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RELAT IONSHIPS BET WEE N HYDROGENAT ION AND HYDROLYSIS
OF DIETARY FAT IN TH E BOVINE RUMEN
A thesis presented in partial ful filment o f the requ irements
for the degree of Master of A gricul tural Science in
A nimal Science
by
W illiam Ross SILCOCK
Massey Univ ersity ,
New Zealand.
1 �8
ACKNOWLEDGEMENTS
Throughout the course of this s t udy the author has found the knowledge
and experi ence of his superviso r , Dr . J. C. Hawke , to be inva luable in the
deve lopment of ideas and experimental te chnique s , and expresses his
grat itude for this he lp , readily gi ven .
The aut h or extends his thanks to
Mr.
G . Wilson and the sta ff of No . 2
dairy unit , wh o have not hesitated to provide the animals and facilities
necessary for this study .
The t ech nical assis tance and hel p ful advi ce of Mis s J . Barne tt is
acknowledged with thanks .
The very capable and rapid typing of Mis s May Soo is gra te fully
a c knowledged, as is the expert guidanc e re ceived from Miss D . Scott of the
C entral Photo graphic Uni t .
The auth o r would finally like t o record his thanks to Massey University
and in par ticu lar the Chemistry and Bio chemistry Department for making
possible the circumstances und er wh ich th is study was carried out .
TABLE OF CONTENTS
Chapter
1
Page
I NTRODUCTION
1 . 1.
Introdu c tion
1
1 . 2.
Hydrolysis o f Glyc erides in Rumen Fluid
5
Hydrogenat ion of Unsaturated Fat ty Acids
in Rumen Fluid
7
1 . 4.
Fac tors A ffecting the Degree of Hydrogenation
of Unsaturated Fa tty Acids of the Die t which
takes pla c e in the Rumen , with respect to the
Composi t ion of Depot and Milk Fats
2
THE A IM OF THE PRESENT STUDY
3
MATERIALS AND METHODS
Solvents and Reagents
3.2.
3.3.
3 . 4.
14
20
22
Incuba tion Pro cedures
3.2.1.
Method
22
3.2.2.
Sampling
23
Emulsification
23
Synthesis o f Radioactive Triglyceride
3.3.1.
Synthesis o f glyceryl !-palmitate
24
3 . 3 . 2.
Synthesis of glyceryl !-palmitate2-oleate and glyceryl !-palmitate3-oleate
24
3 . 3 . 3.
I solation o f the pure Diglyceride
I somers
24
3 . 3 . 4.
Synthesis o f gl�ceryl !-palmitate2 -oleate-3- ( 1-1 C ) -linolenate and
glyceryl l-palmitate -2- ( l -1 4c ) l inolenate-3-oleate
25
Extrac tion and Methylation o f Fatty Acids
3. 4. 1 .
Extraction o f Lipid
25
3. 4.2.
Saponi fication and Extraction o f
Fatty Acids
26
Chapter
Methylation of Fatty Acids
3
3.5.
Thin Layer Chromatography
3 . 6.
4
26
Preparat ion o f Plates
27
3.5.2.
Elution Solvents
27
3·5·3·
Chromatographi c Procedure
27
3 . 5 . 4.
Scanning of Thin Layer Chromatograms
containing Radioactive Components
28
Gas-liquid Chromatography
3. 6.1.
Apparatus
28
3.6.2.
Columns
29
3.6.3.
Operating Conditions
29
3 . 6 . 4.
Ident ification of Methyl Esters o f
Fatty Acids
Radiochemical Methods
31
4. 1 .
In vivo Hydrogenation o f Dietary Lipid
32
4. 2 .
In vitro Hydrogenat ion of Dietary Lipid
33
In vitro Hydrolysis of Triglyc eride
34
Relationships Between Hydrogena tion and
Hydrolysis of Unsaturat ed Triglyceride in
Rumen Fluid
36
RESULTS
4. 4.
Fatty Acid Composition of the Total Lipid a fter
Incubation of Synthetic Triglyceride
4.6 .
5
Fatty Acid Composition of the Glyceride and
Free Fat ty Acid Frac tions a fter Incubation o f
Synthetic Triglyceride
39
DISCUSSION
5 . 4.
