Studies on fermentative production of squalene

P. Bhattacharjee, V.B. Shukla, R.S. Singhal* and P.R. KulkarniFood and Fermentation Technology Division, University Department of Chemical Technology (U.D.C.T), Matunga,Mumbai-400 019, India*Author for correspondence: Fax: +91-22-4145614, E-mail: rekha@foodbio.udct.ernet.in

Received 3 May 2001; accepted 12 September 2001

Keywords: Chromatography, fermentative production, lipid extract, spectroscopy, squalene

Summary

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker’syeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yieldof squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 lg g)1 respectively, dry weightof yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain wereachieved chromatographically. The purified squalene was characterized spectroscopically against an authenticstandard.

Introduction

Squalene is a naturally occuring constituent found inplant oils such as olive oil (Roncero & Janer 1962), palmoil, wheat germ oil, amaranth oil (Singhal & Kulkarni1990), rice bran oil, fish oils as well as in human sebum.It is the principal hydrocarbon in human surface lipids,amounting to about 10% of the total surface fat, and isthe biosynthetic precursor of cholesterol. The richestsource is the liver of Aizame (dogfish) shark (Squalasspp) of the southern Pacific oceans of Australia. It is ahighly unsaturated aliphatic hydrocarbon (2, 6, 10, 15,19, 23-hexamethyltetracosa-2, 6, 10, 14, 18, 22-hexaene;molecular formula – C30H50 belonging to the triterpenegroup of oils) isolated from fish oil by vacuum distilla-tion. It is a clear, brilliant and almost colourless oil witha faint odour and taste. It serves as a detoxificationfactor, as a skin and eye antioxidant, in providing cellswith oxygen, and as a bactericidal and fungicidal agent.It has wide applications as an antistatic agent andemollient in cosmetics and pharmaceuticals, and also infine chemicals, magnetic tape and low-temperaturelubricants. Olive oil containing 0.8% squalene is widelyused for cooking and salad dressing. Oryzanol isdissolved in squalene and added to foods as anantioxidant (Ishitani 1980). Squalene is also used anadditive in animal feed.The limited availability of squalene has compelled

manufacturers to switch over to its substitutes such assqualane, squaliformes etc. A 500 mg capsule of squa-lene is priced at 0.125 US$. The present work was aimedat production of squalene using microbial sources.There are only a few reports on microbial production

of squalene: Saccharomyces (Jollow et al. 1968; Kata-yoka et al. 1992; Kawai 1992; Kamimura et al. 1994;Socaciu et al. 1995; Ciesarova et al. 1996), Pseudomonas(Uragami & Koga 1986), Candida (Tsujiwaki et al.1995a, b), the algae Euglena (Kawaura et al. 1995;Kawaura & Matsuda, 1996) and in general yeasts(Mauricio et al. 1993) are the organisms used for thispurpose.The biosynthetic pathway of squalene production in

Saccharomyces spp. starts with the synthesis of meval-onate from acetate, then conversion of mevalonate totwo activated isoprenes, condensation of six activatedisoprene units to form squalene and finally conversionof squalene to the 4-ring lanosterol (Lehninger et al.1993a).Commercially available compressed baker’s yeast

(Saccharomyces cerevisiae) and a strain of Torulasporadelbrueckii isolated from molasses were used for fer-mentative production of squalene. The yield of squalenein mg g)1 of wet biomass, under anaerobic conditions,was studied as a function of time and inoculum size ofthe aerobic culture. Downstream processing of squaleneto purify it was attempted and ascertained spectroscop-ically using an authentic standard.

Materials and Methods

Materials

Glucose, yeast extract powder, malt extract powder,peptone were from Himedia Laboratories, Mumbai,aluminium HPTLC plates coated with silica gel 60 (F254)

World Journal of Microbiology & Biotechnology 17: 811–816, 2001. 811� 2001 Kluwer Academic Publishers. Printed in the Netherlands.

from E. Merck, Germany, silicic acid (100–200 mesh forlipid chromatography) of Spectrochem Pvt. Ltd, Mum-bai. All solvents used were of AR grade. Commerciallyavailable compressed baker’s yeast (S. cerevisiae) and astrain of T. delbrueckii were used for the isolation ofsqualene. Standard squalene was procured from Sigma-Aldrich Corporation, USA.

