Gori Alessandro Tesi
ALMA MATER STUDIORUM – UNIVERSITÀ DEGLI STUDI DI BOLOGNA
FACOLTÀ DI AGRARIA
DOTTORATO DI RICERCA IN BIOTECNOLOGIE
DEGLI ALIMENTI
Tesi per il conseguimento del titolo di Dottore di Ricerca
Triennio Accademico 2006/2008 – XXI Ciclo di Dottorato
Settore scientifico-disciplinare: AGR/15
APPLICAZIONE DI DIVERSE TECNICHE ANALITICHE
STRUMENTALI ALLA VALUTAZIONE SELETTIVA DI
COMPONENTI BIOSENSIBILI IN MATRICI DI
ORIGINE ANIMALE E VEGETALE
-APPLICATION OF DIFFERENT INSTRUMENTAL TECHNIQUES TO THE SELECTIVE EVALUATION
OF BIO-SENSIBLE COMPOUNDS IN FOODSTUFF OF ANIMAL AND VEGETABLE ORIGIN-
Tutor:
Tesi presentata da:
Prof. Giuseppe Losi
Dott. Alessandro Gori
Co-Tutor:
Prof.ssa Maria Fiorenza Caboni
Coordinatore:
Prof. Giuseppe Losi
ESAME FINALE ANNO 2009
“Litterarum radices amarae sunt,
fructus iucundiores”
Cato
PhD’s RESEARCH ACTIVITIES
Publications
Castagnetti GB, Delmonte P, Melia S, Gori A, Losi G (2007). The effect of extruded
whole linseed flour intake on the variation of CLA (Conjugated Linoleic Acid)
content in milk, and OFA (Oxydated Fatty Acid) on cheese obtained - The
Reggiana cattle’s case. Sci. Tecn. Latt-Cas., 58 (6), 363-382.
Carretero AS, Carrasco-Pancorbo A, Cortacero-Ramíreza S, Cañabate-Díaz B, Gori
A, Cerretani L, Fernández-Gutiérrez A (2008). Direct analysis of 4-desmethyl
sterols and two dihydroxy triterpenes in saponified vegetal oils using LC-MS. Eur.
J. Lipid Sci. Technol., 110, 1142–1149
Castagnetti GB, Delmonte P, Melia S, Gori A, Losi G (2008). The effect of extruded
whole linseed flour intake on the variation of CLA (Conjugated Linoleic Acid)
content in milk - The Reggiana cattle’s case - L’effetto dell’integrazione della
razione con farina di lino estrusa sul contenuto in CLA (Acido Linoleico
Coniugato) nel latte - il caso della razza Reggiana. Prog. Nutr., 10, (3), 174-183.
Gori A, Gambini G, Pecorari A, Nocetti M, Losi G (2008). Fatty Acids composition
of Parmigiano Reggiano cheese samples, with particolar interest of trans Fatty
Acids - La Composizione in acidi Grassi in campioni di formaggio Parmigiano
Reggiano, con particolare riferimento agli isomeri trans (TFA). Sc.i Tecn. LattCas., 59 (6).
Meetings
Gori A, Seller M, Delmonte P(September 2006). Comparison of different techniques
for trans fatty acids analysis. Proceedings of the 120° AOAC INTERNATIONAL,
Minneapolis, Minnesota, USA.
Chiavaro E, Vittadini E, Bendini A, Cerretani L, Gori A, Rodriguez-Estrada MT
(September 2007). Detection of hazelnut oil in extra virgin olive oil by DSC.
Proceedings of the 5th EuroFed Lipid Congress, Goteborg, Sweden.
Gori A, Castagnetti GB, Delmonte P, Melia S, Losi G (September 2007). The effect
of extruded whole linseed flour intake on the variation of CLA (Conjugated
Linoleic Acid) content in milk - The Reggiana cattle’s case. Proceedings of the II
International Congress on Conjugated Linoleic Acid (CLA), Cagliari, Italy.
Gori A, Gambini G, Pecorari A, Nocetti M, Losi G (June 2008). Fatty Acids
composition of Parmigiano Reggiano cheese samples, with particolar interest of
trans Fatty Acids”- “La Composizione in acidi Grassi in campioni di formaggio
Parmigiano Reggiano, con particolare riferimento agli isomeri trans (TFA).
Proceedings of the first Italian Dairy Congress (I Congresso Lattiero Caseario),
Bologna, Italy.
Workshops
119th AOAC INTERNATIONAL, Orlando, Florida, September 2005.
12th Workshop on the Developments in the Italian PhD Research on Food Science
and Technology, Università Mediterranea, Facoltà di Agraria, Reggio Calabria
(RC), 12-14 September 2007.
13th Workshop on the Developments in the Italian PhD Research on Food Science
and Technology, Centro Ricerche Soremartec-Ferrero, Alba (CN), 10-12
September 2008
INDEX
1 SUMMARY..............................................................................................................................................5
2 INTRODUCTION ...................................................................................................................................7
2.1. LIPIDS ...........................................................................................................................................9
2.2. CLASSIFICATION......................................................................................................................10
2.3. NOMENCLATURE OF FATTY ACIDS ....................................................................................13
2.4 VEGETABLE OILS AND FATS .................................................................................................15
2.4.1. OLIVE OIL ........................................................................................................................16
2.4.1.1. Fatty Acids ...............................................................................................................18
2.4.1.2. Triacylglycerols, and Partial Glycerides ..................................................................19
2.4.1.3. Hydrocarbons ...........................................................................................................20
2.4.1.4. Tocopherols..............................................................................................................20
2.4.1.5. Pigments...................................................................................................................21
2.4.1.6. Aliphatic and aromatic alcohols...............................................................................22
2.4.1.7. Sterols ......................................................................................................................23
2.4.1.8. Triterpene acids........................................................................................................24
2.4.1.9. Volatile and aroma compounds................................................................................25
2.4.1.10. Other minor constituents ........................................................................................25
2.4.2. DAIRY PRODUCTS .........................................................................................................26
2.4.2.1. Fatty acids ................................................................................................................28
2.4.2.2. Glycerides ................................................................................................................32
2.4.2.3. Hydrocarbons ...........................................................................................................33
2.4.2.4. Tocopherols..............................................................................................................34
2.4.2.5. Sterols ......................................................................................................................34
2.4.2.6. Phospholipids ...........................................................................................................34
2.4.2.7. Free fatty acids .........................................................................................................37
2.4.2.8 Lipo-soluble vitamins................................................................................................37
2.4.2.9 The fat in milk products ............................................................................................39
2.5. CHEMICAL ASPECTS ...............................................................................................................40
2.5.1. LIPOLYSIS........................................................................................................................40
2.5.2. AUTOXIDATION .............................................................................................................40
2.6. PROCESSING OF FATS AND OILS .........................................................................................41
2.6.1. PROCESSING OF FAT AND OIL....................................................................................41
2.6.2. HYDROGENATION .........................................................................................................42
2.6.3. INTERESTERIFICATION ................................................................................................43
2.7. REFERENCES.............................................................................................................................44
3. COMPARISON OF DIFFERENT TECHNIQUES FOR TRANS-FATTY ACIDS ANALYSIS .47
3.1. SUMMARY AND KEYWORDS ................................................................................................48
3.2. INTRODUCTION........................................................................................................................49
3.3. MATERIALS AND METHODS .................................................................................................50
3.3.1. SAMPLES PREPARATION .............................................................................................50
3.3.2. ANALYSIS BY GAS CHROMATOGRAPHY.................................................................50
3.3.3. ANALYSIS BY AG+-HPLC .............................................................................................50
3.3.4. AG+-HPLC FRACTIONATION FOLLOWED BY GC ANALYSIS...............................51
3.3.5. ATTENUATED TOTAL REFLECTION FT-IR ...............................................................51
3.4. RESULTS AND DISCUSSION...................................................................................................51
TABLES..............................................................................................................................................53
FIGURES ............................................................................................................................................54
3.5. REFERENCES.............................................................................................................................58
4. THE EFFECT OF EXTRUDED WHOLE LINSEED FLOUR INTAKE ON THE VARIATION
OF CLA (CONJUGATED LINOLEIC ACID) CONTENT IN MILK, AND OFA (OXYDATED
FATTY ACID) ON CHEESE OBTAINED - THE REGGIANA CATTLE’S CASE .....................59
4.1. SUMMARY AND KEYWORDS ................................................................................................60
4.1. SUMMARY AND KEYWORDS ................................................................................................60
4.2. INTRODUCTION........................................................................................................................61
4.3. MATERIALS AND METHODS .................................................................................................62
4.3.1. SAMPLES COLLECTION................................................................................................62
4.3.2. SAMPLES PREPARATION .............................................................................................63
4.3.3. GAS CHROMATOGRAPHIC QUANTITATION............................................................64
4.3.4. HPLC ANALYSIS.............................................................................................................64
4.3.5. STATISTYCAL ANALISYS ............................................................................................64
4.3.6. CHEESE SENSORY ANALISYS.....................................................................................65
4.4. RESULTS AND DISCUSSION...................................................................................................65
4.5. CONCLUSIONS ..........................................................................................................................68
TABLES..............................................................................................................................................70
FIGURES ............................................................................................................................................73
4.6. REFERENCES.............................................................................................................................80
5. A SIMPLIFIED METHOD FOR HPLC-MS ANALYSIS OF STEROLS IN VEGETABLE OIL
....................................................................................................................................................................85
5.1. SUMMARY AND KEYWORDS ................................................................................................86
5.2. INTRODUCTION........................................................................................................................87
5.3. MATERIALS AND METHODS .................................................................................................89
5.3.1. CHEMICALS.....................................................................................................................89
5.3.2. INSTRUMENTATION......................................................................................................90
2
5.3.3 ANALYTICAL METHOD .................................................................................................90
5.3.3.1. OFFICIAL METHOD..............................................................................................90
5.3.3.2. PROPOSED METHOD ...........................................................................................90
5.3.4. SAMPLES..........................................................................................................................91
5.3.5. SAMPLE PREPARATION FOR STEROLS AND DIHYDROXY TRITERPENES .......91
5.3.5.1. OFFICIAL METHOD..............................................................................................91
5.3.5.2. PROPOSED METHOD ...........................................................................................92
5.4. RESULTS AND DISCUSSION...................................................................................................92
5.4.1. ISOLATION OF STEROLS AND DIHYDROXY TRITERPENES FROM THE
UNSAPONIFIABLE FRACTION USING THE REDUCED METHOD ...................................92
5.4.2. HPLC-MS CONDITIONS .................................................................................................93
5.4.3. IDENTIFICATION OF STEROLS AND DIHYDROXY TRITERPENES IN
DIFFERENT SAMPLES .............................................................................................................94
5.4.4. ANALYTICAL PARAMETERS.......................................................................................96
5.4.5. APPLICATION AND QUANTIFICATION TO REAL SAMPLES.................................97
5.4.6. COMPARATIVE STUDY.................................................................................................98
5.5. CONCLUSIONS ..........................................................................................................................98
TABLES............................................................................................................................................100
FIGURES ..........................................................................................................................................102
5.8. REFERENCES...........................................................................................................................105
6. QUANTITATION OF LONG CHAIN POLY-UNSATURED FATTY ACIDS (LC-PUFA) IN
BASE INFANT FORMULAE BY GAS CHROMATOGRAPHY, AND EVALUATION OF THE
BLENDING PHASES ACCURACY DURING THEIR PREPARATION....................................109
6.1. SUMMARY AND KEYWORDS ..............................................................................................110
6.1. SUMMARY AND KEYWORDS ..............................................................................................110
6.2. INTRODUCTION......................................................................................................................111
6.3. MATERIALS AND METHODS ...............................................................................................113
6.3.1. SAMPLES COLLECTION..............................................................................................113
6.3.2. SAMPLES PREPARATION ...........................................................................................113
6.3.2. GAS CHROMATOGRAPHIC QUANTITATION..........................................................114
6.3.3. STATISTYCAL ANALYSIS ..........................................................................................114
6.4. RESULTS AND DISCUSSION.................................................................................................114
6.5. CONCLUSIONS ........................................................................................................................117
TABLES............................................................................................................................................119
FIGURES ..........................................................................................................................................126
6.6. REFERENCES...........................................................................................................................134
3
7. FATTY ACIDS COMPOSITION OF PARMIGIANO REGGIANO CHEESE SAMPLES, WITH
EMPHASIS ON TRANS ISOMERS (TFA) .....................................................................................135
7.1. SUMMARY AND KEYWORDS ..............................................................................................136
7.2. INTRODUCTION......................................................................................................................137
7.3. MATERIALS AND METHODS ...............................................................................................138
7.3.1 SAMPLES COLLECTION...............................................................................................138
7.3.2. PRELIMINARY ANALYSIS OF THE ORGANIC AND INORGANIC FRACTION ..138
7.3.3. SAMPLE PREPARATION..............................................................................................138
7.3.4. GAS CHROMATOGRAPHIC QUANTITATION..........................................................139
7.4. RESULTS AND DISCUSSION.................................................................................................139
7.5. CONCLUSIONS ........................................................................................................................140
TABLES............................................................................................................................................142
FIGURES ..........................................................................................................................................146
7.6. REFERENCES...........................................................................................................................147
4
1
SUMMARY
This PhD thesis describes the application of some instrumental analytical techniques
suitable to the study of fundamental food products for the human diet, such as: extra
virgin olive oil and dairy products. These products, widely spread in the market and
with high nutritional values, are increasingly recognized healthy properties although
their lipid fraction might contain some unfavorable components to the human health.
The research activity has been structured in the following investigations:
™
“Comparison of different techniques for trans fatty acids analysis”
™
“Fatty acids analysis of outcrop milk cream samples, with particular emphasis on
the content of Conjugated Linoleic Acid (CLA) and trans Fatty Acids (TFA), by
using 100m high-polarity capillary column”
™
“Evaluation of the oxidited fatty acids (OFA) content during the ParmigianoReggiano cheese seasoning”
™
“Direct analysis of 4-desmethyl sterols and two dihydroxy triterpenes in saponified
vegetal oils (olive oil and others) using liquid chromatography-mass spectrometry”
™
“Quantitation of long chain poly-unsatured fatty acids (LC-PUFA) in base infant
formulas by Gas Chromatography, and evaluation of the blending phases accuracy
during their preparation”
™
“Fatty acids composition of Parmigiano Reggiano cheese samples, with emphasis on
trans isomers (TFA)”
Keywords: dairy fats, vegetable oils, separation techniques, fatty acids, TFA, CLA,
OFA.
5
2
INTRODUCTION
The increasing interest regarding the relation between nutrition and wellness, has led the
scientific research to study the importance of nutrients contained in both the raw
material and their derived food products. Extra virgin olive oil and dairy products,
considered as fundamental food products for the diet of million of people, are source of
liposoluble vitamins (A, D, E), anti-oxidant compounds and mineral salt, although are
simply deemed by consumers as “food dressing” and high energetic foods (since they
are predominantly constituted of fat).
Healthy properties are widely recognized to these food products by the scientific panel.
To the extra virgin olive oil, for example, are attributed several beneficial effects: the
high content in mono-unsatured fatty acids, with the oleic acid as the principal
constituent, shows to have gastro-protective properties, inhibits biliary lithiasis and
improves the intestinal transit regulating it, reduces the LDL cholesterol levels (Low
Density Lipoprotein, the potentially harmful cholesterol with atherogenic effects)
leaving unaltered the HDL levels (High Density Lipoprotein, prevent the oxidized
cholesterol sticks to artery walls), it has antioxidant effect on the LDL (1), enforces the
immune system reducing the risk of autoimmune diseases/ breast cancer/colon-recto
cancer ( 2). Extra virgin olive oil contains antioxidant compounds with proved antiinflammatory and anti-carcinogenic actions (3, 4). Moreover, since it does not subjected
to the refinetion process (on the contrary of what happens for all the others edible oils),
it does not meet to the formation of trans fatty acids.
7
Several investigations about milk fat and, as a consequence, butter and cheese fat,
confirmed the high potential in human nutrition of CLA (Conjugated Linoleic Acid), in
particular, of rumenic acid (C18:2 cis-9, trans-11), and consequently, of its forerunner
vaccenic acid (C18:1 trans-11) that represent the principal isomer of the fatty acids
group called TFA (trans Fatty Acid).
At the present, CLA have attracted considerable attention because of their attitude to
contrast the risk of some lifestyle-related diseases, such as: tumors, atherosclerosis,
diabetes, obesity, and hypercholesterolemia. Moreover, they enhance the immune
system modulation (5, 6, 7). These properties have been verified in animal models and
human cellular lines (8, 9).
Unlike the olive oil, dairy products naturally contain significant levels of fatty acids in
trans configuration. Several studies have reported the negative effects of these isomers
to human health. In 2004, the European Food Safety Authority (EFSA) affirmed that the
negative effects of TFA on human health might be worst than those of saturated fatty
acids. The origin of TFA is due to the rotation of the molecule around a “double
bound”, leading the natural configuration cis-cis to cis-trans. This configurational
overthrow has noteworthy negative effects on human health: TFA, besides increasing
LDL levels, make the cell membrane more permeable allowing molecules, even toxics,
to filter inside the cell. They can weaken the immune system and worsen the deficiency
of essential fatty acids, thwarting the production of the prostaglandins, which regulate
the vascular smooth mussels in blood vessels, kidneys functions, and the inflammatory
responses (10). Although trans fats are naturally present in the ruminant fat as the result
of an enzymatic production (formation of specific isomers), they should be
8
distinguished from the “artificial” trans fat industrially produced under catalytic
conditions (more random distribution of isomers) such as Partially Hydrogenated
Vegetable Oils (PHVO). Since the TFA isomers responsible for the negative effects
have not yet been identified, it makes it difficult to asses whether the TFA from PHVO
and ruminant fats present similar risk factor (11). Moreover, according to the results of a
Danish study, not only the intake of TFA from ruminant fats affect the risk of coronary
health disease, but even more, they do not show any correlation (12).
Another concern, relative to the group which CLA belong to, the Poli-unsatured Fatty
Acids (PUFA), is their susceptibility to the oxidation process, especially whether they
are in the free form (13). Nutritionally, such oxidation can be translated as a negative
health effect because of the inclination to increase the LDL levels, meaning a higher
risk of pathologies related to the atherosclerosis disease (14).
Therefore, it raise spontaneous the need to elaborate scientific methods of analysis that
both permit to value, identify, and eventually improve the fatty acid composition of
olive oils and butters (protecting consumers from sophistications and frauds), and to
point out the tight correlation between natural/ anthropic factors correlated to the
production and the chemical-physical-organoleptic properties, that is “the typicality”.
2.1. LIPIDS
Despite the existence of several definition of lipids, due to their complexity and
heterogeneity, they are generally reported as a broad group of compounds that are
soluble in organic solvents (diethyl ether, hexane, benzene, chloroform or methanol) but
only sparingly soluble in water. They are major components of adipose tissue and,
together with proteins and carbohydrates, they constitute the principal structural
9
components of all living cells. The terms fats and oils refer traditionally to glycerol
esters of fatty acids, which make up to 99% of the lipid of plant and animal origin. The
two terms are used interchangeably and the choice of terms is usually based on the
physical state of the material at ambient temperature and tradition. Generally, fats
appear solid at ambient temperatures and oils appear liquid.
Lipids are important components that contribute very significantly to the nutritional and
sensory value of almost all kinds of foods, except for most fruits, sweets and beverages.
Food lipids are either consumed in the form of “visible” fats, such as butter, lard and
shortening or as constituents of basic foods, such as milk, cheese and meat.
The effect on food quality is mainly related to the contents, distribution in the food
matrix, chemical composition and reactivity of the lipids, as well as to their physical
properties (crystalline structure, melting properties) and changes due to processing and
the interactions with other components. Indeed, during the processing, storage and
handling of foods, lipids undergo complex chemical changes (i.e.: lipolysis, oxidation)
and react with other food constituents, producing several compounds both desirable and
deleterious to food quality.
2.2. CLASSIFICATION
Lipid structures can be classified depending on:
• The physical properties at room temperature. For instance, oils appear liquid and fats
appear solid;
• The polarity. Neutral lipids include fatty acids, alcohols, glycerides and sterols, while
polar lipids, glycerophospholipids and glyceroglycolipids;
• Their essentiality for humans (essential and nonessential fatty acids);
10
• The structure, which can be respectively simple or complex.
Based on structure, lipids can be classified as derived, simple or complex. The derived
lipids include fatty acids and alcohols, which are the building blocks for the simple and
complex lipids. Simple lipids, made up of fatty acids and alcohol components, include
acylglycerols, ether acylglycerols, sterols and their esters and wax esters. In general
terms, simple lipids can be hydrolyzed to two different components, usually an alcohol
and an acid. Complex lipids include glycerophospholipids (phospholipids),
glyceroglycolipids (glycolipids), and sphingolipids. These structures yield three or more
different compounds on hydrolysis.
A general classification of lipids based on their structure is proposed in table 2.1. even
though it should be taken as a guide since other classifications may be more useful. The
most abundant class of food lipids is the acylglycerol, which dominate the composition
of depot fats in animals and plants. The polar lipids are found almost entirely in the
cellular membranes (phospholipids being the main components of the bilayer) with only
very small amounts in depot fats. In some plants, glycolipid constitute the major polar
lipids in cell membranes. Waxes are found as protective coating on skin, leaves and
fruits. Edible fats are traditionally classified in different subgroups illustrated in table
2.2.
11
Major classes
Simple lipids
Subclasses
Acylglycerols
Waxes
Descriptions
Glycerol + fatty acids
Long-chain alcohol + long-chain fatty acid
Compound lipids
Phosphoacylglycerols (or glycerophospholipids)
Glycerol + fatty acids + phosphate + another group usually containing nitrogen
Sphingomyelins
Cerebrosides
Gangliosides
Spingosine + fatty acid + phosphate + choline
Spingosine + fatty acid + simple sugar
Spingosine + fatty acid + complex carbohydrate moiety (including salicilic acid)
Lipid materials not simple or compound
Carotenoids, steroids, fat-soluble vitamins
Derived lipids
Table 2.2. – Lipid subgroups
Lipid subgroups
Milk fats
Lauric acids
Vegetable buters
Oleic-linoleic acids
Linolenic acids
Animal fats
Decription of the kind of fat
Fats from the milk of ruminants (dairy cows)
Main fatty acids
Palmitic, oleic, stearic and appreciable amounts of short chain fatty
acids (C4:0 to C12:0), small amounts of branched, odd-numbered and
trans
Fats from certain species of palm (coconut, babasu)
Lauric acid (40-50%), moderate amounts of C6:0, C8:0 and C10:0,
low in unsaturated acids
Fats from the seed of various tropical trees: vegetable butters (cocoa Saturated fatty acids
butter) used in the manufacture of confections
Oils of vegetable origin: cottonseed, corn, peanut, sunflower, saflower, Oleic and linoleic acid, less than 20% saturated fatty acids
olive, palm and sesame oils
Soybean, rapeseed, flaxsed, wheat germ, hempseed and perilla oils
Substantial amount of linolenic acid
Large amount of C16 and C18 fatty acids, medium amount of
Fats from domestic land animals (lard and tallow), egg lipids
unsaturated acids (oleic, linoleic) and small amount of odd-numbered
acids
12
Table 2.1. – Classification of lipids
2.3. NOMENCLATURE OF FATTY ACIDS
The term fatty acid (FA) refers to any aliphatic monocarboxylic acid that can be
liberated by hydrolysis from naturally occurring fats. Most of FA were originally
described under “trivial” or common name and even after adopting the Internation
Union of Pure and Applied Chemistry (IUPAC) system for nomenclature, the habit of
assigning trivial names to FA acids continues.
In standard IUPAC terminology, the fatty acid is named after the parent hydrocarbon
with the same name of carbon atoms. The terminal letter e in the name of the parent
hydrocarbon is replaced with oic. For example, an 18-carbon carboxylic acid is called
octadecanoic acid, from octadecane, the 18-carbon aliphatic hydrocarbon.
