lllllEIlI II I I r we llllllllllllllllllllllllllllllllllllllllllll W l, LEBMRY Miohfigam filtrate » Univemfiw This is to certify that the dissertation entitled PRODUCTION AND CHARACTERIZATION PENICILLIUM CXEEICOLUM LIPASE presented by Saad Al-Deen M.A. AL-HIR has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in n. It is rapid, accurate and more sensitive than other me‘thods. 25 Temperature Optimum for Lipase Activity The temperature of incubation of the enzyme substrate mixture is important and apparently affects the specificity (Alford and Pierce, 1961). Temperature optimum for lipase activity are listed in Table 3. The optimum temperature varies from 30-600C for various microorganisms and their strains. However, some microbial lipases are capable of hydrolyzing lipids at lower temperature. According to Alford and Pierce (1961) Pseudomonas fragi, Staphylococcus aureus, Geotrichum candidum, Candida lipolytica, Penicillium rogueforti and an unidentified Penicillium gg showed considerable activity within 2-4 days at ~7°C and within a week at -18°C. 26 Table 3. Temperature optimum of microbial lipases. Microorganism Temperature 0C References Serratia marcescens 37 Hugo and Beveridge (1962) Pseudomonas fragi 54 Mencher & Alford(l967), Lu gt gt. (1969) Achromobacter lipolyticum 37 Khan gt gt. (1967) Staphylococcus aureus 45 Vadehara and Harmon (1967) Lactobacillus plantarum 37 Umemoto gt gt. (1968) Lactobacillus casei 37 Umemoto gt gt. (1968) Streptococcus diacetilactis 45 Umemoto _t gt. (1968) Propionibacterium shermanii 47 Oterholm gt _t. (1970) Microbacterium thermos- 35-37 Collins _t gt. (1971) phactum Chromobacterium viscosum 65-70 Sugiura gt gt. (1974) Streptococcus faecalis 4O Chander gt gt. (1979a) Streptococcus thermophilus 45 Demoraes (1981) Aspergillus niger 25 Fukumoto _t _t. (1963) Mgggt pusillus 50 Somkuti gt _t. (1969) Mgggt javanicus 40 Saiki _t gt. (1969) Penicillium roqueforti 37 Eitenmiller _t _t.(1970) Geotrichum candidum 40 Tsujisaka _t _t. (1973) Rhizopus delemar 30, 35, 40 Iwai and Tsujsaka (1974) Penicillium camemberti 25 Belloc gt _t. (1975) Aspergillus wentii 25 Chopra _t _t. (1980) Candida gylindracea 47.5-57.5 Tomizuka (1966) Table 3. (cont'd.). 27 Torpulopsis ernobii Candida mycoderma Debaryomyces kloeckeri Humicola lanuginosa Eremothecium ashbyii 45 35 30 6O 4O Motai gt gt. (1966), Yoshida gt a1. (19 68) Hosona and Tokita, 1970 Hosona and Tokita, 1970 Liu gt gt. (1973) Shen t l. (1974) 28 pH Optimwnfor Lipase Activity According to Lawrence (1967) pH alters rate of lipolysis by changes in activity and stability of the enzyme, the velocity of enzyme-substrate combination and breakdown. In the case of an emulsion substrate the properties of Sub- strate-aqueous phase interface may be affected by pH. Some workers (Nashif and Nelson, 1953; Rottem and Razin, 1964) suggested that optimum pH may depend upon the nature of . substrate being hydrolyzed, the buffer and other conditions of the assay. Hugo and Beveridge (1962) have pointed that the term optimum pH can only be a relative one since the temperature is arbitrarily fixed and all the above factors also affect the optimal lipolysis temperature. pH optimum for various microbial lipase activity are listed in Table 4. Weete _t _t. (1974) stated that fungal lipases appear to have optimum pH around 8.0. A wide range in both acid and alkali pH is reported. Factors affecting pH optimum are: organisms, intra or extracellular lipase, degree of purity, substrate, temperature and the other conditions of the assay. Table 4. pH optimum of various microbial lipases. Microorganism pH Reference Achromobacter lipolyticum 9.0 Shahani gt gt. (1964) (intracellular) Achromobacter lipolyticum 7.0 Khan gt gt. (1967) Staphylococcus aureus 8.3 Vadehra and Harmon (purified) (1967a) Micrococcus freudenrichii 0—8. Lawrence t l. (1967) Pseudomonas fragi Lactobacillus casei Lactobacillus plentarum Lactobacillus helveticus Streptococcus diacetilactis Propionibacterium shermanii Pseudomonas aeroginosa (purified) Microbactrum thermos— phactum Cor nebactrium acnes (partially purified) Lactobacillus brevis Micrococcus caseolyticus Bacillus licheniformis Staphylococcus gp Chromobacterium viscosum Streptococcus faecalis Streptococcus thermophilus Mencher and Alford (1976), Lawrence gt aL, (1967) and Lu gt _t. (1969) Umemoto _t _t. (1968) Umemoto _t gt. (1968) Umemoto t al. 1968) ) __( Umemoto t gt. ( Otterholm t 1. Nadkarni (1971a) Collins t al. (1971) Hassing (1971) Chander t al. (1973) Jonsson and Snygg (1974) Jonsson and Snygg (1974) Jonsson and Snygg (1974) Sugiura et 1. (1974) Chander t l. (1979a) Demoraes (1981) Table 4. (cont'd). 3O Aspergillus niger Penicillium crustosum Mucor javanicus Mucor pusillus Penicillium roqueforti Penicillium roqueforti Penicillium roqueforti Rhizopus arrhizus Geotrichum candidum (crystallized) Mucor lipolyticus Rhizopus delemar Penicillium roqgeforti (intracellular) Aspergillus wentii Torulopsis ernobii Candida cylindracea Candida cylindracea (purified) Candida mycoderma Debaryomyces kloeckeri Humicola lanuginosa Saccharomycopsis lipolytica Eremothecium ashbyii (I) O CID->4:- U'l Fukumoto t al. (1963) Oi gt gt. (1967) Saiki et gt. (1969) Somkuti gt a1. (1969) Fodor and Chari (1949) Shipe (1951) Eitenmiller gt gt. (1970) Semeriva gt a1. (1969) Tsujisaka (1973) Nagaoka and Yamada (1973) Iwai and Tsujisaka (1974) Kornacki gt 1. (1979) Chopra t 1. (1980) 966) and . (1968) 1 Yoshida t 1 Motai et a1. ( a Tomizuka t al. (1966) Ota gt 1. (1970, 1972) Hosono and Tokita (1970) Hosono and Tokita (1970) Liu _£ _t. (1973) Jonsson and Snygg (1974) Shen t al. (1974) 31 Effect of Addition of Bile Saltsand CaCl2 on Lipase Activity Microbial lipase activity may be stimulated by the addi- tion of bile salt. According to Wills (1965),the Nfle of bfle salts appears to involve exact alignment of enzyme mole- cules in the interfacial layer. Benzonana and Desnuelle (1968) stated that in case of pancreatic lipase,bihasaltsare not true activators, but enable the reactions to proceed in a zero order rate. The action of bile salt on lipase acti- vity depends on the degree of enzyme purification, tempera- ture, pH, concentration of bile salt and the nature of the microbial lipase. Several workers reported a stimulatory effect ofime addt— tion of bile salts to the reaction mixture (Nashif and Nelson, 1953; Shahani gt 1., 1964; Oi gt 1., 1969; Ota and Yamada, 1966; Nagaoka and Yamada, 1973; Yamaguchi t 1., 1973; Sugiura t 1., 1974; Wang, 1980). On the other hand, the addition of bile salt had an inhibitory effect as reported by other workers (Fodor and Chari, 1949; Saiki gt 1., 1969; Oterholm t 1., 1970; Finkelstein, 1970; Sugiura gt al., 1974). Other reports indicate no'effect of bile salt on the activity of micro- bial lipases (Nadkarni, l971a;Yamaguchi gt 1., 1973; Sugiura gt gt., 1974). Bashkatova t l. (1976) found the addition of bile salt to the growth medium or(firecth/to Um reaction mixture of lipase of Pseudomonas fluorescens, 32 Serratia marcescens, Mycobacterium mucosum and Escherihia ggtt. Bile salts act in a complicated and probably indi- rect way depending on chemical composition of the salt. The bile salt affected both exo- and endo-lipases of these organisms. Chander gt gt. (1979a) reported that 0.2% bile salts stimulated lipase activity of Streptococcus faecalis. Calcium activation of lipase has been reported by many investigators. According to Shipe (1951), addition of 10 mg of CaC12 to 12 ml of substrate increased the lipase activity of Penicillium roqueforti and Aspergillus niger.. Iwai et 1. (1964) obtained similar results for Aspergillus gtggg lipase by the addition of CaClZ. Oi _t _t. (1969) reported the stimulatory effect of the addition of CaClZ and ferric sul- fate to substrate of Rhizopus lipase. Extracellular lipase of Achromobacter lipolyticum was stimulated by the addition of CaClZ, MgClZ, Na2804, MgS04 and NaCl (Khan _t _t., 1967). Nadkarni (l97la) reported Ca ions stimulated lipase activity of Pseudomonas aeruginosa. Inhibitory effect of calcium chloride on lipase activity has also been reported by Oterholm (1970). Other reports showed no influence of addition of calcium chloride on lipase activity (Rotten and Razin, 1964; Eitenmiller gt gt., 1970). Stimulatory effect of Ca++ results from removal of fatty acids formed during the hydrolysis as insoluble calcium soaps. The calcium soap activates the hydrolysis by changing the interfacial substrate—water relationship 33 to one more favorable for enzyme action (Iwai, 1964). Also, Ca++ may inhibit the resynthesis of ester linkages, which would effectively shift the reaction in the direction of hydrolysis (Lawrence, 1967). Thermostability of Microbial Lipases Lipolytic enzymesand lipolytic organisms contained in foods may survive the food processing treatments. On storage of foods, they may enhance or cause deterioration of lipids (Alford _t__t., 1971; Jonsson and Snygg, 1974). The thermostability of microbial lipase varies considerably among organisms. Nashif and Nelson (l953b) ‘f0und lipase from Pseudomonas fragi degraded fat at -10°C. Tomizuka gt gt. (1966) reported lipase of Candida cylin- deraca to be stable at 15°C. It lost 5% of its activity in frozen state for one month. Motai _t gt. (1966) showed that lipase from Torulopsis gp was stable at pH 3-8 at 370C for 1 h and at pH 5.0 for 10 min. A rapid inactivation was observed above 70°C, 0i gt gt. (1969) studied thermal inactivation of some Rhizopus fungal lipases used in milk flavoring. The lipase did not lose activity at 470C for 15 min. Liu gt 1. (1972) found lipase of Humicola lanuginosa to retain 100% of its activity at 600C for 2 h. Only 35% lipase activity remained at 70°C after 20 min. Geotrichum candidum 34 lipase was stable when stored below 55°C for 15 min (Tsujisaka gt gt., 1973). The purified Mucor lipase was stable below 45°C but was inactivated at 60°C after 1 h. Purified lipase of Humicola lanuginosa was stable under 60°C and retained 55% of activity at 70°C after 20 min (Liu gt 1., 1973). Sugiura t 1. (1974) reported purified lipase A of Chromobacterium viscosum var paralipolyticum less stable than the crude enzyme and was inactivated rapidly above 50°C. Iwai and Tsujisaka (1974) reported crude lipase of Rhizopus delemar to be stable at various temperatures at pH 5.6. Purified enzyme B and C of Rhizopus delemar were stable below 45°C while A was stable even at 60°C. Iwai gt 1. (1975) found two lipases of Penicillium cyclopium were stable at 30°C for 15 min. Jonsson and Snygg (1976) determined lipolytic activity of Pseudomonas fluorescens at various temperatures. The storage stability at 370C de— creased with increasing the age of the culture. The acti- vity disappeared after 10 days. Chander gt gt. (1979) reported purified lipase of Strgptococcus faecalis was stable for 1 month at -18°C and completely inactivated at 90°C after 10 min. Adams and Brawley (1981) found an extra—cellular lipase produced by Pseudomonas gp MC50 to be extremely heat resistant at 100-1500 in water or emulsion. Lamberet and Lenoir (1976) found Penicillium caseicolum lipase was 35 stable at 30°C within pH 7.0-8.5. The stability was low at 37°C and pH 8.0. In general, thermostability of a lipase varies with the type of lipase, or microorganism, its strain, degree of enzyme purification, substrate and pH. Table 5 presents a summary of thermal stability of various microbial lipases. It appears that fungal lipases are relatively stable when compared to pancreatic lipase (Weete t 1., 1974). :FE oF oo oo mzoch>ew Loos: A ova .H1 Ml Fo :FE mF om om FF vcm F EsmOFmscu 53F :FFochm :3: l Wm 8.32sz FE 2 mm om o llllleeeeFee flair; Fmova .Hl Ml opossxza cFE mF om om euer szFFmeeam< FmFmFV .Hm mm consecu omm mF F.FF ooF mF>wLn szFFuenOFoop $3: .lFl flu. 3:28 FE: Om om 8F 2:; 2:62:33 :3: mcmvsozwmpm ucm :memmFo 8mm oF Nmiom ooF mFFomFoomF> chme FFmoF< FOFmFV .H1 m1 EFoccwuo cFE om mo mF FFcoEmem EchmpomnFcoFroa 6 memva :oELaI use mczwum> :FE om mo om mzmezm msooooonggmpm 3 3;: .fl fl 5:; FFE 3 mm 2: 53:3an FF 382052;: 11 ll FFszFFmomcpxmv FFomFV .F p gang :FE om m.FF Fe EzoFFzFanF cmpumnoEocgu< Fog 3 mme4 new :4 uceFmeFvu uFOFF< ccm cmcucwz :FE om o.mm m.FN mecF mmcosouswma Femmva comsz new Fchmz :FE om m.FF om FmoFF mmcoEouzmmm & u FFF>FFUe mocmcmem «EFF waspmcmaEmF Fo mmoo Echmmcoocon .mwmea FanocoFE mzoFLm> Fo xFFFanmemELmse .m an©F 37 FNFQF .OFmFV Fwevam nee Fe Femva Feova FoFmFV .am FoFva .Hm FcFmFV .F Fmeva .F mm eFo .F mF .Hm .Hm mm MM p .Fw e le ewwemeF : 0 Fa pm Fmpoz F estFEOF MM eszFEOF LmFFFEFEuFm LwFFFECmFFm eeFFFEeeFFm MM w>FFmEmm om oF om Om oF om om ma mm mm mw om ov mm om mq go Nm ooF om mo FeeFFFeeav monxFan Fm mm mcFucmu FFaoccm mquonFOH FF amoeechFww mchcmu F mmomcncFFxo acFvceo FFLOszmdL EzF FFFFchwa FFLOFwsmOF EsFFFFocha FFFOFummoc EsFFFFocha m3~Fccem mJQONch .F.e.FeeeV .m eFeeF Action of Microbial Lipases on Natural and Synthetic Lipids According to Alford gt _t. (1964) Penicillium roqueforti lipase preferentially hydrolyzed short chain fatty acids flpm butter fat. Eitenmiller gt gt. (1970) found the same organism to be more specific for tributyrin. The rate of hydrolysis in decreasing order was: tributyrin, tricaprin, tripropionin and triolein. Hassing (1971) observed that lipase activity of Coryggbacterium acnes was highest against tributyrin. Yamaguchi gt 1. (1973) studied the action of Chromobac- terium visc05um on a variety of natural fats and oils. The lipase was more active against lard and butter than against olive oil. Chander t l. (1973) found lipase activity of Lactobacillus brevis to be greater for simple trigly- cerides. Tripropionin was attacked more easily than tri- caprion, tricaprylin and more readily than butter oil and coconut oil. Sugiura gt a1. (1974) used different sub- strates for purified lipase A of Chromobacterium viscosum var paralipolyticum. The lipase was more active toward water insoluble triglyceride such as tributyrin or tri- propionin, but exhibited a weak activity toward water soluble substrate such as triacetin or tweens. Iwai t al. (1975) found the activity of both A and B lipases of Penicillium cyclopium was high against vegetable oils such 39 as olive oil and popy oils. The action of lipase on tung oil and linseed oil was remarkably low. Chander _t _t.