W~rfd Journal
of microbiology
and 6ioi~bno/ogy
9, 51 l-513
Arachidonic acid production by ~~~~ie~e//~ alpina grown on solid substrates S. Stred’anskB,*
D. Slugeti, M. Stred’anskp and J. Grego
Mortierelfa alpina grown in solid state fermentations on cereal substrates gave up to 16% lipid in the final biomass. Arachidonic was at 50% of total fatty acids, with a yield of 36 mg/g of original substrate. Microbial lipid production was successfully scaled up to use S-kg dry substrate batches. Key words: Arachidonic
acid, cereals, microbial
lipid, ~ortierella
Polyunsaturated fatty acids (PLJFA) have been successfully used in the prevention and treatment of many diseases (Das et al. 1988; Horrobin 1990). The fungus Morfierelh alpha is able to produce these biologically active components, mainly arachidonic acid (ARA, 20:4, o - 6)‘ in large quantities in submerged culture using glucose as substrate (Suzuki & Yokochi 1984; Suzuki et al. 1988; Shinmen et al. 1989; Yamada et al. 1989; Bajpai et al. 1992). However, the fungus used by Totani et al. (1987) produced the highest yields of ARA in surface cultivation on a potato/dextrose paste. These results indicate that solid state fermentation (SSF) couid be useful in fungal lipid production. In the present study, M. aipina CCF 185 was grown on solid substrates (moistened with water to a relative humidity of about 100% but in the absence of free water) in attempts to impove the fungus’ production of lipid rich in ARA or other PUFA.
Materials
and Methods
Substrates Cracked barley, peeled barley, dehulled millet, polished rice and wheat were bought on the local market. One vol of each substrate was soaked in three vols water at 50°C for 5 h. Any residual water was then removed. Microorganism Mortierella
Collection
and
Cdture
alpha CCF of Fungi,
Charles
Conditions
185
was obtained from the Culture University, Prague. Flask experiments
S. Stred’ansktt is with the Research Institute of Gerontology. 901 01 Malacky, Slovak Republic. D. SlugeZI, M. Stred’anskq and J. Grego are with the Department of Biochemical Technology, Slovak Technical University, 812 37 Brat&lava, Slovak Republic; fax: (42) 7 493 198. ‘Corresponding author. @ 1993 Rapid Communications
of Oxford
a&a.
were carried out in 300-ml Erlenmeyer flasks, each with 20 g (dry wt) of moist substrate, sterilized at 120°C for 15 min, inoculated with 1 ml of spore suspension (@/ml) and then incubated at 28°C for 7 to 21 days in a water-saturated atmosphere. Scale-up
experiments were performed in microporous polyethyIene plastic bags of base areas 0.09, 0,21 or 0.49 m”, respectively. Moist substrate was added to the small, medium and large bags (0.5, 1.5 and 5 kg dry wt, respectively) in layers (2.0, 3.5 and 5.0 cm thick), the bags sterilized with hot steam at 0.12 MPa for 45 min and then inoculated with 25, 75 and 250 ml of spore suspension (106/ml), respectively. The bags were kept at 28 to 30°C for 14 days and aerated by passing in sterilized, water-saturated air (0.5 Urnin. kg of substrate). Extra&on Original
of Lipid substrate
atid ~ete~j~a~i~ or the cultured
of Fatty Acid
mycelium with residual substrate were dried and crushed. Lipid was extracted in chloroform/ methanol (2:l v/v) and the solvent evaporated under vacuum (Folch et al. 1957). The lipids were suspended in n-hexane and converted to methyl esters with sodium methoxide in 10% (w/v) HCl in methanol (Christopherson & Glass 1969). The faatty acid composition of these esters was then analyzed by GLC (Kobayashi ef nl. 1987) on a column packed with 10% diethyleneglycolsuccinate.
