PROPERTIES OF DOCOSAHEXAENOIC ACID ENRICHED
DAIRY PRODUCTS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
HONG WANG
In partial fulfillment of requirements
for the degree of
Master of Science
July, 1999
O Hong Wang, 1999
11411
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ABSTRACT
PROPERTIES OF DOCOSAHEXAENOIC ACID ENRICHED DAIRY PRODUCTS
Hong Wang
University of Guelph, 1999
Advisor:
Professor Arthur Hill
The purpose of this siudy is to investigate the physical, chernical, sensory and processing
properties of docosahexaenoic acid (DHA) enriched dairy products (milk, Cheddar
cheese and butter). DHA etuiched milk was obtained by feeding fish meal supplement to
Holstein cows. DHA level in the milk was increased to 0.4% of total fatty acids, while
the total fat level in the milk was reduced to 2.43% due to fish meal's inhibition effects on
fat synthesis. As dnnking milk, no difference was found between DHA milk and control
milk. Cheddar cheese made fiom DHA enriched milk ripened faster and developed a
more desirable texture and stronger flavor. In butter making, chuming time of DHA
cream was longer than that of control crearn. DHA butter showed a softer texture and
better cold spreadability.
ACKNOWLEDCMENT
My sincerest gratitude goes io my advisor, Dr. Arthur Hill, for his encouragement,
support, expertise and guidance through the research. 1 wouid also like to express my
special thanks to al1 my advisory cornmittee members, Dr. Alejandro Marangoni and Dr.
Bruce J. Holub for their support, constructive advice, and for making doing this research
both a challenge and a great pleasure.
1 would like to thank Dr. Massimo Marcone, for his technical help and his
friendship through the past three years. 1 am also gratefùl to Tom Wright, Sandy Smith.
Looknauth Ramsahoi, Kim Calamusa and Connie Avrarnis. Without their help,
completing this research would have never corne me.
I appreciate al1 the help and fnendship from my fellow graduate students.
rspecially Ayako Ueda. Kevin Segall, Karla Montoya, Susan Tosh and Cybelle
Fernandez.
Last but not least, I would like to thank my farnily for their undentanding and
patience. 1 especially thank my wonderhl sister, Ping, for her love, for always being
there for me.
To My
Mom,
Qing-Hua
TABLE OF CONTENT
TABLE OF CONTENT ...............................................................................
i
..
LIST OF TABLES.. ................................................................................ v u
...
LIST OF FIGURES ................................................................................
v111
CHAPTER 1 INTRODUCTION ...................................................................1
CHAPTER 2 LITERATURE REVEWS
2.1 ESSENTIALITY AND BENEFITS OF OMEGA-3 FATTY ACIDS ....................-2
2.1.1 Omega-3 Fatty Acids and Normal Growth and Development ..................2
2.1.2 Omega-3 Fatty Acids and Cardiovüscular Diseiise ...............................3
3
2.1.3 Omcga-3 Fütt y Acids and Cancer...................................................
4
2.1.4Omega-3 Fatty Acids and Inflammation ...........................................
2.1.5 Recommended Daily Intake .........................................................4
2.2 METABOLISM OF OMEGA-3 AND OMEGA-6 FATTY ACIDS .....................6
2.3 FOOD SOURCES OF OMEGA-3FATTY ACIDS .........................................8
2.3.1 Fish and Fish Med ...................................................................8
2.3.2 Plant Sources.........................................................................1O
2.3.3 Red Meat and Poultry ...............................................................10
2.3.4 Milk ....................................................................................10
2.4 INCORPORATING OMEGA-3 FATTY ACIDS INTO EGGS AND MEAT
PRODUCTS ..................................................................................... 1 1
2.4.1. Incorporation of Omega-3 Fatty Acids into Chicken Meat ................... 1 1
2.4.2. incorporation of Omega-3 Fatty Acids into Eggs ..............................12
2.4.3. Incorporation of Omega-3 Fatty Acids into Pork .............................. 14
2.4.4. incorporation of Omega-3 Fatty Acids into Ruminant ........................15
2.4.5 Incorporation of Omega-3 Fatty Acids into Bovine Milk .....................16
2.5 BOVINE MILK
FAT - BIOSYTHESIS. POTENTIAL FOR CHANGE ...............19
2.5.1 Milk Structure......................................................................2 0
2.5.2 Biosynthesis of Milk ................................................................
21
2.5.3 Factors Affect Milk Fat Content and Composition ..............................27
CHAPTER 3 CHEMICAL AND PHYSICAL PROPERTES OF DHA ENRICHED
BOVINE M L K
3.1 INTRODUCTION ..............................................................................
30
3.2 MATERLALS AND METHODS .............................................................30
3.2.1 DHA Enriched Bovine Milk and Regular Bovine Milk ........................30
3.2.2 Chernical Propecties of DHA Milk ................................................31
3.2.2.1 Fat. Protein. Lactose and Somatic Cells Count .......................31
3.2.2.2 Milk Protein Distribution by SDS-PAGE .............................31
3.2.2.3 Fatty Acid Composition.................................................33
3.2.2.4 Minerals ...................................................................33
3.2.3 Physical Properties of DHA Milk .................................................34
3.2.3.1 Density .....................................................................34
3.2.3.2 Fat Globule Size Distribution ..........................................35
3.2.3.3 Dynamic Light Scattering - Photon Correlation Spectroscopy
(PCS).......................................................................36
3.2.4 Sensory Evaluation..................................................................38
3.2.4.1 Descriptive Test ..........................................................39
3.2.4.2 Difference Test ............................................................39
3.2.5 Statistical Analysis ..................................................................
40
3.3 RESULTS AND DISCUSSIONS............................................................JO
3 .3.1 Chemicül Properties of DHA Milk and Control Milk ..........................4O
.
.
3.3.1.1 Fat Protein Lactose and Somaiic Ce11 Count ........................JO
3.3.1.2 SDS- PAGE...............................................................42
3.3.1.3 Fütty Acid Profiles of DHA and Control Milk by GLC ...........A 3
3.3.1.4 Minerats....................................................................
43
3.3.2 Physical Properties.................................................................-47
3.3.2.1 Density .....................................................................47
3.3.2.2 Diameter Distribution of Fat Globules ................................47
3.3.2.3 Particle Size Distribution of Cüsein Micelles by Photon
Correlation Spectroscopy ( PCS) .......................................51
3.3.3 Sensory Evaluation.................................................................. 52
CHAPTER 4 CHEESE MAKING PROPERTES OF DHA ENRICHED MILK
4.1 INTRODUCTION..............................................................................54
4.2 MATERiALS
AND METHODS .............................................................54
4.2.1 Coagulation of Milk .................................................................54
4.2.1.1 Acid Coagulation of Milk (Yogurt Culture)..........................55
4.2.1.2 Remet Coagulation of Milk .............................................56
4.2.2 Cheese Making Properties .........................................................57
4.2.2.1 Cheese Müking ............................................................57
4.2.2.2 Cheese Yield ...........................................................- 3 9
61
1.2.2.3Composition Analysis of Cheese .......................................
4.2.3. Cheese Ripening ....................................................................
64
4.2.3.1 pH...........................................................................
64
4.2.3.2 Free Amino Acids ........................................................
61
4.7.3.3 Microstructure by Scanning Electronic Microscopy (SEM).......65
4.2.4 Sensory Evaluation.................................................................-66
4.3 RESULTS AND DISCUSSIONS............................................................ 67
4.3.1. Coagulation ..........................................................................67
4.3.1.1 Acid Coagulation Tests (Yogurt Culture).............................67
4.3.1.2 Rennet Coagulation......................................................70
4.3.2 Cheese Y ield .........................................................................
74
4 3 . 3 Cheese Composition................................................................
75
4 3 . 4 Cheese Ripening .....................................................................
76
4.3.4.1 pH changes during ripening............................................. 77
4.3.4.2 Frec Amino Acids ........................................................78
4.3.4.3 Microstnicture by Scanning Electronic Microscopy................78
4.3.5 Sensory Evaluation..................................................................79
CHAPTER 5 BUTTER MAKING PROPERTIES OF DHA ENRICHED MILK
5.1 JNTRODUCTION ............................................................................. 83
5.2 MATERIALS AND METHODS .............................................................
83
5.2.1. Butter Making .......................................................................83
5.2.2 Chernical and Physical Properties of DHA Butter..............................87
5.2.2.1 Moisture in Butter ........................................................ 87
5.2.2.2 Fat in Butter...............................................................87
5.2.2.3 Hardness ...................................................................88
5.2.3 Sensory Evaluation of Butter ......................................................88
89
5.2.4 Properties of Extracted Butter Oil .................................................
89
5.2.4.1 Extraction of Butter Oil .................................................
5.2.4.2Solid Fat Content by Pulsed Nuclear Magnetic Resonunce
(pNMR)...................................................................$89
5.2.4.3 Dropping Point ............................................................
90
5.2.4.4 Melting Properties by Differential Scanning Calorimetery
(DSC).....................................................................-92
5.2.4.5 Lodine Value ...............................................................
93
5.3 RESULTS AND DISCUSSION ..............................................................
93
5.3.1 Butter Making........................................................................93
5.3.2 Cornposition and Hardness.........................................................95
5.3.3 Sensory Evaluation
...............
5.3.4 Physical Properties of Extracted Butter Oil ......................................99
5.3.4.1 Dropping Point and Iodine Value ......................................99
5.3.4.2 Solid Fat Content by Pulsed Nuclear Magnetic Resonance
(pNMR) ..................................................................10 1
5.3.4.3 Melting Propenies by Differential Scanning Calorimeuy
(DSC)
.....................................................................101
CHAPTER 6 GENERAL CONCLUSION. .................................................... 106
REFERENCES ....................................................................................
108
APPENDK
Appndix 3.1 .............................................................................
-129
Appendix 3.2..............................................................................130
Appendix 4.1 ..............................................................................
131
LIST OF TABLES
TABLE 2.1 Recomrnended intake of omega-3 and omega-6 fatty acids based on
encrgy expressed as daily rates by Health and Welfare Canada.................5
TABLE 2.2 Content of Omega-3 fatty acids in fish ............................................-9
21
TABLE 2.3 Main fatty ücids in milk gl ycerides .................................................
TABLE 3.1 Formulation of fish meal supplement of treatment feed .........................31
TABLE 3.2 Composition and pH of the calcium buffer .......................................38
TABLE 3.3 Fat. Protein and lactose concentration and somatic cell count .................41
TABLE 3.4 Fatty acid profile of DHA and control milk (% of fat)..........................J6
47
TABLE 3.5 Minerais in rnilk ......................................................................
TABLE 3.6 Diameters of fat globules in DHA milk and control milk .......................48
TABLE 3.7 Diameters of casein micelles in DHA milk and control milk ..................52
TABLE 3.8 Scores of descriptive tests of control and DHA milk ............................52
TABLE 4.1 Y ields of control cheese and DHA cheesc .......................................$74
TABLE 4.2 Cheese composition..................................................................
75
TABLE 5.1 Chuming time (seconds) of control butter and DHA butter ....................95
TABLE 5.2 Composition and hardness of control butter and DHA butter ..................98
TABLE 5.3 Physical properties of control and DHA butter oil ............................. 100
vii
LIST OF FIGURES
FIGURE 2.1 Metabolism of omega-3 (left) and omega-6 (right) fatty acids.................7
FIGURE 2.2 Pathway of bio-hydrogenation of linoleic acid to steiiric acid by rumen
Microorganisms......................................................................25
FIGURE 3.1 Protein distribution of contro! and DHA milk .................................A 4
FIGURE 3.2 Typical SDS-PAGE of control and DHA milk ................................-45
FIGURE 3.3 Fat globule size distribution of control and DHA milk ........................49
FIGURE 3.4 Fat globule size distribution of Holstein milk samples from Ontario ........5O
FIGURE 4 .I Flow diagram of Cheddar cheese making ......................................A0
FIGURE 4.2 pH profile of control milk and DHA milk during acid coagulation..........71
FIGURE 4.3 Consistency profiie of control and DHA milk during acid coagulation ......72
FIGURE 4.4 Consistency profile of control and DHA milk during rennet
coagulaiion ..........................................................................
-73
FIGURE 4.5 pH profile of control cheese and DHA cheese during ripening...............80
FIGURE 4.6 Changes in amino acid concentration of control cheese and DHA
.
.
cheese during ripening..............................................................81
FiGURE 4.7 Micrographs of control and DHA cheese during ripening .....................82
FIGURE 5.1 Flow diagram of butter making ................................................... 85
FIGURE 5.2 Schematic diagram of butter churn................................................ 86
FIGURE 5.3 Schematic diagrarn of the measuring principles of a Metiler dropping
point apparatus.......................................................................91
FIGURE 5.4 Solid fat content of DHA and control butter oils by pNMR .................103
viii
FIGURE 5.5 Volume percentage of melted fat of DHA and control butter oil versus
temperature......................................................................
-104
FIGURE 5.6 Typical DSC curves of DHA butter oil and control butter oil ............... 105
CHAPTER 1 INTRODUCTION
Incorporating essential fatty acids (omega-3 series and omeag-6 series) into food
products has been extensively studied recently. Of the omege-3 Fatty acids series,
eicosapentaenoic acid (EPA, 205 03) and docosahexaenoic acid (DHA, 22%0 3 ) are the
most important. a-linolenic acid (18:3 w3) c m be found in some oil seeds, such as canola
seed and flax seed etc. It c m be metabolized into EPA and DHA by undergoing
desaturation and enlongation, but the etriciency is very low. Supplementing food produci
with high amount of EPA and DHA rather than their precursoa is recommended.
Fatty fish are rich in EPA and DHA. Using fish meal as dietary supplements to
dairy cows was proved to incrcase EPA and DHA concentrations in bovine milk. A
special diet with fish meal supplement was developed by Wright and McBride from
Animal and Poultry Science Department at University of Guelph. The dietary supplement
consisted of a combination of hemng meal and feather meal. Different levels of
supplement were used and the corresponding transfer efficiencies of DHA from diet to
milk were investigated. In CO-operationwith Animal and Poultry Science Department,
experiments were conducted to study the chernical, physical and processing propert ies of
the DHA enriched milk.
Objectives:
1. Study the chemicai, physical and sensory properties of DHA milk
2. Study the coagulation and cheese making properties of DHA milk
3. Study the butter making properties of DHA milk
C W E R 2 LITERATURE REVIEWS
2.1 ESSENTIALITY AND BENEFITS OF OMEGA-3 FATTY ACIDS
Polyunsaturated fatty acids are defined as fatty acids that have two or more
double bonds. Omega-3 fatty acid family is a group of polyunsaturated fatty acids with its
first double bond at carbon 3 counting tiom the omega terminus - the methyl end of the
chain. Similarly, omega-6 fatty acid family refers to a group of polyunsaturated fatty
acids whose first double bond, counting from the omega terminus, is located at carbon 6.
Both omega-3 and omega-6 fatty acid series are considered essential fatty acids because
animal tissues are unable to introduce double bonds in positions prior to carbon 9 from
the omega terminus (Uauy-Dagach and Valenzuela, 1992). Thus these fatty acids can not
be synthesized by humans and must be obtained from the diet. Omega-3 fatty acids are
not only essential for normal growth and development, but also play an important role in
treating and protecting cardiovascular disease, arthritis, cancer, hypertension and
inflammation (Sirnopoulos, 1991; 1997).
2.1.1 Omega-3 Fatty Acids and Normal Growth and Developrnent
Snidies performed on monkeys showed that Docosahexaenoic acid (22%03,
DHA) deficiency pre-and pst-natal1y caused abnormalities in visual Function and
learning (Neuringer, 1989; Uauy-Dagach and Valenzuela, 1992). It was also proved that
visual acuity and habituation learning for infants were reiated to DHA status (Jorgensen
et al., 1996; Gibson et al., 1996). DHA is one of the most abundant fatty acids in the
brain's structural lipids and retinal lipids OJutr. Res., 1985). It is essential for the
development of animal newous systems. Infants' requirement for DHA is the highest in
the 1st trimester of pregnancy, because the rate of brain growrh is the fastest in that
period. For preterm infants, this critical growth period is shortened, so they are ofien bom
with lower DHA stores (Newton, 1997). DHA status is also critical for infant growth and
development in the first 1 1 months after birth (Newton, 1997) because animal nervous
systems are incompletely developed at birth and up to 50% of the final amount of DHA
accumulates postpartum (Nutr. Res., 1985). Thus, the availability of dietary DHA
becomes important. DHA status in both pre- and postnatal infants is fully dependent on
the mother's intake and stores of essential fany acids, so increasing dietary omega-3 fatty
acids before, during and afier pregnancy is recommended.
2.1.2 Omega-3 Fatty Acids and Cardiovascular Disease
Omega-3 fatty acids contribute to cardiovascular health mainly by helping
maintaining normal serurn triglyceride levels and normal blood platelet reactivity
(bienleton, 1995). They reduce the risks for cardiovascular disease (Harris et al., 1997;
Leaf, 1989 & 1990). Fish oils rich in omega-3 fatty acids were found to drarnatically
levels by inhibiting the
decrease very low density lipoprotein triglyceride (VLDL-TG)
VLDL-TGsynthesis (Harriset al., 1988a 1989 & 1990). They are also becoming
potentially important agents in the treatment of hypemiglyceridemia without causing
serious side effects (Harriset al., 1989).
2.1.3 Omega-3 Fatty Acids and Cancer
Dietary omega-3 fatty acids were found to have anti-tumor effects in several
animal studies with various tumor models (Karmali, 1989). The results have shown that
omega-3 fatty acids delayed tumor appearance, and decreased the rate of growth, the size
and number of tumors (Simopoulos, 199 1). In human studies, Eskimos and the Japanese
whose diet contain lots of fish, showed a low incidence of breast cancer compared to their
ethnic counterparts in Western countries (Zamula, 1986).
2.1.4 Omega-3 Fatty Acids and Inflammation
A study by Crosby (1996) showed that omega-3 fatty acids can induce antioxidant
enzymes, protecting cells and tissues against oxidation and inflammatory damage. like
arthritis. Omega-3 fatty acids in fish oil exert an important role of anti-inflammation by
modulating the synthesis of eicosanoids (Watkins, 1997). Dietary omega-3 fatty acids can
help reducing the symptoms of inflammation by decreasing production of leukotrienes.
several interleukins. and superoxide formation by monocytes and neutrophils (Leaf,
1991).
2.1.5 Recommended Daily Intake
Separate daily dietary recommendations for omega-3 and omegad fatty acids
shown in table 2.1, were issued by Health and Welfare Canada in 1990.
The quantity of 0-6 or the ratio of 06: 0 3 should be considered when making
dietary recommendations for 0-3 fatty acids (Simopoulos, 1989). Japanese who are
known to have the longest life expectancy in the world, consume relatively large amounts
TABLE 2.1. Recommended intake of omega-3 and omega-6 fatty acids based on energy
expressed as daily rates by Health and Welfare Canada (1990)
Age and sex
0-4 month (M, F)
5- 12 months (M. F)
Energy (kcal)
600
0 3 PUFA (g)
0.5
0 6 PUFA (g)
3
450
0.25
1.5
1 year (M,F)
2-3 years (M. F)
4-6 years (M, F)
7-9 years
M
F
10- 1 2 years
M
F
13-15 years
M
F
16- 1 8 years
M
F
19-24 years
M
F
25-49 years
M
F
50-74 years
M
F
75+ years
M
F
Pregnancy (additional)
1'' trimester
2" trimester
trimester
Lactation (additional)
of omega-3 fatty acids fiom fish and vegetable oils. The average ratio of omega-6
fatty acids to omega-3 fatty acids in their diet is 4: 1 (Sugano, 1996) which is close to 1 :1
.
the dietary ratio on which humans evolved (Eaton et al., 1997). Even though evolutionary
considerations are not a basis upon which to make nutritional recornmendations,
information derived from this aspect may provide valuable understanding about hurnan
dietary needs and about the relation of diet to infant development, adult health and
disease (Simopoulos. 1997). The ratio of omega-6 fatty acids to omega 3 fatty acids in
current western diets is about 10-20:1 which is considered to be unbaianced (Simopoulos.
1997).
Some side effects should also be considered when applying omega-3 fatty acids as
nutritional supplements or for clinical purposes. Ornega-3 fatty acids were observed to
increase bleeding time slightly (Sanders, l985), especially when combined with aspirin
treatment (Hams et al., 1990). Low density lipoprotein (LDL) levels were usually not
affected by fish oil supplements in the studies performed on normolipidemic human
subjects, but fish-oil-induced increases in LDL cholesterol levels in hypertriglyceridemic
patients have been reported (Harris et al., l988b; Rambjor et al., 1996). Another concem
of the safety of consuming omega-3 fatty acids supplements is the potential of these
unsaturated fatty acids to undergo oxidation in the body. Supplementing diet with
antioxidant, such as vitamin E or p-carotene, should be considered for the individuals
who are taking omega-3 fatty acids supplement (Leaf, 1991).