Sampling of Rumen Contents
41
In vivo Hydrogenation of Dietary Lipid
42
Formation o f Palmitic Acid in the Rumen
43
In vitro Hydrogenation of Dietary Lipid
44
Chapter
5
6
Hydrolysis of Triglyceride in Rumen Fluid
45
Relationships Between Hydrogena tion and
Hydrolysis of Unsaturated Triglyceride in
Rumen Fluid
50
Fatty Acid Composition o f the Total Lipid
Obtained a fter Incubation with Rumen Fluid
o f Synth et i c Triglyceride
54
Fatty Acid Composition of the Glyceride and
Free Fatty Acid Fractions Obtained a fter
Incubation o f Synthetic Triglyc e ride with
Rumen Fluid
55
SUMMARY
59
REFERENCES
61
LIST OF TABLES
Table
1
2
The e ffect o f triglyceride composition on degree o f
hydrolysis in rumen fluid
5
The fatty acid composition of the free fatty acid and
"residual glyceride" fractions obtained following 32%
hydrolysis o f l inseed oil in rumen fluid
6
3
Fatty acids forme d following incubation of unsaturate d
fatty ac ids with sheep-rumen c ontents
F.P . 8
4
The radioactive fatty acids obtained during incubation
of u-1 4 c-linolenic acid in rumen fluid
9
The products of oxidation o f the non-conjugated acids
obtained after incubation of U-1 4C-linolenic acid with
rumen fluid
10
The produc ts o f oxidation o f the oc tadecenoic acids
4
obtained after incubation o f u- 1 C-linolenic acid with
rumen fluid
11
7
The e ffect o f diet on Iodine Number of subcutaneous ,
visceral , and body fats of s t e er calves
15
8
Fatty acid composition of samples obtained during
in vivo hydrogenation of dietary lipid
F . P . 32
Comparison of the fatty acid c omposit ion of the r esidues
and the fluid samples ob tained by straining rumen
cont ents ( t rial J )
F . P . 33
Fatty acid compos ition o f samples obtained during
in vivo hydrogenation of dietary lipid ( trial H )
F . P . 34
Percentage distribution of radioactivity during hydrolysis
by rumen fluid o f synthet ic radioac tive triglyceride
F . P . 35
Radioac tive fat ty acids obtained a fter incubation of
synthet ic triglyc eride
F . P.36
Radioactive fat ty acids from free fatty acid and
glyceride fractions after incubation of glycery1
1-pa1mitate-2-o1eate-3- ( l-1 4 c ) -linolenate
( exper iment 5 )
F . P . 3?
5
6
9
10
11
12
13
14
Radioactive fatty acids o f fractions isolated after
rein9ubat ion of glyceryl l-palmitate-2-oleate-3(l-1 4c ) -lino1enat e for 24o minutes
38
Tabl e
15
16
Fat t y acid composition of the total lipid
extrac ted after incubation in rumen fluid o f
synthetic triglyceride
38a
Fatty acid composit ion of the glyc eride and free
fatty ac i d fractions obtained a fter incubation in
rumen fluid of synthe tic triglyceride
( experiment 5)
39a
LIST OF FIGURES
Figure
Oc tade cadienoic acids formed after incubation o f
linolenic acid for 20 minutes w ith sheep-rumen c ontents
( Ward e t al , 1964 )
F . P. 9
Pathways of hydrogenation of linolenic acid by rumen
microorganisms ( Wilde and Dawson , 1966 )
F . P . l2
Pathways of hydrolysis o f glyceryl l-palmitate-2( u-1 4c )-linolenate-3-oleate in rumen fluid
( experiment 1 )
F . P . 46
4.
Pathways o f hydrolysis in rumen fluid o f glycer ides
recovered from experiment 2 ( experiment 3 )
F . P . 49
5
Scan o f free fatty acid and glyceride fractions after
reincubation of glyc eryl l-palmitate-2-oleate -3( l-1 4c ) -linolenate for 2 4o minutes
49a
Scan of fr ee fatty acid and glyceride fractions after
reincubation o f glyceryl l-palmitate-2- ( l-1 4c ) ­
l inolenate-3-oleate for 2 40 minut es
49b
Scan of free fatty acid and glyceride fractions a fter
reincubation o f glyceryl l-palmitate-2-( l-1 4c ) ­
linolenate-3-oleate for 180 minutes
49c
1
2
3
6
7