(1979a)purified Streptococcus faecalis lipase. The enzyme hydrolyzed tributyrin > tricaprion > tricaprylin > triolein. The rate of hydrolysis of natural oils was as follows: butter oil > olive oil > linseed oil > coconut oil. On the other hand,Chopra t al. (1980) reported lipase of Aspetgillus wentii preferentially hydrolyzed tricaprylin as compared to tricaproin, tripropi- onin and tributyrin. They reported the hydrolysis of butter oil was greater than olive oil, followed by coconut oil, mustard oil, and cottonseed oil. Nature and Specificity of Lipolytic Enzymes Lipolytic enzymes, Such as lipases and esterases are involved in degradation and metabolism of fat. Shahani (1975) described the lipolytic enzyme as a hydrolase, catalyzing hydrolysis of the carboxylic acid ester bonds. Desnuelle (1972) differentiated lipases from esterases on the basis of physical state of the substrate. A lipase attacks substrate molecules in emulsion or insoluble form. An esterase attacks the substrate present in true aqueous solution. Wills (1965) and Jensen (1971) also defined lipase as an enzyme hydrolyzing the ester bond in emulsi- fied glycerides at an oil-water interface in an insoluble 40 or heterogeneoussystem. According to the International Union of Biochemistry (1961) lipase is a common name for "glycerol ester hydro- lase” E.C. 3.1.1.3 that catalyses the deacylation of acylyglycerides and phospholipids. The action of lipase on triglycerides is illustrated in Figure l. 41 Slim 5 .s..>> .30: use 6.0 c2325 ".0... .0325 Hogsom 83.00sz Fo cozmuocmou omen: F 939". Newloioax Newbie“: :06 I O _ O _ UH" _ a 1.010: 14.1 1.0.02 20.255559 zlololw m _ :0-“me F _ _ o role“: m mwlolonz :lwlolo o o :o. l .0. :oloam 40585.0 m 40505956. zolouz «twelve: nzwoloaz malololoa I _ o _ o _ :oloFm _ a 11010: 14: 1101 O: J IIUIOI = Ii QIOIw m _ :06. m _ :olwlum _ o _ o :06“: o 10.0“: o «mwoloez m 0.010 I o o IOIWIME IOImv PK 0 TBA—on: gin—UN: mlolo o I .0- I N I II' I I m I 0.0: 29:55.28. 0 a we n : n_. o w _ _ o :06. m _ o a l l a a .. a F. mo 0 : :olo : o :010 x O 42 Lipases differ in relation to substrate, positional and fatty acid specificity (Desnuelle and Savary, 1963; Brockerhoff and Jensen, 1974; Shahani, 1975; Weete _t _t., 1974). The hydrolysis of monoglycerides to glycerol and fatty acids is generally very slow due to the isomeriza- tion of 2 monoglycerides to position 1 and 3 (Borgstrom and Ory, 1970). Shipe (1951) and Eitenmiller gt _t. (1970) reported that lipase of Penicillium roqueforti was specific for short chain fatty acids. Alford gt _t. (1964) reported that most lipases from fungi preferentially cleave fatty acids from C-l position of triglycerides. Aspergillus flavus showed no positional 9 specificity. Geotrichum candidum had specificity for A unsaturation regardless of its position on triglyceride molecule. Jensen (1974) investigated the specificity of a purified extracellular lipase of Geotrichum candidum had no absolute specificity for fatty acids containing cis 9 or Cis 9,12 unsaturation. The enzyme hydrolyzed both the fatty acids regardless of position in triglycerides. Rhizopus arrhizus lipase has specificity for position C-1 and C-3 on triglycerides. Under certain conditions the C-2 acyl group migrates to position 1 and 3 (Weete gt gt., 1974; Semeriva _t _t., 1967). The extracellular lipase of Mgggt pusillus shows maximal activity toward C12 fatty acid (Somkuti and Babel, 1968). Mucor javanicus preferen- tially cleavaged the ester bonds of C—1 and C—3 position 43 of triglycerides (Weete and Weber, 1980). Lactic acid bacteria show preference for short chain fatty acids (Fryer gt 1., 1967a;Umemoto t 1., 1968; Oterholm gt al., 1970; Dovat t al., 1970; Chander gt_al., 1973; Formisano _t _t., 1974; Chander gt _t., 1979a). Also these bacteria are reported to hydrolyze complex and neutral lipids with higher fatty acids (Umemoto gt gt., 1968; Angeles and Marth, 1971; Chander gt gt., 1979). Role of Mold in Cheese Flavor During cheese ripening,a number of chemical reactions proceed. Many of the reactions are mediated by microbial and native milk enzymes (Scott, 1972a; Dwivedi and Shahani, 1973). Proteins, fats and carbohydrates are degraded to varying degrees to yield a complex mixture of compounds, which contribute to cheese flavor (Pack g fl” 1968). Scott (1972) stated that ripening cheese is essentially an enzymatic process, at death, of bacteria and molds assist in the generation of the final flavor. In the mold-ripened cheeses,Roquefort, Gorgonzola, Stilton, and blue cheese, the Penicillium roqueforti enzymes play important rolesin flavor development. The blue-green mold produces substantial amount of lipase which leads to accumulation of free fatty acids, mainly caprylic, capric and caproic acids which in turn are involved in the formation of 44 flavoring compounds (methyl ketones) (Margalith and Schwartz, 1970). The methyl ketones, especially 2—heptanone and 2-nonanone are generally considered as the key flavor components of blue cheese (Day, 1976). Hawke (1966) stated that the formation and metabolism of methyl ketones by fungi in mold ripened cheeses consist of four main enzymatic mechanisms: 1) Liberation of free fatty acids from the triglycerides of milk fat by lipases. 2) Oxidation of the free fatty acids to B-keto acids. 3) Decarboxylation of B—keto acids to methyl ketone. 4) Reduction of methyl ketone to secondary alcohol. Some workers report Penicillium roqpeforti spores as well as mycelium are capable of producing methyl ketones from fatty acids (Gehrig and Knight, 1958; Lawrence, 1968; Dwivedi and Kinsella, 1974). Some investigators studied the lipase of Penicillium caseicolum. Lamberet and Lenoir (1976) found the optimum pH of the crude lipase to be 8.5 at 30°C. The purified lipase had optimum pH of 9.0-9.6 and optimum temperature of 35°C (Lamberet and Lenoir, 1976a). Moinas _t _t. (1975) reported contribution of certain minor compounds in Camembert cheese flavor. Such compounds are aromatic, hydrocarbons and ketones, unsaturated alipha- tic ketones, aromatic esters, an aldehyde containing sulfur and a nitrite. Proks and Cingrosova (1962) showed that 45 Penicillium caseicolum lipase attacked preferentially the lower chain saturated fatty acids. Little activity towards the unsaturated acids was observed. According to Kornacki t l. (1979) lipase of Penicil- lium rogueforti and Penicillium candidum released fatty acids with chain length of C4 - C10 at both pH 6.6 and 8.6. The amount of C4 and C6 acids liberated were up to 10 times as high as their content in milk fat which was used as the substrate. Adda t l. (1978) reported Penicillium casei- ggtgm plays a major role in Camembert cheese flavor. The free fatty acids present in large quantity contribute to the overall cheese flavor and serve as a substrate for the formation of methyl ketone and 2-alkanols. It was found that oct-l-en-3-ol was specifically involved in the plea- sant mushroom flavor in Camembert cheese (Moinas t al., 1973; Dumont _t _t., 1974; Groux and Moinas, 1974). Oct—l- en-3-ol was shown to be produced by several Penicillium gp by oxidation of unsaturated fatty acids. An atypical off- flavor has been observed in cheese with some strains of Penicillium caseicolum which produce large amount of oct-l- en-3-ol. The proteolytic activity of Penicillium camemberti or Penicillium caseicolum is also fundamental for cheese ripening and the typical flavor of Camembert cheese. The proteolytic activity of Penicillium caseicolum was reported by several workers (Proks and Cingrosova, 1962; Lenoir and Choisy, 1971; Gripon t 1., 1977). 46 Soltys _t _t. (1973) found that, during Camembert cheese ripening, there was a decrease in the level of casein to 50% of the original level. Simultaneously, there is an increase in nonprotein nitrogen to 25% of total nitrogen, ammonium nitrogen to 20% of soluble nitrogen, and amino nitrogen to 10% of soluble nitrogen. Also triglycerides decreased from 90% to 70% and free fatty acids increased from 1 to 10%, with very little increase in mono- and di- glycerides. MATERIALS AND METHODS Four strains of Penicillium caseicolum were procured from G. Roger Laboratories (B.P. 20, 77260 La Ferte Sous Jouarre, France), and one strain was a gift from Centro Sperimeatale Del Latte (Via Salasco 4, Italy). Roger Laboratories recommend the mold growth on solid media containing sugar and peptone at 23°C and 70% relative humidity. The specification data show that the first germination appears within 6—9 h. After 11 h of culti- vation, 70-95% of sporesgerminate. The mold grows at pH 4—8. The time for growth varies from one strain to another. Penicillium caseicolum is a halophilic organism capable of tolerating salt concentration used in cheese making. The main characteristics of different strains of Penicillium caseicolum are listed in Table 6. Spore Count The spores of Penicillium caseicolum were counted by using improved Neubauer Haemocytometer (Thomas, A.H. Co. 1981). Penicillium caseicolum was grown on Czapek-Dox agar slant for one week at 25 i 1°C. The spores were scraped from the slant under aseptic conditions using sterilized 0.1 M phosphate buffer, pH 7.0. The spores were suspended 47 48 Table 6. Characteristics of different Penicillium casei— colum strains*. Mold strain Main characteristics Penicillium caseicolum Produces aerial and dense mycelia. C1 and C2 Givesmedium mycelia layer on cheese. The growth is more diffi— cult on curd poor or low in lactose content, leading to an earlier sporulation. Penicillium caseicolum Requires low energy. Produces B5 dense layer of mold and regular growth in ripening room. Grows well in poor or low lactose curd. Penicillium caseicolum A rustic strain that produces K5 dense mycelia. Growth is not influenced by composition of media. It should be mixed with other strains in cheese making. *G. Roger Laboratories Manual. 49 in 100 m1 of the phosphate buffer. One drop of the suspen- sion was placed on Haemocytometer. The spores were counted under a microscope and the total count calculated as follows: 1. The average spore count of 10 small squares multi- plied by 16 x 25, to get the count in large square. 