Methods

Aerobic culture of microorganismsCompressed baker’s yeast (S. cerevisiae). Two millilitreof 10% suspension of compressed yeast in steriledistilled water, was inoculated into 100 ml growthmedium having composition – glucose 20 mg ml)1,yeast extract powder 5 mg ml)1, peptone (bacteriolog-ical) 5 mg ml)1 and malt extract powder 5 mg ml)1 (pH4–5) in 250 ml shake flask. The flask was incubated at30 ± 2 �C for 48 h on a rotary shaker at 44 · g.

T. delbrueckiiMolasses serially diluted with sterile distilled water wasinoculated into growth medium (Long & Ward 1989)having the composition: glucose 20 mg ml)1, yeastextract powder 10 mg ml)1 and peptone 20 mg ml)1

(pH 5.5) and incubated at 30 ± 2 �C for 48 h on arotary shaker at 44 · g. Growth was plated on solidmedium containing 29 mg agar ml)1. The organismsfrom plates were selected and isolated on the basis ofcolony and morphological characteristics. As many as 16isolates were obtained. One of the isolates, which wasused for biotransformation of benzaldehyde to L-Pheny-lacetylcarbinol (Shukla & Kulkarni 2000) and identifiedas T. delbrueckii was deposited in Microbial TypeCulture Collection, Institute of Microbial Technology,Chandigarh, India and was given culture number MTCC3417. The culture had many characteristics similar to S.cerevisiae which is known to be a squalene producer.Hence, this isolate was chosen for a comparative study.One loopful of active culture from the slant was

inoculated into 100 ml growth medium having compo-sition the same as the above, in a 250 ml shake flask.The flask was incubated at 30 ± 2 �C for 48 h on arotary shaker at 44 · g.

Anaerobic production of squalene

The procedure for both the cultures chosen in the studywas similar. After 48 h, 1, 2, 3 and 5% of active culturefrom the aerobic medium were inoculated into fresh100 ml medium having the composition: glucose40 mg ml)1, yeast extract powder 10 mg ml)1 andpeptone 20 mg ml)1 (pH 5.5) (Lodder 1970) containedin 250 and 100 ml conical flasks for both speciesseparately. To maintain anaerobic conditions, a layerof sterile light liquid paraffin about 1 cm thick wasspread on the surface of the medium and the flasks weretightly sealed. The culture was left standing underanaerobic conditions for 24–72 h.

Recovery and detection of squalene from the biomass

After 24, 48 and 72 h, the culture was centrifuged at20,000 · g for 20 min at 28 ± 2 �C, to collect the cellmass. As chloroform is reported to cause yeast cell lysisand is the solvent commonly used for extraction ofneutral lipids, it was used to recover the intracellularsqualene from the biomass after lysis. But chloroformdid not allow quantitative recovery of the biomass fromthe centrifuge tubes owing to the stickiness of the yeastcell on the walls of the tubes. Petroleum ether, reportedto be one of the best solvents for the extraction of oils,was also tested. But the yeast cells formed an agglo-merate in the centrifuge tube which could not be scrapedoff. Microscopic observation indicated a complete lossof individuality of the yeast cells. Finally, the cell masswere dispersed in chloroform/methanol (2:1, v/v) mix-ture. This solvent has been reported for squalenerecovery from Euglena (Kawaura & Matsuda 1996).With this mixture, quantitative recovery of the biomasswas ensured. The commonly reported method for lipidextraction using chloroform/methanol/water (1:2:0.8, v/v) (Lehninger et al. 1993b) was not used as the aim wasto selectively extract only the neutral lipid. Methanol–water mixture is polar and could have extracted alltriglycerides and other sterol esters. About 150 ml of thechloroform/methanol solvent mixture was used forquantitative transfer of the biomass from each anaero-bic culture flask. The dispersed cells were subjected toshaking extraction in 250 ml shake flasks on a rotaryshaker at 34 · g at 30 ± 2 �C for 8–10 h to allowcomplete cell lysis. The biomass was removed byfiltration using non-absorbent cotton–wool and thefiltrate was passed through activated molecular sievesto remove the residual moisture. The biomass on thecotton was washed 2–3 times with fresh chloroform–methanol solvent system (�20 ml each time). Solventfrom the extract was then removed by a rotary evapo-rator operating at 500 mm Hg at 45 �C to yield aviscous oily substance. The crude extract was observedto be a bilayered product – the upper layer, a whitegreasy substance and a pale yellow coloured oilysubstance in the lower layer. The extract was character-ized by gas chromatography (GC) using a capillarycolumn, FT-IR HPTLC and UV analysis, using stan-dard squalene for comparison.

