MINIMIZATION OF EXTRACELLULAR ENZYME PRODUCTION BYPseudomonas fluorescensIN A MODEL MILK SYSTEMBY CONTROLLED OXYGEN ATMOSPHERESBYKAROLINE K. LEEB. Sc. , The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER IN SCIENCEinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF FOOD SCIENCE)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1993© Karoline K. Lee, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department oThe University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)ABSTRACT1. Pseudomonas fluorescens, the predominant aerobic spoilage microorganism in rawmilk, proliferate and produce heat resistant extracellular enzymes during storage. Thecritical upper 02 level which would minimize the synthesis of proteinases and lipases byP. 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 litrecarboys 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 104CFU rriL-1 to reflect the psychrotrophic populations normally encountered in milk andstored under controlled 02 atmospheres at 4 °C up to 18 days. The atmosphericcomposition was analyzed with gas chromatography. Milk samples were collected everytwo 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 duringmid-exponential growth phase of P. fluorescens when populations reached 5.0 - 6.5Logi() CFU mL4 under all atmospheric 02 levels tested.The growth rate of P. fluorescens was enhanced during the first four days of storageunder decreased atmospheric 02 concentrations at 1.2, 4.8, 5.3, and 13.4 % whencompared to the aerobic control. Lower levels of proteinase and lipase activities weredetected 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, and20.9 % 02). As a result, lower degree of proteolysis and lipolysis were observed in milkstored under decreased atmospheric 02 conditions. Deviation from initial milk pHreflecting the predominant effect of either proteolysis or lipolysis was minimized when theinitial dissolved 02 tension in milk was decreased. Greater inhibitory effect on proteinasethan lipase production by P. fluorescens was observed under decreased 02 atmospheres.P. fluorescens under 10 % atmospheric 02 concentration consistently showed slowgrowth rates during initial storage, low proteinase and lipase activities and as a result, lowdegree 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 thelevels tested, which minimized extracellular enzyme production by P. fluorescens, aswell as delaying its growth rate during initial storage, in UHT-sterilized milk.Compared to storage of raw milk under aerobic atmosphere, decreased atmospheric02 conditions may provide a better environment for storage of raw milk when thepredominant flora consists of aerobic P. fluorescens.2. Conditions for an improved f3-naphthyl caprylate assay for lipase activity wereestablished at final concentrations of 15 mM sodium taurocholate and 8 mM 13-naphthylcaprylate in the reaction mixture and a solvent system of 60:40 ethyl acetate to ethanol. Afinal concentration of ethylenediamine tetraacetic acid between 2 and 5 rnM minimizeddegradation of lipases by proteinases and thus enhanced lipase activity during the assayprocedure.CONTENTS PageABSTRACT^CONTENTS ivLIST OF TABLES^ ixLIST OF FIGURESAPPENDICES^ xiiACKNOWLEDGEMENTS^ xiiiPART I^ 1INTRODUCTION^ 2LITERATURE REVIEW 4A. Psychrotrophs in Raw Milk^ 41. Predominance of Pseudomonas fluorescens^ 52. Extracellular Enzymes as Spoilage Causing Agents^ 5A. Heat Stability^ 5B. Enzymatic Degradation of Milk Constituents 63. Relation Between Population Numbers and Enzymatic Activity^ 74. Production of Proteinase and Lipase as Function of Growth Phase^ 75. Drop in Dissolved Oxygen Tension in Milk ^ 86. Dissolved Oxygen as Indicator of Potential Spoilage^ 9B. Role of Oxygen in Aerobic Microorganisms 101. Energy Production^ 10A. Aerobic Respiration 10B. Anaerobic Respiration 112. Metabolism Under Decreased Oxygen Concentrations^ 123. Energy Status and Extracellular Enzyme Production 134. Critical Dissolved Oxygen Tension (CDOT)^ 15C. Factors Effecting Enzyme Production^ 16iv1. Temperature^ 162. Ions^ 173. Oxygen 18A. Oxygen Inhibits Enzyme Production^ 18B. Oxygen Enhances Enzyme Production 194. Regulation of Extracellular Enzyme Synthesis 21A. Proteinase^ 211. C Catabolite Repression^ 222. N Catabolite Repression 23B. Lipase^ 24C. Direct Regulation by Oxygen^ 24D. Potential Disadvantages of Decreased Oxygen Atmospheres^ 25MATERIALS AND METHODS^ 27A. Oxygen Controlled Atmospheres 27B. UHT Milk^ 29C. Culture Conditions^ 29D. Antibiotic Testing of UHT Milk^ 30E. Enumeration 30F. Proteinase Activity Assay 31G. Proteolysis^ 32H. Lipase Activity Assay^ 33I. Lipolysis 34J. Investigation of High ADV 35a. Induced Lipolysis by Mechanical Agitation^ 35b. Metabolism of P. fluorescens biotype A (ATCC 17397)^ 351. Carbohydrate Utilization^ 362. Nitrate Reduction 36c. Carbon Dioxide Interference 37K. Statistical Analysis (Trial III)^ 37RESULTS AND DISCUSSION 39A. TRIAL I^ 411. Growth 41a. Alternate Electron Acceptors^ 43vib. Growth on Solid Medium^ 442. Oxygen^ 44a. Atmospheric Oxygen 44b Dissolved Oxygen^ 44c. Oxygen and Energy Production^ 483. Proteinase Activity 49a. Carbon and Nitrogen Catabolite Repression^ 51b. Proteinase Synthesis and Growth Phase 51c. Factors Influencing Protemase Production 52i. Oxygen^ 52ii. Population Density^ 52d. Proteinase Activity and Inhibition of Cell Lysis^ 53e. The Inhibitory Effect of Nitrogen^ 534. Proteolysis^ 545. Lipase Activity 546. Lipolysis 577. Investigation of High ADV^ 58a. Induced Lipolysis by Mechanical Agitation^ 58b. Carbohydrate Utilization of P. fluorescens biotype A^ 58c. Carbon Dioxide Interference^ 598. Summary of Trial I^ 60B. TRIAL II^ 611. Growth 61a. Low Population Numbers^ 61b. Growth Under Lower Atmospheric Oxygen^ 63c. Oxygen and Nitrate as Electron Acceptors 632. Oxygen^ 64a. Atmospheric Oxygen^ 64b. Dissolved Oxygen 64c. Carbon Dioxide Production 643. Proteinase Activity^ 67a. Aerobic Conditions 67b. Lower Atmospheric Oxygen^ 70c. Relation Between Population Density and Proteinase Activity^704. Proteolysis^ 715. Lipase Activity 716. Lipolysis 747. pH^ 76vii8. Summary of Trial II^ 78C. TRIAL III^ 801. Growth 802. Oxygen 80a. Atmospheric Oxygen^ 80b. Dissolved Oxygen 83c. Carbon Dioxide Production 833. Proteinase Activity^ 87a. Lower Atmospheric Oxygen^ 87b. Drop in Oxygen Tension 904. Proteolysis^ 915. Lipase Activity 91a. Lower Atmospheric Oxygen^ 91b. Drop in Oxygen Tension 946. Lipolysis^ 947. Correlation Coefficients^ 948. pH 989. Summary of Trial III 100D. CONTROL OF OXYGEN CONCENTRATION DURING STORAGE OFRAW MILK^ 102CONCLUSION 103PART II^ 105INTRODUCTION^ 106LITERATURE REVIEW 107A. Lipase Degradation by Proteinases^ 1071. During Storage^ 1072. During Lipase Assay Conditions 108B. Inhibition of Proteinase Activity in Lipase Assay with EDTA^ 108C. Lipase Activity Assay Conditions^ 109MATERIALS AND METHODS 111viiiA. Improvement of the Lipase Assay^ 1111. Sodium Taurocholate^ 1112. P-naphthyl Caprylate 1113. Solvent Clarification 111B. Lipase Activity Enhancement^ 1121. Proteinase Activity Under Lipase Assay Conditions^ 1122. Addition of EiDTA 112RESULTS AND DISCUSSION^ 113A. Improvement of the Lipase Assay 1131. Sodium Taurocholate^ 1132. 11-naphthyl caprylate 1133. Solvent Clarification 113B. Lipase Activity Enhancement^ 1161. Proteinase Activity Under Lipase Assay Conditions^ 1162. Lipase Activity with the Addition of EDTA 116CONCLUSIONS^ 120REFERENCES 121APPENDICES^ 134ixLIST OF TABLES TABLE Page1. Atmospheric oxygen levels tested^ 402. Initial populations of P. fluorescens 401-. Metabolism of P. fluorescens 454. Dissolved oxygen tension of stirred and unstirred samples^ 855. Correlation coefficients of parameters at 4.8 % 02^ 966. Correlation coefficients of parameters at 10.3 % 02 967. Correlation coefficients of parameters at 13.4 % 02^ 978. Correlation coefficients of parameters at 20.2 % 02 979. Clarification of lipase reactions with solvents...^ 11710. Proteinase activity under lipase assay conditions 117xLIST OF FIGURES PART IFIGURE^ Page1. Experimental setup in 4 °C coldroom^ 282. Modification of titration setup 38TRIAL I3. Growth of P. fluorescens^ 424. Profile of oxygen controlled atmospheres^ 465. Dissolved oxygen tension 476. Proteinase activity^ 507. Degree of proteolysis 558. Lipase activity 56TRIAL II 9. Growth of P. fluorescens^ 6210. Profile of oxygen controlled atmospheres^ 6511. Dissolved oxygen tension 6612. Carbon dioxide production ^ 6813. Proteinase activity^ 6914. Degree of proteolysis 7215. Lipase activity 7316. Degree of Lipolysis^ 7517. Milk pH^ 77TRIAL III18. Growth of P. fluorescens^ 8119. Profile of oxygen controlled atmospheres^ 8220. Dissolved oxygen tension 8421. Carbon dioxide production^ 8622. Proteinase activity^ 8823. Degree of proteolysis 9224. Lipase activity 9325. Degree of lipolysis^ 9526. Milk pH^ 99xiPART H FIGURE^ Page27. Effect of NaTC on lipase activity^ 11428. Effect of 0-naphthy1 caprylate on lipase activity^ 11529.^Effect of EDTA on proteinase and lipase activities 118xiiAPPENDICES FIGURE1. Degree^of lipolysis^ 1352. Effect of mechanical agitation on lipolysis^ 1363. Removal of atmospheric CO2 on lipolysis 1374. a-d Drop in 02 tension and increase in proteinase activity^ 1385. a-d Drop in 02 tension and increase in lipase activity 140ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my advisor Dr. Brent J. Skura forhis endless support and guidance. His insightful wisdom and always encouraging wordshave made these past years a pleasure to work under his supervision. Thank you forbelieving in me.I would also like to thank the members of the reviewing committee for theirsuggestions 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 veryimportant 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 whosesupportive words carried me through dark days. This one's for you Grandma.PART IDetermination of Critical Oxygen ConcentrationRequired To Minimize Extracellular Enzyme Production byPseadomonasfluvrescens12INTRODUCTIONStorage of raw milk on farms, during transport and at dairy processing plants in ahighly oxygenated state at refrigeration temperature selects for aerobic, gram negativepsychrotrophs. These aerobic microorganisms cause quality problems in milk and dairyproducts not directly but rather through their production of extracellular enzymes,primarily heat resistant proteinases and lipases which survive most heat treatments andremain active during storage. Proteinases hydrolyze milk proteins and cause bitternessand gelation problems while lipases cause lipolytic, off-flavours from hydrolysis of milkfat.The predominant psychrotroph isolated from raw milk is Pseudomonas fluorescens.These microorganisms produce proteinases and lipases concomitantly during lateexponential - stationary growth phase. Usually a drop in dissolved oxygen tension in themedium occurs just prior to enzyme production. There appears to be a relationshipbetween oxygen and extracellular enzyme production whether causal or casual.Extracellular enzyme production by P . fluorescens is dependent on many factors suchas nutrient availability, growth phase, temperature, pH, and aeration. There arecontradictions in the literature about the role of oxygen in extracellular enzyme productionby psychrotrophs. Some studies show enhancing effects while others show inhibitoryeffects.Use of air and mechanical agitation systems in bulk storage silos in dairy processingplants may inadvertently be promoting extracellular enzyme synthesis by aerobic3psychrotrophs. A better understanding of the role of oxygen in the production ofextracellular enzymes by aerobic psychrotrophs may enable the development of improvedmethods of raw milk storage. The use of controlled oxygen atmospheres for raw milkstorage not only promises to delay the onset of extracellular enzyme production but alsoreduce 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 whichwould minimize extracellular proteinase and lipase production by P. fluorescens in amodel system using UHT-sterilized milk as the growth medium. The critical oxygenconcentration which greatly reduces the synthesis of extracellular enzymes would bebeneficial in providing higher quality raw milk. Several additional days of raw milkstorage in silos at dairy processing plants without risking quality of dairy products madefrom such raw milk may have substantial economic significance.4LITERATURE REVIEWA. Psychrotrophs in Raw MilkThere are many problems associated with extended storage of raw milk in bulk tankson farms, in transport and in processing plants. The use of refrigeration temperatures hasreduced spoilage by lactic acid bacteria and growth of most pathogens but has selected forpsychrotrophs capable of growth at 7 °C or below (Cousin, 1982). An increase in theaeration of raw milk by pumping and agitation has selected for growth of aerobic, gramnegative 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, undesirablebiochemical and sensory changes could result from microbial activity which would effectprocessed milk and other dairy products (Cousin, 1982; Bishop and White, 1986).Possible sources of microbial contamination of raw milk are numerous sincepsychrotrophs are ubiquitous in nature. One major concern would be inadequatelysanitized milking machines and pipelines in milking plants. Psychrotrophic counts of rawmilk can be as high as 103 CPU mL4 from milking machines, 104 to 105 CPU mL4 atcollection depots and 106 to 108 CFU mL-1 at milk plants (Cousin, 1982). Similarnumbers (4.4 x 103 CFU mL-1 from a farm and 9.6 x 103 to 2.9 x 104 CPU mL4 from adairy plant) were reported by Adams et al. (1975). Psychrotrophic counts from a dairyplant 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 lipaseduring prolonged storage at refrigeration temperatures. Stead (1987) stressed the5importance of adequately sterilizing dairy equipment to avoid inoculating fresh raw milkwith the microorganisms and their concentrated enzymes.1. Predominance of Pseudomonas fluorescens Ewings et al. (1984) showed that the predominant psychrotroph from raw milk on thefarm was Pseudomonas at a frequency of 70 % of the total psychrotrophic count. Garciaet al. (1989) showed that 62-70 % of the psychrotroph count was Pseudomonas, ofwhich 73 % were fluorescent. The most frequently occurring psychrotroph from rawmilk is reported to be P seudomonas fluorescens (Griffiths et al., 1981; Brandt andLedford, 1982; Cousin, 1982; Griffiths and Phillips, 1984; Kwan and Skura, 1985)belonging to biotype A (Poffe and Mertens, 1988). Fluorescent pseudomonadspredominated the lipolytic flora at 64 % (Shelley et al., 1987). Other gram negative rodshaped bacteria isolated from raw milk are Achromobacter, Acinetobacter, Aeromonas,Enterobacter, Klebsiella and Serratia (Cousin, 1982).2. Extracellular Enzymes as Spoilage Causing AgentsA. Heat Stability It is not the presence of the microorganisms per se which is detrimental to the qualityof milk but it is their production of extracellular enzymes. These psychrotrophs produceextracellular proteinases and lipases which hydrolyze proteins and lipids to provide readilymetabolizable substrates for growth. Most studies have reported a single extracellularlipase and a single proteinase for P. fluorescens but some pseudomonads may producemore than one proteinase (McKellar, 1989). P. fluorescens showed the highest6proteinase activity when compared to other isolates including P. fragi (Kohlmann et al.,1991).Heat treatments effectively decrease microbial