Fungal strategies for detoxification of medium chain fatty acids

International Biodeterioration & Biodegradation 32 (1993) 213-224

Fungal Strategies for Detoxification of Medium Chain Fatty Acids

Judith L. Kinderlerer Food Research Centre, Sheffield Hallam University, Sheffield, UK, S1 1WB

ABSTRACT Fungi can degrade free fatty acids and triacylglycerols to provide a carbon and energy source. Medium chain fatty acids (those with a carbon length between six and 12 carbon atoms) are relatively uncommon. They are found in three oils of commercial importance--coconut, palm kernel and butter. The ability of fungi to convert medium chain fatty acids or triacylglycerols containing these acids to methyl ketones one carbon atom less has been known for a long time. Five genera can undertake these bioconversions--Aspergillus and Penicillium, and their corresponding teleomorphic genera Trichoderma, Cladosporium and Fusarium. Medium chain fatty acids inhibit fungal growth. The degree of inhibition depends on the concentration of the un-ionised acids. A number of fungal bioconversions of medium chain fatty acids can occur leading to the production of volatile metabolites. These include a partial fl-oxidation to give the methyl ketone one carbon atom less than the parent fatty acid, reduction of the ketone to give the secondary alcohol, hydroxylation of the ketone to give a mono-hydroxy ketone, esterification and decarboxylation. It is suggested that the production of these volatile metabolites is a fungal strategy to eliminate metabolites which would otherwise be toxic.

INTRODUCTION The bioconversion of medium chain fatty acids (hexanoic C6:0, octanoic

C8:0, decanoic C10:0, and dodecanoic C12:0) to the methyl ketones by 213 International Biodeterioration & Biodegradation 0964-8305/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain.

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certain filamentous fungi was described as early as 1924 (St/irkle, 1924). The ability of many penicillia to convert sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene is less well known (Kinderlerer & Hatton, 1990). Conversion of fatty acids to esters has been recognised as a detoxification as well as a route for the production of flavour chemicals (Kinderlerer et al., 1988: Engel et al., 1989). These reactions represent oxidation, reduction, decarboxylation and esterification, and result in the carboxyl group of the medium chain fatty acid becoming less polar. One may speculate that the conversion of medium chain fatty acids to volatile metabolites is a fungal strategy for the elimination of molecules which otherwise would be toxic.

EVIDENCE FOR TOXICITY OF M E D I U M C H A I N FATTY ACIDS Saturated medium chain fatty acids The toxic effects of medium chain fatty acids on fungi have been described by many workers (Stokoe, 1928; Lawrence & Hawke, 1968; Lewis & Darnall, 1970; Hatton & Kinderlerer, 1991). Medium chain fatty acids are known to inhibit cell growth, cell division, uptake of inorganic phosphate and substrate oxidation (Freese et al., 1973; Hufikov/t & Fencl, 1978). Some workers have suggested that short chain lipophilic acids affect substrate transport across cell membranes and electron transport in the mitochondrion (Baird-Parker, 1980). The metabolic effects of fatty acids are dependent on pH, concentration and chain length. The C10:0 and C12:0 fatty acids have greater inhibitory effects than the C6:0 and C8:0 acids (Lawrence & Hawke, 1968). The toxicity of the acid was shown to increase with a decrease in pH (Lawrence & Hawke, 1968; Lewis & Darnall, 1970). Medium chain fatty acids are relatively insoluble in water and relatively soluble in oils and fats (Small, 1986). They act as weak acids and are transported across cell membranes in the undissociated state (Lawrence, 1966). Two mechanisms exist which reduce the concentration of these acids in the aqueous phase: (1) bioconversion to give volatile metabolites which can diffuse into the atmosphere; (2) partitioning between the aqueous and fat or lipid phase. Medium chain fatty acids are far more soluble in the fat than water. During normal metabolism, fatty acids exist as esters, usually of glycerol as a triacylglycerol or as the thioester in the acyl-Coenzyme A derivative or as the carnitine derivative for transport into the mitochondrion for fatty acids > C12:0 in a mammalian system (Gurr & Harwood, 1990).

