing condition, and finally an inactivation temperature of 95 C. Th e remainder of the HHP setup and rheometer equipment were described Chapter 2. Methods Sample preparation and HHP processing Pectin was diluted to 1.5% (w/v) solution in 0.2 M acetate buffer pH 4.5 and ionic strength of 0.1 M The pectinase solution was diluted in 0.5 M citrate buffer (pH 4 and

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85 ionic strength of 0.844M ) to 0. 05 % (1 5 unit mL 1 ) and place d on ice before treatments. Of the un treated enzyme sample 132 L were added to 1,800 L of pectin solution and stirred for 40 s with a miniature magnetic bar and stirrer in an ice bath The pectinase and p ectin solution were placed in 1 mL plastic pouches, heat sealed and placed in the high pressure cell held at 4 C to minimize enzyme activity. For treatment, the HHP cell was closed and a modified L abVIEW program was started exactly 2 min and 30 s after the pectinase had been added to the pectin solution. The constant hold time was to minimize variation in viscosity reduction that occurred before treatment. Pressure was raised to the process set point. Th en, temperature was raised to the incubation set point and when 90% of the change in temperature was reached, processing time started. After processi ng, the pressure cell was heated to over 89 C for a three minute period while the cell was depressurized t o inactivate the enzymes. After the inactivation period, the cell was cooled to 8 C and the sample wa s placed on ice before viscosity analysis. Enzyme activity was assayed on the same day as the HHP treatment. Processing conditions Samples were treated at 0.1 MPa (control) or 200 to 300 MPa at 25 MPa increments, 42.4 to 62.4 C with increments close to 5 C and for a processing time of 0 to 30 min with 7.5 min increments The processing temperatures were 42.4 C, 47.1 C, 52.1 C, 5 7 .1 C, and 62.4 C; t emperature increments were chosen for even distribution of T 1 for the calculation of activation energy with the Arrhenius equation. Come up and come down times were accounted for as f ollows. Ramp up time included pressurizing and heating the cell to 90% of set point temperature. Ramp down time included inactivation, depressurization, and cooling of the cell. Figure 3 1 shows the

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86 pressure and temperature profile for a sample treated at 62.4 C, 250 MPa, and 15 min. Samples were processed in duplicates. Th e study was performed in a randomized block design, with temperature blocks. Pressure and process times were randomly selected. Activity measurements To assess the activity of the enzyme cocktail, the pectinase and pectin solution were placed in a water ba th to raise the sample temperature to 20 C. The viscosity of pectin was recorded every 1.2 s for 5 min with a maximal viscomet er rotational speed of 20 rpm and at a temperature of 20 C Samples that contained pectin only or pectin with only citrate buffe r were run for 10 and 5 min respectively to check pectin consistency. The rate of viscosity reduction measurements differ from the ex situ m easurements made for the viscosity reduction observed in Chapter 2. For this experiment, the viscosity reduction oc curred inside the pressure cell ( in situ ) Therefore the extent of in situ viscosity reduction was determined after processing at selected temperatures followed by enzyme inactivation. Then viscosity was determined ex situ Full enzyme inactivation was ver ified by a constant viscosity of the reaction mixture after treatment. An average viscosity measurement of the last 50 measurements of ea ch viscosity reading was used as the viscos ity value for each time point. For this study, the rate of visco sity reduct ion at high pressure was determined again with same pseudo second order rate Error was reported using the standard error of the slope from the linear regression from the pseudo second order rate of viscosity reduction plot of inverse viscosity versus proc ess time. Additional calculation s for this experiment include the percent viscosity reduction (Equation 3 1)

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87 (3 1 ) Eyring and Arrhe nius equations were used as in Chapter 2 to determine the apparent activation volumes and activation e nergies of viscosity reduction. Experimental error derived from linear regression experiments was reported as standard error. Results and Discussion Visco sity Reduction Pectin viscosity decreased with the increase of process ing time as seen in Figure 3 2. The initial mean pectin viscosity for initial processing time of zero min utes was 0.012 at longer processing periods of 22.5 and 30 min suggesting the enzymes ha s cleaved a majority of the pectin molecules. The lowest viscosity obtained after 30 min treatment was achieved for each pressure by the highest temperature block of 62.4 C with the exception of 57.1 C temperature block for 200 MPa However, the difference from lowest viscosity values at 57.1 and 62.4 C probably can be at tributed to experimental error. During the experimentation there were some slight variations between pectin solutions as well as mixing due to the high viscosity. Also a small temperature gradient was likely to occur during mixing and could have also affect ed enzyme activity before the sample was treated with high pressure. All samples treated at high pressures, 200 to 300 MPa, for 30 min at 62.4 C achieved at lea st a 60% viscosity reduction At atmospheric pressure only a 54.6% reduction was achieved. The reduction at atmospheric pressure was 14.4% smaller than greatest reduction of 6 9% at 200 MPa and 62.4 C. The average viscosity reduction for all temperatures for 30 min treatments is summarized in Table 3 1. At