populations but proteinases and lipasesretain 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 afterheat treatments such as high temperature short time (HTST,77 °C, 17 s) and ultra-hightemperature (UHT, 135-150 °C, 2-5 s) (Griffiths et al., 1981). Extracellular proteinasesof P. fluorescens biotype A were reported to have Di400c of 131 s (Kroll andKlostermeyer, 1984c). These enzymes retained 55-65 % activity after HTST treatmentand 20-40 % activity after UHT treatment (Griffiths et al., 1981). The inferior quality ofUHT milk made from raw, milk stored for 4 days at 6°C compared to that made from milkstored 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 etal., 1976). Psychrotrophic lipases retained 53 % of their activity after pasteurizationtreatment at 72 °C for 15 s although all of the native milk lipase was completelyinactivated (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 ConstituentsCommercial raw milk is composed of approximately 90 % moisture, 3.2 % proteinand 3 % fat. Proteolytic activity on milk proteins results in bitter peptides and gelation ofUHT-milk (Law, 1979; Fairbairn and Law, 1986). K and 13-caseins are reported to be7most 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 areassociated with rancid flavours and off-odors characteristic of cheeses with fruity off-flavours (Law, 1979; Cousin, 1982; IDF, 1991). There were significant lipolytic off-flavours associated with cheese made from milk with high psychrotrophic count althoughlow proteolysis and lipase activity were detected (Law et al., 1979). Free fatty acidsliberated during lipolysis are measured and expressed as acid degree value (ADV) toassess lipolysis of dairy products. Acid degree values exceeding 1.5 - 2.0 were found tobe 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 ofenzyme activity (Rowe and Gilmour, 1986; Skura et al., 1986). Generally, populationsof 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). Thesepopulation numbers were the level at which post pasteurization problems due to enzymescould be expected in dairy products made from such raw milk (Muir and Phillips, 1984).4. Production of Proteinase and Lipase as Function of Growth PhaseExtracellular enzymes are produced by microorganisms after low molecular weightnutrients in milk have been depleted during the exponential growth phase. These enzymesdegrade large molecular weight components and provide smaller molecular weight8substrates such as amino acids, peptides, and free fatty acids during stationary growthphase (Rowe and Gilmour, 1982). Extracellular enzyme production ensures a supply ofcarbon to maintain the microorganism's growth rate. P. fluorescens producedextracellular 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 stationaryphase when population density reached 108 CFU mL-1 (Rowe and Gilmour, 1982; Spohrand Schutz, 1990). Ninety-nine percent of the total phospholipase was produced duringlate exponential phase by Serratia liquefaciens (Givskov and Molin, 1992). Someauthors reported proteinase detection in early log phase (Adams et al., 1975) while othersshowed it was maximum when cultures were in death phase (Malik et al., 1985). Thesedifferences would most likely have reflected the effect of culture conditions and thevarying sensitivities of proteinase activity detection methods.Enzyme synthesis can be expressed as a function of growth in terms of differential rateof synthesis (McKellar, 1989) or specific proteinase producing power (Kroll andKlostermeyer, 1984a). The greatest amount of proteinase was produced during periodswhen 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 andceased when the P. fluorescens population entered the stationary phase.5. Drop in Dissolved Oxygen Tension in MilkThere may be a relationship between enzyme synthesis and oxygen stress since a large9drop in oxygen tension in milk was observed when the P. fluorescens population reached105 - 106 CFU mL4 (Spohr and Schutz, 1990) and 106 - 107 CPU mL4 (Rowe andGilmour, 1982; Griffiths and Phillips, 1984; Rowe and Gilmour, 1986) just prior toenzyme production. The drop in oxygen tension in milk results as the demand for oxygenby microbial populations exceeds the rate at which aeration and agitation could replace theconsumed oxygen. A small increase in demand then could result in a large decrease inoxygen tension (Rowe and Gilmour, 1986).6. Dissolved Oxygen as Indicator of Potential SpoilageDissolved oxygen tension is measured with a polarographic electrode consisting of agold cathode and silver anode (Luck, 1991) and is expressed as parts per million (ppm) oras a percentage of saturation. Oxygen is primarily responsible for a positive redoxpotential (Eh) value which is a measure of the capacity of a system to give up electronsand to be oxidized/reduced (Costilow, 1981). The Eh value can be expressed in volts andthe 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 arisesince oxygen tension of milk is influenced by many factors and continually changesduring storage and transport. Difference in milking procedures, time elapsing betweenmilking and testing, agitation systems, surface area of milk in contact with air, and depthof milk tank would all influence the dissolved oxygen content in milk (Schroder, 1982;Luck, 1991). Rowe and Gilmour (1986) demonstrated that oxygen tension cannot bedirectly correlated to population numbers but have suggested the correlation between10dissolved 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 andapproximately 78 % (v/v) of chemically inert N2 gases (Weast, 1984). Oxygen is anessential nutrient or cosubstrate for many aerobic microorganisms. In obligate aerobes,molecular oxygen functions primarily as a terminal electron acceptor in aerobicrespiration. Microaerophilic microorganisms grow best under lower partial pressures ofoxygen between 2-10 % (v/v). Some microorganisms known as facultative anaerobesare 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 andalkanes via oxygenases as well as dissimilation and assimilation of fatty acids. Theseenzymes oxidize organic substrates by direct incorporation of one or two oxygen atoms.Aromatic compounds or alkanes are catalyzed by such oxygenases and providePsetdomonas with a source of carbon and energy (Clarke and Ornston, 1975b).1. Energy ProductionA. Aerobic Respiration and MetabolismP. fluorescens is an obligate aerobe. These microorganisms obtain most of theirenergy through aerobic respiration and oxygen serves as a terminal electron acceptor.Pseudomonads generate ATP by two biochemical mechanisms known as substrate levelphosphorylation and electron transport (Clarke and Ornston, 1975a). ATP is generatedby transporting electrons through a chain of carrier molecules which undergo reversible11oxidation and reduction. The resulting gradient of protons and electrical charge across themembrane known as the protonmotive force (pmt) provides energy for motility, operationof 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 showvariation among species and environments. According to Stanier et al. (1986) bacteria ofthe same strain show different cytochrome compositions when grown aerobically andanaerobically.Pseudomonas catabolize sugars to yield pyruvic acid via Entner-Doudoroff andpentose phosphate pathways but lack the Embden-Meyerhof pathway (Clarke andOrnston, 1975b). Pyruvate is either oxidatively decarboxylated to yield acetyl-CoA toprovide precursor metabolites for biosynthesis or completely oxidized via the tricarboxylicacid cycle (TCA) to generate ATP when coupled to ETC. Aromatic compounds andaliphatic acids are oxidized via the p-ketoadipate pathway to yield acetyl-CoA which fuelTCA. The tricarboxylic acid cycle manifests its central position through cataboliterepression by its intermediates on other catabolic enzymes (Clarke and Ornston, 1975a;Krieg and Holt, 1984).B. Anaerobic RespirationIn the absence of oxygen or under low oxygen tension, alternate electron acceptorssuch as nitrate are utilized by Pseudomonas (Krieg and Holt, 1984). The samebiochemical pathways are used as aerobic respiration differing only in the terminalelectron acceptor of ETC. Reduction of nitrate (NO3-) yields nitrite (NO2-) then12dinitrogen oxide (N20) and finally nitrogen gas (N2). Nitrate reductase A which isinvolved in nitrate respiration in dissimilatory reactions is suppressed by high 02 levels(Clarke and Ornston, 1975a; Postgate, 1978). Nitrate and nitrite reductase activities wererepressed by 20 % oxygen but when the 02 level was decreased to 6 %, the Bacteriumdenitrificans (P. stutzeri) culture was able to utilize both oxygen and nitritesimultaneously (Kefauver and Allison, 1957).Nitrate can also function as a N source in assimilatory reactions where it is convertedinto NH3 by nitrate reductase B (Clarke and Ornston, 1975a).2. Metabolism Under Decreased Oxygen ConcentrationsRespiratory metabolism of Pseudomonas can decrease dissolved oxygen tension inthe medium very quickly when population numbers are high and its metabolic rate is at itsmaximum during exponential growth (Oblinger and Kraft, 1973). A detectable drop inelectrical potential (Eh) was observed when population numbers were between 105 to 106CFU 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 anaerobicmicroorganisms (Kroll, 1989). Growth rate and biomass of P.fluorescens 378 (biotypeA) 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 ofsubstrates and accumulation of metabolites which reflect the different behavior of cellsunder varying oxygen concentrations (Griffiths and Phillips, 1984; Spohr and Schutz,1990). Utilization of glucose by P. fluorescens was inferior at low (< 20 % of13saturation) oxygen levels but no differences in utilization were shown between medium(25 - 60 %) and high (> 80%) oxygen levels (Spohr and Schutz, 1990). The difference inmetabolism of cells grown in aerated and nonaerated milk was also indicated by changesin concentration of the pools of the intermediary metabolite L-lactate and pyruvate in P.fluorescens (Griffiths and Phillips, 1984). Extracellular enzyme production has beencorrelated 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 toanaerobic state when oxygen is limiting. A decrease in energy charge would favor ATPgenerating catabolic processes and an increase would favour biosynthetic processes toexpend the energy (Harrison, 1976). In Bacillus subtilis, transition-state regulatorscontrol metabolism and energy production when nutrients are depleted to preventexpression of detrimental functions when they are not needed and during suboptimalenvironments, so that alternative pathways are utilized for available nutrients (Strauch andHock, 1993).Growth rate and extracellular enzyme production are regulated in accordance with theenergy status of the cell. There is evidence of an inverse relationship between enzymesynthesis and metabolic state in many microorganisms. Wiersma et al. (1978) suggested14that proteinase was synthesized when growth was limited by low oxygen tension. It wassuggested that growth rate and/or a factor associated with energy production regulatedsynthesis 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 nutrientlimitation (Whooley et al., 1983). This metabolic repression reflected the low energystatus of the cell since available energy, as metabolizable substrates, was limiting. Anincrease in proteinase production by Vibrio was also observed when growth was limitedby low dissolved oxygen tension (Whooley et al., 1983). Aeromonas hydrophila alsoshowed maximal proteinase production under nutritional stress despite poor growth(O'Reilly and Day, 1983). Interestingly enough, regulation in Serratia liquefaciensinvolved dual promoters where distal promoter Px was induced by anaerobiosis andproximal promoter PA was growth-phase regulated (Givskov and Molin, 1992).The relationship between energy and enzyme levels could then be used to explain theincrease in enzyme production during stationary phase when energy level would be low(McKellar, 1989). When growth rate and enzyme activity are low, induction ofproteinase synthesis would result in more readily available substrates, from proteinaseactivity, for growth enhancement.There is also a mathematical model for enzyme production : RE = f(environment) p. Xwhere RE is the overall rate of enzyme synthesis, g is specific growth rate, X is thebiomass concentration, and f(environment) is the function describing the influence ofenvironment on "open doors" period, the time for enzyme synthesis, which is15proportional to a certain portion of a cell cycle (Votruba et al., 1986). A slower growthrate would then ensure longer duration in the "open doors" period and result in increasedenzyme production.4. Critical dissolved oxygen tension (CDOT) The effect of dissolved oxygen tension (DOT) on growth and respiration was similarfor aerobes and facultative anaerobes when respiration provided most of the energy(Harrison, 1976; Costilow, 1981). Dissolved oxygen can be considered a nutrient just asany carbon or nitrogen source but unlike other substrates, it is relatively insoluble (< 10mg/L) so it becomes limiting during intense growth (Costilow, 1981). Growth rate andgrowth efficiency are independent of DOT above the critical DOT (CDOT) level, but whendissolved oxygen is below that level, a decrease in maximum growth rate and growthefficiency was reported (Costilow, 1981).Metabolism of aerobes and facultative microorganisms was not affected by changes indissolved oxygen tension between the range of 20-150 mm Hg (Harrison, 1976). Asdissolved oxygen tension (DOT) decreased from the saturation point, respiration activitywas 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 whichdifferent pathways for synthesis of extracellular products were reported (MacLennan etal., 1971). As DOT decreased, respiration rate of facultative anaerobes increased and thendecreased at very low oxygen tension (Harrison, 1976). This oscillation between high16and low respiration rate was important for the microorganism to switch rapidly from a lowrespiration rate at high oxygen tension to a higher respiration rate at a lower oxygentension to maintain energy status in the cell (Harrison, 1976).Extracellular enzyme production by micloorganisms appears to be under cataboliterepression which is induced by appropriate limiting factors such as oxygen or nutrients.There must, however, be sufficient available energy and substrates to initiate and maintainits synthesis. McKellar and Cholette (1984) reported that maximal proteinase productionoccurred under excess carbon, nitrogen and orthophosphate but when any one of thesewere limiting, proteinase production was lower.C. Factors Affecting Enzyme Production1. TemperatureOptimum growth and extracellular enzyme synthesis did not occur at the sametemperature and optimum enzyme synthesis temperature was usually below optimumgrowth temperature (McKellar, 1989). Fairbairn and Law (1986) hypothesized thatpsychrotrophs, to maintain their growth rate at the expense of cell yield, produced higherenzyme levels at lower temperatures to compensate for the lower enzymatic activity, aswell as decreased cellular processes. The effects of temperature on enzyme synthesis bypsychrotrophs 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 butspecific activity per unit growth was much higher at the lower temperature. On thecontrary, Roussis et al. (1988) reported higher proteinase activity as a function of growth17at 23 °C than at 4 °C for