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Minimization of extracellular enzyme production by Pseudomonas fluorescens in a model milk system by… Lee, Karoline K. 1993

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MINIMIZATION OF EXTRACELLULAR ENZYME PRODUCTION BY Pseudomonas fluorescens  IN A MODEL MILK SYSTEM BY CONTROLLED OXYGEN ATMOSPHERES BY KAROLINE K. LEE  B. Sc. , The University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER IN SCIENCE in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF FOOD SCIENCE)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1993 © Karoline K. Lee, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department o The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ABSTRACT  1. Pseudomonas fluorescens, the predominant aerobic spoilage microorganism in raw  milk, proliferate and produce heat resistant extracellular enzymes during storage. The critical upper 02 level which would minimize the synthesis of proteinases and lipases by P. fluorescens in a model milk system was determined in our study.  Two litre volumes of Ultra-High Temperature sterilized milk (2 % m.f.) in 4 litre carboys were equilibrated with a continuous supply of atmospheric oxygen between 1 21 %. Milk was then inoculated with P. fluorescens biotype A to approximately 1 x 104 CFU rriL-1 to reflect the psychrotrophic populations normally encountered in milk and stored under controlled 02 atmospheres at 4 °C up to 18 days. The atmospheric composition was analyzed with gas chromatography. Milk samples were collected every two days for determination of dissolved 02, P. fluorescens numbers, proteolysis, lipolysis, pH, proteinase and lipase activity. The dissolved 02 tension in milk decreased despite a constant supply of 02 during mid-exponential growth phase of P. fluorescens when populations reached 5.0 - 6.5 Logi() CFU mL4 under all atmospheric 02 levels tested. The growth rate of P. fluorescens was enhanced during the first four days of storage under decreased atmospheric 02 concentrations at 1.2, 4.8, 5.3, and 13.4 % when compared to the aerobic control. Lower levels of proteinase and lipase activities were detected in milk stored under all decreased atmospheric 02 conditions (1.2, 4.8, 5.3, 9.6, 10.2, 10.3, 13.4, and 16.9 % 02) when compared to the aerobic control (20.2, 20.7, and  20.9 % 02). As a result, lower degree of proteolysis and lipolysis were observed in milk stored under decreased atmospheric 02 conditions. Deviation from initial milk pH reflecting the predominant effect of either proteolysis or lipolysis was minimized when the initial dissolved 02 tension in milk was decreased. Greater inhibitory effect on proteinase than lipase production by P. fluorescens was observed under decreased 02 atmospheres. P. fluorescens under 10 % atmospheric 02 concentration consistently showed slow  growth rates during initial storage, low proteinase and lipase activities and as a result, low degree of proteolysis and lipolysis in milk. Initial dissolved 02 tension of 4.1 ppm (unstirred sampling) and 6.1 ppm (stirred sampling) were measured in milk under 10 % atmospheric 02 concentration which was observed to be the critical upper limit, of the levels tested, which minimized extracellular enzyme production by P. fluorescens, as well as delaying its growth rate during initial storage, in UHT-sterilized milk. Compared to storage of raw milk under aerobic atmosphere, decreased atmospheric 02 conditions may provide a better environment for storage of raw milk when the predominant flora consists of aerobic P. fluorescens. 2. Conditions for an improved f3-naphthyl caprylate assay for lipase activity were established at final concentrations of 15 mM sodium taurocholate and 8 mM 13-naphthyl caprylate in the reaction mixture and a solvent system of 60:40 ethyl acetate to ethanol. A final concentration of ethylenediamine tetraacetic acid between 2 and 5 rnM minimized degradation of lipases by proteinases and thus enhanced lipase activity during the assay procedure.  iv CONTENTS Page  ABSTRACT ^ CONTENTS ^ LIST OF TABLES ^  iv ix  LIST OF FIGURES ^ APPENDICES ^  xii  ACKNOWLEDGEMENTS ^  xiii  PART I ^ INTRODUCTION^  1 2  LITERATURE REVIEW ^  4  A. Psychrotrophs in Raw Milk ^ 4 1. Predominance of Pseudomonas fluorescens^ 5 2. Extracellular Enzymes as Spoilage Causing Agents ^ 5 A. Heat Stability ^ 5 B. Enzymatic Degradation of Milk Constituents ^ 6 3. Relation Between Population Numbers and Enzymatic Activity ^ 7 4. Production of Proteinase and Lipase as Function of Growth Phase ^ 7 5. Drop in Dissolved Oxygen Tension in Milk ^ 8 6. Dissolved Oxygen as Indicator of Potential Spoilage ^ 9 B. Role of Oxygen in Aerobic Microorganisms^ 10 1. Energy Production ^ 10 A. Aerobic Respiration ^ 10 B. Anaerobic Respiration ^ 11 2. Metabolism Under Decreased Oxygen Concentrations ^ 12 3. Energy Status and Extracellular Enzyme Production^ 13 4. Critical Dissolved Oxygen Tension (CDOT) ^ 15 C. Factors Effecting Enzyme Production ^ 16  1. Temperature ^ 16 2. Ions ^ 17 3. Oxygen ^ 18 A. Oxygen Inhibits Enzyme Production ^ 18 B. Oxygen Enhances Enzyme Production ^ 19 4. Regulation of Extracellular Enzyme Synthesis ^ 21 A. Proteinase ^ 21 1. C Catabolite Repression^ 22 2. N Catabolite Repression ^ 23 B. Lipase ^ 24 C. Direct Regulation by Oxygen ^ 24 D. Potential Disadvantages of Decreased Oxygen Atmospheres ^ 25 MATERIALS AND METHODS ^  27  A. Oxygen Controlled Atmospheres ^ 27 B. UHT Milk ^ 29 C. Culture Conditions ^ 29 D. Antibiotic Testing of UHT Milk ^ 30 E. Enumeration ^ 30 F. Proteinase Activity Assay ^ 31 G. Proteolysis ^ 32 H. Lipase Activity Assay ^ 33 I. Lipolysis ^ 34 J. Investigation of High ADV^ 35 a. Induced Lipolysis by Mechanical Agitation^ 35 b. Metabolism of P. fluorescens biotype A (ATCC 17397) ^ 35 1. Carbohydrate Utilization ^ 36 2. Nitrate Reduction ^ 36 c. Carbon Dioxide Interference ^ 37 K. Statistical Analysis (Trial III) ^ 37 RESULTS AND DISCUSSION ^  39  A. TRIAL I ^ 1. Growth ^ a. Alternate Electron Acceptors^  41 41 43  vi 44 b. Growth on Solid Medium ^ 2. Oxygen ^ 44 a. Atmospheric Oxygen ^ 44 44 b Dissolved Oxygen ^ c. Oxygen and Energy Production ^ 48 3. Proteinase Activity ^ 49 a. Carbon and Nitrogen Catabolite Repression ^ 51 b. Proteinase Synthesis and Growth Phase ^ 51 c. Factors Influencing Protemase Production^ 52 i. Oxygen ^ 52 ii. Population Density ^ 52 d. Proteinase Activity and Inhibition of Cell Lysis ^ 53 e. The Inhibitory Effect of Nitrogen ^ 53 4. Proteolysis ^ 54 5. Lipase Activity ^ 54 6. Lipolysis ^ 57 7. Investigation of High ADV ^ 58 a. Induced Lipolysis by Mechanical Agitation ^ 58 b. Carbohydrate Utilization of P. fluorescens biotype A^ 58 c. Carbon Dioxide Interference^ 59 8. Summary of Trial I ^ 60 B. TRIAL II ^ 61 1. Growth ^ 61 a. Low Population Numbers^ 61 b. Growth Under Lower Atmospheric Oxygen ^ 63 c. Oxygen and Nitrate as Electron Acceptors ^ 63 2. Oxygen ^ 64 a. Atmospheric Oxygen ^ 64 b. Dissolved Oxygen ^ 64 c. Carbon Dioxide Production ^ 64 3. Proteinase Activity ^ 67 a. Aerobic Conditions ^ 67 b. Lower Atmospheric Oxygen ^ 70 c. Relation Between Population Density and Proteinase Activity ^ 70 4. Proteolysis ^ 71 5. Lipase Activity ^ 71 6. Lipolysis ^ 74 7. pH ^ 76  vii 8. Summary of Trial II ^ C. TRIAL III ^ 1. Growth ^ 2. Oxygen ^ a. Atmospheric Oxygen ^ b. Dissolved Oxygen ^ c. Carbon Dioxide Production ^ 3. Proteinase Activity ^ a. Lower Atmospheric Oxygen ^ b. Drop in Oxygen Tension ^ 4. Proteolysis ^ 5. Lipase Activity ^ a. Lower Atmospheric Oxygen ^ b. Drop in Oxygen Tension ^ 6. Lipolysis ^ 7. Correlation Coefficients ^ 8. pH ^ 9. Summary of Trial III ^  78 80 80 80 80 83 83 87 87 90 91 91 91 94 94 94 98 100  D. CONTROL OF OXYGEN CONCENTRATION DURING STORAGE OF RAW MILK ^ 102 CONCLUSION^  103  PART II ^ INTRODUCTION ^  105 106  LITERATURE REVIEW ^  107  A. Lipase Degradation by Proteinases ^ 107 1. During Storage ^ 107 2. During Lipase Assay Conditions ^ 108 B. Inhibition of Proteinase Activity in Lipase Assay with EDTA ^ 108 C. Lipase Activity Assay Conditions ^ 109 MATERIALS AND METHODS ^  111  viii A. Improvement of the Lipase Assay ^ 111 1. Sodium Taurocholate ^ 111 2. P-naphthyl Caprylate^ 111 3. Solvent Clarification ^ 111 B. Lipase Activity Enhancement ^ 112 1. Proteinase Activity Under Lipase Assay Conditions ^ 112 2. Addition of EiDTA ^ 112  RESULTS AND DISCUSSION ^  113  A. Improvement of the Lipase Assay ^ 113 1. Sodium Taurocholate ^ 113 2. 11-naphthyl caprylate ^ 113 3. Solvent Clarification^ 113 B. Lipase Activity Enhancement ^ 116 1. Proteinase Activity Under Lipase Assay Conditions ^ 116 2. Lipase Activity with the Addition of EDTA ^ 116 CONCLUSIONS ^  120  REFERENCES ^  121  APPENDICES ^  134  ix LIST OF TABLES  TABLE 1. 2. 1-. 4. 5. 6. 7. 8. 9. 10.  Atmospheric oxygen levels tested ^ Initial populations of P. fluorescens^ Metabolism of P. fluorescens ^ Dissolved oxygen tension of stirred and unstirred samples ^ Correlation coefficients of parameters at 4.8 % 02 ^ Correlation coefficients of parameters at 10.3 % 02 ^ Correlation coefficients of parameters at 13.4 % 02^ Correlation coefficients of parameters at 20.2 % 02 ^ Clarification of lipase reactions with solvents... ^ Proteinase activity under lipase assay conditions ^  Page 40 40 45 85 96 96 97 97 117 117  x  LIST OF FIGURES PART I FIGURE^ 1. Experimental setup in 4 °C coldroom^ 2. Modification of titration setup ^ TRIAL I 3. Growth of P. fluorescens ^ 4. Profile of oxygen controlled atmospheres ^ 5. Dissolved oxygen tension ^ 6. Proteinase activity ^ 7. Degree of proteolysis ^ 8. Lipase activity ^ TRIAL II 9. Growth of P. fluorescens^ 10. Profile of oxygen controlled atmospheres ^ 11. Dissolved oxygen tension ^ 12. Carbon dioxide production ^ 13. Proteinase activity ^ 14. Degree of proteolysis ^ 15. Lipase activity ^ 16. Degree of Lipolysis ^ Milk pH ^ 17. TRIAL III Growth of P. fluorescens^ 18. 19. Profile of oxygen controlled atmospheres ^ 20. Dissolved oxygen tension ^ 21. Carbon dioxide production ^ 22. Proteinase activity ^ 23. Degree of proteolysis ^ 24. Lipase activity ^ 25. Degree of lipolysis ^ 26. Milk pH ^  Page 28 38 42 46 47 50 55 56 62 65 66 68 69 72 73 75 77 81 82 84 86 88 92 93 95 99  xi PART H  FIGURE^ Page 27. Effect of NaTC on lipase activity^ 114 28. Effect of 0-naphthy1 caprylate on lipase activity^ 115 29.^Effect of EDTA on proteinase and lipase activities ^ 118  xii APPENDICES  FIGURE 1. 2. 3. 4. a-d 5. a-d  Degree^of lipolysis ^ Effect of mechanical agitation on lipolysis ^ Removal of atmospheric CO2 on lipolysis ^ Drop in 02 tension and increase in proteinase activity^ Drop in 02 tension and increase in lipase activity^  135 136 137 138 140  ACKNOWLEDGEMENTS  I would like to express my deepest appreciation to my advisor Dr. Brent J. Skura for his endless support and guidance. His insightful wisdom and always encouraging words have made these past years a pleasure to work under his supervision. Thank you for believing in me. I would also like to thank the members of the reviewing committee for their suggestions and constructive criticism in preparation of this thesis: Dr. B. Dill (Department of Microbiology), Dr. E. Li-Chan (Department of Food Science) and Dr. T. Durance (Department of Food Science). No research would have been done without the tremendous help of two very important figures in the Department of Food Science: Val Skura and Sherman Yee. Special thanks to Dairyworld Foods for analyzing milk samples for antibiotics. Greater thanks to Allan Sawchenko of Medigas Ltd. for persevering my endless nagging. I would like to thank our research team especially Michel, Gemia and Mawele whose supportive words carried me through dark days. This one's for you Grandma.  1  PART I  Determination of Critical Oxygen Concentration Required To Minimize Extracellular Enzyme Production by Pseadomonasfluvrescens  2 INTRODUCTION  Storage of raw milk on farms, during transport and at dairy processing plants in a highly oxygenated state at refrigeration temperature selects for aerobic, gram negative psychrotrophs. These aerobic microorganisms cause quality problems in milk and dairy products not directly but rather through their production of extracellular enzymes, primarily heat resistant proteinases and lipases which survive most heat treatments and remain active during storage. Proteinases hydrolyze milk proteins and cause bitterness and gelation problems while lipases cause lipolytic, off-flavours from hydrolysis of milk fat. The predominant psychrotroph isolated from raw milk is Pseudomonas fluorescens. These microorganisms produce proteinases and lipases concomitantly during late exponential - stationary growth phase. Usually a drop in dissolved oxygen tension in the medium occurs just prior to enzyme production. There appears to be a relationship between oxygen and extracellular enzyme production whether causal or casual. Extracellular enzyme production by P . fluorescens is dependent on many factors such as nutrient availability, growth phase, temperature, pH, and aeration. There are contradictions in the literature about the role of oxygen in extracellular enzyme production by psychrotrophs. Some studies show enhancing effects while others show inhibitory effects. Use of air and mechanical agitation systems in bulk storage silos in dairy processing plants may inadvertently be promoting extracellular enzyme synthesis by aerobic  3 psychrotrophs. A better understanding of the role of oxygen in the production of extracellular enzymes by aerobic psychrotrophs may enable the development of improved methods of raw milk storage. The use of controlled oxygen atmospheres for raw milk storage not only promises to delay the onset of extracellular enzyme production but also reduce the rate of production once it has been initiated. Removal of oxygen from highly oxygenated milk could be time consuming and costly. The objective of our study was to determine the critical upper level of oxygen which would minimize extracellular proteinase and lipase production by P. fluorescens in a model system using UHT-sterilized milk as the growth medium. The critical oxygen concentration which greatly reduces the synthesis of extracellular enzymes would be beneficial in providing higher quality raw milk. Several additional days of raw milk storage in silos at dairy processing plants without risking quality of dairy products made from such raw milk may have substantial economic significance.  4 LITERATURE REVIEW  A. Psychrotrophs in Raw Milk There are many problems associated with extended storage of raw milk in bulk tanks on farms, in transport and in processing plants. The use of refrigeration temperatures has reduced spoilage by lactic acid bacteria and growth of most pathogens but has selected for psychrotrophs capable of growth at 7 °C or below (Cousin, 1982). An increase in the aeration of raw milk by pumping and agitation has selected for growth of aerobic, gram negative rod shaped bacteria (Law, 1979; Cousin, 1982). During storage of raw milk, which may be as long as 4 to 6 days before receiving any thermal processing, undesirable biochemical and sensory changes could result from microbial activity which would effect processed milk and other dairy products (Cousin, 1982; Bishop and White, 1986). Possible sources of microbial contamination of raw milk are numerous since psychrotrophs are ubiquitous in nature. One major concern would be inadequately sanitized milking machines and pipelines in milking plants. Psychrotrophic counts of raw milk can be as high as 103 CPU mL4 from milking machines, 104 to 105 CPU mL4 at collection depots and 106 to 108 CFU mL-1 at milk plants (Cousin, 1982). Similar numbers (4.4 x 103 CFU mL-1 from a farm and 9.6 x 103 to 2.9 x 104 CPU mL4 from a dairy plant) were reported by Adams et al. (1975). Psychrotrophic counts from a dairy plant showed seasonal variation with a peak in the summer (Garcia et al., 1989). Psychrotrophic flora can survive, grow and produce high levels of proteinase and lipase during prolonged storage at refrigeration temperatures. Stead (1987) stressed the  5 importance of adequately sterilizing dairy equipment to avoid inoculating fresh raw milk with the microorganisms and their concentrated enzymes. 1. Predominance of Pseudomonas fluorescens Ewings et al. (1984) showed that the predominant psychrotroph from raw milk on the farm was Pseudomonas at a frequency of 70 % of the total psychrotrophic count. Garcia et al. (1989) showed that 62-70 % of the psychrotroph count was Pseudomonas, of which 73 % were fluorescent. The most frequently occurring psychrotroph from raw milk is reported to be P seudomonas fluorescens (Griffiths et al., 1981; Brandt and Ledford, 1982; Cousin, 1982; Griffiths and Phillips, 1984; Kwan and Skura, 1985) belonging to biotype A (Poffe and Mertens, 1988). Fluorescent pseudomonads predominated the lipolytic flora at 64 % (Shelley et al., 1987). Other gram negative rod shaped bacteria isolated from raw milk are Achromobacter, Acinetobacter, Aeromonas, Enterobacter, Klebsiella and Serratia (Cousin, 1982).  2. Extracellular Enzymes as Spoilage Causing Agents A. Heat Stability It is not the presence of the microorganisms per se which is detrimental to the quality of milk but it is their production of extracellular enzymes. These psychrotrophs produce extracellular proteinases and lipases which hydrolyze proteins and lipids to provide readily metabolizable substrates for growth. Most studies have reported a single extracellular lipase and a single proteinase for P. fluorescens but some pseudomonads may produce more than one proteinase (McKellar, 1989). P. fluorescens showed the highest  6 proteinase activity when compared to other isolates including P. fragi (Kohlmann et al., 1991). Heat treatments effectively decrease microbial populations but proteinases and lipases retain their activities and cause quality defects in processed milk and dairy products (Law, 1979; Cousin, 1982). These enzymes pose problems in longterm storage of milk after heat treatments such as high temperature short time (HTST,77 °C, 17 s) and ultra-high temperature (UHT, 135-150 °C, 2-5 s) (Griffiths et al., 1981). Extracellular proteinases of P. fluorescens biotype A were reported to have  Di400c  of 131 s (Kroll and  Klostermeyer, 1984c). These enzymes retained 55-65 % activity after HTST treatment and 20-40 % activity after UHT treatment (Griffiths et al., 1981). The inferior quality of UHT milk made from raw, milk stored for 4 days at 6°C compared to that made from milk stored at 2 °C was reported to be mainly due to bacterial proteinases (Griffiths et al. 1988). Lipases from P. fluorescens and P. fragi survived 10 mins at 100 °C (Law et al., 1976). Psychrotrophic lipases retained 53 % of their activity after pasteurization treatment at 72 °C for 15 s although all of the native milk lipase was completely inactivated (Stead, 1983). Lipase retained 70 % of its activity in nonfat dry milk (NDM) and may cause degradation when NDM is used as an ingredient in other food products (Shamsuzzaman et al., 1987). B. Enzymatic Degradation of Milk Constituents Commercial raw milk is composed of approximately 90 % moisture, 3.2 % protein and 3 % fat. Proteolytic activity on milk proteins results in bitter peptides and gelation of UHT-milk (Law, 1979; Fairbairn and Law, 1986).  