2. Large square count multiplied by 10,000 to get the counts in 1 ml. Fungal Maintenance and Inoculation The commercial lyophilized spores of Penicillium casei- ggtgm were suspended in 10 ml of sterilized distilled water and transferred to sterilized Czapek-Dox agar slant. The slant was incubated at 25 i 1°C for 1 wk. The culture was transferred daily to new slants. In general, Penicillium caseicolum culture grown for 1 wk was used in all stages of this investigation. The growth from a slant was suspen- ded in 100 ml sterilized 0.1 M phosphate buffer, pH 7.0. One percent of this suspension was inoculated in the broth media used in this study. Growth Media Five media commonly used for growth of fungi were inves- tigated for growth studies. They were: Czapek—Dox broth, Sab0uraud Maltose broth, Sab0uraud Liquid medium, Maltose extract broth and mycological broth (Difco Lab). The composition of these media are listed in Table 7. 50 Table 7. Composition of growth medium (per liter). Medium Composition pH Czapek-Dox Broth Saccharose Difco 30 g Sodium Nitrate 3 g Dipotassium phosphate 1 9 Magnesium sulfate 0.5 g 7.3 Potassium chloride 0.5 g Ferrous sulfate 0.01 9 Sab0uraud Maltose Neopeptone 10 g broth 5.6 Maltose Difco 10 9 Sab0uraud Liquid Neopeptone 10 9 medium 5.7 Bacto—Dextrose 20 g Mycological broth Bacto soytone 10 g Bacto Dextrose 40 g Malt extract broth Special extract of malt by Difco 4.7 Source: Difco manual of dehydrated culture and media reagents (1972, Ninth edition, Difco Laboratories). 51 The media were sterilized at 121°C (15 psi) for 15 min. Spores and mycelia Suspension of Penicillium caseicolgg (5 ml) were inoculated in 500 ml media. The media were incubated at 23 i 1°C under stationary conditions. The growth was harvested after 4, 7, 10 and 14 days of incuba- tion. Dry cell weight were used to evaluate the growth of Penicillium caseicolum. Cell growth in Czapek-Dox broth and mycological broth was better than in other media. Therefore these media were used in subsequent work. Influence of Stationary and Submerged (Shaking) Condition on Growth and Lipase Production of Penicillium caseicolum This investigation was carried out using 100 ml each of Czapek-Dox broth and mycological broth. The media were sterilized at 121°C for 15 min (15 psi) and inoculated with 1% suspension of Spores and mycelia of Penicillium casei- ggtgg. The media were incubated under stationary as well as submerged (shaking)conditions at 25 i 1°C. The flasks were shaken at 120 rpm (Shaker Model G-25 New Brunswick Scientific Co., New Brunswick, NJ). The fungal growths were harvested at 4, 7, 10 and 14 day intervals. The lipase activity was determined in cell—free broth. Influence of Oil Addition to the Growth Media of Penicillium caseicolum Butter oil, corn oil and olive oil were chosen for this study. The investigation was carried out using 100 ml each 52 of Czapek-Dox broth and mycological broth containing 1% of the oil in 250 m1 Erlenmeyer flasks. The media were sterilized at 121°C for 15 min under 15 psi. One percent suspension of Penicillium caseicolum spores and mycelia were inoculated in the media. Incubation under submerged (shaking)condition was at 25 i 1°C (room temperature) and 120 rpm. The lipase activity in cell-free broth were deter- mined at 4 and 7 days of culturing, also on daily basis. Influence of Incorporating Soytone into Czapek—Dox Broth on Lipase Production by Penicillium caseicolum Mycological broth contains soytone while the Czapek—Dox broth does not contain any organic N source. The experi- ment was designed to determine the effect of incorporating soytone and different oils into Czapek-Dox broth on lipase production. To 100 ml of Czapek-Dox broth 1% each of soytone and the oil was added in 250 ml Erlenmeyer flasks. Growth conditions were similar to those described earlier. Influence of Medium Composition on Growth and Lipase Pro- duction of Penicillium caseicolum Mycological broth (composition: 4% dextrose, 1% soytone) was used as a basal medium for studying the influence of various components on growth and lipase production by Penicillium caseicolum. Corn oil (1%)was added to the 53 medium in all the studies, unless otherwise indicated. Effect of Nitrogen Sources In this phase of the study, soytone in mycological broth was replaced with equivalent concentration (1%) of casein, casamino acid, trypticase, proteose peptone, peptone, sodium caseinate, whey protein concentrate Q9% or whey protein concentrate (20%) as a source of nitrogen. Effect of Carbon Sources The dextrose was replaced with the same level of lactose, fructose, maltose, galactose and sucrose in the basal medium. Growth and harvesting procedures were the same as described earlier. Influence of MineralsAddition to Growth Medium An experiment was conducted to study if incorporation of selected minerals in the growth medium would influence growth and lipase production by the organism. Various minerals were added to mycological broth medium supplemented with 1% corn oil. The minerals were: NaN03, 0.3%; KZHP04—7H20, 0.1%; M9504, 0.05%; KCl, 0.05%; FeSO47H20, 0.001%; and CaSO4, 0.01%. The sterilized media were inocu- lated with 1% suspension of the culture of Penicillium caseicolum under aseptic conditions. For growth, submerged (shaking) condition at 120 rpm and 25 i 1°C was used. The media were harvested after 3 days for evaluation of growth and lipase production. 54 Influence of pH, Temperature and Agitation on Growth and Lipase Production of Penicillium caseicolum The influence of these factors was studied by cultiva— tion in a 7-liter bench-top fermentor (Microferm Fermentor, New Brunswick, NJ) equipped with agitation, aeration and automatic temperature control. The fermentor was connected to a constant pH controller (Virtis Digital pH controller, model 43 DPH CR) which was operated with a Virtis pH pump, model 43 PH-PE (Virtis Company, Gardiner, NY) and autoclava- ble pH electrode (Ingold Electrodes, Inc., Andover, MA). When the pH of the culture medium fell below the desired pH, the system delivered appropriate amount of l N NaOH to keep it at the desired pH. The culture vessel, air filter and tubings were autoclaved under 15 psi and at 121°C for l h. Three liters of mycological broth containing 4% dextrose, 1% soytone and 1% corn oil were prepared and placed in the culture vessel. The medium was sterilized in the culture vessel under 15 psi and at 1210C for 20 min, cooled to room temperature and inoculated aseptically with 1% suspension of Spores and mycelia of Penicilligg caseicolgm. 1) Influence of pH was investigated at pH 4.7, 5.5, 6.5, 7.0, 7.5 and 8.5. The temperature was maintained at 25 i 1°C, aeration at 200 cc/min and agitation at 120 rpm. 2) Influence of temperature. Experiment was carried out at 25°C and 30 : 1°C. Other conditions of growth were: agitation 120 rpm, aeration 200 cc/min, and pH 7.0. 55 3) Influence of agitation of the medium. This study was conducted at constant pH 7.0, and at 25 t 1°C. The aeration rate was 200 cc air/min. Agitation rates were 120, 200 and 500 rpm. In all these investigations, the pH electrode was auto— claved at 15 psi and 121°C for 15 min. It was also chemically sterilized using lysol and alcohol before placing it in growth medium. The NaOH solution was also sterilized at 121°C/20 min. All other parts were chemically sterilized before their assembly to the fermentor. The evaluation of growth and lipase production was carried out on daily basis for 5 days. Measurement of Growth The growth of Penicillium caseicolum was evaluated at all stages of this investigation by dry cell weight. The method was adopted from the procedures of Calam (1969) and Mallette (1969) to establish specific conditions to obtain constant weight of dry cells. Drying of the mycelial cells was done on a Whatman filter paper #5 which was itself dried at 100°C under vacuum (30 inch) for 5 h. A constant weight was reached under these conditions. The dried filter papers were stored in a desiccator. To measure fungal growth, filter paper was weighed and 100 m1 of the culture was filtered through the filter paper on a Buchner funnel. The cells were washed twice with distilled water. The filter paper containing the growth was dried at 100°C 56 under vacuum 30 inch for 5 h to reach constant weight. Evaluation of Lipase Activity 1. Silica Gel Assay Method. Lipase activity in cell free broth of Penicillium caseicolum was assayed by silica gel chromatographic method. The technique was established by Harper gt gt. (1956) and modified by Chandan (1962). Free fatty acids liberated by the action of lipase on substrate are extracted chromatographically and titrated by a standard alcoholic KOH. Materials and Reagents a. Chromatographic column: 38 mm in diameter and 230 mm in length, with a fritted glass disc sealed into 34/28 standard taper joint. b. Silicic acid: 100 mesh powder. c. 2 M phosphotase buffer pH 6.4. Stock solution of 2 M NaH2P04 (27.2 g/100 ml) and 2 M NaZHPO4 (34.8 g/100 ml) were prepared and two solutions mixed to obtain pH 6.4. d. Buffered silica gel slurry. Fifty grams of dry Silicic acid were mixed thoroughly with 30 m1 of 2 M phos- phate buffer and 200 m1 U.S.P. chloroform. The slurry was stored in a tightly stoppered brown bottle under refrigera- tion. e. Eluant: Five parts of n—butanol in 95 parts of chloroform (v/v). 57 f. Titrating reagent. 0.01 N ethanolic KOH. g. Phenol red indicator (0.1%). Phenol red (100 mg) was ground in 0.1 m1 of 0.1N KOH and made to 100 ml with absolute ethyl alcohol. h. 20% (V/V) sulfuric acid. Preparation of Assay Sample The substrate buffer oil (20%) was mixed with 10% gum arabic solution and homogenized at 60-65°C five times in a hand homogenizer. The pH of the emulsion was adjusted to 6.5. Five milliliters of the substrate and 5 ml of cell-free broth were mixed and incubated at 25°C for 2 h. Following incubation the enzyme—substrate mixture was acidified to pH 1.8—2.0 with 0.3 ml of 20% H2504 to stop the reaction. Silica gel (18-20 g) was added to the reaction mixture and thoroughly mixed and ground. Preparation of Chromatographic Silicilic Acid Column The column contained two sections: a. Bottom Section. The column was attached to 500 m1 suction flask. A filter paper disc (Whatman #5) was placed on the fritted glass disc bottom of the column. Silica gel slurry (25 ml) was placed on the top of this section. Another filter paper disc was placed over the slurry. b. Top Section. Silica gel-acidified lipolyzed sample was slurried in 50 m1 of 5% n—butanol in chloroform, then transferred quantitatively to the top of the column. This 58 was repeated two times with 50 ml each of the eluant. In order to extract the free fatty acids, suction was applied to give an eluate rate of 30 ml/min. After elution, 0.3 ml of the phenol red indicator and 15 m1 of absolute alcohol were added to the eluate. The eluate was titrated with 0.01 N alcoholic KOH. The titer value was corrected for the initial free fatty acid content by extraction of control sample containing cell free broth (enzyme) and substrate without incubation. Lipase Activity The activity of lipase was expressed as micromoles of free fatty acids liberated/5 m1 of cell free broth of Penicillium caseicolum. The resultsexpressed in this dissertation represent average values for a mimimum of 3 trials, unless otherwise indicated. 2. pH-Stat Method pH-Stat equipment was composed of four basic modules: 1. Titrator (E526 Metrohm Herisau) 2. Motor Drive Piston Burette (E525 Metrohm Herisau) 3. Potentiometer Recorder (Servogor 210) 4. Constant temperature circulator Model 80 (Fisher Scientific Company). The substrate was an emulsion of 10% butter oil or tributyrin in 10% gum arabic solution. The substrates were 59 prepared at 60-65°C and homogenized 5 times with a hand homogenizer. The pH value was adjusted to desired pH with 0.1 N NaOH. The pH-Stat was standardized at the desired pH and temperature. Five milliliters of the substrate were placed in the reaction vessel and allowed to equilibrate for 2-3 min. Cell free broth (0.2 ml) of Penicillium caseicolum was then added to the substrate. AS a result of lipase action, free fatty acids were liberated in the reaction mixture. Accordingly, the pH tended to drop below the set value. However, it was automatically adjusted to the set pH with 0.02 N NaOH solution. The amount of alkali utilized with respect to reaction time was registered in the potentiometric recorder chart. The time for the test was varied from 5-10 min and the slope of the chart curve was used in calculation of lipase activity. The lipase activity was expressed as micromoles of free fatty acids or butyric acid liberated/min. Lipase production was expressed as micromoles of free fatty acids/0.2 cell free broth/min. There are some advantages of the pH—stat over silica gel method. In the pH-stat method, no extraction of free fatty acids is necessary and no manual adjustment of pH is required. Also no indicator color change is required to be detected. It is a direct method for determination of lipase kinetics as initial velocities are measured (Brockerhoff and Jensen, 1974). According to Parry t al. (1966) this method is more sensitive than silica gel method. It has some disadvantages. 