2.2 METABOLISM OF OMEGA-3 AND OMEGAd FATTY ACIDS
The two most important omega-3 fatty acids, EPA and DHA, are rarely found in
plants, vegetables and terrestrial meat, but a-linolenic (1 8:3 0-3) and linoleic (1 8:2 0 - 6 )
are found in many kinds of oil seeds. Corn seed is rich in linoleic acid. Canola seed and
flax seed are rich in a-linolenic acid. a-linolenic acid c m be converted into EPA and
DHA by undergoing desaturation and elongation. Fany acid chah desaturation and
elongation in mammals occurs between the fatty acid carboxyl goup and the nearesr
double bond. The number of carbons from the methyl end to the first double bond
remains fixed. So there is no conversion between the ornega-6 and ornega-3 fatty acid
linoleic (LA)
C l8:î
a-linolenic (ALA)
C 18:3
1
A6-desaturase
J
elongase
AS -desaturase
eicosapentaenoic (EPA)
C20:S
1
1
arachidonic (AA)
C20:4
elongase
docosapentaenoic (DPA)
C22:S
.1
A
dihomo-gamma-linolenic (DGLA)
C20:3
eicosatraenoic
C20:4
1
A
gamma-linolenic (GLA)
Cl 8:3
octadecatetraenoic
CM4
1
adrenic
C22:4
A4-desaturase
docosahexaenoic (DHA)
C22:6
1
docosapentaenoic
C225
FIGURE 2.1 Metabolism of omega-3 (lefi) and omega-6 (right) fatty acids (UauyDagach and Valenzwla, 1992)
families. The metabolism of a-linolenic acid (1 8:3 0 - 3 ) and linoleic acid (18 2 0-6)is
shown in figure 2.1.
It c m be seen clearly from figure 2.1, that omega-6 fatty acids cornpete with
omega-3 series for A6-desaturase in the first step of desaturation. Omega-6 fatty acids are
fond to be more favored by A6-desaturase. High doses of 18:2m6 when given
in~avenouslywere found to have a strong inhibithg effect on the (03 series. A6-
desaturase also decreases with age and its activity may Vary among individuals (de
Gomez and Brenner, 1975). A4-desaturase is another rate-limiting enzyme in the
conversion of 18:303 to 22:603. The efficiency of converting LNA to EPA and DHA is
very low (Nettleton. 1991& 1993; Harris et al., 1990). An ideal dietary supplement should
contain high amounts of EPA and DHA rather than the precursors of EPA and DHA.
2.3 FOOD SOURCES OF OMEGA-3 FATTY ACIDS
2.3.1 Fish and Fish Meal
Fish is the best food source for long chah omega-3 fatty acids. Fatty acid
composition of fish varies fiom species to species and seasonally. Usually cold water fish
and fatty fish contain more omega-3 fany acids, mainly EPA and DHA (Sanders, 1985).
Table 2.2 shows the omega-3 fatty acid content of some common fish species (Barton
and Emerson, 1986).
Fish meal is an important by-product of the fish industry. About 95% of the raw
material not used for direct hurnan consumption are processed into fish meal (Hussein
and Jordan, 1990). The composition of fish med varies arnong different types, but it
usually contains higher amounts of long-chain (20 carbon atoms or more)
polyunsaturated fatty acids than vegetable fat. It is a rich source of omega-3 fatty acids.
mainly EPA and DHA, which may represent over 30% of the total fatty acids present
(Bimbo and Crowther, 1992).
TABLE 2.2 Content of Omega-3 fatty acids in fish (Barton and Emerson, 1986)
Species
% Oil in flesh
% Omega-3 fatty acids
in oila
% Omega-3 fatty acids
in flesh
Haddock
0.5
39.6
O. 198
Tuna, canned
1 .O
30.0
0.300
Shnrnp
1.1
28.5
0.314
Cod, Atlantic
0.7
45.9
0.32 1
Squid
1.O
53.3
0.533
Mullet
3 .O
19.1
0.573
Halibut
2.O
36.0
0.720
Whiting, Pacific
3.O
33.3
0.999
Trout, Rainbow
7 .O
17.6
1.232
Tuna, Raw
5.1
30.0
1.530
Whitefish, Lake
7.O
22.2
1.554
Sardine, Canned
6.3
26.8
1.688
Salmon
9.3
23 .O
2.139
Mackerel, Atlantic
13.0
19.0
2.470
Hemng
15.0
18.4
2.760
O,
the total percentages of 18:3~03,20:5~~13,22:5a3
and 22:603.
2.3.2 Plant Sources
Food plants are poor sources of EPA and DHA. Some oil seeds, such as flax seed
and canola seed, are rich in ALA, but as we have noted in section 2.2, ALA is
inefficiently converted to EPA and DHA by hurnans and most animals. Purslane is an
exception. It is the only higher plant known to produce EPA. The presence of DHA has
also been reported in purslane (Omara-Alwala et al., 1991). It was found that fatty acids
in purslane varied with the age of the plant, the longer chain 0 - 3 fatty acids (20:5.2:5.
225) increasing with age (Sirnopoulos, 1992). More longer chain fatty acids were found
in leaves than in stems. The predominant fany acid was 18:3 0 3 which was about 7 times
higher than that found in spinach. Purslane is a widely distributed wild plant in the world.
100 grams of fresh purslane contains about 300-400 mg of 18:3 0 3 , 12.2 mg of atocopherol, 26.6 mg of ascorbic acid, 1.9 mg of p-carotene and 14.8 mg of glutahione
(Salonen et al., 1991). It is a wonderful dietary source for both 0 - 3 fatty acids and
antioxidants.
2.33 Red Meit and Poultry
Both pork and beef are poor sources of omega-3 fatty acids. Poultry are also
generally a poor source of omega-3 fatty acids, but on some f m s , fish meal is included
in regular feed as pmtein supplements. In this case, poultry meat can be a good source for
dietary 0 - 3 fatty acids.
2.3.4 Milk
Bovine milk contains very little of omega-3 fatty acids. EPA and DHA are present
in bovine milk only in trace amounts. Infant formulae in North America generally contain
fats derived from vegetable oils and thus do not contain polyunsaturated fatty acids with
more than 18 carbons (Kathryn and Gibson, 1989).
Both EPA and DHA are present in breast milk, but their concentrations Vary
considerably among individuals. Fatty acid composition in breast milk is largely affected
by the fatty acids in the diet. Breast milk fiom lactating Inuit women whose diet contains
lots of fish was found to have 3-4 times more EPA (205 03) and 2 times more DHA
(22:6 03) relative to the breast milk collected in Vancouver (Sirnopoulos, 1989). DHA
concentration was lower in vegan or vegetarian human milk, but was still more than that
in cow milk formula (Sanders et al., 1992). DHA and total omega-3 fatty acids content of
human milk can be readily increased by supplementing the mothers' diets with omega-3
fatty acids (Nutr. Rev., 1985; William et al., 1984).
2.4 INCORPORATINC OMECA-3 FATTY AClDS INTO ECGS AND MEAT
PRODUCTS
Foods modified to provide health benefits beyond the nutrients they traditionally
contain, have been referred to as designer or functional foods. Fortifying foods with
omega-3 fatty acids by incorporating them at an earlier stage in the food chah has been
intensively studied recently.
2.4.1. Incorporation of Omega-3 Fatty Acids into Chicken Meat
Effects of full-fat flax seed (8% and 16%). canola seed (16%)and fish meal (2%)
on the omega-3 fatty acids composition of white and dark chicken meat and whole
carcasses were investigated (Ajuyah et al.. 1992). Elevated levels of omega-3 fatty acids
(total of 18:3 03, 2 0 5 0 3 , 2 2 5 03 and 22% 03) were ail increased in white and dark
meats and whole carcasses of hens fed the oil seeds. Fish meül when fed at 2% of the diet
did not significantly increase the total omega-3 btty acids content in the wholr carcüss.
Tissue concentration of l8:3 0 3 and its metabolites (20503, 2 2 5 0 3 and 2 2 6 0 3 ) were
greatly influenced by their levels in the diet. The total omega-3 fütty ücids content in the
2% fish meal experimentd diet was 4.01 4 which was the lowest among al1 the trertment
diets, while the omega-3 fatty acids contents of other oil seed diets rünged from 7.59% to
40.30%. It also had the highest ratio of omega-6 fatty acids to omega-3 fatty acids ût 5.06
while the other treatment dieis were at the ratios of 0.57.0.3and 2.70. Low omega-3 fatty
acids concentration and high ratio of omegü-6 fütty acids to omegu-3 fatty acids could
have explained why 2% fish meal diet did not increüse the total omega-3 fatty acids level
in the chicken meat successfully as the other treatment diets. In a later study, Ajuyüh et
al. ( 1993) concluded that feeding broiler c hicken wit h wtioxidant produced higher
concentration of omegü-3 fatty acids in the meat.
2.4.2. Incorporation of Omega-3 Fatty Acids into Eggs
Egg yolk lipid composition was found to be readily influenccd by diets including
high levels of polyunsaturated oils. such as safflower. corn and soybean (Cruickshank,
1934; Yu and Sim. 1987; Hulan, 1988; Oh et al.. 1988; Adams et al.. 1989). Eggs are
considered a highly atherogenic food d w to the relatively high yolk cholesterol content.
But eggs are very important ingredients for cooking or baking. Altering fatty acid
composition in eggs can be an opportunity to influence the diet of a great population.
Effects of feeding laying hens with significant amounts of fiax seed (10%.20% and 30%)
were investigated by Caston and Leeson ( 1990) at University of Guelph. a-linolenic.
EPA and DHA levels in eggs were al1 increased significantly. Later. Caston et ;il.(1994)
investigated hen performance when fed diets containing 0. 10 and 20% ground tlax seed.
Both egg production and egg-shell deformation were not affected by dietary flax seed
supplement while the omegr-3 fatty acids levels. especidly a-linolenic acid in the
treatment eggs were significantly increased. Cherian et al. ( 1991) also investigated the
incorporation of omega-3 fatty ücids from the laying hen diet into the egg and into the
developing progeny. Ground flax seed (8% and 16%) and ground canola seed ( 16%) were
used as supplernents to the diet. The predominant omega-3 fatty acid in the egg yolk wüs
found to be linolenic acid (LNA) while EPA, DPA and DHA were also observed in the
yolk, confirming that ALA could be converted io EPA. DPA and DHA through
desaturation and elongation (Anderson et al.. 1989; Farrell. 1990; Nwokilo et al., 1989;
Sim et al., 1990). 3% menhaden oil fed to laying hens was found to significantly increase
the levels of omega-3 fatty acids in egg yolk (Van Elswyk, 1992). Average increases of
LNA and DHA were 75.8% and 356% respectively (Van Elswyk. 1997).Cooking did not
significantly alter levels of omega-3 polyunsaturated htty acids in the egg. No off-flüvor
was observed in hard cooked eggs, but a fish-like flavor was observed in scrambled eggs.
The volatility of flavor compounds in fish oils may have been enhanced due to the
mixing of egg samples prior to scrambling, as well as to exposure to high heat during
scrambling (Van Elswyk, 1992). Further studies showed that flax seed supplements
enrich both the egg yolk lipids and the tissues of the hatched chicks with omega-3 fatty
acids (Cherian and Sim, 1992). Nash et al. ( 1996) studied the effect of dietary menhaden
meal (4%. 8% and 12%) on the omega-3 fatty acids and sensory attributes of egg yolk in
laying hens. Menhaden meül when fed ai 12% of the diet could increase the totai omega3 fatty acids level in the yolk to 3%. Both EPA and DHA level in the yolk were elevated.
But a slight "fishy" off-flavor in the yolk of the soft-cooked eggs was observed when
menhaden level increased to 8% or higher. The health effects of omega-3 fatty acids
enriched eggs were investigated by Oh et al. ( 1991 ). The mean plasma triglyceride
concentration of the 12 human subjects who consumed 4 omegü-3 eggs daily for 4 weeks.
was decreased and the meün plasma cholesterol concentration rernilined unchünged. On
the contrwy. both the plasma triglyceride concentration and the plasma cholesterol
concentration of the subjects who consurned control eggs were increased. Omega-3 eggs
were iilso recommended as a good weaning food for infants (Sirnopoulos and Jr.. 1992).
2.4.3. Incorporation of Omega-3 Fatty Acids into Pork
Methods of enriching pork with omega-3 Fütty ücids have been extensively
reviewed by Howe ( 1998). Flax seed dietary supplement was used to increase the omega-
3 fatty acids content of the pork (Romans et al., 1995). ALA and EPA levels in the
muscle and adipose tissue increased up to 5 fold after 4 weeks, but DHA level remained
unchanged. This may suggest that the conversion of ALA to DHA is very inefficient.
Feeding the pigs pre-formed dietary long chah omega-3 fiitty acids, such as fish or fish
meal, is the most effective wsy to increase their levels in pork. But these fish by-products
must be limited in the pig rations andor rernoved from the rations ai a suitable tirne
before slaughter, otherwise they will cause taint in pork (Howe. 1998).Leskanich et al.
(1996) studied the effects of fish oil and rapeseed oil on the fatty acids and organoleptic
chuacteristics of pig meat. Treatment diets supplemented with 2% rapeseed oil. 1% fish
oil and two levels of a-tocopheryl acetate was used. The levels of EPA and DHA in the
muscle of the pigs fed treatment diets were increased 3 to 4 folds. Reduction in fat
firmness and an increase in meai tendemess were observed. The oxidative stability of the
tissues and sausages from the pigs fed the treatment diets were lower than for the control.
but the overall orgrnoleptic propenies were not adversely affected. The transfer of DHA
from fish oil enriched materna1 diet to the sow milk, and then to the tissues of milk-fed
piglets was dso studied by Arbuckle ünd Innis (1993) and Fritsche et a1 (1993). Both
dernonstrated the succcssful transfer of DHA from the diet to the sow milk and the tissue
of the suckling piglets.
2.4.4. Incorporation of Omega-3 Fatty Acids into Ruminant
Unlike poultry and swine whose tissue fatty acid composition reflects the dietary
fatty acid composition, tissue fatty acid composition of ruminants is largely influenced by
the ability of ruminal microorganisms to hydrolyze and hydrogenate the dietiuy
unsaturated faity acids. Manipulation of fatty acid concentration in ruminants may only
be achieved by alteration or bypass of the rumen environment (Van Elswyk, 1993).It wüs
found that rumen microflora could not hydrogenate 205 w3 and 22:6 03 to a significant
extent when fish oil preparations were incubated with strained rumen liquor. Steric
factors or lack of specific enzymes are possible explmations for the inability of rumen
micrmrgiuiisms to hydrogenate 205 03 and 22:6 03 (Ashes et al.. 1992). Effects of fish
meal supplements on fatty acid composition of beef cattle carcasses were studied by
Mandel1 et al. (1998). Fish meal at 10% of diet increased DHA concentration in muscle
to I 1 rng1100g. Fish taint was observed by the taste panel. The lowest concentration of
fish product required for significünt 0 3 fatty acid enrichment without developing off
flavor still await further research.
2.4.5 Incorporation of Omega.3 Fatty Acids into Bovine Milk
There are three generül approaches to modify the fatty acid profile of mil k.
narnely. special diets, genrtic manipulation. or fatty acid supplements to milk or milk
products.
Adding target fatty acids directly into milk has the advantage that. theoret ically
.
any level of desired fatty acids could be added into milk fat. This method requires
blending fatty acids with milk fat first, and then homogenizing the biend into skimmed
milk. For some products such as most vürieties of cheese, fatty ücid supplements must be
transformed to a water dispersible form, to avoid the necessity of homogenization.
Homogenization of milk reduces the curd tension (Jana and Upüdhyüy. 1992). Raising
coagulation temperature and rennet quantity. allowing more time for rennet action or
incrersing the acidity and adding calcium chloride do not restore the curd tension of
homogenized milk (Maxdy, 1955). In Cheddar cheese making, hornogenization of milk
causes poor curd fusion during Chedduing and gives rise to mechanicd openness defect
in the product (Emmons et al., 1980; Green et al., 1983). Further, EPA andlor DHA
supplements must be refined to reduce undesirable flavors, and micro-encapsulation or
anti-oxidants are frequently required to stabilize long-chah unsaturated fatty ücids
against oxidation, especially in foods that require cooking. lncorporating target fatty
acids directly into milk and other dairy products seems to be an expensive approach.
Drinking milk supplemented with odorless forms of the omega-3 fatty acids EPA and
DHA was recently developed by the international dairy firm. P m r l a t (Howe. 1998).
However, the Company is still working to increase the stability of omega-3 fatty acids in
drinking milk.
Tliere is littla evidcnce for within-breed genetic variation in milk fat composition.
while modest differences ümong breeds and subsiantial differences mong species were
observed (Gibson, 1991).Thesc observations are evidences that genetic change in fat
composition of milk is possible. Genetic changes can be üccornplished by conventionül
breeding methods, or by transgenic alteration (Gibson, 1991 ). Only relatively luge
changes in milk fat composition can meet today's consumen' needs. so conventional
breeding may not be effective becruse the rates of change are very slow. Both
conventional breeding and genetic manipulation have the üdvüntüge that genetic change
is cumulative. But this also means genetic changes cm only aim at long-ierm
improvement rather than short-term solutions.
Comparatively, milk fat composition is more readily altered by changing dietüry
fat to dairy cows. Introducing omega-3 fatty acids ai an exlier stage in the food chain.
such as by feeding fish meal to dairy cows. has several potential advantages. It could
reduce the cost because dietary supplements to dairy cows miy not require the high
degree of refinement required for direct human consumption. It may also enhance the
conversion of ALA to DHA by providing an additional stage for the conversion to occur
(Howe, 1998). Enriching milk with omega-3 fatty acids through changing d i e t q fat
composition has been intensively investigated. Dietary supplements of omega-3 or
omega-3 precursors include fish meal. pure fish oil, dgae and plant sources. Menhaden
oil (Hagemeister et al.. 1991). linseed oil (Hagemeister et al.. 1988).canolü oil (Jenkins et
al.. 1992; Ashes et al., 1997; Khorasani et al. 1991) and fresh grass (Dorothea et al..
1991) were al1 proved to increase omega-3 fatty acid concentrations in milk. Micro-dgae
and algae-like microorganisms as the primary producers of EPA and DHA in the marine
environment. were also studied. A new rnerhod wns developed to produce DHA by
fermentation of drkd sclricoclrytrium sp (a kind of algae) (Barclay et al.. 1997). DHA
produced by this method was very stable at high temperatures used for poultry feed
processing. No losses in DHA content were obsrrved in dried Schizocl~ytririnrsp when it
was pelletized at temperatures of 160-1 9 0 in
~ a~siündürd broiler ration. Once pelletized.
the DHA content remained stable over the 30 düy period evüluüted. It hüs an advüntüge
over fish oil dietary supplement becnuse the high surfüce areü in most of these feeds
readily induces oxidation of the fatty acids. causing taste and odor problems in the
resulting food products. DHA naturally present in micro-algae provides a stable source of
DHA. and meets the needs of vegetarian diets.
Manipulation of fatty acid concentrations in ruminants is üchieved by alteration or
by p u s of the rumen environment (Elswyk, 1993).Fish meal is cooked during
processing. it has a high "by-pass" value. and tends to be relatively resistant to
degradation by rumen microorganisms. permitting those proteins to escape the rumen and
provide the animal with a source of high-quality protein (Hussein and Jordan, 1990). Fish
meal also has an excellent arnino s i d profile. close to that required for growth and milk
production (Stem and Mansfield. l989), but it suppresses milk fat production (Sutton,
1989). Fish meal used as supplements in the diet of dairy cows was proved to increase
DHA concentration in bovine milk (Spain and Polan. 1995; Wright. 1998). lncreased
DHA concentrations in milk were measured with increased fish m e d percentage in diets
based on corn silage (Spain and Polan. 1995). In the diet developed by Wright and
McBride (Wright et al., 1998). Department of Animd Science, University of Guelph. the
rumen-undegradable protein (RUP)supplement was composed of soft-white wheüt,
hemng meal and feather meal. The experirnent was designed as a 6x6 Latin square with a
3x2 faciorial arrangement of ireatment. The 5 factors were 3 levels of rumen-
undegradable protein and 2 levels of overall feed intake. Three protein treatments
(designated low. medium and high) were included at 4.5%. 14.9% and 29.1 % of dry
matter intake (DMI). The correlation between different levels of RUP and its
corresponding DHA level in milk w u investigated. Fish meal supplement was found to
increase milk and milk protein yield which agreed with other studies (Burke et al.. 1997;
Akayezu et al.. 1997).When the rumen-undegradable protein level was increased. there
was a linear decrease in milk fat yield. The efficiency of transfer of DHA from diet io
milk declined linearly from 34.4% to 20.2% to 10.9%as level of RUP increased from
low to high respectively (Wright, 1998).
-
2.5 M L K FAT BIOSYNTHESIS AND POTENTIAL FOR CHANGE
2.5.1 Milk Structure
Milk is a colloidal system in which serum proteins. cascin micelles and fat
globules are dispened in an aqueous solution of lactose, salts and many other dissolved
elements.
