SINGH et ale-MYCOLOGICAL FORMATION OF FAT. IV 697

a plant becomes established, it is likely to yield the same weight of cob whatever the treatment given the seed from which it is derived.

It is hoped to store the samples of seed used in this investigation for some years, to study the effects of prolonged storage, and to link the damage so produced with viability assessments made with tetrazolium salt, and sodium chlorate, hoping ultimately to discover the rBle of methyl bromide in the delay and inhibition of germination.

Acknowledgment

interest is gratefully acknowledged. This work has been supported by a grant from the Agricultural Research Council, whose

Imperial College Field Station Sunninghill

Berks. Received 6 March, r g j 7

References Winteringhani, F. P. \V., J . Sci. Pd Agric., 1955,

6, 269 Lubatti, 0. F., & Blackith, R. E., J. Sci. Fd Agvic.,

1956, 79 149, 343 Lubatti, 0. F., & Smith, B., J . SOC. chenz. I n d .

LoPZd., S948, 67, 297 Wellington, P. S., Report of the Commee on the

Effect of Toxic Substances in Seed Testing,

Proc. A'Ith int. Seed Test. Coizgv., Paris, June, 1956

Page, A. B. P., & Blackith, R. E., Rep. Progv. uppl. Chew., 1956, 41, 535

6 Alexander, 17. E. S., & Clifford, H. T., Nature, Lond., 1957, 179, 109

7 Cochran, \V. G., & Cox, G. M., ' Experimental Designs ', 1950, p. 80 (New York : U'iley)

MYCOLOGICAL FORMATION OF FAT. IV.*-The Component Fatty Acids of the Fat produced by Penicillium soppii Zal.

By J. SINGH,? (Mrs.) SUDHA E . PHILIP and T. K. WALKER

The fat produced by Penicilliuwz soppii when grown in surface culture on a medium containing sucrose and mineral salts has a very low content of free acid (0.67; as oleic acid). The component acids of this fat have been found to be : myristic, 0.3 ; palmitic, 22 .0 ; stearic, 7.6 ; arachidic, 0.9 ; hexadecenoic. 3'3 ; oleic, 45.2 ; linoleic, 20.0 ; lino- lenic, 0.3 ; eicosenoic, 0.476 (w/w). In its content of linoleic acid the fat resembles ground- nut oil.

Introduction

found to be favourable to formation of fat in the mould Penicilliurn sopPii Zal. now been analysed and particulars of its component fatty acids are reported.

Experimental methods The strain of P. so$pii was that employed in the former communication and it was cultivated

in the most favourable of the media described in that paper. This medium contained (g./Ioo ml.) : FeCl,,GH,O, 0.016 ; ZnSO,,7H,O, 0.005 ; K,SO,, 0.022 ; NaH,PO,,zH,O, 0.036 ; MgS04,7H,0, 0.050 ; NH,NO,, 0.15 ; corn-steep liquor, 0.50, and sucrose, 17.0. These con- stituents were made up to volume with tap-water. The medium was dispensed in portions each

In Part I11 of this series of papers, details were given of the media and cultural conditions This fat has

* Part I11 : J . Sci. F d Agric., 1956, 7, 237 t Present address : Chemistry Department, Punjab Eniversity, Hoshiarpur, India

J. Sci. Food Agric., 8, December, 1957

698 SINGH et al.-MYCOLOGICAL FORII/fATION OF FAT. I V

of 2 j o ml. in Glaxo flasks and after sterilization at 10 lb. pressure for I j min. the flasks were cooled and inoculated with spores of the mould. Incubation was conducted a t 27' for 12 days. The felts were then harvested and the fat contents extracted by the procedures described by Woodbine et a1.l It was a pale yellow oil and on examination by the methods of Hilditchz showed saponification equivalent 292.j, free fatty acids (as oleic acid) 0.6% and iodine value 83.1. The unsaponifiablc matter, as estimated by the method of Kolton & W i l l i a m ~ , ~ was 1.6%.