60 However, lipase activity at pH 7.00 is difficult to measure due to incomplete titration of fatty acids with low dissociation constants. Also there is interference with titration from a buffer or protein present in the reaction mixture. This method was used for characterization of the lipase under various conditions as well as for assaying the lipase activity of various cell free extracts. The assay pH was 9.0 to insure complete titration of butyric acid. Characterization of Lipase i Several experiments were conducted to characterize the lipase of Penicillium caseicolum. The mold was grown in 100 m1 mycological broth containing 1% corn oil under shakingcondition at room temperature (25 i 1°C, 120 rpm) for 3 days. The growth was harvested and the cell-free broth was assayed for lipase by the pH-stat method described above. a. Optimum temperature. Temperatures tested were: 25, 30, 35, 40, 45, 50 and 55°C for determining the optimum temperature of lipase activity. The pH of substrate was 9.0 and 0.2 ml of cell-free broth was added to 5 m1 of the substrate. The substrate contained 2 mM CaClZ. Both butter oil and tributyrin were used as substrates. b. Optimum pH. The investigation was conducted at optimum temperature of 35°C and pH was varied from 5.0-11.0. The substrates were butter oil and tributyrin. 61 c. Relationship of lipase activity and enzyme concen- tration. The reaction was carried out using butter oil and tributyrin as substrate at 35°C and pH 9.0. The enzyme concentration varied from O-O.8 ml. d. Addition of certain salts. In this experiment 1-10 umoles of each of sodium taurocholate, sodium desoxy- cholate and CaClz were added individually or in combina- tions to the reaction mixture. The substrates were butter oil and tributyrin. The temperature of reaction mixture was 35°C and the pH was 9.0. e. Thermostability of the enzyme. Table 8 outlines the time-temperature used to determine stability of the lipase. The cell-free broth obtained from 3 days of growth (.2 ml) was added to the reaction mixture maintained at 35°C and pH 9.0. The pH—stat Method was used to determine the enzyme activity. Substrate Specificity Lipase enzyme (cell free broth) was obtained after 3 days of growth in mycological broth containing 1% corn oil. The growth was carried out under submerged condition at 120 rpm and at 25 i 1°C. The lipase activity was deter- mined by pH—Stat method. Substrate contained 10% natural and synthetic lipids. The pH of the reaction mixture was maintained at 9.0 and at 35°C. The assay was carried also by the addition of 8 umole sodium taurocholate and 8 mM 62 Table 8. Time-temperatures used in the determination of stability of Penicillium caseicolum lipase. Temperature (°C) Time -26.6 0, l, 2, 3, 7 and 30 days -15 0, 1, 2, 3, 7 and 30 days 0 0, l, 2, 3 days 4-5 0, l, 2, 3 days 25 0, 6, 12, 18, 24, 48 and 72 h 37 O, 6, 12, 18, 24, 48 and 72 h 45 O, 6, 12, 18, 24 and 48 h 55 O, 6, 12, 18 and 24 h 62 7 30 m1n 100 2 4 and 6 min 121 15 min 63 CaCl2 to the substrate. The lipase activity was calculated as pmole free fatty acids liberated/0.2 m1 cell free broth. Concentration of the Enzyme To the cell free broth containing the enzyme, ammonium sulfate was added to 70% saturation at 4°C and held for 2 h to ensure complete precipitation. The suspension was centrifuged at 11,000 rpm for 2 h at 4°C in Sorvall RC ZB Automatic Refrigerated Centrifuge (Dupont Co., Newtown, CT). The pellet was dissolved in 20 ml distilled water. The con- centrated material was dialyzed in a cellulose acetate dialy- sis tubing with a molecular weight cut off 16,000-18,000 daltons. The sealed tubing was placed in cold, distilled, demineralized water, stirred and kept at 4°C for 24 h. The water was changed after 8 h. The concentrated enzyme was kept frozen. Protein Determination Protein in the concentrated enzyme preparation was determined by Bio—Rad protein assay method (Bio-Rad Laboratories). This colorimetric method was developed by Bradford (1976). An acidic solution of Coomassie brilliant blue G-250 was used to bind the protein. The maximum absorbance for the dye solution shifts from 465 nm to 595 nm following binding to the protein (Resisner gt gt., 1975; Sedmak and Grossberg, 1977). A lyophilzed bovine gamma globulin was used to prepare the standard protein solution. 64 The absorbance of the protein dye mixture was read at 595 nm in a spectronic 20 spectrophotometer (Bausch and Lomb), following the color development for 10 min. In some stages of this investigation, protein in the dry cell weight was determined by Microkjeldahl procedure (AOAC, 1970). Procedure for Makigg Butyl Esters for GLC Analysis Butter oil substrate emulsion containing 10% fat was prepared as described earlier. The pH was adjusted to 9.0 with 0.1 N NaOH. The pH stat method was used for enzyme substrate reaction. The reaction mixture consisted of 9.0 ml substrate and 1.0 ml concentrated enzyme. The mixture was incubated at 35°C for 15 min and acidified to pH 1.9 with 50% H2504. The free fatty acids were isolated by a column chromatography procedure used by Blakely (1970). Butyl esters of the free fatty acid were prepared according to Supelco method (1979). The butyl esters were chromato- graphed on HP584OA gas chromatograph equipped with flame ionization detector (F10) and a Hewlett Packard 18850A GC Terminal was used for the analysis of the fatty acid butyl esters. The glass column (2 m x 2 mm i.d.) was packed with 15% diethylene glycol succinate (DEGS) on Chromosorb W80/ 100 mesh with acid wash. The instrument was operated under the following conditions: Initial temperature (TI) 40°C Time at T1 (t1) 0 min 65 Rate of temperature increase 8°C/min Final temperature (T2) 185°C Time at T2 25 min Injection temperature 210°C FID temperature 350°C Chart speed 1 cm per min Attenuation 10 Nitrogen carrier gas 30 ml per min Hydrogen flow rate 30 ml per min Air flow rate 200 ml per min' Standard fatty acid butyl esters were prepared under identical conditionsand used for identification of samples. Fatty acids were identified by their retention time as compared to standards. Procedure for Making Methyl Esters for GLC Analysis Butter oil was transesterified according to the proce- dure described by Shehata _t _t. (1970) as follows: One milligram of butter oil was dissolved in 2 ml petroleum ether andthe solvent was evaporated under a stream of nitrogen. The transesterifying reagent containedl.5 ml 0.5NNaOCH3 in methanol, six ml of petroleum ether and 2.5 m1 diethyl ether. Transesterifying reagent (25 pl) was added to the vial and quickly capped. It was shaken gently to insure complete mixing, and left at room tempera- ture for 2 min. Then 25 ul petroleum ether was added 66 quickly to wash the inner wall of the vial, capped imme— diately and left for 2 min at room temperature. The reaction mixture was ready for direct injection into GLC. Standard fatty acid methyl esters were prepared accor- ding to the procedure of Morrison and Smith (1964) and used for identification of fatty acids. RESULTS AND DISCUSSION Culture Media In the first phase of this investigation, an experiment was conducted to determine the best media for growth of Penicillium caseicolum Cl. Five fungal media were chosen for this experiment. The growth was carried out under stationary conditions at 23 i 1°C for 4, 7, 10 and 14 days. The growth was evaluated by dry cell weight measurements. ReSultS are presented in Figure 2. Under the conditions studied, the best growth media for Penicillium caseicolum was Czapek Dox broth, followed by mycological broth, Sab0uraud maltose broth, Sab0uraud Liquid medium, and malt extract broth. The variation in growth of Penicillium caseicolum appears to be mainly due to the availability of nutrients for growth and synthesis activity. Czapek-Dox broth contains carbohydrate, inorganic nitrogen and miner- als. 0n the other hand mycological broth contains organic nitrogen, soytone. Soytone is a product of an enzymatic hydrolysis of soybean meal. The other media contain neo— peptone and a SOurce of carbon. The composition of soybean meal and neopeptone are presented in Table 9. It appears that soybean meal contains more nutrient to Support Penicillium caseicolum growth than is provided by 67 68 Figure 2. Growth of Penicillium caseicolum in different fungal media under stationary conditions at 23 321°C. DRY CELL WEIGHT (g/L) 69 INCUBATION TIME (DAYS) 70 Table 9. Composition of soy meal and neopeptone. Soy meal1 Neopeptone2 Difco Total Nitrogen % 14_3 NaCl 4.4 - Na 0.45 Ca 0.05 0.198 Fe 0.02 0.0041 Cl - 0.84 K 3.99 0.85 Mg 0.19 0.051 P 0.38 0.19 S 0.39 - Carbohydrates % 37 4 Amino Acids % - Arginine 4.6 — Aspartic Acid 5.8 - Cystine 0.5 39 Glycine 2.8 _ Glutamic Acid 9.3 - Histidine 1.6 - Isoleucine 2.5 - Leucine 3.2 - Lysine 3.6 - Methionine 0.6 — Phenylalanine 3.6 - Proline 3.4 — Threonine 1.8 _ Tryptophan 0.7 0.73 Tyrosine 1.9 4.72 Valine 2.0 — 71 Table 9. (cont'd). Vitamins mcg/g Biotin Choline Cyanocobalamin Folic Acid Niacin Pantothenic Acid Pyridoxine Riboflavin Thiamine (A) U‘I—l-b-P-NUOOOOOO .35 05 .00115 #000001 1Source: BBL Manual (1973) p. 2Difco Manual (1972) p. 265, _Not reported. 163, Detroit, Cockeysville, MD. 72 neopeptone. Czapek—Dox broth has most of the nutrient requirements for growth. These results are in agreement with the reports of several workers that Czapek Dox broth is an excellent medium for fungal growth (Raper and Thom, 1949). Nevertheless, mycological broth is shown by this work to be a reasonably good medium from the standpoint of the growth of the organism in stationary State. Accordingly, both Czapek-Dox broth and mycological broth were selected for lipase production studies. 73 Growth and Lipase Production of Various Strains of Peni— cillium caseicolum Table 10 Shows results of growth, lipase production and spore count of different Penicillium caseicolum strains. It was noticed that on the average pH of media dropped from 7.1 to 4.87 with different strains. Growth of different strains ranged from 8.146 to 12.092 g/l. The variation in the growth among the strains was very little except in strain K5 which showed appreciable lower growth. The spore counts were 8.4x106 to l x 107/m1. There was no relationship between the growth and spore count. For example, strain C2 had lowest Spore count, but showed highest growth. Lipase activity was determined by pH—stat method. Butter oil and tributyrin were used as substrates. The highest lipase activity was with strain 85, followed by strains C1, C2, Italian source and K5. Strain B5 had lipase activity 3-16 times more than the other strains. The results indicate no relationship between the lipase production and growth. Little information about Spore count of Penicillium caseicolum is available in the literature. In this work the Spore count was determined after 1 wk of growth in Czapek—00x agar Slants, using a Haemocytometer. The Spore counts through all stages of this investigation ranged from 1.16x107 to 1.44x107 Spores/ml. Eitenmiller 74 SOUY‘CE Table 10. Growth, lipase production and Spore count of various strains of Penicillium caseicolum. Penicillium Lipase activity umoles Growth Spore caseicolum free fatty acids/0.2 ml/min Dry cell count strain Butter Butter Tributyrin weight /ml oil oil + 9/1 8 uM sodium taurocholate + 8 mM CaClg/ml C1 2.165 3.511 6.367 11.745 9.2x106 02 2.360 3.439 6.135 12.092 8.0x10° 35 4.500 6.867 17.079 11.597 8.4x10° K5 0.367 0.720 1.060 8.146 96x106 Italian 0.950 1.229 4.083 11.073 1x107 75 t 1. (1970) used 2x107 spores/ml in Penicillium rogue- forti work. Chander gt _t. (1980, 1981) inoculated Penicillium chrysogenum and Rhizopus nigricans at a rate of 2% of 1x107 spores/ml. Influence of Stationary and Submerged (Shaking) Conditions on Penicillium caseicolum Growth and Lipase Production Czapek-Dox broth and mycological broth were employed in this investigation. Growth was evaluated by dry cell weight and lipase production by silica gel chromatographic method. In Czapek-Dox broth (Figure 3), under stationary state the growth of Penicillium caseicolum