Unsaturated FA can be named after the parent unsaturated hydrocarbon and replacement
of the terminal anoic by enoic indicates unsaturation and di, tri and so on represent the
number of double bonds (i.e.: hexadecenoic acid for 16:1, octadecatrienoic acid for
18:3).
The simplest way to specify the location of double bonds is to put, before the name of
the acid, one number for each unsaturated linkage (∆ configuration) representing the
distance from the carboxyl carbon. Oleic acid is, for example, named ∆9-octadecenoic
acid or simply 9-octadecenoic, with one double bond between carbons 9 and 10
(carboxyl group is regarded as carbon 1). Nevertheless, unsaturated FA are often
distinguished by the location of the first double bond from the methyl end of the
molecule, that is, the omega (ω) carbon (shorthand identification). The methyl group is
number 1 (the last character in the Greek alphabet is ω, hence the end): linoleic acid
(cis-9,12-octadecadienoic acid) is therefore 18:2ω6 (or n-6) acid. Fig. 2.1. illustrates the
difference between IUPAC ∆ and shorthand numbering systems.
13
Outside of molecule ∆ numbering
Inside of molecule ω numbering
1
HOOC
18
17
2
16 3
18 1
17
15
2
16
15
4
14 5
3
13
4
12
14
13
5
6
11
7
12
8
11
6
7
8
10
9
10
9
18:3cis-6,cis-9,cis-12
18:3ω6
Figure 2.1. – IUPAC ∆ and common ω numbering system.
The geometric configuration of double bonds is usually designated by the use of terms
cis (Latin, on this side) and trans (Latin, across), indicating whether the alkyl group are
on the same or opposite sides of the molecule (Figure 2.2.). The prefixes cis and trans
can be abbreviated as c and t in structural formulas. In shorthand notation, the
unsaturated fatty acids are assumed to have cis bonding and, if the fatty acid is
polyunsaturated, double bonds are in the methylene interrupted positions.
R1
R1
R2
H
H
H
H
R2
trans-
cis-
Figure 2.2. – Example of cis/trans nomenclature.
In the following page a list of some of the most common FA found in natural fats is
reported (Table 2.3.), indicating both systematic and common name for each FA.
14
Table 2.3. – Nomenclature of some common fatty acids.
Abbreviation
4:0
6:0
8:0
10:0
12:0
14:0
16:0
16:1 n-7
18:0
18:1 n-9
18:1 n-7
18:2 n-6
18:3 n-3
20:0
20:4 n-6
20:5 n-3
22:1 n-9
22:5 n-3
22:6 n-3
Systematic name
Butanoic
Hexanoic
Octanoic
Decanoic
Dodecanoic
Teradecanoic
Hexadecanoic
cis-9-Hexadecenoic
Octadecanoic
cis-9-Octadecenoic
cis-11-Octadecenoic
cis-9,12-Octadecadienoic
cis-9,12,15-Octadecatrienoic
Eicosanoic
cis-5,8,11,14-Eicosatetraenoic
cis-5,8,11,14,17-Eicosapeantaenic
cis-13-Docosenoic
cis-7,10,13,16,19-Docosapentaenoic
cis-4,7,10,13,16,19-Docosahexaenoic
Common or trivial name
Butyric
Caproic
Caprylic
Capric
Lauric
Myristic
Palmitic
Palmitoleic
Stearic
Oleic
Vaccenic
Linoleic
α-Linolenic
Arachidic
Arachidonic
EPA
Erucic
DPA
DHA
2.4 VEGETABLE OILS AND FATS
Reference for this section: 15, 16
Most fats and oils consist of triacylglycerides which differ in fatty acid composition to a
certain extent. Other constituents which make up less than 3% of fats and oils, are the
unsaponifiable fraction (phospholipids, tocopherols, sterols, resins, carbohidrates,
pesticides, proteins trace metals, and pigments) and a number of acyl lipids (traces of
free fatty acids, mono and diacylglycerols). Their composition in fatty acids can differ
greatly, and depends for several factors: plant fat is affected by the cultivar and growth
environment, such as climate and location of the plant; animal fat depends by the kind
and breed of animal and by the feed.
15
Oils and fats naturally occur in a wide range of sources, each one providing a separate
and distinctive material. Hundreds of seeds and fruits bear oil, all animal produce fat,
and marine sources also provide oils; however, only a few of these sources are of
economic importance. The factors that have always influenced their distribution and
eating habit of our ancestors result to be, unequivocally, climate and availability. For
instance, in northern European countries, consumers generally obtain their edible fats
from animals, whereas people in southern Europe, Asia, and Africa acquire their edible
oils from vegetable sources. The food products developed in these different regions use
the available fats and oils products, and consequently, the cuisine of central and
northern European countries developed around the use of solid fats such as butter and
lard, while the diets of inhabitants from warmer climates around the use of liquid oils
for their food products. Since the second half of last century, food products from the
Mediterranean countries have definitely increased of importance in food science due to
their beneficial effects on human health, especially for their principal source of fat: olive
oil and dairy fat.
2.4.1. OLIVE OIL
Reference for this section: 15, 16
Between the vegetable oils, olive oil is the one that unequivocally have ever played an
important role in the word market of oils. As reported in figure 2.3 and 2.4, more than
90% of the world’s olive harvest takes place in the Mediterranean region, due to the
unique agronomic and climatic factors of this area. Olive oil consumption has been for
centuries restricted to the Mediterranean people. For the rest of the word olive oil was
an unfamiliar oil, much more expensive than other vegetables (seed) oils. Due to
16
nutritional and economical factors, this tendency begun to change since the second half
of last century, where non-producing countries (U.S.A., Australia, Canada, Brazil,
Japan, France) increased olive oil consumption, and nowadays, this trend is still
growing.
EU
80.0%
Spain-Italy-Greece
78.7%
Tunisia-SyriaTurkey-Morocco,
15.3%
Rest of the world,
4.7%
2001/05 (tons %)
Figure. 2.3. – The world olive oil production (quantities in tons %)
Spain
43.7%
Italy
25.9%
Greece
14.2%
Syria
5.1%
Tunisia
4.7%
Turkey
4.1%
Morocco
2.4%
2001/05 (tons %)
Figure. 2.3. – The olive oil production in the Mediterranean area (quantities in tons %)
17
2.4.1.1. Fatty Acids
Olive oil fatty acid composition may differ from sample to sample, depending on the
area of production, the latitude, the climate, the variety, and the fruit maturity.
As reported in table 2.4, the main fatty acids are: palmitic (C16:0), palmitoleic
(C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic (C18:3). Others
fatty acids are present in trace amount.
Table. 2.3. – Olive Oil Fatty Acid Composition (%)
Oleic
Palmitic
Linoleic
Stearic
Linolenic
Palmitoleic
Arachidic
Margaric
Margaroleic
Lignoceric
Gadoleic
Behenic
C18:1 n-9
C16:0
18:2 n-6
C18:0
18:3 n-3
16:1 n-7
C20:0
C17:0
C17:1 n-8
C24:0
C20:1 n-11
C22:0
55 to 83
7.5 to 20
3.5 to 21
0.5 to 5
<0.9
0.3 to 3.5
<0.6
<0.3
<0.3
<0.3
0.1 to 0.4
<0.2
Myristic
C14:0
<0.1
Greek, Italian, and Spanish olive oils are low in linoleic and palmitic acids and they
have a high percentage of oleic acid. Tunisian olive oils are high in linoleic and palmitic
acids and lower in oleic acid. On the basis of the analysis of samples from various
countries olive oils are classified in two types, one with a low linoleic-palmitic acid and
high oleic acid content, and the other with a high linoleic-palmitic acid and low oleic
acid content. This effect has been associated with the stage of maturity of the fruit, and
an antagonistic relationship between oleic and palmitic, palmitoleic and linoleic acids
have been observed.
18
2.4.1.2. Triacylglycerols, and Partial Glycerides
The triacylglycerols found in significant proportions in olive oil are OOO (40-59%),
POO (12-20%), OOL (12.5-20%), POL (5.5-7%) and SOO (3-7%). Smaller amounts of
POP, POS, OLnL, LOL, OLnO, PLL, PLnO and LLL are also encountered. Fully
saturated moieties have not been reported and the same applies for the tri-unsaturated
ones containing linolenic acid. Stearic and palmitic acids are absent from the 2-position
of unsaturated species (tri- and tetraunsaturated) or from the molecule when there are
more than five double bonds. Trilinolein or ECN 42 triacylglycerol content (as
corrected recently), which is used as an authenticity marker by the EU, is the sum of the
amounts of LLL, PoPoPo, SLnLn, PoPoL, PPoLn, OLLn, PLLn and PoOLn (positional
isomers included).
The presence of partial glycerides in olive oil is due either to incomplete triacylglycerol
biosynthesis or hydrolytic reactions. In virgin olive oil, concentration of diacylglycerols
(DG) range from 1 to 2.8%. In the diacylglycerol fraction C-34 and C-36 compounds
prevail. Monoacylglycerols are present in much smaller quantities (less than 0.25%)
whereas 1-species are considerably higher than the respective 2-monoglycerides. Their
ratio depends on oil acidity, and the storage conditions affect the distribution of fatty
acids. 1.2-Diacylglycerols present in fresh oil tend to isomerize to the more stable 1.3diacylglycerols. This rearrangement gives information about the age of the oil and
storage conditions. The ratio of 1.3-/1.2-DG is considered as a useful ratio to monitor
quality.
19
2.4.1.3. Hydrocarbons
Two hydrocarbons are present in considerable amounts in olive oil, squalene and βcarotene that will be discussed in the pigments section. Squalene, or 2,6,10,15,19,23hexamethyl-2,6,10,14,18,22-tetracosahexaene, is the last metabolite preceding sterol
ring formation. Its presence is regarded as partially responsible for the beneficial health
effects of olive oil and its chemo-preventive action against certain cancers. It is the
major constituent of the unsaponifiable matter (referring to the whole quantity of
substances present in the oil or fat which after saponification by an alkaline solution
extraction by a specific solvent, are not soluble in aqueous alkali and non-volatile under
the condition of test) and makes up more than 90% of the hydrocarbon fraction. It
ranges from 200 to 7500 mg/kg of oil, even though higher levels up to 12,000 mg/kg
have been also reported in literature. Squalene content depends on olive cultivar, oil
extraction technology, and it is dramatically reduced during the process of refining.
Except for squalene, the hydrocarbon fraction of virgin olive oil is composed of
diterpene and triterpene hydrocarbons, isoprenoidal polyolefins, and n-paraffins.
2.4.1.4. Tocopherols
These compounds are present in olive oil in concentrations of about 150 – 250mg/Kg,
Normally present are the α, β, γ and δ forms of which α (vitamin E) is the most
abundant (90 – 95% of the total tocopherol content). Low amounts of the homologues
β-tocopherol (~10 mg/kg), δ-tocopherol (~10 mg/kg) and γ-tocopherol (~20 mg/kg) are
usually reported. The level of α-tocopherol may be related to the high levels of
chlorophyll pigments and the concomitant requirement for singlet oxygen deactivation,
20
while it seem to reduce with the fruit ripeness. Tocopherols carry out an anti-oxydant
action in oils exposed to “light”(ultaviolet radiation).
2.4.1.5. Pigments
Olive oil color is the result of green and yellow hues due to the presence of chlorophylls
and carotenoids. It is influenced by olive cultivar, maturation index production zone,
extraction system, and storage conditions. Therefore it is considered as a quality index
though no standardized method exists for its measurement. Chlorophylls are
encountered as pheophytins. Among the latter pheophytin α (Pheo α) is predominant,
while pheophytin β is also present though in minute amounts. The presence of Pheo α
is related to processing conditions and enzymatic or enzymatic-like activity. Handling
and duration of storage cause further changes in pheophytin α content. It has been
reported that the presence of pheophytin degradation products (such as: epimers, pyroforms and allomers) can be related with the storage condition. These products, on the
basis of previously reported findings, were identified as pyropheophytin α, 151-OH
lactone pheophytin α and 132-OH-pheophytin α. Under light exposure green pigments
degrade causing oil bleaching.
The main carotenoids present in olive oil are lutein and β-carotene
The presence of carotenoids in olive oil is closely related to that of green pigments and
is influenced by the same factors. The carotenoid fraction may also include several
xanthophylls (violaxanthin, neoxanthin, luteoxanthin, antheraxanthin, mutatoxanthin,
and β-cryptoxanthin). The ratio between the two major carotenoids seems to
be cultivar dependent.
21
2.4.1.6. Aliphatic and aromatic alcohols
Aliphatic and aromatic alcohols present in olive oil are found in free and esterified
form. The most important are fatty alcohols and diterpene alcohols. Alkanols and
alkenols with less than ten carbon atoms in their molecule, which are present in free and
esterified form, and some aromatic alcohols (benzyl alcohol and 2-phenylethanol) are
constituents of the olive oil volatile fraction. Benzyl esters of hexacosanoic and
octacosanoic acid have been also reported in olive oil.
Fatty Alcohols are a class of minor constituents consisting of linear saturated alcohols
with more than 16 carbon atoms which are present in the free and esterified form. The
main fatty alcohols present in olive oil are docosanol, tetracosanol, hexacosanol, and
octacosanol. Others fatty alcohols with odd carbon atoms (tricosanol, pentacosanol, and
heptacosanol) may be found in trace amounts. Virgin olive oil total fatty alcohols level
is affected by cultivar, crop year, fruit ripeness, and processing, even though is not
usually higher than 250 mg/kg. The most abboundand fatty alcohols were found to
be tetracosanol and hexacosanol. Esters of fatty alcohols with fatty acids (waxes) are
also classified as minor olive oil constituents, and they can be used as a criterion to
differentiate various olive oil types (EC Regulation 2568, 1991). The main waxes
detected in olive oil are esters of oleic or palmitic acid with 36, 38, 40, 42, 44, and 46
carbon atoms. Virgin olive oils contain waxes at levels lower than 150 mg/kg and their
content and composition is also affected by cultivar, crop year, and processing.
Phytol and geranylgeraniol are two acyclic diterpenoids present in the aliphatic alcohol
fraction of olive oil in the free and esterified form. Phytol, which probably originates
from chlorophyll, has been found in monovarietal virgin olive oils at levels ranging
from 25 to 595 mg/kg. Geranylgeraniol is reported to be present in virgin olive oil from
22
a new olive cultivar (I-77) at levels lower than 50 mg/kg. Its levels are used in the
calculation of the alcoholic index, a useful parameter for detecting solvent extracted
olive oil in virgin olive oil. Esters identified in the wax fraction of extra virgin olive oil
are oleate, eicosanoate, eicosenoate, docosanoate, and tetracosanoate, mainly as phytyl
derivatives.
2.4.1.7. Sterols
Sterols are important lipids related to the quality of the oil and broadly used for
checking its genuineness. Four classes of sterols occur in olive oil: common sterols (4desmethylsterols), 4α-methylsterols, triterpene alcohols (4, 4-dimethylsterols), and
triterpene dialcohols.
Common Sterols (4α-desmethylsterols) contained in Olive oil are mainly in free and
esterified form, although they have also been found as sterylglucosides and lipoproteins.
The main components of this sterol fraction are β-sitosterol, ∆5-avenasterol, and
campesterol. Other sterols present in smaller quantities or in trace amounts are
stigmasterol, cholesterol, brassicasterol, chlerosterol, ergosterol, sitostanol,
campestanol, ∆7-avenasterol, ∆7-cholestenol,∆7-campestenol, ∆7-stigmastenol, ∆5,23stigmastadienol, ∆5,24-stigmastadienol, ∆7,22-ergostadienol, ∆7,24-ergostadienol, 24methylene-cholesterol, and 22,23- dihydrobrassicasterol. Total sterol content of virgin
olive oils varies mainly between 1000 mg/kg, which is the lower limit set by the
European Union Commission (EC Regulation 2568, 1991), and 2000 mg/kg. Lampante
olive oils contain higher amounts of total sterols, while refined olive oils contain lower
levels because the refining process gives rise to significant losses of sterols, which may
be as high as 25%. Total sterol content of solvent extracted olive oils is up to three
23
times higher than that of virgin olive oils. Studies on olive oil sterol composition show
that β-sitosterol makes up 75 to 90% of the total sterol fraction, ∆5-avenasterol usually
ranges between 5% and 20%, campesterol and stigmasterol make up 4% and 2%
respectively (campesterol levels are always higher than those of stigmasterol). The rest
of the sterols occur in minute quantities. The levels of ∆5- and ∆7-avenasterol, ∆7stigmastenol, stigmasterol, and chlerosterol are used to determine whether virgin,
refined, and solvent extracted olive oils. Sterol composition and total sterol content are
affected by cultivar, crop year, degree of fruit ripeness, storage time of fruits prior to oil
extraction, processing, and also by geographic factors.
4-Methylsterols are intermediates in sterol biosynthesis, and they are present in olive oil
in small quantities in free and esterified form. The predominating components are
obtusifoliol, gramisterol, cycloeucalenol, and citrostadienol. They are ∆7- or ∆8-sterols
except cycloeucalenol which has a 9,19-cyclopropane ring in the steroid skeleton. The
levels of total 4α-methylsterols are lower than that of common sterols and triterpene
alcohols and vary between 50 and 360 mg/kg.
2.4.1.8. Triterpene acids
Hydroxy pentacyclic triterpene acids are important olive fruit constituents. They are
biologically active compounds and are present at trace amounts in olive oil.
The main triterpene acids present in virgin olive are Oleanolic (3β-hydroxyolean-12-en28-oic acid) and maslinic acid (2α, 3β-dihydroxyolean-12-en-28-oic acid) are the main
triterpene acids present in virgin olive. Both compounds and traces of ursolic acid
(3β-hydroxyurs-12-en-28-oic acid) are located in the reticular lipid layer of olive skin.
24
Total triterpene acid content of extra virgin olive oils obtained from fruits of different
olive cultivars was found to range between 40 and 185 mg/kg. Recently, the main factor
influencing the level of hydroxy pentacyclic triterpene acids in olive oil was studied.
Olive oil acidity resulted to be the principal contributor while olive cultivar, olive
ripeness, and oil extraction system have less influence on the levels of these acids.
2.4.1.9. Volatile and aroma compounds
Approximately two hundred and eighty compounds have been identified in the volatile
fraction of virgin olive oils. They are hydrocarbons (more than 80 compounds), alcohols
(45 compounds), aldehydes (44 compounds), ketones (26 compounds), acids (13
compounds), esters (55 compounds), ethers (5 compounds), furan derivatives (5
compounds), thiophene derivatives (5 compounds), pyranones (1 compound), thiols
(1 compound), and pyrazines (1 compound). From this large number of compounds,
only 67 were found to be present at levels higher than their odor threshold contribute to
the flavor of virgin olive oils with sensory defects. The potent odorants of olive oil have
been evaluated by applying aroma extract dilution analysis (AEDA) and gas
chromatography-olfactometry analysis of headspace.
2.4.1.10. Other minor constituents
Some classes of minor constituents are present only in the crude oil. Filtration reduces
the initial levels to a great extent whereas refining process leads to their removal.
Olive oil contains a small amount of phospholipids and, even if experimental work for
their identification is rather limited, phosphatidylcholine, phospatidylethanolamine,
phosphatitylinositol, were reported to be the main constituent of this fraction.
25
2.4.2. DAIRY PRODUCTS
Reference for this section: 15, 17, 18, 19.
Milk and dairy products are major components of the human diet in Western countries,
providing about 30% of dietary proteins and lipids and about 80% of dietary calcium.
Current annual production of milk is about 600 x 106 tons, of which about 85% are
bovine (Figure 2.4).
Bovine
85%
Buffalo
11%
Caprine
2%
Ovine
2%
Annual milk production (tons %)
Figure. 2.4. – Current annual milk production.
Although some raw milk is still consumed, the vast majority of milk is processed to at
least some extent. Liquid (beverage) milk is a major food item in all developed dairying
countries, representing about 40% of total milk production, while the remainder is
processed into one of several thousand products (Figure 2.5). For this reason the dairy
industry is probably the most diverse and flexible sector of the food industry. The
flexibility of milk as a raw material resides in the chemical and physical-chemical
26
properties of its constituents, many of which are unique. The principal constituents of
milk can be modified by enzymatic, chemical and/or physical methods, permitting the
production of new products. The natural function of milk is to supply the neonatal
mammal, of which there are about 4500 species, with its complete nutritional and some
of its physiological requirements. Because the nutritional requirements are speciesspecific and change as the neonate matures, the composition of milk shows very large
interspecies differences. Inter-species differences in the concentrations of many of the
minor constituents are even greater than those of the macro-constituents. Milk from
domesticated animals has been used by humans since at least 8000 BC. Although sheep
and goats were the first domesticated dairy animals, because they are more easily
managed than cattle, the latter, especially certain breeds of Bos taurus, are now the
dominant dairy animals.
Figure 2.5. – Schematic presentation of milk processing.
Therefore, to avoid misunderstanding, this section will concentrate on the properties of
bovine milk or simply “milk”, as term used today as synonymous. Milk is a very
27
flexible raw material from which several thousand types of dairy products are produced
around the world in a great diversity of flavors and forms, including thousand varieties
of cheese. The total world milk yield is used for the production of the principal dairy
products, that are in order: liquid (beverage) milk, cheese, butter, whole milk powder,
skimmed milk powder, concentrated milk products, fermented milk products, casein and
infant formulae. This flexibility and diversity are a result of the properties, many of
them unique, of the constituents of milk, the principal of which are easily isolated from
milk, permitting the production of valuable food ingredients. Moreover the
processability and functionality of milk and milk products are determined by the
properties and concentrations of its principal constituents: proteins, lipids, lactose and
salts. The lipids occur as globules, 0.1–20 µm in diameter, surrounded by the milk fat
globule membrane (MFGM), which serves as an emulsifier. Their concentration varies
with species, breed, individual animal, stage of lactation, mastitis infection, plane of
nutrition, interval between milkings, and point during milking when the sample is take.
2.4.2.1. Fatty acids
Ruminant milk fat contains a wider range of fatty acids than any other lipid system, up
to 400 fatty acids have been reported in bovine milk fat; the principal fatty acids are the
homologous series of saturated fatty acids, C4:0, C18:0 and C18:1. The outstanding
features of the fatty acids in milk fat are a high concentration of short and medium chain
acids (ruminant milk fats are the only natural lipids that contain butanoic acid) and a
low concentration of polyunsaturated fatty acids (PUFA). In ruminants, the fatty acids
for the synthesis of milk lipids are obtained from triglycerides in chylomicrons in the
blood or synthesized de novo in the mammary gland from acetate or β–hydroxybutyrate
28
produced by microorganisms in the rumen. The triglycerides in chylomicrons are
derived from the animal’s feed or synthesized in the liver. Butanoic acid (C4:0) is
produced by the reduction of β-hydroxybutyrate which is synthesized from dietary
roughage by bacteria in the rumen and therefore varies substantially with the animal’s
diet. All C6:0– C14:0 and 50% of C16:0 are synthesized in the mammary gland via the
malonyl-CoA pathway from acetyl-CoA produced from acetate synthesized in the
rumen. Essentially 100% of C18:0, C18:1, C18:2 and C18:3 and 50% of C16:0 are
derived from blood lipids (chylomicrons) and represent about the 50% of total fatty
acids in ruminant milk fat. Unsaturated fatty acids in the animal’s diet are hydrogenated
by bacteria in the rumen unless they are protected (encapsulation). Seasonal variation
can cause very significant changes in the fatty acid profile of milk fat. A grass-based
diet is rich in PUFA (see Figure 2.6), and these are subjected to the bio-hydrogenation
process by bacteria in the rumen (e.g.: Butyrivibrio fibrisolvens) and then converted to
mono-unsatured fatty acids in cis and trans configurations, that they will be found
consequently in milk fat and the relative dairy products obtained.