K and 13-caseins are reported to be  7 most susceptible to proteinases of Pseudomonas (Adams et al., 1976; Law et al., 1977; Murray et al., 1983). Lipolytic activity on milkfat produces short chain fatty acids (C4 - Ci 2) which are associated with rancid flavours and off-odors characteristic of cheeses with fruity offflavours (Law, 1979; Cousin, 1982; IDF, 1991). There were significant lipolytic offflavours associated with cheese made from milk with high psychrotrophic count although low proteolysis and lipase activity were detected (Law et al., 1979). Free fatty acids liberated during lipolysis are measured and expressed as acid degree value (ADV) to assess lipolysis of dairy products. Acid degree values exceeding 1.5 - 2.0 were found to be unacceptable (Hausler, 1972; Deeth et al., 1975; Rowe et al., 1990). 3. Relation Between Population Numbers and Enzymatic Activity Many studies suggest a lack of correlation between bacterial numbers and the level of enzyme activity (Rowe and Gilmour, 1986; Skura et al., 1986). Generally, populations of 5 x 106 - 107 CFU mL-1 of psychrotrophic bacteria were associated with proteolysis, lipolysis and sensory changes (Shelley et al., 1986; Garcia et al., 1989). These population numbers were the level at which post pasteurization problems due to enzymes could be expected in dairy products made from such raw milk (Muir and Phillips, 1984). 4. Production of Proteinase and Lipase as Function of Growth Phase Extracellular enzymes are produced by microorganisms after low molecular weight nutrients in milk have been depleted during the exponential growth phase. These enzymes degrade large molecular weight components and provide smaller molecular weight  8 substrates such as amino acids, peptides, and free fatty acids during stationary growth phase (Rowe and Gilmour, 1982). Extracellular enzyme production ensures a supply of carbon to maintain the microorganism's growth rate. P. fluorescens produced extracellular proteinase and lipase concomitantly during late log and early stationary phase (McKellar, 1982; Rowe and Gilmour, 1982; Murray et al., 1983; Stead, 1987; Griffiths, 1989; Kohlmann et al., 1991). Pseudomonas populations usually entered stationary phase when population density reached 108 CFU mL-1 (Rowe and Gilmour, 1982; Spohr and Schutz, 1990). Ninety-nine percent of the total phospholipase was produced during late exponential phase by Serratia liquefaciens (Givskov and Molin, 1992). Some authors reported proteinase detection in early log phase (Adams et al., 1975) while others showed it was maximum when cultures were in death phase (Malik et al., 1985). These differences would most likely have reflected the effect of culture conditions and the varying sensitivities of proteinase activity detection methods. Enzyme synthesis can be expressed as a function of growth in terms of differential rate of synthesis (McKellar, 1989) or specific proteinase producing power (Kroll and Klostermeyer, 1984a). The greatest amount of proteinase was produced during periods when P. fragi were not actively growing (Myhara and Skura, 1989). Mikel'saar et al. (1982), however, reported that increase in proteinase activity paralleled growth rate and ceased when the P. fluorescens population entered the stationary phase. 5. Drop in Dissolved Oxygen Tension in Milk There may be a relationship between enzyme synthesis and oxygen stress since a large  9 drop in oxygen tension in milk was observed when the P. fluorescens population reached 105 - 106 CFU mL4 (Spohr and Schutz, 1990) and 106 - 107 CPU mL4 (Rowe and Gilmour, 1982; Griffiths and Phillips, 1984; Rowe and Gilmour, 1986) just prior to enzyme production. The drop in oxygen tension in milk results as the demand for oxygen by microbial populations exceeds the rate at which aeration and agitation could replace the consumed oxygen. A small increase in demand then could result in a large decrease in oxygen tension (Rowe and Gilmour, 1986). 6. Dissolved Oxygen as Indicator of Potential Spoilage Dissolved oxygen tension is measured with a polarographic electrode consisting of a gold cathode and silver anode (Luck, 1991) and is expressed as parts per million (ppm) or as a percentage of saturation. Oxygen is primarily responsible for a positive redox potential (Eh) value which is a measure of the capacity of a system to give up electrons and to be oxidized/reduced (Costilow, 1981). The Eh value can be expressed in volts and the more positive the Eh value, the higher the oxidant to reductant ratio. Measurement of dissolved oxygen tension cannot directly indicate the bacterial load (Luck, 1991) nor predict enzyme production by psychrotrophs in milk. Difficulties arise since oxygen tension of milk is influenced by many factors and continually changes during storage and transport. Difference in milking procedures, time elapsing between milking and testing, agitation systems, surface area of milk in contact with air, and depth of milk tank would all influence the dissolved oxygen content in milk (Schroder, 1982; Luck, 1991). Rowe and Gilmour (1986) demonstrated that oxygen tension cannot be directly correlated to population numbers but have suggested the correlation between  10 dissolved oxygen tension and nature and rate of catabolism by microbial populations. B. Role of Oxygen in Aerobic Microorganisms Earth's atmosphere consists of approximately 20-21 % 02, 0.03 % CO2 and approximately 78 % (v/v) of chemically inert N2 gases (Weast, 1984). Oxygen is an essential nutrient or cosubstrate for many aerobic microorganisms. In obligate aerobes, molecular oxygen functions primarily as a terminal electron acceptor in aerobic respiration. Microaerophilic microorganisms grow best under lower partial pressures of oxygen between 2-10 % (v/v). Some microorganisms known as facultative anaerobes are able to grow in the presence or absence of molecular oxygen (Stanier et al., 1986). Oxygen also functions as a cosubstrate in dissimilation of many aromatic compounds and alkanes via oxygenases as well as dissimilation and assimilation of fatty acids. These enzymes oxidize organic substrates by direct incorporation of one or two oxygen atoms. Aromatic compounds or alkanes are catalyzed by such oxygenases and provide Psetdomonas with a source of carbon and energy (Clarke and Ornston, 1975b).  1. Energy Production A. Aerobic Respiration and Metabolism P. fluorescens is an obligate aerobe. These microorganisms obtain most of their  energy through aerobic respiration and oxygen serves as a terminal electron acceptor. Pseudomonads generate ATP by two biochemical mechanisms known as substrate level phosphorylation and electron transport (Clarke and Ornston, 1975a). ATP is generated by transporting electrons through a chain of carrier molecules which undergo reversible  11 oxidation and reduction. The resulting gradient of protons and electrical charge across the membrane known as the protonmotive force (pmt) provides energy for motility, operation of certain permeases, and synthesis of ATP from ADP. The electron transport chain (ETC) is composed of heme proteins such as cytochromes (a ,b ,c and d) which show variation among species and environments. According to Stanier et al. (1986) bacteria of the same strain show different cytochrome compositions when grown aerobically and anaerobically. Pseudomonas catabolize sugars to yield pyruvic acid via Entner-Doudoroff and pentose phosphate pathways but lack the Embden-Meyerhof pathway (Clarke and Ornston, 1975b). Pyruvate is either oxidatively decarboxylated to yield acetyl-CoA to provide precursor metabolites for biosynthesis or completely oxidized via the tricarboxylic acid cycle (TCA) to generate ATP when coupled to ETC. Aromatic compounds and aliphatic acids are oxidized via the p-ketoadipate pathway to yield acetyl-CoA which fuel TCA. The tricarboxylic acid cycle manifests its central position through catabolite repression by its intermediates on other catabolic enzymes (Clarke and Ornston, 1975a; Krieg and Holt, 1984). B. Anaerobic Respiration In the absence of oxygen or under low oxygen tension, alternate electron acceptors such as nitrate are utilized by Pseudomonas (Krieg and Holt, 1984). The same biochemical pathways are used as aerobic respiration differing only in the terminal electron acceptor of ETC. Reduction of nitrate (NO3-) yields nitrite (NO2-) then  12 dinitrogen oxide (N20) and finally nitrogen gas (N2). Nitrate reductase A which is involved in nitrate respiration in dissimilatory reactions is suppressed by high 02 levels (Clarke and Ornston, 1975a; Postgate, 1978). Nitrate and nitrite reductase activities were repressed by 20 % oxygen but when the 02 level was decreased to 6 %, the Bacterium denitrificans (P. stutzeri) culture was able to utilize both oxygen and nitrite  simultaneously (Kefauver and Allison, 1957). Nitrate can also function as a N source in assimilatory reactions where it is converted into NH3 by nitrate reductase B (Clarke and Ornston, 1975a). 2. Metabolism Under Decreased Oxygen Concentrations Respiratory metabolism of Pseudomonas can decrease dissolved oxygen tension in the medium very quickly when population numbers are high and its metabolic rate is at its maximum during exponential growth (Oblinger and Kraft, 1973). A detectable drop in electrical potential (Eh) was observed when population numbers were between 105 to 106 CFU mL4 (Oblinger and Kraft, 1973) and this coincided with the drop in oxygen tension (Spohr and Schutz, 1990). Oxygen limitation affects the metabolism of the aerobic and facultative anaerobic microorganisms (Kroll, 1989). Growth rate and biomass of P.fluorescens 378 (biotype A) in a continuous culture study were limited by rate of oxygen transfer and aeration (Grafe, 1982; Persson et al., 1990). There are reports of differences in utilization of substrates and accumulation of metabolites which reflect the different behavior of cells under varying oxygen concentrations (Griffiths and Phillips, 1984; Spohr and Schutz, 1990). Utilization of glucose by P. fluorescens was inferior at low (< 20 % of  13 saturation) oxygen levels but no differences in utilization were shown between medium (25 - 60 %) and high (> 80%) oxygen levels (Spohr and Schutz, 1990). The difference in metabolism of cells grown in aerated and nonaerated milk was also indicated by changes in concentration of the pools of the intermediary metabolite L-lactate and pyruvate in P. fluorescens (Griffiths and Phillips, 1984). Extracellular enzyme production has been  correlated with pyruvate assimilation and acetate accumulation (Spohr and Schutz, 1990). Acetate accumulation was reported to reflect oxygen deficiency in aerobically cultured P. fluorescens (Spohr and Schutz, 1990) and K. aerogenes (Teixeira de Mattos et al.,  1982). 3. Energy Status and Extracellular Enzyme Production In microorganisms, ATP level is regulated during the transition from aerobic to anaerobic state when oxygen is limiting. A decrease in energy charge would favor ATP generating catabolic processes and an increase would favour biosynthetic processes to expend the energy (Harrison, 1976). In Bacillus subtilis, transition-state regulators control metabolism and energy production when nutrients are depleted to prevent expression of detrimental functions when they are not needed and during suboptimal environments, so that alternative pathways are utilized for available nutrients (Strauch and Hock, 1993). Growth rate and extracellular enzyme production are regulated in accordance with the energy status of the cell. There is evidence of an inverse relationship between enzyme synthesis and metabolic state in many microorganisms. Wiersma et al. (1978) suggested  14 that proteinase was synthesized when growth was limited by low oxygen tension. It was suggested that growth rate and/or a factor associated with energy production regulated synthesis of enzymes (Wiersma et al., 1978). In support of Wiersma and Harder (1978), it was reported that P.aeruginosa synthesized proteinase after a lag phase due to nutrient limitation (Whooley et al., 1983). This metabolic repression reflected the low energy status of the cell since available energy, as metabolizable substrates, was limiting. An increase in proteinase production by Vibrio was also observed when growth was limited by low dissolved oxygen tension (Whooley et al., 1983). Aeromonas hydrophila also showed maximal proteinase production under nutritional stress despite poor growth (O'Reilly and Day, 1983). Interestingly enough, regulation in Serratia liquefaciens involved dual promoters where distal promoter Px was induced by anaerobiosis and proximal promoter PA was growth-phase regulated (Givskov and Molin, 1992). The relationship between energy and enzyme levels could then be used to explain the increase in enzyme production during stationary phase when energy level would be low (McKellar, 1989). When growth rate and enzyme activity are low, induction of proteinase synthesis would result in more readily available substrates, from proteinase activity, for growth enhancement. There is also a mathematical model for enzyme production : RE = f(environment) p. X where RE is the overall rate of enzyme synthesis, g is specific growth rate, X is the biomass concentration, and f(environment) is the function describing the influence of environment on "open doors" period, the time for enzyme synthesis, which is  15 proportional to a certain portion of a cell cycle (Votruba et al., 1986). A slower growth rate would then ensure longer duration in the "open doors" period and result in increased enzyme production. 4. Critical dissolved oxygen tension (CDOT) The effect of dissolved oxygen tension (DOT) on growth and respiration was similar for aerobes and facultative anaerobes when respiration provided most of the energy (Harrison, 1976; Costilow, 1981). Dissolved oxygen can be considered a nutrient just as any carbon or nitrogen source but unlike other substrates, it is relatively insoluble (< 10 mg/L) so it becomes limiting during intense growth (Costilow, 1981). Growth rate and growth efficiency are independent of DOT above the critical DOT (CDOT) level, but when dissolved oxygen is below that level, a decrease in maximum growth rate and growth efficiency was reported (Costilow, 1981). Metabolism of aerobes and facultative microorganisms was not affected by changes in dissolved oxygen tension between the range of 20-150 mm Hg (Harrison, 1976). As dissolved oxygen tension (DOT) decreased from the saturation point, respiration activity was reported to increase as requirement for ATP also increased; therefore, phosphorylation efficiency was dependent on the level of DOT (MacLennan et al., 1971). If DOT fell below a critical level (CDOT) then respiration rate decreased (Harrison, 1976). For the obligate aerobe Pseudomonas AM1, CDOT was 28 mm Hg, below which different pathways for synthesis of extracellular products were reported (MacLennan et al., 1971). As DOT decreased, respiration rate of facultative anaerobes increased and then decreased at very low oxygen tension (Harrison, 1976). This oscillation between high  16 and low respiration rate was important for the microorganism to switch rapidly from a low respiration rate at high oxygen tension to a higher respiration rate at a lower oxygen tension to maintain energy status in the cell (Harrison, 1976). Extracellular enzyme production by micloorganisms appears to be under catabolite repression which is induced by appropriate limiting factors such as oxygen or nutrients. There must, however, be sufficient available energy and substrates to initiate and maintain its synthesis. McKellar and Cholette (1984) reported that maximal proteinase production occurred under excess carbon, nitrogen and orthophosphate but when any one of these were limiting, proteinase production was lower. C. Factors Affecting Enzyme Production 1. Temperature Optimum growth and extracellular enzyme synthesis did not occur at the same temperature and optimum enzyme synthesis temperature was usually below optimum growth temperature (McKellar, 1989). Fairbairn and Law (1986) hypothesized that psychrotrophs, to maintain their growth rate at the expense of cell yield, produced higher enzyme levels at lower temperatures to compensate for the lower enzymatic activity, as well as decreased cellular processes. The effects of temperature on enzyme synthesis by psychrotrophs were reported to be strain specific (Griffiths, 1989). McKellar (1982) reported that only 55 % of the total proteinase activity at 20 °C was detected at 5 °C but specific activity per unit growth was much higher at the lower temperature. On the contrary, Roussis et al. (1988) reported higher proteinase activity as a function of growth  17 at 23 °C than at 4 °C for P. fluorescens GR83. P. fluorescens A32 showed maximum proteolytic activity at 17.5 °C which was not its optimal growth temperature (Gugi et al.,1991). Lipase activity of P. fluorescens in whole milk was higher at 4 °C than at 25 °C which may not be due to an increase in enzyme production (Bucky et al., 1986). The authors suggested that degradation of lipases by coexisting proteinase during storage at 25 °C may have been the reason why the observed activity at 25 °C appeared to be lower. On the contrary, Griffiths (1989) reported that lipase production by P. fluorescens was suppressed at 2 °C and 6 °C but not at 10 °C and 21 °C. 2. Ions There are reports of many ions involved in the production of extracellular enzymes by P. fluorescens. Calcium and zinc are essential for proteinase activity and synthesis  (McKellar and Cholette, 1986b). Phosphate was also required for maximum proteinase synthesis but was detrimental at an excessive level (McKellar and Cholette, 1985). High iron concentrations were reported to repress lipase production (Ishihara et al., 1989) so it was postulated that there was a direct role of iron on the regulation of enzyme production by psychrotrophs (McKellar et al., 1987). It was also reported that limiting iron conditions at the end of exponential growth phase inhibited cytochrome synthesis and thereby enhanced proteinase synthesis by Vibrio (Wiersma et al., 1978). A decrease in cytochrome synthesis would have affected the energy generating status of microorganisms and thus would have had the same effect as limiting oxygen on growth (Wiersma et al., 1978).  