increased during incubation time, but lipase activity reached maximum after 4 days and increased very little after 7 days. In submerged (Shaking) culture, relatively more growth was observed as compared to that in stationary state. The lipase activity was highest after 4 days of growth, then declined after 7 days. The lipase activity was 13.0 and 12.7 umoles FFA/5 ml cell free broth after 4 and 7 days of culturing under sta— tionary condition. For the same incubation period under submerged condition, lipase activity was much higher (29.49 and 15.74 umoles FFA/5 m1 enzyme). There was decrease in lipase production after 4 days in submerged (shaking) cul- ture, but it increased very little in stationary condition. hug; . .1 76 Figure 3. Growth and lipase production of Penicillium casei- colum in Czapek-Dox broth under stationary and submerged conditions at 25 i 1°C and 120 rpm. Lipase activity is expressed as micromoles of free fatty acids liberated/5 m1 enzyme/2 h. 77 Emu. mo_o_2 iv>h_>_.r0< mw> ._i_m0 >m0 INCUBATION TIME (DAYS) 78 In mycological broth, growth and lipase production of Penicillium caseicolum in both phases are presented in Figure 4. In stationary condition growth was 0.56 g/l and 3.26 g/l after 4 and 7 days,respectively. In the same period of time in submerged culture,the growth was higher (2.45 g/l and 5.84 g/l). The growth was higher 4.4 and 1.8 times after 4 and 7 days, respectively in submerged culture than in stationary culture. The lipase production was 11.62 and 13.67 pmole FFA/5 m1 cell free broth after 4 and 7 days, respectively in stationary culture. For the same period in submerged culture,lipase production was 28.10 and 16.8 umole FFA/5 ml of cell free broth. The lipase activity was appreciably higher in submerged culture than sta- tionary culture. The higher growth observed in submerged culture reflects the aerobic nature of the mold° The results obtained are in agreement with many workers (Calam, 1969; Belloc gt gt., 1975; Wang gt gt., 1979; Weete _t_gt,, 1974, Weete and Weber, 1980). There was no substantial difference between lipase activity produced in mycological broth and Czapek Dox broth. The highest lipase production was obtained in both media after 4 days, declining after 7 days in submerged culture. The loss in lipase activity may be due to storage inactivation of the enzyme. Figure 4. 79 Growth and lipase production of Penicillium caseicolum in mycological broth under stationary and submerged conditions at 25 i 1°C and 120 rpm. Lipase activity is expressed as micromoles of free fatty acids liberated/5 ml enzyme/2 h. 80 FE". 8.05. 3 >t>Fo< uni... T30 2 1 :\mv 5.7—Gm; 4.50 >mo INCUBATION TIME (DAYS) 81 Influence of LipidsAddition to the Growth Media on Lipase Production of Penicillium caseicolum The effect of oil addition to Czapek Dox broth on lipase production of Penicillium caseicolum is presented in Figure 5. In the absence of an oil, the lipase produc- tion increased during first 4 days of growth after which it declined. Addition of oils to the medium resulted in lower lipase production. As compared to control (without oil) the relative decrease in lipase production of Eggt; cillium caseicolum by oil addition is shown in Table 11. Considering lipase production withOut oil addition as 100 after 4 days, lipase production was 11.7, 45.6 and 71.7, respectively when butter oil, corn oil and olive oil were added to the medium. Apparently, the fatty acid composition of oil may be involved in this effect. In mycological broth the observations are shown in Figure 6. Addition of butter oil, corn oil and olive oil showed a stimulatory effect on lipase production. The lipase reached maximum activity after 4 days and declined almost to the same level as with butter oil and corn oil. The enzyme was higher with olive oil at 7 days of growth. The relative increase in lipase production by oil addition to mycological broth is listed in Table 12. Corn oil addition gave the highest increase in lipase production, followed by olive oil and butter oil. The dramatic stim- ulatory effect may be due to the linoleic acid content of Figure 5. 82 Lipase production by Penicillium caseicolum in Czapek-Dox broth containing various oils, under submerged conditions at 25 i 1°C and 120 rpm. Lipase activity is expressed as micromoles of free fatty acids liberated/5 ml enzyme/2 h. LIPASE ACTIVITY(,u Moles FFA) 83 1 l l l 2 3 4 5 INCUBATION TIME (DAYS) 1 6 I 7 84 Table 11. Influence of oil addition to Czapek—Dox broth on lipase production of Penicillium caseicolum after 4 days of growth under submerged condi- tion. Medium Lipase activity Relative lipase umoles FFA/5 ml/2 h production Czapek Dox broth 29.46 100 Czapek Dox broth 3.46 11.7 + butter oil Czapek Dox broth 13.43 45.6 + corn oil Czapek Dox broth 21.13 71.7 + olive oil 1c. 1. . .. . .0 in. II - l .. . . . . . .I.JI-1-¢VT‘;J£- ‘- Figure 6. 85 Lipase production by Penicillium caseicolum in mycological broth containing various oils under submerged conditions at 25 i 10C and 120 rpm. Lipase activity is expressed as micromoles free fatty acids liberated/5 m1 enzyme/2 h. LIPASE ACTIVITY( [u Moles FFA) 750 500 250 86 INCUBATION TIME (DAYS) 87 Table 12. Influence of oil addition to mycological broth on lipase production of Penicillium caseicolum after 3 days of growth. Media Lipase activity Relative lipase uMoles FFA/5 ml/2 h activity Mycological broth 36.11 1 Mycological broth 769.26 21.3 + corn oil Mycological broth 341.18 9.4 4 + olive oil W Mycological broth 74.96 2.1 + butter oil 88 the oil. Corn oil has 53% linoleic acid. Olive oil and butter oil contain 7% and 3% linoleic acid, respectively. Several workers found addition of lipids to the growth medium enhance lipase production of many microorganisms (Nashif and Nelson, l953a;Khan t 1., 1967; Ota t 1., 1968; Yoshida t 1., 1968; Iwai t 1., 1973; Akhtar et al., 1974; Umemoto and Sato, 1978; Chander _t gt., 1979). On the other hand, some investigators observed the inhibitory effect on lipase production (Smith and Alford, 1966; Eitenmiller gt_gL, 1970;Jonsson and Snygg, 1974; Chander gt_gt:, 1980, '1980a). In the next phase of this study, lipase production was investigated on a daily basis using mycological broth con- taining either butter oil, corn oil or olive oil. The results are shown in Figure 7. Lipase production was highest in the presence of corn oil after 3 days of growth. When butter oil was used, highest lipase production was observed after 4 days of growth. Using olive oil in the medium,the lipase activity was highest on the second and fifth days of Penicillium caseicolum growth. These results indicated that stimulation of lipase production varied widely with the type of oil in the medium. Under the experimental condition,corn oil stimulated the enzyme pro- duction more than olive oil or butter oil. It was noticed,after 3 days of growth,the pH of media dropped from 7.1 to 6.44, 5.92 and 5.83 when butter oil, olive oil and corn oil were added to the growth medium. 89 Figure 7. Lipase production by Penicillium caseicolum on a daily basis, in mycological broth containing various oils, under submerged condition at 25 i 1°C and 120 rpm. Lipase activity is expressed as micromoles of free fatty acids liberated/5 ml enzyme/2 h. 1 000 LIPASE ACTIVITY( ,u. Moles FFA) 500 9O A / \\ +OLIVE OIL /A\ l \ L-‘ // \\ 7“ \e \ \ + BUTTER OIL \ ‘/EL.\ \ ,’D_'_CI/ .n.\fl—u_¥s 1 l 1 1 l L 1 2 3 4 5 6 7 INCUBATION TIME (DAYS) 4 91 Mycological broth contains soytone as a source of nitro- gen. Czapek—Dox contains inorganic nitrogen and additional minerals. A study was conducted to include soytone in Czapek-Dox broth to investigate its effect on lipase pro- duction. Results are presented in Figure 8. It may be seen that addition of soytone to Czapek Dox broth increased lipase production from 13.43 to 452.65 umoles after 5 days of growth. However, the addition of soytone to Czapek Dox broth resulted in only 61% increase in lipase production as compared to the enzyme production in mycological broth. after 4 days with corn oil. The stimulatory affect could be due to the carbohydrates, minerals and vitamins in soytone. The relative increase in lipase production as a result of soytone addition is shown in Table 13. It appears that addition of soytone and corn oil to Czapek-Dox broth increased the lipase production 13.5 times. In case of olive oil and soytone addition, the increase in the lipase production was 11.1 times. Butter oil and soytone stimula- ted lipase production only 2.90 times. Corn oil was the most effective oil in lipase production of Penicillium caseicolum in mycological broth and in Czapek— Dox broth supplemented with soytone. A comparison of lipase production monitored on daily basis in both media is shown in Table 14. Lipase production in mycological broth was higher in the first 5 days. On last 2 days of incubation lipase production decreased considerably. 92 Figure 8. Lipase production by Penicillium caseicolum on a daily basis in Czapek—Dox broth containing various oils and soytone, under submerged condition at 25 i 10C and 120 rpm. Lipase activity is expressed as micromoles of free fatty acids liberated/5 ml enzyme/2 h. LIPASE ACTIVITY (,u. Moles FFA) 500 eh O O 300 200 —l O O 93 ./. \U #11" 1 l 1 l I 1 2 3 4 5 6 7 INCUBATION TIME (DAYS) 94 Table 13. Influence of addition of soytone to Czapek Dox broth on lipase production of Penicillium caseicolum (after 4 days growth . Media Lipase activity Relative lipase umoles FFA/5 ml/ production 2 h Czapek Dox broth _ 29.46 1 without oil Czapek Dox broth 398.55 13.5 + Soytone + corn oil Czapek Dox broth 326.40 11.1 + Soytone + olive oil Czapek Dox broth 85.48 2.9 + Soytone + butter oil 95 Table 14. Comparison of lipase production of Penicillium caseicolum on daily basis in Czapek Dox broth and mycological broth. Incubation Czapek Dox broth + Mycological broth time, soytone + corn oil + corn oil days Lipase activity, umoles FFA/S m1/2 h l 0.72 14.81 2 129.15 669.79 3 381.97 769.26 4 398.55 634.11 5 452.26 566.42 6 452.93 436.04 7 280.38 70.8 96 Influence of Different Nitrogen Sources on Growth and Lipase Production of Penicillium caseicolum The results of this study are presented in Table 15. The pH of the media dropped by 0.57 to 2.08, depending on nitrogen source. The dry cell weight (growth) was highest with soytone followed by proteose peptone, 29% whey protein concentrate; peptone, 20% whey protein concentrate, tryp- ticase, mycological broth without oil, casamino acid, casein and sodium caseinate. The growth differences seem to reflect A varying degree of amino acid uptake from different sources of nitrogen. The protein content in the growth medium appeared to be approximately related to the growth pattern. Lipase activity was highest with soytone, followed by peptone, trypticase, proteose peptone, sodium caseinate, 29% whey protein concentrate, casein, casamino acid, 20% whey protein concentrate and mycological broth without oil. It may be concluded that soytone supports excellent growth and best lipase production by Penicillium caseicolum. Also there was no relationship between the level of growth and lipase production. Several workers found peptone to be a good source of nitrogen for lipase production by certain microorganisms (Imamura and Kataoka, 1963; Hosono and Tokita, 1970; Chander _t _t., 1977, 1980a; 1981) In this investigation, soytone and peptone were generally comparable in their effect. Soytone produced slightly higher lipase than pemmne. 