P. fluorescens GR83. P. fluorescens A32 showed maximumproteolytic activity at 17.5 °C which was not its optimal growth temperature (Gugi etal.,1991).Lipase activity of P. fluorescens in whole milk was higher at 4 °C than at 25 °Cwhich may not be due to an increase in enzyme production (Bucky et al., 1986). Theauthors 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 wassuppressed at 2 °C and 6 °C but not at 10 °C and 21 °C.2. IonsThere are reports of many ions involved in the production of extracellular enzymes byP. fluorescens. Calcium and zinc are essential for proteinase activity and synthesis(McKellar and Cholette, 1986b). Phosphate was also required for maximum proteinasesynthesis but was detrimental at an excessive level (McKellar and Cholette, 1985). Highiron concentrations were reported to repress lipase production (Ishihara et al., 1989) so itwas postulated that there was a direct role of iron on the regulation of enzyme productionby psychrotrophs (McKellar et al., 1987). It was also reported that limiting ironconditions at the end of exponential growth phase inhibited cytochrome synthesis andthereby enhanced proteinase synthesis by Vibrio (Wiersma et al., 1978). A decrease incytochrome synthesis would have affected the energy generating status of microorganismsand thus would have had the same effect as limiting oxygen on growth (Wiersma et al.,1978).183. Oxygen There are conflicting reports on the role of oxygen in extracellular enzyme productionby microorganisms. Some reports show enhancing effects while others show deleteriouseffects of oxygen but there appears to be a causal relationship between oxygen tension andenzyme production.Studies of the influence of oxygen on subsequent extracellular enzyme synthesis havebeen made using different controls of oxygen tension (McKellar,1989). Someinvestigators have compared agitated and static cultures (Hare et al., 1981; Fairbaim andLaw, 1987) and others have employed nitrogen flushing (Murray et al., 1983) andoverlays (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 aVibrio culture was aerated to increase dissolved oxygen tension, proteinase productionwas subsequently inhibited (Wiersma et al., 1978). A drop in oxygen tension wasobserved by Rowe and Gilmour (1982) just prior to proteinase and lipase production.Rowe and Gilmour (1982) forcibly decreased oxygen tension of a P. fluorescensculture after 3 days from 85 to 12 % within a 6 hour period. This resulted in an earlieronset 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 aerationtreatments to prevent the normal rapid drop in oxygen tension. An increase in lipolysiswas, however, reported which may have been the result of the decrease in proteinase19activity on co-existing lipases. Lu and Liska (1969), however, reported inhibition oflipase production by P. fragi when the culture was aerated.Another way of examining the effect of oxygen is to observe its effect during non-aerated or static culture conditions. Lipase activity was higher in statically grown than inagitated P. fluorescens cultures (Roussis et al., 1988). Proteinase activity per unit dryweight was also higher in static than in agitated cultures although the growth rate wasslower (Fairbairn and Law, 1987). A similar observation about extracellular proteinaseactivity from a non-aerated P. aeruginosa culture was made, even though biomass waslower (Whooley et al., 1983). Limiting oxygen conditions may decrease growth rate butincrease extracellular proteinase production.B. Oxygen Enhances Enzyme ProductionThere are reports that aeration causes more rapid and enhanced proteinase synthesis byP. lachrymans (Keen and Williams, 1967) and by Micrococcus GF (Garcia de Fernandoand 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 therepressive effects of ammonia which otherwise would not have been localized duringagitation (Whitaker et al., 1965). Static conditions were reported to inhibit proteinaseproduction by Vibrio alginolyticus (Hare et al., 1981) and lipase production by P.fluorescens (Fox and Stepaniak, 1983; Bucky et al., 1986). Likewise, no proteinase20activity was detected in nitrogen-flushed raw milk after 18 days of storage althoughproteolytic 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 psychrotrophsfrom raw milk stored under nitrogen atmosphere were able to produce proteinase on skimmilk agar plates during aerobic but not anaerobic incubation (Murray et al., 1983).Normal spoilage pattern was resumed when the nitrogen-overlay of UHT milk inoculatedwith P.fluorescens was disrupted, resulting in an increase of bacterial population similarto that of the aerobic control. An increase in proteinase activity was also observed butmaximum activity in the milk with the disrupted nitrogen-overlay was only 50 % of theaerobic control (Skura et al., 1986). P. fluorescens NC3 showed low proteinaseproduction 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 lowerenzymatic activity during non-aerated conditions. Hare et al. (1981) reported inhibition ofcollagenase and proteinase production with the removal of oxygen and decrease inexponential 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 usingnitrogen to decrease oxygen from 9-12 ppm to 1-3 ppm at 3 °C caused an 83 % increasein 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 flushed21raw milk (Murray et al., 1983). On the contrary, lower biomass for non-aerated culturesof P. aeruginosa showed higher proteinase activity (Whooley et al., 1983). In support ofthis, Fairbaim and Law (1987) reported that aeration resulted in faster growth rate andearlier proteinase production but static culture showed higher proteinase production perunit dry weight although growth rate was lower.4. Regulation of Extracellular Enzyme SynthesisA. ProteinaseProteinase production by P. fluorescens was reported to be inducible (McKellar,1982) and regulated (Vilu et al., 1981). Proteinase production occurred de novo wheretranslation and secretion of extracellular proteinase by P. fluorescens were almostsimultaneous (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 rateof enzyme production. This model assumes that microorganisms produce low basallevels of extracellular enzyme in the absence of the inducer but as the low molecularweight inducer enters the cell, proteinase synthesis is induced. Proteinase ensures asupply of carbon for energy so it is repressed by simple carbon sources, pyruvate andgrowth limiting substrates (McKellar, 1982).221. Carbon Catabolite Repression The delay in proteinase production until late log-early stationary phase is probably theresult of repression of proteinase synthesis by the presence of easily metabolized carbonsources, a process known as catabolite repression (McKellar, 1982). Control systems ntonly ensure the use of simple carbon sources first but also ensure that growth has priorityover less important processes such as differentiation and secondary metabolism (Viningand Chatterjee, 1982). Using continuous cultures, an increase in dilution rate from verylow values was shown to increase proteinase production but was inhibitory at very highdilution rates due to catabolite repression (Wiersma and Harder, 1978).Easily metabolized carbon sources such as tricarboxylic acid cycle intermediatesrepress proteinase synthesis (Hare et al., 1981; McKellar, 1989). Citrate, however,which is the second most available low molecular weight carbon source next to lactose inmilk, 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 handinduced proteinase synthesis (McKellar, 1982; Griffith and Phillips, 1984). It was alsoreported that the lack of a glycolytic (Embden Meyerhof) pathway in Pseudomonas madeglucose a poor source of carbon and a poor repressor of proteinase production (Palleroni,1975; Griffiths and Phillips. 1984). Inducers of proteinase synthesis can be generalizedas proteins, peptides (< 5000 MW), and amino acids such as glutamic acid, glutamine andasparagine (McKellar, 1982; Fairbaim and Law, 1987). Repressors can be generalized assugars, organic acids, and amino acids such as cysteine (Himelbloom and Hassan, 1986).232. Nitrogen Catabolite Repression Since protein is a source of both carbon and nitrogen for Pseudomonas, regulation ofproteinase synthesis may be associated with nitrogen metabolism and ammoniaassimilation (Fairbairn and Law, 1986). During ammonia limitation, production ofcatabolic enzymes for nitrogen and carbon utilization as well as nitrate reduction wereinduced (Grafe, 1982). It was reported that glutamic acid and glutamine were the bestamino acid inducers when used as sole nitrogen sources (McKellar, 1982). Fairbairn andLaw (1986) suggested that it may be valuable for proteinases to be under the control ofnitrogen 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 Joseet al., 1987). It was reported that uptake of these substrates from the externalenvironment regulates metabolism of microorganisms (Clarke and Ornston, 1975a).Ammonium ions have been reported to either repress, by feedback inhibition, or supportproteinase 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 byammonia, transport systems of amino acids, and several anabolic and catabolic reactionsare mediated by glutamine synthetase in enteric microorganisms (Magasanik andNeidhardt, 1987). Ammonia provides a nitrogen source and is assimilated as glutamatefor the synthesis of amino acids. Glutamine is required for the synthesis of nucleotides,24amino sugars, and some amino acids (Reitner and Magasanik, 1987).B. LipaseLipase regulation is usually grouped together with proteinase regulation. Lipase is notessential for growth so it may only be produced in stress-free growth conditions (Fox andStepaniak, 1983). There is some evidence that lipase production by P. fluorescens maybe 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 milkalthough the authors suggested that milk fat interfered with the lipase activity detectionmethods with the whole milk (Bucky et al., 1986; Griffiths, 1989). The addition of oliveoil delayed the growth rate of P. fluorescens but its final population and lipase activitywere unchanged (Andersson, 1980b). Inhibition of lipase production by P. fluorescenswas observed when free fatty acids (FFA) were added to milk which suggested that FFAlevel may repress lipase production through feedback inhibition (Bucky et al., 1986).C. Direct Regulation by OxygenThere is no direct evidence that proteinase or lipase synthesis by P. fluorescens isregulated by oxygen concentration. Genetic evidence has shown that certain functions insome microorganisms are oxygen regulated. In facultatively photosynthetic Rhodobactercapsulatus, pigment protein complexes are under oxygen concentration and light intensitycontrol (Wellington et al., 1991). This microorganism can respire aerobically using theelectron transport chain but under anaerobic or decreased oxygen concentrations it canswitch 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 that25extracellular phospholipase was regulated by two promoters, PA under growth phaseregulation and Px under anaerobic induction (Givskov and Molin, 1992). InSaccharomyces cerevisiae, certain oxygen dependent functions such as alternatecytochrome subunits, oxidases and desaturases in heme and sterol biosynthesis wereinduced 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 toearlier off-flavour development than the non-deoxygenated milk samples despite relativelylow total psychrotroph count (Schroder, 1982). One main reason for this observationwas the higher coliform count in deoxygenated milk. Since coliforms are facultative,these microorganisms were not as sensitive to oxygen content as aerobic microorganismsand proliferated. These decreased oxygen tension environments selected forpsychrotrophic, facultative anaerobes, principally coliforms (Schroder, 1982) and lacticacid bacteria (Murray et al., 1983). Another concern would be the potential growth offacultative pathogens such as Salmonella, Listeria, and Bacillus.A decrease in extracellular proteinase activity of P. fluorescens may inadvertentlyresult in accumulation of intracellular or cell-associated proteinase or peptidases. Keoghand Pettingill (1984) suggested that intracellular proteinases of Pseudomonas may be26significant when population numbers are high. Other studies showed negligible concernfor intracellular proteolytic activity (Kohlmann et al., 1991; Shamsuzzaman and McKellar,1987). There was, however, bitterness associated with the growth of proteinase deficientP. 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 duringstationary growth phase (Torrie et al., 1983). Another concern of decreasing proteinasesynthesis is the possible enhancement of lipase activity in the absence of proteinases.27MATERIALS AND METHODS A. Controlled Oxygen AtmospheresThe 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) werecontrolled with the use of flowmeters (Series 50 M for N2 and Series 150 for 02, LindeUnion 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 airstones into a flask containing sterilized 0.5 % (w/v) NaCl solution with the same ionicstrength as milk (Murray et al., 1983). The mixed gas flowing at — 300 mL min-1continuously overlay 2 L of UHT-sterilized milk (2 % m.f.) which was stirred withmagnetic stir bars at 300 rpm in hypochlorite-sterilized Nalgene carboys (4 L capacity,Sybron/Nalge, Rochester, NY). The oxygen content of the controlled atmosphere wasmeasured just prior to the entry into carboys and monitored using a Shimadzu GasChromatograph-14A (Shimadzu Corporation, Kyoto, Japan) by taking duplicate ortriplicate air samples. The gas chromatograph was set up with 80/100 molecular sieve 5astainless 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 wasused. Data was collected using a Chromatopac CR501 integrator (Shimadzu, Kyoto,Japan).The atmospheric oxygen concentration is reported as a percentage (%). The dissolved02 content was measured in parts per million (ppm) using a portable Clark electrodeFigure 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 in0.5 % (w/v) NaC1 solution. Atmospheric oxygen and carbon dioxideconcentrations were determined using gas chromatography. Each trial consisted offour of these setups under four controlled oxygen atmospheres.2902 probe (YSI, Yellow Springs, OH). The 02 meter was calibrated using water saturatedair at 4 °C as representing 100 % 02 saturation. Since more accurate measurement ofdissolved 02 is made when liquid is stirred (Kotters, personal communications 1992) themilk samples were stirred with a small magnetic bar at slow rpm and values measuredwithin 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 milkin 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 anexpiry shelf-life of 3 months.Tetrapak milk cartons were wiped with 70 % ethanol solution and opened asepticallywith ethanol flamed scissors. Two litres of milk were poured aseptically into carboyswhich had previously been sterilized with a 200 ppm hypochlorite solution andthoroughly rinsed with sterile distilled water. A magnetic stir bar was included in thecarboy before sterilization. Milk was then stirred under appropriate flowing controlledoxygen atmospheres and equilibrated before the inoculation of P. fluorescens.C. Culture ConditionsPseudomonas fluorescens biotype A ATCC 17397 (American Type CultureCollection, Rockville, MD) was maintained on trypticase soy agar (TSA) slants at 4 °Cand transferred monthly. Trypticase soy broth (25 mL TSB in 250 mL flask) wasinoculated