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Unsaturated medium chain fatty acids Sorbic acid is the only unsaturated fatty acid which is currently permitted as a food preservative (Eklund, 1989). It is more effective against moulds and yeasts than against bacteria (Liewen & Marth, 1985; Eklund, 1989). Marth et al. (1966) demonstrated that many Penicillium spp. could degrade sorbic acid to give 1,3-pentadiene. A number of Eurotium spp., however, failed to bring about this bioconversion (Kinderlerer & Hatton, 1990). Eklund (1989) summarised the mechanisms by which sorbic acid may inhibit the growth of microorganisms. These include the inhibition of many enzymes which are involved in the glycolytic pathway and the tricarboxylic acid cycle, as well as inhibition of protein, RNA and DNA synthesis. Other workers have stressed that medium chain fatty acids such as sorbic acid may inhibit substrate transport across the cell membrane and proton gradients in the mitochondrion during oxidative phosphorylation (Freese et al., 1973). ~

TRIACYLGLYCEROLS AS A CARBON SOURCE FOR F U N G A L GROWTH A major cause of loss of quality of oils is fungal growth. The ability of a fungus to utilise a triacylglycerol as a substrate depends on the fatty acid composition and the slip-point. A decrease in the chain length of the constituent fatty acid (in the triacylglycerol) will cause a decrease in the slip-point. An increase in the proportion of unsaturated fatty acids in the triacylglycerol will also cause a decrease in the slip-point. Medium chain fatty acids are unusual in oils and fats of plant and animal origin (Kinderlerer, 1986). They are found in the lauric acid oils (coconut and palm kernel) as well as in milk fat. Vegetable oils such as olive oil contain a high concentration of oleic acid (C18:1) and as a consequence are liquid, whereas other fats (cocoa butter and beef tallow) with a high concentration of saturated long chain fatty acids (between 16 and 18 carbon atoms in length) are solid at ambient temperatures. The data in Table 1 demonstrate that more growth takes place with a liquid substrate than a solid one (compare olive oil and triolein with cocoa butter or beef tallow). However, lower yields were obtained after growth on simple triacylglycerols containing medium chain fatty acids (trihexanoin, trioctanoin, tridecanoin and tridodecanoin) than on mixed triacylglycerols containing medium and long chain fatty acids (coconut and palm kernel). Lower growth yields were obtained when a fungus was grown on triacylglycerols containing saturated long chain fatty acids (beef tallow and cocoa butter) than on olive oil.

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Judith L. Kinderlerer TABLE 1

Growth Yield for Penicillium crustosum IMI 342844 with Various Substrates a Substrate

Liquid triacyiglycerol Olive oil Solid triacylglycerol Beef tallow Cocoa butter

Slip-point (°C)

mg dry biomass g-i substrate

mg dry biomass per substrate (rag k J- ~)

-6

219 4- 27

4.8 4- 0-6

38.9--44.2 28-0-31.0

74 4- 15 57 + 8

1.7 4- 0-4 1.3 4- 0-2

Triacylglycerols containing medium chain fatty acids Coconul oil 23.7-26-1 76 -4-40 Palm kernel oil 24.5-27-3 31 + 4 Trihexanoin -25 23 + 6 Trioctanoin 8.3 72 4- 18 Tridecanoin 31.5 14 4- 1 Tridodecanoin 46.5 6 4- l Tritetradecanoin 57-0 No growth Carbohydrate Sucrose