97 Table 15. Influence of different sources of nitrogen on Penicillium caseicolum growth and lipase produc- tion after 3 days (submerged condition) at ‘ 25 i 1°C and 120 rpm. Medium Dry weight Protein Lipase Relative Drop cell in funqal activity lipase in g/1 growth uMoles/ activity pH mg/l FFA/5 ml/ % 2 h Soytone 10.393 2247.00 702.19 100 1.03 Peptone 8.805 1966.00 689.52 98.20 1.03 Trypticase 6.496 1914.9 543.74 77.43 0.57 Proteose peptone 10.082 2029.2 539.87 75-88 1-17 Sodium caseinate 2.621 761.5 362.4 51 61 1.90 29% whey protein 8.985 1420.9 220.53 31 41 1.70 concentrate Casein 2.965 773.85 187.70 26.64 1.41 Casamino acids 3.047 915.7 158.52 22 62 1.20 20% whey protein 7.484 1533.3 75.114 10.70 2.08 concentrate Mycological broth 3.440 946.0 36.11 5.14 1.46 (no oil) 98 Influence of Different Carbon Sources on Growth and Lipase Production of Penicillium caseicolum Table 16 shows the effect of different sources of carbon in the medium on growth and lipase production of Penicillium caseicolum. It was noticed that pH of these media dropped 1.2 to 1.94 with different sources of carbon. The dry cell weight (growth) was highest with lactose followed by galac- tose, fructose, dextrose, sucrose and maltose. The protein ‘, content of dry cell weight was highest with fructose fol— Ea lowed by dextrose, galactose, sucrose, maltose and lactose. The lipase activity was highest with dextrose followed by sucrose, maltose, galactose, lactose and fructose. Lipase activity was 51.61, 51.32, 33.37, 30.12, 7.16 and 4.35 umoles FFA/mg of growth with dextrose, sucrose, mal— tose, galactose, lactose and fructose, respectively. Although the addition of different sources of carbon to the media increased the fungal growth in some cases, it does not necessarily increase the lipase production. No rela- tionship between the fungal growth and lipase production was evident. However, dextrose and sucrose were the best sources of carbon for both growth and lipase production stand points. These results are in agreement with the reports of some workers. Weete and Weber (1980) stated glucose was the most important source of carbon for growth of fungi. Chander gt gt. (1977, 1980a, 1981) found glucose to be a good source of carbon. Certain workers found 99 Table 16. Influence of different carbon sources on Peni— cillium caseicolum growth and lipase production (submerged condition) at 25 t 1°C and 120 RPM after 3 days. Carbon Dry cell Protein Lipase Lipase Relative source weight in the activity activity lipase 9/1 fungal uMole FFA uMoleS FFA activity growth /5 ml/2 h /mg 0f % mg/l growth Dextrose 10.103 2527.86 521.44 51.61 100 Sucrose 10.080 1770.98 517.30 51.32 99.4 Maltose 9.654 1640.87 322.20 33 37 64.7 Galactose 12.213 2416.68 367.82 30.12 58.4 Lactose 12.663 1060.25 90.72 7.16 13.9 Fructose 11.973 2899.41 52.14 4.35 8.4 100 glucose to inhibit lipase production (Imamura and Kataoka, 1963; Mates and Sudakevitz, 1973). Others reported no significant change in lipase production with different carbohydrates (Iwai and Tsujisaka, 1974). 101 Influence of Addition of Different Minerals on Penicillium caseicolum Growth and Lipase Production Results of this study are presented in Table 17. In general, the addition of minerals to mycological broth had inhibitory effect on the lipase production. The growth of Penicillium caseicolum was not appreciably affected by the addition of minerals. 102 Table 17. Effect of selected minerals on Penicillium caseicolum growth and lipase production after 3 days of growth under submerged condition at 25 i 1°C and 120 rpm. Medium % Dry Protein Lipase Relative mineral cell in activity activity in weight fungal uMoles FFA / media g/l growth /5 ml C611 mg/l free broth MB - 10.977 2245.9 697.02 100.0 MB+(K2HPO4) 0.1 10.459 2702.8 686.75 98.5 MB+KCl .05 10.765 2368.47 513.63 73.7 MB+Fes04 0.001 10.834 2158.4 506.21 72.6 MB+NaN03+K2HP04 0.3, 11.066 2401.14 476.02 68.3 +MgSO4+KCl+FeSO4 8'05 0. 05, 0. ooi MB+NaN03 0.3 10.985 3379.0 457.00 65.6 M8+Mgso4 0.05 11.368 2001.8 372.86 53.5 M8+6as04 0.1 10.052 1828.18 19.27 2.8 MB=Mycological broth —_H?=_M_~—yd 103 Influence of pH on Penicillium caseicolum Growth and Lipase Production The effect of pH on Penicillium caseicolum growth and lipase production was studied in a fermentor under pH-stat conditions. The lipase production was determined by pH-stat method using tributyrin and butter oil substrates with and without sodium taurocholate and CaCl2 addition. The growth of Penicillium caseicolum at different media pH levels is presented in Figure 9. In general, the lower the pH the better was the growth. On the fifth day at pH 5.5 and 6.5, the growth was higher than at pH 4.7. Lipase activity of Penicillium caseicolum against different sub- strates is presented in Figure 10, 11, and 12. At lower pH levels, the lipase production was very low. The lipase production increased with increase in the pH of media. However, lipase production was also low at a higher pH, such as 8.5. The lipase production reached a maximum after 4 days of growth at all pH levels except at pH 7.5 lipase pro- duction peaked at 3 days of growth. The lipase activity at pH 7.5 after 3 days of culturing was lower than at pH 7.0 after 4 days of growth. The relationship between lipase activity and pH was similar where tributyrin or butter oil was used as substrate. These results indicated pH 7.0 is the optimum pH for lipase production by Penicillium caseicolum. The results Figure 9. Effect of different pH on growth of Penicillium caseicolum at 25 i 1°C and 120 rpm. 105 11- 10- _ 8 .)7.0 A75 _ _ _ _ _ 7 6 5 4 3 33:19:, .33 En - 8.5 INCUBATION TIME (DAYS) Figure 10. 106 Effect of pH on lipase production by Penicillium caseicolum at 25 i 1°C and 120 rpm. Lipase activity expressed as micromoles of free fatty acids liberated/0.2 ml enzyme/min, was determined by pH—stat method at pH 9.0 and temperature 35°C toward tributyrin. LIPASE ACTIVITY (,1. Moles FFA) 107 INCUBATION TIME (DAYS) Figure 11. 108 Effect of pH on lipase production by Penicillium caseicolum at 25 i 1°C and 120 rpm. Lipase activity expressed as micromoles of free fatty acids liberated/0.2 m1 enzyme/min., was deter— mined by pH-stat method at pH 9.0, and temperature 35°C toward butter oil. LIPASE ACTIVITY (“Moles FFA) 109 INCUBATION TIME (DAYS) Figure 12. 110 Effect of pH on lipase production by Penicillium caseicolum at 25 i 1°C and 120 rpm. Lipase activity expressed as micromoles of free fatty acids liberated/0.2 ml enzyme/min., was deter- mined by pH-stat method at pH 9.0 and temperature 35°C toward butter oil containing 8 umole sodium taurocholate and 8 mmole CaC12/ml. 111 0 5. 5. 5 7. v7 6 M 4 l \.\. \ . \ w \. ... s .f/ .. a, a 1 o o. ’ o J /./. z _ ‘ul /./.l.c ID _ 'l I... ’I. / m l.../’ /./ .7... .y . D/l AMrD/ ”1 // ..//./ 7A”. fix 1 I .. . I I _ _ _ 3 2 1 2n."— m$.05. 3; >.:>_._.0< mwt>Fo< mean... 4 3 INCUBATION TIME (DAYS) 2 119 Characterization of Penicillium caseicolum Lipase Optimum pH of Penicillium caseicolum Lipase Optimum pH of Penicillium caseicolum lipase towards tri- butyrin and butter oil was determined by the pH-stat method. Resul ts are presented in Figure 15. The optimum pH of.Renjci'llium caseicolum lipase was at pH 9.0 toward both tributyrin and butter oil. As the pH increased or decreased in relation to the optimum pH, the rate of hydrolysis decreased. With ~M tributyrin there was a sharper decrease in the lipase acti- 2% vity than with butter oil. When using butter oil as a substrate there was very little activity at pH 5 and 11. Lambert and Lenoir (1976) reported the optimum pH of Penicillium caseicolum lipase to be around 9.0 to 9.6. 120 Figure 15. Effect of pH on lipase activity of Penicillium caseicolum, was determined by pH-stat method at 35°C toward tributyrin and butter oil. 121 // TRIBUTYRIN _ _ _ .— — _ .- 8 7 6 5 4 3 2 ENE 8.65.1 V >t>fio< mean... 0 BUTTER OIL 11 10 pH 122 Optimum Temperature of Penicillium caseicolum Lipase pH stat method was employed to determine the optimum tem- perature of Penicillium caseicolum lipase. Tributyrin and butter oil were used as substrates. The results are presented in Figure 16. The optimum temperature of the lipase activity was 35°C for both substrates. When compared to butter oil, there was very sharp decrease in the rate of hydrolysis of tributyrin as the temperature increased. Microbial lipases have been reported to be active over 1% a wide range of temperatures, but the optimum temperatures ‘ range is 30-400C (Hugo and Beveridge, 1962; Tomizuka, 1966; Vadehra and Harmon, 1968; Khan t 1., 1967; Umemoto gt 1., 1968; Eitenmiller gt al., 1970; Mosona and Tokito, 1970; Collins gt gt., 1971; Tsujisaka t 1., 1972; Iwai and Tsujisaka, 1974; Chander _t gt., 1979). Other investi— gators reported optimum temperatures for lipase of certain microorganisms to be higher than 400C (Tomizuka, 1969; Lawrence, 1967; Somkuti gt gt., 1969; Oterholm t 1., 1970; Liu _t_gt., 1973). Chopra _t gt. (1980) has reported optimum temperature for lipase activity of Aspergillus wentii as 25°C. Belloc gt g1. (1975) studied lipase activity of Penicil- ligg camemberti at 25°C. The results obtained in this inves- tigation are in agreement with those obtained by Lamberet and Lenoir (1975) who reported 35°C as an optimum temperature for lipase activity of purified Penicillium caseicolum lipase. 123 Figure 16. Effect of temperature on lipase activity of Penicillium caseicolum was determined by pH-stat method at pH 9.0 toward tributyrin and butter oil. LIPASE ACTIVITY ( ,u. Moles FFA) 124 I /A\ "11 ’3 MI’ TRIBUTYRIN\ \ \n surren OIL I I 4 I #L l L 25 30 35 40 45 50 55 TEMPERATURE °C 125 Influence of Bile Salts and Calcium Chloride on Lipase Activity of Penicillium caseicolum The influence of bile salts and CaCl2 on the lipase activity was investigated by the pH-stat method using tributyrin and butter oil as substrates. Different concen— trations of sodium taurocholate, sodium desoxycholate and CaCl2 individually or in mixtures were used. Cell free broth was used as the source of enzyme. When tributyrin was used as substrate (Figure l7),sodium taurocholate, sodium ldesoxycholate and CaCl2 had inhibitory effect on lipase activity. On the other hand,when butter oil was used as sub- strate (Figure l8),sodium taurocholate and sodium desoxy- cholate had stimulatory effect on lipase activity at all concentrations. Sodium taurocholate stimulated the lipase activity more than sodium desoxycholate. However, at concentration of 10 uMole sodium desoxycholate stimulated the initial rate of hydrolysis more than sodium taurocholate did. Calcium chloride had inhibitory effect on the rate of hydrolysis, except at 2 mmole concentration where it slightly increased the rate of hydrolysis. Mixtures of sodium taurocholate, sodium desoxycholate and CaCl2 in different concentrationswere studied for their effect on the rate of hydrolysis of tributyrin and butter oil. The results obtained in case of tributyrin are pre- sented in Figure 19. Sodium taurocholate, sodium desoxy- cholate and CaCl2 in any concentration of mixtures had Figure 17. 126 Effect of different concentration of sodium taurocholate, sodium desoxycholate and Ca612 on lipase activity of Penicillium caseicolum, Lipase activity expressed as micromoles of free fatty acids liberated/0.2 ml enzyme/ min., was determined by pH-stat method at pH 9.0 and temperature 35°C toward tributyrin. LIPASE ACTIVITY( M. Moles FFA) 127 LIIIIIIIIL 012345678910 CONCENTRATION p. Mole SODIUM TAUROCHOLATE AND DESOXYCHOLATE/ml or mMoIe CaCIz/ ml l Jfifliwl . Figure 18. 128 Effect of different concentration of sodium taurocholate, sodium desoxycholate and CaClz on lipase activity of Penicillium caseicolum, Lipase activity expressed as micromoles of free fatty acids liberated/0.2 ml enzyme/ min., was determined by pH-stat method at pH 9.0 and temperature 35°C toward butter oil. LIPASE ACTIVITY (”Moles FFA) 129 1 2 3 4 5 6 7 8 9 10 CONCENTRATION ,uMoIe SODIUM TAUROCHOLATE AND DESOXYCHOLATE/ml on mMOLE CaCIz/Ml Figure 19. Effect of different mix concentration of sodium taurocholate, sodium desoxycholate and Ca612 on lipase activity of Penicillium caseicolum. Lipase activity expressed as micromoles of free fatty acids liberated/0.2 m1 enzyme/min., was determined by pH-stat method, at pH 9.0 and temperature 35°C toward tributyrin. LIPASE ACTIVITY( )1. Moles FFA) 131 SODIUM TAUROCHOLATE +CaCI2 \ """ 0.. SODIUM TAUROCHOLATE + 4 _ \ SODIUM DESOXYCHOLATE \ +CaCI2 \ '0. \ \ \ x .. 2 — Ax“ a. SODIUM DESOXYCHOLATE +Cac12 ‘0‘\\ \ _ ‘A I I I I I I I I L I 012 345 6 7 8910 CONCENTRATION I“ Mole SODIUM TAUROCHOLATE, DESOXYCHOLATE AND mMole CaCIz/ ml 132 inhibitory effect on the initial rate of hydrolysis. Figure 20 shows the effect of mixtures of these salts on the initial rate of hydrolysis of butter oil. Sodium taurocholate + CaClZ and sodium taurocholate + sodium desoxycholate + CaCl2 in all concentration tested had a stimulatory effect as compared to no salts. With sodium taurocholate + CaClz, the highest increase in the initial rate of hydrolysis was at 8 umole and 8 mmole, respectively. On the other hand sodium desoxycholate + CaClz and sodium 1 taurocholate + sodium desoxycholate + CaClZ had stimulatory effect at lower concentrations and inhibitory effect at higher concentration. The results are in agreement with Belloc gt. 1. (1975) who used sodium taurocholate in assaying Penicillium camem— berti lipase. Lamberet and Lenoir (1976) used 2 mM CaClZ in the reaction mixture of Penicillium caseicolum. Lamberet + and Lenoir (1976a) found Ca+ ions to be required for maximum rate of lipase activity of Penicillium caseicolum. The results presented in this work indicate varying effects of the addition of the salts according to the substrate used. Figure 20. 