PUFA are considered to be nutritionally desirable and, consequently, there has been
interest in increasing their concentration in bovine milk fat, e.g. linoleic acid, that is an
essential fatty acid and must be supplied in the diet since it cannot be synthesized by
mammals. This can be done by feeding encapsulated PUFA-rich lipids or crushed
PUFA-rich oil seeds to the animal.
29
Dietary PUFA
Linoleic acid
(C18:2 c9,c12)
Linolenic acid
(C18:3 c9,c12,c15)
isomerase
CLA
(C18:2 c9,t11)
Linolenic acid
(C18:3 c9,t11,c15)
reductase
Vaccenic acid
(C18:1 t11)
(C18:2 t11,c15)
reductase
Stearic acid
(C18:0)
Figure. 2.6. – Biohydrogenation of Dietary Poliunsatured Fatty acids in the rumen
Unsaturated fatty acids may occur as cis or trans isomers (Figure 2.7); trans isomers,
which have higher melting points than the corresponding cis isomers, are considered to
be nutritionally undesirable. Bovine milk fat contains a low level (5%) of trans fatty
acids in comparison with chemically hydrogenated (hardened) vegetable oils, in which
the value may be 50% due to non-stereospecific hydrogenation.
COOH
cis
trans
Oleic acid
(9c-18:1)
COOH
Elaidic acid (9t-18:1)
Figure. 2.7. – Fatty acids in cis and trans configuration.
Another group of fatty acids that have attracted very considerable attention recently, is
the CLA (Figure 2.8). It is a mixture of eight positional and geometric isomers of
30
linoleic acid which have a number of health-promoting properties. Of the eight isomers
of CLA, only the C18:2 cis 9, trans 11 isomer is biologically active.
Methylene interrupted
Conjugated
COO
Linoleic acid
(9c,12c-18:2)
COO
COO
Linolenic acid
(9c,12c,15c-18:2)
9c,11t-CLA
(9c,11t-18:2)
Figure. 2.8. – Conjugated and non-conjugated fatty acids.
This compound is effective at very low concentrations, 0.1 g per 100 g diet. Fatcontaining foods of ruminant origin, especially milk and dairy products, are the
principal sources of dietary CLA which is produced as an intermediate during the
biohydrogenation of linoleic acid by the rumen bacterium, Butyrivibrio fibrisolvens, and
isomerized by delta-9 desaturase in the mammary gland from vaccenic acid (trans-11
C18:1), that is an intermediate of polyunsaturated fatty acid biohydrogenation in the
rumen (Figure 2.9). Since CLA is formed from linoleic acid, it is not surprising that the
CLA content of milk is affected by diet and season, being highest in summer when
cows are on fresh pasture rich in PUFA and higher in the fat of milk from cows on
mountain than on lowland pasture. The concentration of CLA in milk fat can be
increased 5–7 fold by increasing the level of dietary linoleic acid, e.g., by duodenal
infusion or by feeding a linoleic acid-rich oil, e.g., sunflower oil.
31
Figure. 2.9. – Pathways of rumenic acid in rumen and mammary gland
2.4.2.2. Glycerides
Triacylglycerols, called triglycerides for short, make up the bulk (generally more than
98%) of the lipids and accordingly, largely determine the properties of milk fat (Table
2.4). These properties vary with the fatty acid composition. Because the number of
different fatty acid residues is great, the number of different triglycerides is much
greater. The distribution of fatty acid residues over the position in the triglyceride
molecule is far from random: Butanoic and hexanoic acids are esterified almost entirely,
and octanoic and decanoic acids predominantly, at the sn-3 position; as the chain length
increases up to C16:0 an increasing proportion is esterified at the sn-2 position (more
marked for human than for bovine milk fat, especially in the case of palmitic acid);
stearic acid (C18:0) is esterified mainly at sn-1; unsaturated fatty acids are esterified
mainly at the sn-1 and sn-3 positions, in roughly equal proportions. The position of the
fatty acid residues in the triglyceride molecules considerably affects the crystallization
behavior of milk fat.
32
Some of di- and monoglycerides occur in fresh milk fat. Lipolysis increases their
quantities. Diglycerides are predominantly apolar and do not differ much from
triglycerides in properties. Monoglycerides, present in far smaller quantities, are
somewhat polar; they are surface active and thus accumulate at an oil–water interface.
Most lipolytic enzymes, including that of milk, especially attack the 1- and the 3position of the triglyceride molecule. This means that most monoglycerides have a fatty
acid residue at the 2-position, and that most of the free fatty
acids formed originate from the other positions, including the short-chain types that are
predominantly in the 3-position.
Table. 2.4. – Percentage of glycerides in milk fat.
% in milk fat
Neutral glycerides
Tryglycerides
Diglycerides
Monoglycerides
98.7
98.3
0.3
0.003
2.4.2.3. Hydrocarbons
Several hydrocarbons occur in milk in trace amounts. Of these, carotenoids are the most
significant. In quantitative terms, carotenes occur at only trace levels in milk but they
contribute 10-50% of the vitamin A activity in milk and are responsible for the yellow
colour of milk fat. The carotenoid content of milk varies with breed and very markedly
with season. The latter reflects differences in the carotenoid content of the diet (since
they are totally derived from the diet); fresh pasture, is much richer in carotenoids than
hay or silage (due to oxidation on conservation) or cereal-based concentrates. The
higher the carotenoid content of the diet, the more yellow will be the colour of milk and
milk fat.
33
2.4.2.4. Tocopherols
Vitamin E is a generic term used to indicate tocopherols and tocotrienol, and αtocopherol has the greatest activity for humans. Vitamin E is a very effective
antioxidant, protects the lipids (particularly polyunsaturated fatty acids) and membranes
in the body against damage caused by free radicals. The role of vitamin E is of
particular importance in the lungs where exposure of cells to oxygen is greatest.
Vitamin E also exerts a protective effect on red and white blood cells. It has been
suggested that the body has a system to regenerate active vitamin E (perhaps involving
vitamin C) once it has acted as an antioxidant. The concentration of vitamin E in cows'
milk is quite low (0.09mg per l00g) and is higher in summer than in winter milks.
2.4.2.5. Sterols
These compounds are found in the unsaponifiable fraction of milk lipids and consist
mostly of cholesterol with some ∆7-cholesterol and β-Sitosterol.
They are polycyclic alcohols having a secondary –OH group at position 3, and the
presence of this group makes sterols more polar than triglycerides. Moreover, in milk
fat globules, cholesterol, both free (90%) and esterified, is one of the constituents of
the globule membrane. The occurrence of sterols in milk fat is about 0.3%.
2.4.2.6. Phospholipids
The phospholipids comprise approximately 1 % of the total lipid in bovine milk. While
quantitatively minor, the ability of the phospholipids to form stable colloidal
suspensions or emulsions in aqueous solution cause them to be important in the
34
formation and secretion of milk fat. They consist of a glycerol backbone on which one
or two fatty acids and a phosphate residue with different organic groups may be linked
Their physical properties as bipolar molecules and their relatively high concentration of
unsaturated fatty acids also make them an important factor to consider during the
storage and processing of milk. They are relatively susceptible to oxidation because of
their polyunsaturated fatty acid content. As the total milk lipid increases in a milk
product, so does the phospholipid concentration. However, the ratio of phospholipid to
total lipid varies greatly. Skim milk contains the smallest concentration of phospholipid
but the highest ratio of phospholipid to total lipid. The opposite relationship is seen in
cream and butter. Most milk lipid exists as fat globules suspended in the aqueous phase
of milk. The size of the milk fat globules varies from 0.1 to 2 p in diameter. The core of
the globule is primarily TG, which is surrounded by the milk fat globule membrane
(MFGM), as reported in figure 2.10. This membrane contains protein, glycoproteins,
enzymes, phospholipids, and other polar materials. It is a major source of cholesterol
and phospholipid in milk. The major glycerophospholipids are phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Sphingolipids
are a group of phosholipids that consist of an a-polar sphingosine backbone on which a
fatty acid is bound to form a ceramide, and it can contain a similar organophosphate
group like choline (sphingomyelin) or a mono- or disaccharide (glycosphingolipids).
Important dairy sphingolipids are sphingomyelin, glucosylceramide and
lactosylceramide.
35
Figure. 2.10. – Schematic representation of a milk fat globule.
Sphingolipids are known to exhibit various biological properties and are therefore
important in human nutrition, such as, the capability to reduce the cholesterol uptake,
and their inhibitory effect on colon
cancer.
Table. 2.5. – Percentage of Phospholipids, Cerebrosides, Gangliosides in milk fat
Alcohol Residue + Other Costituent
Phospholipids
% in milk fat
Phospho group
0.8
Ph. Choline
Glycerol + Choline
0.27
Ph. Ethanolammine
Glycerol + Ethanolammine
0.26
Ph. Serine
Glycerol + Serine
0.03
Ph. Inositide
Glycerol + Inositol
0.04
Sphingomyelin
Sphingosine + Choline
0.2
Cerebrosides
Sphingosine + Hexose
0.1
Gangliosides
Sphingosine + Hexose
0.01
36
2.4.2.7. Free fatty acids
They occurrence of free fatty acids in milk fat is about 0.1% and lipolysis increases their
amount. Especially the shorter acids are somewhat soluble in water. In water, the acids
can, of course, dissociate into ions; their pK is about 4.8. In milk plasma, they are thus
predominantly in the ionized form (i.e., as soaps), and these are much more soluble in
pure water than the pure fatty acids. Fatty acids dissolve well in oil, though only in the
nonionized form. Moreover, they tend to associate into dimers, by forming hydrogen
bonds. The partition of the acids over the oil and water phases is rather intricate. All in
all, the shorter acids (C4:0 and C6:0) are predominantly in the plasma, the longer ones
(from C14:0 on) in the fat. The other acids are distributed between both fractions,
though more go into the fat with decreasing pH. This is even more complicated because
the fatty acids, especially the long-chain ones, are surface active and tend to accumulate
in the oil–water interface. The distribution over the phases is of much importance
because acids dissolved in the aqueous phase (in the form of soaps) — hence, the
shorter acids — are responsible for the soapy-rancid flavor perceived after lipolysis.
2.4.2.8 Lipo-soluble vitamins
The fat-soluble vitamins are retinol (vitamin A), tocopherols (and related compounds,
vitamin E), calciferols (vitamin D), and phylloquinone (and related compounds,
vitamin K). Since vitamin A and E were previously treated (see Hydrocarbon and
Tocopherols), this part will concern on vitamin D and K.
Unlike other vitamins, cholecalciferol (vitamin D,) can be formed from a steroid
precursor, 7 dehydrocholesterol, by the skin when exposed to sunlight; with sufficient
exposure to the sun, no preformed vitamin D is required from the diet.UV light (280-
37
320 nm) causes the photoconversion of 7-dehydrocholesterol to pre-vitamin D3. This
pre-vitamin can undergo further photoconversion to tachysterol and lumisterol or can
undergo a temperature-dependent isomerization to cholecalciferol. At body temperature,
this conversion requires about 28 h to convert 50% of previtamin D3, to vitamin D3.
Thus, production of vitamin D, in the skin can take a number of days. Preformed
vitamin D, is obtained from the diet. This latter, must undergo two hydroxylations to
become fully active in liver and kidneys respectively. However, at least 37 metabolites
of vitamin D, have been identified, but only 1,25-dihydroxycholecalciferol
1,25(OH) 2D3 is the most biologically active metabolite of vitamin D,. The major form
of vitamin D in both cows’ and human milk is 25-hydroxycholecalciferol 25(OH) 2D3.
Whole cows’ milk contains only about 0.03 µg vitamin D per 100g. The principal
physiological role of vitamin D in the body is to maintain plasma calcium by
stimulating its absorption from the gastrointestinal tract, its retention by the kidney and
by promoting its transfer from bone to the blood. Vitamin D acts in association with
other vitamins, hormones and nutrients in the bone mineralization process. In addition,
vitamin D has a wider physiological role in other tissues in the body, including the brain
and nervous system, muscles and cartilage, pancreas, skin, reproductive organs and
immune cells.
The structure of vitamin K is characterized by methylnaphthoquinone rings with a side
chain at position 3.Menaquinones are synthesized only by bacteria (which inhabit the
human gastrointestinal tract and thus provide some of the vitamin K required by the
body).Whole cows’ milk contains 0.4-1.8 µg vitamin K per 100g. The physiological
role of vitamin K is in blood clotting and is essential for the synthesis of at least four of
the proteins (including prothrombin) involved in this process. Vitamin K also plays a
38
role in the synthesis of a protein (osteocalcin) in bone. Vitamin K deficiency is rare but
can result from impaired absorption of fat. Vitamin K levels in the body are also
reduced if the intestinal flora is killed (e.g. by antibiotics).
2.4.2.9 The fat in milk products
Because the various lipids are unevenly distributed among the physical fractions of milk
(Table 2.8), the fat composition of different milk products varies. The largest
differences originate from variations in the amount of material from the fat globule
membranes. Examples are given in table 2.6. Anhydrous milk fat is prepared from
butter by melting it, and by separating and drying the oil layer obtained; its composition
is virtually equal to the fat in the core of the milk fat globules.
Table. 2.6. – Approximate content of lipids in some milk products
39
2.5. CHEMICAL ASPECTS
Reference for this section: 20, 21.
2.5.1. LIPOLYSIS
Hydrolysis of ester bonds in lipids may occur by enzyme action or by heat and
moisture, resulting in the liberation of free fatty acids from glycerides. Since edible
animal fats are not usually refined, prompt rendering is of particular importance. The
temperatures commonly used in the rendering process are capable of inactivating the
enzymes responsible for hydrolysis.
The release of short-chain fatty acids by hydrolysis is responsible for the development
of an undesirable rancid flavour (hydrolytic rancidity) in raw milk. On the other hand,
certain typical cheese flavors are produced by deliberate addition of microbial and milk
lipases (endogenous). In contrast to animal fats, olive oils may have undergone
substantial hydrolysis by the time the fruits are harvested, giving rise to significant
amounts of free fatty acids; so, it may start while the fruit is still on the tree. The
endogenous lipase does not manifest its activity until the fruit starts turning purple.
Bacteria, yeasts and molds (that may grow on the fruit) elaborate their own lipases. If
the fruit is stored before processing and especially if the storage results unsatisfactory,
then the combined effect of the endogenous and microbial lipases may result in
considerable rise of the acidity of the oil to the detriment of its quality.
2.5.2. AUTOXIDATION
Lipid oxidation in food systems is a detrimental process and is one of the major causes
of food spoilage. It deteriorates the sensory quality and nutritive value of a product,
40
poses a health hazard and presents a number of analytical problems. It leads to the
development, in edible oils and fat-containing foods, of various off flavours and off
odours generally called rancid (oxidative rancidity, which render these foods less
acceptable. In addiction, oxidative reaction can decrease the nutritional quality of food,
and certain oxidation products are potentially toxic. On the other hand, a limited degree
of lipid oxidation is sometimes desirable, as in aged cheeses.
The term autoxidation is referred to the reaction with molecular oxygen via a selfcatalytic mechanism, which is the main reaction involved in oxidative deterioration of
lipids. Although photochemical reactions have been known for a long time, only
recently the role of photosensitized oxidation and its interaction with autoxidation
emerged. In food systems lipids can be oxidized both by enzymic and non enzymic
mechanisms.
2.6. PROCESSING OF FATS AND OILS
Reference for this section: 20, 21.
2.6.1. PROCESSING OF FAT AND OIL
Apart from some oils obtained by cold pressing, most of the oils obtained using
expeller, screw or hydraulic presses, solvent extraction or by melting at elevated
temperatures are not suitable for immediate consumption. Depending on the raw
material and the oil recovery process, the oil contains polar lipids especially
phospholipids, free fatty acids, some odor- and taste-imparting substances, waxes,
pigments (chlorophyll, carotenoids and their degradation products), phenolic
compounds, trace metal ions, contaminants and autoxidation products.
41
A refining process may comprise the following steps: vegetable lecithin removal,
degumming, free fatty acid removal, bleaching and deodorization. All the undesired
compounds and contaminants are removed. In practice, the refining steps used, depend
on the quality of the crude oil and its special constituents. Moreover, the absence of
oxygen, the avoidance of heavy metal contamination and the maintaining of processing
temperatures as low and duration as short as possible, are precautionary misures that
must be taken during the refining in order to avoid undesirable autoxidation and
polymerization reaction.
2.6.2. HYDROGENATION
Liquid oils are supplied mostly from natural sources. However a great demand exists for
fats which are solid or semi-solid at room temperature (such as shortenings and
margarine). The process that convert liquid oil into solid fat is called “fat hardening”
and consists in the addiction of hydrogen to double bonds in the fatty acid chains. In
practice, the oil is first mixed with a suitable catalyst (e.g. nickel), heated to the desired
temperature (140°-225°), then exposed, while stirred, to hydrogen at pressure up to 60
psig. The course of the hydrogenation reaction is usually monitored by determining the
change in the refractive index, strictly related to the degree of saturation of the oil.
When the desired end point is reached, the hydrogenated oil is cooled and the catalyst is
removed by filtration. During hydrogenation, not only some of the double bonds are
saturated, but some may also be relocated and/or transformed from the usual cis to the
trans configuration. Partial hydrogenation thus may result in the formation of a
relatively complex mixture of reaction products, depending on which of the double
42
bonds are hydrogenated, the type and degree of isomerization, and the relative rates of
these various reaction.
2.6.3. INTERESTERIFICATION
It has been mentioned that natural fats do not contain a random distribution of fatty
acids among the glyceride molecules. The tendency of certain acids to be more
concentrated at specific sn position varies from one species to another an is influenced
by factors such as environment and location in the plant or animal. The physical
characteristic of a fat are greatly affected not only by the nature of constituent fatty
acids but also by their distribution in the triacilglycerol molecules. Indeed, unique fatty
acid distribution patterns of some natural fats may limit their industrial application.
Interesterification is one of the processes that can be applied to improve the consistency
of such fats and to improve their usefulness, involving a fatty acids rearrangement so
they become distributed randomly among the triacylglycerol molecules of the fat.
43
2.7. REFERENCES
1)
Alarcon de la Lastra C, Barranco MD, Motilva V, Herreiras JM (2001).
Mediterranean diet and health: biological importance of olive oil. Curr. Pharm.
Des., (7), 933-950.
2)
Perona JS, Canizares J, Montero E, Sanchez-Dominguez JM, Catala A, RuizGutierrez V (2004). Virgin Olive oil reduces blood pressure in hypertensive elderly
subjects, Clin. Nutr., (23), 1113-1121.
3)
Beauchamp GK, Keast RS, Morel D, Lin J, Pika J, Han Q, Lee CH, Smith AB,
Breslin PA, (2005). Ibuprofen-like activity in extra-virgin olive oil. Nature. 437,
45-46.
4)
Hamdi HK, Castellon R (2005). Oleuropein, a non-toxic olive iridoid, is an antitumor agent and cytoskeleton disruptor. Biochem. Biophys. Res. Commun., 334,
(3), 769-778.
5)
Banni S, Heys CSD, Wahle KWJ (2003). Conjugated linoleic acid as anticancer
nutrients: Studies in vivo and cellular mechanisms. In J.Sebedio, W.W. Christie
and R. Adolf, Advances in Conjugated Linoleic Acid Research, AOCS Press, (2),
267-281.
6)
Belury MA (2003). Conjugated linoleic acids in type 2 diabetes mellitus:
implications and potential mechanisms. In J. Sebedio, W.W. Christie and R. Adolf,
Advances in Conjugated Linoleic Acid Research, AOCS Press, Champaign, IL,
USA, (2), 302-315.
7)
Cook ME, Butz D, Li G, Pariza M, Whigham L, Yang M (2003). Conjugated
linoleic acid enhances immune responses but protects against the collateral damage
of immune events. In: J. Sebedio, W.W. Christie and R. Adolf, Advances in
Conjugated Linoleic Acid Research, AOCS Press, Champaign, IL, USA, (2), 283291.
8)
Bauman DE, Barbano DM, Dwyer DA, Griinari JM (2000). Technical Note:
Production of butter with enhanced conjugated linoleic acid for use in biomedical
studies with animal models, J. Dairy Sci., (83) 2422–2425.
9)
Yoon CS, Ha TY, Rho JH, Sung KS, Cho IJ (1997). Inhibitory effect of conjugated
linoleic acid on in vitro growth of human hepatoma. FASEB J., (11), 578.
10) Sebedio JL and Christie W W (1998). Trans Fatty Acid in Human Nutrition, The
oily press, PJ Barnes & Associates, Bridgwater, UK.
11) Weggemans RM, Rudrum M, Trautwein EA (2004). Intake of ruminant versus
industrial trans fatty acids and risk of coronary heart disease - what is the evidence?
Eur. J. Lipid Sci. Technol., 106, (6), 390-397.
12) Jakobsen MU, Overvad K, Dyerberg J, Heitmann BL (2008). Intake of ruminant
trans fatty acids and risk of coronary heart disease, Int. J. Epidemiol., 37, (1), 17382.
13) During A, Mazette S, Combe N, Entressangles B (2000). Lipolysis and oxidative
stability of soft ripened cheeses containing vegetable oils. J. Dairy Res., 67, 461466.
44
14) Khan-Merchant N, Penumetcha M, Meilhac O, Parthasarathy S (2002). Oxidized
fatty acids promote atherosclerosis only in the presence of dietary cholesterol in
low-density lipoprotein receptor knockout mice. American Soc. Nut. Sci., 32563262.
15) O’Brien RD (2004). Formulating and processing for application. Fats and Oils, 2nd
Ed., CRC Press, Boca Raton, London, New York, Washington D.C., USA.
16) Boskou D, Blekas G, Tsimidou M (2006). Olive oil composition. In: D Boskou ,
Olive Oil Chemistry and Technology, AOCS Press, Champaign, IL, USA, (4) 4172.
17) Walstra, P, Wouters JM, Geurts T (2006). Milk Components. In: Dairy Science and
Technology, 2nd Ed., CRC/Taylor & Francis.(2) 1-92
18) Fox PF (2003). The major constituents of milk. In: Smith G, Dairy Processing:
Improving Quality. CRC/Woodhead Publishing. (2), 2-38.
19) Fox PF, McSweeney PLH (1998). Dairy Chemistry and Biochemistry. Blackie
Academic & Professional. (3) 1-37.
20) Nawar WW (1996). Lipids. In: Fennema OR, Food Chemestry, 3rd Ed., Marcel
Dekker, Inc, New York, NY, USA. (5) 226-314.
21) Belitz HD, Grosch W (1987). Edible fats and Oils. Springer-Verlag Berlin. (14)
472- 493.
45
46
3
COMPARISON OF DIFFERENT TECHNIQUES FOR
TRANS-FATTY ACIDS ANALYSIS
3.1. SUMMARY AND KEYWORDS .......................................................................................................48
3.2. INTRODUCTION...............................................................................................................................49
3.3. MATERIALS AND METHODS ........................................................................................................50
3.3.1. SAMPLES PREPARATION .......................................................................................................50
3.3.2. ANALYSIS BY GAS CHROMATOGRAPHY ..........................................................................50
3.3.3. ANALYSIS BY AG+-HPLC.......................................................................................................50
3.3.4. AG+-HPLC FRACTIONATION FOLLOWED BY GC ANALYSIS ........................................51
3.3.5. ATTENUATED TOTAL REFLECTION FT-IR.........................................................................51
3.4. RESULTS AND DISCUSSION..........................................................................................................51
TABLES.....................................................................................................................................................53
FIGURES ...................................................................................................................................................54
3.5. REFERENCES....................................................................................................................................58
47
3.1. SUMMARY AND KEYWORDS
FDA's final rule on trans fat labeling requires that amounts of trans fat per serving be
listed on the nutrition facts panel. AOCS Official Method of Analysis Ce 1h-05 can be
used for analysis of trans fatty acids (TFA) in vegetable or non-ruminant animal oils
and fats. Difficulties in quantitating low amounts of TFA and partial co-elution of cisand trans- 18:1 fatty acids suggest the need for fractionating TFA before gas
chromatographic (GC) analysis. Silver ion-HPLC (Ag+-HPLC) and silver ion solid
phase extraction (Ag+-SPE) have been used for separating the trans-18:1 fatty acid
methyl esters (FAME). Representative samples of fats and oils were studied. Trans 1118:1 fatty acid n-butyl ester (FABE) was included as an internal standard. The TFA
content was also measured by direct Ag+-HPLC analysis, using 3 ChromSpher 5 Lipids
silver ion loaded columns in series with 0.1 % MeCN in hexane mobile phase at 1.0
mL/min. and 196 nm UV detection. Ag+-HPLC fractionation was carried out using the
same mobile phase at 3 mL/min and a ChromSpher 5 Lipids semipreparative column.