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88 lower temperatures of 42.4 C or 47.1 C atmospheric conditions produc ed similar viscosity reduction in which the stabiliz ation of pressure is not yet observable with the sh ort processing times. Figure 3 2 plots the viscosity reduction for the studied extreme temperatures of 42.4 C and 62.4 C. Temperatures of 52.1 to 62.4 C, achieved greater percent viscosity reduction res Samples that were treated with 0.1MPa, 200, or 300 MPa, with 62.4 C showed high percentages of viscosity reduction compared to other treatments while samples at 2 25 MPa to 275 MPa showed high viscosity reductions at temperatures of 52.1 or 57.1 C. The variation in these results between temperature blocks is likely due to the differences in the initial viscosity between samples and possibl y due to inadequate mixing this ended up in the process of using the rate of viscosity reduction as the parameter for measurement of pectinase activity. Rate of Viscosity Reduction In our previous study (Chapter 2) pectin viscosity reduction with the addition of pectinase cocktai l was best fitted as pseudo second order reaction For this study, t he same pseudo second order model was used with inverse viscosity plotted against time and the linear relationship showed with a regre ssion line as seen in Figure 3 3 The rate of viscosi ty reduction for atmospheric and high pressure conditions increased with the increase in processing temperatures from 42.4 C to 6 2.4 C. The second order rate constant of viscos ity reduction along with the standard er ror of the average of the two replicat es is shown in Table 3 2. Deviations that occurred from the general increase were at 52.1 C for 300 MPa, 57.1 C for 0.1 MPa, 250 MPa, and 300 MPa, and finally 62.4 C with 300 MPa. There was no obviou s trend of the rate of viscosity reduction with

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89 pressu re. Larger variations were observed at higher temperatures of 52.1 to 62.4 C. However, at those temperature blocks samples treated at atmo spheric pressure had higher rates of viscosity reduction than at lower temperatures. The largest rate of viscosity r eduction of 0.0960 Pa 1 2 occurred at 62.4 C and 300 MPa. The variation could be due to inconsistencies in initial pectin viscosity, mixing, or to the difference in behavior of the different enzymes in the cocktail with temperature. Pressure has been r eported to have different effects on individual pectinase enzymes. While tomato PME has been noted to be stable up to 700 MPa at room temperature, two forms of tomato PG, a heat labile and heat stable form, have been inactivated within 15 min at pressures from 300 to 500 MPa (Rodrigo and others 2006) For orange juice t he acti vity of PME has been reported to decrease with increased pressure, te mperature and processing time. For example, s amples treated at atmospheric pressure for 15 min at 37.5 C had a residual activity of 89.9%, while samples treated for 12 min at 50 C only retained 65% of residual activity where s amples treated at 400 MPa, 50 C for 12 min had a residual activity of 50.1% (Nienaber and Shellhammer 2001) The two enzymes, PME and PG, also affect the behavior of the other enzyme s Verlent and others, (2007) reported th at tomato PG in the presence of highly esterified pectin and PME at pH 4.4 had peak activity at 50 C and 200 MPa. Tomato PME had an optimum activity at 60 C and 400 MPa without PG present, however with the presence of PG, optimal PME activity was at 0.1 MPa and at lower tempera tures (Verlent and others 2007) While high pressures inhibit activity of some forms of pectinases, HHP increased activity o f the p ectinase cocktail at moderate temperatures as it had do ne with othe r fungal pectinases Fungal pectin methylesterase

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90 (PME) has been stabilized with high pressure. Optimal condition s for PME from A. acu l eatus were found at 200 MPa and 45 C and 300 M Pa and 50 C, while activity decreased for atmospheric conditions over the optimal temperature of 45 C (Fraeye and others 2007) Taken together, it seems that environment al factors (pressure, temperature and process time), type of enzymes, and the enzyme composition of the solutio n all interact to determine the extent that high pressure stabilize enzyme activity. The increase in viscosity reduction can also be quantified w ith the percent increase of activity with respect to samples treated to atmospheric pressure and at 45 C which are the recommended conditions for use of the cocktail used in this study. The rate of viscosity reduction at 45 C and 0.1 MPa was 0.0471 0. 0026 Pa 1 2 High pressure started generating an increase in activity with respect to activity at 45 C and 0.1 MPa, at 47.1 C for pressures of 200 to 250 MPa. The greatest increase in activity with respect to th e recommended conditions was at 62.4 C with all pressures having increase in activity from 60 to 10 4 %. However, the standard error calculated from the linear regression used to calculate the rate constants, suggests that there was no significant difference among all high pressure levels at 62.4 C. The largest rate of viscosity reduction obtained at atmospheric pressure was also at 62.4 C; the percent increase s in activity for high pressures in comparison to the 0.1 MPa sample at the same temperature were between 15.7 to 47%. At temperatures gr eater than 52 .1 C activity generally increases with temperature compared to the standard of 45 C and 0.1 MPa. High pressure stabilized samples from temperature inactivation as shown in Table 3 2 and previously discussed in Chapter 2. At atmospheric pres sure, the percent incre ase wa s from 3.3% to 38.6%

