A STUDY OF VOLATILE SUBSTANCES FROM OXIDIZED MILK FAT

67 Pages·03.084 MB·English

by  KEENEY, MARK

THE PENNSYLVANIA STATE COLLEGE
The Graduate School
Department of Dairy Husbandry
A STUDY OF VOLATILE SUBSTANCES FROM OXIDIZED MILK FAT
A Thesis
by
MARK KEENEY
Submitted in partial fulfillment
of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
June 1950
Approved:
j-,
8f
Head, Depar Dairy Husbandry
Professor of^Dairy Manufacturing
TABLE OF CONTENTS
Page
INTRODUCTION 1
HISTORICAL 5
Definition of Oxidation 5
Concepts of Fat Oxidation 5
Secondary Decomposition Compounds 8
Oxidized Flavor in Milk 12
EXPERIMENTAL AND RESULTS 16
Preliminary Experiments 16
Preparation of Lipid Fractions 16
Preparation of Oxidized Milk Fat 22
Steam Distillation of Oxidized Fat 22
Vacuum Distillation of Oxidized Fat 24
Chemical Properties of Volatile Material 26
Separation of Volatile Material into Neutral and .Acid
Fractions 26
Neutral Fraction 27
Acid Fraction 27
Derivative Preparation from Neutral Fraction 28
Postulated Structure of Carbonyl Derivatives 30
Study of Volatile Substances from Oxidized Milk Fat 30
Higher Melting Solid Fat Fraction- 31
Fractional Distillation of Volatile Neutral Material 32
2,4-Dinitrophenylhydrazones from Neutral Fraction 35
Postulated Identity of 2,4-Dinitrophenylhydrazones 3&
Odor and Flavor Characteristics of Neutral Fraction 40
Liquid Fat Fraction 43.
Use of Girard's Reagent 43
Acids and Lactones 45
Ether Soluble Acids 4&
Water Soluble and Bicarbonate Soluble Fractions 47
Ketone Fraction 47
The Kreis Test and Ferric Chloride Test 47
Flavor Imparted to Milk by Certain Fractions 49
Control Experiments 49
Page
DISCUSSION
51
CONCLUSIONS
56
ACKNOWLEDGEMENTS
57
BIBLIOGRAPHY
58
INTRODUCTION
The dairy industry is confronted with flavor problems, some of the
more serious of which are either directly or indirectly associated with
the oxidative deterioration of the milk fat of dairy products.
Some investigators (13, 38, 58, 64) have obtained data which suggest
that the phospholipid portion of the dairy product is associated with
oxidized flavor while others (11, 12, 39) are of the opinion that the
triglyceride portion is involved. The possibility is also present that
the pigments in milk fat may be involved in flavor development in the
early stages of oxidation (8).
Environmental factors which have been studied in their relation to
oxidized flavor development are; oxidation-reduction potential, pas­
teurizing temperature, homogenization, exposure to air, removal of air,
exposure to light, as well as the presence or absence of such substances
as ascorbic acid, mereapto compounds, carotenoid pigments, riboflavin,
organic acids, proteins, lactose and salts.
The object of the major part of the research reported on this pro­
blem has been to devise ways of preventing the flavor development in
various dairy products. This object has been accomplished fairly well
in some cases. However, oxidized flavor is still a sporadic problem in
the market milk and ice cream industries. Dry whole milk made by the
most modem of methods has a flavor which is atypical. This flavor is
generally considered to be due, in part, to lipid deterioration.
Greenbank (27) has compiled a review of the information available
on the subject of oxidized flavor in dairy products. This review has
2
covered the subject through 194-7. Cursory examination of Greenback* s re­
view shows the conflict and confusion which has existed among workers on
this subject. The reasons for this are many in number. First of all,
many workers have not used proper scientific methods. Conclusions often
have been drawn on data which were inadequate or obtained from poorly
controlled experiments. Secondly, the human taste mechanism is not
infallible and experiments in which it has been used as a tool should be
interpreted with considerable caution. A third and perhaps the main rea­
son for the chaos has been our ignorance of the chemical mechanism of
lipid oxidation and its apparent complexity.
Within the past ten years vast changes have taken place in the ac­
cepted concepts of the mechanism of fat oxidation. Beginning in 1942
Farmer and co-workers (17, 18, 19), in Great Britain, have published the
results of some fundamental studies on the mechanism of fat oxidation.
These studies have led to the so-called hydroperoxide theory of fat oxi­
dation. This work has done much to clear up the confusion which existed
as to the mechanism of original oxygen attack in fats, and has opened up
new vistas of research in fat oxidation.
Another valuable development has been the recent availability of
precise spectrophotometric instruments to workers on fat oxidation. Ultra­
violet spectroscopy has resuited in detailed studies of double bond con­
jugation in fats. Infra red spectroscopy is presently being adapted for
use in this field and in time to come these techniques will probably
help to elucidate some of the, now obscure, changes occurring in the
oxidation of fats and fatty substances.
3
In the light of these developments and because none of the oxidized
flavor compounds have ever been isolated from milk products, the investi­
gation reported here was undertaken. Briefly, the study has involved
removal of the volatile material from oxidized milk fat, chemical
characterization of this material and correlation of chemical structure
with flavor and odor.
At the present time many workers in the field of dairy products flavor