18 3. Oxygen There are conflicting reports on the role of oxygen in extracellular enzyme production by microorganisms. Some reports show enhancing effects while others show deleterious effects of oxygen but there appears to be a causal relationship between oxygen tension and enzyme production. Studies of the influence of oxygen on subsequent extracellular enzyme synthesis have been made using different controls of oxygen tension (McKellar,1989). Some investigators have compared agitated and static cultures (Hare et al., 1981; Fairbaim and Law, 1987) and others have employed nitrogen flushing (Murray et al., 1983) and overlays (Skura et al., 1986) as well as carbon dioxide flushing (King and Mabbit, 1982). A. Oxygen Inhibits Enzyme Production There are many reports of oxygen inhibiting extracellular enzyme production. When a Vibrio culture was aerated to increase dissolved oxygen tension, proteinase production  was subsequently inhibited (Wiersma et al., 1978). A drop in oxygen tension was observed by Rowe and Gilmour (1982) just prior to proteinase and lipase production. Rowe and Gilmour (1982) forcibly decreased oxygen tension of a P. fluorescens culture after 3 days from 85 to 12 % within a 6 hour period. This resulted in an earlier onset of enzyme production. In support of this observation, Griffiths and Phillips (1984) reported a lower level of proteolysis when raw milk was subjected to different aeration treatments to prevent the normal rapid drop in oxygen tension. An increase in lipolysis was, however, reported which may have been the result of the decrease in proteinase  19 activity on co-existing lipases. Lu and Liska (1969), however, reported inhibition of lipase production by P. fragi when the culture was aerated. Another way of examining the effect of oxygen is to observe its effect during nonaerated or static culture conditions. Lipase activity was higher in statically grown than in agitated P. fluorescens cultures (Roussis et al., 1988). Proteinase activity per unit dry weight was also higher in static than in agitated cultures although the growth rate was slower (Fairbairn and Law, 1987). A similar observation about extracellular proteinase activity from a non-aerated P. aeruginosa culture was made, even though biomass was lower (Whooley et al., 1983). Limiting oxygen conditions may decrease growth rate but increase extracellular proteinase production. B. Oxygen Enhances Enzyme Production There are reports that aeration causes more rapid and enhanced proteinase synthesis by P. lachrymans (Keen and Williams, 1967) and by Micrococcus GF (Garcia de Fernando  and Fox, 1991), and collagenase by Achromobacter isophagus (Reid et al., 1978). Higher proteolytic and lipolytic activities were detected in agitated cultures of P. fluorescens (Fox and Stepaniak, 1983) and Pseudomonas sp. B-25 (Malik et al., 1985).  Static culture conditions may have decreased proteinase production due to the repressive effects of ammonia which otherwise would not have been localized during agitation (Whitaker et al., 1965). Static conditions were reported to inhibit proteinase production by Vibrio alginolyticus (Hare et al., 1981) and lipase production by P. fluorescens (Fox and Stepaniak, 1983; Bucky et al., 1986). Likewise, no proteinase  20 activity was detected in nitrogen-flushed raw milk after 18 days of storage although proteolytic psychrotrophs grew to a cell density of 107 CFU mL1 (Murray et al., 1983) and in nitrogen-overlayed UHT-milk (Skura et al., 1986). Proteolytic psychrotrophs from raw milk stored under nitrogen atmosphere were able to produce proteinase on skim milk agar plates during aerobic but not anaerobic incubation (Murray et al., 1983). Normal spoilage pattern was resumed when the nitrogen-overlay of UHT milk inoculated with P.fluorescens was disrupted, resulting in an increase of bacterial population similar to that of the aerobic control. An increase in proteinase activity was also observed but maximum activity in the milk with the disrupted nitrogen-overlay was only 50 % of the aerobic control (Skura et al., 1986). P. fluorescens NC3 showed low proteinase production in anaerobic conditions when nitrate was used as an electron acceptor (Himelbloom and Hassan, 1986). Slower growth rate and low population density may have been correlated with lower enzymatic activity during non-aerated conditions. Hare et al. (1981) reported inhibition of collagenase and proteinase production with the removal of oxygen and decrease in exponential growth. Likewise, the population numbers reported by Skura et al. (1986) may not have been sufficiently high to produce detectable proteinase activity by P. fluorescens in nitrogen atmosphere. Decreasing the aerated state of milk by using  nitrogen to decrease oxygen from 9-12 ppm to 1-3 ppm at 3 °C caused an 83 % increase in generation time of P. fluorescens (Brandt and Ledford, 1982). Longer lag times, slower growth rates and low proteinase activity were also observed for microflora (psychrotrophs, proteolytic psychrotrophs, and lactic acid bacteria) in nitrogen flushed  21 raw milk (Murray et al., 1983). On the contrary, lower biomass for non-aerated cultures of P. aeruginosa showed higher proteinase activity (Whooley et al., 1983). In support of this, Fairbaim and Law (1987) reported that aeration resulted in faster growth rate and earlier proteinase production but static culture showed higher proteinase production per unit dry weight although growth rate was lower. 4. Regulation of Extracellular Enzyme Synthesis A. Proteinase Proteinase production by P. fluorescens was reported to be inducible (McKellar, 1982) and regulated (Vilu et al., 1981). Proteinase production occurred de novo where translation and secretion of extracellular proteinase by P. fluorescens were almost simultaneous (Mikel'saar et al., 1982). An Induction-Repression model has been used to explain proteinase regulation in P. fluorescens. There must be a balance between induction and repression for optimal rate  of enzyme production. This model assumes that microorganisms produce low basal levels of extracellular enzyme in the absence of the inducer but as the low molecular weight inducer enters the cell, proteinase synthesis is induced. Proteinase ensures a supply of carbon for energy so it is repressed by simple carbon sources, pyruvate and growth limiting substrates (McKellar, 1982).  22 1. Carbon Catabolite Repression The delay in proteinase production until late log-early stationary phase is probably the result of repression of proteinase synthesis by the presence of easily metabolized carbon sources, a process known as catabolite repression (McKellar, 1982). Control systems nt only ensure the use of simple carbon sources first but also ensure that growth has priority over less important processes such as differentiation and secondary metabolism (Vining and Chatterjee, 1982). Using continuous cultures, an increase in dilution rate from very low values was shown to increase proteinase production but was inhibitory at very high dilution rates due to catabolite repression (Wiersma and Harder, 1978). Easily metabolized carbon sources such as tricarboxylic acid cycle intermediates repress proteinase synthesis (Hare et al., 1981; McKellar, 1989). Citrate, however, which is the second most available low molecular weight carbon source next to lactose in milk, is spared even when populations reached 109 CFU mL-1 (Griffiths and Phillips, 1984; San Jose et al., 1987). Pyruvate, a key intermediate metabolite, on the other hand induced proteinase synthesis (McKellar, 1982; Griffith and Phillips, 1984). It was also reported that the lack of a glycolytic (Embden Meyerhof) pathway in Pseudomonas made glucose a poor source of carbon and a poor repressor of proteinase production (Palleroni, 1975; Griffiths and Phillips. 1984). Inducers of proteinase synthesis can be generalized as proteins, peptides (< 5000 MW), and amino acids such as glutamic acid, glutamine and asparagine (McKellar, 1982; Fairbaim and Law, 1987). Repressors can be generalized as sugars, organic acids, and amino acids such as cysteine (Himelbloom and Hassan, 1986).  23 2. Nitrogen Catabolite Repression Since protein is a source of both carbon and nitrogen for Pseudomonas, regulation of proteinase synthesis may be associated with nitrogen metabolism and ammonia assimilation (Fairbairn and Law, 1986). During ammonia limitation, production of catabolic enzymes for nitrogen and carbon utilization as well as nitrate reduction were induced (Grafe, 1982). It was reported that glutamic acid and glutamine were the best amino acid inducers when used as sole nitrogen sources (McKellar, 1982). Fairbairn and Law (1986) suggested that it may be valuable for proteinases to be under the control of nitrogen catabolite repression as a part of a group of nitrogen degrading enzymes. Proteinase synthesis may be under endproduct repression where nitrogenous endproducts (ammonia, urea) and certain amino acids directly affect the rate of its synthesis (San Jose et al., 1987). It was reported that uptake of these substrates from the external environment regulates metabolism of microorganisms (Clarke and Ornston, 1975a). Ammonium ions have been reported to either repress, by feedback inhibition, or support proteinase synthesis of P. fluorescens depending on the concentration (Whooley et al., 1983; McKellar and Cholette, 1984). There is increasing evidence that many processes such as repression of proteinase by ammonia, transport systems of amino acids, and several anabolic and catabolic reactions are mediated by glutamine synthetase in enteric microorganisms (Magasanik and Neidhardt, 1987). Ammonia provides a nitrogen source and is assimilated as glutamate for the synthesis of amino acids. Glutamine is required for the synthesis of nucleotides,  24 amino sugars, and some amino acids (Reitner and Magasanik, 1987). B. Lipase  Lipase regulation is usually grouped together with proteinase regulation. Lipase is not essential for growth so it may only be produced in stress-free growth conditions (Fox and Stepaniak, 1983). There is some evidence that lipase production by P. fluorescens may be constitutive, since triglycerides were not required for synthesis (Fox and Stepaniak, 1983; McKellar, 1986). In fact, lipase activity was higher in skim milk than whole milk although the authors suggested that milk fat interfered with the lipase activity detection methods with the whole milk (Bucky et al., 1986; Griffiths, 1989). The addition of olive oil delayed the growth rate of P. fluorescens but its final population and lipase activity were unchanged (Andersson, 1980b). Inhibition of lipase production by P. fluorescens was observed when free fatty acids (FFA) were added to milk which suggested that FFA level may repress lipase production through feedback inhibition (Bucky et al., 1986). C. Direct Regulation by Oxygen  There is no direct evidence that proteinase or lipase synthesis by P. fluorescens is regulated by oxygen concentration. Genetic evidence has shown that certain functions in some microorganisms are oxygen regulated. In facultatively photosynthetic Rhodobacter capsulatus, pigment protein complexes are under oxygen concentration and light intensity  control (Wellington et al., 1991). This microorganism can respire aerobically using the electron transport chain but under anaerobic or decreased oxygen concentrations it can switch to cyclic phosphorylation due to the presence of an oxygen regulated promoter (Adams et al., 1989; Wellington et al., 1991). In Serratia liquefaciens, it was shown that  25 extracellular phospholipase was regulated by two promoters, PA under growth phase regulation and Px under anaerobic induction (Givskov and Molin, 1992). In Saccharomyces cerevisiae, certain oxygen dependent functions such as alternate  cytochrome subunits, oxidases and desaturases in heme and sterol biosynthesis were induced at low oxygen tension so that the oxygen could be utilized more efficiently (Zitomer and Lowry, 1992). There are reports of cloning and sequencing of P. fluorescens B52 lipase (Tan and Miller, 1992) but not of proteinase.  D. Potential Disadvantages of Decreased Oxygen Atmospheres Conditions of limiting oxygen content in milk may pose potential problems. Deoxygenated pasteurized milk samples (0.8 mg 02 L-1 milk) were rated lower due to earlier off-flavour development than the non-deoxygenated milk samples despite relatively low total psychrotroph count (Schroder, 1982). One main reason for this observation was the higher coliform count in deoxygenated milk. Since coliforms are facultative, these microorganisms were not as sensitive to oxygen content as aerobic microorganisms and proliferated. These decreased oxygen tension environments selected for psychrotrophic, facultative anaerobes, principally coliforms (Schroder, 1982) and lactic acid bacteria (Murray et al., 1983). Another concern would be the potential growth of facultative pathogens such as Salmonella, Listeria, and Bacillus. A decrease in extracellular proteinase activity of P. fluorescens may inadvertently result in accumulation of intracellular or cell-associated proteinase or peptidases. Keogh and Pettingill (1984) suggested that intracellular proteinases of Pseudomonas may be  26 significant when population numbers are high. Other studies showed negligible concern for intracellular proteolytic activity (Kohlmann et al., 1991; Shamsuzzaman and McKellar, 1987). There was, however, bitterness associated with the growth of proteinase deficient P. fluorescens RM14 in milk, detected on day 7 when cell density was 108 CFU mL4,  which was probably a consequence of the production of metabolic products during stationary growth phase (Torrie et al., 1983). Another concern of decreasing proteinase synthesis is the possible enhancement of lipase activity in the absence of proteinases.  27 MATERIALS AND METHODS  A. Controlled Oxygen Atmospheres The experimental design shown in Figure 1 was set up in a 4°C coldroom. Nitrogen (N2) and oxygen (02) gases (USP grade, Pacific Medigas Ltd., Vancouver, BC) were controlled with the use of flowmeters (Series 50 M for N2 and Series 150 for 02, Linde Union Carbide, Somerset, NJ) and sterilized with 0.30 gm bacterial air vent filters (Gelman Sciences, Ann Arbor, MI). The gases were humidified by sparging through air stones into a flask containing sterilized 0.5 % (w/v) NaCl solution with the same ionic strength as milk (Murray et al., 1983). The mixed gas flowing at — 300 mL min-1 continuously overlay 2 L of UHT-sterilized milk (2 % m.f.) which was stirred with magnetic stir bars at 300 rpm in hypochlorite-sterilized Nalgene carboys (4 L capacity, Sybron/Nalge, Rochester, NY). The oxygen content of the controlled atmosphere was measured just prior to the entry into carboys and monitored using a Shimadzu Gas Chromatograph-14A (Shimadzu Corporation, Kyoto, Japan) by taking duplicate or triplicate air samples. The gas chromatograph was set up with 80/100 molecular sieve 5a stainless steel column (6' x 1/8") and 80/100 Porapak N, 1/8" stainless steel column (Supelco Canada Ltd., Oakville, ON). Thermal Conductivity Detector (TCD) system was used. Data was collected using a Chromatopac CR501 integrator (Shimadzu, Kyoto, Japan). The atmospheric oxygen concentration is reported as a percentage (%). The dissolved 02 content was measured in parts per million (ppm) using a portable Clark electrode  Figure 1. Experimental setup in 4°C coldroom. Oxygen (02) and nitrogen (N2) gases were sterilized through 0.3 gm air filters prior to mixing and humidifying in 0.5 % (w/v) NaC1 solution. Atmospheric oxygen and carbon dioxide concentrations were determined using gas chromatography. Each trial consisted of four of these setups under four controlled oxygen atmospheres.  29 02 probe (YSI, Yellow Springs, OH). The 02 meter was calibrated using water saturated air at 4 °C as representing 100 % 02 saturation. Since more accurate measurement of dissolved 02 is made when liquid is stirred (Kotters, personal communications 1992) the milk samples were stirred with a small magnetic bar at slow rpm and values measured within 30 seconds during measurements in Trial III but not in Trials I and II. B. UHT Milk  To avoid variation of milk within each trial, one case of 12 DairyMaid 2 % UHT milk in 1 L tetrapak cartons of the same batch were picked up from Dairyworld Foods (Burnaby, BC) and stored at 4 °C. The UHT-milk was recently processed and had an expiry shelf-life of 3 months. Tetrapak milk cartons were wiped with 70 % ethanol solution and opened aseptically with ethanol flamed scissors. Two litres of milk were poured aseptically into carboys which had previously been sterilized with a 200 ppm hypochlorite solution and thoroughly rinsed with sterile distilled water. A magnetic stir bar was included in the carboy before sterilization. Milk was then stirred under appropriate flowing controlled oxygen atmospheres and equilibrated before the inoculation of P. fluorescens. C. Culture Conditions  Pseudomonas fluorescens biotype A ATCC 17397 (American Type Culture  Collection, Rockville, MD) was maintained on trypticase soy agar (TSA) slants at 4 °C and transferred monthly. Trypticase soy broth (25 mL TSB in 250 mL flask) was inoculated with P. fluorescens and incLbated at 21 °C for 18 hours in a shaking water  30 bath at 160 rpm (Lab-Line, Melrose Park, IL) and then subcultured. After 18 hours of incubation, 10 mL of the culture was centrifuged at 12 100 x g for 20 minutes at 4 °C in an RC2-B Sorvall Superspeed centrifuge (Dupont Sorvall, Newtown, CT). The supernatant was removed, the pellet resuspended in 10 mL 0.1 % peptone and the centrifugation process was repeated. Optical density was measured at 660 nm to estimate the population of the resuspended pellet. Two litres of DairyMaid 2 % UHT milk (Dairyworld Foods, Burnaby, BC) was inoculated with P. fluorescens to an initial population of approximately 104 CFU mL-1. After 30 minutes of stirring, samples were collected for plating of P. fluorescens to provide day 0 cell numbers. Samples were collected for plating and tested every 2 days for P. fluorescens cell number, proteinase and lipase activities. Milk samples of 30 mL were frozen at -20 °C or below and analyzed on a later date for proteolysis and lipolysis. D. Antibiotic Testing of UHT Milk  Uninoculated milk samples used in Trials II and III were sent to Dairyworld Foods (Burnaby, BC) for testing of antibiotic residues: 13-1actams, sulfa drugs, and tetracyclines via the Charm II methodology. E. Enumeration  Growth of P. fluorescens was monitored by spiral plating (Spiral Plater Model D, Spiral Systems, Inc., Bethesda, MD) on TSA plates in triplicate followed by incubation at 21 °C for 24-36 hours.  