Fractionation of TFA prior to GC analysis simplified the interpretation of
chromatograms, but provides quantitation only of trans-18:1 FAME. Results of the
analysis of 10 samples of different oils and fats obtained by the different techniques will
be described.
Keywords: trans-fatty acids, gas chromatography, Ag+-HPLC, Ag+-SPE, ATR-FT-IR
48
3.2. INTRODUCTION
Interest in quantitation trans fat has increased following addition of the trans fat content
to the Nutrition Facts panel on food labels. Trans fat is calculated by adding the content
of all the fatty acids with at least one trans double bond (TFA), excluding fatty acids
with conjugated double bonds as conjugated linoleic acid (CLA). After extracting the
lipid fraction from food, TFA are derivatized into fatty acid methyl esters (FAME) and
quantitated by gas chromatography (GC) using long polar capillary columns (1). The
interpretation of the FAME separation is often particularly complex, because TFA are
not completely separated from other fatty acids present in oil and fats. An alternative
approach is the application of silver ion high performance liquid chromatography (Ag+HPLC). The TFA as FAME can either be directly quantitated by Ag+-HPLC or
fractionated prior to GC analysis (2, and 3). This technique is currently limited to the
quantitation or fractionation of the trans-18:1 fatty acids that generally constitute more
than 95% of the trans fat in fats and oils (3). Attenuated total reflection FT-IR (ATRFT-IR), measuring the specific IR absorption of trans double bonds at 966 cm-1,
provides quantitation of total trans fat without giving any information on the content of
individual fatty acids (4). An advantage ATR-FT-IR is that it can be applied to the
direct quantitation of TFA in neat fats and oils, thus eliminating the derivatization step.
In this study, we compared the analysis of 10 different fats and oils using different
techniques of analysis.
49
3.3. MATERIALS AND METHODS
3.3.1. SAMPLES PREPARATION
Ten samples of common fats and oils were obtained from the American Oil Chemist
Society. The samples were methylated according to Official Method AOCS Ce 2-66.
For Ag+-HPLC analysis and Ag+-HPLC fractionation, 1 mg of t11-18:1 FABE as
internal standard was added to c.a. 20-30 mg neat FAME of each sample. t11-18:1
FABE was purchased from Nu Chek Prep, as a special preparation.
3.3.2. ANALYSIS BY GAS CHROMATOGRAPHY
Samples (Figure 3.1.) were analyzed with an Agilent 6890N gas chromatograph
equipped with an FID detector and a Varian CP-Sil 88 capillary column (Varian, 100 m
x 0.25 mm i.d., 0.2 µm thickness). The oven was maintained at 180°C, the detector at
300°C, and the injection port at 250°C. Hydrogen as carrier gas was eluted at 1.0
ml/min and the split ratio was maintained at 1:200.
3.3.3. ANALYSIS BY AG+-HPLC
Analysis were performed with a Waters 2695 separations module equipped with a
Waters 2996 PDA detector and a Waters 2420 ELSD detector. Three silver ion HPLC
columns (ChromSpher 5 Lipids, 4.6 x 250 mm, 5 µm particle size, Varian) were used in
series and maintained at 20°C in a water circulating bath. The mobile phase was 0.15%
MeCN in hexane at 1.0 ml/min., and the injection volume was 5 µl. The UV signal was
acquired between 190 and 300 nm. Chromatograms for purpose were extracted at 203
nm (Figure 3.2.).
50
3.3.4. AG+-HPLC FRACTIONATION FOLLOWED BY GC ANALYSIS
Fractionation was achieved using a Waters Delta Prep 4000 preparative HPLC equipped
with a Waters 717 Plus autosampler and a Waters 2996 PDA detector.
A semi-preparative ChromSpher 5 Lipids column (10 x 250 mm, 5 µm particle size,
Varian) was maintained at room temperature. The mobile phase was 0.1% MeCN in
hexane at 3.0 ml/min. Fractions were collected manually. Collection times were
adjusted to compensate for the drift in the TFA retention times (Figure 3.3.).
3.3.5. ATTENUATED TOTAL REFLECTION FT-IR
Samples as pure fat or oil were analyzed with a Varian (Randolph, MA) FTS 7000e IR
spectrometer controlled by Resolution Pro software (Figure 3.4.). Fourier transform
(FT) IR spectra were collected between 4000 to 600 cm-1 at a resolution of 4 cm-1.
256 scans were acquired for each sample (approximately 4 min), and the signal
averaged. A reference background spectrum was measured for air. A PIKE (Madison,
WI) heated single reflection diamond ATR cell was used, maintained at 65°C. The
height of the negative second derivative of the 966 cm-1 band was measured. The
instrument was calibrated in the interval 0-100% TFA using mixtures of neat tri-elaidin
(TE) in tri-palmitin (TP). TE and TP were supplied by Nu Check Prep, Inc. (Elysian,
MN).
3.4. RESULTS AND DISCUSSION
Table 3.1. shows the comparison of the quantitation of TFA in 10 samples
representative of the lipid fraction of foods, fats and oils available in North American
51
markets. ATR-FT-IR over-estimated trans fat content, compared with GC, in samples
with less than 1% trans fat. Direct analysis by Ag+HPLC can quantify only trans-18:1
FAME, and is not suitable for determining the total trans fat content according to
current food labeling regulations. The Ag+HPLC fractionation followed by GC is also
limited to the quantitation of trans-18:1 FAME and showed higher limits of detection
compared to direct GC analysis. At this time, for samples containing 1% or less trans
fat, GC appears to be the most suitable technique.
52
TABLES
53
Table 3.1 - Comparison of the Trans fat contentas % of total fat in ten samples determined by four teqniques.
54
FIGURES
Figure 3.1. - Partial gas chromatogram of a partially idrogenated oil sample.
55
Figure 3.2. – Ag+-HPLC analysis of a partially hydrogenated oil sample.
56
Figure 3.3. - Ag+-HPLC fractionation of a partially hydrogenated oil sample, followed by GC analysis.
57
Figure 3.4. - Attenuated Total Reflection FT-IR spectra of the calibration solution in the interval 1-5% trans fat. The height of the negative
second derivatative of the 966 cm¯¹ band was measure
3.5. REFERENCES
1)
AOCS official Method Ce 1h-05 (2005). Determination of cis-, trans-, Saturated,
Monounsaturated and Polyunsaturated Fatty Acids in Oils and Fats by Capillary
GLC, in Official Methods and Recommended Practices, AOCS Press, Champaign,
IL, USA.
2)
Delmonte P, Kramer JKG, Banni S, Yurawecz MP (2006). “New developments in
silver ion and reverse phase HPLC of conjugated linoleic acid” in Advances in
conjugated linoleic acid research, Vol. 3, edited by Yurawecz M.P., Kramer
J.K.G., Gudmondsen O., Pariza M.W. and Banni S., AOCS Press, Champaign, IL,
USA.
3)
Cruz-Hernandez C, Deng ZY, Zhou JQ, Hill AR, Yurawecz MP, Delmonte P,
Mossoba MM, Dugan MER, Kramer JKG (2004). J. AOAC Int., 87, 2, 545-562.
4)
Mossoba MM, Yurawecz MP, Kramer JKG, Delmonte P, Rader JI (2003). AOCS
Monograph: Official methods for Trans Fat Labeling, AOCS Press, Champaign,
IL, USA.
58
4
THE EFFECT OF EXTRUDED WHOLE LINSEED FLOUR
INTAKE ON THE VARIATION OF CLA (CONJUGATED
LINOLEIC ACID) CONTENT IN MILK, AND OFA
(OXYDATED FATTY ACID) ON CHEESE OBTAINED THE REGGIANA CATTLE’S CASE.
4.1. SUMMARY AND KEYWORDS .......................................................................................................60
4.2. INTRODUCTION...............................................................................................................................61
4.3. MATERIALS AND METHODS ........................................................................................................62
4.3.1. SAMPLES COLLECTION .........................................................................................................62
4.3.2. SAMPLES PREPARATION .......................................................................................................63
4.3.3. GAS CHROMATOGRAPHIC QUANTITATION .....................................................................64
4.3.4. HPLC ANALYSIS ......................................................................................................................64
4.3.5. STATISTYCAL ANALISYS......................................................................................................64
4.3.6. CHEESE SENSORY ANALISYS ..............................................................................................65
4.4. RESULTS AND DISCUSSION..........................................................................................................65
4.5. CONCLUSIONS .................................................................................................................................68
TABLES.....................................................................................................................................................70
FIGURES ...................................................................................................................................................73
4.6. REFERENCES....................................................................................................................................80
59
4.1. SUMMARY AND KEYWORDS
The experiment was conducted on Reggiana cattle over an eight months period in 2005.
By integrating extruded whole linseed flour (300 g/d) to the basal ration (fresh or
preserved forage) which changes according to the season, the possibility to modify both
the milk fatty acid composition in general, and the CLA content (Conjugated Linoleic
Acids) in particular, has been confirmed. The principal and statistically significant
results find that Rumenic Acid (C18:2 c9-t11) and Vaccenic Acid (C18:1 trans-11)
have risen up to 45% and 47% respectively on the milk fat content, subsequent to the
extruded whole linseed flour administration. Moreover the results confirm the
correlation between these two fatty acids in milk. However, saturated and
monounsaturated fatty acids (SFA, MUFA) are not suitable to be modified, with the
exception of the monounsaturated Oleic Acid that increases during the extruded whole
linseed flour integration and decreases when it is suspended. In conclusion, it is possible
to increase the CLA content in milk and dairy products and to improve their nutritional
values in human nutrition through an appropriate and targeted cattle’s ration integration.
Keywords: cow’s milk, Conjugated Linoleic Acid (CLA), rumenic acid (C18:2 c9t11),
vaccenic acid (C18:1 trans-11).
60
4.2. INTRODUCTION
It is well known how many efforts have been made so far to improve the fatty acids
composition in vegetable oils. However, not enough research concerning animal fat and
dairy products has been conducted, despite the concern of consumers. Only in the last
few years a serious investigation to enhance the fatty acid profile in milk and dairy fat)
has been started even in our country, achieving interesting results that lead the research
to a prosecution of this route. In fact, an increasing number of studies and researches
aimed to the beneficial effects played from some of the Essential Fatty Acids have been
risen. One of the most important in this group is represented by Linoleic Acid (C18:2
cis-9, cis 12); such fatty acid has to be introduced through a proper diet cause of human
incapacity for an endogenous production. Linoleic Acid is considered the forerunner of
all the CLA (Conjugated Linoleic Acids), a generic term used to describe positional and
geometric isomers of octadecadienoic fatty acids containing conjugated double bonds
(1). The cis-9, trans-11 CLA (rumenic acid) is “the one that unequivocally shows
anticarcinogenic activity with animal models” (1-7) as well as positive biological effects
against others pathologies (6-17). Furthermore, unlike other isomers that have
exogenous origin because formed by Butyrivibrio Fibrisolvens, which is widely
presents in ruminant diet, rumenic acid shows to have an endogenous source (in the
mammary gland and adipose tissue) from the conversion of vaccenic acid t-11 C, 18:1
as a consequence of Delta-9 desaturase enzyme (3-6, 12, 15, 18-22). CLA content in
ruminant milk and dairy products appears to be higher than in beef, and the cis-9, cis 12
configuration represents the main isomer from 80% to 90% (3, 6, 7, 14, 15, 18, 21, 2326). The natural variation of CLA content depends on several factors including the
seasonal trend (1, 7, 27-30) the diet, lactation phase, and the breed (4, 6, 15, 22, 31, 32).
61
Thus, animal scientists have set up diets and food strategies for ruminants oriented to
increase the content of these fatty acids in milk and, thereby, in dairy products (4, 6, 10,
15, 19-2131, 33-36), most likely modifying the food composition (14,15, 18, 22, 25, 26,
28, 31, 35, 37-42). Then The purpose of the present work has been to verify the
possibility to modify both the milk fatty acid composition and the CLA content, by
changing the diet as the basal ration (fresh or preserved forage), by integrating extruded
whole linseed flour, and by comparing results we achieved with analogous experiments.
4.3. MATERIALS AND METHODS
4.3.1. SAMPLES COLLECTION
The present work was conducted on a Reggiana cattle farm, located in the district of
Reggio Emilia, in the Parmigiano Reggiano yield area. The normal and usual cows
rationing has been modified by integrating extruded whole linseed flour (300 g/die) to
both the constant concentrated fodder (1kg/3L milk yield) and to the basal ration (fresh
or preserved forage) which changes according to the season, and achieved with the
collaboration of the farm personnel. In table 4.1. the experimental scheme reports what
is explained above
According to the different diets we adopted in the respective experimental periods, from
the milk yield obtained and delivered to the cheese factory later, 150 mL of outcrop
cream has been collected in a specific container after has being mixed for 15 minutes.
Moreover, Parmigiano Reggiano cheese was produced in a dairy factory from the
massal milk yield following the production's technical policy. The sampling were
carried out following the specific policy FIL-IDF (44), and at 6,12, and 24 month of
seasoning
62
4.3.2. SAMPLES PREPARATION
Milk cream samples were collected and stored at -20°C until analysis. About 0.5 g of
cream was placed in a 1000 ml separatory funnel and 50 ml diethyl ether (DE) was
added. The solution was mixed one minute, 50 ml of 50:50 petroleum ether (PE) /DE
(% by volume) was added, was mixed for one minute, then 50 ml of PE was added
before one minute of energetic mixing. Two hundred ml of saturated sodium chloride
solution was added and the separator funnel was gently mixed for one minute. The
organic phase (upper) was recovered and filtered over anhydrous sodium sulphate. The
extraction was repeated, and the extracts were combined. The organic solvent was
removed in a stream of nitrogen, and the extract was stored at -20°C under nitrogen.
The preparation of the methyl esters was performed following the modified procedure
of Cruz-Hernandez et al. About 20 mg of dry fat was put in a test tube and 2 ml of
hexane was added, followed by 40 µl of methyl acetate and 300 µl of 0.5N sodium
methoxyde in methanol ( #33080, Supelco Inc., Bellefonte, PA). The tube was purged
with nitrogen, mixed, heated 10 minutes at 50C in a silicon oil bath, and then frozen.
180 µl oxalic acid (0.5 g in 15 mL di ethyl ether) was added and thoroughly mixed, then
the sodium-oxalate precipitate was separate by centrifugation and the hexane fraction
was filtered through anhydrous sodium sulphate.
The cheese samples obtained from cows fed with two sperimental diet (Fresh
Forage/Fresh Forage and extruded whole linseed flour), were sampled and stored at
-40˚C until analysis. Fat was extracted following the Rose-Gottlieb method modified by
Secchiari (45). According to Rovellini and Cortesi (46) fat samples were transesterified
with 1.0 M sodium benzyloxyde in benzyl alcohol before the analysis by HPLC-DAD.
63
4.3.3. GAS CHROMATOGRAPHIC QUANTITATION
FAME were analyzed with an Agilent 6890 gas chromatograph (Hewlett Packard,
Wilmington, DE) equipped with a CP Sil 88 fused silica capillary column (100 m x0.25
mm (i.d.), 0.2-µm film thickness; Varian, Inc.), a FID detector, and a split injector.
Hydrogen was used as carrier at the constant flow rate of 1.0 min/min. The FID detector
was maintained at 300°C with air flow rate of 400 ml/min, hydrogen flow rate of 30
ml/min, and helium (make up gas) flow rate of 30 ml/min. The split injector was
maintained at 250°C with the split ratio of 1:100. The temperature program was as
follow: 4 minutes at 80°C, ramp 7°C/min to 180°C maintained 30 minutes, ramp 4°C
/min to 225°C maintained 20 minutes. The quantitation was based on FID theoretic
response factors, from AOCS Ce-1h05 Official Method, and the fatty acids percent
composition was calculated considering 100% the sum of the corrected areas.
4.3.4. HPLC ANALYSIS
Cheese samples were analyzed by HPLC-DAD equipped with a Spherisorb ODS-2
column (5 µm, 4,6 mm x 25 cm, 100 Å), 1 ml/min flow rate. Chromatograms were
recorded at 255nm.
4.3.5. STATISTYCAL ANALISYS
Calculation and statistics were performed with the Tukey test (version 6.0) to evaluate
the different levels of fatty acids in cream samples, according to the diet we adopted.
64
4.3.6. CHEESE SENSORY ANALISYS
After 24 month of seasoning, a trained panel examinated cheese samples by sensory
analisys. The panel was composed of 7 persons, members of TINVAL society, spin-off
Alma Mater Studiorum University of Bologna. Cheese samples were tested by judges at
16°C following a random order, and served in a parallelepiped (L:50/80 mm; h:15mm;
w:15mm) (47). Each judge evaluated either the samples structural parameters and the
sense of smell/taste. Mineral water and crackers were used as means of counteraction
between each test. The sensory profile was defined applying the Etana model (48), and
processing the data with Microsoft Excell 2003, ANOVA, and SPSS for Windows
(vers. 13.0, SPSS Inc., Chicago, Illinois, USA).
4.4. RESULTS AND DISCUSSION
Tables 4.1.-4.4. show the results obtained from the samples analysis carried out in
duplicate. Sixty seven fatty acid have been separated, identified and quantified; nineteen
are saturated, 17 monounsaturated, 19 poliunsaturated and 12 are mono and
diiunsaturated trans. From table 4.2. rise that 17 of the 19 fatty acids identified and
quantified as saturated do not change their content, while the C4 and the C6
significantly decrease their percentage parameters (P<0,05) when the linseed flour is
introduced. As reported in table 4.3. most of the 17 monounsaturated fatty acids do not
undergo a change with the linseed flour integration while the only exception is
represented by the linoleic acid which increases considerably. Table 4.4. shows the
variation of the 19 poliunsaturated fatty acids. By administering a linseed flour
enriched diet, a percentage gain of rumenic acid (C18:2 c9-t11) and a downward trend
of arachidonic acid (C20:4) can be observed, while normalize their self when the linseed
65
flour integration is suspended. The variation of rumenic acid appears to be statistically
significant (P<0,05). Relatively to the unsaturated fatty acids with a trans configuration,
as reported in table 4.5., the results point out another relevant matter: an increase of
vaccenic acid (C18:1 trans-11) level when preserved forage is administered instead of
fresh forage, and even when the extruded whole linseed flour is integrated to the fresh
forage ration; the trans vaccenic level normalizes itself when the linseed integration is
suspended. The latter observation is also valid for the elaidinic acid (C18:1 trans-9).
Figure 4.1. shows the full chromatogram with the overall fatty acids separation and
identification, while figure 4.2. and 4.3. report the chromatograms with the cis/trans
C18:1 fatty acids elution area (i.e. CLA), which is our principal purpose in this
investigation. On figure 4.4. the course of the content of rumenic and vaccenic acid
during the test period can be noticed. Moreover, figure 4.5. reports the principal
variations of the 4 fatty acid groups observed. Thus, this investigation demonstrates the
possibility to increase noticeably the CLA content in milk fat, by integrating extruded
whole linseed flour (300 g/die) to the basal ration of the cows. Higher levels of CLA in
milk fat have been found by M. Monici et al (14), who carried out an experimental
dietary study over 2000 cows to increase the ω3 (EPA and DHA) levels in milk fat.
They were able to achieve 1,308 g of CLA/100 g of fat, versus 1,160 g of CLA/100 g of
fat obtained in our research. We have to point out 2 investigations of Strocchi et al. (28,
29) from 1967 (40 years ago), where 200 butter samples from Emilia has been studied;
they firstly separated and identified more than 30 fatty acids, and afterwards they
established a positive correlation between the content in trans-monounsaturated
(vaccenic acid) and conjugated-diens (i.e. CLA). In these studies, the authors noticed
how the suchlike fatty acids composition of butters from the Parmigiano Reggiano yield
66
area differs from the fatty acid profile of butters collected in others district of Emilia
Romagna, even though it does not coincide with the most recent study from Chiavari et
al. (49). Lastly, Capella et al. (50) in 1974 reported the most important acquisition
about 71 fatty acids identified with different analytical techniques.
Moreover, a preliminary study on the content of the OFA in Parmigiano Reggiano
cheese sample collected at 6, 12, 24 month of seasoning was conducted. In this case
cheese samples from two experimental diets were analyzed: 1) Fresh Forage 2) Fresh
Forage with extruded whole linseed flour integration .
Since the CLA diary fat enrichment of the cows under study, during the processing was
equally distributed between butter and cheese, we analyzed the OFA in the Parmigiano
Reggiano cheese during the ripening. The data obtained are reported in figure3. More
precisely, the purpose of this investigation was to verify whether the extruded whole
linseed flour might be involved in the increase of the OFA content, because it could
modify the lipid class in general and, more precisely the MONO- and POLI- unsatured
fraction, which is strongly sensible to the oxidation (51). The lipid oxidation products
are involved on the development of atherosclerosis process even though not much is
known for the human health about their assimilation through the diet (52, 53). The
results obtained are reported in table 4.6. and figure 4.6.
Furthermore, a sensory evaluation of the same Parmigiano Reggiano cheese samples
from the two experimental diets (with and without integration) was conducted. The
results did not show so relevant differences between samples. Although, between the
sensory descriptor considerated in the Etana model (figure 4.7.), two of them showed
significant differences (P>0,001) relatively to the sour and to the friability. Their values
67
resulted to be lower in cheese samples from the experimental diet with extruded whole
linseed flour integration.
4.5. CONCLUSIONS
Over the last eight years, more than 2,600 scientific articles about CLA in dairy
products have been published. The increasing interest in CLA arise from the remarkable
health and dietary effects in human nutrition.
In this work, conducted over an 8 months period on a Reggiana cow farm (located in the
district of Reggio Emilia in the Parmigiano Reggiano yield area), important
considerations can be made by examining the statistical results.
First of all, according to the literature (25, 26, 40) by administering linseed flour
enriched diet to dairy cows, the possibility to increase the rumenic acid content in milk
has been confirmed. In order to point out the tight correlation between rumenic acid and
trans-vaccenic acid when linseed flour is integrated to the cows ration, an increment of
47% and 49% respectively has been seen in milk fat (3, 6, 15, 18-20, 22, 36, 40). This
first result is particularly interesting especially for the entity of rumenic acid
quantitative variation. On the other hand, the increment in trans-vaccenic acid has to be
seen negatively since both USA and EU health care are debating on low total trans fatty
acid (TFA) intake, even though we believe that is an important value to be pointed out.
TFA are generally present in food products containing hydrogenated fat and, as a
consequence, they influence both the low and high density lipoprotein levels (LDL,
HDL), increasing the risk coronary heart disease (54-56).The second result, closely
related with the first, concern an increment of 36% of the total TFA in milk by
68
introducing linseed flour in cow’s diet, while it undergoes a decrease of 22% when the
linseed integration has been suspended.