About 80% of milk proteins are casein which are defined as the proteins
precipitated by acidifying skim milk to pH 4.6 at
IOOC.
Casein is present in the form of
micelles composed of caseins honded together as "calcium caseinate". Three major
components of casein complex are 41-,B- and ccasein. ai-casein is a single-chain
polypeptide with 199 amino acid residues and a molecular weight of 23,600 daltons. The
molecule contains 8 phosphate residues. a,,-casein has little or no recognizable
secondary structure. B-casein is the second most abundant milk protein. Its molecule is ü
single-chain with a molecular weight of 24,500 daltons. It has little or no secondary
structure. K-casein is the third major component of casein complex. It is soluble over a
broüd range of calcium-ion concentration. Its presence in the casein micelles is very
important because it stabilizes the calcium-insoluble Q- and p-caseins. it is also readily
split by rennin, so the colloid will be destablized and coagulation will occur.
Milk fat exists in milk as fat globules. Each fat globule is surrounded by a thin
hyer called fat globule membrane thai protects the fat globules from flocculation and
coalescence. Proteins. phospholipids and iron are al1 present in fat globule membrane.
The published data of fat globule membrane compositions are not consistent because
different sepûrating and washing methods were used in different studies. The main fatty
acids composition of milk fat is shown in Table 2.3.
TABLE 2.3 Main fatty acids in milk glycerides (Brochu et al.. 1985)
Fatty acids
Average contents C weight base
Saturated fatty acids
Butyric acid (4:O)
3.4
Caproic acid (6:O)
1.3
Cnprylic acid @:O)
1.2
Capric acid ( 10:O)
2.2
Lauric acid ( 120)
3.9
Myristic acid ( 11:O)
13.1
Palmitic acid ( l6:O)
25.3
Stearic acid ( 18:O)
10.6
Arüchidic acid (20:O)
1.3
Behenic acid (22:O)
Tr
Mono-unsaturated fatty acid
Caproleic acid ( 10: 1)
0.2
Lauroleic acid ( 12: 1 )
0.3
Myristoleic acid ( 14: 1 )
1.3
Palmitoleic acid ( 16: 1 )
3.7
Oieic acid ( 18: 1 )
30.8
Polyunsaturatd fatty acids
Linoleic acid ( 182)
3.2
Arachidonic acid (20:4)
1.1
C22 acids
Tr
2.6.2 Biosynthesis of Milk
Polysaccharides, proteins. lipids and a11 other fermentable subsirates in the feed
are h ydrol yzed into volatile faity acids, methane. carbon dioxide. and ammonia during
fermentation in the rumen by the rumen microflora (Phillipson, 1970). Acetate,
propionaie. and B-hydroxybutyrate are major end-products of ruminal fermentation and
account for 70-80% of the total energy intake (Dougherty. 1965).Nutrients produced by
the extensive fermentation of feed in the rumen are absorbed into circulüting blood across
the rumen wall and become available to the animal. The absorption of most nutrients
from rumen into blood is accomplished by diffusion. Thus the concentrütion gradient is
iiir iiioii iiiiportüiii h i o r iii deieriiiining rüiab of irünskr (Philiipson. 1970).
Proteins are extensively degraded in the rumen to ammonia. Peptides and amino
ücids are intermediates in protein breakdown in the rumen. but their concentrations in the
rumen are nonnûlly low. Ammonia is absorbed into the circulating blood by simple
diffusion. An ideal ration with respect to nitrogen utilization is one thüt allows a
significant fraction of good quality protein to pass through the rumen to the abomasum,
allowing proteins to be hydrolyzed in the srnall intestine (Phillipson. 1970).The resulting
amino acids are absorbed into the blood as both the nutrients for the animal and the
precursors for milk synthesis. Lipids from the feed are hydrolyzed in the rumen frorn
their esteritied form into free fatty acids. Some unsaturated fatty acids will go through
bio-hydrogenation in the rumen and producc high yields of stearic acid and smaller
amounts of isomers (Phillipson. 1970).Trans and positional isomers of the dietary
polyunsüturated CIg fûtty ücids are formed as intermediates in the conversion of these
acids to stearic acid. Stearic acid is the major free fatty acid that passes from the rumen
to the lower digestive tract. The resulting fatty acids are re-esterrified in the intestinal
mucosa and transferred to biood via the lymph (Walstra and lenness, 1984).
The biosynthesis of milk takes place in the mammary gland. The precursors of
milk components are absorbed from blood by epithelial cells. and then converted into
milk components (Rosenthal. 1991). Some of the milk constituents iw trünsferred
direcily from blood, while most of the constituents of milk are synthesized in the
mammary gland. There are three factors influencing intracellular availability of substrates
for milk synthesis: concentration in the blood. blood flow and membrane transport. The
Iützr IWO üre &lx
niost iniportiint (Davis aiid Collier, 1985).
Lactose and the principal milk proteins are not synthesized in any tissue other
thün the mammary gland (Walstra and Jenness, 1984).Casein, P-lüctoglobulin and alacialbumin are synthesized frorn amino ücids. Lactose and citrate are produced from
glucose which is büsically synthesized from propionate and ürnino acids by
gluconeogenesis in the liver (Walstra and Jenness, 1984). Vitamins, certain minerül salts,
urea. and vürious flavoring compounds from the feed apparently pass from the blood io
the milk without change.
Fatty acid precursors in the blood are readily transferred to the mamrnary gland.
Fatty acids taken up by the gland reflect the fatty acid composition of dietary fat
(although rnodified in ruminants by micmbial hydrogenation) and of the fatty acids
synthesized by the tissues of the body and released into the circulation (Dils, 1986).
Ruminant milk lipids are characterized by relatively large amounis of short- and mediumchain fatty acids. Shon chain fatty acids could be fomed by continwd condensation of
acetrte molecules and could be intermediate stages in the synthesis of long-chain latty
acids. Most fatty acids up to 16 carbon aioms in length. are synthesized from the two
shorter building blocks. acetate and P-hydroxybutyrate. Acetaie contributes increments to
al1 of the C4-C 14 acids and B-hydroxybutyrate is used primarily for the intitial fourcarbon "primer" unit of most fatty acids synthesized. Only a portion of the C 12 to C 16
faity acids are from blood fatty acids (Dils, 1986).Medium-chah (Ca:oand Ci
fatty
acids incorporate directly into milk triacylglycerol and are neither desaturated nor
elongated into longer chah fatty acids (Dils. 1986). Ruminant m a m m q gland has an
ability to desaturate and elongate fatty acids absorbed from the blood (Dils, 1986).C 18:O
can be desatured into C 18: 1 and C 16:0 can be desatured into C 16: 1, but C 12:O and C 14:O
will remain unaffected (Dils. 1986).The ruminant mammary gland does not enlongate
(Dils, 1986).
Conjugated linoleic acid (CLA) is found predominately in food products from
ruminant animals. CLA is a mixture of positional and geometric isomen of linoleic ücid
involving double bonds at positions 9 and 1 1, 10 and 12 or 1 1 and 13. Geometric
variations of cis-cis, cis-trans. trans-cis or trans-trms müy occur. The cis-9, trans- I 1 and
triins-9, cis- 1 1 isomers have been üssociüted with important biological activities,
including anticarcinogenic activity and inhibition of development of üiherosclerosis in
animals. It may also act as a growth promoting agent (Chin et al., 1994).CLA have been
recognized for their ability to inhibit cancer. It is also an effective antioxidant with
activity in a linoleic acicüphosophate buffer/ethanol system greater than that of vitamin E
and similar to butylated hydroxytoluene (Ha et al.. 1990).
CLA is a result of incomplete bio-hydrogenrtion of dietÿry fatty acids in the
rumen (Griinari et al. 1996).The bio-hydrogenütion pathway of linoleic acid involves an
initial isomerization step resulting in the formation of cis-9. trans-1 1 CLA that undergoes
sequeniid reduction steps yielding trans- 1 1-octadecenoicacid and then stearic acid.
When bio-hydrogenation is not complete. CLA can escape the rumen and be absorbed
from the gastrointestinal tract. thereby providing the mammary gland with the source of
CLA that is found in milk fat (Griinari et al. 1996).
Linoleic Acid
(cis-9,cis- 12 octadecadienoic ücid)
+
CLA (conjugated linoleic acid)
(cis-9, trans- 1 1 conjugated diene)
6
Trans Fatty Acid
(trans- 1 I octadecenoic acid)
+
Stearic Acid
(octadecanoic acid)
FIGURE 2.2 Pathway of bio-hydrogenation of linoleic acid to stearic acid by rumen
microorganisrns (Harfoot ünd Hazlewood, 1988).
Dairy products are the major dietary sources of CLA (Jiang et al., 1994). The cis9, trans- 1 1 and trans-9. cis- 1 1 isomers are major forms in milk and dairy products.
representing 92% of total octadecadienoic acid isomers (Stanton et al., 1997). The CLA
content of dairy products is dependent on the initial CLA content of the raw milk. formed
thmugh microbiül enzymatic reactions in the rumen. and further isomerization reactions
during processing (Lin et al.. 1995).
The effects of processing and storüge on CLA concentrations of various dairy
products were studied by Shantha et al. ( 1995).CLA concentrations incrcased 1.33 fold
in salted butter and 1.27 in unsalted butter during processing. Non-fat yogurt ülso showed
an increüse in CLA content during processing, while ice cream (low-fat and regulrr), sour
c r e m and cheeses (Mozzarella, Gouda and Cheddar) showed no change in CLA content.
The stability of CLA wüs also monitored dunng storage. No changes in CLA content
were observed over the storage period in üII simples, including butter and sour creiim
stored at 4 ' for
~ 6 weeks. butter and ice cream stored ai - 2 0 ' ~for 6 weeks and cheeses
stored at 4°C for 30 - 32 weeks (Shüntha et al.. 1995).
Human milk contains measurable arnounts of CLA, and the majority is present as
cis-9,trans-1 l which is considered to be the biologically active form. It is suspected that
the variation of CLA concentrations in the human milk samples is due io the variation in
materna1 intake of CLA. because data suggest that humans may not have the inherent
ability to synthesize CLA (McGuire et al., 1997).CLA was not detectable in most infant
formula because it usually contains vegetable oil as the principle source. The chainlength distribution of the fatty acids synthesized differs among species (Walstra, 1984).
Proper proportions of short- and medium-chain fatty acids give bovine milk a proper
melting point. This ensures the fat synthesized by the mammary gland has a melting point
sufficiently low to be readily secreted as liquid droplets to meet the need of suckling
Young. Apparently an increased portion of short-chain and medium-chain fatty acids and
an increased degree of unsaturation of the long-chain fatty acids cm decreüse the melting
point of milk fat. Most of the glycerol is synthesized from blood glucose and only a small
part cornes from blood triglycerides (Walstra. 1984). Triglycerides in blood plasma that
serve as a precursor pool for milk fat synthesis are largely in the chylomicrons and lowdensity lipoproteins of blood plasma. There is evidence that not al1 triglycerides in
plasma are readily available for milk fat synthesis. and this fraction is probably that in
high-density lipoproteins. Uptake of triglyceride by the mammary gland is dependent
upon activity of lipoprotein lipase in the mümmary cüpillary endothelium. The provision
of long chain fatty acids to the mümrnary gland in an acceptable form depresses
production of short and medium chain fatty acids by a mechünism thought to be mediated
by inhibition of aceiyl coenzyme A carboxylase (Davis and Collier. 1985).
2.6.3 Factors Affecting Milk Fat Content and Composition
Milk fat content is affected by many factors, such as in4iividud characterist ics,
breed, lactation period, feed, season, health, age and milking etc (Webb, 1974). Jersey
cows produce milk higher in fat than Holstein cows. Evening milking usually has higher
fat content than morning milking. During the whole period of lactation, the fat content of
milk decreases in the first 2 months p s t panum. then increases to a level just below the
level at the beginning of the lactation. Milk fat content also varies with season. It reaches
its maximum in winter. Milk fat content decreases with age, poor health and environment
changes.
As discussed before. milk fat content and milk fitty acid composition are largely
affected by the diet of dniry cows. Grain intake, dietary protein intake. amount and
composition of dietary fat, transfer of dietary fat to milk fat and energy intake dl readily
change milk fat content and milk fatty acid composition (Palmquist and Beaulieu, 1993).
Fermentable starch fed ai high levels (> 50% of feed dry matter) will depress milk fat
percentage and change milk fatty scid composition (Palmquist and Beaulieu. 1993).
Increüsed dietary fat from 1 io 5% of feed dry mütter was shown to decrease milk fat
synthesis (Grummer. 1991).Trüns fütty acids from dietary sources or incomplete ruminal
biohydrogenation are believed to inhibit milk fat synthesis (Palmquist and Beaulieu.
1993).CLA concentrdrion in milk is affected by the prescnce of unsaturated fat in the diet
(Griinari et al. 1996). Full-fat rapeseed supplements resulted in substantiül increases in
CLA in milk (Stanton et al., 1997). As CLA is produced naturülly in ruminant animüls.
elevating dietary intakes of unsüturated firtty acids. the substrate for CLA synthesis, müy
be a rational approach to increase milk fat CLA levels (Stanton et al., 1997).
Theoretically. eight possible geometric isomers of 9, 1 1- and 10, 12-octadecadienoicücid
exist, but the cis 9-, crans- 1 1-octadecadienoicacid (c9. t 1 1- 18:2) is the major derivative
of ruminai bio-hydrogenation of polyunsatured fat (Jahreis et al.. 1997). Fresh pasteur
which is rich in polyunsturated fatty acids, increased the formation of CLA in milk fat
(lahreis et al., 1997). The transfer efficiency of dietary fat to milk fat is influenced by
ruminal bio-hydrogenation. absorption and deposition in adipose tissue (Palmquist and
Beaulieu, 1993). In fact, occurrence of bio-hydrogenation, synthesis and degradation of
fatty acids in niminal mammary gland makes it almost impossible to cdculate the real
transfer efficiency of individual or total fatty acids.
CHAPTER 3 CHEMICAL AND PHYSICAL PROPERTIES OF DHA ENRICHED
BOVINE MILK
3.1 INTRODUCTION
In this experiment, fish meül was used as rumen undegradable protein
supplements ( RUP) to increase DHA content in bovine milk. Chernical composition,
physicai and sensory properties of the DHA enriched milk were investigated and
compared with regular bovine milk (control mil k).
3.2 MATERIALS AND METHODS
3.2.1 DHA Enriched Bovine Mil k and Regular Bovine Mil k
DHA enriched bovine milk (DHA milk) was obtained by feeding six Holstein
cows at Elorü Düiry Cattle Reseürch Center (University of Guelph). Elora. Ontario with ü
combination of basal meal and fish meal which was added at the level of 4.5% (diet
developed by Wright and McBride. see section 2.4.5) of dry matter intake (DMI)as RUP
supplement. DHA level in milk was incrrased to 0.4% of the total fatty acids. The
efficiency for transferring DHA from the treatment diet to milk was about 34.4%..
Formulation of the fish merl supplement is shown in Table 3.1 (Wright et al., 1998).
Control milk was obtained from the sarne reseiuch center. DHA milk and control
milk were picked up ai the same time twice or three times a week from March to M y for
physicd and chernical analysis.
TABLE 3.1. Formulation of fish meal supplement
of treatment feed (Wright et ai., 1998)
Herring meal
60%
Feather meül
15%
Wheat
25%
3.2.2 Chernical Properties of DHA Milk
3.2.2.1 Fat, Protein, Lactose and Somatic CeII Count
DHA milk and control milk were picked up by using sünitized 30 L stainless steel
milk cans. Milk wüs pooled respectively in cheese vais and agitated gently for 15
minutes. Sümples of DHA milk and control milk were taken immediately after mixing,
stored in the dark iit 4 ' and
~ tested w ithin 3 hours üfter pick up for fat. protein and
lactose concentration by infrared analysis (Foss System 4000, Hillerod, Dentnark) at
Laboratory Services Division. University of Guelph. Somatic ce11 count was meüsured by
the build-in ceIl counter (Fossomatic System 440).
3.2.2.2 Milk Protein Distribution by SDS-PAGE
Electrophoresis refen to the movement of charged colloidal particles and
macromolecular ions under the influence of ÿn electric field. Their mobility is related to
iheir molecular characteristics. Thus components of a mixture can be separated and
quantified based on the differences in their migration velocities. In gel electrophoresis.
macromolecules are forced by an electric field through a gel with specific porosity.
Polyacrylamide gel electrophoresis (PAGE) is widely used in protein ünalysis becüuse
the pore size of the gel cm be made of molecular size and varied by the ratios of the
chemicals used (Pomeranz and Meloan. 1987). Sodium dodecyl sulfonate (SDS) used in
PAGE is both a potent protein denaturant and solubilizing agent. It neutralizes the
charges of the protein molecules and converts them to a rodlike shape whose lengths vary
with the molecular weight. In SDS-PAGE.sepüration is üchieved on the basis of
moleculÿr weight.
Protein distributions in DHA milk and control milk were deterrnined by SDS-
PAGE electrophoresis. a-lactalbumin. P-lüctoglobulin. u-crsein. p-casein. as 1-casein.
askasein. blood serum albumin and immunoglobulins were quantified.
S m ~ p l ePrepnration
Milk was skimmed by a disk cream separator at 5 0 ' ~ Skimmed
.
milk was
collected. pooled. sampled and stored at - 2 5 ' ~ until use. 75 pl of skimmed milk sample
was measured and mixed with 75 pl of MilliQ waier, 250 pl of 20% SDS solution, 100 pl
of 2-mercaptoethanel and 100 p1 of 5% bromophenol blue solution. The sample mixture
was heated ai 9 5 ' ~for 5 minutes with simultaneous vigorous shaking to ensure complete
protein denaturation.
SDS-PAGE
1 pl of sample prepared as above was loaded ont0 a 20% polyacrylamide gel
( P h m a c i a Biotech) and run in a ~ h a s t s ~ s t esystem
m ~ ~ (Pharmacia LKB-PhüstSystem.
Quebec. Canada) at 1 5 ' ~ .The end point was 101 Avh. The gel was then stained with
Coomassie Blue staining solution (30% methanol. 10% acetic acid. O. 1% phüst-gel blue-
R) and then destüined with 10% acetic acid and 301 methanol. A glycerol solution wüs
used in the last step of the developrnent procedure to preserve the gel from drying out.
Scann ing
The gels were dried and then scanned in a gel scanner (UltraScan XL; Pharmücia
Biotech). The intensity of color and the area of individual protein bands were integrated
and calculated as raw data. By comparing the raw data of samples with the raw data of
standards (skim milk powder) of known composition. concentrütions of individual
proteins of the sümplcs could be quantified.
3.2.2.3 Fatty Acid Composition
Fütty acid composition of milk samples was determined by GLC (Varian 3800 gas
chromütograph. Pa10 Alto. CA) with a 30 m DB-23 capillary colurnn (0.32 mm intemül
diameter) at Research Park. University of Guelph.
3.2,2,4 Minerals
Milk sarnples were tested for total nitrogen, P. Ca. K. Mg. Na ai Analytical
Services Laboratory. Department of Land Resource Science, University of Guelph.
Approximately 0.250g of sarnple was weighed into a 250 ml phosphoric acid
tlask. Then 5 ml of concentrated sulfuric acid was üdded. The flask was placed on a
hotplüte at high temperature in a fume hood for 1 hour. then removed from heat and
allowed to cool slightly. 4-6 drops of 30% hydrogen peroxide were added. The flask was
tnnsferred back to the hotplate for üpproxirnütely another 10 minutes. The addition of
H 2 0and short heütingkooling penods were repeated until the solution became clear and
colorless. The concentration of P in the digestion solution wüs measured by Technicon
Auto Analyzer. Concentrations of Ca, Mg and K were measured by atomic absorption
spectrophotometer.
3.2.3 Physical Properties of DHA Milk
3.2.3.1 Density
Density of raw rnilk samples w u deterrnined ai 4 ' ~by a DMA 45 Digital Density
Meter (Anton Püar K.G.,Austria) üt Laboratory Service Division, University of Guelph.
The measuring principle of the instrument is based on the change of the nütural frequency
of a hollow oscillütor when filled with different liquids or gases. The oscillator ce11
mounted in the center of a double walled c ylinder is electronically excited. The frequency
of the oscillator was influenced by the fraction of volume of the liquid sarnple in the
oscillator ce11 and it changes when filled with different liquids. By comparing with the
frequency of the oscillator filled with liquid of known density (such as water). the density
of any liquid sample at that specific temperature could be cdculated from the
corresponding frequency of the oscillator when filled with that liquid sample. Densities
of samples were calculated automatically by the density meter according to the equütion:
where A and B are apparatus constants which are temperüture dependent, T is the period
of thc çÿstcrn.