--

220 ± 8.3

1.7 4- 0.9 0.7 4- 0-1 0.8 4- 0.2 2.2 -4-0.6 0-4 4- 0.1 0.17 4- 0-1

13.0 4- 8.3

aGrowth in liquid suspension culture (25 ml Czapek medium) at 25°C and pH 7.0 for 72 h and 200 rev rain -~ (1 g substrate, inoculum 2-5 x 107 spores). Data are means and standard deviations for four fermentations. Data derived from Hatton and Kinderlerer (1991). K a w a g u c h i et al. (1990) d e m o n s t r a t e d t h a t a 2 4 - k D a p r o t e i n w a s p r o d u c e d in the culture m e d i u m a f t e r g r o w t h o f T r i c h o d e r m a S M - 3 0 o n s o y b e a n oil w h i c h c o u l d b i n d t r i a c y l g l y c e r o l b u t did n o t h a v e lipase activity . It w a s t h o u g h t t h a t this p r o t e i n w a s i n v o l v e d in t r a n s p o r t o f triacylglycerol across the cell m e m b r a n e . T r i a c y l g l y c e r o l s p r o v i d e a c a r b o n s o u r c e w h i c h is e n e r g y rich a n d c a r b o n p o o r . T h i s c a n be seen in T a b l e 1. T h e g r o w t h yields f o r s u c r o s e are h i g h e r ( k J -~) t h a n t h o s e o b t a i n e d w h e n t r i a c y l g l y c e r o l w a s u s e d as the sole c a r b o n source.

DEGRADATION

OF MEDIUM CHAIN FATTY ACIDS TO METHYL KETONES

T h e ability o f fungi to c o n v e r t m e d i u m c h a i n f a t t y acids o r t r i a c y l g l y c e r o l s c o n t a i n i n g t h e s e acids to m e t h y l k e t o n e s h a s b e e n k n o w n f o r a l o n g time. W o r k b e f o r e 1948 h a s b e e n r e v i e w e d b y F o s t e r (1949) in his classic w o r k o n the c h e m i c a l activities o f fungi. T h r e e lines o f r e s e a r c h h a v e b e e n

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217

undertaken. The first involves studies on rancidity and spoilage in oilseeds such as coconut and palm kernel or in products containing them (Stokoe, 1928; Kellard et al., 1985; Kinderlerer & Hatton, 1991). The second involves a study on flavour development in mould-ripened cheeses. Excellent reviews have been provided by Hawke (!966), Kinsella & Hwang (1976) and Lenoir (1984).The third line of research considers the use of these bioconversions as a source of fine or flavour chemicals. In this area the papers by Yagi et al. (1990b) and Engel et al. (1989) are useful.

Fungi Two systematic surveys have been undertaken to determine the distribution of fungi capable of converting free medium chain fatty acids or triacylglycerols composed of these acids to the corresponding ketone (Franke & Heinen, 1958; Yagi et al., 1990b). Five genera are important-Aspergillus and Penicillium, and their corresponding teleomorphic genera Trichoderma, Cladosporium and Fusarium. Of the five main divisions of the Fungal Kingdom the ability to bring about these bioconversions has been found in two--the Deuteromycotina and the Ascomycotina. Some members of the Mucorales, however, do appear to have a limited ability to bring about these bioconversions (Franke & Heinen, 1958). Fungal spores, vegetative hyphae and cell-free extracts can bring about the conversion of medium chain fatty acids to methyl ketones (Hatton & Kinderlerer, 1991). Fungal spores require additional factors such as glucose, proline, alanine and serine before this conversion can take place (Lawrence, 1966).

Isolation of methyl ketones Early work on the isolation of methyl ketones was by extraction using steam distillation methods. This method preferentially isolated the lower homologues, 2-pentanone and 2-heptanone (see Stokoe, 1928; Kinderlerer & Kellard, 1984), unlike the later work where 2-undecanone and 2-tridecanone were also isolated using direct solvent extraction with dichloromethane or chloroform (Kinderlerer, 1987; Yagi et al., 1989). Methyl ketones may be identified by direct gas chromatography of the original solvent extract using a polar column (e.g. Carbowax 20 M or OV 17).