Lastly, the investigation about the OFA content in the Parmigiano Reggiano cheese
samples, shows an interesting result: as it might be thought, instead of being constant or
increasing in the amount, the OFA content decreased during the ripening, and it reduced
further in cheese samples where cows were fed with extruded whole linseed flour
integration. These results can be explained considering that the state of ripening induce
to create such a strong reducing system inside the cheese, preventing the formation of
the oxidized fatty acids.
69
TABLES
Table 4.1. – Experimental scheme
Period
Food ration
Observations
25 March - 29 April
Preserved forage (hay + MCI*)
4
30 April - 31 May
Fresh forage + MCI
4
Fresh forage + MCI + extruded whole linseed
flour (300 g/d)
Fresh forage + MCI after suspension of
extruded whole linseed flour integration
15 August - 30 September
12 October - 9 November
7
3
Table 4.2. – Percentage composition of saturated fatty acids and branched chain by
administering different diets.
Preserved forage
Fresh forage
Linseed flour
integration
Fresh forage
C4:0
3,55 b ± 0,10
3,52 b ± 0,04
3,20 a ± 0,08
3,26 a ± 0,05
C5:0
0,02 a ± 0,01
0,02 a ± 0,00
0,02 a ± 0,00
0,02 a ± 0,00
C6:0
2,12 b ± 0,03
2,08 b ± 0,07
1,90 a ± 0,02
1,97 a ± 0,03
C7:0
0,02 a ± 0,00
0,02 a ± 0,00
0,02 a ± 0,00
0,02 a ± 0,00
C8:0
1,25 b± 0,02
1,22 b ± 0,05
1,11a ± 0,03
1,19 b ± 0,03
C9:0
0,02 a ± 0,00
0,02 a ± 0,00
0,02 a ± 0,00
0,02 a ± 0,00
C10:0
2,73 b ± 0,05
2,67 b ± 0,13
2,38 a ± 0,07
2,64 b ± 0,05
C11:0
0,31b ± 0,01
0,30 a,b ± 0,01
0,28 a ± 0,01
0,31 a,b ± 0,00
C12:0 +11:1
3,21b ± 0,06
3,13 b ± 0,16
2,79 a ± 0,09
3,12 b ± 0,05
Unsatured
b
b
a
C13:0 +12:1
0,17 ± 0,01
0,17 ± 0,01
0,15 ± 0,01
0,17 b ± 0,00
C14:0
11,15 b ± 0,08
10,96 b ± 0,49
10,20 a ± 0,22
10,94 b ± 0,11
C15:0
1,26 a ± 0,02
1,27 a ± 0,04
1,20 a ± 0,05
1,26 a ± 0,03
C16:0
29,07 b ± 0,66
28,64 b ± 1,08
25,74 a ± 0,85
27,76 b ± 0,07
C17:0
0,76 a,b ± 0,02
0,79 b ± 0,02
0,73 a ± 0,02
0,77 a,b ± 0,01
C18:0
10,54 a ± 0,22
11,52 b ± 0,31
12,56 c ± 0,33
11,17 a,b ± 0,19
C19:0
0,03 a ± 0,05
0,11 a ± 0,00
0,15 a ± 0,18
0,08 a ± 0,03
C20:0
0,20 a,b ± 0,01
0,21 b,c ± 0,01
0,22 c ± 0,01
0,18 a ± 0,01
C22:0
0,08 a ± 0,01
0,10 a ± 0,01
0,09 a ± 0,01
0,08 a ± 0,01
C24:0
0,07 a ± 0,00
0,07 a ± 0,00
0,07 a ± 0,01
0,07 a ± 0,00
Different letters indicate significant differences (Tukey test with p<0,05).
70
Table 4.3. – Percentage composition of mono-unsaturated fatty acids (cis + trans) by
administering different diets.
Mono-unsatured Preserved forage
C13:1
C14:1
C15:1
C16:1 c9
C17:1 c
C18:1 c9 +t15
C18:1 c11
C18:1 c12
C18:1 c13
C18:1 c15
C19:1 c7
C20:1 c5
C20:1 c8
C20:1 c11
C22:1
C24:1
0,00 ± 0,00
0,90 a,b ± 0,03
0,01 a ± 0,01
1,31 b ± 0,02
0,27 a,b ± 0,01
20,91 a ± 0,34
0,55 a ± 0,01
0,26 a ± 0,01
0,07 a ± 0,01
0,11 a ± 0,01
0,11 b ± 0,00
0,01 a ± 0,01
0,14 a ± 0,01
0,05 a ± 0,01
0,01 a ± 0,00
0,01 a ± 0,00
Fresh forage
Linseed flour
integration
Fresh forage
0,00 ± 0,00
0,87 a,b ± 0,03
0,01 a ± 0,01
1,26 a,b ± 0,03
0,27 a,b ± 0,01
21,44 a ± 0,54
0,56 a ± 0,03
0,27 a ± 0,01
0,08 a ± 0,00
0,12 a ± 0,00
0,11 b ± 0,00
0,01 a ± 0,01
0,15 a ± 0,01
0,05 a ± 0,00
0,01 a ± 0,01
0,02 a ± 0,01
0,00 ± 0,00
0,83 a ± 0,05
0,01 a ± 0,00
1,17 a ± 0,06
0,25 a ± 0,01
22,62 b ± 0,50
0,55 a ± 0,02
0,31 a ± 0,02
0,09 a ± 0,01
0,14 a ± 0,06
0,06 a ± 0,12
0,01 a ± 0,02
0,12 a ± 0,05
0,06 a ± 0,03
0,02 a ± 0,00
0,01 a ± 0,01
0,00 ± 0,00
0,93 b ± 0,03
0,01 a ± 0,00
1,32 b ± 0,04
0,28 b ± 0,01
21,85 a,b ± 0,57
0,45 a ± 0,20
0,32 a ± 0,16
0,05 a ± 0,04
0,07 a ± 0,05
0,03 a,b ± 0,06
0,02 a ± 0,00
0,14 a ± 0,01
0,05 a ± 0,00
0,02 a ± 0,01
0,01 a ± 0,00
Different letters indicate significant differences (Tukey test with p<0,05).
Table 4.4. – Percentage composition of poly-unsaturated fatty acids by administering
different diets.
Poly-unsaturated
18:2 c9-c12
C18:2 c9-t11
C18:2 t9-c11
C18:2 t11-c13
C18:2 t11,t13
C18:2 t9,t11,t10,t12
C18:2 c9-t13
C18:3 c-c-c n-3
C18:3 c-c-c n-6
C20:2
C20:3 n-3
C20:3 n-6
C20:4
C20:5
C22:2
C22:3
C22:4
C22:5
C22:6
Preserved forage
a
2,15 ± 0,18
0,80 a ± 0,08
0,04 a ± 0,01
0,01 a ± 0,01
0,00 a ± 0,01
0,02 a ± 0,01
0,18 a ± 0,02
0,75 a ± 0,07
0,05 b ± 0,00
0,03 a ± 0,00
0,02 a ± 0,00
0,11 a ± 0,01
0,15 b ± 0,00
0,06 a,b ± 0,00
0,03 a,b ± 0,00
0,00 a ± 0,01
0,02 a ± 0,00
0,12 a ± 0,00
0,00 a ± 0,00
Fresh forage
a
2,13 ± 0,05
0,80 a ± 0,02
0,05 a ± 0,03
0,02 a ± 0,01
0,01 a ± 0,02
0,01 a ± 0,01
0,19 a ± 0,01
0,72 a ± 0,05
0,04 b ± 0,00
0,03 a ± 0,00
0,02 a ± 0,00
0,11 a ± 0,01
0,15 b ± 0,01
0,07 b ± 0,00
0,04 b ± 0,01
0,00 a ± 0,00
0,03 a ± 0,00
0,12 a ± 0,00
0,01 a ± 0,01
Linseed flour
integration
a
2,14 ± 0,08
1,16 b ± 0,07
0,04 a ± 0,02
0,06 b ± 0,03
0,00 a ± 0,01
0,03 a ± 0,02
0,26 b ± 0,02
0,96 b ± 0,10
0,03 a ± 0,01
0,03 a ± 0,01
0,01 a ± 0,01
0,10 a ± 0,01
0,11 a ± 0,04
0,05 a ± 0,02
0,03 a ± 0,02
0,00 a ± 0,00
0,02 a ± 0,01
0,11 a ± 0,01
0,01 a ± 0,02
Fresh forage
2,19 a ± 0,02
0,94 a ± 0,06
0,04 a ± 0,01
0,03 a ± 0,00
0,00 a ± 0,00
0,01 a ± 0,01
0,21 a ± 0,01
0,78 a ± 0,05
0,04 a,b ± 0,00
0,03 a ± 0,00
0,02 a ± 0,00
0,11 a ± 0,01
0,14 a,b ± 0,01
0,06 a,b ± 0,00
0,04 b ± 0,01
0,00 a ± 0,00
0,02 a ± 0,00
0,12 a ± 0,01
0,01 a ± 0,00
Different letters indicate significant differences (Tukey test with p<0,05).
71
Table 4.5. – Percentage composition of mono and di-unsaturated trans fatty acids by
administering different diets.
Unsatured trans
Preserved forage Fresh forage
Linseed flour
Fresh forage
integration
C16:1 t
0,08 a ± 0,01
0,08 a ± 0,00
0,11 a ± 0,04
0,10 a ± 0,00
C18:1 t4
0,02 a ± 0,00
0,02 a ± 0,00
0,03 b ± 0,00
0,02 a ± 0,00
C18:1 t5
0,02 a ± 0,01
0,02 a ± 0,00
0,02 a ± 0,01
0,01 a ± 0,01
C18:1 t6-8
0,21 a ± 0,01
0,22 a ± 0,00
0,28 b ± 0,01
0,23 a ± 0,02
a
C18:1 t9
0,30 ± 0,01
0,32
± 0,00
0,35 ± 0,03
0,31 a ± 0,01
C18:1 t10
0,32 a ± 0,03
0,34 a ± 0,03
0,31 a ± 0,03
0,32 a ± 0,01
C18:1 t11
1,68 a ± 0,06
1,80 a,b ± 0,03
2,65 c ± 0,18
2,06 b ± 0,02
C18:1 t12
0,26 a ± 0,02
0,27 a ± 0,00
0,36 b ± 0,03
0,26 a ± 0,01
C18:1 t16
0,30 a ± 0,14
0,32 a,b ± 0,01
0,51 b ± 0,06
0,30 a,b ± 0,16
C18:2 t9,t12
0,00 a ± 0,00
0,00 a ± 0,00
0,02 a ± 0,02
0,00 a ± 0,00
C18:1 t13+t14 (+c6-8)
0,62 a ± 0,02
0,68 a ± 0,01
0,89 b ± 0,06
0,66 a ± 0,03
a
a,b
a
b
a
C18:2 c9-t12
0,24 ± 0,10
0,10 ± 0,01
0,19 ± 0,14
0,16 a ± 0,13
C18:2 t9-c12
0,10 a ± 0,01
0,12 a ± 0,01
0,28 b ± 0,13
0,20 a ± 0,04
Different letters indicate significant differences (Tukey test with p<0,05).
Table 4.6. – Trend of oxidized fatty acids (OFA) in 4 moulds of Parmigiano Reggiano
cheese at 6,12 and 24 months of ripening in relation to food ration (mg OFA /100
mg of lipids).
Months
Food ration
6
12
24
Diet with fresh forage
4,15 ± 1,15
0,81 ± 0,04
1,39 ± 0,24
Diet with fresh forage
4,44 ± 1,26
0,93 ± 0,01
0,92 ± 0,06
Extruded whole linseed flour integration
3,23 ± 0,92
0,64 ± 0,60
0,49 ± 0,38
Extruded whole linseed flour integration
3,38 ± 0,91
0,75 ± 0,88
0,59 ± 0,22
72
73
FIGURES
Figure 4.1. - Chromatogram of fatty acid
74
Figure 4.2. – Partial chromatogram of cis/trans-18:1 acids.
75
Figure 4.3. - Partial chromatogram of CLA.
76
Figure 4.4. - Effect of linseed flour integration: vaccenic acid and rumenic acid variations by varying cow’s diet (■ trans vaccenic acid
percentage; ♦ rumenic acid percentage).
77
Figure 4.5. - Effect of linseed flour integration: variation of acidic composition: ( a: preserved forage; b): fresh forage; c) enriched
extruded whole linseed flour; d) fresh forage, non enriched fresh forage).
78
Figure 4.6. - Intake trend of oxidized fatty acids (mg OFA /100 mg of lipids) in Parmigiano Reggiano cheese samples collected at different
ripening times referred to two experimental batches (two samples for batches: ■ - ♦ diet with fresh forage; ○ -▲ extruded whole
linseed flour integration).
Figure 4.7. - Etana profiles of two experimental batches (—— extruded whole linseed
flour integration; ------ control).
79
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depression in dairy cows results in increased trans-10,cis-12 CLA in milk fat and
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an extended period of time. J. Dairy Sci., 87, (6), 1758-1766.
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synthesis of cis-9, trans-11 conjugated linoleic acid in dairy cows fed fresh pasture.
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(2006). Milk production and composition from cows fed small amounts of fish oil
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cultures. J. appl. microbiol, (85), 95-102.
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and linolenic fatty acids to lactating ewes on cheese yield and on fatty acid
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lactating ewes on milk composition, cheese yield, and fatty acid composition of
milk and cheese. Small Ruminant Res., 63, 233-241.
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H, Sébédio JL (2006). Conjugated linoleic acid (CLA) content of French Emmental
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cis-9, trans-11 conjugated linoleic acid content in milk fat from Nordic Countries.
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Prodution responses of dairy cows to dietary supplementation with conjugated
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(Conjugated Linoleic Acids) nel latte: significato biologico, prospettive analitiche
ed applicative. Atti Conv. “Metodologie avanzate di ricerca e tematiche strategiche
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umana”, Firenze, 6 marzo 2002.
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Consumer acceptability of conjugated linoleic acid-enriched milk and Cheddar
cheese from cows grazing on pasture. J. Dairy Sci., 88, (5), 1837-1847.
35) Perfield JW, Lock AL, Pfeiffer AM, Bauman DE (2004). Effects of amideprotected and lipid-encapsulated conjugated linoleic acid (CLA) supplements on
milk fat synthesis. J. Dairy Sci., 87, (9), 3010-3016.
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in milk fat in response to dietary supplementation with calcium salts of trans-18:1
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3836-3844.
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salts and formaldehyde-protected conjugated linoleic acid in inducing milk fat
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38) Dhiman TR, Helmink ED, Mcmahon DJ, Fife RL, Pariza MW (1999). Conjugated
linoleic acid content of milk and cheese from cows fed extruded oilseeds. J. Dairy
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conjugated cis-9, trans-11-ocatadecadienoic acid in bovine milk because of dietary
supplementation. J. Dairy Sci., 81,(12), 3259-3267.
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and conjugated fatty acids and bovine milk fat yield due to dietary concentrate and
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5
A SIMPLIFIED METHOD FOR HPLC-MS ANALYSIS OF
STEROLS IN VEGETABLE OIL
5.1. SUMMARY AND KEYWORDS .......................................................................................................86
5.2. INTRODUCTION...............................................................................................................................87
5.3. MATERIALS AND METHODS ........................................................................................................89
5.3.1. CHEMICALS ..............................................................................................................................89
5.3.2. INSTRUMENTATION ...............................................................................................................90
5.3.3 ANALYTICAL METHOD...........................................................................................................90
5.3.3.1. OFFICIAL METHOD ............................................................................................................90
5.3.3.2. PROPOSED METHOD .........................................................................................................90
5.3.4. SAMPLES ...................................................................................................................................91
5.3.5. SAMPLE PREPARATION FOR STEROLS AND DIHYDROXY TRITERPENES.................91
5.3.5.1. OFFICIAL METHOD ............................................................................................................91
5.3.5.2. PROPOSED METHOD .........................................................................................................92
5.4. RESULTS AND DISCUSSION..........................................................................................................92
5.4.1. ISOLATION OF STEROLS AND DIHYDROXY TRITERPENES FROM THE
UNSAPONIFIABLE FRACTION USING THE REDUCED METHOD .....................................92
5.4.2. HPLC-MS CONDITIONS...........................................................................................................93
5.4.3. IDENTIFICATION OF STEROLS AND DIHYDROXY TRITERPENES IN DIFFERENT
SAMPLES .....................................................................................................................................94
5.4.4. ANALYTICAL PARAMETERS ................................................................................................96
5.4.5. APPLICATION AND QUANTIFICATION TO REAL SAMPLES ..........................................97
5.4.6. COMPARATIVE STUDY ..........................................................................................................98
5.5. CONCLUSIONS .................................................................................................................................98
TABLES...................................................................................................................................................100
FIGURES .................................................................................................................................................102
5.8. REFERENCES..................................................................................................................................105
85
5.1. SUMMARY AND KEYWORDS
A liquid-chromatographic method using atmospheric pressure chemical ionization
(APCI)-mass spectrometry (MS) detection in positive mode has been developed. This
method was used to separate and identify 15 sterols and 2 dihydroxy triterpenes in
saponified oils, enabling the analysis of these compounds directly from saponified
samples without recourse to thin-layer chromatography; this fact thus significantly
simplifies the process. The analyses were made using a Waters Atlantis 5 mm dC18
15062.1 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow
rate of 0.5 ml/min and a column temperature of 30°C. The quantification of several of
these compounds in soybean oil, palm oil, seed oil, sunflower oil, olive-pomace oil and
virgin olive oil was carried out using their commercial standards, and the results were
compared satisfactorily with the official method.
Keywords: High-performance liquid chromatography, Mass spectrometry, Oils, Olive
oil, Sterols.
86
5.2. INTRODUCTION
Sterols are widely occurring natural substances and make up the greatest proportion of
the unsaponifiable fraction of lipids (1, 2). Plant fats and oils contain phytosterols as
naturally occurring constituents (3). The most important natural sources of plant sterols
in the human diet are oils and margarines, although they are also found in a range of
seeds, legumes, vegetables and unrefined vegetable oils (4–6). Their composition
depends on the plant species (7) and, in oils, it may vary according to agronomic and
climatic conditions, the quality of the fruits or seeds, extraction and refining procedures
and storage conditions. Phytosterols can be classified on a structural or biosynthetic
basis as 4-desmethyl sterols, 4α-monomethyl sterols and 4,4-dimethyl sterols. In
addition, the 4-desmethyl sterols may be subdivided into ∆5-sterols, ∆7-sterols and
∆5,7-sterols, depending on the position of the double bonds in the B ring (8). The
predominant phytosterol is β-sitosterol; minor components are campesterol,
stigmasterol, ∆5-avenasterol, ∆7-avenasterol and brassicasterol. These compounds are
biogenetic precursors of numerous metabolites, including plant steroid hormones, and
have anti-inflammatory, antibacterial, antifungal, antiulcerogenic and antitumoral
activity (9).
The characterization and quantification of phytosterols can be carried out by a wide
variety of chromatographic techniques including column chromatography, thin-layer
chromatography (TLC), gas chromatography (GC), normal phase high-performance
liquid chromatography (HPLC), reverse-phase HPLC, capillary electrochromatography
(10) and also online-coupled HPLC-GC. These compounds can be detected with flame
ionization detection (FID), UV detection (UV), evaporative light scattering detection,
infrared detection, nuclear magnetic resonance detection, and mass spectrometry (MS).
87
GC using FID or MS (11–21) and HPLC with different detection systems are the more
widely used techniques (22–25), although the most accurate identifications have been
performed by MS. Recently, some papers on the determination of sterols with HPLCMS (26–29) have been published; in the article of Cañabate et al. (29), a liquid
chromatographic method for the identification and quantification of seven phytosterols
in olive oil and the sterols was developed and the compounds were quantified by liquid
chromatography with MS detection in positive atmospheric pressure chemical
ionization (APCI) mode. This is due to the fact that, in HPLC-MS analysis, baseline
separation of peaks is not always necessary because of the high specificity and
selectivity of the detection system. Because sterols are highly lipophilic and have few
polar functional groups, they are difficult to ionize by conventional electrospray
methods (30); APCI is the most widely used ionization technique for sterol analysis.
HPLC-MS with APCI has been found to be suitable for sterol analyses in different
sample matrices. It has been used to identify sterols in soybean oil (26), to characterize
phytosterols in spelt (30), to determine ergosterol levels in bulrush (31) and to measure
cholesterol oxides in various foods (32).
The official method (33–35) for the analysis of total sterols from oils involves the
saponification of the lipids, extraction of the unsaponifiable matter with diethyl ether
and washing the extract with water (liquid-liquid extraction), followed by separation by
TLC on silica gel plates, derivatization of the sterols with trimethylsilyl derivatives and
subsequent GC analysis. Therefore, sample preparation is laborious, the technique is
highly wasteful in terms of reagents, and the separation of sterols by TLC is deceptive.
Herein is described a simple HPLC-APCI-MS method to analyze 4-desmethyl sterols
and two dihydroxy triterpenes in oil samples after saponification and a simple liquid-
88
liquid extraction. This proposed method is useful as it permits to identify 17 compounds
(4-desmethyl sterols and two dihydroxy triterpenes) and quantify seven of them (vs.
their commercial standards) which are relevant in olive oils and other oils that are
recognized by the International Olive Oil Council and included in European legislation.
The use of MS as detection system provides an accurate identification of the compounds
under study.
5.3. MATERIALS AND METHODS
5.3.1. CHEMICALS
Cholesterol came from Riedel-de Haën (Seelze, Germany). β-Sitosterol (90%),
erythrodiol (97%) and stigmasterol (95%) were from Fluka (Buchs, Switzerland).
Sitostanol (95%), uvaol (95%), ∆5-avenasterol (95%), 2,7-dichlorofluorescein,
anhydrous pyridine, hexamethyl disilazone and trimethylchlorosilane were from SigmaAldrich (St. Louis, MO, USA). Stocks solutions containing 50 mg/mL of sterols were
prepared in HPLC-grade methanol and stored in the dark at 4°C. The concentration of
the final stock solution was calculated taking into account the purity of commercial
standards. Working standard solutions were prepared from these solutions and diluted
with methanol prior to analysis. HPLC-grade solvents (ethanol, methanol, hexane,
diethyl ether, 2-propanol, toluene, acetone, hexane, ethyl ether and chloroform) were
from Panreac (Barcelona, Spain). Potassium hydroxide and anhydrous sodium sulfate
were from Sigma, and Silicagel 60 TLC plates (20x20 cm) from Merck (Barcelona,
Spain). Water was deionized with a Milli-Q system (Millipore, Bedford, USA).
89
5.3.2. INSTRUMENTATION
GC analyses were done with a Fison GC 8000 series (Fison Instrument, France) using
for the separation a WCOT-fused silica 30 m x 0.25 mm i.d. coating CP FIL 8CB, DF =
0.25 µm column (J&W Scientific Inc. Agilent, Spain), and a flame ionization detector
(Fison Instrument, France). The carrier gas was helium. A 10 µl GC microsyringe with
a hardened needle was used.
HPLC analyses were carried out using an HPLC system from Thermo Separation
Products (UK) equipped with an autosampler, a degasser and a heated column.
Separation was done on an Atlantis dC18 150 column (2.1 mm i.d., 5 mm; Waters) under
gradient conditions at an injection volume of 10 µL at 30°C. The mass spectrometer
system was a Finnigan AQA (ThermoQuest, USA) with an APCI interface. Data were
collected by Xcalibur data system software on a personal computer.
5.3.3 ANALYTICAL METHOD
5.3.3.1. Official Method
To assure that our results are reliable, the samples were analyzed with the official
method. The operating conditions of this method are as follows: column temperature,
260 ± 5°C; injector temperature, 280°C; detector temperature, 290°C; linear velocity of
the carrier gas: helium 20 cm/s, hydrogen 30 cm/s; splitting ratio of 1 : 75; amount of
substance injected, 1 µl of trimethylsilyl (TMSE).