Determination of component f u t t y acids To obtain the free fatty acids, the fat was completely saponified by boiling under reflux for

3 h. with 5 parts by weight of alcohol containing 6% (w/v) of potassium hydroxide. The bulk of the alcohol was then distilled off and the soaps dissolved in water and unsaponifiable matter was removed by ether. The acids were liberated by treatment of the aqueous alkaline solution with dilute H,SO, and extracted with ether. The ethereal solution was washed repeatedly with water to remove all traces of mineral acid. The ether was removed by distillation and the acids dried by heating on a steam-bath under vacuum. This mixture of acids was resolved by low- temperature crystallization, using the technique of Hilditch.2 A solution (1094 y ' v ) of the mixed acids in acetone was cooled to -65" in a bath of ethanol and solid CO, and maintained a t that temperature until crystals separated. After 6 h., these were removed by filtration with continued strong cooling, the material remaining in solution at this low temperature constituting group C. The crystals were recrystallized from solution (10% w/v) in ether by cooling to -30' and by this means were obtained two further fractions, group B, recovered from solution, and those acids insoluble in ether a t -30' (group A). The constituents of these three groups were : A, mainly saturated ; B, mainly monoethenoid, and C, mainly polyethenoid acids. Each group of mixed acids was examined spectrophotometrically by the stacdard method of Hilditch et uZ.* and Table I shows the quantities, iodine values and spectrophotometric data for the three groups.

A total of about 200 g. of crude fat was collected.

Table I L o w t e m p e r a t u i ~ e cvystall isation of mixed f a t t y acids

Group Properties -kids ~'7; I cm. after alicaii

\Vt . , q/, ; I val. - ~- g. W/W 234 mp 2W3 nip

isomerization

A

13

Insoluble in 10 vol. of acetone a t -6.j' and

Insoluble in 10 vol. of acetone a t -6j", in 10 vol. of ether a t -30° 40.29 3 2 . 0 1 2 . 3 7'4

soluble in 10 vol. of ether a t -30' 57'47 4.545 94'7 116.8 ~.

C Soluble in 10 vol. of acetone a t -65" 28.24 22.4 r.jo.1 604.2 s.0

Each group of acids was converted to the methyl esters by the method of Bjarnason Sr Mearaj and fractionally distilled through an electrically-heated column as described by Longe- necker.G The column was packed with stainless steel Stedman gauze and the distillation was carried out under reduced pressure (0-1 mm. Hg). It was possible, b y taking conveniently small fractions of the distillate, to obtain not more than two homologous saturated acids and two unsaturated acids of the same series in each fraction. The saponification equivalent and the iodine value of each fraction were then determined (Table 11).

From the analytical data shown in Tables I and I1 the composition of each acid group and finally of the whole fat was calculated according to the method of Hilditch' (Table 111).

Tdentijcation of f a t t y aczds Palmitic acid, recovered from fractions AI-A~, was crystallized from ethanol and had

n1.p. 62.S0, unchanged on admixture with pure palmitic acid. From the saturated acids of later fractions of group A, after Lapworth-Mottram7 oxidation, stearic acid was isolated and recrystallized from ethanol, when the m.p. was 69.4-69.8", alone or in admixture with an authentic specimen of stearic acid. The presence of oleic acid in other appropriate fractions

J. Sci. Food Agric., 8, December, 1957

SINGH et al.-MYCOLOGICAL FORMATION OF F A T . I V 699

Table I1 Fractionation of methyl esters of acids of groups -4 , B and C

Methyl esters of acids of group A Fraction Wt., g. Sap. equiv. I Val.

1.61 2.61 3'74 3.80 3'27 4.10 3.06 3.13 2'75 1'73 1'55

270.2

270.7 270.8 272'5 273'4 273'7 276.4 296.3 298.4 299.0 311.1*

Methyl esters of acids of group R I 1.27 285.0 B2 2'71 288.2

3.35 287.2 3.10 291.0

B3

290.8 B4

3.36 3'51 291 '.j 4.1 I 291.8 R7

B8 3.38 293'4 B9 3.80 296.1"

z

5'9 6.5 6.9 8.9

11.8 13.8 16.4 20.7

7'3

10'2

21'2

R 84.9 88.2 89.9 91.0 92'9 93'7

95'0 91'4

9 4 4

Methyl csters of acids of group C C I I '07 270.6 112'4 0 2 I .64 282.8 133.4

4 3'75 290.6 147'0 5 3.08 292'2 '47.8 6 3'55 292'5 148.5

C8 2'94 296.7 149'5

3'93 '84.5 146.2

7 3.63 296.3 149.1

c9 1'23 343.5* 132.8 * Equivalents of cstcrs (freed from unsaponifiable matter) AII, 309.8 ; H9, 295.6 ; Cg, 311.2

was confirmed by oxidation to g : 10-dihydroxystearic acid, m.p. 13o.3-131', while that of linolejc acid was established by conversion to tetrabromostearic acid, m.p. 113.4-114'. In neither of the two last mentioned cases was the m.p. depressed on admixture with autheniic 9 : ro-dihydroxystearic acid and tetrabromostearic acid, respectively.