Results
and Discussion
Solid state fe~entations (SSF) simulate the fe~entation reactions which occur in nature (Lonsane et af. 1985). The moist substrates act as a source of C, N, minerals, water and other nutrients (Mitchell et al. 1986). Cereals, which contain starch, proteins and low amounts of lipids are easily fermented by M. alpina (Slugei5 1992). In the present experiments, significant changes in substrate composition were observed (Tabie I). The surfaces of all substrates were covered by fungal mycelium 3 days after inoculation. The
Ltd World ~mmal of Microbiology and Biotechnology, Voi 9, 1993
511
S. Stredbnskh et al. Table
1. Lipid
content
and comparison
x + RS’ (8)
Substrate
Fermented mass Dehulled millet Cracked barley Peeled barley Polished rice Wheat Unfermented mass Millet Barley Rice Wheat
of tatty Ltpldt (8)
12.2 12.6 12.9 13.3 14.9
1.9 1.2 1.6 1.3 0.8
20.0 20.0 20.0 20.0
0.8 0.8 0.5 0.5
* Biomass (X) with residual substrate (RS). T 75% of total lipid are fatty acids. $ mg of ARA per g of dry weight of initial substrate §Sum of 14:0, 16:l and a-18:3.
acid
profile
in fermented
ARA$
Relative
Substrate
Millet
Barley
2. Eilect
of cultivation Time (day@
7 14 21 7 14 21
time
x+i?s* &I)
15.1 13.4 13.1 15.4 13.9 13.6
on lipid yield Lipid (91
1.6 1.9 2.1 1.4 1.7 2.1
fatty
acid
cereals. composition
(% w/w)
(w&f 16:0
16:0
16:l
16:2
16:3
20~3
20~4
Others5
35.7 20.6 24.9 17.8 9.1
12.9 14.6 17.3 16.3 14.5
5.7 6.6 5.5 7.0 6.6
7.1 5.6 7.0 14.6 12.3
13.2 13.2 15.5 8.9 23.5
2.6 2.5 2.2 4.2 3.0
1.8 2.3 0.8 8.0 3.4
49.1 45.2 44.3 36.5 34.6
7.6 10.0 8.0 4.5 2.1
-
12.0 22.1 30.8 19.3
1.0 2.6 1.1 3.2
22.7 16.4 22.2 20.8
64.0 55.6 43.8 51.3
-
-
-
0.3 3.3 2.1 5.4
calculated
as lipid (mglg
sum of the biomass and the residual substrates (X + RS) decreasedduring cultivation. The predominant fatty acid in all the original substrates was linoleic acid (18: 2). Mdierella aipj~ yielded 40.0 to 95.0 mg lipid/g initial substrate, which corresponded to 54 to 156 mg lipid/g fermented substrate (X + RS). Of the estimated lipid, 75% was estimated to be fatty acid. The yield of arachidonic acid ranged from 9 to 36 mg/g initial substrate. Previously the best yield of ARA which we have obtained was 3.1 g/l in submersed culture in a 20-l fermenter using hydrolysed whey (unpublished data). (Totani et al. (1987) reported a yield of 11.8 mg of ARA/g medium on surface cultivation of M. alpha on potato/dextrose paste. Millet and barley gave the highest yields of ARA in the fermented mass, so they were used in the subsequent experiment, in which the changes in lipid accumulation and composition with time were evaluated (Table 2). The rate
Table
and unfermented
and fatty
* Biomass (X) with residual substrates (RS). T mg of ARA per g of dry weight of initial substrate *Sum of 14:0, 16:l and a-18:3.
x 0.75 x % ARA in total fatty acid.
of decrease of X + RS observed during first 14 days was markedly faster than in the following week. ARA was again determined as the major fatty acid and its content in the lipid continued to rise. The highest ARA production was reached in the first week. The quantities of the ARA precursors, y-linoleic (18:3) and dihomo-y-linoleic (20:3) acid, changed insignificantly during the fermentation but the contents of palmitic, palmitoleic and linoleic acids after the fermentation were considerably lower than those in cereal lipids. In the SSFprocess, many scale-up problems have to be solved before a laboratory-scale process can be transferred to commercial production (Lonsane et a/. 1992). In scale-up experiments, the effect of the depth of layer was investigated (Table 3). The utilization of cereals decreased with scale because of the diffusion of 0,. Consequently, both total lipid and ARA contents decreased.Layers deeper
acid
profile
ARAt (m&a)
18.6 32.2 38.0 20.3 25.6 32.9
biomass)
in fermented Relative
fatty
cereal acid
substrate.