with P. fluorescens and incLbated at 21 °C for 18 hours in a shaking water30bath at 160 rpm (Lab-Line, Melrose Park, IL) and then subcultured. After 18 hours ofincubation, 10 mL of the culture was centrifuged at 12 100 x g for 20 minutes at 4 °C inan RC2-B Sorvall Superspeed centrifuge (Dupont Sorvall, Newtown, CT). Thesupernatant was removed, the pellet resuspended in 10 mL 0.1 % peptone and thecentrifugation process was repeated. Optical density was measured at 660 nm to estimatethe population of the resuspended pellet. Two litres of DairyMaid 2 % UHT milk(Dairyworld Foods, Burnaby, BC) was inoculated with P. fluorescens to an initialpopulation of approximately 104 CFU mL-1. After 30 minutes of stirring, samples werecollected for plating of P. fluorescens to provide day 0 cell numbers. Samples werecollected for plating and tested every 2 days for P. fluorescens cell number, proteinaseand lipase activities. Milk samples of 30 mL were frozen at -20 °C or below and analyzedon a later date for proteolysis and lipolysis.D. Antibiotic Testing of UHT MilkUninoculated 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 tetracyclinesvia the Charm II methodology.E. EnumerationGrowth 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 at21 °C for 24-36 hours.31F. Proteinase Activity AssayProteinase activity was assayed as described by Kohlmann et al. (1991) with somemodifications. The reaction mixture consisted of 1.0 mL of 0.1 M Tris-HC1 pH 7.5buffer 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 filteredthrough Whatman #1 filter paper to remove any undissolved lumps. Thimerosal wasadded to a final concentration of 0.05 % in 1.0 mL of milk to inhibit extracellular enzymeproduction. Milk samples were then microcentrifuged (Eppendorf Centrifuge 5415 C,Brinkmann Instruments, Rexdale, ON) at 2040 x g for 10 minutes at room temperature toremove microorganisms as well as milkfat. The cell free supernatant was removed assource of proteinase and chilled in an icebath until ready for use. Fifty Ill. of the cell freesupernatant was used for Trial I but was later increased to 200 111, for Trials II and III inan attempt to increase sensitivity of the assay. The contents of the reaction mixture werevortexed and incubated at 40 °C for 1 hour with shaking at 160 rpm. Reactions werestopped with the addition of 2.0 mL of 24 % (w/v) trichloroacetic acid (TCA) andvortexed immediately and placed in an icebath for 15 min. Sample blanks (addition ofTCA to azocasein prior to the addition of enzyme) and substrate blanks (azocasein withoutthe 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 (BeckmanInstruments Inc., Palo Alto, CA) to remove the precipitate. The absorbance of thesupernatant was measured at 366 nm with a UV-160 Shimadzu spectrophotometer(Shimadzu Corporation, Kyoto, Japan). Proteinase activity was reported after subtraction32of controls. All reactions were performed in triplicate. One azocasein unit of proteinaseactivity was defined as an increase in absorbance of 0.01 at 366 nm per mL of milk perhour. Proteinase activity was also checked using 1 % skim milk agar plates containingfilter discs soaked in supernatant followed by incubation at 35 °C for 24 hours. Theplates were flooded with 10 % TCA to check for zones of clearing as an indication ofproteolysis.Skim milk agar (S MA) plates were also used to check whether the absence ofproteinase activity during the assay was a result of loss of ability by P. fluorescens tosynthesize proteinase enzymes during storage due to mutation. P. fluorescens from milksamples were streaked on SMA plates and incubated at 21 °C for 24-36 hours to evaluategrowth and proteolytic activity.G. Proteolysis Proteolysis was determined using absorbance at 280 nm to measure aromatic aminoacids and peptides soluble in 12 % TCA. To each tube containing 2.0 mL of distilledwater and 2.0 mL of 24 % TCA, 100 !IL of each milk sample was added and vortexedand put in an ice bath for 15 min. The tubes were centrifuged at 2910 x g for 15 min andthe supernatant absorbance measured at 280 nm. A tyrosine standard curve was used toconvert absorbance values to tyrosine equivalents. A stock solution of 4 mM tyrosinewas made by dissolving in 10.0 mL 1 N NaOH and then making the volume up to 200mL with distilled water in a volumetric flask. Tyrosine standard solutions were made upin a final volume of 4.1 mL. Degree of proteolysis was expressed as ilmole 12 % TCA33soluble tyrosine equivalents at 280 nm mL-1 milk.H. Lipase Activity Assay Chromogenic phenolic ester fi-naphthyl caprylate was used as a substrate to measurelipase activity in uncentrifuged milk sample. Thimerosal was added to a finalconcentration 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 reactionmixtures. 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-aminoethane-sulfonic acid (BES, Sigma, St. Louis, MO) pH 7.2 buffer, prewarmed to 40 °C. InTrials 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 500mM 13-naphthy1 caprylate (crystalline, Sigma, St. Louis, MO) in dimethylsulfoxide(DMSO) (Fisher Certified, Fisher, Vancouver, BC) was added and vortexed. The tubeswere incubated at 40 °C for 1 hour shaking at 160 rpm to ensure proper mixing. Freshlyprepared 500 mM Fast Blue BB salts (practical grade, Sigma, St. Louis, MO) dissolvedin DMSO was added in 20 pL aliquots to the reaction mixtures. The tubes were incubatedat 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 acetateto ethanol (2.71 mL) was used to clarify the reaction mixtures for Trial I but 60:40 ethylacetate to ethanol (2.51 mL) was used for Trials II and HI since the volume of BES bufferhad been adjusted. Sample (no substrate) and substrate (no enzyme source) blanks were34included. All reactions were performed in quadruplicate. Lipase activity was reportedafter subtraction of controls. Absorbance was measured at 540 nm.A stock solution of 10 mM [3-naphthol (Sigma, St. Louis, MO) was prepared bydissolving in DMSO. Serial dilutions of 13-naphthol were made and added to reactionmixtures in the absence of the enzyme source and incubated along with the otherreactions. A standard curve of concentration of 13-naphthol vs Abs540 was plotted inorder to convert absorbance values into 1.tmoles of 13-naphthol. One 13-naphthol unit wasdefined as one gmole 13-naphthol released mL4 milk h-1.I. LipolysisThe degree of lipolysis of the milk fat was measured by using the titration method withdilute methanolic KOH (Deeth et al., 1975). Five mL of extraction mixture consisting ofisopropanol, petroleum ether and 4 N H2SO4 (40:10:1 v/v) were vortexed with 1.5 mL ofmilk in a 25 mL graduated test tube. Three mL of petroleum ether was added and thereaction mixture was washed with 2.0 mL distilled water. The top organic layer wastitrated, 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 atmosphericCO2 which has been reported to neutralize KOH (IDF, 1991). The KOH concentrationwas determined after standardizing it against 0.01 N HC1. Transition from green to bluerepresented an endpoint of pH 8.7. A control blank without the addition of milk was usedto obtain a background titration volume which was subtracted from samples. Free fattyacids (FFA) were converted into acid degree values (ADV) (Deeth et al., 1975; IDF,1991).35FFA (gequivalent mL -1 milk) = T * N x 103P * Vwhere 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 layerV = volume of milk (mL)ADV (mmoles of FFA per 100 g fat) = (FFA- 0.07) / 0.62 (calculations from Deeth etal., 1975)J. Investigation of High ADVa. Induced Lipolysis by Mechanical AgitationTwo litre volumes of UHT-sterilized milk in 4 L carboys were stored at 4 °C for 7days under aerobic atmospheres, one stirred at 350 rpm and the other unstirred, todetermine the effect of mechanical agitation on high ADV. Milk samples were collectedand 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 ofcarbohydrates unique to biotype A. An increase in acid production may have contributedto the high acid degree values. Nitrate reduction was also checked to confirm a change inmetabolism of P. fluorescens although it may not directly be related to the high aciddegree values.361. Carbohydrate Utilization Hugh Leifson media (Krieg and Holt, 1984) were supplemented with the followingsugars (adonitol, L-arabinose, D-trehalose, D-xylose, D-glucose, and lactose) to checkfor mutations in catabolism during storage and culturing of the P. fluorescens stockstrain. The strain was compared to a new strain of P. fluorescens biotype A purchasedfrom ATCC then subcultured three times in TSB. All sugars were sterilized byautoclaving with the exception of xylose, lactose and D-glucose which were sterilizedthrough 0.45 pm hydrophilic cellulose acetate membrane filter (Pro-X filter unit, DiamedLab Supplies, Mississauga, ON) and added to a final concentration of 0.2 %. HughLeifson 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 24hours at 25 °C. To 3.0 mL of the culture, 0.2 mL of Reagent A (sulfanilic acid) andReagent B (a-naphthol) were added. A red colour indicated the presence of nitritereduced from nitrate.The method of Stanier et al. (1966) was used to determine the denitrifyingcharacteristics. P. fluorescens was grown statically for 24 hours at 25 °C in 5 mL ofBacto nitrate broth supplemented with a final concentration of 1 % glycerol. A loopfulwas transferred to 10 mL of fresh nitrate broth then topped with 3 mL of 1 % molten agarand cooled. The tubes were incubated statically at 25 °C up to 5 days to monitor growthand gas production.37c. Carbon Dioxide InterferenceThe set-up was modified to eliminate the effects of atmospheric CO2 as shown inFigure 2. Either a vacuum or a gentle flow of nitrogen gas (USP grade, Pacific MedigasLtd., Vancouver, BC) was effective in removing the atmospheric CO2 in the titrationvessel. The tip of the volumetric pipette was extended down as far as possible over thefree 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 % atmospheric02 were obtained using SYSTAT (1989). Correlation coefficient is the measure of thestrength of the relationship between two variables x and y (Ott, 1988).380.01 N KOHNitrogen gasFigure 2. Experimental setup for titration of free fatty acids. The ether extractscontaining free fatty acids and indicator were added to the titration flask and flushedwith a gentle flow of nitrogen gas. The rubber stopper was replaced to maintain thecarbon dioxide-free atmosphere during titration.39RESULTS AND DISCUSSIONData 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. Atleast 24 to 36 hours were required to equilibrate milk with the atmosphere in each carboybefore inoculation of P. fluorescens. In the literature, the time required for equilibriumwas reported to be dependent on the atmosphere, flow rate, speed of agitation and themedium 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-1required 48-72 hours (Oblinger and Kraft, 1973), and only 10 minutes were required forN2 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-1to reflect the population normally encountered in raw milk (Cousin, 1982). Initialpopulations 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) ofpsychrotrophs 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 SanJose et al. (1987).The use of UHT-sterilized milk provided a model system where only the activities ofP. fluorescens were observed without interference of competing microorganisms which40Table 1. Atmospheric oxygen levels tested% Oxygen in Atmosphere aSAMPLES^1^2^3^4TRIALI^1.2 + 0.1 b^5.3 + 0.2^10.2 + 0.6^20.9 + 1.0II 13.4 + 0.2^16.9 + 0.2^9.6 + 0.9^20.7 + 0.8III^13.4 + 0.4^4.8 + 0.2^10.3 + 0.7^20.2 + 0.6 ca^Nitrogen gas as balanceb^The mean of triplicate values + standard deviationc^Medical air was used as aerobic control instead of mixing 02 and N2Table 2. Initial population of P.fluorescensLOG 10 CFU mL-1SAMPLES^1^2^3^4TRIALI^4.18 + 0.09 a 4.15 + 0.07^4.13 + 0.06^4.18 + 0.05II^4.16 + 0.00^4.21 + 0.00^4.17 + 0.07^4.16 + 0.03III^4.39 + 0.02^4.38 + 0.02^4.39 + 0.03^4.40 + 0.02a^The mean of triplicate values + standard deviation41would certainly have existed in a raw milk system. UHT-sterilized milk was reported tobe 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 IThe atmospheres tested in the preliminary run were repeated in Trial I and monitoredusing 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 at20.9 % oxygen showed a longer lag phase than those at 1.2 and 5.3 % but by day 8 itexceeded all the other populations as it entered stationary phase. This observation wasdifferent from that reported by Skura et al. (1986). In their study, P. fluorescens underaerobic conditions showed virtually no lag phase and a faster growth rate than populationsunder nitrogen atmosphere. The absence of a lag phase by P. fluorescens in UHT milkwas also reported by Spohr and Schutz (1990) and by Rowe and Gilmour (1983) evenwhen a 25 °C culture was inoculated at 7 °C. The population under 10.2 % oxygenbehaved much like that of aerobic control during storage. It reached very high populationnumbers exceeding 108 CFU mL-1 on day 8 as it also entered stationary phase. Itspopulation density was slightly lower than the aerobic control throughout 14 days ofstorage. The P . fluorescens populations under 1.2 % and 5.3 % oxygen surprisinglyAtmospheric Oxygen1.2%—A— 5.3 %—11— 10.2%—0— 20.9% 1042Figure 3. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlledoxygen atmospheres at 4 °C. Each point represents the mean of triplicate bacterial counts+ standard deviation.0^2^4^6^8^10^12^14^16Storage Time (Days)43showed the fastest growth rate up to day 4 which was also observed in our preliminarywork. 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 washighly unexpected that an aerobic microorganism would show a faster growth rate underlow 02 rather than under aerobic atmosphere. In the literature only one study (King andMabbit, 1982) with P. fluorescens under nitrogen reported a faster growth rate thanuntreated bulk milk during the first 2 days of incubation. In that study, the aerobicpopulation eventually exceeded the population number of that under nitrogen atmosphereon day 3 (King and Mabbit, 1982).a. Alternate Electron AcceptorsP. 