31 F. Proteinase Activity Assay Proteinase activity was assayed as described by Kohlmann et al. (1991) with some modifications. The reaction mixture consisted of 1.0 mL of 0.1 M Tris-HC1 pH 7.5 buffer and 1.0 mL of 1.5% azocasein (Sigma, St. Louis, MO) solution prewarmed to 40 °C. Azocasein had been dissolved by boiling in 0.1 M Tris-HC1 pH 7.5 and filtered through Whatman #1 filter paper to remove any undissolved lumps. Thimerosal was added to a final concentration of 0.05 % in 1.0 mL of milk to inhibit extracellular enzyme production. Milk samples were then microcentrifuged (Eppendorf Centrifuge 5415 C, Brinkmann Instruments, Rexdale, ON) at 2040 x g for 10 minutes at room temperature to remove microorganisms as well as milkfat. The cell free supernatant was removed as source of proteinase and chilled in an icebath until ready for use. Fifty Ill. of the cell free supernatant was used for Trial I but was later increased to 200 111, for Trials II and III in an attempt to increase sensitivity of the assay. The contents of the reaction mixture were vortexed and incubated at 40 °C for 1 hour with shaking at 160 rpm. Reactions were stopped with the addition of 2.0 mL of 24 % (w/v) trichloroacetic acid (TCA) and vortexed immediately and placed in an icebath for 15 min. Sample blanks (addition of TCA to azocasein prior to the addition of enzyme) and substrate blanks (azocasein without the addition of enzyme and incubated with the reaction mixtures) were run concurrently. The tubes were centrifuged at 2910 x g for 15 min in GS-6 Beckman centrifuge (Beckman Instruments Inc., Palo Alto, CA) to remove the precipitate. The absorbance of the supernatant was measured at 366 nm with a UV-160 Shimadzu spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Proteinase activity was reported after subtraction  32 of controls. All reactions were performed in triplicate. One azocasein unit of proteinase activity was defined as an increase in absorbance of 0.01 at 366 nm per mL of milk per hour. Proteinase activity was also checked using 1 % skim milk agar plates containing filter discs soaked in supernatant followed by incubation at 35 °C for 24 hours. The plates were flooded with 10 % TCA to check for zones of clearing as an indication of proteolysis. Skim milk agar (S MA) plates were also used to check whether the absence of proteinase activity during the assay was a result of loss of ability by P. fluorescens to synthesize proteinase enzymes during storage due to mutation. P. fluorescens from milk samples were streaked on SMA plates and incubated at 21 °C for 24-36 hours to evaluate growth and proteolytic activity. G. Proteolysis Proteolysis was determined using absorbance at 280 nm to measure aromatic amino acids and peptides soluble in 12 % TCA. To each tube containing 2.0 mL of distilled water and 2.0 mL of 24 % TCA, 100 !IL of each milk sample was added and vortexed and put in an ice bath for 15 min. The tubes were centrifuged at 2910 x g for 15 min and the supernatant absorbance measured at 280 nm. A tyrosine standard curve was used to convert absorbance values to tyrosine equivalents. A stock solution of 4 mM tyrosine was made by dissolving in 10.0 mL 1 N NaOH and then making the volume up to 200 mL with distilled water in a volumetric flask. Tyrosine standard solutions were made up in a final volume of 4.1 mL. Degree of proteolysis was expressed as ilmole 12 % TCA  33 soluble tyrosine equivalents at 280 nm mL-1 milk. H. Lipase Activity Assay Chromogenic phenolic ester fi-naphthyl caprylate was used as a substrate to measure lipase activity in uncentrifuged milk sample. Thimerosal was added to a final concentration of 0.05 % in 1 0 mL of milk to inhibit extracellular lipase production. Modification of the lipase assay by Versaw et al. (1989) was made to clarify the reaction mixtures. In Trial I, 0.2 mL of 200 mM sodium taurocholate (NaTC) (Sigma, St. Louis, MO) was added to 1.8 mL of 50 mM of N,N-bis[2-hydroxyethy1]-2-aminoethanesulfonic acid (BES, Sigma, St. Louis, MO) pH 7.2 buffer, prewarmed to 40 °C. In Trials H and III, 0.2 mL of 100 mM NaTC was added to 1.0 mL of 50 mM BES buffer. Fifty pL of enzyme source was added and vortexed. Twenty p,L of freshly prepared 500 mM 13-naphthy1 caprylate (crystalline, Sigma, St. Louis, MO) in dimethylsulfoxide (DMSO) (Fisher Certified, Fisher, Vancouver, BC) was added and vortexed. The tubes were incubated at 40 °C for 1 hour shaking at 160 rpm to ensure proper mixing. Freshly prepared 500 mM Fast Blue BB salts (practical grade, Sigma, St. Louis, MO) dissolved in DMSO was added in 20 pL aliquots to the reaction mixtures. The tubes were incubated at 40 °C for 5 minutes and reactions were stopped with the addition of 0.2 mL of 10 % TCA to a final concentration of approximately 1 %. A solvent ratio of 50:50 ethyl acetate to ethanol (2.71 mL) was used to clarify the reaction mixtures for Trial I but 60:40 ethyl acetate to ethanol (2.51 mL) was used for Trials II and HI since the volume of BES buffer had been adjusted. Sample (no substrate) and substrate (no enzyme source) blanks were  34 included. All reactions were performed in quadruplicate. Lipase activity was reported after subtraction of controls. Absorbance was measured at 540 nm. A stock solution of 10 mM [3-naphthol (Sigma, St. Louis, MO) was prepared by dissolving in DMSO. Serial dilutions of 13-naphthol were made and added to reaction mixtures in the absence of the enzyme source and incubated along with the other reactions. A standard curve of concentration of 13-naphthol vs Abs540 was plotted in order to convert absorbance values into 1.tmoles of 13-naphthol. One 13-naphthol unit was defined as one gmole 13-naphthol released mL4 milk h-1. I. Lipolysis The degree of lipolysis of the milk fat was measured by using the titration method with dilute methanolic KOH (Deeth et al., 1975). Five mL of extraction mixture consisting of isopropanol, petroleum ether and 4 N H2SO4 (40:10:1 v/v) were vortexed with 1.5 mL of milk in a 25 mL graduated test tube. Three mL of petroleum ether was added and the reaction mixture was washed with 2.0 mL distilled water. The top organic layer was titrated, with — 0.01 N KOH (methanolic) and 2 drops of 1 % a-naphtholphthalein (Sigma, St. Louis, MO) indicator, in an enclosed vessel in the absence of atmospheric CO2 which has been reported to neutralize KOH (IDF, 1991). The KOH concentration was determined after standardizing it against 0.01 N HC1. Transition from green to blue represented an endpoint of pH 8.7. A control blank without the addition of milk was used to obtain a background titration volume which was subtracted from samples. Free fatty acids (FFA) were converted into acid degree values (ADV) (Deeth et al., 1975; IDF, 1991).  35 FFA (gequivalent mL -1 milk) = T * N x 103 P*V  where T = net titration volume (inL) N = normality of methanolic KOH (N) P = proportion of upper layer titrated  (ie.) volume of aliquot withdrawn relative to total volume of upper layer V = volume of milk (mL) ADV (mmoles of FFA per 100 g fat) = (FFA- 0.07) / 0.62 (calculations from Deeth et al., 1975) J. Investigation of High ADV a. Induced Lipolysis by Mechanical Agitation  Two litre volumes of UHT-sterilized milk in 4 L carboys were stored at 4 °C for 7 days under aerobic atmospheres, one stirred at 350 rpm and the other unstirred, to determine the effect of mechanical agitation on high ADV. Milk samples were collected and checked for degree of lipolysis. b. Metabolism of P.fluorescens biotype A (ATCC 17397)  P. fluorescens biotype A (ATCC 17397) was checked for changes in catabolism of  carbohydrates unique to biotype A. An increase in acid production may have contributed to the high acid degree values. Nitrate reduction was also checked to confirm a change in metabolism of P. fluorescens although it may not directly be related to the high acid degree values.  36 1. Carbohydrate Utilization Hugh Leifson media (Krieg and Holt, 1984) were supplemented with the following sugars (adonitol, L-arabinose, D-trehalose, D-xylose, D-glucose, and lactose) to check for mutations in catabolism during storage and culturing of the P. fluorescens stock strain. The strain was compared to a new strain of P. fluorescens biotype A purchased from ATCC then subcultured three times in TSB. All sugars were sterilized by autoclaving with the exception of xylose, lactose and D-glucose which were sterilized through 0.45 pm hydrophilic cellulose acetate membrane filter (Pro-X filter unit, Diamed Lab Supplies, Mississauga, ON) and added to a final concentration of 0.2 %. Hugh Leifson medium was used to check for acid production with bromothymol blue indicator. 2. Nitrate Reduction P. fluorescens was statically incubated in 10 mL of Bacto nitrate broth (Difco) for 24  hours at 25 °C. To 3.0 mL of the culture, 0.2 mL of Reagent A (sulfanilic acid) and Reagent B (a-naphthol) were added. A red colour indicated the presence of nitrite reduced from nitrate. The method of Stanier et al. (1966) was used to determine the denitrifying characteristics. P. fluorescens was grown statically for 24 hours at 25 °C in 5 mL of Bacto nitrate broth supplemented with a final concentration of 1 % glycerol. A loopful was transferred to 10 mL of fresh nitrate broth then topped with 3 mL of 1 % molten agar and cooled. The tubes were incubated statically at 25 °C up to 5 days to monitor growth and gas production.  37 c. Carbon Dioxide Interference The set-up was modified to eliminate the effects of atmospheric CO2 as shown in Figure 2. Either a vacuum or a gentle flow of nitrogen gas (USP grade, Pacific Medigas Ltd., Vancouver, BC) was effective in removing the atmospheric CO2 in the titration vessel. The tip of the volumetric pipette was extended down as far as possible over the free fatty acid extract to minimize evaporation of methanolic KOH. K. Statistical Analysis (Trial ifi) Correlation coefficients (r) of P. fluorescens cell numbers, proteinase activity, proteolysis, lipase activity and lipolysis under 4.8, 10.3, 13.4 and 20.2 % atmospheric 02 were obtained using SYSTAT (1989). Correlation coefficient is the measure of the strength of the relationship between two variables x and y (Ott, 1988).  38 0.01 N KOH  Nitrogen gas Figure 2. Experimental setup for titration of free fatty acids. The ether extracts containing free fatty acids and indicator were added to the titration flask and flushed with a gentle flow of nitrogen gas. The rubber stopper was replaced to maintain the carbon dioxide-free atmosphere during titration.  39 RESULTS AND DISCUSSION  Data from three trials composed of four treatments in each trial are presented. Conditions were established after preliminary work using atmospheres of 1, 5, 10, and 20 % oxygen. The atmospheric oxygen levels which were tested are shown in Table 1. At least 24 to 36 hours were required to equilibrate milk with the atmosphere in each carboy before inoculation of P. fluorescens. In the literature, the time required for equilibrium was reported to be dependent on the atmosphere, flow rate, speed of agitation and the medium itself. Air flowing at 25 mL min-1 flushing through trypticase soy broth (TSB) required 24-48 hours for Eh values to stabilize, N2 flowing through milk at 75 mL min-1 required 48-72 hours (Oblinger and Kraft, 1973), and only 10 minutes were required for N2 sparging at 100 mL min-1 through raw milk to deplete 02 below 1 ppm (Murray et al., 1983). P. fluorescens was inoculated at an initial cell density of approximately 104 CFU mL-1  to reflect the population normally encountered in raw milk (Cousin, 1982). Initial populations ranged between 4.13 to 4.40 Login CFU mL-1 throughout Trials I to III (Table 2). A geometric mean of 1.6 x 104 CFU mL-1 (4.2 Logic, CFU mL-1) of psychrotrophs in raw milk was reported by Griffith et al. (1988) and counts between 103 -104 CFU mL-1 in raw milk stored at 7 °C for less than 24 hours were reported by San Jose et al. (1987). The use of UHT-sterilized milk provided a model system where only the activities of P. fluorescens were observed without interference of competing microorganisms which  40 Table 1. Atmospheric oxygen levels tested % Oxygen in Atmosphere a SAMPLES^1^2^3^4 TRIAL I^1.2 + 0.1 b^5.3 + 0.2^10.2 + 0.6^20.9 + 1.0 II^13.4 + 0.2^16.9 + 0.2^9.6 + 0.9^20.7 + 0.8 III^13.4 + 0.4^4.8 + 0.2^10.3 + 0.7^20.2 + 0.6 c a^Nitrogen gas as balance b^The mean of triplicate values + standard deviation c^Medical air was used as aerobic control instead of mixing 02 and N2  Table 2. Initial population of P.fluorescens LOG 10 CFU mL-1 SAMPLES^1^2^3^4 TRIAL I^4.18 + 0.09 a 4.15 + 0.07^4.13 + 0.06^4.18 + 0.05  II^4.16 + 0.00^4.21 + 0.00^4.17 + 0.07^4.16 + 0.03 III^4.39 + 0.02^4.38 + 0.02^4.39 + 0.03^4.40 + 0.02  a^The mean of triplicate values + standard deviation  41 would certainly have existed in a raw milk system. UHT-sterilized milk was reported to be more sensitive to the actions of crude proteolytic enzymes than pasteurized milks (McKellar, 1981) and contained less Maillard reaction products from thermal processing (Burton, 1988). A. TRIAL I The atmospheres tested in the preliminary run were repeated in Trial I and monitored using gas chromatography. The atmospheres were 1.2, 5.3, 10.2, and 20.9 % oxygen (Table 1). 1. Growth The growth pattern of P .fluorescens in Trial I is shown in Fig. 3. The population at 20.9 % oxygen showed a longer lag phase than those at 1.2 and 5.3 % but by day 8 it exceeded all the other populations as it entered stationary phase. This observation was different from that reported by Skura et al. (1986). In their study, P. fluorescens under aerobic conditions showed virtually no lag phase and a faster growth rate than populations under nitrogen atmosphere. The absence of a lag phase by P. fluorescens in UHT milk was also reported by Spohr and Schutz (1990) and by Rowe and Gilmour (1983) even when a 25 °C culture was inoculated at 7 °C. The population under 10.2 % oxygen behaved much like that of aerobic control during storage. It reached very high population numbers exceeding 108 CFU mL-1 on day 8 as it also entered stationary phase. Its population density was slightly lower than the aerobic control throughout 14 days of storage. The P . fluorescens populations under 1.2 % and 5.3 % oxygen surprisingly  42 Figure 3. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate bacterial counts + standard deviation.  10  Atmospheric Oxygen  1.2% —A—  5.3 %  —11— 10.2% —0— 20.9%  0^2^4^6^8^10^12^14^16  Storage Time (Days)  43 showed the fastest growth rate up to day 4 which was also observed in our preliminary work. The populations under 1.2 and 5.3 % oxygen entered stationary phase on day 4, four days earlier and at lower cell densities than populations at 10.2 and 20.9 % oxygen. The final population numbers were, however, lower than the aerobic control. It was highly unexpected that an aerobic microorganism would show a faster growth rate under low 02 rather than under aerobic atmosphere. In the literature only one study (King and Mabbit, 1982) with P. fluorescens under nitrogen reported a faster growth rate than untreated bulk milk during the first 2 days of incubation. In that study, the aerobic population eventually exceeded the population number of that under nitrogen atmosphere on day 3 (King and Mabbit, 1982). a. Alternate Electron Acceptors P. fluorescens did not behave like an obligate aerobe during early storage. P. fluorescens was not only able to grow but its growth rate was even enhanced under  decreased atmospheric oxygen conditions during initial storage. The fast growth rate under 1.2 and 5.3 % oxygen was typical of microaerophilic microorganisms which have been reported to grow best at 2-10 % 02 (vol/vol) and are inhibited at air saturation levels (Costilow, 1981; Stolp and Starr, 1981; Stanier et al., 1986). It is known that many species of Pseudomonas can use nitrate as an alternate electron acceptor. Nitrate is reduced to nitrite and completely to nitrogen gas by denitrifying bacteria. According to Bergey's Manual of Systematic Bacteriology (Krieg and Holt., 1984), P. fluorescens b'otype B, C and D are reported to reduce nitrate but not biotype A. The ability of P.  44 fluorescens biotype A to grow under severely diminished oxygen atmospheres reflects  their ability to compensate for the lack of oxygen by using an alternate electron acceptor (Table 3). Deviations of these results from the expected denitrification and nitrate reduction results in Bergey's Manual of Systematic Bacteriology (Krieg and Holt., 1984) may be due to differences between strains although the biotypes may be the same and may be due to mutation of the strain during storage in the lab. b. Growth on Solid Medium P. fluorescens under decreased oxygen atmospheres showed two different colony  sizes when enumerated aerobically on TSA plates. The smaller sized colonies which were only 25 % the size of the larger sized colonies were more prevalent from samples under low oxygen atmospheres. P. fluorescens under low oxygen were probably more stressed and nutritionally fastidious on solid medium. 