133 Effect of different mix concentration of sodium taurocholate, sodium desoxycholate and CaClg on lipase activity of Penicillium caseicolum, Lipase activity expressed as micromoles of free fatty acids liberated/0.2 ml/enzyme/min., was determined by pH-stat method at pH 9.0 and temperature 35°C toward butter oil. Ll PASE ACTIVITY (M Moles FFA) 134 4 _ SODIUM TAUROCHOLATE +CaC|2 ’A\ ” f P SODIUM \\ I TAUROCHOLATE \ + A DESOXYCHOLATE \ SODIUM DESOXYCHOLATE . + CaCI2 O, . o \. 1c \. a. \. SODIUM TAUROCHOLATE ' D"-~.‘D + DESOXYCHOLATE + CaCI2 ...... .‘ ....... ..... . IIIIIIIIII 12345678910 CONCENTRATION p. Mole SODIUM TAUROCHOLATE, DESOXYCHOLATE AND mMole CaCl/ ml 135 Stability of Penicillium caseicolum Lipase Stability of Penicillium caseicolum lipase was studied under different conditions of temperatures and storage time using pH-stat method for lipase assay. The results presented in Table 18 indicated that the lipase was stable at -26.60 and -15°C for one month. The enzyme showed no loss on storage for 72 h at 00 and 4°C. The enzyme lost considerable activity at higher temperatures. After 72 h of storage at 25°, 370 and 45°C, the residual h enzyme activity was 98.7, 42.0 and 13%, respectively. At 55°C, it lost all its activity after 24 h of storage. When the enzyme was exposed to pasteurization treatment, it retained 1.0 of its activity. The enzyme lost its activitycompletely on boiling for 6 min. Autoclave treat- ment retained 1.5% of the enzyme activity. The relative percent of remaining lipase activity at 37, 45, 55, 63 and 100°C was plotted on semi log paper, as shown in Figure 21, and the D-values were derived from the graph. The D—values are presented in Table 19. It was observed that the D—values for experimental tem- peratures below 37°C approached infinity. The Decimal—Reduction-Time (DRT) curve for lipase heated at 37-1000C is presented in Figure 22. A ZD value (the change in temperature yielding a ten fold change in D value) of 15.80C was derived from the DRT curve. 136 Table 18. Stability of Penicillium caseicolum lipase as function of storage temperature and time. TemperatureOC Storage time Percent of residual activity -26.6 1 month 100 -15.0 1 month 100 0 72 h 100 4 72 h 100 25 24 h 99.0 72 h 98.7 37 18 h 98.0 24 h 93.2 48 h 68.0 72 h 42.0 45 6 h 90.6 12 h 74.0 18 h 52.0 24 h 38.0 48 h 21.0 72 h 13.0 55 6 h 1.4 12 h 0 9 18 h 0 5 63 30 min 1.0 100 2 min 1.6 4 min 0 4 121 15 min 1.4 Figure 21. 137 Time-survivor curve for Penicillium caseicolum lipase incubated at different temperature. Lipase activity determined by pH-stat method at pH 9.0, temperature 35°C toward tributyrin. 138 o o ,_ i O —O N O 40 ‘— O ~O _1- _O In S) a in I!) o v- FF 1' o' NEOHEd ALIAILOV SSVd 11 TIME (h) 4.5.»: < . 139 Table 19. D-valuesof heat treatment of Penicillium casei- colum lipase at var1ous temperature. TemperatureOC D-value 37 200 45 . 78 55 ' 13 63 0.25 100 0.02 Figure 22. Decimal-Reduction Time curve for Penicillium caseicolum lipase incubated at different temperature. 1000 " 100 10 D VALUE (h) .01 141 l l 60 80 TEMPERATURE 0C l 100 120 142 Several workers reported ZD values for some microbial lipases. Driessen and Stadhouders (1974) found that the ZD value of Pseudomonas fluorescens 22F lipase was 8.90C. Kishonti (1975) reported that the ZD value of Pseudomonas 218 lipase was 55°C. Adams and Brawley (1981) determined the Z0 value of Pseudomonas gtt_MC50 lipase to be 36°C when heated in H20. The lipase exhibited greatest survival at pH 8.5. Below pH 6.5 survival was less than 10% of the survival at pH 8.5. The ZD value of lipase could be dependent on the micro- organism, strain, type of oil, oil concentration, heat treat- ment of Substrate, composition of the substrate, method of lipase assay, temperature and pH of assay. Previous workers reported microbial lipases to be stable at lower temperature but lost significant activity at higher tempera- ture (Nashif and Nelson, 1953; Frinkelstein gt _t., 1970; Chander, 1979). Various microbial lipase vary in their resistance to heat treatment. According to Anderson gt gt. (1979) the factors involved in the heat resistance of enzyme are primarily due to molecular structure and specific com- ponents in the molecule such as polysaccharides. Divalent cations appear to stabilize the molecule. Anderson t l. (1979) and Liu gt gt. (1973, 1977) stated a high content of hydrophobic amino acids in the enzyme molecule, disulfide bridges and other bonds play a role in stabilizing the enzyme nunlecule. Frieden (1971) reported that the inactivation of 143 lipase at a low temperature may be due to dissociation of monomer into subunits. The dissociated and inactivedenzyme may be slowly converted to different forms. Reassociation may occur after rewarming, but the enzyme is still inactive. Cooper (1977) stated that the freezing and thawing of some proteins may decrease the stability of the enzyme. Lamberet and Lenoir (1976) reported crude Penicillium caseicolum lipase to be stable within pH 7.0 to 8.5 at 30°C. Seventy percent of initial activity was lost after 30 min of storage at 30°C at pH 6.0. Lamberetand Lenoir (1976a) studied the stability of purified Penicillium caseicolum lipase which appeared to be more stable than the crude enzyme. The puri- fied enzyme was stable for 15 min below 3500. The enzyme lost 60% of activity on storage for 5 min at 40°C and pH 8.5, but lost 90% of its activity when stored at 40°C for 60 min. The results obtained in this investigation are in dis- agreement with the work of Lamberet and Lenoir (1976). This may be due to different strains, media composition, pH of media, and the environmental growth factors. 144 Substrate Specificity of Penicillium caseicolum Lipase The effect of Penicillium caseicolum lipase activity on natural and synthetic lipids was investigated by pH- stat method. The results are presented in Table 20. The highest initial rate of hydrolysis was toward tributyrin. The relative ratesof hydrolysis were 47.3, 39.8, 33,7, 25.6, 22.9, 15.9, 9.0% with tricaproin, tricaprylin, tristearin, triolein, trilaurin, trimyristin and tripal- ‘ matin, respectively. On the other hand,the relative rate 14 of hydrolysis in natural lipids ranged from 17.8 to 34.5% :1 with various lipids. Previous studies show addition of 8 umole of sodium taurocholate and 8 mmoles CaClg to reac- tion mixture enhanced the rate of hydrolysis by Penicillium caseicolum lipase. A study was conducted to investigate the effect of these salts on the various lipids. The reSults are presented in Table 21. It appears that there were inhibitory effect with some lipids and stimulatory, effects on the other lipid substrates. In general, the reSults obtained show Egtttttttgm caseicolum lipase preferentially hydrolyzed short chain fatty acid glycerides. These results are similar to the results obtained by some workers for certain microbial lipases (Umemoto gt gt., 1968; Otherholm _t _t., 1968, 1970; Angeles and Marth. 1971: Yamaguchi t 1., 1973; Chander gt gt., 1973; Formisano t al., 1974; Chander gt 1., 1979a. However, Khan gt 1. (1967) 145 Table 20. Relative activity of Penicillium caseicolum lipase toward natural and synthetic lipids. Natural and Lipase activity Relative rate synthetic lipids umoles FFA/ of hydrolysis 0.2 ml/min. % Tributyrin 6.24 100 Tricaproin 2.95 47.3 Tricaprylin 2.49 ‘ 39.8 Tristearin 2.10 33.7 Triolein 1.60 25.6 Trilaurin 1.43 22.9 Trimyristin 0.99 15.9 Tripalmitin 0.56 9.0 Corn oil 2.15 34.5 Sunflower oil 2.07 33.2 Grape seed oil 1.91 30.5 Almond oil 1.82 29.2 Safflower oil 1.79 28.7 Soy oil 1.73 27.7 Peanut oil 1.73 27.7 Butter oil 1.52 24.4 Sesame oil 1.48 23.7 Lard 1.42 22.8 Hazelnut oil 1.35 21.6 Walnut oil 1.31 21.0 Olive oil 1.11 17.8 146 Table 21. Effect ofadding sodium taurocholate and CaClg on activity of Penicillium caseicolum lipase toward different natural and synthetic lipids. Natural and Lipase Lipid Activity _ % synthetic Activity with 8 um sodium increas (+) 11p1d FFA/0.2 m1 taurocholate or /min. and 8 mm CaC12 amoiesdecreaseI-l FFA/0.2 m1/min. Tributyrin 6.24 - ‘ Tripalmitin 0.56 1.18 +110-7 Tricaprytin 2.49 3.63 + 45-8 Trilaurin 1.43 1.93 + 35.0 Trimyristin .99 1.33 + 34.3 Tricaproin 2,95 3.87 + 31.2 Tristearin 2.10 2.50 + 23.8 Triolein 1,50 1.38 - 13.8 Butter oil 1.52 3_51 +137.5 Olive oil 1.11 2.55 +129.7 Lard 1.42 3.02 +112-7 Sesame oil 1.48 1.82 + 23.0 Grape seed oil 1.91 1.62 - 15-2 Walnut oil 1.31 1.04 ' 20-5 Hazelnut oil 1.35 0.91 ' 32 6 Peanut oil 1_74 0999 - 42.8 Soy oil 1.73 0.95 - 45.1 Safflower oil ]_75 0.96 - 54.9 Cor” 011 2.15 0.88 - 59 1 Almond oil 1.82 0.67 - 63.2 Sunflower oil 2,07 0.65 — 68.6 147 reported that an extracellular lipase of Achromobacter lipolytica hydrolyzed triolein more rapidly than tributyrin. Oi t 1. (1969) found that an intracellular lipase of Rhizopus hydrolyzed triolein more efficiently than tributy— rin. The results obtained in this investigation are in agreement with those obtained by Alford et 1. (1964). Oi _t gt. (1969), Eitenmiller gt gt. (1970). Belloc _t gt. (1975) found lipase activity of Penicillium camemberti was relatively higher toward tributyrin. According to Lambert and Lenoir (1976), the relative activity of Penicillium caseicolum lipase toward tributyrin, butter oil and triolein was 100, 87 and 22%, respectively. In this investigation,re1ative lipase activity of Penicil- liEfl caseicolum toward tributyrin, triolein and butter oil was 100, 25.66 and 24.44%,respectively. These differences may be related to the strain, growth factors, method of assay, percentage of oil in the substrate, heat treatment of substrate and other componentsof substrate emulsion. 148 Concentration of Penicillium Caseicolum Lipase Protein content and lipase activity of different fractions of Penicillium caseicolum lipase are presented in Table 22. The degree of lipase concentration was 49-fold. This concentrated enzyme was used for the specificity studies of the lipase. 149 Table 22. Protein content and lipase activity of different fractions of Penicillium caseicolum lipase. Fraction Total Lipase Specific Concen- protein activity activity tration ug/O.2 ml pM FFA/ 0M FFA enzyme 0.2.m1/ 09 protein m1n Supernatant 5.91 0.07 0.012 1 fraction Concentrated 24.49 14.56 0.59 49 lipase 150 Fatty Acid Specificity of Penicillium caseicolum Lipase Gas liquid chromatographic analyses of fatty acids are presented in Table 23. The table shows the free fatty acids hydrolyzed from butter oil substrate as a result of lipase action. Also the free fatty acid composition of butter oil substrate prior to lipase action and fatty acid composition of transesterified butter oil are shown. The results indicate butyric acid was preferentially released by the action of the lipase. Little or no increase in the 1 other short chain fatty acids (C6 and 08) was observed. It is concluded that Penicilligg caseicolum lipase has a high specificity toward butyric acid glycerides of butter oil. Typical chromatogram of free fatty acids in lipolyzed sample, free fatty acids in butter oil substrate and fatty acid composition of butter oil are presented in Figures 23.24 and 25, respectively. The free fatty acid profiles of Penicillium caseicolum lipase shown here are generally in agreement with the work of Kornackl gt _t. (1979). The results also confirm the observations in the previous study of this investigation that the enzyme has a specificity for short chain fatty acids. 151 Table 23. Free fatty acidsliberated by action of Penicil- lium caseicolum lipase and fatty acid composition of butter oil. Fatty FFA in lipolyzed FFA in butter oil Fatty acids acid butter oil substrate composition substrate % of butter oil by % transesterification % C4 19.01 0 3.34 C6 1.80 1.15 2.41 C8 1.60 0.36 1.44 C10 2.25 3.72 3.79 C12 4.02 4.10 4.10 C14 12.74 13.62 12.10 014:] 6.59 7.45 2.92 C16 22.05 18.82 29.25 C16:1 7.29 12.84 4.49 C18 7.98 14.06 10.67 C18zl 12.96 17.50 22.15 C 1.54 6.39 3.34 18:2 152 Figure 23. Gas chromatogram of free fatty acids in lipolyzed butter oil as a result of Penicillium caseicolum lipase action. 