5.3.3.2. Proposed Method
The analyses were made using a Waters Atlantis 5mm dC18 150 x 2.1 mm column with
a gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 0.5 mL/min and a
90
column temperature of 30°C. The MS conditions were: a nebulizer temperature of
450°C, source temperature of 120°C; corona discharge of 10 µA; accelerating voltage of
25 V; cone gas flow rate of 50 L/h; and a desolvation gas flow rate of 350 L/h.
5.3.4. SAMPLES
Different vegetable oils (soybean, palm, seed, sunflower, olive-pomace and virgin olive
oil) were acquired on a local market and used as received.
5.3.5. SAMPLE PREPARATION FOR STEROLS AND DIHYDROXY
TRITERPENES
5.3.5.1. Official Method
In the official method (33–35), the isolation of the sterol fractions in olive oils involves:
(1) Saponification with KOH in ethanol solution: Usually, 5 g of oil is saponified by
refluxing with 50 ml of an ethanolic solution of 2 M KOH for 1 h.
(2) Extraction of the unsaponifiable fraction with diethyl ether: 100 ml water is added
after cooling to room temperature and, subsequently, after phase separation in a
separatory funnel, the aqueous phase is washed three times with diethyl ether.
(3) Washing with water: The diethyl ether fractions are collected, washed with water
and dried with anhydrous sodium sulfate.
(4) Separation by TLC on silica gel plates, derivatization of the sterols and subsequent
chromatographic analysis: 100 ml of a 5% solution of the unsaponifiable fraction in
chloroform is streaked on a chromatographic plate as thinly and uniformly as possible
and then allowed to elute until the solvent front is approximately 1 cm from the upper
91
edge of the plate. The plate is removed from the developing chamber and the solvent
evaporated either in a flow of hot air or by being left for a short while under a hood.
5) Recovery of the sterols by means of scratching and their extraction with chloroform:
The plate is sprayed lightly and uniformly with the 2,7-dichlorofluorescein solution and
the silica gel in the marked area is scraped off with a metal spatula.
6) Derivatization of sterols: The silylation reagent is added to the test tube, which is
then stopped. It is shaken carefully until the sterols are completely dissolved and then
centrifuged for a few minutes. The clear solution is then ready for GC analysis.
7) Analysis by GC-FID.
5.3.5.2. Proposed Method
We describe here a simplified way of pre-treating the sample, which is complete after
the third step. The diethyl ether fractions are collected, washed with water and dried
with anhydrous sodium sulfate. They are then filtered and evaporated to dryness using a
rotary evaporator at reduced pressure. The residue is dissolved in methanol. Sample
extracts are filtered through a membrane filter (0.45 µm) before being analyzed by
HPLC-APCI-MS.
5.4. RESULTS AND DISCUSSION
5.4.1. ISOLATION OF STEROLS AND DIHYDROXY TRITERPENES FROM
THE UNSAPONIFIABLE FRACTION USING THE REDUCED METHOD
The sterol fractions in virgin olive oil and other oils were tested by HPLC-APCI-MS
with and without using TLC as described by the official method. We succeeded in
92
reducing the time needed to isolate these compounds in comparison to the official
method. The official method was simplified prior to separation by TLC. At this point,
the solvent was evaporated and the extract re-dessolved in methanol. Figure 5.1. shows
the differences found between the two extracts of virgin olive oil with and without the
use of TLC. As it can be seen, the simplified extraction method (without the use of
TLC) gave a similar profile to that obtained by the official extraction protocol with
TLC.
5.4.2. HPLC-MS CONDITIONS
The composition of the mobile phase, the solvent ratio and the flow rate was studied.
After preliminary studies, it was decided that the optimum flow rate should be 0.5
ml/min. Different gradients using water (0.01% acetic acid), acetonitrile and methanol,
and different isocratic compositions at a constant flow of 0.5 ml/min were assayed to
obtain the best resolution of the chromatographic peaks. In mixtures containing
methanol, the resolution and intensity of the peaks was worse. After realizing that water
(with 0.01% acetic acid) (as phase A) and acetonitrile (as phase B) were the most
appropriate mobile phases to achieve our aim, we checked a wide number of gradients,
taking into account the resolution among peaks, the intensity in the mass spectrometer
and the analysis time as analytical parameters.
In general, we observed that an increase in the percentage of phase A at the beginning
of the analysis slowed down the analysis time. Using the following gradient, from 0 to
40 min, from 90% B/10% A to 100% B, we obtained a clear resolution between peaks,
so we focused our experimental work upon the study of linear gradients, i.e. 85 to
100%, 90 to 100% and 95 to 100% of phase B, all over a period of 40 min. In this study,
93
we observed that the resolution between peaks decreased concomitantly with an
increase in the initial percentage of acetonitrile. When using an initial percentage of
acetonitrile of 85%, the resolution improved, but the peaks were retarded considerably.
Therefore, we studied in detail the variation of the initial acetonitrile concentration from
90 to 95% over 60 min in order to obtain clear resolution between peaks. We finally
found that with a C18 stationary phase and a gradient between 90% acetonitrile/10%
water + 0.01% acetic acid and 92% acetonitrile/8% water + 0.01% acetic acid, from 0 to
60 min at a flow rate of 0.5 ml/min and a column temperature of 30 °C, we achieved the
best separation.
Optimum MS conditions were: a nebulizer temperature of 450 °C, a source temperature
of 120 °C; corona discharge of 10 µA; accelerating voltage of 25 V; cone gas flow of 50
L/h; and a desolvation gas flow of 350 L/h. HPLC-MS determinations were obtained by
operating the mass spectrometer in the positive ion mode. Full-scan mass spectra were
acquired within the 300–450 m/z range.
The identification of each compound was carried out using the migration time and the
fragmentation, and for the quantification the area of each ion extracted was used.
Figure 5.2. shows a chromatogram of a standard solution of five sterols and two
dihydroxy triterpenes under the optimum conditions chosen.
5.4.3. IDENTIFICATION OF STEROLS AND DIHYDROXY TRITERPENES IN
DIFFERENT SAMPLES
Samples obtained by using the new simple extraction procedure were used for the
quantification of sterols and dihydroxy triterpenes by HPLC-APCI-MS and samples
obtained by the official procedure extraction for the quantification by GC-FID. The
94
presence of erythrodiol, uvaol, cholesterol, ∆5-avenasterol, stigmasterol, β-sitosterol
and sitostanol was confirmed by comparing their mass spectra and retention times with
the data obtained from the standard compounds. Peak identification was also performed
with spiked real samples at different concentration levels. For the identification of the
other sterols, the analyses carried out by GC-FID on each of the oil samples in question
were took into account. The different oils studied contained specific quantities of some
of the sterols, as they were determined previously by GC-FID. Although the migration
time of sterol by GC-FID is different than by HPLC-APCI-MS, the elution order is
known; such results were helpful to identify them in the samples analyzed by HPLC. In
other words, when the official method (GC-FID) is used, the sterols elution order result
to be as follow: cholesterol, brassicasterol, 24-methylene cholesterol, campesterol,
campestanol, stigmasterol, ∆7-campesterol, ∆5,23-stigmastadienol, chlerosterol, βsistosterol, sitostanol, ∆5-avenasterol, ∆5,24-stigmastadienol, ∆7-stigmasterol, ∆7avenasterol. This means all the mentioned compounds can be identified and quantified
by GC-FID, using the integrated area and the external standards. Thus, these results and
the relative concentrations of the different compounds can be used to identify those
analytes in the HPLC profiles.
β-Sitosterol was the principal component in all the oils studied. Cholesterol was more
abundant in palm and seed oils, while brassicasterol only appeared in seeds and olivepomace oils (in minor quantities in the latter); campesterol, campestanol, stigmasterol
and sitostanol were predominant in soy oil, and erythrodyol and uvaol were very
abundant in olive pomace oil. The percentage of each component was determined in
each sample of oil analyzed, in order to assign the corresponding component to each
peak in the chromatogram obtained by using HPLC-APCI-MS.
95
The spectra were characterized by the protonated molecules of the analytes and an
abundant signal corresponding to fragment ions due to the loss of a water molecule (see
Table 5.1.).
5.4.4. ANALYTICAL PARAMETERS
The detection limits (DL = 3S0/b) and quantification limits (QL = 10S0/b) of the method
were tested against erythrodiol, uvaol, cholesterol, ∆5-avenasterol, stigmasterol, βsitosterol and sitostanol using the IUPAC method (36), where S0 is the standard
deviation of the blank using as the blank the signal to- noise (S/N) ratio and b is the
slope of the calibration plot.
All the calibration curves showed good linearity: Cholesterol, stigmasterol and
sitostanol were linear from QL (0.25) to 20 mg/L, β-sitosterol from QL (0.15) to 200
mg/L, ∆5-avenasterol from QL (0.25) to 50 mg/L, and erythrodiol and uvaol from QL
(0.50) to 200 mg/L. Each point of the calibration plot was repeated three times in an
independent solution prepared in the same way. The calibration plots indicate good
correlation between peak areas and analyte concentrations. The regression coefficients
(r2) were 0.999 for all the compounds quantified.
The repeatability of the method was checked by analyzing the same sample seven times
in one day and five times on different days. The intraday relative standard deviation
(RSD) of the retention times for uvaol, erythrodiol, stigmasterol and β-sitosterol was
around 1% and the RSD of the areas for the same compounds was around 2%. The
interday RSD of the retention times and areas was around 1.5 and 4.0%, respectively,
for the same compounds.
96
5.4.5. APPLICATION AND QUANTIFICATION TO REAL SAMPLES
Using the described extraction protocol and the HPLC-APCI-MS method, six different
oil samples (soybean oil, palm oil, seed oil, sunflower oil, olive-pomace oil and virgin
olive oil) were analyzed. All samples were injected into the HPLC instrument five times
(n = 5).
Sample preparations of all oils were made as described in Section 5.4.. The analyses
were carried out under the optimum conditions described in Section 5.4.2., and
quantification was carried out using the extracted ion chromatogram at 369, 395, 397,
399 and 425 m/z. Erythrodiol, uvaol, cholesterol, ∆5-avenasterol, stigmasterol, βsitosterol and sitostanol were quantified individually in mg/kg (Table 5.2.), while
erythrodiol and uvaol were quantified together because they eluted at the same time and
provided the same m/z in MS.
The chromatograms of six samples of different oils are shown in figure 5.3.. The main
peaks in the chromatograms can be observed within the retention time range between 20
and 35 min and belong to ∆7-avenasterol, cholesterol, ∆5-avenasterol, ∆7-campesterol,
campestanol, clerosterol, stigmasterol, ∆7-stigmastenol, ∆5,24-stigmastadienol and βsitosterol.
β-Sitosterol was the most abundant sterol in all the oils. 24-Methylene cholesterol was
only present in soybean and palm oil. Brassicasterol was only present in seed and olive
pomace oil. High quantities of erythrodiol and uvaol were also found in olive pomace
oil. This kind of oil is frequently mixed with virgin olive oil and marketed and sold as
olive oil.
Regarding this last point, it is possible to say that our method could be capable of
detecting fraudulent mixtures of virgin olive oils and olive pomace oils, both
97
quantitatively and qualitatively, analyzing the data of dihydroxy triterpenes in a few
minutes, while the official method of the International Olive Oil Council takes more
than 20 min to achieve the same purpose.
5.4.6. COMPARATIVE STUDY
To assure our results are reliable, all the samples were also analyzed with the official
method. The operating conditions were as follows: column temperature, 260 ±5°C;
injector temperature, 280°C; detector temperature, 290°C; linear velocity of the carrier
gas: helium 20 cm/s, hydrogen 30 cm/s; splitting ratio of 1 : 75; amount of substance
injected, 1 ml of TMSE solution.
The results are shown in Table 5.2. as individual sterol concentrations in mg/kg of fatty
material. A comparison of such a data, reveals that the results obtained with the
proposed method are in good agreement with those obtained with the official method.
Erythrodiol and uvaol can be quantified in all oils except in palm oil with the proposed
method, while they can only be quantified in olive pomace and virgin olive oils with the
official method. However, sitostanol can be quantified in all the oils analyzed with the
official method, but only in soybean, olive pomace and virgin olive oils with the
proposed new method.
5.5. CONCLUSIONS
This study describes the use of HPLC-APCI-MS to identify efficiently 17 compounds
and to quantify seven of them that are legislated upon by several regulations and
98
trademarks laid down by the International Olive Oil Council and the European Union.
The advantages of the proposed method are:
1) simplicity in the preparation of sample, 2) robustness: good repeatability taking into
account retention time and areas, and 3) cheaper process of the samples compared to the
official method. The results found with the proposed method for the analytes studied are
in good agreement with the obtained data using the official method.
99
TABLES
Table 5.1. – Ions observed in the APCI mass spectra of sterols in positive mode and
their retention times.
100
101
Table 5.2. – Concentrations of sterols, expressed as mg/kg of the different vegetable oils quantified by HPLC-APCI-MS and the official
GCFID method; (¤Value = \bar X¤ ± SD)
FIGURES
Figure 5.1. - Chromatogram of two extracts of virgin olive oil using isolation of total
sterols both with and without TLC.
102
Figure 5.2. – HPLC-APCI-MS chromatogram of a standard solution of five sterols and
two triterpenic alcohols. Gradient from 90% acetonitrile/10% water + 0.01%
AcOH to 92% acetonitrile/8% water + 0.01% AcOH from 0 to 60 min. Peaks:
(1+2) erythrodiol and uvaol, (3+4) cholesterol and ∆5-avenasterol, (5)
stigmasterol, (6) β-sitosterol, (7) sitostanol.
103
104
Figure 5.3. - Chromatograms of six different vegetable oils. (1) Erythrodiol, (2) uvaol, (3) ∆5,23-stigmastadienol, (4) 24-methylene
cholesterol, (5) brassicasterol, (6) ∆7-avenasterol, (7) cholesterol, (8) ∆5-avenasterol, (9) ∆7-campesterol, (10) clerosterol, (11)
campesterol, (12) campestanol, (13) stigmasterol, (14) ∆7-stigmastenol, (15) ∆5,24-stigmastadienol, (16) β-sitosterol and (17)
sitostanol.
5.8. REFERENCES
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sterols in vegetables and fruits commonly consumed in Sweden. Eur. J. Nutr., 38,
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vegetables, fruits and berries. J. Food. Comp. Anal., 83, 330–337.
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Chapman & Hall, London (UK).
10) Li D, Sinclair AJ (2002). Macronutrient innovations. The role of fats and sterols in
human health. Asia Pac. J. Clin. Nutr., 11, S155–S162.
11) Abidi SL (2001). Chromatographic analysis of plant sterols in foods and vegetable
oils. J. Chromatogr. A, 935, 173–201.
12) Alonso L, Fontecha V, Lozada L, M. Juarez M (1997). Determination of mixtures
in vegetable oils and milk fat by analysis of sterol fraction by gas chromatography.
J. Am. Oil Chem. Soc., 74, 131–135.
13) Medvedovici A, David F, Sandra P (1997). Analysis of sterols in vegetable oils
using off-line SFC/capillary GC-MS. Chromatographia, 44, 37–42.
14) Van Boven M, Daenens P, Maes K, Cokelaere M (1997). Content and composition
of free sterols and free fatty alcohols in jojoba oil. J. Agric. Food Chem., 45, 1180–
1184.
15) Mandl A, Reich G, Lindner W (1999). Detection of adulteration of pumpkin seed
oil by analysis of content and composition of specific Delta 7-phytosterols. Eur.
Food Res. Technol., 209, 400–406.
16) Lechner V, Reiter B, Lorbeer E (1999). Determination of tocopherols and sterols in
vegetable oils by solid-phase extraction and subsequent capillary gas
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17) Verleyen T, Verhe R, García L, Dewettinck K, Huyghebaert A, De Greyt W
(2001). Gas chromatographic characterization of vegetable oil deodorization
distillate. J. Chromatogr. A, 921, 277–285.
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var. Mission) fruit and tree components. J. Food Sci., 66, 278–281.
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“Periyakulam 1”. J Food Comp Anal., 15, 65–77.
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as marker. Eur. J. Lipid Sci. Technol., 104, 756–761.
21) Cercaci L, Rodríguez-Estrada MT, Lercker G (2003). Solid-phase extraction-thinlayer chromatography-gas chromatography method for the detection of hazelnut oil
in olive oils by determination of esterified sterols. J. Chromatogr. A, 985, 211–220.
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cholesterol, phytosterols and tocopherols in foods. Analyst., 115, 1525–1530.
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vegetable-oils by HPLC with evaporative lightscattering detection. J. Am. Oil.
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and conjugated phytosterols in different plant matrices. J. Chromatogr. B, 777, 67–
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27) Parcerisa J, Casals I, Boatella J, Codony R, Rafecas M (2000). Analysis of olive
and hazelnut oil mixtures by high-performance liquid chromatography-atmospheric
pressure chemical ionisation mass spectrometry of triacylglycerols and gas-liquid
chromatography of non-saponifiable compounds (tocopherols and sterols). J.
Chromatogr. A, 881, 149–158.
28) Sánchez-Machado DI, López-Hernández J, Paseiro-Losada P, López-Cervantes J
(2004). An HPLC method for the quantification of sterols in edible seaweeds.
Biomed. Chromatogr., 18, 183–190.
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performance liquid chromatography-mass spectrometry based chemometric
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Garrido Frenich A, Martínez Vidal JL, Duran Martos J (2007). Separation and
determination of sterols in olive oil by HPLC-MS. Food Chem., 102, 593–598.
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NM, Meurens M, Quetin- Leclercq J, Habib-Jiwan JL (2003). Phytosterol analysis
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and characterization in spelt (Triticum aestivum ssp spelta L.) and wheat (T.
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of ergosterol in a prairie natural wetland. J. Chromatogr. A, 958, 149–156.
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in processed food using high performance liquid chromatography-mass
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107
108
6
QUANTITATION OF LONG CHAIN POLY-UNSATURED
FATTY ACIDS (LC-PUFA) IN BASE INFANT FORMULAE
BY GAS CHROMATOGRAPHY, AND EVALUATION OF
THE BLENDING PHASES ACCURACY DURING THEIR
PREPARATION
6.1. SUMMARY AND KEYWORDS .....................................................................................................110
6.2. INTRODUCTION.............................................................................................................................111
6.3. MATERIALS AND METHODS ......................................................................................................113
6.3.1. SAMPLES COLLECTION .......................................................................................................113
6.3.2. SAMPLES PREPARATION .....................................................................................................113
6.3.2. GAS CHROMATOGRAPHIC QUANTITATION ...................................................................114
6.3.3. STATISTYCAL ANALYSIS....................................................................................................114
6.4. RESULTS AND DISCUSSION........................................................................................................114
6.5. CONCLUSIONS ...............................................................................................................................117
TABLES...................................................................................................................................................119
FIGURES .................................................................................................................................................126
6.6. REFERENCES..................................................................................................................................134
109
6.1. SUMMARY AND KEYWORDS
The purpose of this investigation has been the quantitation of Long Chain Polyunsatured Fatty Acids (LC-PUFA), such as arachidonic acid (ARA , C20:4 n-6) and
docosahexaenoic acid (DHA , C22:6 n3), and the evaluation of their stability during the
blending phases for the preparation of base infant formula added with ARA and DHA
less than 0,5%. The investigation has been also oriented to set up a method that can
permit to reduce the uncertainty values associated with the mesurement of ARA and
DHA, to a compatible level with the their certain determination in the samples
examinated.
Keywords: Infant Formula, LC-PUFA, ARA, DHA.
110
6.2. INTRODUCTION
Infant milk formulae are designed to provide infants with the required nutrients for
optimal growth and development. They are formulated to mimic breast milk
composition in order to obtain an ‘‘ideal’’ substitute (infant formula) for babies. Human
breast milk contains a full complement of all polyunsaturated fatty acids (PUFA),
including the two essential PUFA, linoleic acid (LA,18:2 n-6) and α-linolenic acid,
(ALA, 18:3 n-3) and also a range of LC-PUFA that have been shown to have benefits
for both preterm and term infants, such as promoting sensory and neuronal maturation.
LC-PUFA are present mainly in highly specialized membranes, e.g., retina and
synapses, and in membranes of excitable cells. Moreover, like DHA, ARA is an
essential fatty acid for adequate function of the central nervous system (1).
New research has shown that both preterm and term infants can actively convert the
essential fatty acids LA and ALA, to LC-PUFA (2, 3, 4). However, the amount of LCPUFA being produced, particularly of DHA, may not be sufficient to meet the
developmental requirement of the infant. An important question that is still challenging
investigators is whether formula fed term infants can make enough LCPUFA from the
essential fatty acids that are provided in formula or whether they require added
LC-PUFA. Research is ongoing and there is no final answer to this question, although
there is some evidence that a supply of LC-PUFA to the infant may be beneficial.
Most national and international authorities have based their recommendations for the
manufacturing of infant formulae on mature human milk fatty acids composition as the
gold standard. The Commission of the European Union issued regulations on infant
formula composition, including permission for addition of LCPUFA up to 1 and 2%,
respectively, of total fat content as n-3 and n-6 LC-PUFA (5).
111
Infant formulae are usually marketed as spray-dried powders to be reconstituted with
water, or ready-to-feed infant infant formulae. Their manufacture may differ from
producers, but it generally consists in a dry blending process. It begins with the receipt
of the ingredients, and stored until they are tested for conformance to specifications.
Then, dry ingredients are blended in large batches in a ribbon blender or other large
scale blending equipment. The ingredients are blended until the are uniformly
distributed throughout the batch. Later the product is passed through a sifter, and then
transferred to bags for storage, or directly to the packaging line.
Therefore, manufacturers consider of extremely importance to verify the proper
distribution and the stability of some ingredients in the infant formula during the
blending, since may be present in low numbers and may be non-randomly distributed
within the lot. In this particular case, the object of the investigation is a base infant
formula added with ARA and DHA less than 0,5% of total fat, with the purpose to set
up a method of analysis that permits to reduce the uncertainty values associated with the
measurement of these fatty acids, to a compatible level with the certain determination of
such LC-PUFA in the samples examined. Such a requirement rises from the limits in the
application of the methods of analysis UNI EN ISO 5508 (1998) and UNI EN ISO 5509
(2000), which do not allow to fully verify the effective content of ARA and DHA in the
product, because they determine high uncertainty values for the typology of the samples
under investigation, and because ARA and DHA are present in low concentration in the
formulation.
112
6.3. MATERIALS AND METHODS
6.3.1. SAMPLES COLLECTION
In order to carry out the investigation, the following samples of base infant formulae
were collected. Each sample was stored at -20°C until analysis:
™
10 powder samples (450 g each one) corresponding to the “inter-blend phase”, and
collected at the end of each blend phase (Table 6.1.).
™
3 powder samples (450 g each one) corresponding to the “intra-blend phase”, and
collected at the beginning/half/end of 5° blend phase (Table 6.2.).
™
11 powder samples (900 g each one) collected at the end of each blend and then
packaged. They correspond to the commercial products (Table 6.3.).
™
2 powder samples containing the raw material employed for the addiction of ARA
and DHA.
6.3.2. SAMPLES PREPARATION
Before the extraction, base infant formulae were reconstituted following the
specifications reported in the label. In this case, 4,3 g of powder were weighted and
dissolved in 30 ml of water, obtaining a concentration of 0,14 mg/ml. Later, the fat was
extracted in double following the Rose-Gottlieb official method (6), and then, the
preparation of the methyl esters was carried out applying the method of Christopherson
and Glass (7): about 20 mg of dry fat was put in a test tube, 50 µl of KOH MeOH 2N
and 500 µl of hexane were added respectively, and then the tube was mixed for 30
seconds; 0,5 mg of C13:0 (Sigma-Aldrich, St. Louis, MO, USA) were used for the fatty
acids quantitation as internal standard, while the 53 FAME GLC-reference standard
mix-463 (NU-CHEK-PREP, Elysian, MN) for the fatty acids identification. After the
113
phase separation has been acted, 300 µl of surnatant was collected and put in a vial.