research believe that phospholipid oxidation catalyzed by various agents
such as ascorbic acid, copper and sunlight is responsible for most of the
practical oxidized flavor problems in dairy products, even though there
is some evidence that the fats themselves may also be involved. These
individuals may question the value of work on purified milk fat which is
practically phospholipid free.
In support of the reported work and to answer this possible criticism,
the following case is presented for the investigation.
According to our present knowledge of the fatty acids present in
milk, it is inconceivable that any one particular fatty acid is exclu­
sively present in the phospholipid portion of the lipid material. If it
is assumed that phospholipid oxidation is all-important it may also be
assumed that similar oxidation products may be formed from the triglyce­
ride portion.
Ample evidence has been presented to show that milk fat oxidation is
of practical significance in powdered whole milk (4-6) and this probably
is true of other dairy products as well.
Ar
The reconstitution of beverage milk from butter oil and skim milk
solids in some areas of the world in which there is a fluid milk shortage
is being practiced. This alone would make worthwhile a fundamental in­
vestigation of the oxidation products of milk fat.
5
HISTORICAL
Definition of Oxidation
The v/ord "oxidized” has various implied meanings depending upon the
process description in which it is used. It is common practice when de­
scribing certain biological phenomena to use the v/ord to denote loss of
electrons. In the fields of paint, rubber and fat technology the most
common oxidizing agent is atmospheric oxygen. In these fields it is also
understood that the material being oxidized has, as part of its chemical
structure, carbon to carbon unsaturation. In other words, the molecules
have a deficiency of electrons. In this sense they can be considered to
be partially oxidized and therefore extremely susceptible to further
oxida.tion.
The term "autoxidation" has been used widely to describe the oxida­
tion of unsaturated materials with atmospheric (molecular) oxygen.
For the sake of simplicity the term ”oxidation” will be used in this
paper. It is defined for this purpose as follows: "The deterioration of
a fat in the presence of atmospheric oxygen. The process may or may not
be complimented by thermal energy (heat) and radiant energy (sun’s rays)."
Concepts of Fat Oxidation
In the development of the concepts of oxidation much attention has
been given to the mechanism of original oxygen addition to the fat.
6
The first observation of oxidation of a carbon to carbon double bond
has been credited to Schonbein in 1858 (53). The modern theories of fat
oxidation date from 1900. About this time Bach (2) and Engler (16) pro­
posed the theory that oxidation of double bonds was molecular and not
atomic in nature. Prior to this it was widely believed that oxidation re­
sulted from attack by atomic or ’'active” oxygen. Bach and Engler proposed
that a molecule of oxygen added at the double bond to give a peroxidized
compound of the formula, R ^ - O - O - R g , and that this compound, like
hydrogen peroxide, could oxidize another oxidizable substance.
Staudinger (56) in 1925 proposed a modification of the peroxide
theory of Bach and Engler. He proposed that the first step in oxidation
was the formation of a moloxide which subsequently rearranged to a cyclic
peroxide.
fti - C\H - /HG - R R, - CH - DC - IU
N 0/ — - -> 0 0
*»
0 peroxide
moloxide
In 1936 Criegee (10) proposed that cyclohexene oxidized to form a
hydroperoxide having the structure:
H
\
'00H
A year later Rieche (49) suggested that the oxidation of unsaturated
substances may occur through the formation of oxygen activated methylene
groups resulting in products similar to Criegee1s hydroperoxide:
OOH
<
- CH = CH - CH2 - CH = CH - + 02 — - CH = CH - CH - CH = CH -
Farmer and his co-workers (17, IS, 19) have extended the theory of
Rieche to include the oxidation of unsaturated fatty acids. According to
this group of workers the oxidation of practically all unconjugated olefins
proceeds by addition of a molecule of oxygen to the carbon atom adjacent
to the double bond to form a hydroperoxide having an intact double bond.
Ah experimental evidence to support the hydroperoxide theory of oxidation,
Farmer and Sutton (19) oxidized a sample of pure methyl oleate. By mole­
cular distillation of the oxidized ester a product was obtained which on
analysis appeared to be pure methyl peroxide oleate. It was found to con­
tain one mole of oxygen per mole of ester and an intact double bond. The
product was an oily liquid at room temperature and failed to crystallize
at relatively low temperatures.
While these observations of Farmer do not absolutely disprove the
cyclic peroxide theory of oxidation, they do seem to make the cyclic
theory untenable for the explanation of the mechanism of original oxygen
addition to unsaturated fats.
Another theory of oxidation which may have some significance is that
of Fokin (20). He proposed that the first step in oxidation resulted in
the formation of an oxirane rings
- CI-I = CH - -s- 1/2 Op 3» - CK - CH
This type of reaction is well known in the oxidation of ethylenic bonds
with organic peracids. An analogous reaction may take place in an oxi­
dizing fat system due to the presence of peroxides. However the evidence
suggesting that it is the mode of reaction with molecular oxygen is meager.