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91 from 47. 1 to 62.4 C. At high pressures of 275 or 300 MPa at 47.1 C did not increase the rate of viscosity reduction compared to the standard; however, percent activity increases were seen in all remaining pressures at 47.1 C and all high pressures at temperatures from 52.1 to 62. 4 C. F or t omato PG in the presence of tomato PME activity was 71.9% at 200 MPa and 50C in comparison to samples treated at 40 C and 0.1 MPa, while tomato PME in the presence of tomato PG had the greatest percent increase of 33.7% at the same conditions (Verlent and others 2007) Tomato PG did not have an increase in activity at 50 C or 60C for pressures over 200 MPa while PME had increased activity up to 300 MPa. The pectinase cocktail had greater increased activity than tomato PME and PG; showing that pectinase enzymes as a cocktail were more affected than tomato PG and PME. A. acu l eatus PME, however had larger in creases in activity with respect to samples treated at 45 C and 0.1 MPa than the pectinase cocktail. For pressures of 200 to 300 MPa and temperatures of 50 and 55 C, A. acu l eatus PME had increased activity of 146.8% to 198.9%, while the pectinase cocktai l had increased activity of 29.4 to 66.7% for pressures of 200 MPa and 300 MPa and temperatures of 52.1 to 57.1 C. Yet at higher temperatures and 200 to 300 MPa, the pectinase cocktail had greater increases in activity of 60.4 to 103.8% at 62.4 C, while A. acu l eatus PME increased 15.0% at 200 MPa and 70.6% for 300 MPa at 60 C. Temperature and pressure combinations affect pectinases differently; this data shows the necessity of optimization of processing conditions for each enzyme or set of enzymes. Tomat o PG and PME, A. acu l eatus PME, and the pectinase cocktail all had increased activity with moderate high pressure (<300 MPa) and mode rate temperatures of 50 to 60 C with respect to standards at atmospheric pressure

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92 Maximum activity for commercial pectin ase cocktails has been studied for thermal processing at atmospheric conditions; Pectinase 3XL the same cocktail used for this study, had maximum activity at 50 C and had an active range from 30 to 70 C (Ortega and others 2004) Pectinase 3XL had a wider activity range than two other commercial products tested: Rapidase C80, optim al condition at 55 C and Pectinase CCM, optimal condition at 50 C. Pectinex 3XL only retained 10% of initial activity when thermally treated at 50 C for 1 h; other commercial pectinase Pectinase CMM and Rapidase C80 retained 57 and 5 % activity (Ortega and others 2004) These studies support our findings that high pressure can stab ilize pectinase enzymes that are sensitive to high pressures and temperatures. Viscosity reduction has also been used to characterize total pectinase activity of commercial products Pectinex Clear (PC) and Pectinex BE Colour (PB) and extracts from A. niger and A. oryzae for the clarification of apple, butia palm, grape and blueberry juice (Sandri and others 2011) Viscosity reduction was used for total pectinase activity with one unit of activity stated the necessary amount of enzyme required to red uce the viscosity by 50% (Sandri and others 2011) A lower optimal temperature of 40 C for commercial products was observed than the stated manufacturer optima of 50 C for PC and 54 C for PB (though known to researchers). For apple juice clarification, based on turbidity reduction A. niger T0005007 2 and Pectinex Clear had similar results of about 60% clarificati on measured by optical density with treatments of 30 C for 60 min for Pectinex Clear and samples treated at 50 C for periods of 30 and 60 min for A. niger T0005007 2. Sandri and others, (2011) reported that increasing process time, rather than temperature, promoted greater clarification. O ur results

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93 indicate that raising process temperature (42.4 to 62.4 C) and pressure (2 00 to 300 MPa) increases the rate of viscosity reduction. Activation Volume The effect of pressure on enzyme activity was quantified by calculating the apparent activation volume. Only pressures of 0.1, 200, and 225 MPa wer e used for the estimation. Pressures higher than 225 MPa deviate d from linearity. Therefore, Eyri equation no longer applies for pressures from 200 to 300 MPa (Figure 3 4 ). Error wa s likely to be increased without an even distribution between pressures points of 0.1, 200, and 225 MPa Immobilized lipase in hexane had similar results of change in sign; apparent activation volume with negative values for pressures below 200 MPa and positive apparent activation volu mes values from 300 to 500 MPa (Eisenmenger and Reyes De Corcuera 2009a) At higher pressures from 250 to 300 MPa, temperature inactivation and pressure stabilization of the pectinase cocktail were less predictable at temperatures of betwee n 42.4 and 62.4 C. The u se of pressures of 0.1, 200 MPa or 225 MPa, allow ed for a calculated apparent activation volume that c learly showed increased rates of viscosity reduction and pressure stabilization shown in Figure 3 4 The activatio n volumes vari ed from 0.22 0.18 to 5.21 0.39 cm 3 1 for the studied temperatures of 42.4 C t o 62.4 C as shown in Figure 3 5 Apparent activation volumes increased in magnitude as temperature increased to 57.1 C, then decreased in magnitude to 62.4 C. High p ressure had the smallest effects on lower temperatures 42.4 and 47.1 C, and slightly greater effects on higher temperatures of 52.1C to 62.4 C. The negative apparen t activation volumes show that viscosity reduction was favored with high pressure treat ments for all temperature s The apparent activation volumes

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94 indicate that that pressure had the most favorable effects at the temperature range of 52.1 to 62.4 C, with the peak effect of stabilization at 57.1 C. Previously reported apparent activation vo lumes typically fall into the ra nge of 70 to 60 cm 3 1 for enzymes (Michels Peter and Clark Douglas 1992) Apparent activation volumes have also been studied for carrot and orange PME. Purified carrot PME had similar a pparent activation volumes of 7.80 to 5.73 cm 3 1 for temperat ures of 30 to 55 C; the smallest volume was seen at 40 C and the volume increased as temperature increased to 5.73 cm 3 1 (Sila and others 2007) Orange PME apparent activa tion volumes also increased from 35 to 30.9 cm 3 1 as temperature increased from 25 to 50 C (Nienaber and Shellhammer 2001) Results from our experiment differed from carrot and orange PME, in that, appa rent activation volume increased in magnitude as temperature inc reased from 42.4 to 57. 1 C. High pressure appears to have had less effect on the pectinase cocktail than orang e PME Activation Energy The apparent activation energy was also calculated to observe the effect of temperature on the pectinase cocktail at different pressures. The a pparent activation 1 at 0.1 to 225 1 at 250 MPa, then again increased with 1 at 300 MPa (Figure 3 6 ). Though there wa s a small decrease in apparent activation e nergy at 250 MPa, the decrease was not significant for the values for 22 5 MPa and 250 MPa ( 32.22 1 and 31.66 1 respectively) For high pressure samples, the effects of te mperature from 200 to 275 MPa wer e similar 1 ) with temperature having greater influence at 300 MPa (42.4 1 ). The similarity between activation energies was expected due to the narrow