Environmental conditions for production of methyl ketones by fermentation The optimum temperature for conversion of triacylglycerols containing medium chain fatty acids to the methyl ketone was 25°C for Penicillium crustosum (Hatton & Kinderlerer, 1991) and 28°C for Penicillium

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decumbens (Yagi et al., 1990a). Both groups found that the final pH

tended to be pH 7.0 in aerobic shake culture irrespective of the initial pH (Yagi et al., 1990a,b; Hatton & Kinderlerer, 1991). The optimum pH for conversion of triacylglycerol to ketone was higher (pH 7-8) than that found for growth (pH 6.0) (Yagi et al., 1990a,b; Hatton & Kinderlerer, 1991; Kinderlerer & Hatton, 1991). The Japanese group used a complex medium containing all the B-vitamins and a wide range of micro-nutrients, whereas the Sheffield group used a simple Czapek type medium (Kinderlerer, 1987; Yagi et al., 1990b). Methyl ketones were not produced in the absence of oxygen (Yagi et al., 1990a; Hatton & Kinderlerer, 1991). Bioconversion

Table 2 shows that triacylglycerols containing medium chain fatty acids can act as substrates for ketone production. A ketone one carbon atom less than the parent fatty acid was produced by fermentation of simple acylglycerols with vegetative hyphae. Thus 2-pentanone was produced from trihexanoin, 2-heptanone from trioctanoin, 2-nonanone from tridecanoin and 2-undecanone from tridodecanoin (Table 2). An homologous series of methyl ketones, 2-pentanone, 2-heptanone, 2-nonanone and 2-undecanone was produced on fermentation of coconut and palm kernel oil. Coconut oil was a better substrate for the production of ketones than palm kernel oil, as a result of the higher concentration of medium chain fatty acids in the substrate (Table 2). Differences in yields of ketones were seen with different substrates. A higher proportion of decanoic acid was converted to the ketone (2nonanone) on fermentation of tridecanoin and coconut or palm kernel oil compared with the molar conversion for the other medium chain fatty acids (Table 2). Relatively little conversion of dodecanoic acid took place on fermentation of tridodecanoin. This differed from the situation with coconut and palm kernel oil, where 2-undecanone was the main endproduct of fermentation. Dodecanoic acid is the major fatty acid in both substrates (Table 2). The proportion of the medium chain fatty acids converted to the corresponding ketone differed with each substrate. On fermentation of simple glycerides at pH 7-0 and 25°C the order was C10:0 > C6:0 > C8:0 > C12:0, whereas the order on fermentation of mixed glycerides was C10:0 > C12:0 > C8:0. In general, higher yields of ketones were found for slower-growing strains (Yagi et al., 1989; Kinderlerer & Hatton, 1991). Yagi et al. (1990b) found that species of Fusarium and Trichoderma could convert simple glycerides such as tridecanoin to 2-nonanone but not

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219

TABLE 2 Conversion of Triacylglycerols to Methyl Ketones by Fermentation with Penicilliurn crustosum IMI 342844 a

Bioconversion Hexanoate to 2-pentanone Trihexanoin 20°C 25°C 30°C Coconut oil Palm kernel oil

Substrate (l~mol g -I oil) 7 756 7 756 7756

Product (#mol g -I oil) 364 + 99-1 475 4- 75-8 5774- 1 5 - 8 Trace Trace

Yield ( % )

4-7 + 1.3 6.1 ± 1.0 0.74-0.2

Octanoate to 2-heptanone Trioctanoin 20°C 25°C 30°C Coconut oil 20°C 25°C 30°C Palm kernel oil 20°C 25°C 30°C

6 256 6 256 6 256 535 535 535 216 216 216

327-0 4- 28.2 278.3 4- 54-5 78-0 4- 14-8 41-6 4- 54-7 108-8 4- 7-7 49.9 4- 3.5 22.1 + 3.9 51.54-12.3 27.7 4- 5.6