Discussion The fat elaborated by P. sopPii is very low in content of free acid (0.6% as oleic acid) and

in this respect resembles freshly-isolated seed fats. In the triglyceride portion of fats of microbiological origin, palmitic acid usuallv accounts for about 70% of the saturated acids

Table I11 Componeizt .fatty acids of f a t fi~owi Penicillium soppii

Acid groups (oh)* Total fatty acids A B c: excluding unsaponifiable

~- matter, O 0

w/w mol.

0'3 0'4 Myristic 0.8 - Palmitic 61.3 5'0 0 5 2 2 ' 0 23.6

7.6 7'3 - 0.9 0.8

Stearic 22.3 0'9 Arachidic 2.8 -

5.1 4.2 3'3 3.6 Hexadecenoic -

Oleic 12'0 76.6 28.8 45'2 44'1 Linoleic 0.8 ~ 2 . 4 62.9 20'0 19.6

1'4 0'3 0'3 Linolenic - - 0'4 0'3 1'7 Eicosenoic - -

0.5

(32.0$(,)7 ~ (45.6qb)t-- (z2.4Uo)f ~

~

_ _ Unsaponifiablc - -. ~

* Component acids as percentage (w/w) of group Groups as percentage (w/w) of total acids

.J. Sci. Food Agric., 8, December, 1957

700 SINGH et a1.-MYCOLOGICAL FORMATION OF FAT. I V

presents and, in the case of P. soppii alSo, the palmitic acid content (71.4%) of the saturated acids approximates closely to this value. The proportion of hexadecenoic acid, 3-3q:, of the total fatty acids, is low and in accord with previous findingss. in the case of certain moulds which are capable of synthesizing large quantities of fat from carbohydrates. In linoleic acid content (20%) the fat of P. so$$ii resembles that of groundnut oil.

'The component acids of the seed fats are characteristic, to a more or less marked extent, of the different plant families. The fats from phanerogams can be classified broadly on the basis of the major component acids and this arrangement can be related to the botanical classification of the plants yielding the fats. The component acids of thc fats from cryptogams show consider- able variation in their composition. Detailed studies of the component acids of the fats pro- duced by yeasts and by moulds have been carried out in only a few cases, some of which are cited in Table IV, and the data are insufficient to show whether the alignment of these fats on the basis

Table IV Component fat ty acids of fats pvoduced by micvo-ovganisnzs

Peuiicil- Aspev- Pewicil- Penicil- Yeast l thodo- Toi.iilopsis

soppii nidulans lilacinzinz spinulosum No. 72 sp. Rcichert14 liuni gillus ZiUnl liunl strain torula Sp.

I'resent Singh, Singh, Shimill Hilditch & Holm- berg13 Shriva-

tion hfeara* \Valkerg stava12 investiga- Walker & Shah &

Free aciditj- (0; oleic) 0.0 0.8 0 . 2 j.8 33 I 8 , j r .2

Component acids 3iyristic Palmitic Stearic Arachidic Behenic Lignoceric Hexadecenoic Oleic Linoleic Linolenic Unsaturated C,,

1 0.9

3'3 45.2

0.3 0.4

20'0

0'7 20.9 15'9

1 '4

1'2

40'3 I 7.0

2'4 0 ' 2

- 0 '1 32'3 18.0

9'4 11.9

1'4 1 '4

3'4 3.8 38.6 43'3 '3'4 21'1

0'3 1 '4 0 ' 2

-

0'1 2 j .6 5.9

5'1

1'3 54'5 5'7 0'7 1'1

1 . 1

29.8 8.8

1.4

1.8 40.1 11 '2

4.8 1'0

0'3 7'9 3.8

0 ' 2

7'6 2 I '.j

49'7 4'4 -

of their contents of component acids can be related to the botanical status of the organisms which produce them. Moreover, two of us have shown recently,1° in the course of a study of fat formation in Aspergillus niduZans, that the relative amounts of the component fatty acids show considerable variation during the period of fat synthesis and that the largest proportion of saturated acids is formed when the production of fat is maximal. Study of the effects of cultural conditions on the composition of fat in several species of moulds is now in progress in these Laboratories.

Acknowledgments One of the authors (J. S.) desires to thank the Medical Kesearch Council for a personal grant

to enable him to participate in this work. The authors are indebted to Dr. A. R. Thompson of this College for spectroscopic determinations and to Dr. M. L. Meara for helpful discussions.