DHA milk and control milk were sampled right after pick up. Milk sümples were
held in a refrigerator at 4'~and transferred to a @C circulaiing water büth when tested
for density. The sample was injected into the oscillator by using n 5cc syringe. Wnter
from a J'C circulüting water büth was pumped through the spüce between the inner wall
and outer wall of the cylinder. so the sample could reüch its temperature equilibrium
~
out the testing. Readings of density were taken
quickly and be maintained at 4 ' through
directly from the measuremcnts. All meüsurements were done in duplicates.
3.2.3.2 Fat Globule Size Distribution
DHA milk and control milk were sampled right after pick up. Milk samples were
held ai J'C and tested for fat globule size distribution within 3 hours by integrated light
scattering using a Mastersizer X (Mdvern Instruments Inc., Southboro. MA).
Measurements were conducted under room temperature.
Integrated light scattering is a technique widely used in detemûnation of particle
size in micelle and microemulsion systems. The minimum length scale or particle size
that can be seen by an integrated light scattering expenrnent is determined by the
wavelength of the incident radiation and the maximum scattering angle. For helium-neon
incident light scattering from particles in an aqueous environment. the minimum diameter
is about 50nm.In general. integrated light scattering is good for measuring large
particles.
Milk sample was added gradually into the circulating sample systern and diluted
with MilliQ water to an optimum concentration indicated by the instrument. The
scattered light is detected at 3 1 angels and analyzed by an inverse fourier transfomütion
by the instrument. A histogram of the size distribution of the ht globules and the specific
surface area is calculated by moment analysis. The software provided with the instrument
gave out cumulative size distribution curves that showed the volumc percent of particles
in each size interval.
-
3.2.3.3 Dynamic Light Scattering Photon Correlation Spectroscopy (PCS)
Buckgruund of 1)vnrirnic Light Scattering
Dynarnic iight scattering is now a widely used technique for measuring particle
sire and particle size distribution in a suspension. Dynamic light scattering must be used
on a sufficiently diluted systern in which only single scattering tükes place. Thus its
application in foods has been generally restricted to milk and simple emulsion systems
because they c m easily be diluted without disrupting their natural textures (Hallett,
1994). The technique is based on the scattering of light by diffusing particles. Milk is a
liquid that contains colloidal particles that scatter light well. Dynamic light scattering has
been extensively applied in measuring the distributions of casein micelles.
The incident light. 632.8nm. was produced by a helium neon laser. The light
scattered by the sample particles was detected by a photonmultiplier tube (Tosh. 1994).
The relative phase of scattered wavelets differed due to the differing incident phases
which they experienced and due to differeni particle-detector distances (Hüllet. 1994).
The differences resulted in a variation in the number of photons collected by the
photonmultiplier at any given time and were measured by the üutocorrelütor in small
segments called sample t i mes. The average h ydrodynamic diameter of the particle wüs
derived from the autocorrelütor analysis.
Scrmple Preparation anJ Mrusuremenl
Milk was heüted to 60'~and sepiiraicd by a disk creüm sepiiraior into crcam and
skim milk. Skim milk was collected and pooled in a 2OL stainless steel cm. Samples
were tüken into 2Oml amber vials and held üt - 2 5 ' ~ until used.
Samples of skim milk were thawed at roorn temperature and filtered through
Whatman 934-AH glass fiber filter püper to remove residual fat globules. Approximately
3 ml of calcium buffer (composition shown in Table 3.2) wûs filtered through ii 0.22 l m
filter (Milipore Ltd., Mississauga. Ontario. Canada) and transferred into a cuvett. 1 ml of
prepared milk sample was added and mixcd well into the buffer solution, and tested for
average particle size by photon correlation spectroscopy. The diameters of casein
micelles were measured with a set of 12 individual PCS mns, each lasting 20
microseconds and the results were avenged. The scattering angel was set at 90O. samplr
time was 20 microseconds. and the temperature of the samples was controlled at 2 5 ' ~
with a circulating water bath. Al1 rneasurements were done in duplicate.
TABLE 3.2 Composition and pH of the calcium buffer
Calcium chloride
5mM
Sodium chloride
5OrnM
Imidüzole
30mM
PH
7 .O
3.2.4 Sensory Evaluation
DHA milk and control milk were picked up from Elora Dairy Cattle Research
Center (University of Guelph). Elorü, Ontario. and processed on the same day.
Milk was pooled and sampled right üfter pick up. Control milk was heüted to
5 0 ' ~and sepmted into cream and skim milk using a disk c r e m sepürator. Cream and
skim milk were collected, pooled and sampled respectively. Sümples of DHA milk.
control milk. control cream and control skim milk were tested for fat. protein and Iüciose
concentration within 3 houn after pick up by infrared anülysis (Foss System 4000.
Hillerod, Denmark) üt Laboratory Services Division. University of Guelph. Büsed on the
analytical results. control milk was standardized to the same fat concentration as that of
DHA milk by adding calculated volume of crearn back to the skim milk. Standürdizrtion
of control milk ensured the fat contents of control milk and DHA milk used in the
following sensory evaluation were the same. The fat concentration was about 2.43kg/hl
and the protein concentration was about 3.30 kg/hl.
DHA milk and standardized control milk were pasteurized at 8 0 ' ~for 2 minutes
through a UHT system (NO-BAC@UNITHERM IV") at #2 Pilot Plant. Guelph Food
Technology Center. Hot water at 8 0 ' ~and sanitizer were nin through the UHT system
consequentl y between the pasteurization of siandardized control mil k and DHA milk.
Püsteurized milk was bottled and stored in the dark at 4 ' ~ .
3.2.4.1 Descriptive Test
Descriptive tests were conducted by three expert graders on day 1. düy 4. düy 7.
düy 12. düy 15 and dûy 21 after processing. Al1 the samples were coded with randomly
selected 3 digit numbers. The graders were asked to assign a grade to each sümple with 1
k i n g the lowest quulity and 5 k i n g the highest quülity. Three replicates were conducted.
A copy of the questionnaire sheet for the descriptive test of milk is attached as
Appendix 3.1.
3.2.4.2 Dlfference Test
Triangle tests were used to determine if there wüs üny signiticant difference
between DHA milk and control milk. Triangle tests were conducted on day 1 after
pasteurization by an untrained consumer panel composed of graduate students, faculty
and staff of University of Guelph. Al1 the panelists received three samples c d e d with 3
digit random numben. Among the three samples, two were the same and the other was
different. The panelists were asked to pick up the odd sample. They were asked to guess,
if they could not tell any difference. Three replicates were conducted. A copy of the
questionnaire sheet for this triangle test of milk is attached as Appendix 3.2.
33.5 Statistical Analysis
Two-tailed t-tests were adopted in al1 the data analysis that compared the
differences between the means throughout this research. Significant differences were
declared at P < 0.05.
3.3 RESULTS AND DISCUSSIONS
3e3.1 Chemical Properties of DHA Milk and Control Milk
When feeding dniry cows with fish meül supplements. rnilk hi concentrÿtion wiis
changed significmtly while other components in milk remainrd relativcly constant.
3.3.1.1 Fat, Protein, Lactose and Somatic Cell Count
Fat. protein and lactose concentrations. and somütic cell count of DHA milk and
control milk are shown in Table 3.3. Bulk Ontario milk contins 5.03 kg/hl of lactose,
3.33 kg/hf of protein and 3.95 kglhl of fat. Comparing with the published data, the protein
concentrations of both control milk and DHA milk, lactose concentrations of control milk
and DHA milk. and fat concentration of control milk. were al1 within normal ranges.
Fat concentration of DHA milk wüs out of normal range for Holstein rnilk which is 2.8 5.0 g/kg (Walstra & Jenness. 1984). It was significüntly lower than control milk. due to
tïsh mral supplement in the diet. It was observed ihat unprotected fish-oils when fed to
dairy cows decreased milk fat content (Sutton, 1989. Cant et al., 1997). Milk fat
percentage was decreased and plasma long-chain omega-3 polyunsaturated fatty acids
were increased when feeding dairy cows with fish meal supplements (Spain and Polan,
1995). It was reported that the inclusion of polyunsaturated fatty acids in diets of
lactating cows decreased the molar percentage of ruminal acetate and milk fat percentüge
( V m a n et cil., 1968).The change was not associated with changes in patterns of ruminai
volatile fatty acids (VFA) (Spain and Polan. 1995). This suggests that the bypass of
polyunsaturated fatty acids may have ültered postruminal lipid metabolism (Varman et
al.. 1968).
TABLE 3.3 Fat, Protein and lactose concentration and sornsitic ceIl count of control milk
and DHA milk
Protein (kg/hl)
Lactose (kghl) SCC ( l 03/ml)
Minimum value 3.19
3.05
4.40
181
Maximum value 4.48
3.49
4.84
3 14
1 .g2
3.10
4.03
190
Maximum value 3.29
3.9 1
4.69
305
Fat (kghl)
Control milk
DHA enriched milk
Minimum value
', means values with different letten in the sarne column are significantly different at
the level P<O.O5
15 replicates
Somatic cell counts is a routine milk qudity test. Somatic cells are a mixture of
epitheliai cells (2%) and leukocytes (98%).High counts are associated with reduced milk
yields and increased incidence of mastitis (Jensen et al.. 1995).They bring several
enzymes into milk (Jensen et al.. 1995).The proteüses in leukocytes could cause
proteolysis during cheese ripening if not inactivated during processing. The origin of
these proteases is from somatic cells and blood plasma. The most significant proterse
from blood plasma is plasmin which is heat stable and may cause gelation and spoilage of
UHT milk (deKoning et al.. 1985). Individual variation in SCC can be fairly large. It also
increases with time in lactation. Bovine milk normally contains about IO' to 10' cellslml
(Lipkin et al.. 1993: Walstra & Jenness. 1984).The average SCC of bulk Ontario milk
from 1996 - 1997 was 258 x 103/ml(Dairy Fmners of Ontario statistical handbook).
Somatic ceIl counts of both DHA milk and control milk were within normal range.
The data supports the conclusion that feeding fish meal supplements to dairy cows
üt
the level of 4.5% of DM1 lowered the fat content in milk by about 4 0 8 but did not
affect protrin and lactose concentration and somatic ceIl counts in milk.
3.3.1.2 SDS- PAGE
ai-casein. ~t,~-casein,
p-casein. K-casein.a-lactalbumin. P-lactoglubulin, blood
serum albumin and immunoglobulins in DHA milk and control milk were identified
quantitatively by SDS-PAGE. Results are shown in Figure 3.1. A typical SDS-PAGEof
skimmed control and skirnmed DHA milk is shown in Figure 3.2.
There was no significant difference in K-caseinconcentration of DHA milk and
control milk. But oqi-caesin and p-casein concentrations in DHA milk were lower than
that in control milk. The total casein number of DHA milk was also lower than that of
control milk. The differences rnay have negative effects on milk coagulating properties.
such as coagulation time and curd fimness, and cheese yield. The coagulating properties
of DHA milk and control milk will be discussed later in Chapter 4.
3.3.1.3 Fatty Acid Profiles of DHA and Control Milk by GLC
The fatty acid profiles of DHA and control milk are shown in Table 3.4. DHA
content in treatment milk was signitïcantly increased to about 0.4% of total fatty acids. Its
level in control milk was much lower. which was only about 0.03% of total fütty acids.
C l6:O was decreased. while C 18: 1, C l 8 : 3 d , C l8:4& were al1 increased in DHA milk.
3.3.1.4 Minerals
Several minerals and their concentrations in DHA milk and control milk are
prescnted in Table 3.5. No significmt difference between control milk and DHA milk
wüs observed regarding P, Ca, K and Mg concentration. P. K and Mg concentrations of
control and DHA milk were al1 within normal range cornparhg with published data
(Walstra & Jenness. 1984), while their Ca concentrations were higher thün 0.1 15-0.125
glkg, the value reponed in the litenture (Walstra & Jenness. 1984). Also when
comparing with 0.035-0.050 glkg, the published data of sodium concentration in fresh
whole milk (Walstn & Jenness. 1984), both control and DHA milk in this experiment
had higher sodium concentration. This was due to higher sodium level in the feed and
could be easily brought down by changing the sodium concentration in the diet.
FIGURE 3.1 Protein distribution of control and DHA milk as estimated by PAGE
Serum Albumin
Immunoglubulins
Lane 1 . Standard (skimmed milk powder);
Lane 2. Control milk;
Liine 3. Control milk (duplicate);
Lane 4. DHA enriched milk;
Lane 5 . DHA enriched rnilk (duplicate);
Lane 6. Standard (skimmed milk powder).
FIGURE 3.2 Typical SDS-PAGE of skimmed control milk and skimmed DHA milk
TABLE 3.4 Fatty acid profile of DHA and control milk (% of fat)
C8:O
C24: 1
a, b,
Control
1.1 l a
DHA
O
O
1.10"
......x, y, z, means values with different letters in the same row are significantly
different at the level PcO.05
TABLE 3.5 Minerals in milk
Control milk
DHA milk
P
O. 1 1 I0.00"
0.10
Ca
0.16 f 0.00~
0.15 f 0.02~
K
O. 12 1 O.0dC
0.13 IO.OSC
Mg
0.0096 & 0.00~
0.0097 f 0.00~
Na
0.052
i
+ 0.01'
+ 0.01"
0.073 k 0.0 1'
. . , ' means values with different letters in the same row are significantly different
l
b,
c
d,
c
at the level P<0.05
7 replicates
3.3.2 Physical Properties
3.3.2.1 Deasity
The density of control milk was 1.0326~lcm'and 1.O323 &m.' for DHA milk.
-
Both were in normal range of 1 .O2 1 1.O37 gkm3.There was no significant difference
between convoi milk and DHA milk regarding density at 4 ' ~ .Density of milk depends
on its composition. So it can be concluded that the decrease in fat concentration in DHA
milk was not large enough to change the milk density at 4 ' ~ .
3.3.2.2 Diameter Distribution o f Fat Globules
Diameter distribution of fat globules is shown in Table 3.6 and Figure 3.3.
Milk fat exists in the form of an emulsion of spherical globules dispersed in the
milk plasma. Diameters of the milk fat globules range from O. 1 to 22 pm (Jenness and
TABLE 3.6 Diameters of fat globules in DHA milk and control milk
DHA milk (pm)
Control milk (pm)
Meana
1.789 i: 0.056~
2.3 1 8 st 0.007C
Minimum value
1.660
1.920
Maximum value
1.930
2.860
a
, average of
b,c;
10 measurernents, each measurernent was perfomed in duplicate
means values with different letters in the same row are significantly different at the
level Pc0.05
Patton. 1976). The distribution of fat globule size is a breed characteristic. Cows used in
this experiment were al1 Holstein, so the variation in fat globule size distribution caused
by the breed should not be significant. From Figure 3.3, it can be seen that fat globule
particles in DHA milk had smaller diarneter and were more uniform in s i x distribution.
DHA milk had a larger portion of small fat globules with a diameter around 1 pm and a
smaller portion of large fat globules when comparing with control milk. It suggests DHA
milk could be more stable than control milk during storage but more difficult to cream
and churn. More details will be discussed in Chapter 5.
In order to understand better the correlation of fat globule size distribution and
milk fat content, more Holstein milk samples of different milk fat content from different
dairy f m s in Ontario were tested for fat globule size distributions. (Al1 the milk samples
were provided by the Milk Testing Center in Laboratory Services Division at University
of Guelph). The results are shown in Figure 3.4. For milk sarnples of fat contents fiom 3
to 4%, their average fat globule sizes faIl in the range fiom 2 to 2.5 m.For milk samples
. . . - . . . Cont ml
FIGURE 3.3 Fat globule size distribution of control and DHA milk
Data were the mean of I O measurements. The average diameter for DHA
milk fat globules was 1 . 7 8 9 and
~ 2.3 18pm for control milk fat globules.
Fat Content of Hoktein Milk (Oh)
FIGURE 3.4 Fat globule size distribution of Holstein milk samples from Ontario
Each data point was an average of duplicate measurements.
of fat contents higher than 4%, the higher the fat content is, the larger the average fat
globule size is. In this experiment, no other sarnples with a fat content lower than 2.7%.
except DHA enriched milk, was obtained. The average fat globule size of DHA enriched
milk was 1.789 nrn. So it can br assumed that milk lower in fat will most likely have a
smaller average fat globule size.
3.3.2.2 Particle Size Distribution o f Casein Micelles by Photon Correlation
Spectroscopy (PCS)
Photon correlation spectroscopy was used to detemine if fish meal supplement
had any effect on the size distribution of casein micelles in milk. Raw data is shown in
Table 3.7.
About 80% of milk protein is casein which is defined as proteins precipitated
t'rom skim milk by acidification to pH 4.6 to 4.7. Casein exists in milk in the fonn of
large colloidal particles containing protein and salts. The average diameters of casein
micelles in bovine milk are about 120-180 nm (Farrell et al., 1990). but if they exist in a
continuous gradation of sizes or in a series of discrete sizes is still not clear.
In this experiment, the average diameters of casein micelles were 187.51nm for
control milk and 170.78 nrn for DHA milk. It can be seen fiom the results that feeding
dairy cows with fish meal as RUP supplements may have an effect on the casein micelle
size distribution even though the total protein concentration in milk was not changed.
Coagulation properties of DHA milk proteins were studied and will be discussed in
Chapter 4. Whether the changes in casein micelle size distribution will affect other
functional properties of milk proteins needs huther study.
TABLE 3.7 Diameters of casein micelles in DHA milk and control milk
DHA milk
Control milk
176.59 nm
178.38 nm
171.15 nm
185.60 nm
166.55 nm
193.31 nm
166.02 nrn
192.74 nrn
Each value was an average of 12 readings.
3.3.3 Sensory Evaluation
Results fiom the descriptive tests of DHA and control milk are shown in Table
TABLE 3.8. Scores of descriptive tests of control and DHA milk
Cor,trol
Treatment
Day 1
3.50
3.38
Day 4
3.42
3.70
Day 7
3.60
3 $60
Day 12
3 .O
3.3
Day 15
2.63
3
In descriptive tests, no significant difference was found between control milk and
DHA milk regarding flavor. No obvious oxidized flavor was observed in both control
milk or DHA milk before day 15 after pasteurization. Similady, consumer difference
tests showed no significant difference between DHA milk and control milk as drinking
milk. Of 23 panelists in the consumer difference tests, 8 picked up the odd samples
correctly. According to the statistical table for triangle test, for a difference analysis by a
panel composed of 23 panelists, number of correct answen necessary to establish level of
significance at P~0.05is 17. So the difference between control milk and DHA milk was
not significant. It can be concluded that changes in the feed did not affect the quality and
flavor of DHA milk as drinking milk. Incorporating DHA into milk through fish
supplement diet at the level used in this experiment (4.5% of DMI)did not bnng any
fishy or other undesirable flavor. In particular, there was no evidence to suggest that
treatment milk was more susceptible to development of oxidized flavor.
It is possible that the level of fish meal supplement, used in this experiment. was
ioo low to bring any undesirable flavor to the DHA milk as drinking milk. but with
increased fish meal supplement intake, fishy flavor may be observed. Because
undesirable flavors were reported with food products of high level of long-chain
polyunsaturated fatty acids, M e r experiments should be carried out to test the effects O t'
increased fish meal supplement at different levels of DM1 on the quality of DHA milk.
Sensory properties of different kinds of omega-3 fatty acids enriched food
products have been widely investigated recently. It was found that omega-3 fatty acid
enriched eggs had obvious fishy flavor when served scnunbled, but did not have any
unacceptable flavor when boiled and served at room temperature. This may suggest that
no fishy flavor was found in the DHA milk in this experiment, because the sensory
evaluatioa was only conducted on drinking milk served cold at 1 5 ' ~ .The flavor
attributes of DHA milk versus temperature should also be studied.
CHAPTER 4 CHEESE MAKING PROPERTIES OF DHA ENRiCHED MILK
4.1 INTRODUCTlON
As discussed in Chapter 3, fish meal supplement resulted in smaller casein
micelles. smaller fat globule and lower fat concentration. These changes could affect the
functional properties of milk proteins and thus affect the processing properties of DHA
milk. The objectives of this chapter were to study the cheese making properties of DHA
e ~ c h e milk
d and the coagulating properties of proteins of DHA enriched milk.
4.2 MATERIALS AND METHODS
4.2.1 Coagulation o f Milk
The three major components of the casein protein complex are %i -casein. Pcasein and K-casein which are distributed as a,i-casein, 50%; D-casein, 3 3%; and ICcasein, 15% (Farrell and Thompson, 1974). a,,-casein is insoluble under normal
conditions of pH, ionic strength and temperature. p-casein is insoluble at room
temperature in the presence of C d + at concentrations which are lower than that in milk.
K-casein is soluble over a very broad range of ca2+concentrations. So milk as a colloidal
liquid complex, can be considered as a mixture of calcium-insoluble proteins stabilized
by a calcium-soluble protein (Farrell and Thompson, 1974).
Coagulation of the milk system is initiated when casein micelles are destabilized
by enzymatic cleavage of K-casein. Stability of casein micelles are affected by
acidification towards the isolectic region of caseins or charges in ionic and salt
concentrations.
