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Copyright is owned by the Author of the thesis. Permission is given for

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THE BIOSYNT1Ii�SIS OF t·ITErfHYL KETONIDS 'diTH SPECIAL lli':FI!:RENCE TO THEIR

PRIGSBNCE IN C

H8DD.AR CHEESE

being a thes i s

p res ented f o r the Degree of' Doc tor of Philos ophy of the Man�:;ey Universi ty of Manawatu

R .C ^• LAX'iRf�JCE

Mas s ey Univers i t y of Manawat u

1964

ii

PREFACE

Grate ful aclmowl e dgement i s made t o The Dairy

Research I nstitute (N.Z.) �or granti ng l e ave o� abs ence to under take thi s investigation.

Particu l ar thanks are extended to:

Dr .

J.G.

Hawlce �or hi s enc01.1rage ment and gui dance throughou t the c ou rs e o� thi s work;

D r . W .A ^.. McGi l l i vray, D r . P .s. R oberts on and Mr .

G . M . Wal l ac e �or help�ul comments on the manuscr ipt;

Dr .

^F.B.

S ho rland of' The Fats Res earch L ab oratory, Well ington for generous gi�ts of' radi o ac tive

milk fat s ampl es and � or making availab l e apparatus and l ab or atory fac i l i ties;

Mr .

T.

Gers on �or the i s olation of' fatty a c ids fr om radi oactive milk �at , as de s c r ibed in the thes i s , and for help and advi c e in the

estimati on o� the ir s pec i�ic activi ties;

Dr . B. Latch f or the i dent i� i c a t i on o f the �ungus Peni c illium r ogue�orti . Thom, isol ated �rom

N .Z.

Blue c hees e, and for help�ul di s cuss i ons

on the cu ltivati on o� the f'ungus ;

Mr. H. Wenham f'or advice and guidance in laboratory techniques concerning the cultivation of' fungi;

Mist> M.G. Campbell and the Library staff;

Mr. J. Gilles for the manufacture of the experimental cheeses;

Mr. B. Le Heron for milk fat samples;

Mr.

R .

'J�oms for the photographic reproduction of'

the gP8.phs;

Miss

D. Jackson for typing the manuscript.

Early work on the formation of methyl kctones as artifacts during the steam distillation of' Cheddar cheese and milk fat has been published

(

J. Dair•y Hesearch

(1963).

2.Q,

161).

^A note on the origin of methyl ketones using radioactive milk fat was published in co-m1thorship �ith Dr. J .C. Hawke

(

^Nature

( 1 963). ill, 1276). A I>8.lJer V!il:l be presented to the N.Z.I.C. Conference in Augm:;t 196!�

on the r elationship of milk f'at s;y-nthesis to· the methyl ketones :round in steam distillates of milk fat. Early results on the oxidation of' fatty acids by Penicillium rogueforti were cow.municated to the H.Z.I.G. Conference

(

^August,

1963)

and more recent Vlork will be presented, in eo-authorship with Dr. J.C. Hawke, at the 6th

International Biochemical Congress

(

^July,

1964)

in New York.

j_v

CONTENTS

Page

INTRODUCTION

1

Chemical nature o� cheese �l&vour ₂

Importance of milk fat in Cheddar flD.vour formation 3

Flavourful substances in Cheddar cheese 5

Flavourful substances in Blue cheese 9

Simulation of Cheddar flavour 1 0

Mechanisms of methyl ketone formation

1 1 Biosynthesis of fatty acids in milk lipids 13

Micro-organisms in Cheddar cheese 20

Fatty acid oxidation 211•

Oxidation of fatty acids by �1ngi 2

6

Toxicity o� fatty acids towards fungi 32 Oxidation of triglycerides by :fungi 35 Formation of methyl carbinols by fungi

37

AIMS OF THE

PRESJ�:N'r

INVESTIGATION l1-0

PART I. THE ORIGIN OF METHYL KETONES IN STEAM

DISTILLATES FROM CIEDDDAR CHEESE _AND Iv!ILK FAT

MATERIA LS

MJ�THODS

Isolation of radioactive fatty acids Determination of specific activities c:ollection. of methyl ketone s

46

1;..6

47

49

Page Purification of radioactive methyl ketones 50

Chromatographic separation and identification of

2,l,_-Dini trophenylhydrazones 52

EXPERIMJ.:a�

TAL

Methyl ketone patterns in steam distillates of cheeses manufactured under controlled

bacteriological conditions 54

Isolation of methyl ketones from Gheddar cheese and milk fat by steam distillation at

atmospheric pressure 56

Identification of methyl ketones with an even

number of carbon atoms 59

Exhaustive steam distillation of cheese and milk fgt 59 Isolation of methyl ketones by steam distillation

under reduced pressure 60

Isolation of methyl ketones by stripping cheese

extracts with nitrogen 64

Isolation of carbonyls from coconut oil 66

Comparison of radioacti vi ties in methyl lcetones

and fatty acids from radioactive milk fat 67

PART

II.