Gas chromatographic analysis of the extract

The white and yellow portions of the extract wereanalysed by GC separately. The two fractions afterthorough drying were redissolved in n-hexane andanalysed by GC using Chemito 8510 model equippedwith a flame ionization detector connected to Oracle 1computing integrator. A BP-5 capillary column (2.2 mmo.d. · 50 m length) with the following specifications wasused. The carrier gas was Iolar grade hydrogen with aflow rate of 40 ml min)1 at room temperature. Purge andsplit were 5 and 60 ml min)1 respectively. About 0.4 ll of

812 P. Bhattacharjee et al.

the extract was used for analysis. The column was heatedfrom 120 to 280 �C at 10 �C min)1 and held at 280 �C for30 min. Injector and detector temperatures were 250 and260 �C respectively. Squalene standard and that in theextract had a retention time of 27.76 ± 2 min. Since thepure compound could not be flushed out completely in asingle run from the capillary column but was carried overto the consecutive runs; GC could not quantify thesqualene in the extract. Various temperature program-ming were tried with no success. Squalene could bedetected only in the yellow portion of the extract. Thoughquantification of squalene using gas-liquid chromato-graphy is reported as a rapid and accurate technique forvegetable oils (Lanzon et al. 1995), such as olive oils(Leonardis et al. 1998) and in amaranth oils (Singhal1989). It could only be used for qualitative detection ofsqualene with the microbial lipid extract.

UV scan of extracts for kmax

Pure squalene of 0.5% w/v and the yellow portion of thecrude extracts, dissolved in chloroform (Spectro grade)were scanned in the UV region of 400–200 nm by theHitachi Spectrophotometer (Model U 2001). kmax of272 nm obtained for both pure and the extracts con-firmed presence of squalene in the extracts (Singhal &Kulkarni 1990).

Colorimetric estimation of squalene

The above extract was thoroughly dried by purgingcommercial grade nitrogen and quantification of squa-lene was carried out colorimetrically at 400 nm asdescribed by Rothblat et al. (1962) using Elico Spectro-photometer (Model CL-27).

Densitometric estimation of squalene

For densitometric assay, several solvent systems weretried. Cyclohexane was found to give the best resolution,in which squalene recorded an Rf value of 0.60 ± 0.02.The extracts were spotted on aluminium plates coatedwith silica gel 60 (F254) by use of Camag Linomat IV.The extracts, dissolved in n-hexane (AR grade), wereapplied to the plates in the form of bands, each 6 mmwide, spacing between consecutive bands being 8 mm.Nitrogen gas was used at a low flow rate of 4 bar forspotting. The plates were developed at 26 ± 2 �C in aglass chamber containing cyclohexane. The spot corre-sponding to squalene could be easily detected onexposure to iodine vapours. Spectrum scanning of thespots after development was carried out in CamagHPTLC unit (TLC scanner II). Squalene recorded akmax value of 200 nm (95% transmission which de-creased to 86% at 203–204 nm). Densitometric studieswere performed at 200 nm and the area under the curvefor squalene was recorded. A standard curve was plottedfor pure squalene at 200 nm (area under the curve as

recorded by Camag HPTLC vs. lg of pure squalenespotted).

Isolation and purification of squalene from the lipidextract by chromatographic technique