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 underdecreased atmospheric oxygen conditions during initial storage. The fast growth rateunder 1.2 and 5.3 % oxygen was typical of microaerophilic microorganisms which havebeen 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 manyspecies of Pseudomonas can use nitrate as an alternate electron acceptor. Nitrate isreduced to nitrite and completely to nitrogen gas by denitrifying bacteria. According toBergey's Manual of Systematic Bacteriology (Krieg and Holt., 1984), P. fluorescensb'otype B, C and D are reported to reduce nitrate but not biotype A. The ability of P.44fluorescens biotype A to grow under severely diminished oxygen atmospheres reflectstheir 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 nitratereduction 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 maybe due to mutation of the strain during storage in the lab.b. Growth on Solid MediumP. fluorescens under decreased oxygen atmospheres showed two different colonysizes when enumerated aerobically on TSA plates. The smaller sized colonies which wereonly 25 % the size of the larger sized colonies were more prevalent from samples underlow oxygen atmospheres. P. fluorescens under low oxygen were probably morestressed and nutritionally fastidious on solid medium.2. Oxygen a. Atmospheric Oxygen The oxygen level in the atmospheres were monitored throughout 14 days of storageand only minor fluctuations were observed (Fig. 4).b. Dissolved OxygenDespite a constant supply of oxygen and agitation, there was a decrease in dissolvedoxygen tension in milk (Fig. 5). The levels of dissolved oxygen were inversely related toexponential growth since the demand for oxygen increased as population numbersincreased. The initial decrease in oxygen tension occurred when population numbers45Table 3. Metabolism of P.fluorescens biotype A ATCC 17397 inHugh Leifson Medium aMetabolic ResponseOld P. fluorescens 0^New P. fluorescensSugars^Growth^Acid^Growth^Acidadonitol (+ 8 )^++ Y^ ++^-L-arabinose (-) ++ +^++ +D-glucose (+)^++^+ ++^+lactose (-) _D-trehalose (+)^++ ++^-D-xylose (+) ++^+^++ +Denitrification (- C)^++ +Reduction ofNO3 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 173975 "expected" results for growth or acid production or both according to Bergey'sManual of Systematic Bacteriology, (Krieg and Holt, 1984)£ growth and gas production under denitrifying conditionsy - no growth or no acid production+ minimal growth or low acid production+ growth or acid production++ heavy growth0^1^2^4^6^8^10^12^14250201510546Figure 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 themean of triplicate gas samples.Atmospheric Oxygen• 1.2%131 5.3%10.2%121 20.9%Storage Time (Days)Atmospheric Oxygen—*-- 1.2%—6— .3%—0— 10.2%—{a— 20.9%1210--47Figure 5. Dissolved oxygen concentration in UHT-sterilized milk (2 % m.f.) inoculatedwith P .fluorescens during storage under controlled oxygen atmospheres at 4°C.0^2^4^6^8^10^12^14^16Storage Time (Days)48reached 105 and 106 CFU mL-1. Other studies have shown decreases in 02 tension atpopulation 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 % oxygenand by day 8 under 10.2 and 20.9 % oxygen. The drop in oxygen tension would beprimarily due to the demand for oxygen during exponential growth when metabolic rate ismaximal (Oblinger and Kraft, 1973).The dissolved oxygen measurements indicated that utilization of oxygen by thesemicroorganisms exceeded the rate at which atmospheric oxygen dissolved in the milk.The dissolved oxygen measurements do not represent the rate at which oxygen isconsumed by these microorganisms. The degree of oxygen limitation in the aerobiccontrol would not have been as severe as in milk stored under decreased 02 atmospheresalthough less than 1.0 ppm dissolved oxygen was measured. There would have been ahigher efficiency of oxygen transfer when the partial pressure of oxygen in the gas phasewas high and the dissolved oxygen tension was low (MacLennan et al., 1971). Thus, therate of oxygen transfer would have been higher and more oxygen would have beenavailable to microorganisms in the aerobic control than in low oxygen atmospheres despitethe very low tensions measured by the oxygen probe.c. Oxygen and Energy ProductionGrowth and metabolism of P. fluorescens would be affected by the decreased 02levels at 1.2, 5.3 and 10.2 % which as a result might alter the microorganisms ability to49produce extracellular enzymes. Oxygen is primarily responsible for the positive redoxpotential (Eh) which would determine cellular processes (Kroll, 1989). For obligateaerobes, where respiration provides most of the energy, dissolved oxygen tension wasreported to have a similar effect on growth and respiration (Harrison, 1976; Costilow,1981). It was reported that when the dissolved oxygen tension exceeded a criticaldissolved oxygen tension (CDOT) level, growth rate and growth efficiency wereindependent of oxygen concentration but when the level fell below CDOT, both growthrate and efficiency decreased (Costilow, 1981). Likewise, Persson et al. (1990) reportedthat aeration and the rate of oxygen transfer affected growth rate and biomass of P.fluorescens 378 biotype A.3. Proteinase ActivityProteinase production by populations under decreased oxygen atmospheres werelower than under aerobic atmosphere throughout 14 days of storage (Fig. 6). Thedecrease in proteinase activity in samples under low atmospheric oxygen was alsoobserved in preliminary work. The absence or decrease in proteinase activity was not dueto mutation in synthesis since large zones of proteolysis were observed on 1 % skim milkagar plates inoculated with milk samples when aerobically incubated.Other researchers have reported similar effects of deoxygenation and its influence onextracellular proteinase synthesis. Murray et al. (1983) showed that removal of oxygenin raw milk with nitrogen flushing resulted in no detectable proteinase activity throughout18 days of storage. It was also observed that when the nitrogen-flushed milk was1000^2^4^6^8^10^12^14^168020604050Figure 6. Proteinase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) undercontrolled oxygen atmospheres at 4 °C using azocasein as a substrate. Each pointrepresents the mean of triplicate proteinase activity values + standard deviation.Atmospheric Oxygen—•"--- 1.2%—aer— 5.3%—.— 10.2%—0— 20.9 %Storage Time (Days)51incubated anaerobically on skim milk agar plates, no proteinase activity was detectedwhereas aerobic incubation promoted its synthesis. Skura et al. (1986) showed thatremoval of oxygen with nitrogen inhibited proteinase production by P. fluorescens.Disruption of the nitrogen overlay by introducing aerobic conditions restored the ability ofP. fluorescens to grow and produce proteinase.a. Carbon and Nitrogen Catabolite RepressionProteinase activity was first detected on day 10 in the aerobically grown culture andincreased (Fig. 6). Proteinase steadily accumulated during storage in other studies (Keenand Williams, 1967; Hare et al., 1981; Stead, 1987). The reason for the delay in enzymeproduction until day 10 might have been due to catabolite repression. Enzymes arethought 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 acidspresent 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 aminoacids for biosynthesis.b. Proteinase Synthesis and Growth PhaseIncrease in proteinase activity of the aerobic control started between days 8 and 10during early stationary phase. Other studies also reported proteinase production duringearly stationary growth phase (McKellar, 1982; Rowe and Gilmour, 1982; Murray et al.1983; Stead, 1987; Griffiths, 1989; Kohlmann et al., 1991). Proteinase activity increasedbetween day 8 and 14 but greater increase was observed between days 8 and 10.52c. Factors Influencing Proteinase Productioni. OxygenProteinase is synthesized during stationary phase when growth is limited by lowoxygen tension (Rowe and Gilmour, 1982; Wiersma et al., 1978). There was evidenceof 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 indirect 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 processesand result in decreased proteinase production. P. fluorescens at < 20 % of saturation 02was shown to utilize glucose and L-lactate at lower rates and its malate dehydrogenaseactivity was lower (Spohr and Schutz, 1990). If oxygen is limiting, then lower metabolicactivity would result in fewer metabolite precursors necessary for synthesis ofextracellular enzymes.ii. Population DensityThere may be other factors besides the decrease in the initial dissolved 02 tension inmilk which may have affected proteinase production. One factor may be populationdensity. 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 producingproteinases. The population number which is often associated with enzymatic activity is108 CFU mL4 during early stationary growth phase (Rowe and Gilmour, 1982, Spohr53and 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 beenenough 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 studyalthough P. fluorescens cell numbers exceeded 108 CFU mL-1 . There is a consensusamong researchers that a lack of correlation exists between bacterial numbers and the levelof enzyme activity (Rowe and Gilmour, 1986; Skura et al., 1986; Skura, 1989).d. Proteinase Activity and Inhibition of Cell LysisThe 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 aconsequence of the lower proteinase activity. Proteinases provide necessary smallmolecular weight products when nutrients are depleted (Rowe and Gilmour, 1982) andare beneficial for growth. It was reported that Bacillus subtilis deficient in proteinaseproduction tended to lyse more readily at the end of exponential growth than a proteinaseproficient culture due to the actions of autolytic enzymes which would otherwise havebeen 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, thepopulation numbers near the end of storage on days 12 and 14 were lower than theaerobic 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 the54literature. Unlike carbon dioxide which shows antimicrobial activity (Rowe, 1988),nitrogen is not known to have any direct inhibitory action on cellular components exceptin its role of replacing oxygen in the atmosphere.4. Proteolysis The cumulative effect of proteinase activity in milk throughout 14 days of storage wasmonitored with 12 % trichloracetic acid (TCA) soluble components where absorption ofproteins was maximal at 280 nm. The absorption was mainly due to the presence ofsoluble 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 aslong as nucleic acid content is less than 20 % of the total A280. The release of DNA andRNA materials due to lysis was not measured at A260 and used to correct A280 values inour study.An increase in proteolysis was observed for the aerobic control on day 10 whichcorresponded with the increase in proteinase activity (Fig. 7). There were smalldifferences in proteolysis between 1.2, 5.3 and 10.2 % atmospheric 02 and no dramaticincrease throughout 14 days of storage. A decrease in A280 components may possiblyhave reflected the use of amino acids and peptides in biosynthetic reactions by P.fluorescens.5. Lipase ActivityLipase activity was detected earlier than proteinase activity and as early as day 2 for theaerobic control (Fig. 8). This may have been due to constitutive expression of lipase.1210 -••••■.41:48 -2 -I^•^I^I I^•^•^I^•^i^•0^2^4^6^8^10^12^14^16055Figure 7. Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescensunder controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicateproteolysis values + standard deviation.Atmospheric Oxygen1.2%—tr"--- 5.3 %—4,— 10.2%—0— 20.9%Storage Time (Days)0.60.5 —0.4 —0.1 -1^•^.0^2^4^6^8^10^12^14^160.056Figure 8. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) undercontrolled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicatelipase activity values + standard deviation.Atmospheric Oxygen---*-- 1.2%---tr— 5.3 %—.— 10.2%—0— 20.9 %Storage Time (Days)57There are some reports of constitutive expression of lipase production by P. fluorescenssince triglycerides were not required for synthesis (Fox and Stepaniak, 1983; McKellar,1986). Lipase activity of the aerobic control increased on day 6 and accumulated withtime but decreased on days 10 and 14 which corresponded with high proteinase activity inthe milk. These observations are similar to those made by Stead (1987) who reportedearlier detection of lipase than proteinase and a decrease in lipase activity during prolongedstorage as bacterial and indigenous proteinases degraded the lipases. Rowe and Gilmour(1986) also reported earlier detection of lipase activity than proteinase so only lipase wasmonitored as an indicator of enzyme production.The three milk samples under low oxygen atmospheres showed lipase activity afterday 6. The milk sample under 5.3 % 02 reached its maximum activity of 0.2 units on day10 and then decreased. The decrease in lipase activity may be due to inactivation byproteinases although proteinase activity was detected at a low level. Further synthesis oflipases may have been repressed by free fatty acids (Bucky et al., 1986) or low energylevels which may have been insufficient to sustain the energy-consuming synthesis oflipases. Lipase activity of milk under 10.2 % 02 decreased after day 8 but increased againon day 14 to its maximal level of 0.4 units since no proteinase accumulated throughoutstorage.6. LipolysisThe 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 in58the literature (Appendix Fig. 1). The reason for high titratable free fatty acids (FFA) wasinvestigated.7. Investigation of High ADV There are several possible reasons for the unusually high acid degree values. Theincrease 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 freefatty 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 ofmicrobial lipases which had survived the UHT-treatment. The primary cause of lipolysisduring aeration and excessive agitation is the result of damage to fat globule membraneswhich would make triglycerides more accessible to lipases as well as the release of lipasesfrom their association with milk proteins (Law, 1979). Results from an experiment wheresterile milk was stirred at 350 rpm for 7 days to determine the effect of mechanicalagitation and the possible release of lipases from their association with milk proteins didnot show any dramatic increase in titratable fatty acids (Appendix Fig. 2). The increasesin ADV were not caused by the activity of native nor bacterial lipases which had survivedthe 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 biotypeA between the current laboratory strain used in these trials and a new ATCC biotype A59strain (Table 3). There was a difference between the current and new strain in the abilityto reduce nitrate to nitrite in Bacto nitrate broth. Nitrite was detected in the 24 hour cultureof our lab strain but not in the culture of the new ATCC strain. Both biotype A strainswere however able to dissimilate nitrate and produce detectable nitrogen gas.c. Carbon Dioxide InterferenceIt has been suggested that atmospheric carbon dioxide could neutralize the hydroxidein the titration vessel (IDF, 1991). To eliminate the carbon dioxide interference withKOH, the titration setup was modified (Fig. 2). Either a vacuum or a gentle flow ofnitrogen gas was effective in removing the atmospheric CO2 in the titration vessel.Creating a vacuum was more cumbersome so a gentle N2-flow was preferred. The tip ofthe volumetric pipette was also extended down as far as possible over the free fatty acidextract to minimize the evaporation of methanolic KOH. Stability of the colour at theendpoint of titration was also reported to be improved with the removal of CO2 in theatmosphere (IDF, 1991). Removal of CO2 from the atmosphere resulted in a lower ADVthroughout storage (Appendix, Fig. 3). The acid degree values also did not fluctuate asseverely as in Appendix Figure 2 when CO 2 was not removed from the titrationatmosphere. A larger titration volume of KOH required to neutralize FFA would give theappearance of higher concentration of FFA in the sample which could not be correctedwith titration blanks. Not enough milk samples remained to analyse the extent of lipolysisin Trial I using the modified method.608. Summary of Trial I Atmospheres containing 1.2, 5.3, 10.2, and 20.9 % 02 continuously overlayed 2 L ofmilk inoculated with P. fluorescens at an initial population of 4.13 to 4.18 Logi() CFUmL-1. The populations under 1.2 and 5.3 % 02 showed faster growth rate and higherpopulation density during early storage time.Despite a constant supply of atmospheric oxygen, the dissolved 02 level in milkstarted to decrease when population numbers ranged between 5.0 and 5.5 Logic, CFUmL-1. The dissolved 02 concentration in milk measured < 1.0 ppm when populationsentered stationary phase.Milk samples under 1.2 and 5.3 % 02 atmospheres showed low proteinase activitywhile milk under 10.2 % 02 did not show any proteinase activity throughout 14 days ofstorage. 