composition
(% w/w)
16:0
18:0
16:l
18:2
16:3
2023
20:4
Others$
19.0 12.7 9.6 16.7 16.0 14.0
8.6 6.1 4.5 10.8 10.0 7.9
11.0 8.2 8.9 17.1 9.7 15.2
16.7 23.0 14.2 12.7 13.4 14.3
2.4 2.7 4.3 2.2 2.5 2.2
4.4 5.0 3.7 2.1 2.6 2.7
31.0 44.7 47.3 37.8 40.8 42.8
6.9 7.6 7.5 0.8 5.0 0.8
(calculated
as Table
1).
Arachidonic Table
3. Effect
Substrate
of layer
thickness
Weight (kg)
on microbial
Layer thickness
lipid
x + RS* (kg)
conversion Lipid (9)
of barley ARAt
Millet
2.0 3.5 5.0 2.0 3.5 5.0
0.5 1.5 5.0 0.5 1.5 5.0
0.3 1.0 3.4 0.3 0.9 3.3
30.8 78.5 191.1 41.9 90.8 269.2
* Biomass (X) with residual substrates (RS). T mg of ARA per g of dry weight of initial substrate *Sum of 14:0, 16:l and a-18:3.
during
Relative
SSF in plastic fatty
acid
bags.
composition
(% w/w)
@Wg)
(cm) Barley
and millet
acid from solid substrafes
18.3 12.1 5.3 26.6 13.0 10.2
(calculated
16:0
16:0
16:l
16:2
16:3
20:3
20:4
19.0 20.4 20.9 12.4 18.0 21.7
8.8 8.2 4.8 9.5 7.2 5.4
9.0 10.1 23.4 13.8 15.2 19.1
16.5 16.1 28.4 17.9 20.4 24.5
3.0 2.9 2.1 1.7 1.4 1.3
2.5 2.1 1.2 1.0 0.5
39.7 30.9 17.6 42.3 28.7 25.2
as Table
than 5 cm gave unsatisfactory results. Millet gave better results than barley in all tests. During SSF, we have obtained cereals enriched with ARA of microbial origin (Slugefi et al. 1992). These lipids are suitable as dieteticum and probioticum for human and veterinary applications. The SSF process should therefore provide an inexpensive way of producing ARA and other PUFA for food and feed supplementation.
Others% 1.7 9.3 1.6 1.7 0.1 0.3
1).
composition of Cundidu species by gas liquid chromatography using a polar column. Microbios 52, 37-42. Lonsane, B.K., Ghildyal, N.P., Budiatman, S. & Ramakrishna, S.V. 1985 Engineering aspects of solid state fermentation. Enzyme and Microbial
Technology
7, 258-265.
Lonsane, B.K., Saucedo-Castaneda, G., Raimbault, M., Roussos, S., Viniegra-Gonzales, G., Ghildyal, N.P., Ramakrishna, M. & Krishnaiah, M.M. 1992 Scale-up strategies for solid state fermentation systems. ProcessBiochemistry 27, 259-273. Mitchell, D.A., Grenfield, P.F. & Doelle, H.W. 1986 A model substrate for solid state fermentation. Biotechnology Letters 6, 827-832.
Shinmen, Y., Shimizu, S., Akimoto, K., Kawashima, H. & Yamada, H. 1989 Production of arachidonic acid by A4orfierellu fungi.
Acknowledgements We are grateful valuable help.
to
J. Moravek
and
E. Sturdik
for
References Bajpai, P.K., Bajpai, P. & Ward, O.P. 1991 Production of arachidonic acid by Mortierellu a@na ATCC 32222. ]ownal of Indtrstriul Microbiology
8, 179-186.