Acid coagulation (with yogurt culture and cheese culture) and remet coagulation
were done with both skimmed control milk and skimrned DHA milk to compare the
coagulation properties of their milk proteins.
4.2.1.1 Acid Coagulation of Milk (Yogurt Culture)
Sample Preparat ion
DHA enriched milk and control milk were picked up at the same time from Elora
Dairy Cattle Research Center (University of Guelph), Elora, Ontario. Milk was warmed
to 60'~and then separated to crearn and skim milk by a disk cream separator. Skim milk
was collected, pooled and sampled. Skim milk samples were stored in a cold storage at
4 ' and
~ used in acid coagulation test within 3 days.
Acid Coagulation Test
Skim milk samples were preheated to 3 7 ' ~in a water bath, then inoculated with
0.00035% fieeze-dried yogun culture (YC 380, streptococcus therrnophilus and
lactobacillus bulgaricus at the ratio of 1 :1) at the same level as used in yogurt making and
mixed thoroughly. The mixture was transfened into two 400 ml beakers and then
incubated in the sarne water bath at 37'~.
One of the samples was used for monitoring changes in pH by emersing the
e1ectro.deof a pH meter (Model) into the sample. The pH mrter was c o ~ e c t e dto a
personal computer which collected data every minute and stored them on a floppy disk.
At the end of the experiment, al1 the data collected during the expriment were
compressed to ratio 1 :15 by provided software and converted to corresponding pH
values.
The other sample was used for monitoring changes in curd consistancy during
acid coagulation with a Nametre vibrational sphere viscorneter (Nameter CO..Edison,
N.J.). Vibrational viscometea are surface loaded systems because they respond to a thin
layer of fluid at the surface of the sensor. The sensor is driven at a fixed frequency and
the power required to maintain a precise amplitude is measured (Steffe, 1996). The
vibrating sphere sensor was connected to a personal computer. Data was collected every
5 minutes and saved on floppy disks.
4.2.1.2 Rennet Coagulation of Milk
Rennet coagulation of milk occurs in two sequential stages. In the primary
proteolytic step, k-casein is hydrolysed by calf chymosin to yield para-k-casein micelles;
in the secondary step, paracasein micelles undergo limited aggregaiion at temperature >
2 0 ' ~to form a gel. When about 86% of the total uîasein has been hydrolyzed, the
paracasein micelles begin to aggregate as a result of intermicellar cross-linking via
calcium binding to serine-phosphate groups (Guinee & Wilkinson, 1992). In up to 60%
of visual remet coagulation time (RCT) the micelles are dispersed and individual as in
unrenneted milk. Thereafier the paracasein micelles begin to aggregate gradually, initially
to fonn aggregates of 3-4 at 1 0 % of the RCT.At 200% of the visually obsewed RCT,
strands are formed by the fusion of aggregates. Evennially, at 300% of the visual RCT,a
continuous three dimensional network is formed via the overlapping and cross-lining of
strands.
Sumple Preparation
Skim DHA milk and skim control milk were preheated to 4 2 ' ~in a water bath.
and then inoculated with single strength remet (CHRHANSEN, FPC,Lot# 20067-
2 1630, Mississauga, Ontario), mixed thoroughly and incubated in the water bath at 4 2 ' ~
for 3 hours. Changes in consistency of the sarnple were monitored by a Narnetre
vibrational sphere viscometer. Data was collected every 5 minutes and stored on floppy
disks.
4.2.2 Cheese Making Properties
4.2.2.1 Cbeese Making
Cheese making was a good way to study the real life coagulating properties of
milk protein. Cheddar cheese was chosen in this expriment because long term curing is
required which made it possible to study the stability of DHA and its effects on cheese
flavor development as a function of storage time. A flow diagram of Cheddar cheese
making is presented in Figure 4.1.
DHA milk and control milk were picked up from Elora Dairy Cattle Research
Center (University of Guelph), Elora, Ontario. Control milk was warmed to 6 0 ' ~and
then separated into cream and skim milk by a disk cream separator. DHA milk, control
milk, skim control milk and control cream were sarnpled and tested for fat and protein
concentrations by i n h e d analysis (Foss System 4000, Hillerod, Denmark) at the
Laboratory Services Division, University of Guelph. Based on the analytical results, skim
control miik was standardized to the sarne fat concentration as that of DHA milk by
adding calculated amount of control crearn. DHA milk and standardized control milk
were used in Cheddar cheese making.
DHA enricheci cheese and control cheese were made at the same time in either a
double O vat or a square vat in the #2 Dairy Pilot Plant at Guelph Food Technology
Center. Milk was pasteurized in the cheese vat at 6 3 ' ~for 30 minutes and then cooled to
3 I'C. Starter culture (Superstar@ concentrated cultures@, Madison, WI, USA) was
added to the milk at 3 1OC at the level of 0.1 587% (vtw). Gentle agitation was applied to
ensure thorough mixing. AAer pH decreased by 0.05 units or titritable acidity increased
by 0.0 1%, single strength remet (CHRHANSEN, FPC Lot# 20067-21630, Mississauga.
Ontario) was added to the milk with gentle agitation at the level of O.OOOl9% (dm).
Agitation was then stopped. The curd was allowed to set. When the curd became firm. it
was cut into to cubes with 318 inches knives. Cooking was started afier 15 minutes of
cutting. Temperature was brought up slow1y in the first 1 5 minutes with no more than
I'C increase every 5 minutes in the first 15 minutes and then cooked slowly to 3 9 ' ~ .
Curd was kept at 39'~ with agitation until whey pH reached 6.4, then the whey was
drained. Curd was piled 13-1Scm deep along the sides of the vats and rllowed to mat.
Then it was cut into blocks about 25cm wide and turned every 15 minutes until pH
reached 5.5. Curd was milled into strips by a mechanical curd mil1 and then salted with
2.5% (of estimated yield) of table salt 15 minutes after milling. Salted cheese curd was
filled into 20 lbs hoops lined inside with plastic single use press cloth. Hoops were
pressed overnight. Cheese were weighed next moming and vacuum packed in polyethene
nylon bags. Al1 the cheese samples were stored in a cold storage at 4 ' during
~
ripening.
This experiment adopted low fat Cheddar cheese making procedure in which
whey pH at draining stage was 6.4 instead of 6.2 and curd pH at milling stage was 5.5
instead of 5.4-5.3. Higher pH at both draining stage and milling stage retains more
moisture in the final product, to help reduce the typical rubbery and corky texture of low
fat Cheddar cheese. The target cheese moisture content was 40% in this experiment.
4.2.2.2 Cheese Y ield
Expected cheese yield were calculated by Van Slyke formula:
Where EY = expected yield
F = fat content of milk (w/w)
P = protein content of milk (wlw)
M = moisture fraction
Y ield was calculated as:
DHA enriched mil&
Control milk
I
l
Standardize to proteidfat ratio of DHA milk
l
Pasteurize ( 6 3 ~ " 30
. minutes)
I
Cool to 3 1 C"
i
Add starter culture
1
Ripen till pH drops by 0.05 units
I
Add rennçt
I
Sei till curd is
tlrni
I
Cut (318 inch kinives)
I
Hroi up io 39C" slowly in 30 minutes with grnile stirring
I
Hold at 3 9 ~ until
" pH reaches 6.4
I
Drain whey
I
Pile the curd and allow to mat
I
CUIthe curd inro 25cm wide biocks
1
Turn ai 15 minutes intervals uniil pH rcriches 5.50
t
Mill
I
Add salt (2.5%)
I
Fill hoops
1
Press overnight
I
Vacuum pack
I
Store ût JC"
Figure 4.1 Flow diagram of Cheddar cheese making
Where: Y = yield
W 1 = the weight of cheese
W2 = the weight of milk
Cheese yield efficiency was calculated as:
Where: YE = yield eflciency
Y = yield
EY = expected yield
4.2.2.3 Composition Analysis of Cheese
Fur
Babcock method was used to determine fat content in cheese. Nine gram of
finely ground cheese was accurately weighed into a 50% Babcock bottle. First 10 ml of
distilled water was added at 60'~and then 17.6 ml of sulphuric acid was added in at least
thee increments. The bottle was swirled until al1 cheese particles were well dissolved.
The mixture was centrifuged for 5 minutes at 2000rpm/min, then distilled water was
added at 6 0 ' ~to bnng contents to within one-quarter inch of the base of the neck. The
mixture was centrifuged for another 2 minutes at 2000rpm/rnin and more distilled water
was added at 6 0 ' ~to float the fat into the neck of the bonle. Mixing was avoided from
this point. The mixture was centrifuged for another I minute at 2000rpm/min, then the
bottle was tempered in a 5 5 ' ~water bath for 5 minutes. Four to five drops of glymol
were added and then the length of the fat colurnn was read fiom the demarcation between
fat and glymol to the bottom of the lower meniscus. Percentage of fat was reportea.
Crude Protein
Cnide protein concentrations of cheese were measured by Buchi Kjeldahl
nitrogen analyzing system. Two gram of finely ground cheese were accurately weighed
ont0 filter paper prefolded into an envelop. Sarnples together with the filter paper were
then put into digestion tubes. 25 ml of sulfunc acid and two selenium tablets were added
aAer. Samples were then digested in a &unit digester until 30 minutes afier the samples
had turned completely clear which was about 2 to 3 hours in total. Afler digestion was
complete, the heater was turned off and the samples were left to cool to room
temperature.
Distillation was carried out in a Buchi distillation system. 100 ml of distilled
water was added to the sample tube to dilute the sarnple, followed by 100 ml of sodium
hydroxide solution (30% w/w). The receiving flask contained 100 ml of 2% boric acid
solution and 10 drops of bromocresol-green methyl red indicator. AAer 7 minutes of
distillation, the content of the receiving flasks were titrated with 0.1 N sulfuric acid to the
pink end point.
Salt
Salt in cheese was determined by QUANTAB" Chloride Tirators which were
convenient test devices used to measure the chloride content in aqueous solutions or
dilute aqueous extntcts of solids.
10 gram of finely ground cheese sample were accurately weighed and placed in a
250 ml beaker. Ninety miles of boiling water was added. The mixture was stirred
vigorously for 30 seconds, allowed to set for 1 minute, then stined for another 30 seconds
and allowed to cool to room temperature.
Filter paper (Whatman #1) was folded into a cone-shaped cup and placed into the
sample to allow several ml of filtrate to enter the bottom of the cone. QUANTAB
Chloride Titrator was inserted in the cone to allow the sample fluid to completely saturate
the strip. The height of the white column in Quantab units was read and converted to
corresponding percent sodium chloride with the calibration table.
Moisture
Moisture was measured by vacuum oven method. Flat-bottorn metal dishes with
tight-fit. slip-in cover were pre-dried. cooled in a desiccator and weighed. Approximately
4 grams of tinely ground cheese samples were carefully weighed into the pre-treated
metal dishes. Loosely covered dishes were then put into the vacuum oven immediately
and drkd to constant weight at 1OO'C under pressure not in excess of 100 mm Hg. The
vacuum pump was stopped and air was admitted into the oven carefully. Covers were
tightly pressed into dishes. Dishes were immediately moved out of the oven, allowed to
cool to room temperature in a desiccator and weighed.
Ash
Crucibles were pre-ckied, cooled in a desiccator and weighed. Approximately 5
gram of finely ground cheese sarnples were weighed into pre-treated crucibles and
placed into the furnace not over 4 2 5 ' ~ .Temperature was slowly increased to 5 5 0 to
~ ~
avoid spattering. AAer the ignition was complete, crucibles were moved out of the
fumace, allowed to cool to room temperature in a desiccator, and weighed.
43.3. Cheese Ripening
Cheddar cheese requires several months of aging during which the typicai flavor
and texture are developed. In addition to lactose fermentation, products of proteolysis and
lipolysis are very important. Proteolysis has k e n generally recognized as an acceptable
indicator of cheese aging with the concentration of free amino acids and amines
correlating significantly with flavor development (Puchades et al., 1989). In this
experiment, changes of pH and free amino acids were monitored during cheese ripening
as indicators of the degree of proteolysis.
5.2.3.1 pH
pH of cheese were taken at day 1 and month 1, month 2, month3 and month 4
after manufacture.
4.2.3.2 Free Amino Acids
The fiee amino acids content in cheese were determined at month 1, month 2,
month 3 and month 4 afler manufacture by 2,4,6, trinitrobenzene sulphonic acid (TNBS)
reaction with fiee amine (Shama, 1988).
Reugents
1.OM trinitrobenzene sulphonic acid
O. 1M NaH2P04containing 1SmM N*S03
Borate buffer pH 9.5 (0.1M N a 2 B 4 0 in
~ O. 1M NaOH)
Approximately 2 grarns of finely ground cheese was weighed into 40 ml borate
buffer solution and warmed in a 4 5 ' ~water bath for 10 minutes. The sample mixture was
homogenized using a Polytron for 1 minute and then centrifuged at 20,000 rpm at 1 0 ' ~
for 30 minutes. The supernatant. 0.5 ml was diluted to 10 ml with distilled wüter. The
ünalysis was ciirried out by mixing 0.5 ni1 of the diluted extract with 0.5 ml of borate
buffer in the cuvette md adding 25 pl of TNBS solution. The mixture was allowed to
stand for 5 minutes in the dark for the reaction to take place. The reaction wüs stopped by
adding 2 ml of NaH2P04solution. Ten second intervals were kept between each smple
when adding TNBS and NaH2P04.The blank was prepared with 0.5 ml of distilled water
in place of samplc solution. The absorbante was measured at 420 nm within 20 minutes
after stopping of the reaction. The glycine standard curve was used for determining the
free amino acids expressed as m moles per gram cheese.
4.2.3.3 Microstructure by Scanning Electmnic Microscopy (SEM)
Changes in cheese microstructure during ripening have been extensively studied
and used as an important indicator of the developments of body and texture (Anderson
and Mistry, 1994. Metzger and Mistry, 1995. and Bryant 1995). In this experiment. SEM
technique was used to monitor the changes in cheese microstructure during ripening.
Sample Preparation
Cheese samples were cut into small pieces, then attacheded to the specimen
holders by using Tissue-Tek O.C.T.
compound (Miles Scientific. Napreville, a).
The
sarnples were fixed cryogenically by plunging the specimen holder into nitrogen slush at 2 1 0 ' ~in a Emscope SPZOOOA cryo-unit (I.E.O. Inc.. Houston. TX). Then samples were
freeze-fractured with a cooled macro knife assembl y on the cold stage of the cryo-unit.
sublimated for 45 minutes at - 8 0 ' ~ .and sputter coated with gold to a thickness of ca.300
A. Sarnples were then transferred into a scanning electron microscope (Model: Hitachi S570, Hitachi. Ltd., Tokyo. Japan). The temperÿtures of the microscope and the sümple
' ~ liquid nitrogen. Experimental
preparütion chamber were kept at - 1 4 5 ~ ~ k l 5with
conditions were held constant for al1 samples. Accelerüting voltage was 10 kilo volt;
working distance wiis 12 mm; tilt was O degree; objective aperture dimeter was 50
micrometer. SEM micrographs were generated by a 100 second image sweep on the
photo-CRT and recorded by a Mamiya 6x7 roll, film holder assembly.
4.2.4 Sensory Evaluation
A difference test (triangle test) was conducted on cheese smples rifter 6 months
ripening by a consumer panel composed of the graduate students. staff and faculty at
Food Science Department of University of Guelph. Each panellist received 3 cheese
samples coded with 3 digit randorn number. Two of the sarnples were the same, the other
one is different. They were asked io pick out the odd one and explain in which aspect it
was different from the others. A sample of the questionnaire sheet is attached as
Appendix 4.1.
4.3 RESULTS AND DISCUSSIONS
4.3.1. Coagulation
4.3.1.1 Acid Coagulation Tests (Yugurt Culture)
Starter Cufiirrt'
Yogun culture. a mixture of Lactobucilliis biclguricus and Streptococciis
tliemophilus at the ratio of 1: 1, was used in this enperiment. Both of them use lactose,
the major carbohydrate in milk. as substrate and convert 95% or more of fermented sugar
into lactic acid while producing some other by-products. Lactose is first hydrolyzed into
glucose and galactose. Glucose is then converted into lactic acid. The üccumulütion of
Iactic acid lowers the pH or increases the acidity of milk, which affects the solubility of
caseins. When the pH of milk is lowered to about 4.6, casein becomes destübilized and a
coagulum is fomed (Helferich and Westhoff, 1980).
Lnciohcillus hulgariciis is a rod-shaped
bacterium with an optimum growing pH
;it 5.5. optimum growing temperature at 4 ' 4 3 ' ~ . minimum 2 2 ' ~and maximum 5 2 . 5 ' ~ .
Proteolytic enzymes of Lactobacillus bulgaricus degrade caseins leading to a relatively
high accumulation of free amino acids and low molecular-weight peptides. But it shows a
weak lipase activity (Rasic and Kurmann, 1978). Streptococcus tliermophilus is a
sphericai-shaped baterium with an optimum growing pH at 6.5. It has an optimum
minimum 2 0 ' ~and maximum 5 0 ' ~ .It is very
temperature for growth at 4°-45U~.
sensitive to inhibitory substances. particularly antibiotics. It has very weak proteolytic
übility, depending on peptides originating from the casein hydrolysis (Rasic and
Kunnann, 1978).
The interaction of Lactobacillus bulgnricus and Streptococcus themoplrilus is
mutualistic. When used together. the coagulation time of milk becomes shoner (Rasic and
Kumann, 1978). When growing in milk. lactobacillus bulgüricus releases ümino acids
frorn cüseins which are required by Strrptococciis thermopliilus. In return. Strrptococcits
rlw-mophiiics grow faster in the early part of the incubütion and outnumber the
Liictob~rcillitsbulgaricus quickly. While growing. Streprococcics synthesize forrnic acid
and increases the acidity. both of which favor the growth of Lactobacillus bulgaricus. In
the latter stage. the growth of streptococci is inhibited by the increased acidity of milk and
L«ctobucillus bulgririciis becomes the dominant living culture. Streptococccts is not as
bufgciricus.Strrptococcus tolerate up to 0.8%lüctic ücid.
acid tolerant as L~~fobucillus
while Luctob~icillicsbitlguricus tolerüte up to 1.7% lactic acid. In short. the Streptococcrts
rlrcmtophiliis contribute to the acid production in the first stage of incubütion, while
L~~tob~icillus
bulgoricus contribute in the second stage.
Rrsiei~so{Acid Coagulntion Tests
Serum proteins are not sensitive to changes in the hydrogen ion concentration in
milk (Rasic and Kumann. 1978). but the solubility of caseins can be greatly affected by
changes in ion concentrations in milk.
During the growth of Iactic acid yogurt culture, the calcium and phosphorus are
gradually removed from caseinate particles which are then transformed to a soluble staie.
When pH drops to 5.2 - 5.3, the caseinate particles are destabilized and precipitation
stms. Complete precipitation will be reached when pH drops to the isoelectric point
which is 4.6 - 4.7. At isoelectric point, caseins are free of bound salis. Some of the
displaced calcium will combine with lactic acid as calcium lactate. The average size of
casein micelles remains very constant in a pH range of 6.6 - 5.3. but increases as pH
drops from 5.3 to 4.6 due to destabilization and aggregation of casein particles.
The consistency of the coagulum is affected by the total solids content in milk.
pwticularly the protein content. heat treatment of rnilk. denüturation of the serum
proteins. homogenization. acidity and sdt concentration in milk. Serum proteins are
relatively stable. Denaturation of serum proteins could be ignored in this experiment
because the skim milk samples were not subject to üny heat treatment before testing md
also the testing temperature. 4 2 ' ~ .was not high enough to induce any denaturation.
Increased protein content and denatuntion of the serum proteins can increase the
tïrmness of the coagulum. Protein and lactose concentrations of control and DHA milk
were about the same. but control milk was higher in casein content. This contributed to a
rimer curd of control milk during the late stage of incubation. At the first stage of
incubation. curd consistencies of control milk and DHA rnilk were about the same. This
wüs because the coügulation only stuted at the late stage of incubation when the pH
dropped close to the isoelectric point of casein proteins.
From the changes in pH profile during acid coagulation of control and DHA rnilk.
it can be seen that the pH of DHA milk dropped faster than that of control milk during the
late stage of incubation. But the curd consistency of DHA milk didn't increase as fast as
that of control milk, this could be explained by the lower casein content in DHA milk,
which is probably associated with lower buffer capacity. In yogun making, the soft curd
can be compensated by high heat treatment to increase the denaturation and water holding
capacity of semm proteins; and by increasing solids content with skim milk powder or
condensed milk.
4.3.1.2 Rennet Coagulation
Rennet coagulation is a two phase process. When about 80-908 of K-casein is
hydroylzed, the clestabilized micelles will aggregate by diffusion. Coagulation time
depends on the rüte of K-caseinhydrolysis and micelle diffusion. In the first stage of
firming. the firming rate rises quickly within 12- 15 minutes after observed coagulation
time and then followed by r decrease. In the second stage. the firming rüte will either
remain the same or increase slightly (Storry and Ford. 1982). At the observed coagulation
time. the amount of soluble casein depends on the original casein concentration in milk.