Analysis by gas chromatography has been followed.
6.3.2. GAS CHROMATOGRAPHIC QUANTITATION
FAME were analyzed with a Perkin Elmer Clarus 500 gas chromatograph equipped
with a RTX 2330 (Restek, Bellefonte, PA, USA) fused silica capillary column (30 m
x0.25 mm i.d., 0.2-µm film thickness), a FID detector, and a split injector. Helium was
used as carrier at the constant flow rate of 0.75 min/min. The FID detector was
maintained at 240°C with air flow rate of 400 ml/min, hydrogen flow rate of 40 ml/min,
and helium (make up gas) flow rate of 30 ml/min. The split injector was maintained at
240°C with the split ratio of 1:50. The temperature program was as follow: 1 minute at
60°C, ramp 7°C/min to 240°C and maintained 10 minutes.
6.3.3. STATISTYCAL ANALYSIS
Calculation and statistics were performed with Statistica for windows (1998), and
Microsoft excel for windows (2003).
6.4. RESULTS AND DISCUSSION
The investigation has been focalized on the deepening of the following steps:
a. Characterization of the raw materials.
b. Set up of the method.
c. Fatty acid analysis of the intra-blend samples.
d. Fatty acid analysis of the inter-blend samples.
e. Fatty acid analysis of the commercial products.
114
a. The characterization of the raw materials has permitted to optimize the condition for
the fatty acids separation by gas chromatography, necessary to their identification
and quantification in the complex mixture.
b. A deep verification of the method of analysis has been carried out in order to verify
the steps and the manipulations requested, starting from the matrix under study. In
this case, the matrix was base infant formula, and the lipidic fraction has been
extracted with solvent following the Rose-Gottlieb official method. This method,
which can be also used on liquid milk, takes place in basic condition with the
purpose to break the milk fat globule membrane. In milk powder the fat is not
completely structured such as in non-modified milk, even though it must be stable in
the liquid product when reconstituted. However, the extraction must be conducted
considering the residual globules with membrane, which are more problematic to
break with solvent due to the smaller size and the higher ratio area/volume of the
globules. Therefore, the repetibily of the method has been verified by identifying
and quantifying the fatty acids in one of the commercial samples for five times, and
the results are reported on table 6.4.. The quantification has been carried out on the
principal fatty acids, besides the fatty acids added to the powder for the formulation.
Other minor fatty acids have been grouped under “others”. On figure 6.1. are
reported the chromatograms relative to the samples of raw materials employed for
the addiction of ARA and DHA, and a sample from the inter-blend. The verification
of the method have permitted to calculate the limit of detection (LOD) and the limit
of quantitation (LOQ), that are respectively of 20 and 60 ppm. The error, relatives to
the fatty acids under investigation, have been calculated on the commercial samples
115
according with the equation of William Horwitz (8, 9) that estimate the uncertainty
of measure (Table 6.5.). The equation is:
%RSDR=2(1-0.5logC)
where %RSDR is the inter-laboratory Coefficient of Variation, and C is the
concentration of analyte in the sample as a decimal fraction.
c. 3 powder samples corresponding to the “intra-blend phase” and collected at the
beginning/half/end of 5° blend phase, were analyzed in double by gas
chromatography. In table 6.6. are reported the mean, the standard deviation, and the
variance of the fatty acids identified and quantified in the samples. The fatty acids of
interest are underlined in grey, while fatty acids quantified as less than 0,1g/100g
are classified as “others” at the bottom of the table. The variance analysis of the
fatty acids of interest is reproduced on figure 6.2..
d. Table 6.7. shows the results of the analysis by gas chromatography of the inter-blend
samples carried out in double. The mean, the standard deviation, and the variance of
the fatty acids identified and quantified in the samples have been calculated. The
fatty acids of interest are underlined in grey. Moreover, in figure 6.3. is reported the
variance analysis of the fatty acids under investigation. Fatty acids quantified as less
than 0,1g/100g are classified as “others” at the bottom of the table.
e. The fatty acid analysis of the 11 commercial samples carried out in double, is
showed in table 6.8.. Fatty acids of interest are underlined in grey, while those
quantified as less than 0,1g/100g are classified as “others” at the bottom of the table.
The results include the mean, the standard deviation, and the variance of the fatty
acids identified and quantified in the samples, while in figure 6.4. is reported the
variance analysis of the fatty acids under investigation.
116
6.5. CONCLUSIONS
The investigation, oriented to set up a method that can permit to reduce the uncertainty
values associated with the measurement of ARA and DHA and to verify the effective
content of these fatty acids in base infant formulae, have permitted to achieve the
following goals:
First of all, the elaboration of the uncertainty values associated with the measurement of
ARA and DHA and the other fatty acids under study (Table 6.5.) has been obtained
from the analysis by gas chromatography of a commercial sample. The estimation of the
reproducibility calculated with the equation of William Horwitz can provide the
assessment of the uncertainty value, which can be verified through the comparison with
the single measurement of the fatty acids.
Moreover, as reported in table 6.4., the method of analysis shows a good repeatability
when considering the mean, the standard deviation and the variance of the fatty acids.
Intra-blend samples, collected at the beginning/half/end of 5° blend phase (Table 6.6.,
Figure 6.2.), present values absolutely satisfactory; the variance and the standard
deviation appear low, and the mean values are by far less than the error of the method
(Table 6.5.).
Same consideration can be made for the samples corresponding to the inter-blend
(Table 6.7., Figure 6.3.), even though the standard deviation and the variance result to
be higher, that is may due to the different lot of production.
On table 6.8. and figure 6.4. are reported the results about the commercial samples.
They show to be more homogeneous than the inter-blend samples, probably because the
latter represent an intermediate step in the production; as it can see in table 6.9., the
percent standard deviation results to be less than 10% for all the fatty acids considered.
117
A statistical comparison between intra-blend samples and commercial samples is
reported in table 6.10., where their principal parameters are compared, in particular the
student’s t-test that permits to describe the estimation of the data’s similarity. It rise that
with a level of test significative of 95%, the differences between the mean values
observed are not statistically significatives with p<0.05, indicating how the two groups
are non-different. Figure 6.5. (a to i) graphically shows the comparison of the
considered parameters in table 6.10., about the total lipids extracted and mono-, di-,
poli-unsatured fatty acids. The same figure report the mean, the standard error, and a
95% confidence interval.
118
TABLES
Table 6.1. – Powder samples corresponding to the inter-blend phase.
Number of
blends
1
2
3
4
5
6
7
8
9
10
Name on the can streaker
Meaning
End of 1° Blend
End of 2° Blend
End of 3° Blend
End of 4° Blend
End of 5° Blend
End of 6° Blend
End of 7° Blend
End of 8° Blend
End of 9° Blend
End of 10° Blend
EB1, BB2
EB2 BB4
EB3, BB6
EB4, BB8
EB5, BB10
EB6, BB12
EB7, BB14
EB8, BB16
EB9, BB18
EB10, BB20
Table 6.2. – Powder samples corresponding to the intra-blend phase (5° Blend).
Number of
blends
5
5
5
Name on the can streaker
BB5, BB9
MB5, BB9
EB5, BB10
Meaning
Beginning
Half
End
Table 6.3. – Powder samples corresponding to the commercial products.
Number of blends
2
3
4
5
6
7
8
9
10
Name on the can streaker
FP-BB3 / FP-BB4
FP-BB6
FP-BB8
FP-BB10 / FP-BB9
FP-BB12
FP-BB14
FP-BB16
FP-BB18
FP-BB20
119
Meaning
Obtained from 2° Blend
Obtained from 3° Blend
Obtained from 4° Blend
Obtained from 5° Blend
Obtained from 6° Blend
Obtained from 7° Blend
Obtained from 8° Blend
Obtained from 9° Blend
Obtained from 10° Blend
Table 6.4. – Repeatability of the method of analysis.
(g/100g of powder)
FAT
AG19_2-1 AG19_2-2 AG19_2-3 AG19_2-4 AG19_2-5 mean
SD VAR
22.81
25.00
24.91
25.64
22.86
24.24 ± 1.32 1.73
Satured
7.93
8.43
8.26
8.72
7.76
8.22
± 0.38 0.15
C4:0
C6:0
C8:0
C10:0
C11:0
C12:0
C14:0
C15:0
C16:0
C17:0
C18:0
C20:0
C22:0
C23:0
C24:0
0.27
0.20
0.14
0.30
0.01
0.36
1.07
0.10
3.86
0.07
1.30
0.09
0.08
0.01
0.06
0.32
0.23
0.00
0.31
0.01
0.36
1.10
0.11
4.22
0.08
1.44
0.10
0.08
0.01
0.05
0.30
0.21
0.14
0.29
0.01
0.34
1.06
0.11
4.08
0.08
1.42
0.10
0.08
0.01
0.05
0.32
0.23
0.15
0.31
0.01
0.36
1.12
0.11
4.29
0.07
1.49
0.10
0.09
0.01
0.06
0.28
0.20
0.13
0.27
0.01
0.32
1.00
0.10
3.83
0.07
1.32
0.09
0.08
0.01
0.05
0.30
0.21
0.11
0.30
0.01
0.34
1.07
0.11
4.06
0.08
1.39
0.10
0.08
0.01
0.06
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
Mono-unsatured
9.93
11.08
11.28
11.30
10.09
10.74 ± 0.67 0.45
C14:1
C16:1c
C17:1
C18:1 c 9
C18:1 c 11
C18:1 c 12
C18:1 t16-c14
C20:1 n9
C22:1
C24:1
0.15
0.24
0.05
9.00
0.28
0.02
0.05
0.10
0.03
0.02
0.15
0.26
0.05
10.08
0.30
0.02
0.05
0.10
0.04
0.02
0.14
0.25
0.05
9.86
0.30
0.02
0.50
0.10
0.04
0.02
0.15
0.27
0.04
10.31
0.32
0.03
0.03
0.11
0.04
0.02
0.14
0.24
0.05
9.18
0.28
0.02
0.05
0.10
0.03
0.02
0.15
0.25
0.05
9.68
0.30
0.02
0.13
0.10
0.03
0.02
±
±
±
±
±
±
±
±
±
±
Poli-unsatured
4.94
5.50
5.37
5.62
5.01
5.29
± 0.30 0.09
C18:2 9t 12t
C18:2 C/t
C18:2 n6
C18:3 n6
C18:3 n3
C20:2
C20:3 n6
C20:4 n6 (ARA)
C20:5 n3 (EPA)
C22:6 n3 (DHA)
0.05
0.03
4.18
0.01
0.45
0.01
0.02
0.10
0.02
0.08
0.06
0.03
4.66
0.01
0.49
0.01
0.02
0.11
0.03
0.08
0.04
0.03
4.57
0.01
0.49
0.01
0.02
0.12
0.02
0.08
0.04
0.03
4.78
0.01
0.51
0.01
0.02
0.12
0.03
0.08
0.04
0.02
4.25
0.01
0.45
0.01
0.02
0.11
0.02
0.08
0.05
0.03
4.49
0.01
0.48
0.01
0.02
0.11
0.02
0.08
±
±
±
±
±
±
±
±
±
±
120
0.02
0.01
0.06
0.02
0.00
0.02
0.05
0.00
0.21
0.00
0.08
0.01
0.00
0.00
0.00
0.01
0.01
0.01
0.57
0.02
0.00
0.20
0.01
0.00
0.00
0.01
0.00
0.26
0.00
0.03
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.04
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.32
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
0.07
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Table 6.5. – Error of the method, relatives to the fatty acids under investigation, and
estimated on the commercial samples.
Error of the method
Fatty acids
(g/100g of powder)
C18:1 c9
4.04
C18:2 n6
1.88
C18:3 n3
0.20
C20:4 n6 (ARA)
0.04
C22:6 n3 (DHA)
0.03
Table 6.6. – Fatty acids composition (g/100 g of powder) of the 3 intra-blend samples.
(g/100g of powder)
BB5 BB9 MB5 BB9 EB5 BB10
mean
SD
VAR
FAT
22.17
23.20
22.52
22.63
± 0.52
0.27
Satured
7.35
7.96
7.71
7.68
± 0.30
0.09
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C15:0
C16:0
C18:0
0.28
0.21
0.13
0.26
0.30
0.94
0.09
3.74
1.12
0.31
0.23
0.15
0.31
0.33
1.00
0.10
3.85
1.35
0.29
0.22
0.15
0.29
0.32
0.98
0.10
3.76
1.31
0.29
0.22
0.14
0.29
0.32
0.97
0.10
3.78
1.26
±
±
±
±
±
±
±
±
±
0.01
0.01
0.01
0.02
0.02
0.04
0.00
0.06
0.12
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
Mono-unsatured
9.82
10.07
9.79
9.89
± 0.15
0.02
C14:1
C16:1c
C18:1 c 9
C18:1 c 11
C20:1 n9
0.13
0.23
9.11
0.14
0.10
0.14
0.24
9.25
0.23
0.10
0.14
0.24
9.00
0.22
0.10
0.13
0.24
9.12
0.20
0.10
±
±
±
±
±
0.00
0.00
0.12
0.05
0.00
0.00
0.00
0.02
0.00
0.00
Poli-unsatured
5.00
5.17
5.01
5.06
± 0.10
0.01
C18:2 n6
C18:3 n3
C20:4 n6 (ARA)
C22:6 n3 (DHA)
4.20
0.46
0.11
0.06
4.33
0.47
0.12
0.07
4.21
0.45
0.10
0.06
4.24
0.46
0.11
0.07
±
±
±
±
0.00
0.00
0.00
0.00
0.07
0.01
0.01
0.00
0.58
Others*
Others* < 0.1 g/100g (C11:0; C17:0; C17:1 c; C18:1 c 12; C18:1 t16-c14; C18:2 c/t; C18:2 9t12t; C18:2
c9, t11 (CLA) C18:3 n6; C18:3t; C20:0; C20:2; C20:3 n6; C20:5 n3 (EPA); C21:0; C22:0; C22:1; C24:1;
C23:0; C24:0)
121
Table 6.7. – Fatty acids composition (g/100 g of powder) of the 10 inter-blend samples.
23.89
24.91
21.46
23.2
EB5
BB10
22.52
Satured
7.88
8.40
7.48
8.13
7.71
8.02
8.62
9.39
8.40
8.40
7.49
± 0.54 0.29
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
0.29
0.23
0.15
0.30
0.31
0.97
3.68
1.53
0.31
0.24
0.16
0.32
0.35
1.05
4.05
1.45
0.31
0.23
0.15
0.31
0.34
0.97
3.52
1.22
0.35
0.26
0.17
0.36
0.38
1.07
3.79
1.30
0.29
0.22
0.15
0.29
0.32
0.98
3.76
1.31
0.31
0.23
0.15
0.31
0.33
1.00
3.87
1.37
0.32
0.25
0.16
0.32
0.36
1.08
4.25
1.50
0.31
0.26
0.18
0.35
0.39
1.18
4.60
1.63
0.30
0.24
0.16
0.32
0.35
1.06
4.10
1.45
0.30
0.24
0.16
0.32
0.35
1.06
4.10
1.45
0.28
0.22
0.14
0.29
0.32
0.95
3.61
1.29
±
±
±
±
±
±
±
±
Mono-unsatured
10.45
10.92
9.28
10.01
9.79
10.29
11.24
12.12
10.96
10.96
9.64
± 0.81 0.66
C14:1
C16:1c
C18:1 c 9
C18:1 c 11
0.13
0.21
9.69
0.20
0.14
0.25
10.06
0.23
0.13
0.22
8.43
0.28
0.15
0.24
9.02
0.32
0.14
0.24
9.00
0.22
0.14
0.24
9.46
0.23
0.15
0.25
10.36
0.24
0.16
0.29
11.16
0.25
0.15
0.26
10.05
0.26
0.15
0.26
10.05
0.26
0.13
0.22
8.84
0.23
±
±
±
±
Poli-unsatured
5.56
5.58
4.70
5.05
5.01
5.25
5.76
6.20
5.57
5.57
4.93
± 0.43 0.19
C18:2 n6
C18:3 n3
C20:4 n6 (ARA)
C22:6 n3 (DHA)
4.72
0.47
0.11
0.05
4.70
0.50
0.11
0.07
3.94
0.42
0.10
0.07
4.24
0.46
0.11
0.07
4.21
0.45
0.10
0.06
4.39
0.47
0.12
0.07
4.84
0.52
0.12
0.07
5.19
0.56
0.13
0.08
4.66
0.50
0.12
0.07
4.66
0.50
0.12
0.07
4.14
0.44
0.10
0.06
±
±
±
±
EB1 BB2 EB2 BB4 EB3 BB6 EB4 BB8
EB6
BB12
23.56
EB7
BB14
25.62
EB8
BB16
27.71
EB9
BB18
24.92
EB10
BB20
24.92
mean
SD
VAR
22.06 ± 1.76 3.08
0.02
0.01
0.01
0.02
0.02
0.07
0.31
0.12
0.01
0.02
0.79
0.03
0.36
0.04
0.01
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.10
0.01
0.00
0.00
0.62
0.00
0.13
0.00
0.00
0.00
0.79
Others*
Others* < 0.1 g/100g (C11:0; C15:0; C17:0; C17:1 c; C18:1 c 12; C18:1 t16-c14; C18:2 c/t; C18:2 9t12t; C18:2 c9, t11 (CLA) C18:3 n6; C18:3t; C20:0; C20:1 n9;
C20:2; C20:3 n6; C20:5 n3 (EPA); C21:0; C22:0; C22:1; C24:1; C23:0; C24:0)
122
(g/100g of
powder)
FAT
(g/100g of powder) FP BB3 FP BB4 FPBB6 FP BB8 FP BB9 FP BB10 FP BB12 FP BB14 FP BB16 FP BB18 FP BB20 mean
SD
VAR
FAT
23.16
23.88
21.80
24.19
25.26
22.15
24.60
22.15
26.33
25.28
25.28
24.00 ± 1.52
2.31
Satured
7.74
8.01
7.48
8.41
8.56
7.51
8.47
7.52
8.94
8.55
8.55
8.16
± 0.52
0.27
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C15:0
C16:0
C18:0
0.29
0.22
0.14
0.28
0.31
0.97
0.10
3.84
1.30
0.27
0.20
0.13
0.27
0.31
1.00
0.10
4.00
1.41
0.30
0.23
0.15
0.30
0.33
0.95
0.09
3.56
1.26
0.34
0.26
0.17
0.35
0.39
1.09
0.10
3.96
1.38
0.31
0.23
0.15
0.31
0.35
1.08
0.11
4.21
1.49
0.28
0.21
0.14
0.28
0.31
0.94
0.09
3.68
1.31
0.33
0.25
0.17
0.34
0.37
1.08
0.11
4.04
1.42
0.28
0.21
0.14
0.27
0.30
0.94
0.09
3.70
1.29
0.32
0.25
0.16
0.33
0.37
1.12
0.11
4.37
1.55
0.31
0.24
0.16
0.32
0.35
1.07
0.11
4.20
1.49
0.31
0.24
0.16
0.32
0.35
1.07
0.11
4.20
1.49
0.31
0.23
0.15
0.31
0.34
1.03
0.10
3.98
1.40
±
±
±
±
±
±
±
±
±
0.02
0.02
0.01
0.03
0.03
0.07
0.01
0.26
0.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.07
0.01
Mono-unsatured
10.21
10.52
9.50
10.49
11.07
9.72
10.71
9.72
11.53
11.10
11.10
10.52 ± 0.67
0.44
C14:1
C16:1c
C18:1 c 9
C18:1 c 11
C20:1 n9
0.13
0.23
9.46
0.18
0.10
0.14
0.24
9.75
0.18
0.10
0.13
0.22
8.67
0.28
0.10
0.15
0.25
9.53
0.32
0.11
0.15
0.25
10.20
0.23
0.11
0.13
0.23
8.91
0.24
0.09
0.15
0.25
9.76
0.30
0.11
0.13
0.23
8.98
0.17
0.09
0.15
0.27
10.60
0.26
0.12
0.15
0.25
10.21
0.26
0.11
0.15
0.25
10.21
0.26
0.11
0.14
0.24
9.66
0.24
0.10
±
±
±
±
±
0.01
0.01
0.62
0.05
0.01
0.00
0.00
0.39
0.00
0.00
Poli-unsatured
5.21
5.35
4.81
5.30
5.63
4.92
5.41
4.91
5.86
5.63
5.63
5.33
± 0.34
0.12
C18:2 n6
C18:3 n3
C20:4 n6 (ARA)
C22:6 n3 (DHA)
4.40
0.47
0.10
0.06
4.51
0.48
0.11
0.06
4.05
0.43
0.10
0.06
4.45
0.48
0.11
0.07
4.73
0.51
0.12
0.07
4.13
0.45
0.10
0.06
4.54
0.49
0.12
0.07
4.15
0.45
0.09
0.05
4.92
0.53
0.12
0.08
4.76
0.51
0.12
0.07
4.76
0.51
0.12
0.07
4.49
0.48
0.11
0.07
±
±
±
±
0.08
0.00
0.00
0.00
0.29
0.03
0.01
0.01
0.62
Others*
Others* < 0.1 g/100g (C11:0; C17:0; C17:1 c; C18:1 c 12; C18:1 t16-c14; C18:2 c/t; C18:2 9t12t; C18:2 c9, t11 (CLA) C18:3 n6; C18:3t; C20:0; C20:2; C20:3 n6;
C20:5 n3 (EPA); C21:0; C22:0; C22:1; C24:1; C23:0; C24:0)
123
Table 6.8. – Fatty acids composition (g/100 g of powder) of the 11 commercial samples.
Table 6.9. – Comparison of the mean values of inter-blend and commercial samples
FAT
22.06
Commercial
sample
24.00
Satured
7.49
8.16
8.15
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C15:0
C16:0
C18:0
0.28
0.22
0.14
0.29
0.32
0.95
0.09
3.61
1.29
0.31
0.23
0.15
0.31
0.34
1.03
0.10
3.98
1.40
7.85
5.10
4.93
5.06
6.39
7.94
8.63
9.16
7.71
Mono-unsatured
9.64
10.52
8.33
C14:1
C16:1c
C18:1 c 9
C18:1 c 11
C20:1 n9
0.13
0.22
8.84
0.23
0.09
0.14
0.24
9.66
0.24
0.10
8.12
7.63
8.48
6.89
8.32
Poli-unsatured
4.93
5.33
7.53
C18:2 n6
C18:3 n3
C20:4 n6 (ARA)
C22:6 n3 (DHA)
4.14
0.44
0.10
0.06
4.49
0.48
0.11
0.07
7.76
8.19
4.71
6.24
mean (g/100g)
inter-blend
124
SD (%)
8.09
Intra
blend
24.27100
FAT
8.24300
Satured
Mono-unsatured 10.60200
9.72800
C18:1 c 9
5.42500
Poli-unsatured
4.55500
C18:2 n6
0.48500
C18:3 n3
C20:4 n6 (ARA) 0.11400
C22:6 n3 (DHA) 0.06800
Commercial
samples
t student
value
df
p value
N Intrablend
samples
N commercial
samples
SD
Intrablend
24.00727
8.15818
10.51545
9.66182
5.33273
4.49091
0.48273
0.11000
0.06545
0.369294
0.366968
0.267785
0.215151
0.544885
0.450074
0.147377
0.883504
0.723261
19
19
19
19
19
19
19
19
19
0.715992
0.717698
0.791750
0.831942
0.592172
0.657750
0.884387
0.388007
0.478329
10
10
10
10
10
10
10
10
10
11
11
11
11
11
11
11
11
11
1.753178
0.536678
0.812866
0.785774
0.430278
0.361855
0.039511
0.009661
0.007888
SD
commercial
samples
1.519665
0.521974
0.666984
0.621302
0.344647
0.289774
0.031013
0.010954
0.008202
Variance
ratio F
p
Variances
1.330933
1.057136
1.485277
1.599518
1.558647
1.559370
1.623083
1.285714
1.081169
0.659612
0.924410
0.545361
0.474376
0.498559
0.498120
0.461012
0.715949
0.916107
125
Table 6.10. – statistical comparison between intra blend and commercial sample
126
FIGURES
Figure 6.1. - Chromatograms relative to the samples of raw materials employed for the addiction of ARA and DHA, and a sample from the
inter-blend.