METHYL :KJi::TONES FOUND

ll{

S'l'EAM DISTILLATES FROM CHE.i1SE CONTAINING SYNTH}]TIC TRIGLYCERIDES

DISCUSSION OF PARTS I

_{MiD II}

73

77

vi

PART III. THE OXIDATION OF FATTY ACIDS AND TRIGLYCERIDES

BY

P��ICILLIUM ROQUEFORTI

CULTURES

MATERIALS

rvt:THODS

Growth o� mycelium

Page

92

95

96

96 96

Composition o� synthetic growth medium and subsequent oxygen uptake in the presence o� �atty acids

Estimation o� micro-quanti ties o� methyl ketones

99

as 2 ,4.-DNP hydrazones 1 04.

Instability o� 2,4.-DNP hydrazones o� methyl ketones 1 05

Estimation o� methyl carbinols 1 08

Identi�ication o� �ree �atty acids 1 09

Measurement o� radioactive carbon dioxide 1 1 1

Synthesis o� non-available substrates 1 1 3

EXPER IMJ!}NTAL

CONDITIONS Ali'FECTING THE OXIDATION OF FAT'l'Y ACIDS 1 1 6

Endogenous respiration 1 1 6

Oxidative assimtlation o� �atty acids by mycelium 1 1 7

Pre-starving mycelium in bu��er 1 1 7

Inter-relationship. between pH and concentration o�

�atty acid substrate 1 20

Puri ty o f acid

E�fec t of inorganic i ons ( Mg2+ , c a2+ ,

^PO

h. 3�) Sui tabil i ty of co2 forma t i on as a measure of

fat ty acid oxi dation Oxygen balance sheets

Comparative rates of �ormati on o� methyl ketones by mycelium and s pores

Me tabol ism of oc tanoic ac i d over l ong peri ods Effec t of malonate and diethyl mal onate on

oxidation of fatty ac i ds by mycel ium

SUMMARY OF PRELIMINARY EXPERIMENTS

OXIDATION OF FAT'l'Y ACIDS iJHTH AN

EVEN

NUMBER OF

CARBON ATOMS BY MYCJ.!:LIUM

OXIDAT::=Ol'J O:B' FATTY ACIDS 'NITH AN ODD

:NUMBER

OF

1 21 1 24

1 28 1 31

1 31 1 35 137

138

1 4 0

CARBON ATOMS BY MYCELIUM 1 11-3

OXIDATION OF FATTY ACIDS BY SPORES 1 44

OXIDATION OF OCTANOIC ACID AND ITS METABOLIC PRODUCTS 1 l1-5 Oxidat i on of 1 -c1 4 and 2-c111- oc tan o i c ac ids by

spores 145

Oxidati on of 1 -c1 4 and 2 -c 1 11· oc tanoic ac ids by

mycelium 149

Metabol ism of 2-heptanone by mycelium 1 51 Metabol ism of 2-heptanone by spores 1 53 Formati on of methyl c arb inols from fatty ac ids

by myc elium and spores 1 56

vi ii

Page F ormation of methyl c arbinols from triglycerides

by spores 1 58

INHI BITION OF RESPIRATION OF MYCELIUM BY FATTY ACIDS 1 59 Pre-incubation with a non-toxic acid fol l owed by

a

toxic acid 1 60

Pre-incubation with a toxic acid foll ov1ed by a

non- toxic acid 1 63

R eversibiJ_j_ ty of toxic i ty of oc tanoic acid at l ow pH 1 66 Effect o f -C O

^.

C H2

^.

co- grouping and of glutathione

on oxidation of fa tty acids 1 69

OXIDATION OF TRIGLYCERIDES 1 72

Oxidation of triglyc erides by mycelj_um 1 72 E;f:rect of c onc entration of triglyc eride on methyl

ketone forma t i on by spores 1 72

Induction of l ipolytic ac tivi ty in spores 1 73 Effect of pH on methyl ketone formation 1 78

OXIDATION OF FATTY ACIDS BY CELL

FREE

EXTRACTS

PREPARED FROM tiYCELIUM 1 81

Preparation of cell free extracts 1 82

Succinic dehydr ogenas e ac tivity 1 83

Oxidation of oc tano�c acid by cell free extracts

from the Hughes Press 1 84

Ac t ivity of mycelial debris from Hughes Press 1 8 6

DISCUSSION OF PART III

REFERENCES

ABSTRACT

1 89

205

2 24-