Christopherson, E.W. 81 Glass, R.Z. 1969 Preparation of milk fat methyl esters by alcoholysis in an essentially nonalcoholic solution, ]ownul of Dairy Science 52, 1289-1290. Das, U.N., Horrobin, D.F., Phil, D., Begin, M.E., Huang, Y.S., Cunnane, S.G., Manku, MS. & Nassar, B.A. 1988 Clinical significance of essential fatty acids. Nutrition 4, 337-341. Folch, J., Lees, M. & Sloane-Stanley, G.H. 1957 A simple method for isolation and purification of total lipids from animal tissues. ]ournul of Biological Chemistry 226, 450-497. Horrobin, D.F. 1990 Gamma linoleic acid. An intermediate in essential fatty acid metabolism with potential as an ethical pharmaceutical and as a food. Reviews in Contemporary Phurmucotherupy
Applied
Microbiology
and Biotechnology
31,
11-16.
Slugen, D. 1992 Production of hydrolytic enzymes by Mucoruceue fungi in natural solid state fermentations. In Interbiotech’92 Conference, p. 22. Bratislava: Dom Techniky. Slugen, D., Stred’ansky, M., Stred’anska, S., Certik, M. & Grego, J. 1992 Process of dietetic treatment of cereal substrates using solid state fermentation of Mucoruceue filamentous fungi. Czechoslovak Patent Application 3711/92. Suzuki, 0. & Yokochi, T. (1984) Method for the preparation of fungal body and a lipid therefrom and the use of such lipid. European Patent Application 125 765, 1984. Suzuki, O., Yokochi, T., Amano, K., Sano, T., Seto, S., Ohtu, I., Ishida, S., Iwamoto, S., Morioka, K., Satoh, A. & Uotani, K. 1988 Development in production of fat containing y-linolenic acid by fungi and its industrialisation. Ytkuguh 37, 1081-1096. Totani, N., Watanabe, A. & Oba, K. 1987 An improved method of arachidonic acid production by Mortierellu ulpinu. Yukuguku 36, 328-331.
Yamada, H., Shimizu, S. & Shinmen, Y. 1989 Production of arachidonic acid by Morfierellu elongutu IS-~. Agriculturul and Biological Chemistry 5 1, 785-790.
1, l-45.
Kobayashi, K., Suginaka, H. & Yano, I. 1987 Analysis of fatty acid
(Received in revised form 1 March 1993; accepted 7 March 19%)
of microbiology
and 6ioi~bno/ogy
9, 51 l-513
Arachidonic acid production by ~~~~ie~e//~ alpina grown on solid substrates S. Stred’anskB,*
D. Slugeti, M. Stred’anskp and J. Grego
Mortierelfa alpina grown in solid state fermentations on cereal substrates gave up to 16% lipid in the final biomass. Arachidonic was at 50% of total fatty acids, with a yield of 36 mg/g of original substrate. Microbial lipid production was successfully scaled up to use S-kg dry substrate batches. Key words: Arachidonic
acid, cereals, microbial
lipid, ~ortierella
Polyunsaturated fatty acids (PLJFA) have been successfully used in the prevention and treatment of many diseases (Das et al. 1988; Horrobin 1990). The fungus Morfierelh alpha is able to produce these biologically active components, mainly arachidonic acid (ARA, 20:4, o - 6)‘ in large quantities in submerged culture using glucose as substrate (Suzuki & Yokochi 1984; Suzuki et al. 1988; Shinmen et al. 1989; Yamada et al. 1989; Bajpai et al. 1992). However, the fungus used by Totani et al. (1987) produced the highest yields of ARA in surface cultivation on a potato/dextrose paste. These results indicate that solid state fermentation (SSF) couid be useful in fungal lipid production. In the present study, M. aipina CCF 185 was grown on solid substrates (moistened with water to a relative humidity of about 100% but in the absence of free water) in attempts to impove the fungus’ production of lipid rich in ARA or other PUFA.