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95 range of pressures selected from the results of the previous experiment at inactivating co nditions (Chapter 2). The error range was 1 for pressures of 0.1 to 1 Also, temperature had a greater effect when enzymes were treated at high pressure versus atmosphe ric pre ssure with an increase of 71.0% at 250 MPa to 182.1% at 300 MPa. Temperature with the addition of high pressure appears to be the main stimulus of the increased activity of the pectinase cocktail at moderate temperatures of 52.1 to 62.4 C. Reporte d activation energies for carrot PME were similar but slightly higher to data obtained in this experiment. Apparent activation energies for carrot PME at 0.1 MPa 1 1 for pressures 200 to 500 MPa, with no trend associ ated with increase in pressure for temperatures of 30 to 55 C (Sila and others 2007). Apparent activation energies of orange PME, 13.5 1 had the opposite general trend seen in our experiment as appa rent activation energy decreased with the increase in pressure of 400 to 600 MPa (Nienaber and Shellhammer 2001). The pectinase cocktail, with multiple enzymes, had less variation in activation energies than the carrot PME indicating the mixture may be more stable than individual enzymes alone. Pr eliminary Microbial Inactivation E. coli K12 was inactivated with treatments ranging from 250 to 350 MPa, 60 to 80 C, and a process time of 15 min. A 1 to 2 log reduction was also seen for ramp up and ramp down times (process time of zero minutes). The e xperiment is detailed in the Appendix

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96 Figure 3 s in high pressure cell for a sample treated at 62.4 C, 250 MPa, 15 min. Figure 3 2 V ) MPa -50 0 50 100 150 200 250 300 0 500 1000 1500 2000 Axis Title Axis Title Chart Title Pressure, Replicate 1 Temperature, Replicate 1 0.0E+00 2.0E-03 4.0E-03 6.0E-03 8.0E-03 1.0E-02 1.2E-02 1.4E-02 0 5 10 15 20 25 30 35 Viscosity (Pas) Process Time (min)

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97 Table 3 1. Average viscosity reduction (%) of a 30 min p rocess time relative to t = 0 min. Temperature Pressure (MPa) 42.4 C 45 C 47.1C 52. 1C 57.1C 62.4 C 0 .1 49.6 51.8 51.1 46.0 52.7 54.6 200 50.8 52.5 60.5 59.9 69.0 225 47.4 50.2 58.8 63.3 62.3 250 49.3 49.7 65.3 56.6 62.2 275 45.5 49.5 55.2 61.1 61.0 300 43.4 47.8 61.2 55.2 66.4 Figure 3 3 R ate of viscosity reduction with a pseudo seco nd order rate of reaction plot for pectinase treated ) 42.4 C and 300 MPa, Table 3 2. 0 50 100 150 200 250 300 0 5 10 15 20 25 30 35 (Viscosity) 1 (Pas) 1 Process Time (min)

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98 Table 3 2. Rate of viscosity r eduction standard e rror (Pa 1 s 2 ) n = 2 with R 2 values for different pressure treatments. Temperature (C) 0.1 MP a R 2 200 MP a R 2 225 MP a R 2 42.4 0.0434 0.0032 0.984 0.0446 0.0023 0.992 0.0439 0.0006 0.999 47.1 0.0487 0.0022 0.994 0.0531 0.0028 0.992 0.0506 0.0015 0.997 52.1 0.0487 0.0062 0.954 0.061 0.0056 0.975 0.0677 0.0044 0.987 57.1 0.0505 0.0051 0.970 0.0723 0.0047 0.988 0.0785 0.0039 0.993 62.4 0.0653 0.0084 0.952 0.0829 0.0101 0.958 0.088 0.0095 0.966 (C) 250 MP a R 2 275 MP a R 2 300 MP a R 2 42.4 0.0437 0.0026 0.990 0.0388 0.003 8 0.972 0.0342 0.0025 0.984 47.1 0.0495 0.0028 0.991 0.0457 0.0046 0.970 0.0426 0.0035 0.981 52.1 0.0676 0.0059 0.977 0.0619 0.0043 0.986 0.0763 0.0113 0.938 57.1 0.0653 0.0062 0.974 0.0727 0.0041 0.991 0.0599 0.007 0.961 62.4 0.078 9 0.005 0.988 0.0755 0.0069 0.976 0.096 0.0108 0.963 Note: Each replicate was calculated from the linear regression of the reciprocal of vis cosity vs. time of at least five incubation times.

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99 Figure 3 4 Eyring plot for viscosity reduction for sa Figure 3 5 Effect of pressure on the activation volume of the rate of viscosity reduction of pectin solutions. Error bars represent standard error of linear regression.