2. Oxygen a. Atmospheric Oxygen  The oxygen level in the atmospheres were monitored throughout 14 days of storage and only minor fluctuations were observed (Fig. 4). b. Dissolved Oxygen  Despite a constant supply of oxygen and agitation, there was a decrease in dissolved oxygen tension in milk (Fig. 5). The levels of dissolved oxygen were inversely related to exponential growth since the demand for oxygen increased as population numbers increased. The initial decrease in oxygen tension occurred when population numbers  45 Table 3. Metabolism of P.fluorescens biotype A ATCC 17397 in Hugh Leifson Medium a  Metabolic Response Old P. fluorescens 0^New P. fluorescens Sugars  ^  Growth^Acid^Growth^Acid  adonitol (+ 8 )^++ Y^ ++^L-arabinose (-)^++^+^++^+ D-glucose (+)^++^+^++^+ _ lactose (-)^ D-trehalose (+)^++^ ++^D-xylose (+)^++^+^++^+ Denitrification (- C)^++^ + Reduction of NO3 to NO2 (-)^+ a Hugh Leifson Medium from Bergey's Manual of Systematic Bacteriology,  (Krieg and Holt, 1984) 13 strains of P.fluorescens biotype A ATCC 17397 5 "expected" results for growth or acid production or both according to Bergey's Manual of Systematic Bacteriology, (Krieg and Holt, 1984) £ growth and gas production under denitrifying conditions y - no growth or no acid production + minimal growth or low acid production + growth or acid production ++ heavy growth  46 Figure 4. Profile of controlled oxygen atmospheres overlaying MT-sterilized milk (2 % m.f.) inoculated with P. fluorescens during storage at 4 °C. Each value represents the mean of triplicate gas samples.  25  20  Atmospheric Oxygen • 1.2%  15  131 5.3% 10.2%  10  121 20.9% 5  0 0^1^2^4^6^8^10^12^14  Storage Time (Days)  47 Figure 5. Dissolved oxygen concentration in UHT-sterilized milk (2 % m.f.) inoculated with P .fluorescens during storage under controlled oxygen atmospheres at 4°C.  12  10  Atmospheric Oxygen -  -  0^2^4^6  ^  8^10^12  Storage Time (Days)  ^  14^16  —*--  1.2%  —6—  .3%  —0—  10.2%  —{a—  20.9%  48 reached 105 and 106 CFU mL-1. Other studies have shown decreases in 02 tension at population densities between 5 x 105 and 5 x 106 CFU mL-1 (Spohr and Schutz, 1990) and 106 and 107 CFU mL-1 (Rowe and Gilmour, 1982; Griffiths and Phillips, 1984). Less than 1 ppm of oxygen was dissolved in milk by day 4 under 1.2 and 5.3 % oxygen and by day 8 under 10.2 and 20.9 % oxygen. The drop in oxygen tension would be primarily due to the demand for oxygen during exponential growth when metabolic rate is maximal (Oblinger and Kraft, 1973). The dissolved oxygen measurements indicated that utilization of oxygen by these microorganisms exceeded the rate at which atmospheric oxygen dissolved in the milk. The dissolved oxygen measurements do not represent the rate at which oxygen is consumed by these microorganisms. The degree of oxygen limitation in the aerobic control would not have been as severe as in milk stored under decreased 02 atmospheres although less than 1.0 ppm dissolved oxygen was measured. There would have been a higher efficiency of oxygen transfer when the partial pressure of oxygen in the gas phase was high and the dissolved oxygen tension was low (MacLennan et al., 1971). Thus, the rate of oxygen transfer would have been higher and more oxygen would have been available to microorganisms in the aerobic control than in low oxygen atmospheres despite the very low tensions measured by the oxygen probe. c. Oxygen and Energy Production Growth and metabolism of P. fluorescens would be affected by the decreased 02 levels at 1.2, 5.3 and 10.2 % which as a result might alter the microorganisms ability to  49 produce extracellular enzymes. Oxygen is primarily responsible for the positive redox potential (Eh) which would determine cellular processes (Kroll, 1989). For obligate aerobes, where respiration provides most of the energy, dissolved oxygen tension was reported to have a similar effect on growth and respiration (Harrison, 1976; Costilow, 1981). It was reported that when the dissolved oxygen tension exceeded a critical dissolved oxygen tension (CDOT) level, growth rate and growth efficiency were independent of oxygen concentration but when the level fell below CDOT, both growth rate and efficiency decreased (Costilow, 1981). Likewise, Persson et al. (1990) reported that aeration and the rate of oxygen transfer affected growth rate and biomass of P. fluorescens 378 biotype A.  3. Proteinase Activity Proteinase production by populations under decreased oxygen atmospheres were lower than under aerobic atmosphere throughout 14 days of storage (Fig. 6). The decrease in proteinase activity in samples under low atmospheric oxygen was also observed in preliminary work. The absence or decrease in proteinase activity was not due to mutation in synthesis since large zones of proteolysis were observed on 1 % skim milk agar plates inoculated with milk samples when aerobically incubated. Other researchers have reported similar effects of deoxygenation and its influence on extracellular proteinase synthesis. Murray et al. (1983) showed that removal of oxygen in raw milk with nitrogen flushing resulted in no detectable proteinase activity throughout 18 days of storage. It was also observed that when the nitrogen-flushed milk was  50 Figure 6. Proteinase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C using azocasein as a substrate. Each point represents the mean of triplicate proteinase activity values + standard deviation.  100  80  Atmospheric Oxygen  60  40  20  0^2^4^6^8^10^12^14^16  Storage Time (Days)  —•"---  1.2%  —aer—  5.3%  —.—  10.2%  —0—  20.9 %  51 incubated anaerobically on skim milk agar plates, no proteinase activity was detected whereas aerobic incubation promoted its synthesis. Skura et al. (1986) showed that removal of oxygen with nitrogen inhibited proteinase production by P. fluorescens. Disruption of the nitrogen overlay by introducing aerobic conditions restored the ability of P. fluorescens to grow and produce proteinase.  a. Carbon and Nitrogen Catabolite Repression Proteinase activity was first detected on day 10 in the aerobically grown culture and increased (Fig. 6). Proteinase steadily accumulated during storage in other studies (Keen and Williams, 1967; Hare et al., 1981; Stead, 1987). The reason for the delay in enzyme production until day 10 might have been due to catabolite repression. Enzymes are thought to be under C and N catabolite repression (McKellar, 1982; Fairbaim and Law, 1987). Proteinase activity is required after simple sugars, free amino acids and fatty acids present initially in the medium are exhausted (Rowe and Gilmour, 1986). McKellar (1982) reported that proteinase functions to ensure a C supply rather than to ensure amino acids for biosynthesis. b. Proteinase Synthesis and Growth Phase Increase in proteinase activity of the aerobic control started between days 8 and 10 during early stationary phase. Other studies also reported proteinase production during early stationary growth phase (McKellar, 1982; Rowe and Gilmour, 1982; Murray et al. 1983; Stead, 1987; Griffiths, 1989; Kohlmann et al., 1991). Proteinase activity increased between day 8 and 14 but greater increase was observed between days 8 and 10.  52 c. Factors Influencing Proteinase Production i. Oxygen Proteinase is synthesized during stationary phase when growth is limited by low oxygen tension (Rowe and Gilmour, 1982; Wiersma et al., 1978). There was evidence of an inverse relationship between growth and enzyme synthesis (Whooley et al., 1983). Oxygen primarily acts as a terminal electron acceptor in obligate aerobes as well as in direct oxidation of many compounds by oxygenases (Palleroni, 1975; Harrison, 1976; Stanier et al., 1986). Decreasing the initial dissolved oxygen tension in milk would affect cellular processes and result in decreased proteinase production. P. fluorescens at < 20 % of saturation 02 was shown to utilize glucose and L-lactate at lower rates and its malate dehydrogenase activity was lower (Spohr and Schutz, 1990). If oxygen is limiting, then lower metabolic activity would result in fewer metabolite precursors necessary for synthesis of extracellular enzymes. ii. Population Density There may be other factors besides the decrease in the initial dissolved 02 tension in milk which may have affected proteinase production. One factor may be population density. The reason for low or no detectable proteinase activity in milk under 1.2 and 5.3 % oxygen may have been due to an insufficient number of cells capable of producing proteinases. The population number which is often associated with enzymatic activity is 108 CFU mL4 during early stationary growth phase (Rowe and Gilmour, 1982, Spohr  53 and Schutz, 1990). The population under nitrogen atmosphere reported by Skura et al. (1986) did not show any detectable proteinase activity but there may not have been enough cells to produce a sufficient amount of proteinase for detection. There was, however, no proteinase activity in the milk under 10.2 % oxygen in the present study although P. fluorescens cell numbers exceeded 10 8 CFU mL -1 . There is a consensus among researchers that a lack of correlation exists between bacterial numbers and the level of enzyme activity (Rowe and Gilmour, 1986; Skura et al., 1986; Skura, 1989). d. Proteinase Activity and Inhibition of Cell Lysis  The reason for lower population density of P. fluorescens in milk stored under 1.2, 5.3, and 10.2 % oxygen compared to the aerobic control after 14 days of storage may be a consequence of the lower proteinase activity. Proteinases provide necessary small molecular weight products when nutrients are depleted (Rowe and Gilmour, 1982) and are beneficial for growth. It was reported that Bacillus subtilis deficient in proteinase production tended to lyse more readily at the end of exponential growth than a proteinase proficient culture due to the actions of autolytic enzymes which would otherwise have been inactivated by proteinases (Coxon et al., 1991). Although the population under 10.2 % oxygen did enter stationary phase at a density similar to that of the aerobic control, the population numbers near the end of storage on days 12 and 14 were lower than the aerobic control. The lower numbers may be reflective of a susceptibility of P. fluorescens to cell lysis under those conditions.  e. The Inhibitory Effect of Nitrogen  The inhibitory effect of nitrogen on proteinase synthesis is not usually discussed in the  54 literature. Unlike carbon dioxide which shows antimicrobial activity (Rowe, 1988), nitrogen is not known to have any direct inhibitory action on cellular components except in its role of replacing oxygen in the atmosphere. 4. Proteolysis The cumulative effect of proteinase activity in milk throughout 14 days of storage was monitored with 12 % trichloracetic acid (TCA) soluble components where absorption of proteins was maximal at 280 nm. The absorption was mainly due to the presence of soluble aromatic amino acids, such as tyrosine, and nucleic acids (Hanson and Phillips, 1981). An estimation of the content of soluble proteins in TCA solution can be made as long as nucleic acid content is less than 20 % of the total  A280.  The release of DNA and  RNA materials due to lysis was not measured at A260 and used to correct A280 values in our study. An increase in proteolysis was observed for the aerobic control on day 10 which corresponded with the increase in proteinase activity (Fig. 7). There were small differences in proteolysis between 1.2, 5.3 and 10.2 % atmospheric 02 and no dramatic increase throughout 14 days of storage. A decrease in  A280  components may possibly  have reflected the use of amino acids and peptides in biosynthetic reactions by P. fluorescens.  5. Lipase Activity Lipase activity was detected earlier than proteinase activity and as early as day 2 for the aerobic control (Fig. 8). This may have been due to constitutive expression of lipase.  55 Figure 7. Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate proteolysis values + standard deviation.  12  10 -  Atmospheric Oxygen  ••••■ .4  1:4  8-  1.2%  2-  0  I^•^I^I  I^•^•^I^•^i^•  0^2^4^6^8^10^12^14^16  Storage Time (Days)  —tr"---  5.3 %  —4,—  10.2%  —0—  20.9%  56 Figure 8. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicate lipase activity values + standard deviation.  0.6  0.5 —  Atmospheric Oxygen 0.4 —  0.1 -  0.0  1^•^. 0^2^4^6^8^10^12^14^16  Storage Time (Days)  ---*--  1.2%  ---tr  5.3 %  —  —.—  10.2%  —0—  20.9 %  57 There are some reports of constitutive expression of lipase production by P. fluorescens since triglycerides were not required for synthesis (Fox and Stepaniak, 1983; McKellar, 1986). Lipase activity of the aerobic control increased on day 6 and accumulated with time but decreased on days 10 and 14 which corresponded with high proteinase activity in the milk. These observations are similar to those made by Stead (1987) who reported earlier detection of lipase than proteinase and a decrease in lipase activity during prolonged storage as bacterial and indigenous proteinases degraded the lipases. Rowe and Gilmour (1986) also reported earlier detection of lipase activity than proteinase so only lipase was monitored as an indicator of enzyme production. The three milk samples under low oxygen atmospheres showed lipase activity after day 6. The milk sample under 5.3 % 02 reached its maximum activity of 0.2 units on day 10 and then decreased. The decrease in lipase activity may be due to inactivation by proteinases although proteinase activity was detected at a low level. Further synthesis of lipases may have been repressed by free fatty acids (Bucky et al., 1986) or low energy levels which may have been insufficient to sustain the energy-consuming synthesis of lipases. Lipase activity of milk under 10.2 % 02 decreased after day 8 but increased again on day 14 to its maximal level of 0.4 units since no proteinase accumulated throughout storage. 6. Lipolysis The degree of lipolysis did not relate well with lipase activity. The acid degree values (ADV) were much higher in both Trial I and in preliminary work than those reported in  58 the literature (Appendix Fig. 1). The reason for high titratable free fatty acids (FFA) was investigated. 7. Investigation of High ADV There are several possible reasons for the unusually high acid degree values. The increase in free fatty acids may be due to induced lipolysis by mechanical agitation, changes in metabolism of P. fluorescens which may have produced more titratable free fatty acids, or the interference of atmospheric carbon dioxide during titration. a. Induced Lipolysis by Mechanical Agitation  The high acid degree values could have been the result of increased activity of microbial lipases which had survived the UHT-treatment. The primary cause of lipolysis during aeration and excessive agitation is the result of damage to fat globule membranes which would make triglycerides more accessible to lipases as well as the release of lipases from their association with milk proteins (Law, 1979). Results from an experiment where sterile milk was stirred at 350 rpm for 7 days to determine the effect of mechanical agitation and the possible release of lipases from their association with milk proteins did not show any dramatic increase in titratable fatty acids (Appendix Fig. 2). The increases in ADV were not caused by the activity of native nor bacterial lipases which had survived the UHT-treatment and enhanced with mechanical agitation. b. Carbohydrate Utilization of P . fluorescens biotype A (ATCC 17397)  There was no difference in the utilization of 6 sugars unique to P . fluorescens biotype A between the current laboratory strain used in these trials and a new ATCC biotype A  59 strain (Table 3). There was a difference between the current and new strain in the ability to reduce nitrate to nitrite in Bacto nitrate broth. Nitrite was detected in the 24 hour culture of our lab strain but not in the culture of the new ATCC strain. Both biotype A strains were however able to dissimilate nitrate and produce detectable nitrogen gas. c. Carbon Dioxide Interference It has been suggested that atmospheric carbon dioxide could neutralize the hydroxide in the titration vessel (IDF, 1991). To eliminate the carbon dioxide interference with KOH, the titration setup was modified (Fig. 2). Either a vacuum or a gentle flow of nitrogen gas was effective in removing the atmospheric CO2 in the titration vessel. Creating a vacuum was more cumbersome so a gentle N 2 -flow was preferred. The tip of the volumetric pipette was also extended down as far as possible over the free fatty acid extract to minimize the evaporation of methanolic KOH. Stability of the colour at the endpoint of titration was also reported to be improved with the removal of CO2 in the atmosphere (IDF, 1991). Removal of CO2 from the atmosphere resulted in a lower ADV throughout storage (Appendix, Fig. 3). The acid degree values also did not fluctuate as severely as in Appendix Figure 2 when CO 2 was not removed from the titration atmosphere. A larger titration volume of KOH required to neutralize FFA would give the appearance of higher concentration of FFA in the sample which could not be corrected with titration blanks. Not enough milk samples remained to analyse the extent of lipolysis in Trial I using the modified method.  60 8. Summary of Trial I Atmospheres containing 1.2, 5.3, 10.2, and 20.9 % 02 continuously overlayed 2 L of milk inoculated with P. fluorescens at an initial population of 4.13 to 4.18 Logi() CFU mL-1. The populations under 1.2 and 5.3 % 02 showed faster growth rate and higher population density during early storage time. Despite a constant supply of atmospheric oxygen, the dissolved 02 level in milk started to decrease when population numbers ranged between 5.0 and 5.5 Logic, CFU mL-1. The dissolved 02 concentration in milk measured < 1.0 ppm when populations entered stationary phase. Milk samples under 1.2 and 5.3 % 02 atmospheres showed low proteinase activity while milk under 10.2 % 02 did not show any proteinase activity throughout 14 days of storage. The highest proteinase activity was observed in the aerobic control (20.9 % 02) which corresponded well with the increase in proteolysis. Lipase activity was observed earlier than proteinase activity for the aerobic control. The level of lipase activity appeared to be influenced by the activity level of proteinase, either increasing or decreasing during storage. Unusually high acid degree values appeared to be due to CO2 interference in the titration vessel. A decrease in the initial dissolved 02 concentration in milk resulted in decreased and delayed proteinase and lipase synthesis by P.fluorescens.  