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 valuesappeared to be due to CO2 interference in the titration vessel.A decrease in the initial dissolved 02 concentration in milk resulted in decreased anddelayed proteinase and lipase synthesis by P.fluorescens.61B. TRIAL IIBased on results of Trial I, oxygen levels between 10 and 20 % were tested to find themaximum 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 itentered stationary phase. Its population density remained higher than other P.fluorescens populations throughout storage. The P. fluorescens population under 9.6and 16.9 % 02 showed a similar rate of growth during the early storage time and reachedstationary 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 stationaryphase on day 6 when the population density was less than 108 CFU mL4 which wasunusually low for stationary growth. By day 16, all the populations exceeded 108 CFUmL-1.a. Low Population Numbers It was highly unexpected that all populations entered stationary phase when populationdensity did not exceed 108 CFU mL-1. Poor growth of cultures especially the aerobiccontrol was not seen in any other work and may have been an anomaly of the inoculum orthe batch of milk used for Trial II. It was possible that the presence of antibiotics62Figure 9. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) under controlledoxygen atmospheres at 4 °C. Each point represents the mean of triplicate bacterial counts+ standard deviation.10. Atmospheric Oxygen—0— 9.6%—A— 13.4%16.9%—4"--—Cl-- 20.7 %I•I^I^I^I^.^I^'^I-.^I^•0^2^4^6^8^10^12 14^16^18Storage Time (Days)63inhibited growth but the results from Dairyworld Foods were negative for the presence of13-lactams, tetracyclines and sulfa drugs.b. Growth Under Lower Atmospheric Oxygen Growth of P. fluorescens was enhanced under 13.4 % atmospheric oxygen since itshowed faster growth rate, earlier transition into stationary growth phase and higherpopulation density than the aerobic control during 16 days of storage. As observed inTrial I with 5.3 % oxygen, P. fluorescens behaved more like a microaerophile than anobligate aerobe. However, decreasing the atmospheric oxygen down to 10 % did notresult in similar growth enhancing effects.c. Oxygen and Nitrate as Electron AcceptorsIt was reported that use of nitrate by aerobes and facultative microorganisms yieldsless energy, reduced growth rates and lower cell populations than the use of oxygenwhich is preferentially used as a terminal electron acceptor (Costilow, 1981; Palleroni,1975). In Bacterium denitrificans (P. stutzeri), nitrite reductase activities were repressedat 20 % atmospheric oxygen. At 6 % oxygen however, this microorganism was able toutilize both oxygen and nitrites simultaneously as electron acceptors (Kefauver andAllison, 1957). It is possible then that P. fluorescens were able to use both the availableoxygen and nitrates/nitrites simultaneously at 5 % and 13.4 % atmospheric oxygenconcentrations to maintain their rapid growth rate during early storage time. Unlikeoxygen which is continuously supplied from the atmosphere, nitrate in milk wouldeventually be exhausted during storage. Cellular processes would then decrease if no64other electron acceptors are provided when atmospheric oxygen concentration is low.Under aerobic conditions (20.7 %), P. fluorescens must rely solely on oxygen asterminal electron acceptors and repress nitrate and nitrite reductase activity (Clarke andOrnston, 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 9.6and 20.7 % (Fig. 11). There was no discrepancy between the initial dissolved oxygentension measured for milk under 9.6 and 13.4 % oxygen since similar values of 4.1 and3.7 ppm were measured. Dissolved oxygen tension in the containers of milk dropped andremained low despite constant oxygen supply and agitation. The dissolved oxygentension under 9.6 % and 20.7 % oxygen decreased down to less than 1.0 ppm, four andtwo days earlier than observed in Trial I. This oxygen limitation corresponded withearlier shifts into stationary growth phase despite lower population numbers. Thesepopulations may have been respiring at a faster rate and consuming oxygen more rapidlythan the populations under similar atmospheres in Trial I. These differences reinforce thedifficulty in comparing data between trials.c. Carbon Dioxide ProductionCarbon dioxide is a byproduct of oxidation of organic carbon substrates and reduction• 9.6%El 13.4%16.9 %.Ea 20.7%15102520...$^.0^'000 ,r,',11,^::::..o^04# 0^1 :-#',,..e.^,,• %,r,^'4^.'..,•■•-1^0:4'^.'''..:,, 0-4/ #^01,....^, ., • , •,,I.;^": o^k0/.#•if,'^''4,. 014;..$ 0^i' F^*:41,:1% !A ','4Atmospheric Oxygen500246^10 12 14 16 17 18: 0^< 0065Figure 10. Profile of controlled oxygen atmospheres overlaying UHT-sterilized milk (2% m.f.) inoculated with P. fluorescens during storage at 4 °C. Each value representsthe mean of triplicate gas samples.Storage Tiine (Days)Atmospheric Oxygen—4,-- 9.6%—1k--- 13.4%16.9%—.——13-- 20.7 %66Figure 11. Dissolved oxygen concentration in UHT-sterilized milk (2 % m.f.) inoculatedwith P. fluorescens during storage under controlled oxygen atmospheres at 4 °C.0^2^4^6^8 10^12^14^16^18Storage Time (Days)67of molecular 02. There are many decarboxylating reactions involved in the tricarboxylicacid cycle which has central importance in both catabolism and biosynthesis ofPseudomonas (Clarke and Ornston, 1975a). All populations produced carbon dioxidegas, a byproduct of many decarboxylating reactions involved in the tri:;arboxylic acidcycle (Fig. 12). The earliest detection of measurable CO2 was on day 6 by the populationunder 16.9 % oxygen. The highest CO2 emission was noted for the aerobic control onday 12 at 0.04 %. The CO2 production patterns by populations under variousatmospheric oxygen levels suggest difference in respiration as well as metabolic pathwaysof P. fluorescens. More research would have to be conducted in order to determinewhether the differences are significant.3. Proteinase Activitya. Aerobic Conditions The aerobic control showed proteinase activity on day 10 and levelled off afterreaching its maximum activity of 60 units on day 12 (Fig. 13). Other studies have alsoshown that proteinase activity reached a maximum plateau then levelled off (Rowe andGilmour, 1982; Rowe, 1988) or decreased (Murray et al. 1983; Skura et al., 1986). Onceproduction of proteinase has been initiated, its continual synthesis would not be requiredsince P. fluorescens would benefit from its remaining proteolytic activity. Proteinaseactivity in Trial II was not as high as the maximum of > 90 units seen in Trial I for theaerobic control (Fig. 6). The lower proteinase activity may be related to the earliertransition of P. fluorescens into stationary phase when its population of proteinase-68Figure 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)10080 -0^2^4^6^8^10 12 14 16 18 2020604069Figure 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 pointrepresents the mean of triplicate proteinase activity values + standard deviation.Atmospheric Oxygen9.6%13.4%—9— 16.9%—0— 20.7 %Storage Time (Days)70competent cells was lower. Hare et al. (1981) reported no detectable level of collagenaseand proteinase activity when the growth rate of V. alginolyticus was slower and its celldensity was lower than the aerobic control.b. Lower Atmospheric OxygenThe population under 13.4 % oxygen showed proteinase activity on day 12 which was2 days earlier than the population at the higher oxygen concentration of 16.9 %. Theproteinase activity of the milk under 13.4 % oxygen remained steady at 10 - 15 units untilday 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 thelevel of the aerobic control on day 18. P. fluorescens under 9.6 % atmospheric 02 didnot show proteinase activity until day 16 and had the lowest level of activity throughoutstorage. A decrease in the initial dissolved 02 tension in milk during storage delayedinitiation and decreased the amount of proteinase synthesis by P. fluorescens once it wasinitiated.c. Relation Between Population Density and Proteinase ActivityPopulation numbers showed poor relationship with proteinase activity when the initialdissolved 02 tension was decreased. The population density was higher under 13.4 % 02than under aerobic atmosphere throughout 16 days of storage but the proteinase activitywas much lower. Proteinase activity was detected, however, when populations under9.6, 13.4 and 16.9 % oxygen all exceeded the cell number of 108 CFU mL-1. Kroll andKlostermeyer (1984b) reported detectable proteinase activity with azocasein when71population numbers reached 1.4 - 7.1 x 107 CFU mL-1. Generally, population densitiesof 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 onday 12 which steadily increased throughout 18 days (Fig. 14). The increase inproteolysis in the aerobic control was observed despite a constant level of proteinaseactivity since A280 represents the cumulative effect of proteinase activity as well as theinterference of nucleic acids which would have been released upon cell lysis. Samplesunder 13.4 and 16.9 % oxygen showed high degree of proteolysis starting on days 12and 14 respectively which corresponded with increases in proteinase activities. The highlevel of proteolysis under 13.4 % oxygen suggested that proteinase activity of 10 - 15units 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 toits low proteinase activity.5. Lipase ActivityThe lipase activity of P. fluorescens in the aerobic control was detected on day 12well after the population entered stationary phase on day 6 (Fig. 15). There was no early_•10- Atmospheric Oxygen-9.6%--ir— 13.4%16.9%—41---—0-- 20.7 %-•i-I^•^1^.^i^.^1^.^1^.^1^.^1^.^1^.^I0^2^4^6^8^10 12 14 16 18 200-9 --,•1.^II..iL ......U ,-.4.■••.„.- --, -,.....-z ....it" "r_.4■;#`■1.7%--- -^• -..., ,-1-_-72Figure 14.^Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P.fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents themean of triplicate proteolysis values + standard deviation.—10—Storage Time (Days)__4^6^8^10 12 14 16 18 2073Figure 15. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) undercontrolled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicatelipase activity values + standard deviation.Atmospheric Oxygen--e— 9.6%—A-- 13.4%16.9%—4)——a— 20.7 %Storage Time (Days)74detection of lipase activity as observed in Trial I for the aerobic control. The delay inlipase production may be due to insufficient number of cells required to produce detectablelipase activity since the aerobic population entered stationary phase at an unusually lowdensity of < 108 CFU mL-1 . Lipase activity of the aerobic control steadily increased to itsmaximum level on day 16 but decreased on day 18 possibly due to the degradative actionsof coexisting proteinases. Lipase activity in milk under 16.9 % 02 was slightly lowerthan in milk held under 13.4 % 02 on days 14 and 16 which could be reflective of itshigher proteinase activity. Milk under 9.6 % 02 showed lipase activity starting on day14. There was a small delay in lipase production when the initial dissolved 02concentrations in milk were decreased but by day 18, all samples showed a similar lipaseactivity of 0.2 units.6. LipolysisThe lipolysis graph showed that acid degree values (ADV) for samples fluctuated fromday to day but steadily increased during storage (Fig. 16). Skura et al. (1986) alsoshowed steady increases in ADV for P. fluorescens throughout storage. There wereincreases 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 etal., 1990). An ADV of 1.2 to 1.5 was reported to be the level where off-odors andflavours were detected (Hausler, 1972; Rowe et al., 1990). Milk under 9.6 % 02 and20.7 % reached ADV of 1.5 on day 10 although lipase activity was not detected on thatday. This may be due to the difference between the two methods in the measurement of75Figure 16. Lipolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescensunder controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicatelipolysis values + standard deviation.76the lengths of fatty acids. The 3-NC assay measures lipase activity on a shorter chainfatty acid (C8) while the titration method of Deeth et al. (1975) measures longer chain fattyacids (C14-20). The titration method is reported to be well adapted for the titration of freefatty 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 % andC18:1 at 97 % recovery rates) using the titration method.A second washing of the ether extract was added to ensure the removal of watersoluble acids such as lactic and citric acid from the titatable organic layer since these acidshave 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 sensorydefects, are not extracted with the organic layer (Brathen, 1984), it has been reported thatlipolysis can be measured accurately with the dilution method prior to sensory detection oflipolytic off-flavours (IDF, 1991). The titration method, however, has often beencriticized as being laborious and showing variation between milk samples (Law, 1979).The results in Figure 16 confirm the difficulty in obtaining reproducible data since largeerror bars are shown.7.flAs an indication of the combined effects of proteolysis and lipolysis in milk, pH wasmeasured (Fig. 17). Fluctuations reflect the dynamic system of milk. Proteolysis of milkproteins 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 was6^8 10 12 14 16 18 206.0^•^1^•^1^1^I^•^1^•^1^-^1^I^I^•^02 477Figure 17. pH of 'UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens undercontrolled oxygen atmospheres at 4 °C.Atmospheric Oxygen—11— 9.6%--A--- 13.4%16.9%—0--—1:1-- 20.7 %Storage Time (Days)78reported that pH values increased as a result of ammonia production when amino acidswere used as a C source (San Jose et a., 1987). Lipolysis would decrease pH as fattyacids are liberated. The aerobic control showed a large increase in pH on day 14 sinceproteinase activity was high and its effzzts more prominent than lipase activity. Milksamples under decreased 02 atmospheres showed relatively constant pH valuesthroughout 18 days of storage. Milk under 13.4 % 02 had lower pH values than the othermilk 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 andnitrogen overlayed milk. The smallest decrease in pH was reported for milk stored undernitrogen atmosphere (Skura et al., 1986).8. Summary of Trial IIAtmospheres consisting of 9.6, 13.4, 16.9 and 20.7 % oxygen continuously flowedover 2 L of milk with initial P. fluorescens populations of 4.2 Logic, CFU mL4 for 18days at 4 °C. The dissolved 02 concentration in milk started to decrease when cell densityranged between 5.0-6.5 Logic) CFU mL-1. The dissolved 02 tension measured < 1.0ppm when all populations entered stationary phase. P. fluorescens showed the fastestinitial growth rate and reached the highest population density under 13.4 % atmospheric02.The lowest proteinase activity and degree of proteolysis were observed in milk under9.6 % oxygen. Lipase activity in all milk samples was detected much later and at lowerlevels than observed in Trial I. The reason for the decrease in proteinase activity and79delay 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 densitythan in Trial I.Results from the lipolysis analysis suggest that storage