Materials
and Methods
Substrates Cracked barley, peeled barley, dehulled millet, polished rice and wheat were bought on the local market. One vol of each substrate was soaked in three vols water at 50°C for 5 h. Any residual water was then removed. Microorganism Mortierella
Collection
and
Cdture
alpha CCF of Fungi,
Charles
Conditions
185
was obtained from the Culture University, Prague. Flask experiments
S. Stred’ansktt is with the Research Institute of Gerontology. 901 01 Malacky, Slovak Republic. D. SlugeZI, M. Stred’anskq and J. Grego are with the Department of Biochemical Technology, Slovak Technical University, 812 37 Brat&lava, Slovak Republic; fax: (42) 7 493 198. ‘Corresponding author. @ 1993 Rapid Communications
of Oxford
a&a.
were carried out in 300-ml Erlenmeyer flasks, each with 20 g (dry wt) of moist substrate, sterilized at 120°C for 15 min, inoculated with 1 ml of spore suspension (@/ml) and then incubated at 28°C for 7 to 21 days in a water-saturated atmosphere. Scale-up
experiments were performed in microporous polyethyIene plastic bags of base areas 0.09, 0,21 or 0.49 m”, respectively. Moist substrate was added to the small, medium and large bags (0.5, 1.5 and 5 kg dry wt, respectively) in layers (2.0, 3.5 and 5.0 cm thick), the bags sterilized with hot steam at 0.12 MPa for 45 min and then inoculated with 25, 75 and 250 ml of spore suspension (106/ml), respectively. The bags were kept at 28 to 30°C for 14 days and aerated by passing in sterilized, water-saturated air (0.5 Urnin. kg of substrate). Extra&on Original
of Lipid substrate
atid ~ete~j~a~i~ or the cultured
of Fatty Acid
mycelium with residual substrate were dried and crushed. Lipid was extracted in chloroform/ methanol (2:l v/v) and the solvent evaporated under vacuum (Folch et al. 1957). The lipids were suspended in n-hexane and converted to methyl esters with sodium methoxide in 10% (w/v) HCl in methanol (Christopherson & Glass 1969). The faatty acid composition of these esters was then analyzed by GLC (Kobayashi ef nl. 1987) on a column packed with 10% diethyleneglycolsuccinate.
Results
and Discussion
Solid state fe~entations (SSF) simulate the fe~entation reactions which occur in nature (Lonsane et af. 1985). The moist substrates act as a source of C, N, minerals, water and other nutrients (Mitchell et al. 1986). Cereals, which contain starch, proteins and low amounts of lipids are easily fermented by M. alpina (Slugei5 1992). In the present experiments, significant changes in substrate composition were observed (Tabie I). The surfaces of all substrates were covered by fungal mycelium 3 days after inoculation. The
Ltd World ~mmal of Microbiology and Biotechnology, Voi 9, 1993
511
S. Stredbnskh et al. Table
1. Lipid
content
and comparison
x + RS’ (8)
Substrate
Fermented mass Dehulled millet Cracked barley Peeled barley Polished rice Wheat Unfermented mass Millet Barley Rice Wheat
of tatty Ltpldt (8)
12.2 12.6 12.9 13.3 14.9
1.9 1.2 1.6 1.3 0.8
20.0 20.0 20.0 20.0
0.8 0.8 0.5 0.5
* Biomass (X) with residual substrate (RS). T 75% of total lipid are fatty acids. $ mg of ARA per g of dry weight of initial substrate §Sum of 14:0, 16:l and a-18:3.