61 B. TRIAL II Based on results of Trial I, oxygen levels between 10 and 20 % were tested to find the maximum atmospheric oxygen required to inhibit extracellular enzyme production by P. fluorescens.  1. Growth Growth of P. fluorescens was monitored under 9.6, 13.4, 16.9, and 20.7 % atmospheric oxygen for 16 days (Fig. 9). The P. fluorescens population under 13.4 % 02 did not show a lag phase and showed the fastest growth rate up to day 4 when it entered stationary phase. Its population density remained higher than other P. fluorescens populations throughout storage. The P. fluorescens population under 9.6  and 16.9 % 02 showed a similar rate of growth during the early storage time and reached stationary phase on day 6 when populations were approximately 5 x 107 CFU mL4. P. fluorescens in the aerobic control initially showed a short lag phase and entered stationary  phase on day 6 when the population density was less than 108 CFU mL4 which was unusually low for stationary growth. By day 16, all the populations exceeded 108 CFU mL-1. a. Low Population Numbers It was highly unexpected that all populations entered stationary phase when population density did not exceed 108 CFU mL-1. Poor growth of cultures especially the aerobic control was not seen in any other work and may have been an anomaly of the inoculum or the batch of milk used for Trial II. It was possible that the presence of antibiotics  62 Figure 9. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate bacterial counts + standard deviation.  10  . Atmospheric Oxygen  I•I^I^I^I^.^I^'^I-.^I^•  0^2^4^6^8^10^12 14^16^18  Storage Time (Days)  —0—  9.6%  —A—  13.4%  —4"--  16.9%  —Cl--  20.7 %  63 inhibited growth but the results from Dairyworld Foods were negative for the presence of 13-lactams, tetracyclines and sulfa drugs. b. Growth Under Lower Atmospheric Oxygen Growth of P. fluorescens was enhanced under 13.4 % atmospheric oxygen since it showed faster growth rate, earlier transition into stationary growth phase and higher population density than the aerobic control during 16 days of storage. As observed in Trial I with 5.3 % oxygen, P. fluorescens behaved more like a microaerophile than an obligate aerobe. However, decreasing the atmospheric oxygen down to 10 % did not result in similar growth enhancing effects. c. Oxygen and Nitrate as Electron Acceptors It was reported that use of nitrate by aerobes and facultative microorganisms yields less energy, reduced growth rates and lower cell populations than the use of oxygen which is preferentially used as a terminal electron acceptor (Costilow, 1981; Palleroni, 1975). In Bacterium denitrificans (P. stutzeri), nitrite reductase activities were repressed at 20 % atmospheric oxygen. At 6 % oxygen however, this microorganism was able to utilize both oxygen and nitrites simultaneously as electron acceptors (Kefauver and Allison, 1957). It is possible then that P. fluorescens were able to use both the available oxygen and nitrates/nitrites simultaneously at 5 % and 13.4 % atmospheric oxygen concentrations to maintain their rapid growth rate during early storage time. Unlike oxygen which is continuously supplied from the atmosphere, nitrate in milk would eventually be exhausted during storage. Cellular processes would then decrease if no  64 other electron acceptors are provided when atmospheric oxygen concentration is low. Under aerobic conditions (20.7 %), P. fluorescens must rely solely on oxygen as terminal electron acceptors and repress nitrate and nitrite reductase activity (Clarke and Ornston, 1975a). 2. Oxygen a. Atmospheric Oxygen  The atmospheric oxygen levels throughout 18 days of storage are shown in Figure 10. b. Dissolved Oxygen  The initial dissolved oxygen values were similar to those obtained in Trial I for 9.6 and 20.7 % (Fig. 11). There was no discrepancy between the initial dissolved oxygen tension measured for milk under 9.6 and 13.4 % oxygen since similar values of 4.1 and 3.7 ppm were measured. Dissolved oxygen tension in the containers of milk dropped and remained low despite constant oxygen supply and agitation. The dissolved oxygen tension under 9.6 % and 20.7 % oxygen decreased down to less than 1.0 ppm, four and two days earlier than observed in Trial I. This oxygen limitation corresponded with earlier shifts into stationary growth phase despite lower population numbers. These populations may have been respiring at a faster rate and consuming oxygen more rapidly than the populations under similar atmospheres in Trial I. These differences reinforce the difficulty in comparing data between trials. c. Carbon Dioxide Production Carbon dioxide is a byproduct of oxidation of organic carbon substrates and reduction  65 Figure 10. Profile of controlled oxygen atmospheres overlaying UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens during storage at 4 °C. Each value represents the mean of triplicate gas samples.  25  20  Atmospheric Oxygen  15  0^  ',11  ,^  ,r,  El 13.4%  ::::..o^04 1 :-# # 0^  ',,..e.^,,•  %  r,^'4^.'..,•■ •,-1,^ 0-4  16.9 %.  0:4'^.'''..:,  Ea  , •^,^•,,. / #^01,....^,  5 I.;^":  k0 o^ /.#  •if,'^''4,. ;..$ 0^i'  0  9.6%  ...$^.  0^'00  10  •  <0  : 0^ 0  014  F^*:41,:  1% !A  ','4  0246  ^  10 12 14 16 17 18  Storage Tiine (Days)  20.7%  66 Figure 11. Dissolved oxygen concentration in UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens during storage under controlled oxygen atmospheres at 4 °C.  Atmospheric Oxygen —4,--  9.6%  1k---  13.4%  —  —.— 16.9% —13-- 20.7 %  0^2^4^6^8  10^12^14^16^18  Storage Time (Days)  67 of molecular 02. There are many decarboxylating reactions involved in the tricarboxylic acid cycle which has central importance in both catabolism and biosynthesis of Pseudomonas (Clarke and Ornston, 1975a). All populations produced carbon dioxide  gas, a byproduct of many decarboxylating reactions involved in the tri:;arboxylic acid cycle (Fig. 12). The earliest detection of measurable CO2 was on day 6 by the population under 16.9 % oxygen. The highest CO2 emission was noted for the aerobic control on day 12 at 0.04 %. The CO2 production patterns by populations under various atmospheric oxygen levels suggest difference in respiration as well as metabolic pathways of P. fluorescens. More research would have to be conducted in order to determine whether the differences are significant. 3. Proteinase Activity a. Aerobic Conditions The aerobic control showed proteinase activity on day 10 and levelled off after reaching its maximum activity of 60 units on day 12 (Fig. 13). Other studies have also shown that proteinase activity reached a maximum plateau then levelled off (Rowe and Gilmour, 1982; Rowe, 1988) or decreased (Murray et al. 1983; Skura et al., 1986). Once production of proteinase has been initiated, its continual synthesis would not be required since P. fluorescens would benefit from its remaining proteolytic activity. Proteinase activity in Trial II was not as high as the maximum of > 90 units seen in Trial I for the aerobic control (Fig. 6). The lower proteinase activity may be related to the earlier transition of P. fluorescens into stationary phase when its population of proteinase-  68 Figure 12. Carbon dioxide production by P. fluorescens in UHT-sterilized milk (2 % m.f.) during storage under controlled oxygen atmospheres at 4 °C.  Storage Time (Days)  69 Figure 13. Proteinase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C using azocasein as a substrate. Each point represents the mean of triplicate proteinase activity values + standard deviation.  100  80 -  Atmospheric Oxygen 9.6%  60  13.4% —9— 16.9%  40  —0— 20.7 % 20  0^2^4^6^8^10  12 14 16 18 20  Storage Time (Days)  70 competent cells was lower. Hare et al. (1981) reported no detectable level of collagenase and proteinase activity when the growth rate of V. alginolyticus was slower and its cell density was lower than the aerobic control. b. Lower Atmospheric Oxygen The population under 13.4 % oxygen showed proteinase activity on day 12 which was 2 days earlier than the population at the higher oxygen concentration of 16.9 %. The proteinase activity of the milk under 13.4 % oxygen remained steady at 10 - 15 units until day 18. When the population under 16.9 % oxygen reached 108 CFU mL-1 on day 14, its proteinase activity was greater than under 13.4 % oxygen and steadily increased to the level of the aerobic control on day 18. P. fluorescens under 9.6 % atmospheric 02 did not show proteinase activity until day 16 and had the lowest level of activity throughout storage. A decrease in the initial dissolved 02 tension in milk during storage delayed initiation and decreased the amount of proteinase synthesis by P. fluorescens once it was initiated. c. Relation Between Population Density and Proteinase Activity Population numbers showed poor relationship with proteinase activity when the initial dissolved 02 tension was decreased. The population density was higher under 13.4 % 02 than under aerobic atmosphere throughout 16 days of storage but the proteinase activity was much lower. Proteinase activity was detected, however, when populations under 9.6, 13.4 and 16.9 % oxygen all exceeded the cell number of 108 CFU mL-1. Kroll and Klostermeyer (1984b) reported detectable proteinase activity with azocasein when  71 population numbers reached 1.4 - 7.1 x 107 CFU mL-1. Generally, population densities of 5 x 106 -107 CFU rnL-1 are associated with proteolysis, lipolysis, and sensory changes (Shelley et al., 1986; Garcia et al., 1989) and problems in pasteurized dairy products (Muir and Phillips, 1984). 4. Proteolysis The initial A280 value on day 0 in Trial II (Fig. 14) was higher than in Trial I (Fig. 7). This reflects the difference between batches of milk. There was an increase in the degree of proteolysis for the aerobic control starting on day 12 which steadily increased throughout 18 days (Fig. 14). The increase in proteolysis in the aerobic control was observed despite a constant level of proteinase activity since A280 represents the cumulative effect of proteinase activity as well as the interference of nucleic acids which would have been released upon cell lysis. Samples under 13.4 and 16.9 % oxygen showed high degree of proteolysis starting on days 12 and 14 respectively which corresponded with increases in proteinase activities. The high level of proteolysis under 13.4 % oxygen suggested that proteinase activity of 10 - 15 units may cause a large increase in 12 % TCA soluble components. The milk under 9.6 % oxygen showed only a small increase in proteolysis on day 18 which corresponded to its low proteinase activity. 5. Lipase Activity The lipase activity of P. fluorescens in the aerobic control was detected on day 12 well after the population entered stationary phase on day 6 (Fig. 15). There was no early  72 Figure 14.^Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate proteolysis values + standard deviation.  10  •  9-  -,  • •  „.- --, U ,-.4.■••.  .^II ..iL ......  1  -,.....-z ....it" "r_. • -..., ,4■;#  `■1.7%---^ -  1 0  i  _ - Atmospheric Oxygen  _  -  -  I^•^1^.^i^.^1^.^1^.^1^.^1^.^1^.^I 0^2^4^6^8^10 12 14 16 18 20  Storage Time (Days)  —10—  9.6%  --ir—  13.4%  —41---  16.9%  —0--  20.7 %  73 Figure 15. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicate lipase activity values + standard deviation.  _ _  Atmospheric Oxygen --e—  9.6%  —A-- 13.4% —4)— 16.9% —a— 20.7 %  4^6^8^10 12 14 16 18 20  Storage Time (Days)  74 detection of lipase activity as observed in Trial I for the aerobic control. The delay in lipase production may be due to insufficient number of cells required to produce detectable lipase activity since the aerobic population entered stationary phase at an unusually low density of < 10 8 CFU mL-1 . Lipase activity of the aerobic control steadily increased to its maximum level on day 16 but decreased on day 18 possibly due to the degradative actions of coexisting proteinases. Lipase activity in milk under 16.9 % 02 was slightly lower than in milk held under 13.4 % 02 on days 14 and 16 which could be reflective of its higher proteinase activity. Milk under 9.6 % 02 showed lipase activity starting on day 14. There was a small delay in lipase production when the initial dissolved 02 concentrations in milk were decreased but by day 18, all samples showed a similar lipase activity of 0.2 units. 6. Lipolysis The lipolysis graph showed that acid degree values (ADV) for samples fluctuated from day to day but steadily increased during storage (Fig. 16). Skura et al. (1986) also showed steady increases in ADV for P. fluorescens throughout storage. There were increases in ADV despite the absence of detectable lipase activity between days 4 and 10. It was reported that good quality milk had ADV of 0.4 (IDF, 1991) and 0.5-1.0 (Rowe et al., 1990). An ADV of 1.2 to 1.5 was reported to be the level where off-odors and flavours were detected (Hausler, 1972; Rowe et al., 1990). Milk under 9.6 % 02 and 20.7 % reached ADV of 1.5 on day 10 although lipase activity was not detected on that day. This may be due to the difference between the two methods in the measurement of  75 Figure 16. Lipolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate lipolysis values + standard deviation.  76 the lengths of fatty acids. The 3-NC assay measures lipase activity on a shorter chain fatty acid (C8) while the titration method of Deeth et al. (1975) measures longer chain fatty acids (C14-20). The titration method is reported to be well adapted for the titration of free fatty acids liberated from milk lipids since most are C16 and C18:1. Deeth et al. (1975) reported high recovery rates for long chain fatty acids (C16 at 99 %, C18 at 102 % and C18:1 at 97 % recovery rates) using the titration method.  A second washing of the ether extract was added to ensure the removal of water soluble acids such as lactic and citric acid from the titatable organic layer since these acids have been reported to cause interference with the titration method (BrAthen, 1984). Although most of the short water soluble FFA (C4-12), which are responsible for sensory defects, are not extracted with the organic layer (Brathen, 1984), it has been reported that lipolysis can be measured accurately with the dilution method prior to sensory detection of lipolytic off-flavours (IDF, 1991). The titration method, however, has often been criticized as being laborious and showing variation between milk samples (Law, 1979). The results in Figure 16 confirm the difficulty in obtaining reproducible data since large error bars are shown. 7.fl As an indication of the combined effects of proteolysis and lipolysis in milk, pH was measured (Fig. 17). Fluctuations reflect the dynamic system of milk. Proteolysis of milk proteins would provide P. fluorescens with amino acids as a carbon source. Deamidation of amino acids would then increase pH as basic amines are produced. It was  77 Figure 17. pH of 'UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C.  Atmospheric Oxygen  —11—  9.6%  --A---  13.4%  —0--  16.9%  —1:1-- 20.7 %  6.0^•^1^•^1^1^I^•^1^•^1^-^1^I^I^•^ 02 4 6^8 10 12 14 16 18 20  Storage Time (Days)  78 reported that pH values increased as a result of ammonia production when amino acids were used as a C source (San Jose et a., 1987). Lipolysis would decrease pH as fatty acids are liberated. The aerobic control showed a large increase in pH on day 14 since proteinase activity was high and its effzzts more prominent than lipase activity. Milk samples under decreased 02 atmospheres showed relatively constant pH values throughout 18 days of storage. Milk under 13.4 % 02 had lower pH values than the other milk samples. These results are different from those reported by Skura et al. (1986) where a decrease in pH values was observed throughout storage in both aerobic and nitrogen overlayed milk. The smallest decrease in pH was reported for milk stored under nitrogen atmosphere (Skura et al., 1986). 8. Summary of Trial II Atmospheres consisting of 9.6, 13.4, 16.9 and 20.7 % oxygen continuously flowed over 2 L of milk with initial P. fluorescens populations of 4.2 Logic, CFU mL4 for 18 days at 4 °C. The dissolved 02 concentration in milk started to decrease when cell density ranged between 5.0-6.5 Logic) CFU mL-1. The dissolved 02 tension measured < 1.0 ppm when all populations entered stationary phase. P. fluorescens showed the fastest initial growth rate and reached the highest population density under 13.4 % atmospheric 02. The lowest proteinase activity and degree of proteolysis were observed in milk under 9.6 % oxygen. Lipase activity in all milk samples was detected much later and at lower levels than observed in Trial I. The reason for the decrease in proteinase activity and  79 delay in lipase activity in the aerobic control may have been due to slow growing P. fluorescens population which entered stationary phase earlier and at lower cell density  than in Trial I. Results from the lipolysis analysis suggest that storage up to 10 days under 10 % 02 may not be desirable although there were no detectable increases in proteinase activity, proteolysis, or lipase activity. The combined effects of proteolysis and lipolysis in milk were reflected in pH measurements which either increased or decreased depending on the level of proteinase and lipase activity. Observations made from Trials I and II showed that decreasing the initial oxygen tension in milk would delay and decrease the amount of extracellular enzyme production by P. fluorescens once its synthesis was initiated. The decrease in atmospheric oxygen and initial dissolved oxygen tension in milk had greater effects on proteinase than lipase synthesis by P. fluorescens.  80 C. TRIAL III Trial III was performed to confirm the results of decreasing the initial dissolved 02 tension in milk on enzyme production by P. fluorescens. The critical oxygen level appeared to be 10 % since the growth rate of P. fluorescens was enhanced under 5.3 % and 13.4 % atmospheric oxygen. 