up to 10 days under 10 % 02may not be desirable although there were no detectable increases in proteinase activity,proteolysis, or lipase activity. The combined effects of proteolysis and lipolysis in milkwere reflected in pH measurements which either increased or decreased depending on thelevel of proteinase and lipase activity.Observations made from Trials I and II showed that decreasing the initial oxygentension in milk would delay and decrease the amount of extracellular enzyme productionby P. fluorescens once its synthesis was initiated. The decrease in atmospheric oxygenand initial dissolved oxygen tension in milk had greater effects on proteinase than lipasesynthesis by P. fluorescens.80C. TRIAL IIITrial III was performed to confirm the results of decreasing the initial dissolved 02tension in milk on enzyme production by P. fluorescens. The critical oxygen levelappeared 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 Iand II, populations under 4.8 and 13.4 % oxygen grew markedly faster than populationsunder 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 togrow until day 10 when it entered stationary phase and exceeded the population density ofthe aerobic control. P. fluorescens in milk under 10.3 % oxygen and aerobicatmospheres 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 alsoobserved in Trial H.2. Oxygen a. Atmospheric Oxygen The oxygen levels in the atmospheres were monitored throughout 18 days of storageand the data are shown in Figure 19.10-. Atmospheric Oxygen--er— 4.8%—0--- 10.3%---A— 13.4%—0— 20.2 %0^2^4^6^8 10 12 14 16 18 2081Figure 18. Growth of P. fluorescens in UHT-sterilized milk (2 % m.f.) undercontrolled oxygen atmospheres at 4 °C. Each point represents the mean of triplicatebacterial counts + standard deviation.Storage Time (Days)Atmospheric Oxygen• 4.8%10.3%EI 13.4%Ea 20.2%0 1 2 4 6 7 8 9 10 12 13 14 15 16 1825205151082Figure 19. Profile of controlled oxygen atmospheres overlaying UHT-sterilized milk (2% m.f.) inoculated with P. fluorescens during storage at 4 °C. Each value representsthe mean of triplicate gas samples.Storage Time (Days)83b. Dissolved Oxygen The initial dissolved oxygen tensions measured with the oxygen probe were notablyhigher than in Trials I and II but the pattern of decrease was similar (Fig. 20). Thedifference in sampling method could explain the higher values overall which did notdecrease below 1.0 ppm as seen in previous trials. Instead of measuring non-agitatedmilk, the samples were stirred with a stir bar for more accurate measurements and readwithin 30 seconds (Table 4). The difference in the ppm measurements suggest greatpotential 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. Theinitial drop in oxygen tension in milk corresponded with mid-exponential growth between5.5 - 6.5 Logi() CFU m1-1.c. Carbon Dioxide ProductionThe carbon dioxide profile showed that P . fluorescens in milk under 10.3, 13.4 and20.2 % 02 produced detectable CO2 (Fig. 21). P . fluorescens under 4.8 % atmosphericoxygen did not produce any measurable CO2. This may suggest differences in metabolicrates and pathways for P. fluorescens under 4.8 % 02 compared to those under higheratmospheric 02 concentrations. In general, a decrease in the initial dissolved 02 tensionin milk resulted in lower amounts of carbon dioxide emitted by P 17uorescens.1210-.^I^1-1^.^1^.^1^.^I^•^1^.^1^•^r •^I0^2^4^6^8 10 12 14 16 18 2084Figure 20. Dissolved oxygen concentration in stirred samples of UHT-sterilized milk (2% m.f.) inoculated with P. fluorescens during storage under controlled oxygenatmospheres at 4 °C.Atmospheric Oxygen--"ti— 4.8 %—41— 10.3%—ii--- 13.4%—0— 20.2%Storage Time (Days)85Table 4. Dissolved oxygen tension of stirred and unstirred samples aNon-agitated Sampling Agitated SamplingAtmospheric 02^Dissolved 02(%)^(ppm)Atmospheric 02^Dissolved 02(%)^(ppm)1.2 1.4 -5.3 2.7 4.8 3.69.6 4.1 _10.2 4.1 10.3 6.113.4 3.7 13.4 7.216.9 4.7 -20.9 7.220.7 7.3 20.2 10.1a single sampling of 25 mL of milk86Figure 21. Carbon dioxide production by P. fluorescens in UHT-sterilized milk (2 %m.f.) during storage under controlled oxygen atmospheres at 4 °C.873. Proteinase Activitya. Lower Atmospheric OxygenThe first detectable proteinase activity was observed on day 8 in the aerobic control butnot in milk stored under lowered 02 concentrations (Fig. 22). Milk under 4.8, 10.3 and13.4 % 02 showed proteinase activity starting on day 10 and remained low throughout 18days of storage despite reaching high population numbers.Proteinase production under aerobic conditions was detected during early stationaryphase and increased steadily during storage to its maximum activity of 378 azocasein unitson 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 inTrials I and II which did not show such high proteinase activity. The proteinase activityof 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 atmospheresbut proteinase production was severely decreased. During prolonged storage, energyproduction is expected to decrease when both 02 and alternate electron acceptor(NO3/NO2) may be limiting. A decrease in energy charge would favour ATP generatingcatabolic processes and an increase would favour biosynthesis (Harrison, 1976), but ithas been reported that when there was a limitation in orthophosphate, carbon, or nitrogen,proteinase production by P.fluorescens was decreased (McKellar and Cholette, 1984).88Figure 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 pointrepresents the mean of triplicate proteinase activity values + standard deviation.89Limiting oxygen, an essential nutrient for obligate aerobes, might then have a similareffect 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 notexpended in expressing functions which are detrimental (Strauch and Hock, 1993). Therestriction of enzyme synthesis would result in greater economy for cell growth.Oxygen demanding catabolic and biosynthetic processes may be limited when theinitial dissolved 02 concentration is decreased. This would alter the production ofmetabolite precursors and energy by P. fluorescens for synthesis of extracellularenzymes. In a continuous culture study with P. fluorescens 378 (biotype A), Persson etal. (1990) reported that substrate utilization in oxygen limiting conditions followed thesame nutritional pattern as a carbon source limiting culture. Aeration and oxygen transferrate determined the biomass and when oxygen was limiting, biomass was determined bydilution rate (Persson et al., 1990). Cells behave differently under varying oxygenconcentrations. Utilization of glucose as well as metabolite pools of L-lactate andpynivate were lower in cultures under lower oxygen atmospheres (Griffiths and Phillips,1984; Spohr and Schutz, 1990). Lowering atmospheric oxygen concentration woulddecrease the amount of available electron acceptors during respiration, decrease thecosubstrate supply of many oxidation reactions, and then result in lower energyproduction for biosynthesis and extracellular enzyme production. According to Stanier etal. (1986), bacteria of the same strain show different cytochrome compositions whengrown aerobically and anaerobically. A decrease in cytochrome synthesis may affect the90energy generating status of microorganisms and may have the same effects as limitingoxygen on growth (Wiersma et al., 1978). Proteinase production by P.fluorescens NC3was 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 andthe use of oxygen increased. It is possible that severe oxygen limiting conditions, whenthe demand exceeds the supply, initiated proteinase synthesis in P. fluorescens. Anaerobically grown culture would require a means of adapting to its new 02 stressedenvironment and may require the actions of extracellular proteinases and lipases providingthere is enough oxygen or alternate electron acceptor to maintain its respiration for energyproduction. Rowe and Gilmour (1982) observed an earlier onset of proteinase and lipaseproduction by P. fluorescens when the dissolved 02 tension was forcibly decreased.Cultures under decreased atmospheric oxygen may use both the available oxygen as wellas nitrate as electron acceptors and may not respond to 02 stress in the same manner as anaerobically grown culture. This would result in lower detectable proteinase and lipaseactivities if their regulation was oxygen-dependent. There is no concrete evidence that 02stress initiates proteinase synthesis in P. fluorescens. Phospholipase synthesis by S.liquefaciens, however, was reported to be regulated by two promoters, PA which wasunder growth phase regulation and Px under anaerobic induction (Givskov and Molin,911992).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 ofproteolysis in milk under 13.4 and 20.2 % atmospheric oxygen. The greatest extent ofproteolysis during storage occurred in the aerobic control.5. Lipase Activitya. Lower Atmospheric OxygenLipase activity was generally detected earlier than proteinase activity. Milk under 13.4and 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 constitutiveexpression of lipase. Although there were detectable levels of lipase activity in all milksamples under lower atmospheric oxygen, lipase activity was lower than in the aerobicatmosphere. 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 asa result of increased proteinase activity on coexisting lipases.As with proteinase, a decrease in the initial dissolved 02 tension in milk resulted in adecrease in lipase synthesis by P. fluorescens. Lipase synthesis by P. fluorescensappears to follow the same restrictions of oxygen limitation as proteinase. Inhibition oflipase production by P.fluorescens was not as dramatic as with proteinase when the92Figure 23.^Proteolysis of UHT-sterilized milk (2 % m.f.) inoculated with P.fluorescens under controlled oxygen atmospheres at 4 °C. Each point represents themean of triplicate proteolysis values + standard deviation.93Figure 24. Lipase activity of P. fluorescens in UHT-sterilized milk (2 % m.f.) undercontrolled oxygen atmospheres at 4 °C. Each point represents the mean of quadruplicatelipase activity values + standard deviation.94initial 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 inoxygen tension in all samples (Appendix Fig. 5. a-d). Lipase synthesis could also beregulated by oxygen stress signals.6. Lipolysis Despite fluctuations in lipase activity, there were increases in the degree of lipolysis ofmilk fat throughout storage (Fig. 25). Acid degree values were highest for the aerobiccontrol. Milk under oxygen diminished atmospheres showed similar acid degree valueswhich 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 milkunder 4.8, 10.3, 13.4 and 20.2 % atmospheric 02. Correlation coefficients for cellnumbers and proteinase activity ranged between 0.50 to 0.82 and r values for cellnumbers and lipase activity ranged between 0.43 and 0.76 but not all r values weresignificant. Proteinase activity and degree of cumulative proteolysis in milk showedstrong correlation (r = 0.84-0.96) and were highly significant at all the atmospheric 02levels. Lipase activity and degree of lipolysis did not show strong correlation as r variedbetween 0.06 to 0.72 and were not all significant. Lipase activity may be dependent onproteinase activity as well as its association with milk proteins.95Figure 25. Lipolysis of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescensunder controlled oxygen atmospheres at 4 °C. Each point represents the mean of triplicatelipolysis values + standard deviation.96Table 5. Correlation coefficients of P.fluorescens cell numbers and enzyme activities inmilk stored under 4.8 % atmospheric 02 atmosphereCorrelation coefficients (r)Parameters^Cell numbers Proteinase^Proteolysis^Lipase^LipolysisCell numbers^1.00Proteinase^0.50^1.00Proteolysis^0.27^0.84**^1.00Lipase 0.43^0.01^-0.17^1.00Lipolysis^0.53^0.89***^0.77**^0.06^1.00Table 6. Correlation coefficients of P.fluorescens cell numbers and enzyme activities inmilk stored under 10.3 % atmospheric 02 atmosphereCorrelation coefficients (r)Parameters^Cell numbers Proteinase^Proteolysis^Lipase^LipolysisCell numbers^1.00Proteinase^0.73*^1.00Proteolysis^0.51^0.91***^1.00Lipase 0.76**^0.42^0.19^1.00Lipolysis^0.86***^0.86**^0.64^0.72*^1.00*, **, *** significant at 5 %, 1 % and 0.1 % levels, respectively97Table 7. Correlation coefficients of P.fluorescens cell numbers and enzyme activities inmilk stored under 13.4 % atmospheric 02 atmosphereCorrelation coefficients (r)Parameters^Cell numbers Proteinase^Proteolysis^Lipase^LipolysisCell numbers^1.00Proteinase^0.82**^1.00Proteolysis^0.75*^0.89***^1.00Lipase 0.68*^0.40^0.49^1.00Lipolysis^0.89***^0.90***^0.89***^0.64^1.00Table 8. Correlation coefficients of P .fluorescens cell numbers and enzyme activities inmilk stored under 20.2 % atmospheric 02 atmosphereCorrelation coefficients (r)Parameters^Cell numbers Proteinase^Proteolysis^Lipase^LipolysisCell numbers^1.00Proteinase^0.55^1.00Proteolysis^0.55^0.96***^1.00Lipase 0.70*^0.37^0.47^1.00Lipolysis^0.64^0.81**^0.92***^0.64^1.00*, **, *** significant at 5 %, 1 % and 0.1 % levels, respectively98Proteinase activity and lipolysis showed correlations (r = 0.81-0.90) at highlysignificant 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 relationshipbetween proteinase activity on casein micelles to release lipases from its association andproteolytic degradation of lipases. Complexity of lipase determination warrants moreresearch in this area.8. atThe aerobic control showed a large increase in pH on day 8 which coincided with anincrease in proteinase activity (Fig. 26). In relation to the energy state of the cell, a basicextracellular pH has been reported to have a positive effect on extracellular enzymeproduction by P. aeruginosa (Whooley and McLoughlin, 1983). It was suggested thatthe increase in pH gradient between the cell and its environment could have a decreasingeffect on proton motive force, a decrease in ATP level and therefore, an increase inenzyme production (Whooley and McLoughlin, 1983).Milk samples under lower atmospheric oxygen showed decreases in pH starting onday 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 reflectiveof the overall effects of proteinase and lipase activity in milk. Activity of one enzymehowever could effectively negate the effects of pH changes brought about the other.99Figure 26. pH of UHT-sterilized milk (2 % m.f.) inoculated with P. fluorescens undercontrolled oxygen atmospheres at 4 °C.1009. Summary of Trial IIIInitial 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 4.8and 13.4 % 02 showed faster growth rates than the microorganisms in the aerobic controlduring 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 theaerobic control which corresponded to the increase in proteolysis. Proteinase activity ofP.fluorescens and the degree of proteolysis in milk under oxygen decreased atmosphereswere 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 highas the other samples. Lipase activity appears to be sensitive to proteinase degradationduring assay conditions.Correlation coefficients for proteinase activity and degree of proteolysis (r = 0.84-0.96) 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 andproteinase and lipase activities under 4.8 and 20.2 % atmospheric 02 when activity levelsare either very high or very low.Milk pH values, indicating the combined effects of proteolysis and lipolysis, deviatedthe least from initial pH in milk under 10.3 % oxygen.101A decrease in the initial dissolved 02 tension in milk effectively decreased synthesis ofproteinases and lipases by P. fluorescens in milk. Changes in metabolism due to 02limitation and/or a decrease in the 02 stress signal may be responsible for the minimizationof extracellular proteinase and lipase production by P. fluorescens.102D. CONTROL OF OXYGEN CONCENTRATION DURING STORAGE OF RAWmruc Results from this model system using P. fluorescens suggest that growth andproduction of extracellular enzymes by aerobic spoilage microorganisms may be enhancedunder highly aerobic conditions during storage of raw milk. Many factors such asagitation methods, surface area in contact with air, depth of storage tanks as well astemperature, influence the oxygenated state of milk. Current practice at dairy processingplants include mechanical and air agitation systems in raw milk silos. Agitation andaeration systems in these silos should be investigated for rate of oxygen transfer duringstorage of raw milk to minimize microbial activity.Decreasing the oxygen content of milk by altering the atmospheric oxygen level mayinhibit the detrimental activities of P. fluorescens and other obligate aerobes but mayselect for facultative anaerobes which may emerge as the next major spoilagemicroorganisms.103CONCLUSION1. The dissolved oxygen tension in milk under all atmospheric oxygen levels testeddecreased during mid-exponential growth phase of P. fluorescens when populationsreached 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 enhancedduring the first four days of storage at 4 °C under decreased atmospheric 02concentrations 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 aerobicconditions.3. Aerobic conditions supported:highest proteinase and lipase activity of P. fluorescens when the dissolved 02tension in milk decreased to low levels- greatest degree of milk proteolysis and lipolysis and greatest change in milk pH4. 