acid
profile
in fermented
ARA$
Relative
Substrate
Millet
Barley
2. Eilect
of cultivation Time (day@
7 14 21 7 14 21
time
x+i?s* &I)
15.1 13.4 13.1 15.4 13.9 13.6
on lipid yield Lipid (91
1.6 1.9 2.1 1.4 1.7 2.1
fatty
acid
cereals. composition
(% w/w)
(w&f 16:0
16:0
16:l
16:2
16:3
20~3
20~4
Others5
35.7 20.6 24.9 17.8 9.1
12.9 14.6 17.3 16.3 14.5
5.7 6.6 5.5 7.0 6.6
7.1 5.6 7.0 14.6 12.3
13.2 13.2 15.5 8.9 23.5
2.6 2.5 2.2 4.2 3.0
1.8 2.3 0.8 8.0 3.4
49.1 45.2 44.3 36.5 34.6
7.6 10.0 8.0 4.5 2.1
-
12.0 22.1 30.8 19.3
1.0 2.6 1.1 3.2
22.7 16.4 22.2 20.8
64.0 55.6 43.8 51.3
-
-
-
0.3 3.3 2.1 5.4
calculated
as lipid (mglg
sum of the biomass and the residual substrates (X + RS) decreasedduring cultivation. The predominant fatty acid in all the original substrates was linoleic acid (18: 2). Mdierella aipj~ yielded 40.0 to 95.0 mg lipid/g initial substrate, which corresponded to 54 to 156 mg lipid/g fermented substrate (X + RS). Of the estimated lipid, 75% was estimated to be fatty acid. The yield of arachidonic acid ranged from 9 to 36 mg/g initial substrate. Previously the best yield of ARA which we have obtained was 3.1 g/l in submersed culture in a 20-l fermenter using hydrolysed whey (unpublished data). (Totani et al. (1987) reported a yield of 11.8 mg of ARA/g medium on surface cultivation of M. alpha on potato/dextrose paste. Millet and barley gave the highest yields of ARA in the fermented mass, so they were used in the subsequent experiment, in which the changes in lipid accumulation and composition with time were evaluated (Table 2). The rate
Table
and unfermented
and fatty
* Biomass (X) with residual substrates (RS). T mg of ARA per g of dry weight of initial substrate *Sum of 14:0, 16:l and a-18:3.
x 0.75 x % ARA in total fatty acid.
of decrease of X + RS observed during first 14 days was markedly faster than in the following week. ARA was again determined as the major fatty acid and its content in the lipid continued to rise. The highest ARA production was reached in the first week. The quantities of the ARA precursors, y-linoleic (18:3) and dihomo-y-linoleic (20:3) acid, changed insignificantly during the fermentation but the contents of palmitic, palmitoleic and linoleic acids after the fermentation were considerably lower than those in cereal lipids. In the SSFprocess, many scale-up problems have to be solved before a laboratory-scale process can be transferred to commercial production (Lonsane et a/. 1992). In scale-up experiments, the effect of the depth of layer was investigated (Table 3). The utilization of cereals decreased with scale because of the diffusion of 0,. Consequently, both total lipid and ARA contents decreased.Layers deeper
acid
profile
ARAt (m&a)
18.6 32.2 38.0 20.3 25.6 32.9
biomass)
in fermented Relative
fatty
cereal acid
substrate.
composition
(% w/w)
16:0
18:0
16:l
18:2
16:3
2023
20:4
Others$
19.0 12.7 9.6 16.7 16.0 14.0
8.6 6.1 4.5 10.8 10.0 7.9
11.0 8.2 8.9 17.1 9.7 15.2
16.7 23.0 14.2 12.7 13.4 14.3
2.4 2.7 4.3 2.2 2.5 2.2
4.4 5.0 3.7 2.1 2.6 2.7
31.0 44.7 47.3 37.8 40.8 42.8
6.9 7.6 7.5 0.8 5.0 0.8
(calculated
as Table
1).
Arachidonic Table
3. Effect
Substrate
of layer
thickness
Weight (kg)
on microbial
Layer thickness
lipid
x + RS* (kg)
conversion Lipid (9)
of barley ARAt
Millet
2.0 3.5 5.0 2.0 3.5 5.0
0.5 1.5 5.0 0.5 1.5 5.0
0.3 1.0 3.4 0.3 0.9 3.3
30.8 78.5 191.1 41.9 90.8 269.2
* Biomass (X) with residual substrates (RS). T mg of ARA per g of dry weight of initial substrate *Sum of 14:0, 16:l and a-18:3.
during
Relative
SSF in plastic fatty
acid
bags.
composition
(% w/w)
@Wg)
(cm) Barley
and millet
acid from solid substrafes
18.3 12.1 5.3 26.6 13.0 10.2
(calculated
16:0
16:0
16:l
16:2
16:3
20:3
20:4
19.0 20.4 20.9 12.4 18.0 21.7
8.8 8.2 4.8 9.5 7.2 5.4
9.0 10.1 23.4 13.8 15.2 19.1
16.5 16.1 28.4 17.9 20.4 24.5
3.0 2.9 2.1 1.7 1.4 1.3
2.5 2.1 1.2 1.0 0.5
39.7 30.9 17.6 42.3 28.7 25.2
as Table
than 5 cm gave unsatisfactory results. Millet gave better results than barley in all tests. During SSF, we have obtained cereals enriched with ARA of microbial origin (Slugefi et al. 1992). These lipids are suitable as dieteticum and probioticum for human and veterinary applications. The SSF process should therefore provide an inexpensive way of producing ARA and other PUFA for food and feed supplementation.