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100 Figure 3 6 Effect of temperature on the activation energy of the rate of viscosity reduction of pectin solutions. Error bars represent standard error of linear regression 0 10 20 30 40 50 60 0.1 200 225 250 275 300 Activation Energy, E a (kJmol 1 ) Pressure (MPa)

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101 CHAPTER 4 EFFECT OF HIGH PRESSURE ON PH COLOR INDICATORS IN SOLUTION As hig h pressure becomes more prominent as an alternative to thermal processing in the food industry, its effects on physical factors such as the pH of a food must be considered Knowledge of food pH during processing is important due to its effect on the behavi or of enzymes, microorganisms and other parame ters crucial to the food quality The final portion of this research focused on the pH shift of acid/base color indicators in response to HHP. This research is significant because pH probes for high pressure ar e not commercially available ; therefore, an alternative method must be established to determine shifts in pH with HHP. The objective of this study was to assess color changes of pH indicators at pressures up to 600 MPa for five a cid/base indicators and com pare to calculated pressure shifts. Materials and Methods Materials Five color acid/base indicators were used to provide a color array for ac idic pH range (1.2 < pH < 6.8). Table 4 1 provides a list of the five indicators used, the ir pH range and the col ors associated with the pH range Bromocresol Purple, Bromophenol Blue, Metanil Yellow, Methyl Orange and Thymol Blue were purchased from Fisher Scientific (Pittsburg, PA, USA). The color indicators were each dissolved in distilled water to make a 0.1% (w/ v) stock solution Equipment The laboratory high pressure setup was similar to the equipment setup detailed in Chapters 2 and 3. For this experiment, the Unipress (Warsaw, Poland) High Pressure Optical Vessel U103 with sapphire windows was used instead of Model U111 pressure

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102 cell. A DH 2000 UV VIS NIR light source (Mikropa k) with deuterium and halogen lamps was used as the light source and a HR4000CG UV NR high resolution spectrom eter from Ocean Optics, Inc (Dunedin, FL, USA) was used to collect visible s pectra. The li ght source and the spectrometer were connected to the high pressure opt ical cell through fiber optic cables The temperature of the jacketed high pressure chamber was control led with a water bath model Isotemp 3016D from Fisher Scientific (Pi ttsburg, PA, USA) ; the water bath was used to keep the optical vessel a nd indicator solution at 25 C. OOIBase32 software (Ocean Optics, Inc) was used to measure a nd collect absorbance spectra from 200 to 1200 nm Methods Sample preparation The concentrated indicator stock solution was added to distilled water to produce a lighter dilute indicator/water solutions to be use d for absorbance measurements. The final concentrations are as followed: Bromophenol Blue was 5.9 x1 0 6 M, Methyl Orange 9.5 x 10 6 M, Th ymol Blue 2.0 x 10 5 M, Bromocresol Purple 3.6 x 10 5 M, and Metanil Yellow 5.1 x 10 5 M. Distilled water was used as the blank. The dilute indicator solution was adjusted to the desired pH with addition of HCl or NaOH. The s ample was enclosed in a cylin drical quartz cuvette with two Teflon stoppers. The vial was plugged avoiding the entrapment of air bubbles, and then levelly submerged into the pressurization silicon fluid contained in the optical cell to avoid refraction of the light beam and provide a consistent path length through the cuvette Processing conditions The spect rum of each dye at selected pH was re corded at atmospheric pressure at 25 C ( Table 4 1 ). D yes were pressurized up to 600 MPa in 100 MPa increments at

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103 25 C Spectra of t wo replica tes were collected for each indicator solution The study was performed in a randomized block design, blocked by indicator. Pressure was randomly selected. Peak a rea m easurements Figure s 4 1 and 4 2 display the spectra of the acid or basic form of each ind icator respectively. The data was normalized by adjusting the averag e data from 750 nm to 800 nm in the infra red region to the origin of absorbance data The peak area was calculated for each peak with the adjusted spectrum by integrating absorbance value s against wavelength using Microsoft Excel Calibration c urves and compression compensation As pressure wa s applied, volume decreased causing an increase in concentration of the dy es, resulting in increased large absorbance values. The change in the peak a rea with pressure is therefore due to both an increased concentration and a shift in pH with applied HHP. The effect of temperature on the pH was not compensated for due to the sample being kept at room temperature with the jacketed cell and water bath. Th e effect of pressure compression was compensated for by using th e known compressibility of water. This adjustment allowed us to determine t he pH shift du e to HHP alone The effect of compression of HHP on water was sourced from the NIST Standard Reference Database 69: NIST Chemistry WebBook (USNIST 2008) The compression factor was the percent decrease of water density with HHP treatments. The peak area at atmospheric pressure was divided by the water compression factor to compensate for the concentration effect at each selected pressure as described in equation 4 1.

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104 (4 1) Where PA comp is the pressure compensated peak area, PA 0.1MPa is the peak area at atmospheric pressure and CF is the compression factor. The difference between the p eak area compensated for compression and the observed peak area PA obs wi th pressure was used to assess the shift in equilibrium ( PA shift ) for each indicator as expressed in equation 4 2 (4 2) With the chang e in color, adjusted peak area and the pH calibration curves established for each indicator, an apparent pH shift was calculated The compression factor decreases with the increase in pressure with a value of 1 at atmospheric pressure, 0.96 at 100 MPa and continued to decrease to 0.85 at 600 MPa. Results and Discussion Peak Area For each acid/base indicator, a shift in pH within its specific indicator range led to a color change that was monitored by absorbance in the visible sp ectrum. Some pH indicators had one large absorbance peak that shifted with pH, such as Methyl Orange and Metanil Yellow, while others like Bromocresol Purple, Bromophenol Blue, and Thymol Blue had two peaks that changed in size with the change in pH (Figure 4 3). For Bromocresol Pur ple the peak from 400 to 485 nm was used, while for Bromophenol Blue and Thymol Blue the peaks from 5 00 to 650 nm and 400 to 490 nm were used respectively for pH correlation. With the application of pressure, not only the height of the peak increase d but the peak absorbance wavelength shift ed The peak area