The soluble casein will contribute to the continuing firming process by either aggregating
with other free casein micelles. or by incorporating into the already fomed coagulum. In
the second stage of firming process. the coagulum reaches its final structure. DHA milk
exhibited a softer curd in rennet coagulation tests as shown in Figure 4.4. This c m be
explained by the lower casein content in DHA milk, or may also be caused by the smaller
size of casein micelles in DHA milk. Coagulation propenies of milk can be affected by
many factors. such as pH of milk samples, temperature. buffer capacities of rnilk proteins
etc. DHA milk was observed to have a slightly higher pH value than control milk.
Whether or not the change was due to the dietary RUP supplement. and whether or not
the buffer capacities of the proteins DHA milk was affected still awaits funher study.
O
50 100 150 200 250 300 350 400 450 500
Tirne (minute)
FIGURE 4.2 pH profile of control milk and DHA milk during acid
coügulütion
O
100
200
300
400
500
T h e (minute)
FIGURE 4.3 Consistency profile of control and DHA milk dunng acid coagulation
O
30
60
90
120
150
180
Coagulation T h e (minute)
FIGURE 4.4 Consistency profile of control and DHA milk during rennet coagulation
4.3.2 Cheese Yield
Control cheese and DHA cheese had the sarne cheese yield efficiency as shown in
Table 4.1. No significant differences were observed in cheese making regarding
coügulating time. or cutting time.
Table 4.1 Yieids oC controi chelsr: and DHA cheestt (rneüns F SEM).
Real Yield (%)
Expecied Y ield (%)
Y ield Efficiency
Control Cheese
9.22 f 0.3 lad
8.89 f 0.52"'
96.4
DHA Cheese
8.75 f 0.39~"
8.26 0.27"
+
+ 3.44'
94.4 f 2.26"
ü,b,c,d.e.f,g = rneûns with different superscripts in the same column are significantly
different (P < 0.05).
The protein concentrations of control milk and DHA rnilk were about the s m e as
shown in chüpter 3, so the protein concentration of standardized control milk was
increased and thus was higher than that of DHA milk after standardization due to the
pürtiül removal of fat. Different cüsein concentrations between control and DHA milk
could iilso lead to different real yields because casein concentrations were found to affect
curd firmness even though clotting time was not significantly affected (McMahon and
Brown, 1984). Softer curd cm cause lower recovery of fine parts of the curd after cutting,
thus aiso results in a lower yield in cheese making. DHA milk exhibited a relatively softer
curd at the time of cutting. which negatively affected the cheese yield. Even though the
real yield of control cheese was slightly different from that of DHA milk. no significant
differences were observed regarding the cheese yield efficiencies. The results indicate that
fish med supplement fed to the dairy cows at the experimental level did not affect the
cheese making properties of milk significantly.
DHA milk was collected from six Holstein cows close to the end of their lactation.
On the contrary, control milk was from pooled milk from al1 the rest lactating Holstein
cows at different lactation stages. Late lactation milk was reported to have poorer milkclotting propenies (longer clotting time and reduced curd firmness) (Gruffeny and Fox.
1988: Skigbo et al.. 1985). Plüsmin activity usually increases toward the end of lactation,
but it does not influence clotting time and curd firmness significlintly (Büstirn et al.,
199 1). Cheese making properties of DHA milk should be studied on a larger scüle in the
funhcr study, with more cows on trial and at different lactation stages.
4.3.3 Cheese Composition
The composition of control cheese and DHA cheese is shown in Table 4.2.
TABLE 4.2 Cheese composition
Fat (%)
Protein (%)
Moisture (%)
Regular cheese
27.93
29.98
38.50
DHA cheese
28.28
29.38
39.28
Bot h Cheddar cheese were reduced fat chcese due to lower fat concentration in
milk. Moisture in both cheese were high due to higher pH at draining. Higher moisture
content was türgeted to compensate for the rubbery, corky texture of reduced fat cheese.
4.3.4 Cheese Ripening
Proteolysis contributes to the development of cheese texture and flavor. During
proteolysis, many chernical and bio-chemical changes occur. Proteins, lipids and residual
lactose in cheese are degraded. Four agents are responsible for the proteolysis in Cheddar
cheese. namely rennet. indigenous milk enzymes. starter culture and nonstarter culture
(Fox. 1989).
Proteolysis is influenced by many factors. such as temperature (Aston et al.. 1985:
Grazier et al., 1991 and B o u m et al., 1993), moisture to casein ratio (Creamer and Olson.
1982). residual rennet ( de Jong. 1977) and starter culture. and changes in pH during
ripening (Creamer and Olson. 1982).
Cheddar cheese ripening is a two phase process. In the first stage w hich occun
during the first to the second week during curing, about 20% ml-casein is hydrolyzed by
the residual rennet to as 1-1. in the second stage which may last for months. as 1-cnsein
and other caseins are extensively broken down into smaller peptides and amino acids by
the proteinaselpeptidase of the starter and nonstarter bacteria (Creamer and Olson. 1982).
Most of the rennet is lost during whey drainage, but some remains in the curd.
Lower pH nt draining and higher moisture content in cheese results in higher rennet
retention. In these experiments. moisture content in control and DHA cheese were about
the same. and pH ai draining for both control cheese and DHA cheese were controlled to
be the siune, so residud rennet of both cheese were probably nt the sarne level and
wouldn't cause any difference in the rate of proteolysis.
p-casein decreases in the milk from mid-lactation or late lactation (Donnelley and
Barry, 1983). DHA milk was lower in B-cüsein content as shown from the SDS-PAGE
results. This may be bccüuse the DHA milk was from 6 Holsteins cows close to the end of
lactation.
Cheddar cheese is usually ripened nt the temperature below
IOOC
in order to
minimise the potentid of developing off-flavour (Lawrence et al., 1986). Required rates
of ripening can be achieved by adjusting the sali to moisture ratio and ripening
temperature. in this experiment. both control cheese and DHA cheese were stored in the
same cold room at 4 ' ~ .
4.3.4.1 pH changes during ripening
Changes in pH during cheese ripening is an important indicator of cheese texture
developrnent.
pH profiles of control cheese and DNA cheese are shown in Figure 4.5. In the first
month of ripening. pH dropped because the residud lactose was broken down by the
starter culture to lactic acid. As pH decreases. more colloidal calcium phosphate was
removed from the casein micelles. cheese became more strechable (Lawrence et ai.,
1986).
After one month. pH increüsed slightly due to the small peptides. free amino acids
and other products from more protein break down in further proteolysis.
4.3.4.2 Free Amino Acids
The changes in amino acids content during cheese ripening are usually used as an
indicator of the rate of proteolysis.
The changes of free amino acids in control cheese and DHA cheese during
ripening were shown in Figure 4.6. In the first 3 months of ripening, there was no
significant difference between control cheese and DHA cheese reguding the level of free
amino acids. But üfter 3 months of ripening. the level of free ümino ücids in DHA cheese
went up much fÿster than control cheese. which means the DHA cheese ripened much
füster than control cheese. This agrees with the sensory evaluation result which indicüted
that DHA cheese had stronger cheese flavor and much softer. more desirable texture than
control cheese.
4.3.4.3 Microstructure by Scanning Electronic Microscopy
Caseins form a continous solid net work in cheese. Proteolysis of casein network
results in more water soluble molecules. thus changes the cheese into a smoother, more
homogenous texture. Changes in the microstructure of the casein network occurs during
the process of proteolysis. The texture of cheese is determined by its pH and the ratio of
intact casein to moisture. in the first one to two weeks after manufacture, the texture of
Cheddar cheese chünges mainly due to the hydrolysis of a small fraction of as 1-casein by
the rennet results in a general weakening of the casein network. The texture changes
thereafter are determined by the rate of proteolysis which is determined by the residual
rennei and plasmin in the cheese, sali to moisture ratio, and storage temperature
(Lawrence et al., 1986). The protein matrix of cheese can be seen as a sponge filled with
fat globules and an aqueous phase composed of free water in which various compounds
are dissolved. During ripening. proteins are cleaved at different sites and thus the original
microstructure of the cheese is changed.
Micrographs of microstructure of control cheese and DHA cheese at different
stage of ripening are shown in Figure 4.7. Reduced fat cheese were reported to have poor
texture. They usuülly show a denser protein network under SEM.It can be seen that üt the
beginning of ripening. both control cheese and DHA cheese had typical dense texture of
reduced fat cheddar cheese. But DHA cheese developed a more open structure dunng
ripening than control checse. This agrecs with the results from both TNBS test and
sensory evaluation.
4.3.5 Sensory Evaluation
In the difference test (triangle test) conducted on both control and DHA cheeses
after 6 months ripening, al1 the 20 panellists picked up the odd one correctly. The
summüry of the comments from al1 the panellists suggested that DHA cheese had a softer
and smoother texture and a stronger flavour than control cheese. These findings ügree
with the results from the cheese ripening test.
t
- .-o.-.Control
Ripening Time (month)
FIGURE 4.5 pH profile of control cheese iind DHA cheese during cheese ripening
Ripening Time (month)
FIGURE 4.6 Changes in amino acid concentration of control cheese and DHA cheese
during ripening
A - crinird cliccse rifier I riiorith o t' riprning; B control chccsè atier h nionihs' of ripening
C - DHA chccse after I nionth of' ripening:
D - DH.4 clitxsc iifter 6 rnontli ut' ripeniiig
FIGURE 4.7 Micrographs of control and DHA çheese during ripening
CHAPTER S BUTTER MAKING PROPERTIES OF DHA ENRICHED MILK
5.1 INTRODUCTION
As discussed in Chapter 3. DHA enriched milk had lower fat concentration. an
increased DHA level and a smaller average fat globule size. These changes could affect
some of the processing propenies of DHA enriched milk. especially the creaming
properties. The objectives of this chapter were to study the butter making properties of
DHA enriched milk and some physical and chernical propenics of the butter fat extracted
from DHA enriched milk.
5.2 MATERIALS AND METHODS
5.2.1. Butter Making
Figure 5.1 shows the flow diagram of the butter making procedure. Control milk
and DHA milk were picked up at the same time from Elora Dairy Cüttle Research Center
(University of Guelph). Elora. Ontario. and processed on the samc day.
Milk was heated to 600C in a 20 L stainless steel culture vat equipped with a
steam jacket. and separated immediately through a disk cream separator twice (for control
milk) or three cimes (for DHA milk) until the fat concentration of the cream reached
about 40%. Cream and skimmed milk were collected separately into 20L stainless steel
milk c a s and well mixed by gentle agitation. Samples of both cream and skirnmed milk
were taken into 20ml amber vials and tested within 3 hours for fat concentrations by
infrared anaiysis (Foss System 4000, Hillerod, Denmark) ai Laboratory Services Division,
University of Guelph. Cream was standardized to fat content of 35% by adding a
calculated amount of skimmed milk back to it. Standardized cream was pasteurized in a
four-unit batch pasteurizer (Highland Equipment Limited. Mississauga. Toronto. Ontario)
~
at 8 0 ' ~for 30 seconds, then quickly cooled and stored in a cold storage at 4 ' overnight.
Butter making was carried out the next moming.
The schematic diagram of the butter churn is shown in Figure 5.2. The butter
churn sits in a wooden rack. Shaft A is fixed on the top wooden board of the rack and is
driven by a motor. Shaft B and the screwed cap of the chum are integrated together. Shüft
A and shaft B can be engaged together, so the chum can tightly fit into the spwe between
the top and the bottom wooden boards of the rack. The whole installation c m be
reinforced by joining the semicircular metal ring with the one built in the rack through
two bolts. After switching on the power. shüft B will tum together with the square paddle
which is built as one piece of the shaft. and chuming starts.
Cream w u churned in a 8 ' cold
~ room using a IL butter chum. 600 g of cream
was weighed into the butter churn and churned untii 10 seconds after breaking. The
breaking point is the point when butter granules and free buttemilk becomes evident. The
breaking stage is generally considered complete when small streûks of creüm are washed
clear from the port glass (McDonall. 1953). In this expriment. 10 seconds were
considered long enough to complete the breaking stage. Chumed cream was transferred to
a plastic container with holes on the wall for draining and worked with a plastic spatula
for 5 to 8 minutes until rnoisture content reached 16 to 17%. Moisture content was
checked with a moisture balance. Then 2.5% salt was added to the butter.
DHA milk
control milk
/
\
disc c r e m seperator
r
crem (40% of
ht)
!
standardize to 35% of fat
I
pasteurize at 8 0 ' ~for 30 seconds
I
cool to 4Oc
I
store at 4 ' ovemight
~
I
churn uritil 10 seconds üfter breaking
1
work until moisture content reaches about 15%
I
add salt (2.5%)
1
pack
I
store at 1 " ~
FIGURE 5.1 Flow diagrm of butter making
skim milk
1. top woden rack; 2. build-in shaft A; 3. shaft B; 4. screwed cap; 5. spindle;
6. body of the butter churn; 7. build-in semicirculûr metal ring; 8. bottom wooden
board
FIGURE 5.2 Schematic diagram of butter churn
The butter was worked until salt was well dissolved. Final products were packed in
plastic containers and stored in the dark 4 ' ~ .
52.2 Chernical and Physical Properties of DHA Butter
5.2.2.1 Moisture in Butter
Four grams of butter was weighed into predried and weighed flat-bottom metal
dishes. Loosely covered dishes were plüced in a vacuum oven kept at SO'C and dried to
constant weight. Moisture in butter was reported as percentage of weight.
5.2.2.2 Fat in Butter
Babcock method wüs used to detemine the fat content in butter. Nine grams of
butter was weighed into a 86% Babcock bottle. First. 10 ml of distill water was added at
6 0 ' ~and
~ then 17.5 ml sulphuric acid was added in at least three increments. The bottle
was swirled until the color of the sürnple mixture was uniform chocolate brown and üII
the butter sample was completely dissolved. Then the sample was centrifuged at 2,000
rpm for 5 minutes. Distilled water was added at 6 0 ' ~to bring contents to within onequarter inch of the base of the neck. The sample mixture was centrifuged a i 2,000 for
another 2 minutes. More distilled water was added at 60'~to float flat into the neck of
the bottle. The sample mixture was cenvifuged at 2,000 rpm for another lminute and then
tempered in a 5 5 ' ~water bath for 5 minutes. Four to five drops of glymol was added on
the fat column and allowed to run down the side of the neck. The length of the fat column
from the demarcation between fat and glymol to the bottom of the lower meniscus was
measured. Fat was reported as percentage of weight.
5.2.2.3 Hadness
Hardness of DHA butter and control butter was determined by a cone
pnetrometer according to AOCS official method Cc 16-60.
Al1 the samples were ternpered in a cold room at IO~)C
for 48 hours prior to the
memurement. The testing was conducted in the same cold room ai
IOOC.
The angle of the
cone was 20'. The total weight of the conc assembly and the shaft was 92.5 grüms. The
position of the tip of the cone was adjusted so it just touched the surface of the sample
and then released. The indicetor rack was immediütely lowered üfter 5 seconds of contact
and the penetration depth wüs read on the dial. Hardness index was calculüted according
to the following (Hayakawa and deMan. 1982):
w here:
HI
= hardness index
M
= mass of penetrating assembly in gram
P
= depth of pnetration in 0.0 1 mm
5.2.3 Sensory Evaluation of Butter
Descriptive test and difference test were used in sensory evaluation. Experimental
design was the same as the one described in Chapter 3.
Descriptive tests were done by 3 expert graders. Samples were tested for oxidütive
flavors on day 1, day 3. day 5 and dsy 7 after manufacture. Difference tests were done to
detennine if there was any difference between DHA butter and control butter by an
untrained consumer panel composed of graduate students, faculty and staff of the
University of Guelph.
5.2.4 Properties of Extracted Butter Oil
5.2.4.1 Extraction of Butter Oil
Butter was tempered üt 6 0 ' ~for 30 minutes and then the upper clear portion wüs
collected and centrifuged at 2000 rpmlmin for 3 minutes. The supernütnnt was collected
again and filtered hot through a Whatman #4 1 filter paper. Hdf of each kind of butter oil
was collected and frozen at - 2 5 ' ~ for solid fat content, melting properties analysis by
Differential Scanning Calorimetry (DSC) and dropping point analysis and used within 3
days after extraction. The other half was treated with 0.02% BHT,and frozen at - 2 5 ' ~
for later iodine value determination.
5.2.4.2 SolM Fat Content by Rilsed Nuclear Magnetic Resonance (pNMR)
Measuring Procedure
Solid fat content was determined by AOCS officiai Method Cd 16-81 by pNMR
using a Bruker P U 2 0 Series NMR Analyzer (Miispec, Milton, Canada). Samples were
first tempered at a 6 0 ' ~water bath for 15 minutes, then in ü 2 6 . 7 ' ~water bath for 30
minutes. solidified at O'C for 15 minutes, back to 2 6 . 7 ' ~for another 30 minutes and
solidified again üt O'C for 15 minutes. The sarnples were then conditioned for 30 minutes
from 5 ' ~to 4 5 ' ~ai 5 ' ~intervals and tested for solid fat content at relevant temperature.
5.2.4.3 Dropping Point
Mecisicring Principlrs of Dropping Point
When subjected to heat. fat will start to melt. Dropping point is the temperature at
which fat will start to flow on its own weight. The measuring pnnciples of the Mettler
dropping point apparatus are illustrated in Figure 5.3. Tube A is used to transfer the
sample holders to avoid any direct contact wiih operator's hmds. So tube A should be
chilled ai the same temperature as that of the sample tempering before the measurement.
Tube B ensures the sample holder is placed ai the right position inside the equipment. so
the light from the lamp can go through the two symmetrical fissures and be detected by
the detector. When the temperature reachcs the dropping point, the first drop of fat will
cut off the light travelling from the iümp. When the cut-off is detected by the detector, the
temperature is recorded and dispiayed as the dropping point.
Meusuring Procedure
Dropping point of butter oil was determined by a Mettler dropping point apparatus
(FP83, Mettler, Zurich, Switzerland). Samples were liquefied at 8 0 ' ~for 15 minutes,
1 . rube A; 2. sûmple holdrr; 3. tube B; 4. open fissure; 5 . larnp: 6 . detector
FIGURE 5.3 Schematic diagram of the rneasuring principles of a Mettler dropping point
apparatus
then filled into chilled sample holders and kept at - 1 0 ' ~for 1 hour before measurement.
Parmeters of the experiment were as following:
Starting temperature: 20.0'~
Heating rate: loc/min.
5.2.4.4 Melting Properties by Differential Scanning Calorirnetery (DSC)
Mecrsuring Procedr re
Melting properties of butter oil were determined by differential scruining
calorimetry using a DuPont 1090 di fferential scünning cülorimeter. Calibration before the
measurements was done by using Indium and Gallium as references.
Butter oil samples were tempered in an 8 0 ' ~water bath for 15 minutes to ensure
al1 the fat crystiils were dissolved. Then 0.5 g of liquefied sirmple was carefully weighed
into an irluminum sarnple pan. The pan was then covered, sealed and kept üt 5 " for
~ 48
hours prior to measurement to ensure that al1 the samples had identical temperature
history. The following DSC program was used:
Equlibrate ai S'C for 10 minutes
Data storage: on
Ramp: 5'c/rnin from 5 ' ~to 6 0 ' ~
Equlibrate ai 6 0 ' ~for 2 minutes
Data storage: off
Equilibrate ai 5 ' ~for 2 minutes
5.2.4.5 Iodine Value
lodine value was determined by A.0.C.S official method Cd 1-25. Approxirnately
0.7 g of butter oil sample was weighed accurately into a 500 ml flask. 20 ml of
cyclohexane was added and then followed by 25 ml of Wijs solution. The flask wüs
sealed. swirled to insure an intimate mixture and then immediately tnnsferred to the dark
and stored for 1 hour. Two blanks were prepÿred simultaneously.
The flasks were removed from storage and 20 mL of K1 solution was added.
followed by 100 mL of distilled water. The sample mixture was iitriited with 0.1 N
Nü2S203solution until the yellow color h d almost disrppeared. I to 2 ml of starch
indicator solution wüs added and titration was continued until the blue color had just
disappeared. lodine value was reported as:
where:
B = Titration of blank
S = Titration of sample
N = Norrnality of Na2S203solution
W = Weight of sarnple
5.3 RESULTS AND DISCUSSION
53.1 Butter Making
Chuming time and temperature changes during chuming were measured.
Churning time for control butter and DHA butter is shown in Table 5.1.
Churning time of DHA butter was longer than of control butter. This is due to softer fat.
smaller fat globules and their more uniform distribution in DHA milk. This cün be
corrected by using ü lower chuming temperature. as is common for summer butter.