Manchester College of Science and Technology Manchester, I

Received IS February, I g j l

References TT'oodbine, M., Gregory, Margaret E., & TYalkcr, Bolton, E. K., & \\'illiams, I(. A , , .4nalyst, 1930,

T. K.. 1. exb . Bot.. 1061. 2 . L O A 55. 6 I I I d

Hilditch, $., The Chemical &nstitution of Natural Fats, 2nd Edn, 1947 (London: Chap- man & Hall) ; see also British Standards Institu- tion (1950), British Standard Methods of A4nalysis of Oils and Fats, B.S. 684 Ind., Lond . , 1944, 63, 61

4 ~ i l d i t ~ f ~ , 'r, I,., ~il~! . , J , I>,, & patel, C, R., zgIzalyst,

Bjarnason, 0. B., & Neara, &I. L., J . Soc. chem. 1951, 76, 81

J. Sci. Food Agric., 8, December, 1957

CONNELL-EXPRESSIBLE FLUID OF F I S H FILLETS. I ~ I 701

References (cont.) 6 Longenecker, H. E., J. SOC. chem. I n d . , Lond., 10 Singh, J , , & Walker, T. I<., Biochem. J . , 19j6, 62,

7 Lapworth, h., & Mottram, E. N., J. chem. Soc., 11 Shimi, I. R., Ph.D. Thesis, University of Man-

8 Singh, J., Walker, T. I<., R: Jleara, M. L., Biochem. 1 2 Hilditch, T. P., & Shrioastava, R. I<., Biochinz.

9 Singh, J.. Sudha Shah, & Walker, T. K., Biochewz. l 3 Holmberg, J., Svensk. kenz. T i d s k r . , 1948, 60, 1.1 l4 Reichert, R., Helv . chim. A c f a , 1945, 28, 484

1937. 56, 199 286

1925, 127, 1628 Chester, 19.j5

I . , 1955, 61, 85

J., 1956, 62, 222

biophys. Acta, 1948, 2, So

THE EXPRESSIBLE FLUID OF FISH FILLETS. VI.*-Electro- phoretic Analysis of the Expressible Fluid of Cod Muscle

By J. J. CONNELL

Electrophoretic diagrams of thc expressible fluids of cod muscle have been compared A new component has been observed in the

Its possible sites in the muscle with those of extracts a t low ionic strength. expressible fluids and some of its properties examined. structure and its bearing on the nature of expressible fluid are discussed.

Introduction and Love3. * have described the preparation and nature of the

fluids which can be expressed from fresh, iced or frozen cod muscle. In particular, it has been concluded that the expressible fluid obtained from iced fish is largely derived from the inter- stitial or intercellular fluid, and that after certain conditions of freezing which are conducive to cell damage, intracellular components (eg. deoxypentosenucleic acid) are then observed in the expressible fluid. It seemed likely that additional information would be gained by examining other muscle constituents which were present in expressible fluid, and on the suggestion of Dr. R. M. Love an electrophoretic analysis of expressible fluid obtained under various conditions has been undertaken. The present paper is concerned solely with unfrozen cod ; work on the effect of freezing will be described in a future publication. Apart from these considerations it was of interest to analyse expressible fluid since preparations of some ' myogens ' originate with this type of fluid or what is sometimes known as ' press juice ' 5 and these have never been clearly differentiated electrophoretically from the type of low ionic strength extract which is understood to contain most, if not all, of the sarcoplasmic proteins.

Experimental Material

One was obtained in the manner described by Banks1 from whole fillets cut from 3- to +day iced cod and the other from coarsely minced cod muscle which was pressed in a hydraulic press at 10 tons/sq. in. In this case the flesh was obtained from fish that had been killed only several minutes before since it was desired not only to examine fluid obtained by applying pressure in a different way but also to avoid any changes in the muscle proteins which might occur during the icing and pressing period of approximately 3-5 days.

With hydraulically pressed muscle there was a delay of 10-15 minutes before any fluid could be expressed, corresponding to the period before the onset of rigor mortis. This period is very much shorter than that noted by Banks using whole gutted fish ; but it is known that in minced fish the state of rigor is passed through extremely rapidly.6, Using a dwell of 30-60 minutes, the yield of fluid was about 10 g. per IOO g. muscle ; the protein content of the fluid was about 6%.

In recent papers, Banks1.

Two types of expressible fluid have been used.

* Part V : J . Sci. Fd Agvic., 1957, 8. 238

J. Sci. Food Agric., 8, December, 1957