Variance
varianza
0.02
0.02
C18:1 c 9
0.02
0.01
0.01
0.01
0.01
0.01
C18:2 n6
0.00
0.00
0.00
C18:3 n3
0.00
g/100g di polvere
0.00
C20:4 n6
0.00
C22:6 n3 (DHA)
0.00
g/100g of powder
Figure 6.2. - Variance of the fatty acids under investigation, and relatives to the intrablend samples.
Variance
varianza
0.45
0.40
C18:1 c 9
0.39
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
C18:2 n6
0.08
C18:3 n3
0.00
g/100g di polvere
C20:4 n6
0.00
C22:6 n3 (DHA)
0.00
g/100g of powder
Figure 6.3. - Variance of the fatty acids under investigation, and relatives to the interblend samples.
127
Variance
varianza
0.45
0.40
C18:1 c 9
0.39
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
C18:2 n6
0.08
C18:3 n3
0.00
g/100g
di
polvere
g/100g of powder
C20:4 n6
0.00
C22:6 n3 (DHA)
0.00
Figure 6.4. - Variance of the fatty acids under investigation, and relatives to the
commercial samples.
128
Box & Whisker: Lipids
Box & Whisker: Satured
8.7
25.6
25.4
8.6
25.2
8.5
25.0
24.8
8.4
24.6
Saturi
24.4
24.2
8.3
129
8.2
24.0
8.1
23.8
23.6
8.0
23.4
Mean
±SE
±1.96*SE
23.2
23.0
Intrablend
Intra blend
Prodotto finito
Commercial sample
7.9
Mean
±SE
±1.96*SE
7.8
Intrablend
Intra blend
Prodotto finito
Commercial sample
Figure 6.5a,b. – Lipids and Satured: comparison of the mean, standard error, and a 95% confidence interval.
Box & Whisker: Oleic (C18:1 n-9)
Box & Whisker: Mono-unsatured
10.4
11.0
10.2
10.8
10.0
10.6
9.8
10.4
9.6
10.2
9.4
130
11.2
Mean
±SE
±1.96*SE
10.0
Intrablend
Intra blend
Prodotto finito
Commercial sample
Mean
±SE
±1.96*SE
9.2
Intrablend
Intra
blend
Prodotto finito
Commercial
sample
Figure 6.5c,d. – Mono-unsatured and Oleic acid: comparison of the mean, standard error, and a 95% confidence interval.
Box & Whisker: Poli-unsatured
Box & Whisker: Linolenic (C18:2 n-6)
5.8
4.8
5.7
4.7
5.6
4.6
5.5
131
4.5
5.4
4.4
5.3
4.3
5.2
Mean
±SE
±1.96*SE
5.1
Intrablend
Intra
blend
Prodotto sample
finito
Commercial
Mean
±SE
±1.96*SE
4.2
Intrablend
Intra
blend
Prodotto finito
Commercial
sample
Figure 6.5e,f. – Poli-unsatured and Linoleic acid: comparison of the mean, standard error, and a 95% confidence interval.
Box & Whisker: Linolenic (C18:3 n-3)
Box & Whisker: Arachidonic (C20:4 n-6)
0.52
0.122
0.120
0.51
0.118
0.50
0.116
0.114
0.49
0.48
132
0.112
0.110
0.108
0.47
0.106
0.46
Mean
±SE
±1.96*SE
0.45
IntraIntrablend
blend
Prodottosample
finito
Commercial
0.104
Mean
±SE
±1.96*SE
0.102
Intrablend
Intra blend
Prodotto finito
Commercial sample
Figure 6.5g,h. – Linolenic and Arachidonic acid: comparison of the mean, standard error, and a 95% confidence interval.
Box & Whisker: Docosaesaenoic (C22:6 n-3)
0.074
0.072
0.070
0.068
0.066
0.064
0.062
Mean
±SE
±1.96*SE
0.060
IntraIntrablend
blend
Prodotto finito
Commercial
sample
Figure 6.5i. – Docosahexaenoic acid: comparison of the mean, standard error, and a
95% confidence interval.
133
6.6. REFERENCES
1)
Birch EE, Hoffman DR, Uauy R, Birch DG, Prestidge C, (1998). Visual acuity and
the essentiality of docosahexaenoic acid and arachidonic acid in the diet of term
infants. Pediatr. Res., 44, 201.
2)
Carnielli VP,Wattimena DJL, Luijendijk IHT, Boerlage A, Degenhart HJ, Sauer
PJJ, (1996). The very low birth weight premature infant is capable of synthesizing
arachidonic and docosahexaenoic acids from linoleic and linolenic acids. Pediatr.
Res., 40, 169–74.
3)
Demmelmair H, v.Schenck U, Behrendt E, Sauerwald T, Koletzko B, (1995).
Estimation of arachidonic acid synthesis in fullterm neonates using natural
variation of 13C-abundance. J. Pediatr. Gastroenterol. Nutr., 21, 31–36.
4)
Sauerwald TU, Hachey DL, Jensen CL, Chen HM, Anderson RE, Heird WC,
(1997). Intermediates in endogenous synthesis of C22:6 omega3 and C20:4
omega6 by term and preterm infants. Pediatr. Res., 41 183–7.
5)
Commission on the European Communities (1996). Commission Directive
94/6/EEC of 16 February 1996 amending directive 91/321/EEC on infant formulae
and follow-on formulae. Off. J. Eur.Comm., 39 (L49) 12–6
6)
IDF standard 9C (1987). Determination of fat content of dried milk, dried whey,
dried butter milk and dried butter serum. International Dairy Federation, Brussels,
Belgium.
7)
Christopherson SW, Glass RL (1969). Preparation of milk fat methyl esters by
alcoholysis in an essentially nonalcoholic solution. J. Dairy Sci 52, 1289–1290.
8)
Ramsey MH, Ellison SLR, (2007). Measurement uncertainty arising from
sampling: a guide to methods and approaches. Eurachem, EUROLAB, CITAC,
Nordtest, AMC Guide.
9)
Ramsey MH, Ellison SLR, (2007). Use of uncertainty information in compliance
assessment. Eurachem, EUROLAB, CITAC, Nordtest, AMC Guide.
134
7
FATTY ACIDS COMPOSITION OF PARMIGIANO
REGGIANO CHEESE SAMPLES, WITH EMPHASIS ON
TRANS ISOMERS (TFA).
7.1. SUMMARY AND KEYWORDS .....................................................................................................136
7.2. INTRODUCTION.............................................................................................................................137
7.3. MATERIALS AND METHODS ......................................................................................................138
7.3.1 SAMPLES COLLECTION ........................................................................................................138
7.3.2. PRELIMINARY ANALYSIS OF THE ORGANIC AND INORGANIC FRACTION............138
7.3.3. SAMPLE PREPARATION .......................................................................................................138
7.3.4. GAS CHROMATOGRAPHIC QUANTITATION ...................................................................139
7.4. RESULTS AND DISCUSSION........................................................................................................139
7.5. CONCLUSIONS ...............................................................................................................................140
TABLES...................................................................................................................................................142
FIGURES .................................................................................................................................................146
7.6. REFERENCES..................................................................................................................................147
135
7.1. SUMMARY AND KEYWORDS
A wide debate in the scientific panel regarding the human consumption of food products
containing fatty acids in trans configuration (TFA) have been raised, influencing either
the nutritional recommendations proposed by national health cares, and food laws for
nutritional labeling purpose. Despite the low levels of TFA in foods products from
animal origin, and their particular composition in these products, even dairy products
have been involved in the diatribe. Therefore, due to the lack of specific information
about the TFA content in the Parmigiano Reggiano cheese, in this work the study of the
fatty acid profile with particular emphasis to the TFA in Parmigiano Reggiano cheese
samples has been proposed.
Keywords: trans fatty acids, Parmigiano Reggiano, food labels.
136
7.2. INTRODUCTION
The current investigation, dedicated to describe the fatty acid profile (with particular
interest on trans fatty acids) of 13 samples of Parmigiano Reggiano cheese, collected in
the 4 districts of the yield area (Bologna, Mantova, Parma, Reggio Emilia), and at
different time of seasoning, is related to the increasing concern regarding unfavorable
roles on human health of TFA in coronary heart diseases (CHD) and, lately, in prostate
cancer risk (1). The discovery of their negative health effects concurrently to the
advances in medicine and food science research, sparked a great interest in the scientific
panel, inducing many governments to implement legislation to reduce the total TFA
content of food products by introducing mandatory labeling of total TFA or restricting
the sale of industrially produced fats and oils with more than a certain amount.
Unfortunately, the debate, is still today an object of simplifications and mistakes that
origin perplexity (2).
The predominant dietary sources of TFA in the western diet are: vegetable-oil-based
margarines, shortenings, and cooking oils that have been subjected to the industrial
process called hydrogenation; ruminat fats (dairy products and beef), where TFA are
naturally produced by the process called biohydrogenation and present in a considerable
lower amount. Recently, new studies found an inverse association between ruminant
TFA and risk of CHD, implying that ruminant TFA intake might be innocuous or even
protective against CHD (3, 4). Moreover, the TFA isomers from ruminant and industrial
fat may differ in their relative abundance; the TFA isomers in industrial fats are
produced under catalytic conditions that results in a more random distribution of isomer,
while the isomers in ruminant fats are enzymatically produced resulting in the formation
of specific TFA isomers. Therefore, the new investigations on total TFA negative health
137
effects should consider the differences between TFA isomers from ruminant and
industrial fat, as well as the nutritional information to consumers and the definition of
nutritional profiles that determine the food quality in dairy products should be seen
under a different point of view.
7.3. MATERIALS AND METHODS
7.3.1 SAMPLES COLLECTION
13 samples of Parmigiano Reggiano cheese were collected in different dairy factory
with the collaboration of the farm personnel.
7.3.2. PRELIMINARY ANALYSIS OF THE ORGANIC AND INORGANIC
FRACTION
Each sample was analyzed in its organic and inorganic fraction composition by Food
Scan (Foss)-NIT (Table 7.1.) at the Parmigiano Reggiano cheese Consortium, and then
stored at - 40°C until analysis.
7.3.3. SAMPLE PREPARATION
The fat was extracted in double following the method of Blight and Dyer (5), and then,
the preparation of the methyl esters was carried out applying the method of
Christopherson and Glass (6): about 20 mg of dry fat was put in a test tube, 50 µl of
KOH MeOH 2N and 500 µl of hexane were added respectively, and then the tube was
mixed for 30 seconds; 0,5 mg of C13:0 (Sigma-Aldrich, St. Louis, MO, USA) were
used for the fatty acids quantitation as internal standard. After the phase separation was
138
acted, 300 µl of surnatant was collected and put in a vial. It followed the analysis by gas
chromatography.
7.3.4. GAS CHROMATOGRAPHIC QUANTITATION
FAME were analyzed with a Perkin Elmer Clarus 500 gas chromatograph equipped
with a Supelco 2560 (Supelco Inc., Bellefonte, PA) fused silica capillary column (100
m x0.25 mm i.d., 0.2-µm film thickness), a FID detector, and a split injector. Helium
was used as carrier at the constant flow rate of 0,75 min/min. The FID detector was
maintained at 250°C with air flow rate of 400 ml/min, hydrogen flow rate of 40 ml/min,
and helium (make up gas) flow rate of 30 ml/min. The split injector was maintained at
250°C with the split ratio of 1:67. The temperature program was as follow: ramp 1)
3°C/min from 100°C to 180°C and maintained 10 minutes, then ramp 2) 3°C/min from
180°C to 240°C and maintained 30 minutes. For the fatty acids identification was used
the 53 FAME GLC-reference standard mix-463 (NU-CHEK-PREP, Elysian, MN).
7.4. RESULTS AND DISCUSSION
The fatty acid profile of the Parmigiano Reggiano cheese samples has been determined
(Table 7.2.). Likewise the milk fatty acid distribution, the major fatty acids have
resulted to be the palmitic acid (30,13 %), oleic acid (19,28 %), stearic acid (10,40 %),
and miristic acid (10,39 %). The analysis of TFA content has followed: for definition,
trans fats, are defined as all the unsaturated fatty acids that contain one or more isolated,
non-conjugated, double bonds in a trans geometric configuration. Conjugated fatty acids
with a trans double bond, including CLA isomers, are excluded from the definition of
trans fats. By observing table 7.3. and figure 7.1. the dominant trans isomer in the
139
samples analyzed result to be the vaccenic acid (C18:1 t11), that represent the 74 % of
the overall TFA content, while their total content fluctuates between the minimum of
2,5 % and a maximum of 5.4 % of total fatty acids composition, turning out to be in
accordance with the literature (7). Several studies have observed variation of TFA
among same dairy samples; seasonal variation is probably the most important cause of
differing their fatty acid composition (8). Specifically, during the outdoors feeding
period the intake of polyunsaturated and monounsaturated fatty acids increases and this
leads to an increase in the proportions of oleic acid and trans fatty acids and a
corresponding decrease in saturated fatty acids of milk (9). This trend has also been
observed in the samples examined (Figure 7.2.), even though the cattle diet was
unknown; this is difficult to determine in retrospect but since the samples were collected
during different times, and seasoned differently, seasonal variation probably accounted
for much of the differences found between the 13 samples in the present study.
7.5. CONCLUSIONS
The TFA composition found in the 13 Parmigiano Reggiano cheese samples results to
be in accordance with the literature for dairy products and reflects, logically, the TFA
content of milk. Nevertheless, it has been necessary to obtain such information from
analysis about this unique cheese, in order to support with objective data the food
labeling regulation, whereas Parmigiano Reggiano cheese is exported in countries
where is mandatory the TFA declaration on the food label.
Moreover, from a nutritional point of view, the data have confirmed the specificity of
TFA composition in dairy products, especially, if compared with food products
containing hydrogenated fat. The vaccenic acid (C18:1 t11), the most abundant isomer,
140
is also the forerunner of rumenic acid (C18:2 c9 t11), the principal isomer of the group
of CLA, and the presence of conjugated poli-unsatured fatty acids is very significative,
since nutritional and healthy properties have been attributed.
141
TABLES
Table 7.1. – Organic and inorganic fraction composition of Parmigiano Reggiano
cheese samples.
35
40
41
43
45
47
49
52
53
D5
5
6
7
Umidity
31.26
30.13
31.12
30.04
31.52
31.76
32.35
31.56
31.92
30.62
35.02
35.1
31.12
Fat
31.39
32.58
30.61
33.62
31.64
31.29
31.75
29.43
30.99
33.03
28.83
25.07
30.59
142
Protein
31.82
32.87
32.33
31.29
31.91
31.39
30.92
34.1
32.81
31.07
30.65
33
33.77
Salt
1.6
1.45
1.69
1.88
1.34
1.4
1.87
1.51
1.51
1.43
1.36
1.63
1.61
Sample #
35
Satured (100g of fat)
1.81
c4:0
0.87
c6:0
0.55
c8:0
1.42
c10:0
0.22
c11:0
2.80
c12:0
10.90
c14:0
1.10
c15:0
31.47
c16:0
0.76
c17:0
11.76
c18:0
0.16
c20:0
0.01
c21:0
0.07
c22:0
0.02
c23:0
0.03
c24:0
64.01
Total
g/100g of cheese
20.09
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40
41
43
45
47
49
52
53
5
6
7
D5
2.39
1.79
1.10
2.95
0.22
4.00
13.58
1.52
39.22
0.95
12.55
0.12
0.01
0.06
0.00
0.10
80.57
26.24
1.34
1.17
0.93
2.47
0.28
2.74
11.72
1.40
32.64
0.80
10.38
0.17
0.04
0.09
0.05
0.07
66.36
20.31
1.75
1.23
2.90
1.97
0.22
2.60
10.80
1.36
34.11
0.84
10.39
0.16
0.03
0.00
0.04
0.03
68.53
23.03
1.62
1.31
0.87
2.27
0.23
2.98
11.44
1.24
32.84
0.80
12.14
0.19
0.02
0.08
0.03
0.04
68.11
21.54
1.47
1.25
0.85
2.27
0.26
3.07
11.64
1.44
35.60
0.91
10.75
0.16
0.03
0.08
0.03
0.05
69.85
21.85
1.56
1.23
0.79
2.03
0.14
2.64
9.81
1.06
30.90
0.66
11.62
0.13
0.01
0.05
0.01
0.02
62.67
19.89
1.32
1.18
0.84
2.46
0.09
3.45
11.57
1.52
32.18
0.80
9.65
0.14
0.02
0.12
0.00
0.03
65.37
19.23
1.15
1.06
0.73
1.94
0.19
2.58
10.06
1.17
30.47
0.77
11.35
0.16
0.03
0.07
0.01
0.04
61.78
19.14
1.84
1.42
0.92
2.40
0.24
3.07
11.29
1.23
31.48
0.84
11.42
0.17
0.03
0.08
0.04
0.05
66.52
19.17
1.64
1.27
0.89
2.36
0.16
3.01
10.24
1.10
26.03
0.64
13.22
0.16
0.02
0.07
0.02
0.02
60.85
15.25
1.56
1.19
0.84
2.27
0.20
2.94
10.41
1.09
28.41
0.67
11.48
0.13
0.02
0.08
0.02
0.03
61.34
18.76
1.65
1.29
0.91
2.58
0.23
3.59
12.03
1.39
36.47
0.81
8.87
0.15
0.02
0.07
0.03
0.02
70.10
23.15
143
Table 7.2. – The fatty acid profile of the Parmigiano Reggiano cheese samples.
35
40
41
43
45
47
49
52
53
5
6
7
D5
Mono-ins.cis (100g of fat)
0.80
c14:1
1.86
c16:1c
0.22
c17:1
23.34
c18:1 c 9
0.72
c18:1 c 11
0.30
c18:1 c 12
0.06
c18:1 c 13
0.12
c18:1 c15
0.06
c20:1 n9
Total
27.48
g/100g
8.62
0.50
1.33
0.11
12.07
0.38
0.18
0.01
0.10
0.00
14.69
4.78
1.02
1.86
0.24
20.97
0.48
0.24
0.04
0.16
0.04
25.06
7.67
0.84
1.82
0.22
19.73
0.53
0.21
0.03
0.13
0.02
23.54
7.91
0.81
1.68
0.18
19.51
0.56
1.06
0.01
0.11
0.04
23.95
7.57
0.87
1.93
0.23
18.73
0.54
0.20
0.03
0.12
0.04
22.69
7.10
0.83
1.88
0.19
23.58
0.67
0.36
0.05
0.13
0.05
27.75
8.81
1.05
2.01
0.23
22.29
0.95
0.33
0.06
0.10
0.02
27.03
7.95
0.88
1.98
0.15
23.52
0.72
0.36
0.06
0.14
0.06
27.86
8.63
0.84
1.72
0.21
21.27
0.58
0.35
0.04
0.13
0.05
25.19
7.26
0.73
1.40
0.15
23.71
0.58
0.73
0.07
0.18
0.02
27.55
6.90
3.16
1.62
0.18
22.25
0.68
0.52
0.07
0.18
0.04
28.70
8.77
1.00
1.94
0.20
18.94
0.64
0.21
0.03
0.14
0.02
23.12
7.63
Poli-uns. cis (100g of fat)
2.60
c18:2 n6
0.02
c18:3 n6
0.52
c18:3 n3
0.02
c20:2
0.11
c20:3 n6
0.14
c:20:4 n6
0.00
c22:2
0.03
c20:5 n3
0.00
c22:6 n3
Total
3.44
g/100g of cheese
1.07
1.68
0.00
0.31
0.00
0.06
0.07
0.00
0.01
0.00
2.12
0.69
2.08
0.01
0.75
0.01
0.10
0.13
0.01
0.06
0.00
3.15
0.96
2.17
0.01
0.76
0.02
0.10
0.12
0.00
0.03
0.00
3.20
1.07
2.31
0.02
0.47
0.01
0.10
0.12
0.00
0.03
0.01
3.06
0.96
1.97
0.01
0.54
0.00
0.08
0.11
0.00
0.04
0.01
2.77
0.86
3.22
0.02
0.59
0.02
0.12
0.15
0.00
0.03
0.01
4.18
1.32
2.82
0.02
0.58
0.02
0.15
0.11
0.00
0.03
0.00
3.73
1.09
3.59
0.03
0.58
0.02
0.14
0.16
0.00
0.04
0.00
4.56
1.41
2.36
0.02
0.81
0.02
0.13
0.16
0.00
0.06
0.00
3.56
1.02
4.17
0.02
0.97
0.01
0.15
0.17
0.00
0.02
0.00
5.51
1.38
3.60
0.02
0.97
0.02
0.12
0.13
0.00
0.04
0.00
4.91
1.50
2.59
0.04
0.65
0.00
0.12
0.16
0.00
0.02
0.00
3.61
1.19
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144
Sample #
Sample #
35
40
41
43
45
47
49
CLA (100g of fat)
c18:2 c9, t11
0.77 0.2 0.9 0.75 0.64 0.58 0.69
g/100g
0.24 0.06 0.27 0.25 0.2 0.18 0.21
52
0.4
0.11
53
5
6
7
D5
0.72 0.57 0.69 0.59 0.39
0.22 0.16 0.17 0.18 0.12
TFA (100g of fat)
Total
4.31 2.42 4.54 3.98 4.23 4.11 4.71 3.45 5.08 4.16 5.39 4.46 2.79
g/100g of cheese 1.35 0.78 1.38 1.33 1.33 1.28 1.49 1.01 g 1.57 1.19 1.35 1.36 0.92
Table 7.3. – TFA compositionof the Parmigiano Reggiano cheese samples.
TFA (% FAME)
TOTAL
AGE
SAMPLE
24
23
6
24
24
26
12
26
26
36
24
25
24
#40
#D5
#5
#47
#45
#43
#6
#52
#35
#7
#41
#49
#53
C13:1
0.08
0.09
0.12
0.11
0.09
0.13
0.07
0.04
0.08
0.06
0.16
0.06
0.08
C16:1
0.36
0.30
0.37
0.40
0.39
0.40
0.28
0.28
0.36
0.29
0.45
0.33
0.41
C18:1 t11
1.74
1.85
3.06
3.06
3.23
2.88
4.17
2.47
3.34
3.35
3.24
3.61
3.83
145
C18:2
0.20
0.44
0.47
0.43
0.40
0.44
0.77
0.55
0.39
0.65
0.54
0.59
0.63
C18:3
0.03
0.11
0.14
0.11
0.12
0.13
0.10
0.11
0.13
0.11
0.15
0.12
0.13
2.42
2.79
4.16
4.11
4.23
3.98
5.39
3.45
4.31
4.46
4.54
4.71
5.08
g/100g of
cheese
0.79
0.92
1.20
1.28
1.34
1.34
1.35
1.35
1.35
1.37
1.39
1.49
1.57
MEAN
4.12
1.29
TFA
FIGURES
Figure 7.1. – Partial chromatogram of the TFA elution area.
81%
70%
70%
69%
68%
67%
66%
65%
64%
63%
62%
61%
61%
FA %
Satured
c18:1 c9
TFA
19%
19%
20%
20%
21%
21%
22%
23%
24%
24%
3%
4%
5%
5%
22%
24%
12%
2%
#40
3%
#D5
4%
#47
4%
#43
4%
#45
4%
#5
5%
#41
#52
#35
#49
#53
4%
#7
5%
#6
Samples
Figure 7.2. – Percentual variation of satured FA, oleic acid, and TFA in each sample.
.
146
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147
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