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 storagegreater inhibition on proteinase than lipase production by P.fluorescens1045. Of the atmospheric 02 concentrations tested, 10 % was the critical upper level whichsuppressed:growth rates of P . fluorescens during first four days of storageproteinase and lipase activity of P .fluorescens in UHT-sterilized milkdegree of proteolysis and lipolysischange from initial milk pH during storagePART IIImprovement of the I3-naphthyl caprylateLipase Activity Assay105106INTRODUCTIONMost P. fluorescens produce proteinases and lipases simultaneously during earlystationary growth. During storage experiments, lipase activity fluctuates and usuallydecreases when proteinase activity increases. There is concern of proteolytic degradationof lipases during storage at lower temperatures but there is greater concern during lipaseactivity assays. The optimal temperature for lipase and proteinase activity are similararound 35 - 45 °C. Proteinase activity could then affect the apparent lipase activity.Inhibition of proteinase activity during lipase assay conditions would then result in moreaccurate reflection of lipase activity contained in milk at the time. The objective of thestudy in this part of the thesis was to enhance the sensitivity of the lipase activity byminimizing proteinase, but not lipase activity, with a metal chelator, ethylenediaminetetraacetic acid (EDTA).107LITERATURE REVIEWA. Lipase Degradation by ProteinasesThere are many reports to suggest that lipases may be inactivated through the actionsof co-existing proteinases since both are produced by P. fluorescens. Lipase activitywould then reflect the total amount of functional enzyme after hydrolysis by proteinase(Roussis et al., 1988).1. During StorageDecrease in lipase activity is often observed during prolonged storage and withincreases in proteinase activity. Lipase activity of Micrococcus caseolyticus reached itsmaximum then decreased with an increase in proteolytic activity (Jonsson and Snygg,1974). P. fluorescens showed a decrease in lipase activity when proteinase activityincreased markedly suggesting inactivation of lipase by considerable accumulation ofproteinase (Fox and Stepaniak, 1983; Bucky et al., 1986). Lipase susceptibility wasenhanced 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 arapid inactivation of P. fluorescens lipase at 20 °C (Andersson, 1980a) and addition ofproteinase caused 16 % reduction in lipase activity (Roussis et al., 1988). Stead (1987)suggested that lipase activity probably reflected a dynamic system of degradation of lipaseby proteinase since P. fluorescens AR11 in whole milk at 7 °C with high proteinaseactivity showed a steady decrease in lipase activity over time. A proteinase deficient P.fluorescens AR11M mutant on the contrary, showed higher lipase activity since lipase108degradation by bacterial proteinase would have been less than in the proteinase proficientstrain. In support of this, degree of lipolysis as a measure of cumulative lipase activitywas greater in milk sample under N2-disrupted atmosphere than the aerobic control (Skuraet al., 1986) which may possibly be due to its lower proteinase activity.2. During Lipase Assay ConditionsProteinase activity would be enhanced during incubation of lipase activity assay sinceit 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). Theactivity of P. fluorescens AR11 proteinase at 4 °C was 33 % of that at 35 °C (Alichanidisand Andrew, 1977) and P. fluorescens AFT 36 proteinase at 7 °C was only 30 % ofproteinase 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 ofproteolytic digestion of the lipase at the higher temperature (Jonsson and Snygg, 1976).During incubation at 40 °C for lipase activity, presence of proteinase activity coulddecrease 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 aresensitive 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 ofthese proteinases from psychrotrophic pseudomonads and increases their thermal stability(McKellar, 1989). Activity of typical metalloproteinases are inhibited by a metal chelator109such 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 otherinhibitors tested by Kohlmann et al. (1991). At 1 mM EDTA, 66 % of the proteinaseactivity 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 ofCa2+ 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 at50 °C, pH 7.2, 0.1 mM 0-NC and 17.6 mM sodium taurocholate (NaTC) (Paquette andMcKellar, 1986). Psychrotrophic lipase is activated by the addition of bile salts such asNaTC to release milk proteins which may block its active site (Stead, 1983) or by theaction of trypsin (McKellar and Cholette, 1986a). Lipases are usually adsorbed on milkfat globules and their activity is strongly reduced by presence of milk fat in assay reactionsbecause of competition between fat and the assay substrate (McKellar and Cholette,1986a). The presence of 2 % milk fat inhibited 92 % of lipase activity (McKellar andCholette, 1986a). The removal of milk fat by centrifugation has been suggested but lowerlipase 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, ofaround 100 InM, than are proteinases (Bozoglu et al., 1984).110111MATERIALS 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 responsewas observed for 17.6 mM (Paquette and McKellar, 1986). Concentrations between 0and 20 mM of NaTC were added to the reaction mixture. The difference in volume wascorrected with BES buffer.2. 11-naphthyl Caprylate Versaw et al (1989) used 20 IlL of 200 mM 13-naphthyl caprylate for a finalconcentration of 1.93 mM in each reaction. A final concentration range of 0 to 7 mM ofP-naphthyl caprylate dissolved in DMSO was tested for best concentration of the substratewhile minimizing the volume of DMSO which is added to the reaction mixtures.3. Solvent ClarificationAfter the reactions were stopped with the addition of 0.2 mL of 10 % TCA, thereactions were clarified with the addition of solvent(s). Addition of BES buffer wasdecreased from 1.8 to 1.0 mL for easier pipetting. Attempts were made to extract thediazonium colour compounds from the aqueous layer with 100% ethyl acetate. Solvents(2.51 mL) used to clarify the lipase reaction mixtures were dimethylformamide,112dimethylsulfoxide, acetone, formaldehyde, and varying ratios of ethyl acetate and ethanol.B. Lipase Activity Enhancement1. Proteinase Activity Under Lipase Assay Conditions Proteinase activity was determined using 1.0 mL of 1.5 % azocasein in pH 7.2 BESbuffer 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 mLmilk sample with a final concentration of 0.05 % thimerosal to inhibit extracellular enzymeproduction. 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 preparedfrom a 250 mM EDTA stock solution. The tubes were incubated at 40 °C for 1 h withshaking 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 at2910 x g for 15 minutes in a GS-6 Beckman centrifuge (Beckman Instruments Inc., PaloAlto, CA) and the absorbance of the supernatant was measured at 366 nm. All sampleswere assayed in triplicate. One azocasein unit of proteinase activity was defined as anincrease in absorbance of 0.01 at 366 nm per mL of milk per hour.2. Addition of EDTALipase assay conditions stated in Part I were used with the addition of finalconcentration 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 onegmole 13-naphthol released per tnL of milk per hour.113RESULTS AND DISCUSSIONA. Improvement of the Lipase Assay 1. Sodium TaurocholateLipase activity was enhanced as NaTC concentration was increased. A finalconcentration of 15 mM NaTC was used in future lipase activity assays (Fig. 27).2. 13-naphthyl caprylateAlong with lipases in the 50 III.. aliquot used in the assay, appreciable amounts ofmilk fat in 2 % m.f. UHT milk (as much as 1.0 mg) was possibly carried over to eachreaction mixture and allowed to compete with the substrate. The [3-naphthyl caprylate at afinal concentration of 2 mM was calculated to be 1.08 mg and created a ratio of 1 to 1between milk fat and the substrate. Final concentrations of 0 to 7 tnM of 13 -naphthylcaprylate were tested. An increase in lipase activity was proportional to an increase insubstrate concentration showing maximum activity at a final concentration of greater than5 mM and the ratio of substrate: fat corresponding to 3:1 (Fig. 28). A stock solution of500 mM I3-naphthyl caprylate was used at a final concentration of 8 mM so that thesubstrate concentration was not limiting in the assay.3. Solvent ClarificationThe 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 thereaction mixtures. Turbidity was due to the presence of milk proteins. Also the BESbuffer was decreased from 1.8 to 1.0 mL for easier pipetting volume, changing the›--;•IMMIet-eCFigure 27. Effect of increasing sodium taurocholate (NaTC) concentrations onlipase activity. Each point represents the mean of triplicate values + standarddeviation.1140^5^10^15^20NaTC Concentration (mM)0.80.70.60.50.4030.20.10.0-0.10 1^2 3^4 5^6^7 8Figure 28. Effect of increasing 13-naphthyl caprylate (0-NC) concentrations onlipase activity. Each point represents the mean of triplicate values + standarddeviation.11513-NC Concentration (mM)116proportion of solvents required to clarify the aqueous reaction mixtures. A better solventsystem for clarifying the reaction mixtures was needed and Table 9 shows the solventsystems tested. A 60:40 ethyl acetate to ethanol solvent system completely clarified thereaction mixtures. Phase partitioning occurred with other proportions of ethyl acetate andethanol ard resulted in incomplete extraction.B. Lipase Activity Enhancement1. Proteinase Activity Under Lipase Assay ConditionsProteinase activity under lipase assay conditions was 33.5 % of the activity underproteinase 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 concentrationof 2 to 5 mM EDTA in the reaction mixtures enhanced the activity of lipase quantified andgreatly 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 proteinaseactivity with EDTA. When both proteinases and lipases are present in milk cultures, anaccurate measurement of the lipase activity present in milk at 4 °C would not berepresented by lipase assay conditions set at 40 °C since proteinases would degrade lipaseduring incubation and decrease measurable lipase activity. Proteinase activity showed aplateau at EDTA concentrations exceeding 2 mM and a decrease in lipase was observed at117Table 9: Clarification of Lipase Reactions with SolventsSolvents^Clarification adimethyl formamide (DMF)dimethyl sulfoxide (DMSO)acetoneformaldehydeethyl acetate to ethanol ratio:10:9020:8030:7040:60^50:50^+55:45 +60:40 +65:35^+70:3080:2090:10a^- turbid,^+ some turbidity,^+ clarifiedTable 10: Proteinase activity under lipase assay conditionsSample[EDTA]rnMProteinase activity(Azocasein units + Std Dev)% RelativeActivitywithout NaTC, P-NC 0 542.7 +^5.8 100.0with NaTC, 13-NC 0 181.7 +^3.0 33.520 28.3 +^4.0 5.235 8.0 +^1.4 1.550 0 +^2.3 0118Figure 29. Effect of increasing EDTA concentrations on proteinase and lipaseactivities in lipase assay conditions. Each point represents the mean of triplicatevalues + standard deviation.119EDTA concentrations exceeding 10 mM. 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Each point representsthe mean of triplicate lipolysis values + standard deviation.1350^2^4^6^8^10^12^14^16Atmospheric Oxygen---•— 1.2%---tr— 5.3%—9— 10.2%—0— 20.9%Storage Time (Days)136AppendixFigure 2. The effect of mechanical agitation at 350 rpm on the increase in aciddegree values (ADV) of UHT-sterilized milk (2 % m.f.) stored at 4 °C underaerobic conditions. Each point represents the mean of triplicate lipolysis values +standard deviation.3.0 1.5 -—0— stirred—0— unstirred1 .0 -0.5 -0.0 ^2^4^6^8Storage Time (Day)10.0—0— stirred—0— unstirred3.02.5 -2.0 -15 --1.0 -137AppendixFigure 3. The effect of CO2 removal from the titration atmosphere on the aciddegree values (ADV) of UHT-sterilized milk (2 % m.f.) stored at 4 0C underaerobic conditions. Each point represents the mean of triplicate lipolysis values +standard deviation.o^2^4^6^8Storage Time (Day)138AppendixFigure 4. a - d. The effect of dissolved oxygen and its limitation on proteinaseactivity of P. fluorescens in UHT-sterilized milk (2 %) under controlled oxygenatmospheres during storage at 4 °C. Each point represents the mean of triplicateproteinase activity values and a single dissolved oxygen value.0i. i.i.i.i.i.02 4 6 8 10 12 14 16 18 201210a)—0--- ppm„a1),etc.)Proteinase-300.,"amtg- 2004—) 0ON- 100^.,e!139C>012c. 13.4 % Atmospheric Oxygen--A—50-40.--303ppm- 20 et^c),11 co)ProteinaseN—10 0000 2 4 6 8 10 12 14 16 18 20Storage Time (Days)d. 20.2 % Atmospheric OxygenStorage Time (Days)AppendixFigure 5. a - d. The effect of dissolved oxygen and its limitation on lipase activityof P. fluorescens in UHT-sterilized milk (2 %) under controlled oxygenatmospheres during storage at 4 °C. Each point represents the mean ofquadruplicate lipase activity values and a single dissolved oxygen value.a. 4.8 % Atmospheric Oxygen140 —A— ppm—A-- LipaseOA-^.^1.^-1 .1 .^-^.^0.00 2 4 6 8 10 12 14 16 18 20Storage Time (Days)b. 10.3 % Atmospheric Oxygen12 0.6- 0.5•••••••- 0.4 •-- 0.3 !...; -64.)4!-0.2^<1.) 0.ettCS-0. 1—0— ppm—40-- LipaseO• 0.^•^•I.I.^.1^0.00 2 4 6 8 10 12 14 16 18 20Storage Time (Days)—D— ppm• Lipase—0.3—0.2•—0.1•— •—•—c. 13.4 % Atmospheric Oxygen0.6— 0.5—0.4— 0.3—0.2—0.10.02 4 6 8 10 12 14 16 18 20Storage Time (Days)1210C.C.8>4 6'asc.)42A0141*4-)j ppm•et^.4a4 —A-- Lipasecneta)^0.C.d. 20.2 % Atmospheric Oxygen0.6—0.5—0.4tit••••• •^•^.1 )1^.1^•^0.00 2 4 6 8 10 12 14 16 18 20Storage Time (Days)