Summer butter hüs higher level of unsaturated fatty ücids because most cows are
consuming fresher forages during summer time and ihis results in softer fat. which
requires lower temperature for optimum yield and butter quality.
Besides the properties of milk fat. a heat denüturable protein üdsorbed on cold fiit
globules dso affects the creaming of bovine cow milk. The protein. known as fat
agglutinin, promotes clustering of globules (Farah and Ruegg. 1991 ). After sepuaiion.
skimmed milk is richer in agglutinin protein thün cream. C m e l milk is known to have
poor creaming properties both in raw and heaied milk at refrigerator and room
temperatUres, although the fat globule size distribution of camel milk is similu to ihat of
bovine milk (Farah and Ruegg, 1991). Farah and Ruegg ( 1991 ) compiired the creaming
behavior of carne1 milk fat and bovine milk fat. They found reconstituted milk from
c m e l creüm and bovine skim milk crearned much better than both camel milk and the
mixture of bovine crcam and camel skim rnilk. Their results indicated thnt camel milk
lacked the agglutinating substance required to cluster fat globules. Similarly, buffdo milk
and goat milk were also reported to have poorer creaming properties compared to bovine
milk due to an insufficient quantity of agglutinin (Abo-Elnage. 1966a. AbElnage et
al.. 1966b.Parkash and Jenness. 1968).The level of agglutinin in both DHA rnilk and
control were not studied in these experiments. Whether DHA milk lacks agglutinin or not
still awaits funher study.
Temperatures of cream before and after chuming were determined using a dial
thennometer. Temperature increûse during chuming for control cream fell in the r m p of
5 to 6 CO, and 8 to 10 CO for DHA crearn. Higher temperature increase for DHA creüm
was due to longer churning time.
TABLE 5.1 Chuming tirne (seconds)of control butter and DHA butter
Weight of crem (g) Control butter
DHA butter
Ratio (DHA: control)
500
77.5
155
2 .O
600
96.4
23 1.3
2-4
5.3.2 Composition and Hardness
Many instruments have been used to determine the rheological propenies of
butter. Among al1 the rquipment, cone penetrometer is a simple and economical one.
Interpretation of the results of cone penetrometer is also relütively easy. The depth of
penetration is a function of cone angel, weight of the penetrating assembly and
penetration tirne. When keeping the conditions of cone angle. mass of the cone and shaft,
temperature and time allowed for penetrütion constant, the results cm be simply
interpreted in tenns of the depth of penetration (Dixon and Parekh, 1979).Other factors
that can influence the reading of depth of penetration are, smoothness of the cone,
sharpness of the cone tip. and work hardening and kinetic energy of the cone (Haighton,
1959). It is generally agreed that hardness can be calculated by the following equation
(Hayakawa and Deman. 1982):
where:
H = hardnrss or yield value
C = a constant depending on cone geometry
M = mass of penetrating assembly
P = depth of penetration
n = an exponent
However the hardness or yield value obtained from the übove equation depends
on the mass of the penetrating assembly. It is impossible to compare the hardness data
obtained using different penetrating assemblies of differeni mas. Hayakawü and deMan
(1982) suggested using hardness index (HI) which is independent of cone assembly
weight to mess the difference in hardness of different fats. The hürdness index wüs
defined as:
where:
HI = hardness index
M = rnass of cone assembly
P = depth of penetration.
n = an exponent
Fats with a wide range of hardness values were tested for hardness using a cone
peneirometer with a 2oUcone angle and different assembly weight. Tests were done at
IOOC,
and the penetration time waï set at 5 seconds. Several formulae were derived from
cquation 5.2 by assigning different values to n. Results were evaluated and compared in
order to establish which fomulae showed the leiist dependence on cone assembly rnass.
When the cone of 20' angle as specified in the AOCS method is used exclusively, a
sütisf;ictory conversion of penetration depth to hardness index can be made using
equation 5.1 which is expressed as following:
Fat concentration, moisture concentration and hardness index of control butter and
DHA butter are shown in Table 5.2, DHA butter had a much lower hardness index. This
indicates that DHA butter was much softer than control butter at the cxperimenrül
temperature. Since the hardness index was measured at IOOC,it can be concluded that
DHA butter had a better cold spreadability than control butter.
The structure of butter is made up of both globular and free fat. The free fat
produced during chuming contains both liquid fat and crystdline fat. In physicai
structure. the liquid fat forms the continuous phase of butter, while the crystalline fat.
globular fat. air bubbles, curd particles and water and brine droplets are dispersed in the
continuous liquid phase. The hardness of butter is mostly determined by its composition
and fat propenies. while some other hctors. such as season, cooling and tempering of the
crearn. and storage temperature of butter cm also affect its hardness. In this experiment.
control butter making and DHA butter making were conducted ai the same time
üccording to the same procedure. The finai products were siored under the süme
condition. so the effects from season. processing procedure and storage condition can be
ignored. The difference between the hardness of control butter and DHA butter can be
considered as mainly due to their different fatty acids composition. DHA butter was
enriched in DHA. Usually unsüturated fatty acids are found in greater proportions in the
low nieltiiig fraction. The incorporation of DHA can increüse the arnount of low melting
portion and contribute to softer fat. Similar. but smaller differences are observed between
summer butter, which contains more unsaturated fat than winter butter. In this
experiment. only about 0.4% DHA (of total fatty acids) was incorporated into DHA
butter. but this small increase had a great effect on the hardness of the DHA butter.
TABLE 5.2 Composition and hardness of control butter and DHA butter
Fat (%)
Moisture (%)
Hardness index
Control butter
84.05"
13.87~
13.62'
DHA butter
82-05'
15.07~
6.73d
'. b. '.
means values in the sarnc column with different ktten are significantly different at P<O.OS%
5.3.3 Sensory Evaluation
In both descriptive test and triangle test. flavor difference between control butter
and DHA butter was found to be not significant. but the color difference was significant.
DHA butter was white compared to the yellow control butter. because the DHA diet
lacked p-carotene. Fresh forages are the main source for p-carotene in milk fat. As fresh
forages were not included in the treatment diet. p-cÿrotene concentration in DHA milk
wüs much lower than that of control milk. The color of control butter may v q with
season. Summer butter is typically more yellow than winter butter because most cows are
consuming fresh forages during summer time. It is common commercial practice to
standardize butter color using ünnato.
5.3.4 Physical Properties of Extracteà Butter Oil
Se3D4D1 Dropping Point and Iodine Value
Dropping points and iodine value of DHA and control butter oils are shown in
Table 5.3.
Dropping point is the temperiture at which the fat stiuts to flow under its own
weight. It is a rheological measurement. When the fat is unemulsified, it is also a measure
of the melting of fats (Borwankar et al., 1992). There was a significant difference
between convol butter oil and DHA butter oil. DHA butter oil had lower dropping point
because of the incorporation of polyunsaturated fatty acids. DHA is a long-chain polyunsaturated fatty acids with 6 double bonds. The more double bonds exist in the fatty
acid. the lower melting point the triglyceride has. This can explain the lower dropping
point of DHA butter oil.
Iodine number is an indication of the degree of unsaturation of fats and oil.
Unsaturated fatty acids can absorb iodine atoms and form addition compounds. Iodine
value of butter oil can be affected by factors such as season (Kunz. 1980)and feed.
Usually summer butter has higher iodine value becüuse the fresh forage in the feed is rich
in polyunsaturated fatty acids. The increased level of polyunsaturated fatty acids in the
feed will be refiected in the milk fat and thus increases the iodine value of the butter fat.
Generally the iodine number for pure butter fat falls between 25.7 to 49. In this
experiment, the iodine value is 35.34 for control butter oil and 37.87 for DHA butter oil.
Boih are within normal range. No significant difference was observed between control
butter oil and DHA butter oil regarding iodine number. This may be due to the relatively
low concentration of DHA (only 0.4% of total fatty acids) present in the DHA butter oil.
TABLE 5.3 Physical properties of control and DHA butter oil
Dropping Point (Cu) Iodine Value
Control Butter Oil
34.06 f 0.29'
35.34 $r 2.6OC
DHA Butter Oil
32.89 I 0 . 5 5 ~
37.87 f 2.80'
Mean f SEM
a, b. c, d, e means values in the s m e colume with different letters are significantly
different at the level P d . 5
5.3.4.2 Solid Fat Content by Pulsed Nuclear Mapetic Resonance (pNMR)
The results of solid fat tests of control and DHA butter oil are shown in Figure
5.4. At temperatures below 2 5 ' ~ .DHA butter oil had more melted fat than control butter
oil. This indicates that DHA butter had better spreadability than control butter oil at the
temperatures below room temperature which agrees with the data from hardness index
. DHA
tests. But no significant difference was observed at temperatures lrbove 2 5 ' ~ So
butter will most likely have the süme hudness and spreadability as those of control butter
iit
temperütures übove 2 5 ' ~ .
5.3.43 Melting Properties by Differential Scanning Calorimetry (DSC)
Changes in volume percentage of melted fat versus temperature were plotted
.
butter oil had more melted fat. It
(Figure 5.5). At temperatures below 2 0 ' ~ DHA
indicütes that DHA butter would be softer and have better spreadability thün control
butter at the temperatures below room temperature. This agrees with both the results from
hardness index tests by cone penetrometery and solid fat content tests by pNMR. At
temperatures between 2 0 ' ~and 30'~.control butter oil had slightly more melted fat than
DHA butter. When temperature increased above 3 0 ' ~and became closer and closer to the
dropping points of DHA and control butter oils, the differences in their volume
percentages of melted fat becme srnaller and smaller. At 3 3 ' ~ . the dropping point of
DHA butter oil. the volume percentage of melted fat in DHA butter oil increased to 95%.
When temperature reached 3 4 ' ~ .the temperature of control butter oil, the volume
percentage of melted fat in control butter oil was about 97%. However, the rheoiogical
propenies of DHA and control butter oils were not determined in this experiment. The
differences in solid fat contents of DHA and control butter oils at the temperatures of
their dropping points were not detected by pNMR tests. because the readings of solid fat
content were taken at a SOC interval. No data were available at the temperatures of 3 3 ' ~
and 3 4 ' ~ .
The typical DSC curves of DHA and control butter oils are shown in Figure 5.6.
DSC curve of DHA butter oil showed 3 peiiks. while the curve of control butter oil hüd 2
peaks. The second and the third peaks in the DSC curve of DHA butter oil hüd the süme
shepes of those in the DSC curve of control butter oil and occurred at neÿrly the s m e
tempcratures. The first peak of control butter oil is sharper than the corresponding second
peak of DHA butter oil; while the last p k s of both control butter oil and the fat. The 2%
difference may be due to both the differences in composition and the DHA butter oil were
almost the süme shape and occurred ai the same temperature. This indicütes thüt control
butter oil hüd higher level of medium-melting triglycerides than DHA butter oil, and they
mostly likely had the süme mount of high-melting triglycerides. The first peak in DHA
butter oil is characteristic and similar peük was not observed in control butter oil. This
concludes that DHA butter oil wüs higher in low-melting triglycerides duc to the
incorporation of DHA. Further studies on identifying the triglycerides profile in DHA and
control butter oils is needed to explain the differences in their melting properties in depth.
+Treatment
- - - - Control
@.
--
s
t
20
30
40
Temperature
FIGURE 5.4 Solid fat content of DHA and control butter oils by pNMR
FIGURE 5.5 Volume percentage of melted fat of DHA and control butter oil versus
temperature
Treatment
Cantrol
FIGURE 5.6 Typical DSC curves of DHA butter oil and control butter oil
CHAPTER 6 GENERAL CONCLUSIONS
Supplementing the diet to dairy cows with fish meal enriched milk with DHA. In
this experiment, the physical, chemicd and processing properties of DHA enriched milk
(DHA level at 0.4% of total fütty acids) were investigated. Conclusions are as following:
1. Pasteurized DHA milk was similür to control milk with respect to sensory
quality as drinking milk. Neither consumer nor expert pünelists detected fishy or oxidized
flavors in DHA milk.
2. After 3 months of ripening at 40C. Cheddar cheese made from DHA milk
ripened faster thrn control milk as indicated by accumulation of free amino acids.
Sensory evaluation indicated both softer and smoother texture and stronger flavor than
control cheese.
3. DHA butter was more spreadable thün control butter ût room temperature. This
observation was confirmed by pNMR measurements of solid fat index. dropping point
(melting point). and hardness index (cone penetration).
1.Fat globules in DHA rnilk had reduced average diameter and more uniform
diameters relative to control milk. This probably accounts for increased chuminp time for
butter manufacture from DHA cream. Ice cream processing conditions may also need to
be slightly dtered for DHA ice cream.
5. Casein micelles were significantly smüller in DHA milk ( 170.78 nm, relative to
187.5 lnrn for control milk). DHA milk also contûined less casein and more whey protein
than control milk. Theoretically, this will result in lower cheese yield efficiency for DHA
milk. although yield measurement in our experiments were not sufficiently sensitive to
detect a difference in cheese yield efficiency.
6. Future work is needed to confirm results on more than one set of cows and
during an entire lactütion period. Fat globule size. casein micelle size. and protein
distribution need to be further studied in relation to thc level of DHA supplementation.
Differences in processing properties and quality of milk supplemented wiih DHA by diet
versus by direct supplementation of milk methods need io be investigüted.
REFERENCES:
Abo-Elnage, I.G. l966a. Factor affecting creaming of cow's and bu ffalo's milk.
Mischwissenscha$! 2 1 :429
Abo-Elnaga. I.G.; El-Sadek, G. M.; El-Sokary, A. M. 1966b. Clustering of fat globules in
cow's and buffalo's milk: creaming mechanism and physical arrangement of globules in
grüvity crenm. Milchwissensclicifi 2 1:2 10
Ajuyah. A.O.; Hardin, R.T.;Cheung. K.; Sim, J.S. 1992. Yield, lipid. cholesterol and
Fütty acid composition of spent hens fed full-fat oil seeds and fish meal diets. J of Food
Sci 57(2):338-341
Ajuyah, A.01; Ahn. DU.:Hardin, R.T., and Sim. J.S.1993. Dietary antioxidani s and
storage affect chernical characteristics of w3 fatty acids enriched broiler chicken meats.
J. food Sci. 58: 43-46
Akayezu. J. M.. Hansen. W. P.. Otterby. D.E.. Crooker, B. A., and Marx. G. D 1997.
Yield response of lactating holstein dairy cows to dietary fish meal or meat and bone
meal. J. dairy. Sci 80: 2950-2963.
Anderson. G.J.; Connor, W.E.; Corliss. J.D.; Lin. D.S.1989. Rapid modulation of the
omega-3 docosahexaenoic acid levels in the brain and retina of the newly hatched chick. J
Lipid Res 30:433-441
Ashes J. R.; Siebert BD; Gulati SK; Cuthbertson AZ; Scott TW. 1992. Incorporation of n3 fatty acids of fish oil into tissue and serum lipids of ruminants. Lipids. 27: 1 102-1 103
Aston, J.W.; Giles. LE.; Durward, 1.; Dulley, J.R.; 1985. Effect of elevated ripening
temperatures on proteolysis and flavour development in cheddar cheese. J Dairy Rrs
52:565-572
Biuclay. William; Abril, Ruben; Abril, Patricia; Weaver, Craig; Ashford. Amy. 1997.
Produciin of docosahexaenoic acid from microdgae and its benefits for use in animal
feeds. iN:The Return of w3 fatty acids into the food supply 1. land-büsed animal food
products and their health effects. Edited by A.P. Simopoulos. Karger
Bÿnon, Beverly M.: Emerson. John A. 1986. Seafood in your diet - a choice of recipes. in
Health Effects of Polyunsaturated fatty acids in seafoods. edited by Simopoulos, Artemis
P.; Kifer. Robert R. and Martin. Roy E. Academic Press, iNC. Harcourt Brüce
Jovanovich. Publishers
Bastian, E.D.;
Brown. R.J.; and Emstrom. C.A.; 1991. Plasmin activity and milk
coagulation. J Duiry Sci 74:3677-3685
Bimbo, Anthony P. and Crowther Jane B. 1992. Fish meal and oil: currcnt uses. J of the
Am Oil Chem Soc 69 (3):221-227
Burke, LM; Staples, C.R.; risco. C. A.; De La Scota; R. L.; and Thatcher, W. W. 1997.
Effect of ruminant grade menhaden fish meül on reproductive an dproductive
performance of lactating dairy cows. J. Duiry Sci. 80:3386-3398
Borwankar, R.P.; Frye, L. A.; Blaurock, A. E.; Sasrvich, F. J . 1992. Rheological
characterization of melting of margarines and tablespreads. J ofFood Eng 1655-74
Bouzas, S.; Kantt, C.A.; Bodyfelt. F.W.; Torres, J.A. 1993. Time and temperature
influence on chernical aging indicator for a commercial Cheddar cheese. J Food Sci
58(6): 1307- 1312
Brochu, E.; Dumais. R.; Julien, J.P.; Nadeau, J.P.;Riel, R. 1985. Dais, Science and
Technology: principles and applications. La Fondation de technologie laitiere du Québec
Inc.
Bryant. A.; Ustunol, 2.and Steffe, J. 1995. Texture of Cheddar Cheese as influenced by
Fat reduction. J. Food Sci. vol. 60. p. 12 16-1219
Caston, Linda and Leeson, Steve. 1990. Research Note: Dietary Flax and Egg
Composition. Pou1 Sci 69: 1617-1620
Caston, L.J.; Squires. E.J. and Lesson. S. 1994. Hen Performance, Egg quality, and the
sensory evaluation of eggs frorn SCWL hens fed dietary flax
Carlson S.E.; Rhodes, P.G.;
Rao R.S.; Goldgar D.E.1987. Effect of fish oil
supplementation on the n-3 fatty acid content of red blood ceIl membranes in preterm
infants. Pdicitric Res 2 1 507-5 10
Cherian. G.; Sim J.S. 1991. Effect of feeding full fat flax and cünola seeds to laying hens
on the fatty acid composition of eggs. ernbryos. and newly hatched chicks. P o d r p Sci
70:9 17-922
Cherian. G.and Sirn. J.S. 1992. Omega-3 fatty acid and cholesterol content of newly
hatched chicks from a-linolenic acid enriched eggs. Lipids. 27: 706-7 10
Chin, S. F.; Storkson, J. M.; Albright, K.J.;Cook. M.E.; and Pariza, M. W. 1994.
Conjugale dlinoleci acicd is a growth-factor for rats as shown by enhanced weight-gain
improved feed efticiency. J. Nurri. 124: 2344-2349
Creümer. L.K.;Olson, N.F. 1982. Rheological evaluation of maturing cheddar cheese. J
Food Sci 47:63 1
Cruickshank. E.M.1934. Studies in fat metabolism in the fowl. The composition of the
egg fat and depot fat of the fowl as affected by the ingestion of large amounts of different
fats. Biochem J 28:965-977
Dairy farmers of Ontario: Dairy statitical handbook 1996-1997. 13thedition.
Dalgleish. D.G. 1981. Effect of rnilk concentration on the nature of curd formed during
renneting - a theoreticda discussion. J Dairy Res 48: 65-69
Davis. S. R. and Collier, R. 1. 1985. Mammary blood flow and regulation of substrate
supply for milk synthesis. j. dairy sci. 68: 1041-1058
de Gomez Dumm [NT.;Brenner RR. 1975. Oxidative desaturütion of alpha-linoleniç.
linoleic. and stearic acids by humün liver microsomes. Lipids 10:3 15-317
de long. L. 1977. Protein breakdown in soft cheese and its relation to consistency. 2. The
influence of the rennet concentration. Neth Milk D~tiryJ 3 1: 3 14-327
DeKoning. P.J.; Kaper. J.; Rollema, H.S.;Driessen, F.M. 1985. Age-thinning and
gelation in unconcentrated and concentrated UHT-sterilized skim milk. Effect of native
milk proteinase. Neth Milk Dairy J 39:7 1
Dietary fish oil increases n-3 long-chain polyunsaturated fatty acids in hurniin milk Nutr
Rev October, l985.43( 10):302
Dils. R.R. 1986. Comparative Aspects of Milk Fat Synthesis. J Dairy Sci 69: 904-9 10
Dixon. B. D.; Parekh. I. V. 1979. Use of the cone penetrorneter for testing the f i m e s s of
butter. J of Texture Stud lO:42 1-434.
Donnelly, W.J.; Barry,J.G.1983. Casein compositional studies III. changes in Irish
milk for manufacturing and role of milk proteinase. J Dairy Res 50:433
Dougherty. B.W. 1965. Physiology of digestion in the ruminant: Papers presented at the
second international symposium on the physiology of digestion in the ruminant.
Washington ButterWorths.
Eaton Boyd S.; Eaton II1 Stanley B.; Sinclari Andrew J.; Cordain. Loren; Mann. Neil J.