Since the colorimetric test (Rothblat et al. 1962) basedon H2SO4–formaldehyde chromogen also shows apositive colour reaction with lanosterol or ergosterol(if produced from squalene) attempts were made topurify the cell extract by silicic acid chromatography.Silicic acid (100–200 mesh for lipid chromatography)was washed with methanol and water to remove finesand impurities and activated at 110–120 �C overnight.Approximately 35–38 g of silicic acid was used for thecolumn of length 180–200 mm and ID 25 mm. A slurryof silicic acid in 6% (v/v) benzene in hexane was pouredinto the column. About 1 g of the lipid extract obtainedby cell lysis was applied to the column. The crudeextract after thorough drying in a rotary evaporator wasadsorbed onto 1.5 times its weight of silicic acid andloaded on to the column. Squalene was eluted with 6%benzene in n-hexane (Horning et al. 1960) in the initialfractions itself which was confirmed by HPTLC, butpure squalene was not obtained. Since sterols and sterolesters are eluted along with hydrocarbons in the initialfractions (Weber 1969; Stoller & Weber 1970), theimpurity peaks may have been due to ergosterol and/orlanosterol. The Liebermann–Bucchard test for thepresence of the steroid nucleus was positive for theeluates. So further purification of squalene was attempt-ed by alumina chromatography. The silicic acid columnfractions in which squalene was detected by TLC werecombined, saponified and loaded on to an aluminacolumn as described (Firestone 1995). The hydrocarbonfraction containing squalene was eluted in petroleumether, concentrated by rotary evaporator at 45 �C and500 mm Hg vacuum and further purified by preparativeTLC using silica gel-coated glass plates and cyclohexaneas solvent. The band with Rf value 0.6 was scraped offand characterized by FT-IR, 1H-NMR, 13C-NMR andGC-MS. Squalene isolated from both the microbialsources was purified and characterized. The spectrum ofsqualene isolated from T. delbrueckii had been shown tobe similar to that obtained from S. cerevisiae.

FT-IR spectra of the purified compound

The FT-IR spectra for both authentic and purifiedsqualene from T. delbrueckii were recorded on Perkin-Elmer-783 spectrophotometer using CHCl3 as solvent.IR (neat, cm)1): 2914 (CAH stretching), 2728, 1668

(alkene, non-conjugated), 1446 (alkane, CH2), 1382(alkane, CH3), 1330, 1224, 1151, 1188, 964 (alkene,disubstituted trans), 835 (two adjacent hydrogen atoms),722.1 (due to CHCl3 solvent used for dissolution ofisolated squalene). The FT-IR spectra of the purifiedsqualene compared well with the authentic standard.

Squalene production by yeasts 813

NMR spectra of the purified compound

1H-NMR for both authentic and purified squalene fromT. delbrueckii were recorded in CDCl3 using 300 MHzBrucker ACP-300 model. In 1H-NMR spectra, chemicalshifts are reported in ¶ units (ppm) relative to TMS asinternal standard. 1H-NMR (CDCl3, 300 MHz): ¶ 5.1(t, 6H), 1.93–2.09 (m, 20H), 1.68 (s, 6H), 1.60 (s, 18H).

13C-NMR spectra were recorded in CDCl3. In13C-

NMR spectra, coupling constants (J) are reported inhertz (Hz). 13C-NMR (CDCl3, 125 MHz): 135.00 (d,J = 25.7 Hz), 131.10 (s), 124.42 (d, J = 13.7 Hz),77.24 (t, J = 31.9 Hz; due to CDCl3 solvent), 39.73(s), 28.2 (s), 26.77 (d, J = 14.3 Hz), 25.63 (s), 17.61 (s),15.98 (d, J = 3.36 Hz). Both 1H-NMR and 13C-NMRspectra of the purified squalene showed good correlationwith those of the authentic sample.

GC-MS of the purified compound

The compound was analyzed in a GC-7A Shimadzumodel coupled to a QP-5000 MS equipped with a flameionization detector and electron ionization (EI) detectorrespectively. EI mass spectra was obtained with anionization voltage of 70 eV. The column was a fusedsilica capillary column (0.3 mm o.d. · 50 m length). It isa non-polar column packed with DB-5 (polymethylsiloxane with 5% phenyl modification). The carrier gaswas helium at 21 ml min)1 at 25 �C. The programmingwas as described above. The retention time for squalenewas 24.47 min. EI mass spectra data of squalene(purified from T. delbrueckii) are reported in the formm/z (relative abundance). m/z 41 (M+ , 0.07), 149 (7.14),136 (14.2), 95 (14.5), 81 (43.5), 69 (100), 41 (14.8).