Others% 1.7 9.3 1.6 1.7 0.1 0.3
1).
composition of Cundidu species by gas liquid chromatography using a polar column. Microbios 52, 37-42. Lonsane, B.K., Ghildyal, N.P., Budiatman, S. & Ramakrishna, S.V. 1985 Engineering aspects of solid state fermentation. Enzyme and Microbial
Technology
7, 258-265.
Lonsane, B.K., Saucedo-Castaneda, G., Raimbault, M., Roussos, S., Viniegra-Gonzales, G., Ghildyal, N.P., Ramakrishna, M. & Krishnaiah, M.M. 1992 Scale-up strategies for solid state fermentation systems. ProcessBiochemistry 27, 259-273. Mitchell, D.A., Grenfield, P.F. & Doelle, H.W. 1986 A model substrate for solid state fermentation. Biotechnology Letters 6, 827-832.
Shinmen, Y., Shimizu, S., Akimoto, K., Kawashima, H. & Yamada, H. 1989 Production of arachidonic acid by A4orfierellu fungi.
Acknowledgements We are grateful valuable help.
to
J. Moravek
and
E. Sturdik
for
References Bajpai, P.K., Bajpai, P. & Ward, O.P. 1991 Production of arachidonic acid by Mortierellu a@na ATCC 32222. ]ownal of Indtrstriul Microbiology
8, 179-186.
Christopherson, E.W. 81 Glass, R.Z. 1969 Preparation of milk fat methyl esters by alcoholysis in an essentially nonalcoholic solution, ]ownul of Dairy Science 52, 1289-1290. Das, U.N., Horrobin, D.F., Phil, D., Begin, M.E., Huang, Y.S., Cunnane, S.G., Manku, MS. & Nassar, B.A. 1988 Clinical significance of essential fatty acids. Nutrition 4, 337-341. Folch, J., Lees, M. & Sloane-Stanley, G.H. 1957 A simple method for isolation and purification of total lipids from animal tissues. ]ournul of Biological Chemistry 226, 450-497. Horrobin, D.F. 1990 Gamma linoleic acid. An intermediate in essential fatty acid metabolism with potential as an ethical pharmaceutical and as a food. Reviews in Contemporary Phurmucotherupy
Applied
Microbiology
and Biotechnology
31,
11-16.
Slugen, D. 1992 Production of hydrolytic enzymes by Mucoruceue fungi in natural solid state fermentations. In Interbiotech’92 Conference, p. 22. Bratislava: Dom Techniky. Slugen, D., Stred’ansky, M., Stred’anska, S., Certik, M. & Grego, J. 1992 Process of dietetic treatment of cereal substrates using solid state fermentation of Mucoruceue filamentous fungi. Czechoslovak Patent Application 3711/92. Suzuki, 0. & Yokochi, T. (1984) Method for the preparation of fungal body and a lipid therefrom and the use of such lipid. European Patent Application 125 765, 1984. Suzuki, O., Yokochi, T., Amano, K., Sano, T., Seto, S., Ohtu, I., Ishida, S., Iwamoto, S., Morioka, K., Satoh, A. & Uotani, K. 1988 Development in production of fat containing y-linolenic acid by fungi and its industrialisation. Ytkuguh 37, 1081-1096. Totani, N., Watanabe, A. & Oba, K. 1987 An improved method of arachidonic acid production by Mortierellu ulpinu. Yukuguku 36, 328-331.
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(Received in revised form 1 March 1993; accepted 7 March 19%)