5-2 4- 0.5 4.5 4- 0-9 1-3 4- 0-2 7-8 4- 0.9 20.3 4- 1-4 9.3 4- 0.7 10-2 4- 1.8 23.94-5.7 12-8 4- 1.2

Decanoate to 2-nonanone Tridecanoin 20°C 25°C 30°C Coconut oil 200C 25°C 30°C Palm kernel oil 20°C 25°C 30°C

5 105 5 105 5 105 344 344 344 186 186 185

9-9 -t- 1.4 860-0 4- 139-2 200.4 4- 106.2 43.0 4- 3.5 110.1 4-9.2 67.9 4- 49.3 25-0 4- 3-7 52-2 4- 13.8 40-1 4- 2-1

0-2 4- 0.1 16.8 4- 3-0 3.9 4- 2.1 12-5 4- 1-0 32-04-2.7 19-7 i 1-3 16.6 4- 5-4 35.8 + 6-0 21.5 :t: 1-1

29.7 4- 1.5 34.0 4- 9-6 15.1 4- 4.1 110-8 4- 3.6 509.24-46.0 327.0 4- 22.2 95.2 4- 20.7 293-2 4- 96.1 245.1 4-7.3

0.7 4- 0-3 0-8 4- 0.2 0-3 4- 0.1 4.8 + 0-2 22.1 + 11-4 14.2 4- 1.0 5.0 4- 2.1 15.2 4- 4.9 12-74-0.4

Dodecanoate to 2-undecanone Tridodecanoin 20°C 4473 25°C 4473 30°C 4473 Coconut oil 20°C 2 309 25°C 2309 30°C 2 309 Palm kernel oil 20°C 1 930 25°C 1 930 30°C 1930

~Growth in liquid suspension culture (25 ml Czapek medium) at pH 7.0 for 72 h at 20°C, 25°C and 30°C and 200 rev min-' (1 g substrate, inoculum 2.5 x 107 spores). Data are means and standard deviations for four fermentations. Data derived from Hatton and Kinderlerer (1991) and Kinderlerer and Hatton (1991).

220

Judith L. Kinderlerer TABLE 3

Inhibition of Penicillium crustosum by Free medium Chain fatty Acids Free fatty acid

Hexanoic acid Octanoic acid Decanoic acid Undecanoic acid Dodecanoic acid

Minimum inhibitory concentration mM

mg kg -1

32.5 20-0 10 4.7 15.3

3 776 2 884 1 723 880 3 066

Fermentations carried out in liquid shake culture at 200 rev min -l, 25°C and pH 7.0 for 72 h; sucrose (3% w/v) was the sole carbon source; inoculum 2.5 × 10 7 spores (Hatton & Kinderlerer, 1991). mixed acylglycerols. Species o f Aspergillus, Cladosporium and Penicillium could convert both simple acylglycerols (tridecanoin) as well as mixed acylglycerols (palm kernel oil) to the relevant ketone. This pattern was found for Penicillium c r u s t o s u m (Table 2). A number o f workers have suggested that conversion o f medium chain fatty acids to the ketone one c a r b o n less is a detoxification (Stokoe, 1928; Lawrence, 1966; Lewis & Darnall, 1970; Kinderlerer & Hatton, 1991). In an aqueous culture m e d i u m at p H 7.0 with sucrose as the sole carbon source the order o f inhibition o f spore germination was C11:0 > C10:0 > C12:0 > C8:0 > C6:0 for P. crustosum (Table 3). The first step in conversion o f medium chain fatty acids to the methyl ketone from a triacylglycerol substrate is hydrolysis. The presence o f medium chain fatty acids in the a q u e o u s phase is inhibitory (Table 3). If these acids were dissolved in the lipid phase, their inhibitory action would be reduced. This m a y well account for the difference in the rates of conversion in the simple and mixed triacylglycerols. A similar situation was observed during degradation of n-alkanes by yeasts (Boulton & Ratledge, 1984). It would appear that solid substrates are less well metabolised than liquid ones (Table 1).