Results and discussion

Table 1 shows a comparative data on squalene produc-tion as estimated colorimetrically by S. cerevisiae and T.delbrueckii in 250 ml shake flasks. After 48 h of anaero-bic fermentation, the best yield of squalene was obtainedwith 5% inoculum. A decrease in squalene content wasobserved after 72 h. This could be due to utilization ofsqualene as a carbon source by the yeast cells forsustenance or due to conversion into some otherunknown product. There is a report on utilization ofsqualene as a carbon source by C. famata US-238(Tsujiwaki et al. 1995a, b). Yeast at a glucose concen-tration above 0.4% is known to exhibit the Crabtreeeffect, in which it will forego respiration and carry outfermentation. Yeast will first utilize oxygen to synthesizesterols for cell wall building. So the ergosterol and/orlanosterol generated in 2% glucose medium in aerobicphase gets extracted by solvents later when cell wall isruptured for lipid extraction. Hence, the colorimetricestimation assays both sterols and squalene. Up to 5%inoculum level, the media nutrients are sufficient topromote good growth of yeast cells and consequently

squalene production in anaerobic phase. But 10 and20% inoculum levels are too high to be sustained. Thegrowth of yeast cells may have been arrested in theaerobic phase and the media nutrients in the anaerobicphase have been utilized for maintenance rather thanchannelized for squalene production. Five percent ofinoculum gave the best yield of squalene after 24 h ofanaerobic fermentation for T. delbrueckii. The trend insqualene production is just the reverse with thatobserved with S. cerevisiae. This may be probably dueto greater utilization of squalene by T. delbrueckii ascompared to compressed yeast.Since oxygen is the primary limiting factor in yeast

growth, it was thought that squalene production wouldbe better in 100 ml flasks. No definite trend in squaleneproduction was observed and hence the study wasdiscontinued.For both the organisms, the set that gave the best

yield in shake flask studies was quantified by thedensitometric method as shown in Tables 2 and 3. Thisassay established T. delbrueckii to be a better squaleneproducer than S. cerevisiae. The values for squaleneestimated colorimetrically were higher compared to thatestimated densitometrically as ergosterol and/or lano-sterol contributed to the values in the colour test.Isolation and purification of squalene from the lipid

extracts of both S. cerevesiae and T. delbrueckii bycolumn chromatography has confirmed the authenticityof squalene. This study could be valuable in theproduction of squalene, which due to limited availabilityhas compelled manufacturers to switch over to substi-tutes.

Table 1. Comparative production of squalene by S. cerevisiaea and T.

delbrueckiib in mg g)1 of wet biomassc,d.

Percentage (%) of

inoculum added to

anaerobic medium

from aerobic medium

Time of fermentation (h)

24 48 72

1a 0.22 ± 0.05 0.27 ± 0.05 0.34 ± 0.07

1b 1.07 ± 0.08 0.49 ± 0.09 0.34 ± 0.08

2a 0.27 ± 0.09 0.37 ± 0.08 0.38 ± 0.08

2b 1.09 ± 0.06 0.52 ± 0.08 0.41 ± 0.07

3a 0.40 ± 0.09 0.40 ± 0.10 0.41 ± 0.08

3b 1.30 ± 0.07 0.64 ± 0.08 0.52 ± 0.07

5a 0.42 ± 0.06 1.38 ± 0.06e 0.35 ± 0.06

5b 1.89 ± 0.06f 0.51 ± 0.08 0.34 ± 0.08

10a 1.25 ± 0.08 0.40 ± 0.04 0.36 ± 0.07

10b 1.53 ± 0.08 0.43 ± 0.04 0.30 ± 0.05

20a 0.99 ± 0.09 0.23 ± 0.04 0.22 ± 0.06

20b 1.04 ± 0.08 0.34 ± 0.08 0.34 ± 0.08

a Data for S. cerevisiae.b Data for T. Delbrueckii.c The results are expressed as mean±S.D of four individual

production runs.d Yield of squalene in terms of dry weight of biomass could not be

done as complete removal of the adhered media broth to the

biomass was not possible.e The optimum yield of squalene from S. cerevisiae.f The optimum yield of squalene from T. delbruekii.

814 P. Bhattacharjee et al.

Table

2.Densitometricassayofsqualenefrom

S.cerevisiae.