153 .55. NSC. 20....2mhmm mm on mm on m P o. m o _ _ . _ _ A _ a 0 Ir 9 w I. Ito o 79 m 13 7 U 3 S d 3 O m N S 3 154 Figure 24. Gas chromatogram of free fatty acid, in butter oil. 155 :55 m2: zonuEm BSNOdSEU 156 Figure 25. Gas chromatogram of fatty acids composition of butter oil determined by transesterification. 157 L-QLO 9L 7L 30 25 BSNOdSSU RETENTION TIME (min) SUMMARY AND CONCLUSIONS Growth and Lipase Production by Penicillium caseicolum Penicillium caseicolum C] was grown and maintained in Czapek-Dox agar slant at 25 i 1°C for 7 days. The fungal growth was suspended in 0.114phosphate buffer, pH 7.0, then inoculated into the media at rate 1%. The spore counts in 7 to 1.44x107/m1. the inoculum were 1.16x10 In order to determine the best growth conditions for Penicillium caseicolum, five fungal media were chosen. These media were: Czapek-Dox broth, Sab0uraud Maltose broth, Sab0uraud liquid medium, mycological broth and malt extract broth. The media were sterilized, inoculated with 1% spore and cell suspension and incubated at 23 i 1°C under station- ary conditions. The growth was harvested after 4, 7, 10 and 14 days, and evaluated by dry cell weight measurements. The cultures were filtered through Whatman filter paper #5, and washed twice with distilled water. The dry weight was deter- mined after drying in a vacuum ovum (30 inch) at 100°C for 5 h. These conditions were established to achieve constant weight. The fungal growth in Czapek-Dox broth and mycologi- cal broth was higher, followed by Sabouraud maltose broth, Sab0uraud Liquid medium and maltose extract broth. Czapek-Dox broth and mycological broth were chosen for 158 159 further studies. During all stages of this investigation, lipase activity was determined by silica gel column chromatographic procen dure or pH-stat method. Ten percent tributyrin or butter oil emulsified in 10% gum arabic solution containing 8 uM sodium taurocholate and 8 mM CaC12 was used as substrate. Penicillium caseicolum growth and lipase production in Czapek-Dox broth and mycological broth at 25 i 1°C under stationary conditions as well as under submerged (shaking) condition at 120 rpm were compared. Lipolytic activities were determined by silica gel method. The results indicated that Czapek-Dox broth and mycological broth supported better growth and lipase production under submerged (shaking) than stationary conditions. Five strains were studied for their growth, spore count and lipase production in mycological broth containing 1% corn oil. The data showed there was little difference in the growth among various strains. Lipase activity varied widely and was highest with strain 85. No relationship between growth, spore count and lipase activity was evident. One percent corn oil, butter oil and olive oil were added to mycological broth and Czapek-Dox broth. In Czapek Dox broth, all the oils had inhibitory effect on lipase production. When mycological broth was used, corn oil, olive oil and butter oil had stimulatory effect on lipase production. Maximum activity was obtained after 4 days of 160 growth of Penicillium caseicolum. The lipase activity decreased after 7 days of growth. The lipase production was considerably higher with corn oil incorporation in the medium as compared to the olive oil and butter oil addition. Investigation on lipase activity of Penicillium caseicolum on daily basis in mycological broth showed that the highest lipase activity was obtained after 3 days of growth after which it declined. Using olive oil in the medium, the lipase activity was highest on the second and fifth day of 1 growth. In the next study, addition of soytone into Czapek— Dox broth resulted in an increase of 61% in lipase production as compared to mycological broth after 4 days of fungal growth. Different sources of nitrogen were used in the growth media of Penicillium caseicolum. The pH of the medium dropped between 0.57 and 2.08, depending on the source of nitrogen, reflecting the degree of amino acid uptake. Soytone was found to be the best source of nitrogen and showed slightly higher lipase activity than peptone. The lowest lipase activity was with 20% whey protein concen- trate. Replacing dextrose with different sources of carbon in the growth media of Penicillium caseicolum showed a decrease in pH by 1.2 to 1.94. The growth rate was highest with lac- tose followed by galactose, fructose, dextrose, sucrose and maltose. Dextrose showed highest lipase activity, followed 161 by sucrose, maltose, galactose, lactose and fructose. Lipase production in media containing dextrose and sucrose was comparable. Also the results showed no relationship between growth and lipase production with different sources of carbon. The addition of certain minerals to mycological broth showed inhibitory effect on growth and lipase production to various degrees. The results indicated no relationship between growth and lipase production. The growth and lipase production of Penicillium casei- ggtgm was markedly affected by pH of the medium. Fungal growth generally increased with incubation period. The optimum pH for growth was 4.7. The highest lipase produc- tion was obtained after 3-4 days of growth. The optimum pH for lipase production was 7.0. A decrease in the lipase production was observed below or above pH 7.0. The optimum growth and lipase production by Penicillium caseicolum was at 250C. Above this temperature, a sharp decrease in the lipase production was observed. Lipase production increased with an increase in the degree of agitation of the medium. The lipase activity reached its maximum after 2 days of fungal growth at 500 rpm. The growth of the fungal was higher at 500 rpm than at 120 and 200 rpm. 162 Characterization of the Lipase The optimum temperature for lipase activity of Penicil- ttgm caseicolum was 35°C, when butter oil and tributyrin were used as substrates. The optimum pH of the enzyme was 9.0. There was a sharp decrease in lipase activity above the optimum pH for both the substrates. The lipase activity was 3-4 times higher with tributyrin as compared to butter oil substrate. Addition of bile salts and calcium chloride to the sub- _ strate affected the reaction to varying degrees. When tri- fl butyrin was used as a substrate, bile salts and CaClZ indi- vidually or in mixed form had inhibitory effect. In case of butter oil, bile salts had stimulatory effect at 1-10 uM concentrations as compared with no salts. Calcium chloride stimulated the rate of hydrolysis at low concentrations (2 mM) and had an inhibitory effect at higher concentra- tion. Stimulatory effect was highest with a mixture of 8 uM sodium taurocholate and 8 mM CaClZ/ml with butter oil sub- strate. Lipase from Penicillium caseicolum was stable at —26.6 and -150C for one month. It was stable up to 3 days at 00C and 40C. The initial rate of hydrolysis decreased by 1, 58, and 87% on storage for 3 days at 25, 37 and 450C, respectively. The enzyme was completely inactivated after 24 h of storage at 550C. Upon pasteurization, boiling and autoclaving, the lipase was 98-100% inactivated. The ZD 163 value was 15.80C. Penicillium caseicolum lipase showed specificity toward simple triglycerides containing short chain fatty acids, especially C4. The rate of hydrolysis of tributyrin was 4 times higher than that of triolein. In case of natural lipids, lipase activity was higher toward lipids containing high polyunsaturated fatty acids. Corn oil and sunflower oil were hydrolyzed 1.5 times faster than butter oil. Gas chromatographic analyses showed that the lipase was highly specific for the release of butyric acid from butter oil. X I D N E DI P A 164 Table A1. List of chemicals used in this study. Chemical Reference Company Number Acetone 2440 Mallinckrodt Ammonium hydroxide 1177 Mallinckrodt Ammonium sulfate 3512 Mallinckrodt BF3-Butanol (14% w/v) 3-3125 Supelco BF3-Methanol (14% w/v) 3-3020 Supelco Bromocresol green 5-983-5 Fisher Scientific Butyl Alcohol (normal) 2990 Mallinckrodt Calcium caseinate Liberty Enterprise Calcium chloride 1-1332 Baker Calcium sulfate 4300 Mallinckrodt Casein C-203 Fisher Scientific Casamino acid B-23O Difco Chloroform 4440 Mallinckrodt Dextrose 4912 Mallinckrodt Dipotassium phosphate 7092 Mallinckrodt, Ethanol Aaper Alcohol and Chemical Ethyl ether 0844 Mallinckrodt Ethyl glycol 5001 Fisher Scientific Ferrous sulfate 2070 Baker Fructose L-95 Fisher Scientific Galactose B163 Difco Gum Arabic 69752 Sigma Table A1. (cont'd). 165 Gamma globulin Hexane Hydrochloric acid Iso-propyl alcohol Lactic acid Lactose Magnesium sulfate Maltose Methanol Neutralizing agent Phenolptalein Phenol red Phosphoric acid Potassium acid phtalate Potassium chloride Potassium hydroxide Proteose peptone Silicilic acid Sodium caseinate Sodium chloride Sodium dibasic phosphate Sodium desoxycholate Sodium hydroxide Sodium monobasic phosphate 5189 2612 5-9084 1-0194 8-156 1-2506 B-169 3024 3-3052 2788 6704 6858 6984 B-120 2847 7581 3824 B—248 7708 7892 BioRad Laboratories Mallinckrodt Mallinckrodt Baker Baker Difco Baker Di fco 11 Mallinckrodt 1 Supelco Mallinckrodt United States Bio- chemical Corp. Mallinckrodt Mallinckrodt Mallinckrodt Mallinckrodt Difco Mallinckrodt Liberty Enterprise Mallinckrodt Baker Difco Mallinckrodt Mallinckrodt Table A1. (cont'd). 166 Sodium nitrate Sodium taurocholate Sucrose Sulfuric acid 20, 29% whey protein 1-3780 $07270 1-4072 2468 Baker Pfaltz & Bauer Baker Mallinckrodt Sheffi Ltd. 167 Table A2. List of microbiological media used in this study. Media Reference Company number Agar B-l40 Difco, Detroit,MI Czapek-Dox broth B-338 " " Sab0uraud Liquid medium B-109 " " Malt extract broth B-113 " " Peptone B-118 " u Mycological broth B-406 " " Sab0uraud Maltose broth B-429 " " Soytone B-436 " n Trypticase 02-148 BBL, Cockeysville, MD Table A3. List of naturali&and synthetic lipids used in this study. Lipid Purity % Reference Company number Tributyrin 96-98 T4637 Sigma Chemical Co. Tricaproin 90+ T4137 Sigma Chemical Co. Tricaprylin 90+ T9126 Sigma Chemical Co. Trilaurin 90+ T3127 Sigma Chemical Co. Trimyristin 90+ T7252 Sigma Chemical Co. Tristearin 90+ T6628 Sigma Chemical Co. Triolein 75+ T7752 Sigma Chemical Co. Tripalmitin 90+ T8127 Sigma Chemical Co. Almond oil Butter oil Corn oil Grapeseed oil Hazelnut oil Lard Olive oil Peanut oil Sesame oil Soy oil Safflower oil Etsguenard-France Made from Land 0' Lakes butter Best Foods, CPC International Inc., NJ Soleillou-France G. Viver-France Armour, Phoenix, AZ Pompeian, Baltimore, MD Hunza, distributed by M and J Foods, L.A., CA Hunza, " Hunza, " Hunza, ” Table A3. (cont'd). 169 Sunflower oil Walnut oil Hunza, distributed by M and 0 Foods, CA Rougre-France L.A., *Commercial oil no purity. 170 Table A4. List of free fatty acids standard* used in this study. Fatty acid Reference Company number Butyric acid FAOO4O Alltech Association Caproic acid FA0060 ” ” Enanthic acid FA0070 ” " Caprylic acid FAOO8O " " Capric acid FA0100 " " Lauric acid FA0120 ” " Myristic acid FA0140 ” " Myristoleic acid M6129 Sigma Palmitic acid FA0160 Alltech Association Palmitoleic acid P0875 Sigma Margaric acid FA0170 Alltech Association Stearic acid FA018O Alltech Association Oleic acid FAOl81C ” " Linoleic acid FA0182C " " *Purity 99+%. 171 Table A5. List of instruments used in this study. Instrument Company Autoclave (Type 20) Wilmot Castle Company, Roches- ter, NY Balance Mettler H30 Mettler Instrument Corporation, Highstown, NJ Balance (Mettler top Mettler Instrument Corporation, load Type 120) Highstown, NJ Centrifuge RCZb Sorvall, Newtown, CT (Automatic refrigerator) Digestor Microneldahl FF699 Lab. Con Co., Kansas City, MO Distillator (Microkjeldahl) Fisher Scientific, Pittsburg,PA Fermentor (Microform) New Brunswick Scientific Co., MF107 New Brunswick, NJ Gas chromatography Model Hewlett Packard, Avondale, PA 5840A Haemocytometer (improved Thomas and Arthur Co. Neubauer 2936—010) Hand Homogenizer Fisher Scientific, Pittsburg, PA (ll-504-200) Incubator Model 4 (31480) Precision Scientific Co., Chicago, IL Incubator Blue M (Model 200A) Blue M Electric, Blue Island, IL pH Controller Model 43DPHCR Virtis, Gardiner, NY pH-electrode (Autoclavable) Ingold Electrode Inc., Andover, (XPHD-700) MA pH-Meter CHEMTRIX6OA Chemtrix Inc., Killboro, 0R pH-Pump Model 43pH-PE Virtis Co., Gardiner, NY pH-Stat Titrator-E526 Metrohm, Herisau, Switzerland Motor Drive Piston Metrohm, Herisau, Switzerland Burette E-525 172 Table A5. (cont'd.). 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