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105 calculated provided the best relationship at atmospheric pressure with the change in pH because peak area accounted for changes in peak absorbance as well as shifts in peak absorbance. Calibration Cur ves Peak area correlated linearly with pH at atmospheric pressure in the stud ied range as shown in Figure 4 4 and Table 4 2. Table 4 2 provides the parameters of the linear relationships used to correlate pH with peak area found with linear regression I n some cases, deviations from linearity occurred at edges of the reported pH indication range for each indicator as shown for Bromophenol B lue Figure 4 3 A t t he edge of the color indication range, change in color is minimal in comparison to the working pH i range and ei ther the acidic or basic form of the indicator dominates the solution limiting color change This explains the leveling off from the linear relationship. The correlation coefficie nts support the adequacy of the calibration curves for estimation of pH shifts. Effect of Pressure on pH With the application of pressure, a reversible change was seen in peak areas for each indicator. For indicators that produced two distinct peaks, the peak with the area that increased in size with pressur e was chosen to represent the indicator. As pressure was applied, volume decreased causing an increase in concentration due to compression. After compensation for compression, predictions of the pH shift were obta ined using the correlation of peak area an d pH at atmospheric pressure For all dyes, experimental results indicate d an apparent acidic pH shift when pressur ized between 0 .1 and 600 MPa, as shown in Figure 4 5 Research has reported in creases in water ionization constan t that create an acidic shif t pH (El'yanov and Hamann 1975) At 600

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106 MPa four indicators had smaller apparent pH shifts of 0.12 to 0.29 pH units than Bromocresol purple which had an apparent shift of 0.98 pH units as shown in Figure 4 6 Although t his may indicate that the pH shifts are more pronounced at a high er pH, closer to neutral pH the largest difference suggests that the equilibrium of the dye is itself affected by pressure. Literature reports the decrease in pH with HHP as a common effect for water and some buffers with fluorescence and acid/base indicators (Stippl and others 2004; Quinlan and Reinhart 2005) F or example, f luorescence has been used to determine the effect of pressure on buffers (Stippl and others 2004) Carboxylate buffers showed a decrease in pH with the application of pr essure, while cationic buffers had an increase in pH Acetate pH decreased a t a ra te of 0.08 pH units/ 100 MPa, from an initial pH 7, while other carboxylate buffers were more sensitive to pressure. Blending buffers that have opposing pH responses to pressure has led to pressure stable combinations, such as Tris/tricarballylate and Tris /ph osphate, which had an estimated o f less than 0.025 pH units/100 MPa (Quinlan and Reinhart 2005) Yet reported changes in pH with pressure do vary. Acetic acid, an acidic buffe r with initial pH of 4.1, also has been reported to drop by 0.22 and 0.40 pH units at 100 and 200 MPa respectively. Distilled water with an initial pH of 5.8 had pH shifts of 0.30 units at 100 MPa and 0.31 units at 200 MPa (Hayert and others 1999) These results were similar to values seen in previous literature (Owen and Brinkley 1941; Distche 1972) but higher than predicted by our apparent shift reported here suggesting that the shift in the equilibrium constant of the color indicator is antagonist to the dissociation of wat er An acid shift has also been noted in CO 2 pressurized systems; the observed pH of a CO 2 water system

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107 decreased from the initial pH by 2.53 pH units with an increase in pressure of 0 to 5.516 MPa. However, the observed pH only decrea sed a n additional 0.1 4 pH units with an increase in pressure of 5.516 to 34.49 MPa indicating that the solvation of CO 2 is the main cause in the pH shift not pressure Systems that contained ascorbic acid and/or citric acid showe d even smaller initial decrease in pH with the i ncrease in pressure This result shows that the systems component conce ntrations also have an effect on the pH of a system (Meyssami and others 1992) The acid shift in pH with water and carboxylates acids observed i n these studies agrees with the apparent decrease of acid/bas e indicators in water. Table 4 1. Acid/base color i ndicators Indicator pH Range Low Acid Color High Acid Color pH Points Bromocresol Purple 5.2 6.8 Purple Yellow 5.2, 5.5, 6, 6.5, 6.8 Bromoph enol Blue 3 5 Purple Yellow 3, 3.5, 4, 4.5, 5 Metanil Yellow 1.2 2.4 Orange Hot Pink 1.2, 1.8, 2.4 Methyl Orange 3.1 4.4 Orange Red Orange 3.1, 3.5, 4, 4.4 Thymol Blue 1.8 2.8 Orange Pink 1.2, 2, 2.8 Figure 4 1. A djusted absorbance spectra for acid ic pH points. A) Bromocresol Purple ( ----) pH 5.2, B) Bromophenol Blue ( ) pH 3, C) Metanil Yellow ( --0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 400 450 500 550 600 650 700 Adjusted Absorbance Wavelength (nm)