Lipid

extractapplied

ontheplate(lg)

Y1a

Y2a

X1b=

Y1/

m·10)3

X2b=

Y2/

m·10)3

Mean·10)3

SD

(n)1,

n=

2)·10)6

%RSD

=

(S.D/m

ean)·100

Squalene/gof

lipid

extract(lg)

Mean

lgofsqualene/

gm

oflipid

extract

Squalene/gofdry

biomass(lg)

55.4

236.3

235.1

0.73

0.73

0.74

2.65

0.36

28.38

27.47

41.16

234.5

236.1

0.72

0.73

0.73

3.54

0.49

28.33

110.8

488.5

467.6

1.51

1.44

1.48

45.96

3.11

28.78

475.2

481.2

1.47

1.49

1.48

1.27

0.86

28.77

166.2

699.6

697.7

2.16

2.16

2.16

3.82

0.18

28.04

698.5

697.1

2.16

2.15

2.16

2.12

0.10

27.99

221.6

841.5

834.7

2.60

2.58

2.59

15.56

0.60

25.22

836.4

838.7

2.58

2.59

2.59

4.24

0.16

25.20

277.0

1120.8

1119.1

3.46

3.46

3.46

3.75

0.11

26.96

1121.2

1122.8

3.46

3.47

3.47

3.54

0.10

27.02

Thewetweightofthebiomassobtained

bycentrifugationwas5.863gwithamoisture

contentof75.43%.

Concentrationoflipid

extracttaken

forHPTLCanalysis:27.7

lgll

)1.

aY1andY2are

areasunder

thecurves

forsqualenewhen

thelipid

extractisspotted

inreplica

inoneTLCplate;twosetsofY1andY2are

twoindependentrunsontwoTLCplates.

bX1andX2are

concentrationofsqualenein

theextractevaluatedfrom

standard

curveofpure

squalenewithslope,m

=323830,R2=

0.9927.

Squalene production by yeasts 815

References

Ciesarova, Z., Sajbidor, J., Smogrovicova, D. & Bafrncova, P. 1996

Effect of ethanol on fermentation and lipid composition in S.

cerevisiae. Food Biotechnology 10, 1–12.

Firestone, D. 1995 Squalene in oils and fats: Titrimetric analysis. In

Official Methods of Analysis of AOAC International, 16th edn, Vol

II, ed. Cunniff. P. p. 30 Virginia: AOAC International ISBN 0-

93558454-4.

Horning, M.G., William, E.A. & Horning, E.A. 1960 Separation of

tissue cholesterol esters and triglycerides by silicic acid chromato-

graphy. Journal of Lipid Research 1, 482–485.

Ishitani, A. 1980 Oryzanol antioxidant for food. Japan Kokai Tokkyo

Koho JP 80, 50, 094 (C.A.- 93:112560).

Jollow, D., Kellesman, G.M. & Linnane, A.W. 1968 The biogenesis of

mitochondria. III The lipid composition of aerobically and

anaerobically grown S. cerevisiae as related to the membrane

systems of the cells. Journal of Cell Biology 37, 221–230.

Kamimura, N., Uozumi, T. & Hidaka, S. 1994 Preparation of squalene

with sterol-dependent S. cerevisiae mutant. Japan Kokai Tokkyo

Koho JP 06, 90, 473 (C.A.-121:33289).

Kataoka, H., Matsumura, M., Myamoto, K. & Myabe, H. 1992

Simultaneous manufacture of squalene and ethanol with Saccharo-

myces. Japan Kokai Tokkyo Koho JP 04, 179, 487 (C.A.-

117:210767).

Kawai, H. 1992 Manufacture of squalenes with yeast mutant strains.

Japan Kokai Tokkyo Koho JP 04, 311, 393 (C.A.-118:123141).

Kawaura, S. & Matsuda, N. 1996 Squalenes manufacture with

Euglena. Japan Kokai Tokkyo Koho JP 08, 89, 260 (C.A.-

125:8714).Kawaura, S., Matsuda, N. & Kobayashi, N. 1995 Squalene manufac-

ture with Euglena. Japan Kokai Tokkyo Koho JP 07, 115, 981

(C.A.-125: 8714).

Lanzon, A., Guinda, A. & de la Osa, C. 1995 A rapid method for

squalene determination in vegetable oils. Grasas y Aceites 46, 276–

278.

Lehninger, A.L., Nelson, D.L. & Cox, M.M. 1993a Principles of

Biochemistry, pp. 262–264. USA: Worth Publishers. ISBN

0-87901-500-4.

Lehninger, A.L., Nelson, D.L. & Cox, M.M. 1993b Principles of

Biochemistry, pp. 669–694. USA: Worth Publishers. ISBN

0-87901-500-4.