B I O C O N V E R S I O N O F M E T H Y L K E T O N E S TO S E C O N D A R Y ALCOHOLS AND ESTERS Kinderlerer and Kellard (1984) demonstrated that secondary alcohols were formed on fermentation o f desiccated coconut with Eurotium spp. and Penicillium citrinum under conditions where oxygen was limiting.

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2-Pentanol, 2-heptanol, 2-nonanol and 2-undecanol were isolated and identified by gas chromatography-mass spectroscopy (GC-MS) (Kinderlerer & Kellard, 1984). Engel et al. (1989) demonstrated that aliphatic methyl ketones C7-11 were reduced to the corresponding secondary alcohols in shake culture by P. citrinum. The degree of reduction depended on the chain length of the ketone. Maximum conversion of 2-nonanone to 2-nonanol was observed. The secondary alcohols were chiral. Two other series of compounds were formed, 2,(5, 6, 7 or 8)-dihydroxynonanes as well as the 7- and 8-hydroxy-2-nonanones (from 2nonanones). Similar monohydroxy-2-nonanones were produced by Fusarium avenaceum from tridecanoin (Yagi et al., 1991a). In a non-aqueous environment, lipase enzymes, which would normally catalyse the hydrolysis of triacylglycerol to free fatty acids and glycerol, can catalyse the reverse reaction, i.e. esterification of free fatty acids (Nishio & Kamimura, 1988). In a 9-month solid-state fermentation of desiccated coconut by Chrysosporium xerophilum, the 2-hexyl and 2-heptyl esters of hexanoic, octanoic, decanoic and dodecanoic acids were formed (Kinderlerer et al., 1988).

PATHWAY FOR CONVERSION OF MEDIUM CHAIN FATTY ACIDS TO METHYL KETONES Triacylglycerols can be degraded by extracellular lipases to give free fatty acids. The principal route for fatty acid degradation in all cells is by floxidation. Although some degradation occurs in the mitochondrion of yeasts and moulds growing on fatty acids, it is thought that 90% of the degradation occurs in the peroxisomes (Gurr & Harwood, 1990). The difference between peroxisomal degradation and degradation in the mitochondria is that peroxisomes use an acyl-Coenzyme A oxidase enzyme for conversion of acyl-Coenzyme A to ~, E-unsaturated acylCoenzyme A, unlike the mitochondrial system where a dehydrogenase catalyses this reaction. When E-oxidation takes place in the peroxisome, hydrogen peroxide rather than adenosine triphosphate (ATP) is formed. Evidence for the production of hydrogen peroxide was found in fungal mycelia grown on triacylglycerols as the sole carbon source (Hatton & Kinderlerer, 1991). Methyl ketone formation involves two hydrolytic steps (Lawrence, 1967). The initial hydrolysis of triacylglycerols as well as deacylation of the ~-ketoacyl Coenzyme A to give the corresponding ~-ketoacyl acid. One could speculate that fl-ketoacyl Coenzyme A deacylase is found in the peroxisome of certain fungi. The presence of two enzymes ~-ketoacyl

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Coenzyme A deacylase and fl-ketoacyl acid decarboxylase leads to the production of methyl ketones, fl-Ketoacyl acid decarboxylase enzymes were isolated from fungi by Franke et al. (1961) and Hwang et al. (1976). Yagi et al. (1991b) suggested that an alternative pathway to fl-oxidation was involved in the conversion of m e d i u m chain fatty acids to the corresponding methyl ketone. They demonstrated that a cell wall, membrane fraction (which sedimented at low speed, 1500 g) derived from a 5-day culture of Penicillium decumbens grown on glucose converted decanoic acid to 2-nonanone in high yields in the presence of calcium but not in the mitochondrial or microsomal fraction. More work is needed to establish the pathway for the conversion of medium chain fatty acids to metabolites which are volatile. Many suggestions have been made in an attempt to understand why some filamentous fungi convert medium chain fatty acids to metabolites which are volatile. Methyl ketones may act as insect pheromones and aid in the dispersal of fungi. They may inhibit the growth of other microorganisms which otherwise would be competitors. The loss of the carboxyl group by decarboxylation, partial fl-oxidation, esterification or reduction may be a strategy for elimination of molecules which would otherwise be toxic.