1. Growth  Growth of P. fluorescens populations was monitored under atmospheres of 4.8, 10.3, 13.4, and 20.2 % oxygen. None of the populations showed a lag phase and P. fluorescens all grew exponentially during early storage (Fig. 18). As observed in Trials I  and II, populations under 4.8 and 13.4 % oxygen grew markedly faster than populations under 10.3 % and 20.2 % oxygen during early storage. P. fluorescens under 4.8 % oxygen entered stationary phase on day 6 but the population under 13.4 % continued to grow until day 10 when it entered stationary phase and exceeded the population density of the aerobic control. P. fluorescens in milk under 10.3 % oxygen and aerobic atmospheres showed similar growth rates up to day 4. P. fluorescens in milk under 13.4 % oxygen produced the highest cell density after 18 days of storage which was also observed in Trial H. 2. Oxygen  a. Atmospheric Oxygen The oxygen levels in the atmospheres were monitored throughout 18 days of storage and the data are shown in Figure 19.  81 Figure 18. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate bacterial counts + standard deviation.  10  -  . Atmospheric Oxygen  --er—  4.8%  —0---  10.3%  ---A—  13.4%  —0— 20.2 %  0^2^4^6^8 10 12 14 16 18 20  Storage Time (Days)  82 Figure 19. Profile of controlled oxygen atmospheres overlaying UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens during storage at 4 °C. Each value represents the mean of triplicate gas samples.  25  20  Atmospheric Oxygen  • 4.8%  15  10.3% EI 13.4%  10  Ea 20.2%  5  0 1 2 4 6 7 8 9 10 12 13 14 15 16 18  Storage Time (Days)  83 b. Dissolved Oxygen The initial dissolved oxygen tensions measured with the oxygen probe were notably higher than in Trials I and II but the pattern of decrease was similar (Fig. 20). The difference in sampling method could explain the higher values overall which did not decrease below 1.0 ppm as seen in previous trials. Instead of measuring non-agitated milk, the samples were stirred with a stir bar for more accurate measurements and read within 30 seconds (Table 4). The difference in the ppm measurements suggest great potential risk for inter-operator variability. By day 4, dissolved oxygen was limiting for samples under 4.8 and 10.3 % oxygen. The samples under 13.4 and 20.2 % oxygen showed limitation on days 6 and 8. The initial drop in oxygen tension in milk corresponded with mid-exponential growth between 5.5 - 6.5 Logi() CFU m1-1. c. Carbon Dioxide Production The carbon dioxide profile showed that P . fluorescens in milk under 10.3, 13.4 and 20.2 % 02 produced detectable CO2 (Fig. 21). P . fluorescens under 4.8 % atmospheric oxygen did not produce any measurable CO2. This may suggest differences in metabolic rates and pathways for P. fluorescens under 4.8 % 02 compared to those under higher atmospheric 02 concentrations. In general, a decrease in the initial dissolved 02 tension in milk resulted in lower amounts of carbon dioxide emitted by P 17uorescens.  84 Figure 20. Dissolved oxygen concentration in stirred samples of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens during storage under controlled oxygen atmospheres at 4 °C.  12  10  Atmospheric Oxygen  -  .^I^1-1^.^1^.^1^.^I^•^1^.^1^•^r •^I 0^2^4^6^8 10 12 14 16 18 20  Storage Time (Days)  --"ti—  4.8 %  —41—  10.3%  —ii---  13.4%  —0—  20.2%  85 Table 4. Dissolved oxygen tension of stirred and unstirred samples a  Non-agitated Sampling Atmospheric 02^Dissolved 02 (%)^(ppm) 1.2 5.3 9.6 10.2 13.4 16.9 20.9 20.7  1.4 2.7 4.1 4.1 3.7 4.7 7.2 7.3  a single sampling of 25 mL of milk  Agitated Sampling Atmospheric 02^Dissolved 02 (%)^(ppm) -  4.8  3.6  10.3 13.4  6.1 7.2  _  20.2  10.1  86 Figure 21. Carbon dioxide production by P. fluorescens in UHT-sterilized milk (2 % m.f.) during storage under controlled oxygen atmospheres at 4 °C.  87 3. Proteinase Activity a. Lower Atmospheric Oxygen The first detectable proteinase activity was observed on day 8 in the aerobic control but not in milk stored under lowered 02 concentrations (Fig. 22). Milk under 4.8, 10.3 and 13.4 % 02 showed proteinase activity starting on day 10 and remained low throughout 18 days of storage despite reaching high population numbers. Proteinase production under aerobic conditions was detected during early stationary phase and increased steadily during storage to its maximum activity of 378 azocasein units on day 19. Stead (1987) showed a steady increase in enzyme production by P. fluorescens, as did Keen and Williams (1967) for P. lachrymans and Hare et al. (1981)  for V. alginolyticus. This accumulation of proteinase was different than the results in Trials I and II which did not show such high proteinase activity. The proteinase activity of 80 units on day 10 in Trial III was comparable to the activity seen on day 10 in Trial I (Fig. 6) and day 12 in Trial II (Fig. 13). P. fluorescens populations flourished under 4.8, 10.3, and 13.4 % 02 atmospheres  but proteinase production was severely decreased. During prolonged storage, energy production is expected to decrease when both 02 and alternate electron acceptor (NO3/NO2) may be limiting. A decrease in energy charge would favour ATP generating catabolic processes and an increase would favour biosynthesis (Harrison, 1976), but it has been reported that when there was a limitation in orthophosphate, carbon, or nitrogen, proteinase production by P.fluorescens was decreased (McKellar and Cholette, 1984).  88 Figure 22. Proteinase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C using azocasein as a substrate. Each point represents the mean of triplicate proteinase activity values + standard deviation.  89 Limiting oxygen, an essential nutrient for obligate aerobes, might then have a similar effect on the production of extracellular enzymes as well as other biosynthetic reactions. In B. subtilis, transition-state regulators control cellular processes so that energy is not expended in expressing functions which are detrimental (Strauch and Hock, 1993). The restriction of enzyme synthesis would result in greater economy for cell growth. Oxygen demanding catabolic and biosynthetic processes may be limited when the initial dissolved 02 concentration is decreased. This would alter the production of metabolite precursors and energy by P. fluorescens for synthesis of extracellular enzymes. In a continuous culture study with P. fluorescens 378 (biotype A), Persson et al. (1990) reported that substrate utilization in oxygen limiting conditions followed the same nutritional pattern as a carbon source limiting culture. Aeration and oxygen transfer rate determined the biomass and when oxygen was limiting, biomass was determined by dilution rate (Persson et al., 1990). Cells behave differently under varying oxygen concentrations. Utilization of glucose as well as metabolite pools of L-lactate and pynivate were lower in cultures under lower oxygen atmospheres (Griffiths and Phillips, 1984; Spohr and Schutz, 1990). Lowering atmospheric oxygen concentration would decrease the amount of available electron acceptors during respiration, decrease the cosubstrate supply of many oxidation reactions, and then result in lower energy production for biosynthesis and extracellular enzyme production. According to Stanier et al. (1986), bacteria of the same strain show different cytochrome compositions when grown aerobically and anaerobically. A decrease in cytochrome synthesis may affect the  90 energy generating status of microorganisms and may have the same effects as limiting oxygen on growth (Wiersma et al., 1978). Proteinase production by P.fluorescens NC3 was lower when nitrate was used as an electron acceptor during anaerobic conditions (Himelbloom and Hassan, 1986). b. Drop in Oxygen Tension The increase in proteinase activity in samples coincided with a drop in oxygen tension (Appendix Fig. 4. a-d). Dissolved oxygen tension decreased as population density and the use of oxygen increased. It is possible that severe oxygen limiting conditions, when the demand exceeds the supply, initiated proteinase synthesis in P. fluorescens. An aerobically grown culture would require a means of adapting to its new 02 stressed environment and may require the actions of extracellular proteinases and lipases providing there is enough oxygen or alternate electron acceptor to maintain its respiration for energy production. Rowe and Gilmour (1982) observed an earlier onset of proteinase and lipase production by P. fluorescens when the dissolved 02 tension was forcibly decreased. Cultures under decreased atmospheric oxygen may use both the available oxygen as well as nitrate as electron acceptors and may not respond to 02 stress in the same manner as an aerobically grown culture. This would result in lower detectable proteinase and lipase activities if their regulation was oxygen-dependent. There is no concrete evidence that 02 stress initiates proteinase synthesis in P. fluorescens. Phospholipase synthesis by S. liquefaciens, however, was reported to be regulated by two promoters, PA which was  under growth phase regulation and Px under anaerobic induction (Givskov and Molin,  91 1992). 4. Proteolysis Milk under 13.4 and 20.2 % 02 showed increases in proteolysis after day 8 (Fig. 23). There were small differences in proteolysis between milk samples under 4.8 and 10.3 % oxygen which increased after day 10 but were markedly lower than the degree of proteolysis in milk under 13.4 and 20.2 % atmospheric oxygen. The greatest extent of proteolysis during storage occurred in the aerobic control. 5. Lipase Activity a. Lower Atmospheric Oxygen Lipase activity was generally detected earlier than proteinase activity. Milk under 13.4 and 20.2 % oxygen showed lipase activity starting on day 4 during exponential growth (Fig. 24) rather than during stationary growth phase as observed with proteinase (Fig. 22). The early detection of lipase activity as seen in Trial I may suggest constitutive expression of lipase. Although there were detectable levels of lipase activity in all milk samples under lower atmospheric oxygen, lipase activity was lower than in the aerobic atmosphere. Fluctuations in lipase activity occurred as proteinase activity increased. Lipase activity in samples under 13.4 and 20.2 % oxygen decreased on day 16 perhaps as a result of increased proteinase activity on coexisting lipases. As with proteinase, a decrease in the initial dissolved 02 tension in milk resulted in a decrease in lipase synthesis by P. fluorescens. Lipase synthesis by P. fluorescens appears to follow the same restrictions of oxygen limitation as proteinase. Inhibition of lipase production by P.fluorescens was not as dramatic as with proteinase when the  92 Figure 23.^Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate proteolysis values + standard deviation.  93 Figure 24. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicate lipase activity values + standard deviation.  94 initial dissolved 02 tension in milk was decreased. b. Drop in Oxygen Tension As observed with proteinase, the increase in lipase activity coincided with the drop in oxygen tension in all samples (Appendix Fig. 5. a-d). Lipase synthesis could also be regulated by oxygen stress signals. 6. Lipolysis  Despite fluctuations in lipase activity, there were increases in the degree of lipolysis of milk fat throughout storage (Fig. 25). Acid degree values were highest for the aerobic control. Milk under oxygen diminished atmospheres showed similar acid degree values which were about 50 % lower than the aerobic control. 7. Correlation Coefficients  Tables 5 to 8 show correlation coefficients (r) for P. fluorescens cell numbers, proteinase and lipase activities and cumulative degree of proteolysis and lipolysis in milk under 4.8, 10.3, 13.4 and 20.2 % atmospheric 02. Correlation coefficients for cell numbers and proteinase activity ranged between 0.50 to 0.82 and r values for cell numbers and lipase activity ranged between 0.43 and 0.76 but not all r values were significant. Proteinase activity and degree of cumulative proteolysis in milk showed strong correlation (r = 0.84-0.96) and were highly significant at all the atmospheric 02 levels. Lipase activity and degree of lipolysis did not show strong correlation as r varied between 0.06 to 0.72 and were not all significant. Lipase activity may be dependent on proteinase activity as well as its association with milk proteins.  95 Figure 25. Lipolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate lipolysis values + standard deviation.  96 Table 5. Correlation coefficients of P.fluorescens cell numbers and enzyme activities in milk stored under 4.8 % atmospheric 02 atmosphere Correlation coefficients (r) Parameters^Cell numbers Proteinase^Proteolysis^Lipase^Lipolysis Cell numbers^1.00 Proteinase^0.50^1.00 Proteolysis^0.27^0.84**^1.00 Lipase^0.43^0.01^-0.17^1.00 Lipolysis^0.53^0.89***^0.77**^0.06^1.00  Table 6. Correlation coefficients of P.fluorescens cell numbers and enzyme activities in milk stored under 10.3 % atmospheric 02 atmosphere Correlation coefficients (r) Parameters^Cell numbers Proteinase^Proteolysis^Lipase^Lipolysis Cell numbers^1.00 Proteinase^0.73*^1.00 Proteolysis^0.51^0.91***^1.00 Lipase^0.76**^0.42^0.19^1.00 Lipolysis^0.86***^0.86**^0.64^0.72*^1.00  *, **, *** significant at 5 %, 1 % and 0.1 % levels, respectively  97 Table 7. Correlation coefficients of P.fluorescens cell numbers and enzyme activities in milk stored under 13.4 % atmospheric 02 atmosphere Correlation coefficients (r) Parameters^Cell numbers Proteinase^Proteolysis^Lipase^Lipolysis Cell numbers^1.00 Proteinase^0.82**^1.00 Proteolysis^0.75*^0.89***^1.00 Lipase^0.68*^0.40^0.49^1.00 Lipolysis^0.89***^0.90***^0.89***^0.64^1.00  Table 8. Correlation coefficients of P .fluorescens cell numbers and enzyme activities in milk stored under 20.2 % atmospheric 02 atmosphere Correlation coefficients (r) Parameters^Cell numbers Proteinase^Proteolysis^Lipase^Lipolysis Cell numbers^1.00 Proteinase^0.55^1.00 Proteolysis^0.55^0.96***^1.00 Lipase^0.70*^0.37^0.47^1.00 Lipolysis^0.64^0.81**^0.92***^0.64^1.00  *, **, *** significant at 5 %, 1 % and 0.1 % levels, respectively  98 Proteinase activity and lipolysis showed correlations (r = 0.81-0.90) at highly significant levels, as well as for the degree of proteolysis and lipolysis (r = 0.64-0.92). Results from the correlation matrix tables show that there may be relative relationship between proteinase activity on casein micelles to release lipases from its association and proteolytic degradation of lipases. Complexity of lipase determination warrants more research in this area. 8. at The aerobic control showed a large increase in pH on day 8 which coincided with an increase in proteinase activity (Fig. 26). In relation to the energy state of the cell, a basic extracellular pH has been reported to have a positive effect on extracellular enzyme production by P. aeruginosa (Whooley and McLoughlin, 1983). It was suggested that the increase in pH gradient between the cell and its environment could have a decreasing effect on proton motive force, a decrease in ATP level and therefore, an increase in enzyme production (Whooley and McLoughlin, 1983). Milk samples under lower atmospheric oxygen showed decreases in pH starting on day 6 which coincided with increases in lipase activity. The final pH of milk under 10.3 % 02 deviated the least from initial milk pH during storage. pH values could be reflective of the overall effects of proteinase and lipase activity in milk. Activity of one enzyme however could effectively negate the effects of pH changes brought about the other.  99 Figure 26. pH of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C.  100 9. Summary of Trial III Initial populations of 4.40 Logio CFU mL-1 of P. fluorescens were stored under 4.8, 10.3, 13.4 and 20.2 % atmospheric 02 for 19 days at 4 °C. P. fluorescens under 4.8 and 13.4 % 02 showed faster growth rates than the microorganisms in the aerobic control during initial storage. The highest population density was again observed under 13.4 % 02. The decrease in dissolved 02 was similar to the pattern observed in previous trials. Proteinase activity was first detected on day 8 during early stationary phase for the aerobic control which corresponded to the increase in proteolysis. Proteinase activity of P.fluorescens and the degree of proteolysis in milk under oxygen decreased atmospheres  were severely inhibited despite high population numbers. Lipase activity was first detected earlier than proteinase and increased in all samples. The aerobic control showed the highest lipase activity which was at times, twice as high as the other samples. Lipase activity appears to be sensitive to proteinase degradation during assay conditions. Correlation coefficients for proteinase activity and degree of proteolysis (r = 0.840.96) were highly significant. Lipase activity and lipolysis showed lower correlation (r = 0.06 to 0.72) and were not all significant. Low correlation exists for cell numbers and proteinase and lipase activities under 4.8 and 20.2 % atmospheric 02 when activity levels are either very high or very low. Milk pH values, indicating the combined effects of proteolysis and lipolysis, deviated the least from initial pH in milk under 10.3 % oxygen.  101 A decrease in the initial dissolved 02 tension in milk effectively decreased synthesis of proteinases and lipases by P. fluorescens in milk. Changes in metabolism due to 02 limitation and/or a decrease in the 0 2 stress signal may be responsible for the minimization of extracellular proteinase and lipase production by P. fluorescens.  102 D. CONTROL OF OXYGEN CONCENTRATION DURING STORAGE OF RAW  mruc Results from this model system using P. fluorescens suggest that growth and production of extracellular enzymes by aerobic spoilage microorganisms may be enhanced under highly aerobic conditions during storage of raw milk. Many factors such as agitation methods, surface area in contact with air, depth of storage tanks as well as temperature, influence the oxygenated state of milk. Current practice at dairy processing plants include mechanical and air agitation systems in raw milk silos. Agitation and aeration systems in these silos should be investigated for rate of oxygen transfer during storage of raw milk to minimize microbial activity. Decreasing the oxygen content of milk by altering the atmospheric oxygen level may inhibit the detrimental activities of P. fluorescens and other obligate aerobes but may select for facultative anaerobes which may emerge as the next major spoilage microorganisms.  