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108 --) pH 1.2, D) Methyl Orange ( ----) pH 3.1, and E ) Thymol Blue ( -----) pH 1.2. Figure 4 2 Adjust ed absorbance sp ect ra for low acid pH points. A) Bromocresol Purple ( -----) pH 6.8 B) B romophenol Blue ( ) pH 5 C) Metan il Yellow ( ---) pH 2.4 D) Methyl Orange ( ----) pH 4.4 and E ) Thymol Blue ( ----) pH 2.8 Figure 4 3 Adjusted a b sorba nce s pectra for Bromophenol Blue at 0.1 MPa A) pH 5 ( ) B) pH 4.5 ( ----), C) pH 4 ( ----), D) pH 3.5 ( ----), and E) pH 3 ( -----) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 400 450 500 550 600 650 700 Adjusted Absorbance Wavelength (nm)

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109 Table 4 2 Parameters for linear r elationsh ips c o rrelating pH vs. peak a rea Indicator Sl ope (nm 1 ) Intercept Coefficient of Determination Bromocresol Purple 0.0813 0.00113 8.7515 .9996 Bromophenol Blue 0.034 0.00412 2.8513 .9578 Metanil Yellow 0.0246 0.00564 3.951 .9994 Methyl Orange 0.0275 0.06092 8.3029 .9224 Thymol Blue 0.3 398 0.00059 .9301 .9689 Figure 4 4 The pH calibration plot A) Bromocresol Purple ( ), B) Bromophenol Blue ( ), C) Metanil Yellow (*), D) Methyl Orange ( ), and D) Thymol Blue (X). 0 20 40 60 80 100 120 140 160 180 200 0 1 2 3 4 5 6 7 8 Area peak (nm) pH

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110 Figure 4 5 Apparent effect of p ressure on pH A) Bromocreso l Purple ( ), B) B romophenol Blue ( ) C) Metan il Yellow (*) D) Methyl Orange ( ) and D) Thymol Blue (X) Figure 4 6 Apparent pH shift with p ressure A) Bromocresol Purple ( ), B) B romophenol Blue ( ) C) Metan il Yellow ( ) D) Methyl Orange ( ) an d D) Thymol Blue ( ) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0 100 200 300 400 500 600 700 pH Pressure (MPa) -1.20 -1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0 100 200 300 400 500 600 700 pH Pressure (MPa)

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111 CHAPTER 5 FINAL COMMENTS Overview High pressure treatments of 200 400 MPa stabilized the commercial pectinase cocktail which maintained activity longer than the pectinase cocktail did at atmospheric pressure at t emperature s of 69. 3 to 85 C. Pressure slowed thermal inactivation. At moderate temperatures and high pressure s of 200 300 MPa, the rate of viscosity reduction and pectinase activity increased with temperatures for 42.4 to 62.4 C High pressure can be used to stabilize enz ymes used in fruit juice clarification which might allow the use of higher temperatures than for treatments at atmospheric pressure while minimizing thermal degradation. High hydrostatic pressure assisted thermal processing would be an alternative that cou ld stabilize clarification enzymes at higher temperatures than that possible at atmospheric pressure. Preliminary results confirmed th at HH P treatments at these temperatures can inactivate E. coli K12 Finally acid base color indicators demonstrated an ap parent change in pH with HHP treatments at 25 C. High hydrostatic pressure processing is a relevant alternative to be explored for improved food and beverage processing techniques Future Work The p ectinase cocktail was stabilized with high pressure and m oderate temperature treatments. Future experimentation can be performed to further optimize clarification and pasteurization conditions at HHP to make accurate economic comparisons to thermal processing Because the highest rates of reduction were seen wit h the highest temperature block tested, 62.4 C r esearch at higher temperatures (>62.4 C) would be beneficial and provide more inclusive results Also, research testing the individual

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112 enzymes of the pectinase solution under the same conditions would be a ppropriate to help explain some of the variation in this experimentation which could not be attributed to individual variation of replicates and/or devi ation from second order plots. Future studies at lower pressure s should be considered to improve the acc uracy of the apparent activation volume s and would allow for better quantification of the effects of HHP on the pectinase cocktail Experimentation at pressures between 0.1 MPa and 350 MPa with 50 MPa increments, and temperatures from 60 to 80 C should be performed to determine optimal conditions for juice clarification with HHP Optimal conditions found for pectin solutions should be confirmed in apple juice and with particular attention paid to the microbes pe rtinent to apple juice such as E. coli O157:H 7 and A. acidoterrrestris Lastly HHP research on the change in pH result should be expanded to include the behavior of acid/base indicators at HHP. A possible experiment could be to use indicators with know n peak absorbance wavelengths for the acid a nd b asic forms of the compound ; and a change in the absorbance at these wavelengths with HHP could be correlated with pH and then used to determine if the ionization constant K a changed with the application of pressure.

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113 APPEND IX INACTIVATION OF E.COLI K12 WITH HHP TREATMENT Prelimin ary work was done with E. coli K 12 to confirm inactivation of E. coli at processing conditions for juice clarification with HHP processing The E. coli K12 was used as a substitute for the pathogenic E. coli O157:H7 Materials a nd Methods The E. coli K12 (L.E. Barrett) was inoculated in 10 mL of tryptic soy broth and incubated at 37 C for 24 hours. Cloudy apple juice, pH of 4.2, was made with red delicious apples crushed in a hydraulic press f rom Norwalk Juicers (Lowell, AR, US A) The E. coli broth was mixed with boiled cloudy apple juice to dilute the cells to an initial concentration in the order of 10 8 CFU mL 1 (Mean = 6.01 x CFU mL 1 ) Two milliliters of apple juice and E. coli were heat sealed in plastic pouches. Cont rols i ncluded pouches of untreated samples B roths and agar for this experiment were purchased from Fisher Scientific (Pittsburg, PA, USA). The high pressure equipment had the same setup as the experiment design described in Chapter 2. Two milliliters of inocula ted juice were sealed in plas tic pouches and submerged in HHP cell W ater baths kept the high pressure reactor at either 25 C or the selected incubation temperature. I noculated juice samples were treated between 250 and 350 MPa with 50 MPa increments at 60 to 80 C with 10 C increments and process times of 0 or 15 min. Untreated samples were used as controls. E. coli and apple juice solutions were kept on ice until and after processing. After treatments, samples were serially diluted with Bacto pepton e and plated on MacConkey agar. Plates were placed in an incubator at 37 C for at least 24 h. After incubation, plate counts were performed to estimate the