Leonardis, A.D., Macciola, V. & Felice, M.D. 1998 Rapid determi-

nation of squalene in virgin olive oils using gas-liquid chromato-

graphy. Italian Journal of Food Science 10, 75–80.

Lodder, J. 1970 The Yeasts: A Taxonomic Study, p. 42. London:

North-Holland Publishing Company. ISBN 0-7 204 4054 8.

Long, A. & Ward, O.P. 1989 Biotransformation of benzaldehyde by

Saccharomyces cerevisiae: characterisation of fermentation and

toxicity effects of substrate and products. Biotechnology and

Bioengineering 34, 933–941.

Mauricio, J.C., Ortega, J.M., Rozes, N. & Larue, F. 1993 Effects of

culture conditions on the lipid fractions of various species of wine

yeasts. Cerevisia Biotechnology 18, 54–63.

Roncero, A.V. & Janer, L. 1962 Squalene in olive tree leaves. Grasas y

Aceites 13, 242–243.

Rothblat, G.H., Martak, D.S. & Kritchevsky, D. 1962 A quantitative

colorimetric assay for squalene. Analytical Biochemistry 4, 52–56.

Shukla, V.B. & Kulkarni, P.R. 2000 Review: L-Phenylacetycarbinol

(L-PAC): biosynthesis and industrial applications. World Journal

of Microbiology and Biotechnology 16, 499–506.

Singhal, R.S. 1989 Studies on Amaranths. PhD Thesis, Mumbai,

India.

Singhal, R.S. & Kulkarni, P.R. 1990 Effect of puffing on oil

characteristics of Amaranth (Rajgeera) seeds. Journal of the

American Oil Chemists Society 67, 952–954.

Socaciu, C., Faye, M., Salin, F., Paul, G. & Gleizes, M. 1995 In vitro

yeast (S. cerevisiae) presqualene and squalene synthesis related to

substrate and co-factor availability. Complete Rendus des Seances

de l’ Academie des Sciences. Serie III 318, 919–926 (C.A.-124:

112000).

Stoller, E.W. & Weber, E.J. 1970 Lipid constituents of some common

weed seeds. Journal of Agricultural and Food Chemistry 18, 361–364.

Tsujiwaki, G., Yamamoto, H. & Minami, K. 1995a Docosahexaenoic

acid-high fat manufacture with Candida. Japan Kokai Tokkyo

Koho JP 07, 289, 272 (C.A.-124:53834).

Tsujiwaki, G., Yamamoto, H. & Minami, K. 1995b Manufacture of

squalene with Candida famata. Japan Kokai Tokkyo Koho JP 07,

289, 272 (C.A.-124: 230184).

Uragami, S. & Koga, H. 1986 Bacterial production of squalene. Japan

Kokai Tokkyo Koho JP 61, 212, 290 (C.A.-106: 65872).

Weber, E.J. 1969 Lipids of maturing grain of corn (Zea mays L.):

changes in lipid classes and fatty acid composition. Journal of the

Association of Official Agricultural Chemists 46, 485–488.

Table 3. Densitometric assay of squalene from T. delbrueckii.

Lipid extract applied

on the plate (lg)Y1

a Y2a X1

b = Y1/

m · 10)3X2

b = Y2/

m · 10)3Mean

· 10)3SD (n)1,n = 2) · 10)6

% RSD =

(SD/mean)

· 100

Squalene/g

of lipid

extract (lg)

Squalene/g of

dry

biomass (lg)

1.53 113.4 114.5 0.31 0.35 0.35 2.48 0.70 496.73 237.25

3.06 294.8 292.7 0.91 0.90 0.91 4.60 0.51 640.29

4.59 493.0 487.8 1.52 1.51 1.51 11.60 0.77 712.47

6.12 528.9 521.4 1.63 1.61 1.62 16.26 1.00 572.39

7.65 543.5 540.2 1.68 1.67 1.67 7.07 0.42 472.23

The wet weight of the biomass obtained by centrifugation was 7.3875 g with a moisture content of 80.49%.

Concentration of lipid extract taken for HPTLC analysis: 15.3 lg ll)1.a Y1 and Y2 are areas under the curves for squalene when the lipid extract is spotted in replica in one TLC plate.b X1 and X2 are concentration of squalene in the extract evaluated from standard curve of pure squalene with slope, m = 323830,

R2 = 0.9927.

816 P. Bhattacharjee et al.