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Freese, E., Sheu, C.W. & Galliers, E. (1973). Function of lipophilic acids as antimicrobial food additives. Nature, 241,321-5. Gurr, M.I. & Harwood, J.L. (1990). Lipid Biochemistry An Introduction, 4th edn. Chapman and Hall, London. p. 47. Hatton, P.V. & Kinderlerer, J.L. (1991). Toxicity of medium chain fatty acids to Penicillium crustosum Thom and their detoxification to methyl ketones. Journal of Applied Bacteriology, 70, 401-7. Hawke, J.C. (1966). Reviews of the progress of dairy science, Section D. Dairy chemistry. The formation and metabolism of methyl ketones and related compounds. Journal of Dairy Research, 33, 225-43. Hufikovh, Z. & Fencl, Z. (1978). Toxic effects of fatty acids on yeast cells: possible mechanisms of action. Biotechnology and Bioengineering, 20, 123547. Hwang, D.H., Lee, Y.J. & Kinsella, J.E. (1976). fl-Ketoacyl decarboxylase activity in spores and mycelium of Penicillium roquefortii. International Journal of Biochemistry, 7, 165-71. Kawaguchi, M., Hatano, T., Yagi, T., Miyakawa, T. & Fukui, S. (1990). Triglyceride-binding proteins in the culture fluid of a triglyceride-using fungus stain SM-30. Journal of Fermentation and Bioengineering, 70, 70-84. Kellard, B., Busfield, D.M. & Kinderlerer, J.L. (1985). Volatile off-flavour compounds in desiccated coconut. Journal of the Science of Food and Agriculture, 36, 415-20. Kinderlerer, J.L. (1986). Bacterial and mould spoilage in coconut. In Spoilage and Mycotoxins of Cereals and other Stored Products, ed. B. Flannigan. International Biodeterioration, 22S, 41-7. Kinderlerer J.L. (1987). Conversion of coconut oil to methyl ketones by two Aspergillus species. Phytochemistry, 26, 1417-20. Kinderlerer, J.L. & Kellard, B. (1984). Ketonic rancidity in coconuts due to xerophilic fungi. Phytochemistry, 23, 2847-9. Kinderlerer, J,L. & Hatton, P.V. (1990). Fungal metabolites of sorbic acid. Food Additives and Contaminants, 7, 657-70. Kinderlerer, J,L. & Hatton, P.V. (1991). The effect of temperature, water activity and sorbic acid on ketone rancidity produced by Penicillium crustosum Thorn in coconut and palm kernel oils. Journal of Applied Bacteriology, 70, 502-6. Kinderlerer, J.L., Hatton, P.V., Chapman, A.J. & Rose, M. (1988). Essential oil produced by Chrysosporium xerophilum in coconut. Phytochemistry, 27, 2761-3. Kinsella, J.E. & Hwang, D.H. (1976). Enzymes of Penicillium roquefortii involved in the biosynthesis of cheese flavor. Critical Reviews in Food Science and Nutrition, 7, 191-228. Lawrence, R.C. (1966). The oxidation of fatty acids by spores of Penicillium roquefortii. Journal of General Microbiology, 44, 393-405. Lawrence, R.C. (1967). The metabolism of triacylglycerols by spores of Penicillium roquefortii. Journal of General Microbiology, 46, 65-7. Lawrence, R.C. & Hawke, J.C. (1968). The oxidation of fatty acids by mycelium of Penicillium roquefortii. Journal of General Microbiology, 51, 289-302. Lenoir, J. (1984). The surface flora and its role in the ripening of cheese. International Dairy Bulletin, 171, 3-20.

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