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114 number of cells after treatment; plate counts were limited by the number of colonies. P lates with All experiments were run in triplicate A picture of E. coli colonies grown on a MacConkey agar plate from an untre ated sample i s shown in Figure A 1. Results a nd Discussion One to two decimal reductions of E. coli K12 populations occurred during process come up and come dow n time. An 8 log reduction in E. coli K12 was observed with pressure temperature combinations from 250 to 350 MPa and 60 to 80 C after 15 min incubation. Because init ial population was on the order of 10 8 CFU mL 1 (t he standard deviation between initial concentrations was between 1.5 and 3.1 x10 8 CFU mL 1 ), a 5 log reduction required for pasteurization can b e en sured. Garcia Graells and others (1998) reported treating pressure resistant E. coli A treatment of 15 min at 20 C and 500 MPa, followed by 2 day refrigeration in a juice at pH 4 resulted in a 5 log reduction of microbial populations. Conclusions Operat ion under these conditions have the potential to simultaneously pasteurize and clarify fruit juices as seen with pasteurization confirmed with this experiment. This study was a preliminarily study, that should be followed by more th o rough microorganism pas teurization studies. Once optimization of clarification processing is completed, pasteurization experiments with E.coli O157:H7 and A. a cido terristris will be critical to confirm pasteurization under specific HHP clarifying conditions. Pasteurization exper iments with these microorganisms after acid adaption are needed to test the ability of HHP to pasteurize juices under the proposed conditions

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115 Figure A 1. E. coli K12 colonies growing on a MacConkey agar plate. Samples was not treated with pressure or heat The pink colonies represent the E. coli K12 colonies, while the appearance of white circles is the condensation and reflection on the plate.

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116 LIST OF REFERENCES Alkorta I, Garbisu C, Llama MJ, Serra JL. 1998. Industrial applica tions of pectic enzymes: a review. Process Biochemistry 33(1):21 8. Araya Farias M, Mondor M, Lamarche F, Tajchakavit S, Makhlouf J. 2008. Clarification of apple juice by electroflotation. Innovative Food Science & Emerging Technologies 9(3):320 7. Balas ubramaniam VM, Farkas D, Turek EJ. 2008 Preserving Foods through High Pressure Processing. Food Technology Magazine. Besser R, Lett S, Weber J, Doyle M, Barrett T, Wells J, Griffin M. 1993. An outbreak of diarrhea and hemolytic uremic syndrome from Escher ichia coli O157:H7 in fresh pressed apple cider. Journal of the American Medical Association 269:2217 20. Binning R, Possmann P. 1993. Apple Juice. In: Nagy S, Chen CS, Shaw PE, editors. Fruit Juice Processing Technology. Auburndale, FL: AGSCIENCE, INC. p 271 317. Black EP, Setlow P, Hocking AD, Stewart CM, Kelly AL, Hoover DG. 2007. Response of Spores to High Pressure Processing. Comprehensive Reviews in Food Science and Food Safety 6(4):103 19. Bruins ME, Matser AM, Janssen AEM, Boom RM. 2007. Buffer s election for HP treatment of biomaterials and its consequences for enzyme inactivation studies. High Pressure Research 27(1):101 7. Buckow R, Kastell A, Terefe NS, Versteeg C. 2010. Pressure and Temperature Effects on Degradation Kinetics and Storage Stab ility of Total Anthocyanins in Blueberry Juice. Journal of Agricultural and Food Chemistry 58(18):10076 84. Ceci L, Lozano J. 1998. Determination of enzymatic activities of commercial pectinases for the clarification of apple juice. Food Chemistry 61(1):2 37 41. Deliza R, Rosenthal A, Abadio FBD, Silva CHO, Castillo C. 2005. Application of high pressure technology in the fruit juice processing: benefits perceived by consumers. Journal of Food Engineering 67(1 2):241 6. Demazeau G, Rivalain N. 2011. High h ydrostatic pressure and biology: a brief history. Applied Microbiology and Biotechnology 89(5):1305 14. DePedro HM, Urayama P. 2009. Using lysosensor yellow/blue DND 160 to sense acidic pH under high hydrostatic pressures. Analytical Biochemistry 384:359 61.

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123 BIOGRAPHICAL SKETCH Brittany was born in Birmingham, Alabama and attended high scho ol in Gulf Breeze, Florida. She obtained a Bachelor of Agricultural and Biological Engineering from the University of Florida, and continued her studies in the ABE graduate program at Florida. Brittany presented research at the 6th International CIGR Techn ical Symposium in Nantes, France, 61st Annual Citrus Processors' and Subtropical Technology Conference Meeting in Lake Alfred, FL, National Annual IFT Meeting in Chicago, IL and New Orleans, La, and Florida ASABE Conference in Jupiter Beach, FL. s interests are in food and biological research, development, and juice clarifi to join the industrial wor kforce in a research and development unit.