103 CONCLUSION  1. The dissolved oxygen tension in milk under all atmospheric oxygen levels tested decreased during mid-exponential growth phase of P. fluorescens when populations reached 5.0 - 6.5 Logi() CPU mL4 despite a constant supply of 02.  2.  The growth rate of P. fluorescens in UHT-sterilized milk (2 % m.f.) was enhanced during the first four days of storage at 4 °C under decreased atmospheric 02 concentrations at 1.2, 4.8, 5.3, and 13.4 % 02. The final cell density of P. fluorescens under 13.4 % atmospheric 02 was greater than under aerobic  conditions.  3. Aerobic conditions supported: highest proteinase and lipase activity of P. fluorescens when the dissolved 02 tension in milk decreased to low levels - greatest degree of milk proteolysis and lipolysis and greatest change in milk pH  4.  Decreased atmospheric oxygen concentrations (<21 % 02) caused: a decrease and a delay of onset of proteinase and lipase synthesis by P. fluorescens in UHT-sterilized milk (2 % m.f.) during storage  greater inhibition on proteinase than lipase production by P.fluorescens  104 5. Of the atmospheric 02 concentrations tested, 10 % was the critical upper level which suppressed: growth rates of P . fluorescens during first four days of storage proteinase and lipase activity of P .fluorescens in UHT-sterilized milk degree of proteolysis and lipolysis change from initial milk pH during storage  105  PART II  Improvement of the I3-naphthyl caprylate Lipase Activity Assay  106 INTRODUCTION  Most P. fluorescens produce proteinases and lipases simultaneously during early stationary growth. During storage experiments, lipase activity fluctuates and usually decreases when proteinase activity increases. There is concern of proteolytic degradation of lipases during storage at lower temperatures but there is greater concern during lipase activity assays. The optimal temperature for lipase and proteinase activity are similar around 35 - 45 °C. Proteinase activity could then affect the apparent lipase activity. Inhibition of proteinase activity during lipase assay conditions would then result in more accurate reflection of lipase activity contained in milk at the time. The objective of the study in this part of the thesis was to enhance the sensitivity of the lipase activity by minimizing proteinase, but not lipase activity, with a metal chelator, ethylenediamine tetraacetic acid (EDTA).  107  LITERATURE REVIEW A. Lipase Degradation by Proteinases There are many reports to suggest that lipases may be inactivated through the actions of co-existing proteinases since both are produced by P. fluorescens. Lipase activity would then reflect the total amount of functional enzyme after hydrolysis by proteinase (Roussis et al., 1988). 1. During Storage Decrease in lipase activity is often observed during prolonged storage and with increases in proteinase activity. Lipase activity of Micrococcus caseolyticus reached its maximum then decreased with an increase in proteolytic activity (Jonsson and Snygg, 1974). P. fluorescens showed a decrease in lipase activity when proteinase activity increased markedly suggesting inactivation of lipase by considerable accumulation of proteinase (Fox and Stepaniak, 1983; Bucky et al., 1986). Lipase susceptibility was enhanced in skim milk but not in whole milk, so it was suggested by Bucky et al. (1986) that milk fat provided protection from proteinases. The addition of subtilisin caused a rapid inactivation of P. fluorescens lipase at 20 °C (Andersson, 1980a) and addition of proteinase caused 16 % reduction in lipase activity (Roussis et al., 1988). Stead (1987) suggested that lipase activity probably reflected a dynamic system of degradation of lipase by proteinase since P. fluorescens AR11 in whole milk at 7 °C with high proteinase activity showed a steady decrease in lipase activity over time. A proteinase deficient P.  fluorescens AR11M mutant on the contrary, showed higher lipase activity since lipase  108 degradation by bacterial proteinase would have been less than in the proteinase proficient strain. In support of this, degree of lipolysis as a measure of cumulative lipase activity was greater in milk sample under N2-disrupted atmosphere than the aerobic control (Skura et al., 1986) which may possibly be due to its lower proteinase activity. 2. During Lipase Assay Conditions Proteinase activity would be enhanced during incubation of lipase activity assay since it would be closer to its optimal temperature for activity. Optimal temperature for P. fluorescens proteinase activity was reported to be 35 to 44 °C (Cousin, 1982). The  activity of P. fluorescens AR11 proteinase at 4 °C was 33 % of that at 35 °C (Alichanidis and Andrew, 1977) and P. fluorescens AFT 36 proteinase at 7 °C was only 30 % of proteinase activity at 45 °C (Stepaniak and Fox, 1983). Greater decrease in P. fluorescens lipase activity was observed at 37 °C than at 30 °C due to increased rate of  proteolytic digestion of the lipase at the higher temperature (Jonsson and Snygg, 1976). During incubation at 40 °C for lipase activity, presence of proteinase activity could decrease the sensitivity of the assay if lipases are degraded by the co-existing proteinase. B. Inhibition of Proteinase Activity in Lipase Assay with EDTA Many of the proteinases of P. fluorescens are neutral metalloproteinases and are sensitive to metal inhibitors (Stepanialc and Sorhaugh, 1989). Metal cations such as Ca2+ provide structural integrity while Zn2+ maintains integrity of the active site for many of these proteinases from psychrotrophic pseudomonads and increases their thermal stability (McKellar, 1989). Activity of typical metalloproteinases are inhibited by a metal chelator  109 such as ethylenediamine tetraacetic acid (EDTA) at 1-10 mM (Stepaniak and Sorhaugh, 1989). It was reported that EDTA had the most inhibitory effect compared to the other inhibitors tested by Kohlmann et al. (1991). At 1 mM EDTA, 66 % of the proteinase activity remained while at 10 mM EDTA only 12 % of the proteinase activity remained (Kohlmann et al., 1991). Preformed proteinases cannot be activated with the addition of Ca2+ after EDTA inactivation (McKellar and Cholette, 1985). C. Lipase Activity Assay Conditions The use of super-simplex Gptimization to determine the best assay conditions for P. fluorescens lipase activity on n-naphthyl caprylate (n-NC) showed optimal response at  50 °C, pH 7.2, 0.1 mM 0-NC and 17.6 mM sodium taurocholate (NaTC) (Paquette and McKellar, 1986). Psychrotrophic lipase is activated by the addition of bile salts such as NaTC to release milk proteins which may block its active site (Stead, 1983) or by the action of trypsin (McKellar and Cholette, 1986a). Lipases are usually adsorbed on milk fat globules and their activity is strongly reduced by presence of milk fat in assay reactions because of competition between fat and the assay substrate (McKellar and Cholette, 1986a). The presence of 2 % milk fat inhibited 92 % of lipase activity (McKellar and Cholette, 1986a). The removal of milk fat by centrifugation has been suggested but lower lipase activity is often reported because of concomitant removal of the lipases (Stead, 1983; Versaw et al., 1989). The adsorption of a lipase to substrate : water interface (Downey, 1980) can be increased with the addition of salts such as Na+, K+, Mg2+ and (Roussis et al., 1988) since the salts can precipitate free fatty acids (MacRae, 1983). The activity of lipases are usually inhibited by slightly higher EDTA concentrations, of  110 around 100 InM, than are proteinases (Bozoglu et al., 1984).  111  MATERIALS AND METHODS  A. Improvement of the Lipase Assay The method established by Versaw et al. (1989) based on that published by McKellar (1986) was modified. 1. Sodium Taurocholate  Versaw et al. (1989) added 200 ilL of 200 mM of bile salt sodium taurocholate (NaTC) to give a final concentration of 19.3 mM. It was reported that maximum response was observed for 17.6 mM (Paquette and McKellar, 1986). Concentrations between 0 and 20 mM of NaTC were added to the reaction mixture. The difference in volume was corrected with BES buffer. 2. 11-naphthyl Caprylate  Versaw et al (1989) used 20 IlL of 200 mM 13-naphthyl caprylate for a final concentration of 1.93 mM in each reaction. A final concentration range of 0 to 7 mM of P-naphthyl caprylate dissolved in DMSO was tested for best concentration of the substrate while minimizing the volume of DMSO which is added to the reaction mixtures. 3. Solvent Clarification After the reactions were stopped with the addition of 0.2 mL of 10 % TCA, the reactions were clarified with the addition of solvent(s). Addition of BES buffer was decreased from 1.8 to 1.0 mL for easier pipetting. Attempts were made to extract the diazonium colour compounds from the aqueous layer with 100% ethyl acetate. Solvents (2.51 mL) used to clarify the lipase reaction mixtures were dimethylformamide,  112 dimethylsulfoxide, acetone, formaldehyde, and varying ratios of ethyl acetate and ethanol. B. Lipase Activity Enhancement 1. Proteinase Activity Under Lipase Assay Conditions Proteinase activity was determined using 1.0 mL of 1.5 % azocasein in pH 7.2 BES buffer as a substrate in presence of 0.20 mL of 100 mM sodium taurocholate (NaTC), 0.02 mL of 500 mM 13-naphthyl caprylate in dimethylsulfoxide (DMSO) and 0.05 mL milk sample with a final concentration of 0.05 % thimerosal to inhibit extracellular enzyme production. The enzyme source was the milk sample frozen at -30 °C on Day 14 (20.2 % 02) in Trial III. Final concentrations of EDTA ranging from 0 to 50 mM were prepared from a 250 mM EDTA stock solution. The tubes were incubated at 40 °C for 1 h with shaking at 160 rpm. The reactions were stopped with the addition of 2.0 mL of 24 % trichloroacetic acid (TCA) in an ice bath for 15 minutes. The tubes were centrifuged at 2910 x g for 15 minutes in a GS-6 Beckman centrifuge (Beckman Instruments Inc., Palo Alto, CA) and the absorbance of the supernatant was measured at 366 nm. All samples were assayed in triplicate. One azocasein unit of proteinase activity was defined as an increase in absorbance of 0.01 at 366 nm per mL of milk per hour. 2. Addition of EDTA Lipase assay conditions stated in Part I were used with the addition of final concentration between 0-50 mM EDTA prior to enzyme addition to reaction mixtures. The same controls were used in quadruplicate. One 13-naphthol unit was defined as one gmole 13-naphthol released per tnL of milk per hour.  113  RESULTS AND DISCUSSION A. Improvement of the Lipase Assay 1. Sodium Taurocholate Lipase activity was enhanced as NaTC concentration was increased. A final concentration of 15 mM NaTC was used in future lipase activity assays (Fig. 27). 2. 13-naphthyl caprylate Along with lipases in the 50 III.. aliquot used in the assay, appreciable amounts of milk fat in 2 % m.f. UHT milk (as much as 1.0 mg) was possibly carried over to each reaction mixture and allowed to compete with the substrate. The [3 naphthyl caprylate at a -  final concentration of 2 mM was calculated to be 1.08 mg and created a ratio of 1 to 1 between milk fat and the substrate. Final concentrations of 0 to 7 tnM of 13 naphthyl -  caprylate were tested. An increase in lipase activity was proportional to an increase in substrate concentration showing maximum activity at a final concentration of greater than 5 mM and the ratio of substrate: fat corresponding to 3:1 (Fig. 28). A stock solution of 500 mM I3-naphthyl caprylate was used at a final concentration of 8 mM so that the substrate concentration was not limiting in the assay. 3. Solvent Clarification The addition of 2.71 mL 50:50 ethyl acetate: ethanol recommended by Versaw et al. (1989) to the reaction mixtures for a final volume of 4.0 mL did not completely clarify the reaction mixtures. Turbidity was due to the presence of milk proteins. Also the BES buffer was decreased from 1.8 to 1.0 mL for easier pipetting volume, changing the  114 Figure 27. Effect of increasing sodium taurocholate (NaTC) concentrations on lipase activity. Each point represents the mean of triplicate values + standard deviation.  ›-  -; •IMMI  et -eC  0  ^^ ^ ^ 15 20 5 10  NaTC Concentration (mM)  115 Figure 28. Effect of increasing 13-naphthyl caprylate (0-NC) concentrations on lipase activity. Each point represents the mean of triplicate values + standard deviation.  0.8  0.7  0.6  0.5  0.4  03  0.2  0.1  0.0  -0.1 0  1^2  3^4  5^6^7  13-NC Concentration (mM)  8  116 proportion of solvents required to clarify the aqueous reaction mixtures. A better solvent system for clarifying the reaction mixtures was needed and Table 9 shows the solvent systems tested. A 60:40 ethyl acetate to ethanol solvent system completely clarified the reaction mixtures. Phase partitioning occurred with other proportions of ethyl acetate and ethanol ard resulted in incomplete extraction. B. Lipase Activity Enhancement  1. Proteinase Activity Under Lipase Assay Conditions Proteinase activity under lipase assay conditions was 33.5 % of the activity under proteinase activity conditions without 15 mM NaTC and 8 mM P-naphthyl caprylate (Table 10). This proteinase activity could interfere with the lipase activity assay, especially in cultures with high proteinase concentrations. Proteinase showed more sensitivity towards EDTA than lipase. A final concentration of 2 to 5 mM EDTA in the reaction mixtures enhanced the activity of lipase quantified and greatly reduced the activity of proteinase (Fig. 29).  2. Lipase Activity with the Addition of EDTA The observed increase in lipase activity was probably due to inactivation of proteinase activity with EDTA. When both proteinases and lipases are present in milk cultures, an accurate measurement of the lipase activity present in milk at 4 °C would not be represented by lipase assay conditions set at 40 °C since proteinases would degrade lipase during incubation and decrease measurable lipase activity. Proteinase activity showed a plateau at EDTA concentrations exceeding 2 mM and a decrease in lipase was observed at  ^  117 Table 9: Clarification of Lipase Reactions with Solvents Solvents^Clarification a dimethyl formamide (DMF) dimethyl sulfoxide (DMSO) acetone formaldehyde ethyl acetate to ethanol ratio: 10:90 20:80 30:70 40:60 50:50^+ 55:45^+ 60:40^+ 65:35^+ 70:30 80:20 90:10 a^ turbid,^+ some turbidity,^+ clarified -  Table 10: Proteinase activity under lipase assay conditions Sample  [EDTA] rnM  without NaTC, P-NC 0 with NaTC, 13-NC 0 20 35 50  Proteinase activity (Azocasein units + Std Dev) 542.7 181.7 28.3 8.0 0  +^5.8 +^3.0 +^4.0 +^1.4 +^2.3  % Relative Activity 100.0 33.5 5.2 1.5 0  118 Figure 29. Effect of increasing EDTA concentrations on proteinase and lipase activities in lipase assay conditions. Each point represents the mean of triplicate values + standard deviation.  119 EDTA concentrations exceeding 10 mM. 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A continuous culture study of the regulation of extracellular protease production in Vibrio SAl. Antonie van Leeuwenhoek 44:141155. Zitomer, R. S. and Lowry, C. V. 1992. Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol. Rev. 56:1-11.  134  APPENDICES  135 Appendix Figure 1. Lipolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicate lipolysis values + standard deviation.  Atmospheric Oxygen  1  •^I^•^I^•^I^•  0^2^4^6^8^10^12^14^16  Storage Time (Days)  ---•—  1.2%  ---tr—  5.3%  —9—  10.2%  —0—  20.9%  136 Appendix Figure 2. The effect of mechanical agitation at 350 rpm on the increase in acid degree values (ADV) of UHT-sterilized milk (2 % m.f.) stored at 4 °C under aerobic conditions. Each point represents the mean of triplicate lipolysis values + standard deviation.  3.0  1.5 -  —0— stirred —0— unstirred  1 .0 -  0.5 -  0.0 ^ 2^4^6^8  Storage Time (Day)  137 Appendix Figure 3. The effect of CO2 removal from the titration atmosphere on the acid degree values (ADV) of UHT-sterilized milk (2 % m.f.) stored at 4 0C under aerobic conditions. Each point represents the mean of triplicate lipolysis values + standard deviation.  3.0  2.5 -  2.0 -  —0— stirred 15 -  —0— unstirred -  1.0 -  0.0  o  ^  1  2  ^ ^ ^ 8 4 6  Storage Time (Day)  138 Appendix Figure 4. a - d. The effect of dissolved oxygen and its limitation on proteinase activity of P. fluorescens in UHT-sterilized milk (2 %) under controlled oxygen atmospheres during storage at 4 °C. Each point represents the mean of triplicate proteinase activity values and a single dissolved oxygen value.  139 c. 13.4 % Atmospheric Oxygen 50  12  -40 .-30 30  --A—  C - 20 > 0  Proteinase  et^c),  11  ppm  co) N  —10  00  0 0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days)  d. 20.2 % Atmospheric Oxygen 12 10 -300 .,"  amt  g  - 200  —0--- ppm  a) „a1),  et c.) 4—) 0 ON  - 100^.,e!  0  i. i.i.i.i.i.0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days)  Proteinase  140 Appendix Figure 5. a - d. The effect of dissolved oxygen and its limitation on lipase activity of P. fluorescens in UHT-sterilized milk (2 %) under controlled oxygen atmospheres during storage at 4 °C. Each point represents the mean of quadruplicate lipase activity values and a single dissolved oxygen value. a. 4.8 % Atmospheric Oxygen  —A— ppm —A-- Lipase  OA-^.^1.^-1 .1 .^-^.^0.0 0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days) b. 10.3 % Atmospheric Oxygen 12  0.6 - 0.5 •••••••  - 0.4  •-  4.) -6 4!  - 0.3 !... ; -0.2^<1.) CS  -0. 1 O• 0.^•^•I.I.^.1^0.0 0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days)  0. ett  —0— ppm —40-- Lipase  141  c. 13.4 % Atmospheric Oxygen 0.6 — 0.5 —0.4 — 0.3 —0.2  ppm  *4-) j •et^.4a4 a)^0. cn et C.  —A-- Lipase  —0.1 0.0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days)  d. 20.2 % Atmospheric Oxygen 12  0.6  •  10  —0.5  C.  C.  8— >4 'as c.) A  •  tit  6 4— 2— •  0  —0.4  •  •  —0.2 —0.1  • •^•^.1 )1^.1^•^0.0 0 2 4 6 8 10 12 14 16 18 20  Storage Time (Days)  —D— ppm •  •  • •  —0.3  Lipase  

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