1997. Dietary intake of long-chain polyunsaturüted fatty acids during the püleolithic. LN
The retum of w3 fatty acids into the food supply 1. land-based animal food products and
their health effects. Edited by A.P. Sirnopoulos. Kwger
Elswyk. M.E.; Sams, A.R.; Hargis. P.S. 1992. Composition. functionality. and sensory
evaluation of eggs from hem dietary menhaden oil. J of Food Sci 57(2):342-344
Eisw yk. M.E.V. 1993. Designer foods: manipulation the fatty acid composition of meat
and eggs for the health conscious consumer. Nitsr Todrry. March/April:2 1-28
Emmons, D. B.; Kalab, M.; Larmond, E. and Lowrie, R.J. 1980. Milk gel structure. X.
Texture and microstructure in Cheddar cheese made from whole milk and from
homogenized low fat milk. J. Text. Studies 1 1 : 15-34
Farah. 2.and Ruegg, M. 1991. The creaming propenies and size distribution of fat
globules in came1 milk. J Dairy Sci 74 (9):
290 1-2904
Farrell. IR.; Thompson. M.P.1974. Physical equlibria: Proteins in Fundamentals of dairy
chemistry. 2"dEdition. The AVI publishing Company. WC.
Farrell. D.J.; Gibson, R.A. 1990. Manipulation of the composition of lipid in eggs and
poultry meat. IN proceedings of the inaugural Massey pig and poultry symposium. New
England. NSW. Australia, Nov.26-28
Farrell. H M . ; Pessen. Ir.. Brown, H.; Kamosinski. T.F. 1990. Structural insights into the
bovine casein micelle: Small angle X-wy scattering studies and correlation with
speciroscopy. J Dnirv Sci 73:3XU-3601
Fernandes, G.; Venkatraman, J. T. 1993. Role of omega-3 fatty acids in heülih ünd
disease. Nutr Res 13:s19445
Fox. P.F. 1989. Proteolysis during cheese manufacture and ripening. J Dai- Sci 72: 1379-
1400
Fritsche. Kevin L.; Huang, Shu-Cai. and Cassity, Nancy A. 1993. Enrichment of omega-3
fatty acids in suckling pigs by materna1 dietary fish oil supplementation. J. Anim. Sci. 7 1:
1841-1847
Gibson, R.A.; Neumann, M.A.; Makrides, M. 1996. Effect of dietary docosahexaenoic
acid on brain composition and neural function in term infants. Lipids. 3 1:s 1774 18 1
Grazier. C.L; Bodyfelt. F.W.; McDaniel. M.R.; Torres. S.A. 1991. Temperature effects on
the development of cheddar cheese flavor md uoma. 3 Duiry Sci 74:3656-3668
Green. M. L.; Marshall. R. J. and Glover, F. A. 1983. Influence of homogenization of
concentrüted milk on ihe structure and properties of rennet curds. J. Doiry Res. 50: 34 1 348
Griinari. J.M.; Dwyer, D.A.; McGuire, M.A.; Baumün, D. E. 1996. Partially
hydrogenated fatty acids and milk fat depression. J. Duiry Sci. 79 (suppl. 1): 177
Gruffeny, M.B.;
Fox. P.F. 1988. Functional propenies of casein hydrolyzed by aiküline
milk proteinase. N.Z.J Dtriry Sci Trchnol23:95
Grumer, R.R. 1991. Effect of feed on the composition of milk fat. J Dairy Sci 74:3244.
Ha, Y.L.; Siorkson. J.; and Pariza M. W. 1990. inhibition of knzo(a)pyrene-induced
mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer
Res. 50:1097-1101.
Haighton. A. J. 1959. The measurement of the haidness of margarine and fats with cone
penetrometers. J of the Ameri Oil Chem Soc 36:345-348
Harfoot. C.G. & G. P. Hazlewood. 1988. Lipid nietabolism in the rumen. The rumen
microbial ecosystem (Hobson. P.N.. ed.) pp. 285-322. Elsevier Science Publishers B. V..
Amsterdam, The Netherlands.
Harris, William S.; Connor. William E.; Lindsey. Saralyn. 1984. Will dietüry n-3 fatty
iicids change the composition of human mil k? The Ani J of Clin Nutn' 10:780-785
Harris. William S.; Connor William E.; Alam. Nargis and Illingworth D. Roger 1988.
Reduction of postprandial triglyceridernia in humans by dieiüry n-3 fatty ücids J Lipid
Res 29: 1451-1460
Harris. William S.; Dujovne. Carlos A.; and Zucker, Marjorie. 1988b. Effects of a low
süturated fat, low cholesterol fish oil supplement in hypertriglyceridemic patients. A
placebo-controlled trial. Annnfs of lntern Merl Setp.: 465-470
Harris, William S. 1989. Fish oils and lipoprotein metabolism. J. Lipid Res. 30: 784-807
Harris. William S.; Connor. William E.; Illingworth. D. Roger; Rothrock. Douglas W.;
and Foster David M. 1990. Effects of fïsh oil on VLDL triglyceride kinetics in humans. J
Lipid Res. 3 1 : 1549- 1558
Harris. William S.; Silveira Scott: and Dujovne, Carlos A. 1990b. The combined effecis
of n-3 fatty acids and aspirin on hemostatic parameters in man. Thrombusis Resrarch 57:
5 17-526
Harris. William S.; Rambjor, Gro Siri; Windsor. Sheryl L.; and Diederich Dennis. 1997.
n-3 Fatty acids and urinary excretion of nitric oxide metabolites in humans. Ani J clin
Nutr 65~459-464
Hayükawa, M.; Deman J. M. 1982. Interpretütion of cone penetromrter consistency
measurements of fat. J o f t e . ~ r r esritd l3:2O 1-2 10
Hebeisen, Dorothea F.; Hoeflin Frieder; Reusch. Hans Peter; Junker Edith and huterburg
Bernhard H. 1997. lncreased concentrations of omega-3 fatty ücids in milk and platelet
rich plasma of grass-fed cows.
Helferich. W.; Westhoff. D. 1980. All about yogun. Prentice-hdi. Inc.. Englewood cliffs.
New Jersey.
Howe, Peter R.C. 1998. a3-enriched pork. IN The retum of 03 fatty acids into the food
supply 1. land-based animal food products and their health effects. Edited by A.P.
Sirnopoulos. Karger
Hussein, H.S.; Jordan. R.M. 1990. Fish Meal as a protein supplement in ruminant diets: a
review.
Jahreis, Gerhard, Fritsche, Jm.. Steinhart. 1997. Conjugated linoleic acid in milk Fat: high
variatin depending on production system. Nutr. Res. 17(9): 1479- 1484
h a . A.H. and Upadhyüy. K. G. 1992. The australian journal of dairy technology. 47: 7279
Jenness. Robert; Patton. Stuart. 1976. Principles of dairy chemistry. Robert E. Krieger
Publishing Company. Hungtingon. New York.
Jiang Jin; Bjoerck. Lennürt; Fonden. Rang ne: Emanuelson. Mugareta. 1996. Occurrence
of conjugüted cis-9, trans- 1 1-octüdecadienoic acid in bovin milk : effects of feed and
dietüry regimen. J. h i r y Sci. 79: 438-445
Jrogensen. M.H.;
Hemell, O.; Lund, P.; Holmer. G.; Michüelsen, K.F. 1996. Visual acuity
and erythrocyte docosahexaenoic acid status in breast-fed and formula-fed term infants
during the first four months of life. Lipids. 3 1 :99-105
Kathryn, A.J.; Roben, A.Gibson. 1989. Weaning foods cannot replace breast milk as
sources of long-chain polyunsaturated fatty acids. Ani J Cliri N ï m 50:980-982
K m a l i , R.A. 1989. Dietary n-3 and n-6 fatty acids in cancer. Dietary n-3 and n-6 fatty
acids: biological effects and nutritional essentidity. 35 1-360
Khorasani GR.;Robinson P.H.;Boer G. De and Kennelly J.J. 1991. Influence of canola
fat on yield, fat percentage, fatty acid profile and nitrogen fractions in holstein milk. J
Dliiry Sci 74:1904- 191 1
Kremer. LM.: Jubiz, W.; Michalek. A.; Rynes, R.I.; Bartholomew, L.E.; Bigüoutte. J.:
Timachalk, M.; Beeler. D. and Lininger, L. 1987. Fish-oil h t t y x i d ingestion in active
rheumatoid arthritis: a double-blind, controlled, crossover study. Ann htrrn M d
106:497-503
Kurtz. Floyd E. 1980. The lipids of milk: composition and properties. IN: Fundamentüls
of dairy chemistry. zndEdition. Edited by Webb. Byron H.; Johnson. Arnold H. and
Alford. John A. The AVI publishing compüny, iNC.
Lawrence. R.C.;Creamer. L.K. and Gilles. J. Symposium: cheese ripening technology.
Texture development during cheese ripening. J Dairy Sci 70:1748- 1760
Leiif, David Alexander. 1989.Omega-3 fatty acids and coronary artery disease.
Pusrgraduate Medicine. 85: 237-244
Leaf, David Alexander. 1990. Cardiovascular Effects of fish oils Beyond the platelet.
Circulation 82524-628
Leaf. David Alexander. 1991. Health Claims: Omega-3 fatty acids and cardiovascular
disease. Nutrition Reviews 50 ( 5 ) : 150-154
Leskanick, C.O.; Mattews. KR.; Warkup. CC.; Noble. R.C.; and Hazzledine. M. 1997.
The effect of dietary oil containing (n-3) fatty acids on the fatty acid. physicochemical.
and organoleptic chuacteristics of pig meat and fit. J. Anim. Sci. 75: 673-683
Leskanich. C.O.: Matthews, K.R.;Würkup. C.C.; Noble, R.C. and Hazzledine. M. 1997.
The effect of dietary oil containing n-3 fatty acids on the fütty acids. physicochemicül and
organoleptic characteristics of pig meat and fat. J Anini Sci 75673-683
Lin. H.; Boylston, T.D.;
Chang, M.J.; Luedecke. L.O.and Shultz. T. D. 1995. Survey of
the conjugated linoleic acid contents of dairy products. J. Dcriry Sci. 78: 2358-2365
Mandell. I.B.. Buchanan-Smith, J.G., Holub, B.J.. 1998. Enrichment of beef with 03 fütty
acids. in The retums of 0 3 fatty acids into the food supply 1. Land-based animal food
products and their health effects.
Maxcy, R. B.; Price. W. V. and h i n e . D. M. 1955. Irnproving curd-forming properties of
homogenized milk. J. Dairy Sci. 38: 80-86.
McDonall. F.H.1953. The buttermaker's manual.vol. 1. New Zealand University Press.
McGuire. Michelle K.; Park. Youngsoon; Behre, Rebecca A.; Harrison. Lisa Y.. Shultz,
Terry D.; Mc Guire, Mark A. 1997. Conjuagted linoeic acid concentrations of humanmilk
and infant formula. Nurition Researcli . 17 (8): 1277- 1283.
McMahon, D.J.and Brown. R.J. 1984. Enzymic coagulation of casein micelles: a review.
J Dniry Sci 67 :9 1 9
Nash, D.M.; Hamilton. R.M.G.; Sanford, K.A.; and Hulün, H.W. 1996. The effect of
dirtÿry menhaden meal and storüge on the omega-3 fatty acids and sensory attributes of
egg yolk in laying hens. Cam J. Animal Sci. 76:377-383
Nash D.M.;
Hamilton. R.M.G. and Hulan. H.W. 1995. The effect of herring meül on the
omegü- 3 fatty ücid content of plasma and egg yolk lipids of laying hens. Clin J Anitn Sci
75:247-253
Nash D.M.; Hamilton. R.M.G.;
Smford, KA.; Hulan. H.W. 1996. The effect of dietary
rnenhaden med and storage on the ornega-3 fatty acids and sensory attributes of egg yolk
in laying hens. Cun J ofAnini Sci 377-383
Nettleton. J.A. 1991. c+3 fatty acids: cornparison of plant and s e a f d sources in human
nutrition. J of the Am Dief Assoc 9 1:33 1-337
Nettleton J. 1995.Omega-3fatty acids mdhealth. New York. Chapman&Hall.
Neuringer. M. and Connor. W.E. 1989.Ornega-3 fatty acids in the reinü. Dietary 0 3 and
w6 fatty acids: biological effects and nutritional essentiality. 177- 190
Newton. Ian S. 1997. Global food fortification perspectives of long-chain 03 fatty acids
In: The return of 0 3 fatty acids into the food supply 1. land-based animal food products
and their heiilth effects. Edited by A.P. Simopoulos. Karger
Nwokolo. E. and Sim. 1. 1989. Barley ÿnd full fat crnolü seed in broiler diets. Poitlt Sci
68: 1374.
Oh. Suk Y; ryue. Jehong; Hsieh. Chia-Hong: and Bell. Dean E. 1991. Eggs Enriched in
0 3 fatty acids and alteration s in lipid concentration s in plasma and lipoproteins and in
blood pressure. Ani. J. Clin. Nutr. 54: 689-695
Okigbo. LM.;Richardson. G.H.; Brown, R.J. and Emstrom. C.A. 1985. Casein
composition of cow's milk of different chyrnosin coagulation propenies. J D d r ~Sci
68: 1887
Omara-Alwala T.R.; Mebrahtu. T.; Prior D.E.;Ezekwe. M.O.1991.Omega-3 fatty acids
in purslance (Portulaca oleracea) tissues. J Am Oil Chen, Soc 68: 198- 199
Parkash. S. and Jenness. R. 1968. The composition and characteristics of goat milk. D d r y
Sci Abstr 30:67
Phillipson, A.T. 1970. Physiology of digestion and metabolism in the ruminant. Oriel
Press
Phillips. Lance, G.; McGiff, Mary Lou; Barbano. David, M. and Lawless. Harry T. 1995.
The influence of fat on the sensory properties, viscosity, and color of lowfat milk. I.
Dai- Sci. 78: 1258- 1266.
Porneranz Yeshüjahu and Meloan Clifton E. 1987. Electrophoresis in food analysis:
theory and practicr. 2" edition. Van Nostrand Reinhold Cornp. New York.
Rümbjor, Gro S.; Walen. Ann 1.; Windsor. Sheryl L.; and Harris. William S. 1996.
Eicosapentaenoic Acid is Primarily Responsible for hypotriglyceridemic effect of fish oil
in humans. Lipids. 3 1: S-45449
Rasic, J. Lj. And Kurmann. J. A. 1978. Yoghurt: scientific grounds, technology,
manufacture and prepürations.
Romans, J. Wuff D.; Johnson. R.; Libd, G.;Costello. W. 1995. Effect of ground flaxseed
in sw ine diets on pig performance and on physical and sensory chuacteristics and omegü3 content of pork. II. Duration of 15% dietary flazseed. J Anim Sci 73 : 1987- 1999
Rosenthal. Ionel. 1991. Milk and Dairy Products: Propenies and Processing. Balaban
Publishers.
Salonen. J.T.; Sdonen. R.; Seppanen. K.; Rinta-Kiikka. S.; Kuukka. M.; Korpela, H.:
Alfthan. G.; Kantola. M.; Schalch W. 199 1. Effects of antioxidant supplementation of
plütelet function a randomized pair-matched. placebo-controlled, double-blind trial in
men with low üntioxidant status. Am J Clin Ncrrr 53: 1222- 1229
Sanders. T.A. B. 1985. Influence of fish-oil supplements on man. Proceedings of rhe niitr
soc 4439 1-397,
Sanden. T.A.B. 1989.Omegr-3 fatty acids and lipoproteins. Dietüry 03 and O% fiütty
acids: biological effects and nutritional essentiality. 153-262
Sander. T.A.B.. Reddy. Sheela, 1992. The influence of a vegetariün diet on the b t t y rcid
composition of human milk and the essential fiitty acid status of the infant. The J
Pedi~~tric
April S7 1477
Schukken Ynte H.;Leslie. K. E.; Weersink. A. J. and Martin. S. W. 1992. Ontario bulk
milk somatic ce11 count reduc tion program. 1 . Impact on somatic ceIl counts and milk
quality. J Dairy Sci 753352-3358
Shackleford. S.: Reagan. J.; Haydon. K.; Miller M. 1990.Effects of feeding elevated
levels of monounsaturated fats to growing-finishing swine on acceptability of boneless
h m s . J Food Sci 55:l485-lW.
Shantha. Nalur C.; Ran. Lathr N.; O' Leary. Joe.; Hichks, Clair L. and Decker. Eric A.
1995.Conjugated linoleic acid concentrations in dairy products üs affected by procesing
and storage. J. of Food Sci. 60(4): 695-697
Sims. S.J. 1990. Flÿx seed as a high rnergy/protein/omega-3 fatty ücid feed ingredient for
pouliry. In proceeding of the 53rd Flax institute of the United States. 65.
Simopoulos. Anemis. P. 1989. Summiuy of the NATO ûdvünced reseürch workshop on
dietary 0 3 and 06 fatty acids: biological effects and nutritional essentidity. J of Ani bt of
Nurr 52 1-528
Simopoulos. AP. 1991.Omega-3 fatty acids in health and diseüse and in growth and
development. Am J clin Nutr 54:438-463
Simopoulos, Artemis P.; Norman, Helen A.; Gillaspy. James E.; Duke. James A. 1992.
Common purslane: a source of omega-3 fatty acids and antioxidants. J of the Am College
of Nutr 1 1(4):374-392
Simopoulos. Artemis P; and Jr. Norman Salem. 1992. Egg yolk as a source of long-chain
polyunsaturated fatty acids in infant feeding. Am. J. clin. Niitr. 55: 4 1 1-4 14.
Simopoulos. Artemis P. 1997. Overview of evolutionary aspects of 03 fatty acids in the
diet. [N: The return of 03 fatty acids into the food supply 1. land-based animal food
prodiicts and their health effects. Edited by A.P. Simopoulos. Karger
Spain. LN. and Polan. C.E. 1995. Evaluating effects of fish nieal on milk fat yield of
dairy cows. J Duiry Sci 78: 1 142- 1 153
Stÿnton. C.. Lawless. F.. Kjellmer. G..Harrington. D.. Devery. R.. Connonllly. J.F.. and
Murphy. J . 1997. Dietary influences on bovine milk cis-9. t m s - I 1 conjugütçd linolric
acid content. J. of Food Sei. 62 (5): 1083- 1O86
Steffe. J.F. 1996. Rheological methods in food process engineering. 2"' Edition. Freeman
Press.
Stern. M.D.and H.R. Mansfield, 1989. Feehtuffs 6 1 : 16
Storry, J. e. and Ford, GD. 1982. Development of coagulum firmness in renneted milk - a
two-phase process
Sugano. M. 1996. Characteristics of fats in Japanese diets and current recommendations.
Lipids. 3 1 :S283-S286
Tuorila. H.1987. Selection of milks with varying fat contents and related overall liking,
attitudes. noms and intentions. Appctirte 8: 1
Uüuy-Dagach. R. and Valenzuela. A. 1992. Marine oils as a source of omega-3 fatty acids
in the diet: how to optimize the health benefits. Progras in food nutrition science.
16: 199-243
Ustunol. 2.;Küwachi. K. md Steffe. J. 1995. Rheologicrl properties of Cheddar cheese as
influenced by fat reduction and ripening tirne. J. Food Sci. 60: 1208-12 10
Van Elswyk. Mary. E. 1997. cornparison of n-3 fatty acid sources in Iiiying hen rations
for improvement of whole egg nutritionai quality: a review. BN. J. NUtri 78: s61-~69.
Walstra. Pieier and Jenness. Robert. 1984. Dairy Chemistry and Physics. A WileyInterscience Publication John Wiley & Sons
Watkins, Bruce A. 1997. Regulatory Effects of Polyunsaturates on Bone Modeling and
Cartilage function. [N: The retum of 03 fatty acids into the food supply 1. land-based
animal food products and their health effects. Edited by A.P. Sirnopoulos. Karger
Wright. Tom.; McBride. Brian.; Holub. Bruce. 1998. Docosahexaenoic acid-enriched
milk LN:The retum of 0 3 fatty acids into the food supply 1. land-büsed animal food
products and their hedth effects. Edited A.P. Simopoulos. Karger
Zamula. E.; The Greenland diet: can fish oils prevent heart diseüse? FDA consumer. Oct.
6, 1986
Appendix 3.1
Name:
Product:
Date:
Please taste each sarnple and assign a grade between 1 and 5 with I being the lowest
quaiity and 5 being the highest quality.
Identification Number:
Grade:
Comment:
Appendix 3.2
Name:
Product :
Date:
Two of these three samples are identical. the third sample is different.
Please taste the sarnples in the order indicated and identify the odd sample. If you can not
identiQ the odd sample, please guess.
Product Identification Nurnber
Comments:
Check Odd Sample
Appendix 4.1
Narne:
Product: Cheddar Cheese
Date:
Two of these three samples are identical, the third is different.
1.
Taste the samples in the order indicated and identify the odd sample.
Code
Check Odd Sample
2.
Indicate the degree of difference between the duplicate sarnples and the odd
sample.
Slight
Moderate
Much
Extreme
3.
Acceptability :
Odd sample more acceptable
Duplicates more acceptable
4.
Comments: