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Sanaz Haratifar
The stability of electro-reduced inilk lipids
Thèse présentée à la Faculté des études supérieures de l'Université Laval
dans le cadre du programme de maîtrise en· Sciences et technologie des aliments pour l'obtention du grade de maitre es sciences (M.Sc.)
FACULTE DES SCIENCES DE L'AGRICULTURE ET DE L'ALIMENTATION UNIVERSITE LA V AL
QUEBEC
2009
© Sanaz Haratifar, 2009
Résumé
Le lait est un aliment complexe sujet à de nombreuses ' réactions d 'oxydoréduction, dont
l'oxydation de la matière grasse, la dénaturation des protéines et la dégradation des
vitamines.
Une technique électrochimique a été récemment-proposée pour. contrôler et/ou prévenir les
phénomènes d'oxydation dans le lait. Cette procédure vise la réduction, par électrolyse avec
membrane, d ' espèces électrochimiquement actives présentes dans le lait, ce qui ' entraîne un
abaissement de son potentiel d'oxydoréduction (POR). Le but de cette étude était d ' évaluer
l'impact du traitement d'électroréduction sur la stabilité des lipides du lait, au cours de leur
entreposage à l'obscurité ou sous une lumière fluorescente.
Dans cette recherche, une ' chute significative du POR et une baisse en oxygène dissous
(OD) ont été observées pendant le traitement. Pendant l'entreposage des échantillons de
lait, l'OD diminue alors que le POR a remonté graduellement, ce qui démontre une
instabilité ou une réversibilité des équilibres réactionnels mis en place lors de
l'électroréduction. Pendant l'entreposage à l'obscurité aucun changement significatif n' a .
été observé dans la composition en acides gras, ce qui montre que la baisse en OD n ' est pas
causée par une oxydation des acides gras. L'entreposage sous une lumière fluorescente
entraîne une dégradation des acides gras, alors que l' électroréduction ralentit leur
dégradation oxydative comparativement au lait non traité
Il est envisageable qu'un traitement électrochimique de réduction du lait permettrait
d'augmenter son temps de conservation ainsi que son aptitude à la transformation, en
limitant l'oxydation des acides gras.
- - - - - - - - - ---
Abstract
Oxidation-reduction reactions (redox reactions) are mainly responsible for milk
degradation during processing and storage. These reactions have important impacts on the
. quality of dairy products and cause unwanted modifications in sensitive compounds such as
unsaturated fatty acids, colorants and vitamins as weIl as denaturation of proteins.
Milk electro-reduction is an electro-chemical method put forward to control and/or prevent
oxidation reactions in milk. This process uses electrolysis to reduce electrochemically
active species present in milk, causing a decrease in its redox potential.
The objective of this study was to evaluate the impact of electro-reduction treatment on the
stability of milk lipids during treatment as well as storage.
In this study an electro-reduction process was performed to modify the redox state of milk.
Parameters such as redox potential, dissolved oxygen and pH were measured during the
course of treatment. Significant decrease of redox potential and dissolved oxygen was
observed during treatment. Analysis showed that while the dissolved oxygen continued to
decrease for aH samples throughoùt storage, the low values of redox potential for electro
reduced milk were not stable, and gradually reached positive values showing the instability
of the electro-reduced species of milk. During the storage period the fatty acid composition
of the electro-reduced milk was measured at intervals of one week by
Gas Chromatography. The results showed that a significant change was not seen in the
fatty acid composition of the samples placed in dark. On the contrary, storage under
fluorescent light involved a degradation of the fatty-acids, whereas the electro-reduction
treatment slowed down the oxidative degradation of electro-reduced samples in comparison
to untreated milk samples. Results of this study show that the electro-reduction treatment
can be a potential method of enhancing the shelf-life of products containing unsaturated
fatty acids.
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Acknowledgemen ts
This current research could not have been possible without the support and the contribution
of many people of which they deserve to be mentioned.
Firstly, 1 make a point of expressing my deep gratitude and appreciation towards my
supervisor of research, Dr. Paul Angers, not only for his confidence in me during these 3
years of research, but also for rus support and his warm welcome at the time 1 needed it
most.
1 would also like to thank Dr. Michel Britten, my co-director, for his great help and
guidance.
My sincere thanks are extended to Dr. Laurent Bazinet for providing valuable suggestions
and advice to improve this research work.
1 am also indebted to Monica Araya-Farias., Anne-Françoise Alain, Diane Gagnon and
Ronan Corcuff who offered valuable technical assistance in the laboratory to me.
1 give special thanks to everyone that helped me in the ALN department and staff at
Laval University and the three students that worked with me; Claudia Gonzalez, Nathalie
Manoury and Jérome Péricou.
Last but not least, 1 thank my family for encouraging me to fulfill my dreams even at the
most harde st of times; my parents, my uncles, my sister, my husband and especially my
grandmother.
This dissertation is dedicated ta my mather and grandmather, my twa angels in heaven ...
Every successful persan has a painful stary. Every painful story has a successful ending. Accept·the pain and get ready far success.
Table of contents
Résumé .................................................................................................................................... i .Abstract ........................................................................................ ~ ......... ~ .............................. ii Acknowledgements ..................................................................................... ~ ......................... iii Table of contents ...................................................................... ~ ............................................ v List of Tables ................................................................................... ~ .................................. viii List of Figures ....................................................................................................................... ix
Introduction .................................................................•......................................................... 1
Chapter 1. Literature Review .......................................................................................... 3 1. Milk ............... .. ..... .... ........... ...................................... .. .... .. .... .. ... .. ... ... ...... ......... .. .... 4
1.1.Milk protein ..................................... , ... ............. .... ... .................... ........ ... ; ..... .. 4 1.2 Milk Lipids ........... ... ......................................................... ..................... ..... .... 5 1.3. Carbohydrates of milk .................................................... .... ... ......... .............. . 8 1.4. Other components ofmilk .................. ................................... ..... ................... . 8
2. Oxidation- Reduction reactions ............................................................ .... .......... .... 9 2.1 . Measurement of oxidation -reduction potential (Redox potential) .......... .. .. . 9 2.2. Redox potential of milk ........................ ~ .................. .. ... ............. ..... ..... ........ 1 0
2.2.1 The effect of micro-organism on the redox value ... ................ .. ........... 10 2.2.2. The effect of flavor on the redox value ................... ............................ 11 2.2.3. The effect of dissolved oxygen concentration on the redox value .. ... 11 2.2.4. The effect.ofthe composition ofmilk on the redox value ....... ......... .. 11
2.3 Oxidation- Reduction Reactions in milk ... .. ....................................... ....... .. . 13 2.4 Lipid Oxidation ................. ......................................................... .... .... ........ ... 13
2.4.1. Auto-oxidation Reactions ...... ~ .................... ...... ... ; ...................... ... .. ... 14 2.4.2. Photo-oxidation Reactions ... ......... ...................... .... .... ......... ... ....... .... . 15 2.4.3. Factors that influence lipid oxidation of milk ................. .................... 16 2.4.4. Determining the extent of lipid oxidation ............ · ........................... ... . 17
2.5. Preventing Redox Reactions ............. .............. ~ ............................. .. ... .. ........ 18 3. Electro-reduction procedure ............................................................... .. ..... .. ...... ... 19
3.1 Electrodialysis ......................... ............................................................... .. ....... 19 3.2. Electrolysis Principal ............................................................. ........... ...... ... .. 20 3.3. Applications in food industries ...... .. ..... ................................................. .. .... 20 3.4. Electro-reduction ofmilk .. .................................................................... ....... 21
Hypothesis and objectives .................................................................................................. 23 Hypothesis ........ ; ................. ............................. .... .......... ............... ............................ 23 Objectives: .............................. ............................................................................. ..... 23
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. VI
Chapter 2 ~ ............................................................................................................................ 25 1. Introduction ............................................................................................................. 26
2. Materials and methods ............................................................................................ 28 2.1 Material ........................................................................ ~ ....................................... 28
2.1.1 Preparation of 2% emulsion of canola oil and skim milk powder ............. 29 2.2 Method .......................................................... ................ ...................................... 30 2.3. Analytical methods ............................ : ............................................................... 33
2.3.1 Oxidation-reduction potential measurement ..................... · ......................... 33 2.3.2 Dissolved oxygen (DO) measurement. .................................. .- ................... 33 2.3.3 Conductivity measurement ........................................................................ 33 2.3.4 pH nieasurement ........... ~ ........................ : .................................................. 33 2.3.5 CUITent intensity measurement ..... ~ ............................................................ 33
2.4. Statistical analysis ...................................... , ........................................................ 34
3. Results and Discussion ............................................................................................ 34 3.1 Electro-reduction treatment of oil/water emulsion ................................. , .. .. ....... 34
3.1.2 Effect on Dissolved oxygen (DO): ............................................................ 36 3.1.3 Effect on pH: .......................................... : ..... : ...................... ....................... 37 3.1.4 Effect on Conductivity: .............................................................................. 38 3.1.5 Effect on Current Intensity: ....................................................................... 39
3.2 Storageof electroreduced oillmilk emulsion ...................................................... 40 3.2.1 Effect of storage on redox potential (ORP) ................................................ 40 3.2.2 Effect of storage on dissolved oxyg~n concentration (DO) ....................... 44 3.2.3 Effect of storage on pH .................................................................... ~ ......... 47
Conclusion ................................................................................................................. 50
Chapter 3 .............................................................................................................................. 51 Abstract ................................................................................................................................ 52
1. Introduction ................... · ..... · ..................................................................................... 53 2. Materials and Methods ........................................................................................... 54
2.1 Material ............................................................................................................... 54 2.2 Electroreduction system .......................................................................... ~ ........... 55 2.3 Storage ................................................................................................................ 56 2.4. Analytical methods .............................................................................................. 57
2.4.1 Oxidation-reduction potential measurement: ............................................. 57 2.4.2 Dissolved oxygen (DO) measurement: ...................................................... 57 2.4.3 Conductivity measurement: ....................................................................... 57 2.4.4 pH measurement: ........................................................................................ 57 2.4.5 CUITent intensity measurement: ................................................................. 58 2~4.6 Fatty acid composition measurement: ... ~ ................................................... 58 2.4.6.1 Milk fat extraction .................................................................................. 58 2.4.6.2 Fame methylation .................... ; .............................................................. 58 2.4.6.3 Fatty acid analysis ................................................................................... 58
2.5. Statistical analysis .............................................................................................. 59 3. Results and Discussion ............................................................................................ 59
VI.
vu
3.1 Electroreduction treatment of omega-3 emiched milk .................................. .. ... 59 3.1.1 Effect on redox potential (ORP) ....... .. ...................................................... 59 3.1.2 Effect on Dissolved oxygen (DO) : .............. -.............................................. 60 3.1 .3 Effect on pH: ... ................................................... ................................. ... .... 61 3.1.4 Effect on Conductivity: ..................................... ~ ........................ ........ ........ 62 3.1.5 Effect on Current Intensity: ....................................................................... 63
3.2 Storage of electroreduced omega-3 emiched milk ................. ; .. -........................ . 64 3.2.1 Effect of storage on redox potential (ORP) ................................ .. ..... .. ...... .. .... 64
3.2.2 Effect on dissolved oxygen (PO): ............................................................. 67 3.2.3 Effect on pH: ................................................................................ .............. 70 3.2.4 Effect on fatty acid composition: ............................................................. .. 71
Conclusion ..................... ................................ .......................................... ........... .......... 75
-Bibliographie ....................................................................................................................... 76
vu
List of Tables
Table 1.1 Average composition of milk .............................................. : ........ .................. ........ 4
Table 1.2 Average composition of lipid classes in bovine milk ............................................ 6
Table 1.3 Distribution of the major fatty acids in b<?vine milk fat. ....................................... 6
Table 3. 1 Fatty acid composition <?f omega-3 enriched milk .............................................. 71
IX
List of Figures Figure 2.1 Simplified diagram of emulsion preperation .................................... ..... ............... 29
Figure 2.2 Simplified diagram of electrolysis cell ................................................................ 30
Figure 2.3 Changes in the redox potential during treatment of milk at 4V and OV .... .......... 35
Figure 2.4 Changes in the dissolved oxygen concentration during treatment of milk at 4V and OV ........................................... ~ .................................................................... 36
Figure 2.5' Changes in the pH during treatment of milk at 4V and OV .......... ...... ~ ............ .... 37
Figure 2.6 Changes in the conductivity ofmilk during treatment at 4V and OV .................. 38
Figure 2.7 Changes in the CUITent intensity ofmilk during treatment at 4V ........................ 39
Figure 2.8 Changes in the redox potential value during storage of samples ......................... 43
Figure 2.9 Changes in the dissolved oxygen value during storage of samples ..................... 46
·Figure 2.1 0 Changes in the pH value during storage of samples.· ......................................... 49
. Figure 3.1 Simplified diagram of electrolysis ceI1. .................. .............................................. 55 .
Figure 3.2 Changes in the redox potential during treatment ofomega-3 enriched milk ...... 59
. Figure 3.3 Changes in the dissolved oxygen concentration during treatment of omega-3 . enriched milk at 4V .......................................................................... ~ ....................... -.60
Figure 3.4 Changes in the pH during treatment of omega-3 enriched milk at 4V ................ 62 .
Figure 3.5 Changes in the conductivity of omega-3 emiched milk at 4V ................. · ........... 63
Figure 3.6 Changes in the CUITent intensity of omega-3 enriched milk at 4V ...................... 64
Figure 3.7 Changes in the redox potential value of omega-3enriched milk samples during storage .......................................................................................... .................. 65
Figure 3.8 Changes in the dissolved oxygen value of omega-3 énriched milk samples during storage .................................................................. ~ .......................................... 67
Figure 3.9 Changes in the pH of omega-3 milk samples during storage ...... : ....................... 70
Figure 3.10 Changes in the fatty acid composition of electro-reduced and control milk . . during storage in dark ................................................................................................ 72
IX
x
Figure 3.11 Changes in the fatty acid composition of electro-reduced and control milk during storage in light. ..................... ~ .................................... ~ ............. .. .................... 72
Figure 3.12 Changes in the fatty acid composition of ALA in electro-reduced and control milk during storage ............................................. ........... ......... " ... ...... " ............ 74
x
Introduction
In the calendar year of 2005-2006, the Canadian Dairy Commission announced that
dairy production in Canada ranked fourth after grains, red meats and horticulture.
The total milk production in the year 2005-2006 was 74.8 million hectoliters which
supplied the fluid market (flavored milks and creams) and the industrial market (using
milk to make products such as butter, cheese, yogurt, ice cream and milk powders).
Quebec's production represents 38% of Canada's total dairy production which the dairy
industry is the first agricultural iridustry in this province. It is important for milk producers
to continuously try to improve their efficiency in order to maintain the viability of their
enterprise.
Consumer demand for food that is natural and has higher nutritional values has encouraged
. the food industries to produce products with such qualifications. Functional foods that
contain such needs promise to deliver health and wellness to consumers while having a
tasty formulation as weil. ' Milk is a natural, multi-component, nutrient-rich beverage .
. With these characteristics ~ milk is an ideal delivery . system for newly discovered food
ingredients targeting modern lifestyle diseases. Interest in the omega-3 fatty acids as health
promoting nutrients has expanded dramatically in the recent years.
Milk is a complex reactive liquid in which different modifications occur during its
treatment and storage. One of the main chemical transformations causedby oxidation
reduction reactions in milk is lipid oxidation. Phospholipids are the main fat molecules
sensitive to oxidative stress because of their position on the milk fat globule membrane and
because they ar~ composed of polyunsaturated fatty acids.
As a result of the decomposition of the unstable hydroperoxides into secondary products,
off-flavors and nutritional losses are seen (Çesa, 2004). The same factors that promote
oxidation of unsaturated lipids enhance thedegradation of colorants, vitamins and aroma
concentrates either by direct oxidation or by the effect of free radical formation
(BorIe et al. , 2001).
In recent years, the electro-reduction procedure has also been put forward as a way to
inhibit or reduce the oxidation phenomena in milk by controlling milks redox potential.
According to N emst (Morris, 2000) the redox potential (oxidation - reduction potential
o RP) is a measure between a standard redox potential and the concentration ratio of
oxidizers and reducers. The electro-reduction procedure which is an electrochemical
process has been applied in the food industries for rnany years. This pro cess uses
electrolysis to reduce electrochemically active species causing a decrease in the redox
potential of thetreated product without changing the organoleptic and nutritive values 'of
milk. The principal effect of this treatment is that it inhibitates the oxidation-reduction
reactions that take place in milk (Inoue and Kato, 2003).
Although sorne research has been done to determine the efficiency of the electro- reduction
treatment for milk, no specific research has yet been done to verify the affects of this
procedure on the oxidative stability of milk lipids. Since, one of the main chemical
transformations caused by oxidation-reduction reactions is lipid oxidation; this factor must
be considered as weIl.
In this current research the goal is to determine the effect of electro-reduction treatment on
the oxidative stability of milk lipids. The objective of this study was to verify if the
decrease in the redox potential due to the electro-reduction treatment improves the
oxidative stability of the lipids in a dairy beverage alike milk and as weIl as omega-3
enriched milk during treatrnent and storage. Thus determining the factors of storage which
affect the oxidative stability of the lipids of milk, especially the omega-3 fatty acid, must
also be considered . .
In a more geheral way, the results of this research will allow the acquisition of new
knowledge on the effect of electro-reduction on the oxidative stability of the milk lipids.
2
2
Chapter 1. Literature Review
1. Milk
Mjlk by nature was designed to provide nourishment for the newbom, and therefore is a
highly complex mixture. This is because milk is a mineraI solution, a colloidal dispersion
and an emulsion (Amiot et al., 2002). Milk is known as an oil in water emulsion
(a dispersed phase not solubilized liquid), with fat contents of hydrophobie behaviour
(Cheftel, 1984). The composition of bovine milk is not constant during one year, it is
function of the food' intake (Small, 2002), of the seasons, of the period of lactation, the race,
the age of the cow and the feed (Webb, 1965} The four main chemical classes present in
. milk, irrespeètive of species are fat, protein, carbohydrates. and mineraIs which each
component plays an important nutritional raIe. Also, enzymes, vitamins, pigments, gases,
and micro-organisms are seen in milk in smaller quantities. Table 1.1 shows the average
composition of mid lactation bovine milk (Muir and Banks, 2000).
Table!.! Average composition of milk (Muir and Banks, 2000)
Constituent
Fat Protein:
Casein Whey protein
Non-protein nitrogen Lactose Ash Total solids
1.1. Milk protein
Concentration (gL-1) Proportion solids (%)
37.0 34.0 27.4 6.4 1.9 ~8.0 7.0
127.0
28.9 26.6
1.5 37.5 5.5
100.0
Proteins are polymers made up of several small units called amino-acids, composed of a
carboxyl group (- COOH) and amine group (- NH2). The amino-acids join to structure
proteins by forming amide groups between two amino-acids. Different amino-acids have
different chemical structures. Among those, cystein is provided with a thiol group (-SH),
that is very important for the structure ot proteins since it can form a disulfide bridge
(- S-S-) with another cystein.
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4
The proteins in milk are classified as caseins and whey proteins. Casein consists more than
80% of the true proteins in bovine milk. Caseins are hydrophobic ' having a fairly high
charge, many prolines and a few cystine residues. Caseins are presented in micellar forms
and are classified as as 1-, as2-, ~- , y- and K - caseins. The primary structure of aIl caseins
in bovine milk has been defined and has been shownthat these caseins are modestly sized
without possessing an organized structure. For this reason the caseins can not be denatured
for example by heating (Muir and Banks, 2000). Due to the fact that the casein in milk is
not present in solution but rather in micelles has caused important consequences for the
properties of milk. The casein. micelles determine the physical stability of milk products.
during heat treatment, concentrating and holding. The caseins are phosphoproteins and are
able to interact with ions which is an important aspect of their functionality for example in . .
cheesemaking. The micelles largely determine the rheological properties of sour and
concentrated milk products and the interaction of case in micelles with oil/water interfaces
is of importance with respect to properties of homogenized milk products.
The other group of proteins in milk is the whey proteins, which consist the
~-lactoglobuline, a-lactalbumine, serum bovine albumin and other minor proteins.
Whey proteins are globular proteins stabilized by disulphide bridges which can be
disrupted by heat treatments and as a result the proteins denature. On the contrary,
undenatured whey proteins are not very much effected by multivalent ions and therefore do
not precipitate.
In the c3:tegory of dairy proteins, the enzymes in milk are also important. These enzymes
are generally produced by micro-organisms and are able to react with specific substrates.
The most important in milk are the lactoperoxydase, the plasmine, the catalase, the
xanthine oxydase, phosphatases and lipases (Cayot and Lorient, 1998).
1.2 Milk Lipids
Lipids are esters of fatty acids with the molecule glycerol that are soluble in nonpolar
organic solvents and insoluble in water. Almost aIl the fat in milk is in fat globules.
J\1ilk fat occurs naturally as a complex structure. Milk globules range indiameters from
about 0.1 to 15 Jlm and are characterized by their size distribution (Walstra et al.,1999).
5
5
phospholipids. The milk fat globule membrane (MFGM) is fragile and damaged by
physical treatment. As seen in table 1.2, milk fat consists almost entirely of triglycerides or
triglycerols, phospholipids, cholesterol, diglycerides, monoglycerides, free fatty acids and
other fractions such as cholesteryl esters (Jensen et al. , 1991).
Tablel.2 Average composition of lipid classes in bovine milk (Muir and Banks, 2000)
Lipid class
Triglycerides Phospholipids
Cholesterol Diglycerides
. Monoglycerides Free fatty acids
Cholesteryl esters
Percentage
95.8-97.4 0.56-1.11 · 0.30-0.53 1.01-2.25 0.03-0.08 0.18-0.28 0.02-0.05
The chemical and physical properties oflipids depend mostly on the kind of molecule they
are as well a,s the fatty acid pattern which determines properties such as melting point,
chemical reactivity and nutritional value (Walstra et al. , 1999).
Tablel.3 Distribution of the major fatty acids in bovine milk fat (Jensen, 2002).
Fatty acid carbon
number
4:0 6:0 8:0 10:0 12:0 14:0 15:0 16:0 16: 1 17:0 18:0 18: 1 18:2 18:3
Average range (wt 0/0)
2-5 1-5 1-3 2-4 2-5 8-14 1-2
22-35 1-3
0.5-1.5 9-14
20-30 1-3
0.5-2
6
6
The fatty acids ln milk fat are variable mainly in chain length, degree of saturation, position
of double bonds, configuration of double bonds -and branching. Most fatty acids contain
4-18 carbon atoms and the even numbered are predominant. Milk contains a high
proportion of short chain fatty acids with 4-10 carbons. Milk fat consists -mostly of
saturated fatty acids such as myristic acid (C 14: 0), palmitic acid (C 16: 0) and stearic acid
(C18: 0). The unsaturated fatty-acids in milk are mainly oleic acid (C18: 1) and linoleic
acid (C18: 2) (Jensen et al. , 1991).
The phospholipids are composed of glycerol or of sphingosine connected to fatty-acids and
a phosphate groups. Phospholipids have two charged groups and therefore are mainly polar.
They do not dissolve in water or oil and form micelles. They are highly surface active and
tend to associate with proteins to make lipoproteins. In bovine milk about 600/0 of the
phospholipids are associated in milk fat globule membrane (MFGM) while the res~ is
located in the skim milk phase (Mather LH., 2000). The most abundant phospholipids are
the zwitterionic; phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, while
theanionic forms phosphatidylserine and phosphatidylinositol are present in lower amounts
(Wiking, 2005). The MGFM phospholipids contain high levels of palmitic and oleic acid,
while the short and medium-chain fatty acids are present in very low levels (Mc Pherson
and Kitchen, 1983). The fatty acid composition in the core milk fat can be changed through
the feeding of cows. Simultaneously, the composition of the phospholipids is changed.
Other lipids in milk are the unsaponifiable lipids which largely consist of cholesterol that is
in association with phospholipids. Changes can occur in milk fat by autoxidation, lipolysis
and intense heating.
7
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1.3. Carbohydrates of milk
Bovine milk contains on average 46 ' g/L catbohydrates, which the major carbohydrate. is
Lactose. Milk contains traces of other carbohydrates but polysaccharides are not seen in
inilk. Lactose is a disaccharide which is formed by the union of D-glucose and D-gaiactose
where the aldehyde group of galactose is linked to the C-4 group of glucose through a
p-1,4-glycosidic linkage. Due to the hydrolysis of lactose, these two monosaccharides can
be seen in milk as well as sorne oligosaccharides with low concentrations (between 1 and 2
g/L). Hydrolysis of lactose by acid does not take place easily which requires high
temperatures and low pH. On the contrary lactose can easily be hydrolyzed by the enzyme
lactase .
. The very important Maillard reactions that are due to prolonged-~torage as weIl as heating
occur in the presence -of lactose . which leads to brown color, loss of nutritional value and
off-flavors in milk.
Lactose is known as a reducing sugar because the anomeric carbon of glucose is not
engaged in a glycosidic bond (Amiot et al., 2002). Therefore, suitable reagents or enzymes
can cause mild oxidation of lactose, which the adehyde group is converted to a carboxyl
group .and more vigorous oxidation causés rupture in the glycosidic linkage and produces
carboxyl groups (Walstra et al. , 1999) .
. 1.4. Other components of milk
MineraIs inmilk are mainly found as salts (phosphate, citrate, sulfate, carbonate ... ), bases
or acids (Jennies and Koops, 1962). Milk is a great source of calcium and potassium but
small quantities of chlorine, phosphorus, sodium, magnesium, zinc, iron, copper and other
trace elements are present as weIl.
Milk also contains many vitamins which play a big role from a nutritional point of view . .
For example, vitamins A, D, E and K which are 'lipid soluble vitamins associated with the
fat contents of milk as well as the water-soluble vitamins such as group B vitamins and
vitamin C (ascorbic acid). Milk contains other components ·in ' very small concentrations
(lower than 100 mg/L) such as gases, alcohols, carbonyl compounds, carboxylic acids,
8
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nonproteinic nitrogen, hormones and micro.-organisms. Among gases of milk, carbon
dioxide, oxygen and nitrogen with an average concentration of 90, 15 and 6 mg/L milk are
seen respectively.
Lastly, the microbial flora of milk includes a large variety of yeasts, moulds and bacteria, in
particular of the psychrotrophic bacteria (Lamontagne et al., 2002). These micro-organisms
are responsible for many modifications in milk, due to their production of enzymes, gases,
alcohols and acids.
2. Oxidation- Reduction reactions
Oxidation-reduction reactions, also called redox reactions are chemical reaction in which an
electron is transferred from one molecule to another. The electron-don~ting molecule is the
reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or
oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or
redox pairs.
2.1. Measurement of oxidation -reduction potentiaI (Redox potentiaI)
The aptitude of a system to collect and yield electrons is known as the redox potential.
In order to measure the redox potential of a system, an inert electrode made of gold or
platinurn and an electrode of reference of known potential is usually included (Walstra and
Jenness, 1984). Âccording to the equation of Nernst, the redox potential (Eh) of a system is
related to the concentrations of the oxidized and reduced compounds available in that
system. The equation of Nernst shows that the redox · potential (oxidation -reduction
potential ORP) is a measure between a standard redox potential and the concentration ratio
of oxidizers and reducers (Morris, 2000). The redox potential can determine if a system is
in an oxidized or reduced state. The equation is as below;
RT [o:x] Eh = Eo +-ln-[-]
, nF red
in which Eo is the standard ORP (or ORP at equal concentration of oxidant and reductant),
Ris the molar gas constant, T is the absolute temperature, F is the Faraday constant, n is the
9
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9
number of electrons transferred in the process per molecule, [ox] is the molar concentration
of the oxidized form, and· [red] is the molar concentration of the reduced form. Therefore,
when IEhl > IEol, it is concluded that the oxidized form in the system predominates the
reduced form of the system.
2.2. Redox potential of milk
The redox potential of individual milk samples generally varies between +250 to +350 m V
(electrode ofreference SHE, 25 oC, pH = 6.6-6.7) (Walstra and Jenness, 1984). The feed of
the cows has been proven to have a major role in the redox potential of milk, whereas cows
that nourished on pasture produced milk with a redox potential of 20 m V lower than that of
the silage fed cows (Sherbon, 1999).
Generally, the principal systems of milk that determine its redox potential are dissolved
oxygen, ascorbic acid and riboflavin. The denaturation of whey proteins which releases free
thiol groups from disulfide bonds may also affect the ORP (Walstra and Jenness, 1984).
Other systems that may have an influence on the redox potential of milk such as the lactate
pyruvate system which are present in low quantities in fresh milk are not reversible.
The lactate-pyruvate system is not reversible unless activated by enzymes (Walstra and
Jenness, 1984).
In the dairy processes and procedures, oxidoreduction reactions are infl uenced by heat
treatments, dissolved oxygen concentration, metal ions concentr~tion (such as Cu2+),
exposure to light, as weIl as by micro-organisms (Singh et al., 1987; Walstra and Jenness,
1984).
2.2.1 The effect of micro-organism on the redox value
The initial use of the redox potential indicators in milk has been for the determination of its
microbiological quality where the growth of a micro-organism in a medium such as milk
causes the potential redox to decrease (Jacob, 1979; Morris, 2000). Therefore~ this type of
measurement is · applicable for following-'-up and observing bacterial growth in milk
(Alamo et al., 2006). AIso, the type of bacterium and the fact that it is aerobic or anaerobic
10
10
has an impact on the value of the redox potential (Matthes et al., 2000). This is due to the
fact that when micro-organisms consume oxygen, a decrease is caused in the dissolved
oxygen concentration of the medium and therefore this tends to decrease the redox potential
value. In general, a medium having a negative redox potential is favorable to the growth of
anaerobic bacteria, while if it is positive, it is favorable to the aerobic bacteria.
2.2.2. The effect of flavor on the redox value
In many studies the value of the redox potential has been associated with the organoleptic
quality of milk. Whereas, several studies have èstablished relations between the redox
potential and the flavor of ·milk. Certain reports .observe on several bonds between the
flavor of milkand its redox potential value, but also the interactions due to the heat
treatments, bacteria growth and the composition of milk have been shown as weIl (Webb
and Hileman, 1937; Greenbank, 1940; Swanson and Sommer, 1940b). According to these
results, when the number of oxidized molecules of milk increases, an increase is seen in the
redox potential value which generates an increase in the oxidized flavor of milk.
2.2.3. The effect of dissolved oxygen concentration on the rerlox value
As suggested by several authors the dissolved oxygen concentration of milk contributes a
strong share to its redox potential value (Walstra and Jenness, 1984; Morris, 2000).
It has been shown that the positive redox potential. of fresh milk is partly due to its
dissolved oxygen concentration, since when milk was deaerated by nitrogen its redox
potential quickly decreased to +0,050 V (Walstra and Jenness, 1984). Other authors report
the use of gases such as nitrogen that decrease the dissolved oxygen concentration in milk,
have an impact on decreasing its potential redox, as weIl as slowing down the development
of bad taste and off-flavors (Greenbank and Wright, 1951; Harland et al., 1952;
. Higginbottom and Taylor, 1960),
2.2.4. The effect of the composition of milk on the rerlox value
As mentioned previously, milk has a complex composition as weIl as a complex structure,
but only 'some of its substances can have an impact on the redox potential value.
Il
Il
Pastuschenko et al. (2000) has shown that with the increase of the fat content in milk the
ORP increases as well. Schreyer (2007, PhD Thesis) showed that pasteurized whole milk
(3.25% fat) shows post-electro-reduction variations in ORP that were lower than those for
pasteurized skim milk but they also concluded that fat molecules of milk, do · not have a
significant effect on the ORP value since they are not electro-reductible.
On the contrary the proteins of milk have an important impact on its redox potential value.
Bazinet et al., (1997) showed that the reduction of the disulfides bridges of proteins in
thiols groups were in . part responsible for the variation of the redox potential value.
These same groups could be also responsible for the decrease of the redox potential of
heated milks (Greenbank, 1940). In the case of heated milks, thiol groups are products of
protein denaturation which their antioxydant activity has been shown (Crowe et al., 1948;
Calligaris et al., 2004). Although this antioxydant activity, wh~ch results in a decrease in
the redox potential of heated milks, Ï-s dependant on the thermal conditions applied.
Whereas, during 90°C . and 80°C treatments, the redox potential of milk tends to increase,
while \vith 120°C, the redox potential decreases (Anese et al., 2003).
Among the many mineraIs present in milk, only iron and copper were recognized to have
an oxidation-reduction activity. Many researches have shown the impact ofthe'presence of
copper on the flavor and the redox potential ofmilk (Webb and Hileman, 1937; Greenbank,
1940). A mathematical relation between the redox potential, the concentration of copper,
. iron and ascorbic acid was precisely established, according to the sudden heat treatment by
milk (pasteurized or sterilized), with a coefficient of correlation higher than 99% (Vahcic et
al., 1992).
Conceming the vitamins, the ascorbic acid has been recognized as a powerful antioxydant
and is regarded as being one of the principal chernical species influencing the redox
potential of milk (Alais, 1984; Walstra and Jenness, 1984). It has been shown that an
in~rease of ascorbic acid concentration in milk generates a reduction in its redox potential
value (Greenbank, 1940; Swanson and Sommer, 1940b) as well as an increase in the
oxidative stability of the fatty acid composition of milk (Rosenthal et al., 1993).
Riboflavin was also recognized to have an effect on the redox potential of milk (Alais,
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12
1984; Walstra and Jenness, 1984). The oxidized form of thiamin (BI) was used to
determine the reducing capacity of milk (Harland and Ashworth, 1945) and this study
showed that this vitamin played an important role in the oxidation of the fat contents under
the effect of light (Borie et al. , 2001).
2.3 Oxidation- Reduction Reactions in milk
ln a complex fluid such as milk many modifications arise during its processing and storage,
whereas the oxidation- reduction reactions are dominantly the main reason for the chemical
degradation of milk and dairy products (Jensen et al, 1991). These reactions result in the
alteration of sensitive compounds. One of the main chemical transformations caused by
oxidation-reduction reactions in milk is lipid oxidation. Phospholipids are very sensitive to
oxidative stress, because they are composed of polyunsaturated fatty acids and also are the
main fat on the milk fat globule membrane. As a consequence, undesirable
oxidized/metaIlic flavours can develop in dairy products (Swanson and Sommer, 1940a).
AIso, many vitamins such as A, D, E,K, BI, and B2 are found in J)1ilk and for this reason
milk is known as a good source of vitamins for human nutrition. But the factors that
promote oxidation of unsaturated lipids can enhance the degradation of vitamin A and E.
This· may either be by direct oxidation or by the affect of free radical formation. Another
negative consequence is that the microbial cell metabolism is driven by different oxidation
~eduction systems which may conclude to the spoilage of milk. It has also been shown that
oxidation reduction reactions have a negative impact on the thermal stability of milk
(Vahcic et al., 1992). Colorants and aroma concentrates added to products are also sensitive
to oxidation (BorIe et al., 2001).
2.4 Lipid Oxidation
One of the most important chemical transformations that occur in milk and dairy products
is lipid degradation which generally results in deterioration in sens ory and nutritional value.
The least stable macro-constituents in foods are the lipids. The problem of oxidative
deterioration has great economical importance as weIl as safety importance in the
production of lipid-containing foods. The extent of changes in the appearance, texture,
13
13
flavor and odour of foods affected by oxidative reactions depends on the type of dairy
product as well as the conditions applied on this product. Milk is a complex biological
system containing many factors , which may act as antioxidants and/or pro-oxidants. The
relative amounts of these factors in milk as weIl as processing, heating (Karatapanis et al. ,
2006) and storaging conditions have been shown to influence the rate and extent of lipid
oxidation in milk. These factors include oxygen, light, storage temperature, metals,
enzymes and water activity (O'Conner et al, 2006).
Lipid degradation of food products is caused by complex reactions. which are generally
caIled; hydrolytic rancidity and oxidative rancidity. The basic princip le of oxidative
rancidity is a reaction between unsaturated fatty acids and oxygen. Oxidative rancidity is
explained by two main mechanisms; auto-oxidation and photo-oxidation.
2.4.1. Auto-oxidation Reactions
Auto-oxidation reactions are known to have three maIn stages; the initiation, the
propagation and the termination.
When the hydrogens : at the alpha-position of the double bonds (allylic hydrogens) of
unsaturated fatty acids (RH) are removed, free fatty radicals (Re) are obtained. This . fatty
. acid radical formation is called the initiation stage. For this stage to take place external
energy from heat, light, radiation or catalysts such as metals specially Iron. and Copper
must be present.
Once the fatty acid radicals are formed they can easily react with oxygen and therefore fatty
acid peroxy radicals (ROOe) are obtained. These peroxy radicals are highly reactive and
could react with another unsaturated fatty acid to form a hydroperoxide (ROOH) and a new
fatty acid radical (Re). This stage is called the propagation stage because new radicals are
continuously produced which leads to an acceleration of hydroperoxide formation.
R- + 02 ~ RaO- + RH ~ ROOH + R-
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14
Finally the production of new radicals is terrninated when two radicals react with each other
and non-reactive compounds are made.
Hydroperoxides and free fatty acids are known as the main preliminary products of lipid
oxidation. Since, hydroperoxides are not stable they decompose to other compounds such
as aldehydes, alcohols, cetones and hydrocarbons which are highly volatile and are
responsible for the rancid off-flavors in food products.
2.4.2. Photo-oxidation Reactions
This mechanism of lipid oxidation is based on the generation of highly reactive oxygen
known as singlet oxygen (1 02) from the dissolved atmospheric oxygen· (normally in triplet
state 302). The electron configuration of singlet and triplet oxygen causes their difference.
Singlet oxygen has been seen to enable rapid reactions with the electrons that sUITound the
double bond of unsaturated fatty acids. Singlet oxygen is formed in the presence of light
and a photo seIisitizer from triplet state oxygene Pigments such as chlorophyll and
riboflavin and heavy metals which all naturally occur as minor components in food have
been shown as sensitizers. Also, synthetic dyes such as eosin and erythrosine can also act as
sensitizers (Bradley et and Min, 1992). In these reactions the singlet oxygen generated from
triplet oxygen is highly reactive and can directly react with unsaturated fatty acids and form
hydroperoxides. The position of the double bonds in the fatty acid carbon chain is shifted as
a result of photo-oxidation and the hydroperoxides formed are different from those formed
by fatty acid radicals but since they are highly reactive they can influelfce the initiation
stage of auto-oxidation reactions as welle
. 302 __ Ll....;:;...·gh_t_/P_h_ot_oS_en_s--+)102 + RH ~ ROOH
Another formation of hydroperoxides involves the reaction between oxygen and
unsaturated fatty acids through the catalytic action of enzymes which are known as
lipoxygenases. The hydroperoxides produced via this route have a different nature from
those resulting ftom free radicals or photo-oxidation.
15
15
·As mentioned before, hydroperoxides are the preliminary products of lipid oxidation which
are both odorless and unstable .. They decompose to other compounds such as aldehydes,
alcohols and hydrocarbons. The main characteristic of aU the decomposition reactions of
hydroperoxides is they result in the formation of molecules with shorter carbon chain
length than the original fatty acid carbon chain therefore are much more volatile than the
hydroperoxides and can develop rancid off-flavors in food.
It has been shown that the activation energy in aU three stages of auto-oxidation is not the
same. The first two stages; initiation and propagation require very low activation energ~ for
the auto-oxidation chain reactions to occur (Hamilton, 1994).
The typical time-course of auto-oxidation is nieasured by the concentration of produced
hydroperoxides, whereas it consists of a lag phase (induction period) and then is followed
by a rapid accumulation of hydroperoxides (reaching a maximum) and then there is a
decrease as the hydroperoxide decomposition reactions become more important
(O'Conner and O'Bien, 2006).
Another important aspect of auto-oxidation is that the rates of oxidation reactions are
different, which depends on the number of the double bonds in the carbon chain of
unsaturated fatty aclds. The relative rates measured by Gunstone and Hilditch at 20°C for
:methyl oleate, methyl linoleate and methyl linolenate are 1: 12:25 (Kristott, 2000). This
means at room temperature, polyunsaturated linoleic acid C 18:3 wiU be degraded by
oxidation reactions in a much shorter time than mono-unsaturated oleic acid C18: 1.
Therefore the amount of unsaturated fatty acid composition of a food product is an
important fac10r for the assessment of their stability towards oxidation during storage.
2.4.3. Facto"rs that influence lipid oxidation of milk
A range of environmental and physical factors, processing andstorage conditions,
endogenous and exogenous chemical constituents and enzymes have been shown to
influence the rate and extent of lipid oxidation in milk and milk products. These factors
include oxygen, light, endogenous and exogenous metals, natural 'antioxidants and
pro-oxidants, thiols, proteins, enzymes, browning reaction products, milk fat globule
membrane .(MFGM) constituents, storage temperature and water activity. The balance
between pro-oxidant and antioxidarit factors iscritical for the oxidative stability of milk
16
16
(Stapelfeldt et al., '1999; Morales et al., 2000). Milk is a complex biological system
containing many factors, w~ich may act as antioxidants and/or pro-oxidants. The relative
amounts of these factors in milk are in regards to parameters such as the breed, health,
nutritional status, and stage of lactation of the cow. AIso, storage conditions of milk have a
profound influence on the progress of lipid oxidation. The degree of unsaturation of milk
lipids ~s also another important factor that influences oxidation.
2.4.4. Determining the extent of lipid oxidation
When lipids are oxidized, a complex mixture of primary and secondary products with low
molecular weight- compounds is obtained. The nature and composition of these mixtures
vary according to the conditions and extent of oxidation. Many methods are available to
determine oxidative deterioration as weIl as determining their extent and nature. Sorne of
these methods' are as following;
• Sensory methods: Using trained panels to measure a~d analyze the characteristics of
lipids evoked by the senses of taste, smeIl, sight and mouth feel.
• Peroxide value: ' The standard iodometric measures the iodine produced by
potassium iodide added as a reducing agent to the oxidized sample dissolved in a
choloroform-acetic acid mixture. This method may be by 'titration, colorimetric or
electrometric methods.
• Conjugated dienes: The conjugated diene hydroperoxides produced from
polyunsaturated fatty acids can be determined quantitatively by their strong
absorption maximum at 234nm. This method is done in the early stages of lipid
oxidation in which the hydroperoxides have underwent little or no decomposition.
• The Anisidine test: This test measures high molecular weight saturated and
unsaturated carbonyl compounds in triacylglycerols. This test gives information on
non-volatile carbonyl compounds which is seen in lipids with omega-3
polyunsaturated fatty acids.
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17
• Thiobarbituric acid value (TBA): This colorimetric method is used to me as ure
rancidity based on the pink color formed between TBA and oxidation products of
polyunsaturated fatty acids absorbance at 532-535nm.
• Gas chromatographic methods: These methods are capable of determining volatile
oxidation products that are either directly responsible for or are markers of flavor
development in oxidized lipids. Ge analysis for volatile compounds are correlated
with flavor scores by sensory analysis · and also provide sensitive methods to detect
low levels of oxidation in lipids. The main Ge methods used are; Static headspace
method, Dynamic headspace methèd and the direct injection method.
2.5. Preventing Redox Reactions
Although oxidation-reduction reactions have a significartt effect on the quality of dairy
products, few methods are available to control them without causing other negative results.
Heat treatrnents have been used in this regard for many years but they can promote to
numerous chemical reactions, such as Maillard reactions, hydrolysis and vitamin
degradation, that affects the organoleptic . and nutritional properties of milk (Walstra and
Jenness, 1984; Van Boekel, 1998).
In sorne studies the decrease of the ORP value of bovine milk has been seen as a result of J .
degasification (or splashing) with nitrogen (Greenqank and Wright, 1951; Alais, 1984;
Walstra and J enness, 1984).
Various methods have been proposed to prevent oxidative deterioration of dairy products
such ex?-mples are; use of chelating agents to bind metallic ions (Let et al., 2003); or the
addition of antioxidants (Decker and Ashworth, 1951; Granelli, 1997). Unfortunately, it has
been shown that these processes have a negative impact on the flavor and nutritive value of
milk (Van Boekel, 1998).
Other fairly new methods to prevent the oxidation reduction reactions in milk are the
selection of packaging materials with low oxygen permeability and light transmission
(Mortensen et al., 2004) or vacuum or inert gas packaging to reduce oxygen concentration
(Moyssiadi et al., 2004).
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18
With the increasing interest in non thermal preservation technologies (Manas and Pagan,
2005) and the use of highly sensitive ingredients, preventing oxidative degradation in the
food industry has become a challenge. For instance, tocopherols and other phenolic
antioxidants can be lost during several different processing stages that include heating
techniques. Water soluble antioxidants such as ascorbic acid can be lost by leakage to the
process water in some other processes (Mortensen et al., 2002).
In recent years, the electro-reduction procedure has also been put forward as a way to .
inhibit or reduce the oxidation phenomena in milk.
3. Electro-redu.ction procedure
3.1 Electrodialysis
Electrodialysis is an electrochemical separation process by which ionic charged species
. are transported from one solution to another, by transfer through one or more selective
membranes, under the influence of a De electrical current (Gardais, 1990).
Electrodialysis can be preformed with two main cell . types; the muIti membrane cells and
the electrolysis cells. The multi-membrane cells are for dilution-concentration ·and water
dissociation applications while the electrolysis cells are used for oxidation-reduction
reactions (Gardais, 1990). The multi membra,ne cells are mainly used with monopolar
membranes and are applied to different food industries, whereas in the dairy industry they
are . dominantly used for the demineralization of milk or milk products, protein separation
and acid caseinate production.
In the case of electrolysis cells, two principle applications have been published;
electrochemical coagulation and electro-reduction (Bazinet and Araya-Farias, 2005). The
technology for electrolysis cells is based on the use of one or a combination of ion
permeable membrane, which electrical energy is used to force reactions to take place on the
surface of the electrode immersed in the liquid to be treated. These cells function ln
continuous, semi-continuous and discontinuous modes (Pletcher and Weinberg, 1992).
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19
3.2. Electrolysis Principal
The chemical modifications that take place in the electrolysis cell are determined by three
factors ; the oxidation reactions at the anode, the reduction reactions at the cathode and the
nature of the chemical species circulating through separators between the electrodes.
The separators are generally membranes which can be permeable to the ions.
The membranes allow the passing of the CUITent and are used to bala~ce the load of the
system. During the procedure an electrical potential is applied between the anode and the
cathode, the positively charged cations migrate towards the cathode, ,and the negatively
charged anions migrate towards the anode. The c.ations pass easily through the negatively
charged cation- exchange membranes but are retained by the pbsitively charged anion
exchange membranes, and vice versa. On the other hand, electrolysis cells also exist where
two solutions, one for each electrode compartmènt, are separated by a single membrane.
This application is based on electrode redox reactions which are electrolysis-specific
properties. The electrolysis cell in the dynamic (coptinuous) mode is ' generally separated
into two compartments by an ion exchange membrane. The fluid tO ,be treated can circulate
in contact with an anode where oxidation reactions occur or on the other side, in contact
with a cathode where reduction reactions take place. Electro chemical active compounds of
the liquid to be treated are converted into their reduced form when in contact with the
cathode and these reactions eventually cause a decrease in the redox .potential of the
product treated.
A number of parameters should be taken into account when optimizing the efficiency of
such a process, in'cluding the applied voltage, the conductivity of the ionic species in the
solutions, pH and the water dissociation which occurs when excess CUITent is applied
(Britz and Robinson, 2008).
3.3. Applications in food industries
Electrolysis has already been proposed to reduce oxygen in fruit juice (Hékal, 1983), to
extract cytoplasmic proteins from alfalfa (Labrecque et al., 1990), to coagulate proteins
(Janson and Lewis, 1994) and to redu~e disulfide bonds in ,proteins (Bazinet et al. , 1997).
In more recent years the electro-reduction process has been proposed for milk products
(Bazinet et al., 2006; Schreyer et al., 2007). The electro~reduction process has the
20
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advantage to. decrease the o.xidatio.n -reductio.n value o.f milk witho.ut causing any alteratio.n
o.f the o.rgano.leptic and nutritive values o.f milk. Mo.reo.ver, no. chemical substances are used
during this pro.cess to. decrease the ORP o.f the milk and the electro.lyte used during the
pro.cess co.uld be reused (Schreyer, 2007, PhD thesis) ~
3.4. Electro-reduction of milk
Since milk is a co.mplex fluid with many electro.chemical species, it is therefo.re important
to. o.bserve the impact o.f the redo.x po.tential transfo.rmatio.n in dairy pro.ducts in regard with
two. main aspects; the efficiency o.f the treatment and the quality o.f the treated pro.ducts.
Recently a series o.f experiments have been do.ne concerning the impact o.f the electro.
reductio.n pro.cess o.n milk. The evo.lutio.n o.f the redo.x po.tential o.f electro.-reduced milk
during treatment and sto.rage has been sho.wn by Bo.lduc et al., (2006a) and Schreyer,
(2007, PhD thesis). Bo.lduc et al. , (2006a) applied electro.-reductio.n to. mo.dulate the redo.x
po.tential o.f milk using five ano.de/catho.de vo.ltage differences (2, 4, 6, 8 and 10 V) to.
electro.-requce pasteurized skim milk. Acco.rding to. their pro.to.co.l, 4 V was the best
ano.de/catho.de vo.ltage difference whereas the decrease in the redo.x po.tential value is
pro.po.rtio.nal to. the different vo.ltages used. Altho.ugh negative redo.x :values were reached
during the treatment and the o.xygen co.ncentratio.n decreased, the redo.x values were no.t
stable and returned to. po.sitive values during sto.rage. This demo.nstrated that pro.bably
during sto.rage, o.xygen is o.nce again intro.duced to. the pro.duct which fo.r this reaso.n it is
better to. have sto.rage in an anaero.bic co.nditio.n.
AIso., o.ther recent wo.rk o.n the electro.-reductio.n o.f milk has sho.wn that the fat and pro.tein . .
co.ntents o.f the milk pro.ducts (raw milk, pasteurized milk, pasteurized skim milk and ultra
filtratio.n permeate) and the heattreatments applied have a co.nsiderable effect o.n the redo.x
po.tential after electro.-reductio.n (Schreyer, 2007, PhD thesis). The same trend o.f the
evo.lutio.n o.f redo.x values was seen as the previo.us research while o.xygen reached to.
amo.uilts near to. zero.. The electro.-reductio.n treatment did no.t pro.vo.ke any majo.r physical
changes in milk, where the co.mpo.sitio.n stayed the same thro.ugho.ut the pro.cedure just as ,
the pH had very slight changes (Schreyer et al., 2008, IFSET).
Co.ncerning the effect o.f electro.-reductio.n o.n the micro.bial part o.f milk, it has been sho.wn
that while the redo.x po.tential of electro.-reduced raw milk was very lo.w,. the electro.-
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21
reduction treatment could not affect the growth of the aerobic bacteria during storage.
This could only be obtained if the milk samples were kept in anaerobic storage conditions
or degassing was used. However, the efficiency of the electro-reduction treatment of milk
on the viability of probiotic bacteria has been shown (Bolduc et al. , 2006b).
1t has been observed that the resistance to oxidative stress in electro reduced pasteurized
milk increases up to 16% in comparison with the untreated pasteurized milk. Also the
degradation of Vitamin C (ascorbic acid) in electro reduced milk and untreated milk was
compared, which is respectively about 27% and 43% for storage of 4 days (Schreyer, 2007,
PhD thesis).
Although some research has been done to determine the efficiency of the electro-reduction
treatment for milk, no specific research has yet been done to verify the effects of this
procedure on the oxidative stability of milk lipids.
Since as mentioned before, one of the main chemical transformations caused by oxidation
reduction reactions is lipid oxidation, this factor must be considered as weil.
22
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22
Hypothesis and objectives
Hypothesis
The decrease in the redox potential due to the electro-reduction treatment improves the
oxidative stability of the lipids in milk during storage.
Objectives:
• To determine the modifications that takes place on the oil/water emulsion during
electro-reduction.
• To indentify the factors of storage which affect the oil/water emulsion after electro
reduction.
• To determine the modifications that takes place on the omega-3 enriched
commercial milk during electro-reduction.
• To identify the factors that affect the lipid st-ability of electro-reduced omega-3
enriched commercial milk during storage.
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23
Chapter 2
Electrochemical modification of th·e redox potential .of oil/water emulsion and the affect of storage conditions
. on its evolutioD.
24
24 .
Abstract
Milk is a highly perishable fluid which many modifications arise during its processing and
storage. Milk degradation is dominantly due to oxidation-reduction reactions.
The oxidation- reduction reactions active in milk conclude to negative consequences such
as; Upid oxidation, undesirable flavors , degradation of vitamins and changes in microbial
flora ofmilk.
In recent years, the electro-reduction procedure has been put forward as a way to inhibit or
reduce the oxidation phenomena in milk by controIling milk redox potential. This process
uses electrolysis to reduce electrochemically active species causing a decrease in the redox
potential-of the treated product. The decrease _ of the ORP in milk could then allow a quality
impro:vement of these products.
The main objective of this research was to investigate the modifications that take place on
the oil/water emulsion during electro-reduction as weIl determining the different storage
conditions that affect the electro-reduced emulsion.
The results showed that the electro-reduction treatment significantly reduced the redox
potential of the emulsion samples to negative values and was' also able to decrease their
dissolved oxygen concentration, without causing any major changes in their pH.
Although the electro-reduced samples maintained negative ' redox values throughout the
14 day storage period a significant increase of the redox value was seen. The storage
conditions of headspace and temperature showed to have an important impact on the ORP
value a~d the DO value of ail samples, showing optimal storage conditions in regards to
storage temperature and the absence, of oxygen can maintain the low values of ORP and
DO for a longer storage period.
The results of the mentioned projects can be used in order to improve the quality of dairy
products as weIl as the enhancement of the shelf- life of the dairy products.
1. Introduction
In a complex fluid such as milk many modifications arise during its processing and storage,
whereas· the oxidation- reduction reactions are dominantly the main reason for the
degradation of milk (Jensen et al, 1991). These reactions result in the alteration of sensitive
compounds. One of the main · chemical transformations caused by oxidation-reduction
reactions in milk.is lipid oxidation. Phospholipids, the main fat on the milk fat globule
membrane, are composed of polyunsaturated fatty acids and therefore are very sensitive to
oxidative stress. As a consequence, undesirable oxidized/metallic flavours can develop in
dairy products. AIso, many vitamins such as A, D, E, K, BI , and B2 are found in milk and
for this reason milk is known as a good source of vitamins for human nutrition. But the
factors that promote oxidation of unsaturated lipids can enhance the degradation of vitamin
A and E. This rnay. either . be by direct oxidation or by free radical formation.
Another negative consequence is that the microbial cell metabolism is driven by different
oxidation reduction systems which conclude to the spoilage of milk. It has also been shown
that oxidation reduction reactions have a negative impact on the thermal stability of milk
(Vahcic, 1992) Colorants and aroma concentrates added to products are also sensitive to
oxidation (BorIe et al., 2001).
Recently a series of experiments have been done conceming the impact of the electro
reduction process on milk. The evolution of the redox potential of electro-reduced milk
during treatment and storage has been shown by Bolduc et al. (2006a) and Schreyer (2007,
PhD thesis).
Bolduc et al. , (2006a) applied electro-reduction to modulate the redox potential of milk
using five anode/cathode voltage differences (2, 4, 6, 8 and 10 V) to electro-reduce
pasteurized skim milk. They showed that as the anode/cathode voltage difference increases,
reduction reactions take place in a faster rate (Tallec, 1985) which explains the amount of
foam that forms in the electro-reduction system. No apparent arnount of foam was seen
during electro-reduction at 2V and 4 V while due to the extreme amount of foarn produced
the treatments at 6,8, and 10 V were ended sooner.
They also concluded that the decrease in the redox potential value is proportional to the
different voltages used since the voltage difference of 2 V seemed to be insufficient for the
26
26
electro-reduction of aIl milk reducible species and an anode/cathode voltage differences
equal to or higher than 4 V had to be applied in order to reach low ORP values. Therefore,
according to their results, 4 V was the optimal anode/cathode voltage difference to be
applied for the electro-reduction treatment of milk.
Although negative redox values were reached during the treatment and the oxygen
concentration decreased, the redox values were not stable and retumed to positive values
du~ing storage.
AIso, other recent work on the electro-reduction of milk has shown that the fat and protein
contents of the milk products (raw milk, pasteurized milk, pasteurized skim milk and ultra
filtration permeate) and the heat treatments applied have a considerable effect on the redox
potential after electro-reduction. The same trend of the evolution of redox values was seen
as the previous research while oxygen reached to amounts near to zero. The electro-
. reduction treatment did not provoke . any maj or physical changes in milk, where the
composition stayed the same throughout the procedure (Schr~yer, 2007, PhD thesis).
Concerning the microbial aspect of milk, it has been shown that while the redox potential
of electro reduced raw milk was very low, the electro-redu~tion treatment could not effect
the growth of the aerobic bacteria during storage. This could only be obtained if the milk
samples were kept in anaerobic storage conditions or degassing was used. However, the
efficiency of the electro-reduction treatment of milk on the viability of probiotic bacteria
has been shown (Bolduc et al. , 2006b).
1t has been observed . that the resistance to oxidative stress in electro-reduced pasteurized
milk increases up to 16% in comparison with the untreated pasteurized milk. Also the
degradation of Vitamin C (ascorbic acid) in electro-reduced milk and untreated milk was
compared, which is respectively about 27% and 43% for storage of 4 days (Schreyer, 2007,
PhD thesis).
Although sorne research has been done to determine the efficiency of the electro-reduction
treatment for milk, no specifie research has yet been done to verify the affects of this
procedure on the oxidative stability of milk lipids.
Since, one of the main chemical transformations caused by oxidation-reduction reactions is
lipid oxidation; this factor must be considered as weIl.
27
27
In this current research the goal is to deterrnine the effect of electro-reduction on the
oxidative stability of milk lipid's, a model system must be used so the experiments can be
repeatable due to . the factor of variahility of milk. For the model system, canola oil and
skim milk powder will be used. Canola oil contains a very low level of saturated fats
(approximately 7%), a moderate level of poly unsaturated fats (approximately 32%), and a
high level of mono unsaturated fats (approximately 61 %). Moreover, canola oil contains a
healthy balance of the essential poly unsaturated fatty acids (PUF A' s), linoleic aèid
(omega-6) and alpha-linolenic acid (omega-3). Linoleic acid (C18:2) is a poly unsaturated
fatty acid which is unstable to reacting with light and oxygen .. It is also referred to as
omega-6 because the first double bond occurs after the 6th carbon atom in its fatty acid
chilÎn. Alpha-linolenic acid (C18:3) is also a poly unsaturated fatty acid which is 5 times
more unstable than linoleic acid in reacting with oxygen and light. The first double bond in
alpha-linoleic acid occurs on the 3rd carbon atom in its chain therefore it is also referred to
omega-3 fattyacid.
2. Materials and methods
2.1 Material
The electro-reduction treatments were carried out on a 2 % stable oil/water emulsion made
of Canola and Skim milk powder (model system for the research).
28
28
Preparation of Emulsion
Homogenisation
" Electroduction
Storage
+ 0% } 22° C
100/0 .
50%
Figure 2.1. Simplified diagram of the steps applied.
2.1.1 Preparation of 2 010 emulsion of canola oïl and skim milk powder
In order to make 4L of a 2% stable oil/water emulsion alike milk, 1969 of skim milk
powder (Agropur, Canada) was weighed and dissolved in 1862mL of warm water (54°C)
with agitation inside a thermostated water bath. After the powder had completely dissolved
in water, the mixture was left to rest for 30min. Then 41.44g of canola oil (Merit selection,
Canada) was weighed and while mixingwas added to the dissolved skim milk powder.
Afterwards, the warm (54°C) oil/water emulsion was homogenized using an EF-C5
homogenizer (Avestin, Ottawa, Ca) with pressure of 2500 psi (17 MPa). Homogenization
was repeated two times on each emulsion and then the homogenized emulsion was placed
in 4°C in order to cool down to about 20°C.
29
29
2.2 Method
+ Electrical Source -
H 20- X 0 2 +2H+ +2e-
Electrolyte .
.. l . i0 2 i t02i H2
Treated Milk ..
}'i02 +2H+ ~ H 20
H 20 + 2e- ~ 20H- + H2
. Milk to be treated
Figure 2.2. Simplified diagram of the electrolysis cell. (Source: Bolduc et al., 2006a)
2.2.1 Electro-reduction system .
The electro-reduction system used is the same as the one used by Bolduc et al. , (2006a).
This system contains a dynamic cell where the cell is divided into two different
compartments by a cationic membrane (CMX-SB, Tokuyama Soda Corp, Tokyo, Japan).
In one side of the membrane, the emulsion was in contact with a food-grade stainless steel
cathode, and on the other side of the membrane, the electrolyte (0.1 M H2S04) was in
contact with an anode. In each compartment, one polypropylene spacer (2.02 mm thick)
was placed to allow the liquid to flow through and to have contact with both the membrane
and the corresponding electrode. The assembly was made watertight with rubber gaskets
(1.23 mm thick), placed next to each of the electrodes, spacers and membrane. In order to
have a continuous circulation during the treatment, each of the cell compartrilents were in
contact with their own external tank. Both solutions were circulated by two centrifugaI
pumps (Iwaki Magnet Pump, Iwaki Co, Ltd., Tokyo, Japan), and their flows were
30
30
controlled by two flow meters· (Aalborg Instruments and C"ontrols, Inc., Orangeburg, USA)
and were kept at 300 mL/min.
2.2.2 Treatments
Using the electrolysis cell described above two anode/cathode voltage differences were
-applied between the electrodes (OV and 4V) which was supplied by an electrical power
supply (Mo dei HPD 30-10, Xantrex, Burnaby, Canada). In this study OV was used in order
to investigate the modifications that take place on the emulsion just by passing through the
electrolysis ceIl without having a potential difference. In this case a voltage difference was
not supplied by the electric power supply, but due to the presence of the liquid (oil/water
emulsion) and the electrolysis material a small voltage difference was present.
Each electro-reduction treatment was done in triplicates using 300 mL of the oil/water
emulsion and an equal volume of O.lM sulfuric acid. During each 60 min treatment,
oxidation-reduction potential (ORP), dissolved oxygen (DO), pH ,conductivity and the
CUITent intensity of milk were recorded at intervals of 30 seconds during the first 10 ·
minutes and at intervals of 1 minute thereafter.
2.2.3 Storage
The goal was to determine the storage conditions that have an affect on the modified
parameters of the electro-reduced oil/water emulsion. The three most important storage
factors that impact the oxidative stability of lipids; oxygen, temperature and light were
studied on the electro-reduced emulsion and control samples. After each electro-reduction
treatment, samples of electro-reduced emulsion were poured in glass jars. Sodium azide
(0.02%) was added to aIl modulated samples in order to prevent microbial growth in the
samples during storage. They were placed in conditions of storage as described in table 2.1
in regards to headspace, temperature and dark for duration of 14 days in parallel with
control non electro-reduced milk. The oxidation-reduction potential (ORP), dissolved
oxygen (DO), and pH milk samples were recorded on days 0, 1,2,4, 8 and 14 at ~hich,
analyses were performed on different jars of samples in order toprevent sample and
31
31
'headspace contamination. The experimental design used was full facto rial (2x2x3) for
control and two treated samples at OV and 4V.
a) Oxygen: The factor oxygen was demonstrated by the head space in the sample bottles.
Three main head spaces were selected in portion to the volume of the sample bottles.
• 0%: no oxygen in the headspace of ~he sample
• 10%: typical volume ofheadspace in commercial milk.
• 50%: presence of an important quantity of oxygen.
b) Storage temperature:
• 4°C: refrigerator te~perature
• 22°C: ambient temperature
c) Dark: The samples were studied in the absence of environmentallights.
Table 2.1. Storage conditions for the electro-reduced and control milk samples
days 0 1 2 4 8 CD 3 ::r 0 0 0 0 0 0 0 0 0 0 0 0
"'0 CD ~ m m ~ m m ~ m m ~ m m ~
m m ~ CD ru ro ro ro ro ro ro ro ro ro ro
ru c.. ~ 0 0 ~ 0 0 ~ 0 0 ~ 0 0 ~ 0 0 ~ Cf) en ~ ~ en ~ ~ en ~ ~ en ~ ~ en ~ ~ en Ë "'0 w W al W W al -, ru 3 0 ,t.. 3 0 ,t.. 3 0 ,t.. 3 0 ,t.. 3 · 0 ,t.. 3 CD () "0 < < "0 < < "0 . < < "0 < < "0 < < "0 CD ro ro ro ro ro ro
0%
Dark 4'C 10%
50%
00/0
22'C 10%
50%
14
m ro 0 ~ 0 <
m ro 0 ~ ,t..
<
--
32
32
2.3. Analytical methods
2.3.1 Oxidation-reduction potential measurement
The ORP was measured using a VWR Symphony platinum electrode (VWR Scientific
Products, West Chester, PA, USA) with an internaI Ag/Agel reference electrode and filled
with the recommended solution containing KCI and AgCl. This electrode was connected to
a VWR Symphony portable SP20 pH/ISE meter. The electrode reading was verified with a
homemade solution of potassium ferrocyanide and potassium ferricyanide having an ORP
of+234mV.
2.3.2 Dissolved oxygen (DO) measurement
The DO was measured using a VWR Symphony electrode (VWR Scientific Products)
mounted with the specifiedmembrane and filled with the supplied DO electrolyte solution .
. The electrode was connected to a VWR Symphony SP50D portable DO meter.
The electrode was calibrated every 2 h as described in the supplier's manu al.
2.3.3 Conductivity measurement
The conductivity was measured with an immersible YSI probe (model 3417, K = 1 cm-l,
Yellow Springs Instrument, Yellow Springs, OH) connected to an YSI 3232 adaptor to
allow readings on the YSI 3100 conductivity meter of the same manufacturer.
Since the conductivity varied proportionally with temperature and the values were not
automaticaUy compensated by the conductivity meter, aU readings were corrected to 25 oC
using the method described by Bazinet et al., (2004).
2.3.4 pH measurement
The pH was measured using a VWR Symphony electrode (VWR Scientific Products)
equipped with an automatic temperature compensation de vi ce and connected to a VWR
Symphony SR60lC benchtop pH meter.
2.3.5 Current intensity measurement
The current passing through the electrodes was read from a Mastercraft numerical
multimeter (Mo dei 52-0060-2, Mastercraft, Toronto, Canada).
33
- - ------ - - - - - - -
33
2.4. Statistical analysis
Data obtained during treatments and storage were subjected to multivariate analysis of
variance using JMP IN software (Version 5.1, SAS Institute inc., Cary, Ne).
3. ResuIts and Discussion
3.1 Electro-reduction treatment of oil/water emulsion
3.1.1 Effect on redox potential (ORP)
The repeated measure analysis of variance of the data showed that the anode/cathode
voltage difference (P<O.OOOI), the time (P<O.0037) and the dual interaction time/voltage
difference (P<O.OOO 1) had a significant effect on the ORP value during electro-reduction
applying the voltage difference of 4V. As expectçd, the anode/cathode voltage difference of
OV had no effect on the ORP value during treatment (P>0.1084). In this case a voltage
difference was not supplied by the electric power supply, but due to the presence of the
emulsion and the electrolysis material a smaU voltage difference was present.
The electro-reduction treatment using the 4 V anode/cathode voltage difference performed
. on milk samples resulted in a significant exponential decrease of the ORP value as can be
seen on Figure 2.3. Generally speaking, raw milk has an ORP between +200 and +300 mV
under aerobic conditions (Morris, 2000), while recently Bolduc et al., (2006a) observed an
. average value of + 182 m V for the same brand of pasteurized skim milk. However, ORP
values for the oil/water emulsion of canola oil and skim milk were not found in the
literature. In this experiment, the mean initial ORP value for the oil/water emulsion used
was +85±12 mV.
During electro-reduction by applying an anode/cathode voltage difference of 4 volts, the
general trend was that the redox potential decreased significantly in the first minutes of the
treatment, ta then stabil ize at a constant value corresponding to a plateau.
However, it was possible to decrease the ORP value to -412 m V after 60 min of treatment.
On the contrary, voltage difference of OV did not decrease. the ORP value and maintained
constant throughout the electro-reduction treatment. This decline in the ORP can be
explained by the fact that the operating principle of electro-reduction is to generate the 34
34
electrons needed to reduce electro-active species. This is emphasized by using a 'potential
difference between the electrodes, which speeds . up the transfer of electrons between
electrodes (Tallec, 1985). It appears from these results that the greater the anode/cathode
voltage difference applied the greater the number of electrons generated. These results were
. in accordance with those of Bolduc et al. , (2006a) who observed that a 2V treatment was
not enough to reach the minimum ORP value. As the anode/cathode voltage difference
increases, electrons are transferred more rapidly from the cathode to the reducible species
of milk. As a result, reduction reactions ~re taking place at a faster rate, which cause a
faster decrease of the ORP value Bolduc et al., (2006a).
150 l
100.La~""~N+~~~~~~~~~~~~~~~~ 50 Î .
o _ -50 ~
E -100 ~ -; -150 o -200 ~ -250
[d.' ... '4V
li! • - - .. - . Ov
" -300 -350 -400 -450
• 'ij_ .. .... rj ..
Il il ll ••••
••••••••••••••••• •••••••••••••••••••••••••••••• -500 -l---------,-- ---,---- --,------.----..,-------,
o l'V o
VJ o
Time (min)
(Jl o
0) o
Fig 2.3) Changes in the redox potential (ORP) during treatment of milk samples at 4V and OV. ·
35
35
----------~-------------------------------------------------------- --- -- -
'3.1.2 Effect on Dissolved oxygen (DO):
Based on the results of the repeated measure analysis of variance, the anode/cathode
voltage difference (P<O.0023) and the dual interaction time/voltage difference (P<O.OOOl)
had a significant effect on the evolution of the dissolved oxygen during the electro
reduction treatment applying the voltage difference of 4V (figure 2.4).
Although the DO increased in the first few seconds, a decrease was seen in the DO value of
the emulsion treated at 4V while the DO concentration at OV remained stable throughout
the treatment as shown in Figure 2.4. The increase of the DO concentration may be due to
the ,air that has remained in the tubes of the electrolysis cell. When the emulsion circulates
through the cell compartments via the tubes it may be exposed to the remaining air and
therefore an increase of the oxyge~ concentration of the emulsion can be caused.
6
~. 5 ~ •• ~.-.
, .. • E ~ ..... .
.e: 4 ••••• .
F4 ~
ci r'~~"""'" .............. . :.. . .... : ......... : .. IIIIII.::-•••• :::~::::::::::
o l'V o
VJ o
lime (min)
(]'I o
m o
Fig 2.4) Changes in the dissolved oxygen concentration during treatment of milk samples at 4V and OVe
The DO concentration was brought down from an averaged initial value of 3.8 mg/L to 2.5
mg/L during the course of the electro-reduction treatment at 4V. The decrease in the
concentration of oxygen is directly related to the reduction reac~ions taking place at the
cathode; ~ 02 + 2 H+ + 2 e- --* H20 (Tallec, 1985). Electrons are transferred from the
electric circuit to' the cathode and then to the emulsion alike milk in which they are
accepted by active species, one of them being oxygen. The protons necessary to this
reaction would be provided by sulphuric acid from the anodic compartment which migrates
36
through the cationic membrane to the milk cathodic compartment. These migrated protons
would be consumed by dissolved oxygen to form water or they would simply be reduced in
dihydrogen according to the foUowing equation,
'li02 + ·2H+ + 2e- ~ H20
2H20 + 2e- ~ H2 + 2 OH- (Tallec, 1985).
These results were in accordance with those reported for pasteurized skim milk by ·Bolduc
et al. , (2006a) and Schreyer et al. (2008, IFSET).
3.1.3 Effeet on pH:
6.80
6.75 1- -. - 4V 1 - - ,+- - 'OV
6.70
=a 6.65 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ••••• ~ •••••••••••••••• eeeeee •••••• e ...
••••• ••••••••••••••••••••••••••••••• 6.60
6.55
6.50 0 "-l ~ m ())
~ ~ ~ "-l f\.) W W W ~ ~ Ol Ol Ol "-l m 0 ~ . ()) "-l m 0 ~ ())
Time(min)
Fig 2.5) Changes in the pH during treatment of milk samples at 4V and OVe
Figure 2.5 presents the evolution of pH during the course of treatment of 4v and OV. The
mean initial pH value of the oillmilk emulsion in this study was 6.64. When milk was
treated at 4 V, the pH decreased 0.2 pH units to reach 6.62 although the pH of the emulsion
remained constant at OV throughout the treatment. The repeated measure analysis of
variance of the data showed that the anode/cathode voltage difference does not have a
significant effect on the pH evolution during electro-reduction treatment (P>0.1084).
The slight decrease of pH at 4V could be due the presence of protons in the cathod section
which had migrated through the catonic membrane from the anod compartement. The same
results were observed by Bolduc et al., (2006a) which the pH decreased 0.2 and 0.15 units
37
37
for electro-reduction treatment carried-out at anode/cathode voltage difference of 2 and 4 V
respecti vel y .
~.1.4 Effeet on Conduetivity:
According to the repeated measure analysis of variance, there was not a significant
differences between the changes of conductivity in aH treatments (P>0.2058). ·
Figure 2.6 shows the evolution of conductivity measurements during electro-reduction at
4V and at OVe The mean initial value of conductivity of the pasteurized milk used in this
study was 4493 ~S/cm which remained constant throughout the 60 minutes of treatments at
4V and OVe
The results suggest that the electro-reduction treatment did not change the electrolyte
composition of the emulsion, and only r~dox state of the electro-active species present were
changed which is in accordance with Schreyer (2007, PhD thesis) showing that the electro
reduction treatment has only an impact on the redox state of electro-active ·species of milk
.without causing modifications in the physico-chemical composition of pasteuri~ed skim
milk.
~~j ·· · .··-4V 1 .. .•... av
4500 •. .. . : .-: -- .' " :.''-'-:.-.-, .. ' , ....... , .•.. ' ...... , . ' .. , '.'. . •...•.......•.............. ' .... .
....... .... ...... ........ ..... ......... ...... .. .1 4400
4350
lime (min)
Fig 2.6) Changes in the eonduetivity of milk samples during treatment at 4V and OVe
38
- - - --- -- -- ------ - - - - - - -
38
3.1.5 Effect on Current Intensity:
130~--------------------------------------~
<' 110 .s ~ 'c;; t: (1,)
.Ë 90
Il - • - • -. - • - .... -. - •
o l'V
.. -.- ...... - .. .-.-.-.-.-11
Time (min)
l'V o
0J ~ o ·· 0
01 o
()) o
Fig 2.7) Changes in the current intensity during treatment of milk samples at 4V.
During electrolysis, monitoring of current intensity makes it possible to deterinine the
progress of the reactions taking place at the electrolysis cell (Gardais, 1990). The intensity
measured at the start of electro-reduction treatment reflects the amount of electro-active
substances present in the milk that are likely to become reduced during electro-reduction in
response to the electrolysis potential applied. The flow of electrons transferring to
electroactive species in milk increases as a function of the voltage applied between the
electrodes. Mean values of current intensity measured during milk electro-reduct~on at 4V
varied between 120 and 110 mA (Figure 2.7). The values measured for milk are in
accordance with those of Bazinet et al. (2009). This value remained almost constant during
the treatment. The small amount of decrease-of the current intensity at the end of treatment
may be due to the decrease in the concentration of reducible species of milk.
Bolduc et al., (2006a) also concluded that since the voltage applied between both electrodes
remains constant throughout the electro-reduction treatment, the increase in the global
resistance of the system might also cause the decrease of current intensity (U = RI).
The rise in resistance can be caused by a slight fouling on the membrane which is due to
protein coagulation on the surface of the membrane. These authors concluded that the
acidic pH at the surface of the membrane causes the coagulation of the proteins present in
milk. Bazinetet al (2009) showed that by increasing the flow rate of milk within the
39
39
,electric cell throughout the electro-reduction treatment, fouling at the membrane could be
minimized. Of course, the CUITent intensity remained zero during the treatment of OV.
3.2 Storage of electro-reduced oillmilk emulsion
3.2.1 Eff~ct of storage on r~dox poténtial (ORP)
According to the repeated measure analysis of variance, there was a significant effect of
anode/cathode voltage difference (P<O.OOO 1) and of the double interaction time/voltage
(P<O.OOOl) on the ORP evolution during storage of electro-reduced samples.
The non-electro-reduced control samples and the electro-reduced samples at av presented
the same evolution of ORP during the storage of 14 days, after an increase in the first day
of storage in all conditions their ORP value remained fairly constant throughout the storage
period (Figure 2.8 Band C). Samples that were treated at 0 V maintained a positive ORP
value, with a starting value similar to the values of control samples.
Electro-reduced samples at 4V had initial negative values and although a linear increase
was seen for the samples with 10% and 50% at 4°C and 20°C, all samples maintained their
negative values throughout the 14 day storage periode In the case . of electro-reduced
samples at 4 V the storage factor of headspace of the sample jars had a significant effect on
the redox value after 14 days of storage.As shown on fig. 2.7 A samples with more amount
of headspace had a higher redox value compared to samples with less headspace in their
sample jars. Whereas, samples with 50% headspace had higher redox than samples with
10% headspace and samples with no headspace had the lowest redox values throughout the
storage periode On the contrary, the storage factor of temperature did not seem to have a
significant effecton the redox value of the electro-reduced samples. As shown in fig.(2.8
A) the samples with the similar headspace had similar redox values whether they were
placed in 20°C or 4°C throughout the 14 day storage.
In the case of electro-reduced samples at OV and control samples (fig. 2.8 Band C) the
storage factor of temperature had an important effect on the redox value during the storage
period whereas samples stored in the same temperature had similar redox values. Aiso it
was seen that samples stored in 20°C had higher redox values than the redox values of
samples stored at 4°C. 40
40
The increase in the ORP value of the different electro-reduced milk products reflects the
instabili.ty or reversibility of the changes in redox state of sorne milk species during electro
reduction. l1he main electrochemically active species that determine the redox potential of
milk are Qxygen, ascorbate and riboflavin, found in low concentrations in milk. However,
exposed thiols and Maillard reaction conjugates produced by thermal process could affec~
the redox potential as weIl. According to Jacob (1970), dairy products profit from a
capacity to buffering their ORP, it means that · they are capable to counteract with any
variation in their ORP.
Reoxidation may occur because the compounds that are electro-reduced .during electrolysis
consume the dissolved oxygen ~till present in the mille These results are consistent with
those obtained by Bolduc et al., (2006a) and Schreyer et al., (2008) for pasteurized 2% fat
milk using an identical electro-reduction system. Although in th~se previous studies,
. electro-reduced milk samples were stored in polypropylene jars and therefore the ORP
values of these treated milk increased during storage to reach their initial after less than 7
days of storage. AIso, the milk samples were opened daily for taking measurements.
The exposition to oxygen could be responsible in part for the increase in ORP values of
these samples. In our study glass jars were used instead of polypropylene jars and contained
sodium azide as weIl as an anaerobic atmosphere was used until the last day of storage
which allowed the elimination of the oxygen effect on the increase in ORP. In the absence
of oxygen, the ORP of treated samples maintained stable during the 14 days of storage.
41
41
o -50 -
-100
-150
>' -200 E '-' -250 ~ a -300
-350
-400
-450
-500
---~. _ .. -.,-
- - - - .. - - - - - - - - - - - - - - - - - - -=-~ ~ .-_-: .. _ .. _ .. _ .. _ .. _._.-.-~ ._._._.-'_._.-.t/;.-- - ~-- --- -- à --
Il _. _.
(.: :-::: -.. -.. -.-.-:-. - _ ... . .. --' _. -' -' -' -.-..... -... - - _ ..... -_. _ .. - --- - - _ ... - _ .. . ...• ...... • .. ... . .. . ... • ....
....... . . .. . • .......................................... •
o 2 4 · 6 8 10 12 14
~ '-'
~ 0
··· .. ·- 0%: 4°C
~. '_~: :}OCY~ .. :._ ~~~f. __
Time (days) ···. ·- 0%: 20°C
. ~.~Q~~ .. : ,!~C - -.- - 10%: 4°C :.:_!=._~_. ~oyo _:_ 29~C
A. Redox changes of electro-reduced milk samples at 4V during storage
180
160
140 .. ,: ;;, ... .. .:; .
1 , ,'1
120 l " .. , I .~ './
("
100 - 1,1
r ;; l '
/, '
" 1
80 !. /. /il
60 0 2 4
··· .. ·- 0%: 40C
···. ··- 0%: 200C
- -.- - 10%: 40C
- .• . - 10%: 200C
--0- 50%: 40C
."':..:......... - ... .. 50%: 200C . . :.:~ ''''''~ ....... ...,_.---..-
- - - - - - - - - - ~ '~ '''::'': ::..:.~._- ~ --. ""' -:-' -::- '-: .--: ::-. -:-. :-. :-.--
6 8 10 12
Time (days) 14
B. Redox changes of electro-reduced milk samples at OV during storage.
42
42
180 -
160
>' 140
S ~ 120
o
60 . - -
o 2 4 .
- --- .- ....
6 8 T ime (days)
10
··· .. ·· 0% : 40C
.. ·. · · 0%: 200C
- -.- - 10%: 40C
- .• . - 10%: 200C
50%: 40C
. 50%: 200C
12
c. Redox changes of control milk samples during storage.
14
Fig 2.8) Changes in the redox potential value during storage of milk samples. Where 4°C and 20°C= ternperature of storage, 0%, 100/0, 50%= headspace of sarnple jars
43
43
3.2.2 Effect of storage on dissolved oxygen concentration (DO)
Figure 2.9 presents the changes in dissolved oxygen concentration in the electro-reduced
samples at 4V, samples electro-reduced at OV and control untreated samples during storage
of 14 days. The repeated measure ' analysis of variance showed a significant effect of
anode/cathode voltage difference (P<O.0091) and of time (P<O.OOQ1) . on the dissolv~d
oxygen evolution during storage. In all cases · a decrease ' was observed at the end of the
14 days of storag~ which shows a consumption of the dissolved oxygen still present in the
milk samples. AIso, interactions of the storage factors of headspace and storage temperature
seem to have an impact on the value of DO as seen in fig.2.9.
One reason that can cause the decrease of DO value of milk samples involves the
autoxidation of unsaturated fatty acid which may consume the dissolved oxygen present in
the sample jars. Autoxidation reactions which take place via free radical ,reactions may be
initiated by hydroperoxide decomposition, metal catalysis such as iron or copper, exposure
to light and oxygen. Even though the concentration of lipids in skim milk is ,very low, the
main fat molecules present in skim milkare phospholipids, which .are very sensitive to the
presence of oxygen. AIso, in the emulsion prepared canola oil was used which
approximately co~tains 32% poly unsaturated fats, and approximately 61 % mono
unsaturated fats which are unstable in reacting with oxygen and light.
Another possible reason which causes the decrease of DO value may be due to the growth
of psychrotrophic bacteria. These microorganisms . could have consumed the oxygen present
in the milk during their growth, decreasing its concentration to low values near zero after
14 days. The contaminations might have been introduced from contact with the electro
reduction unit and/or from ambient air and materials during measurements. Although
sodium azide was used to eliminate bacteria in this study the enzymes released during the
destruction of microflora may have remained active and the y could have had a role in the
consumption of remaining oxygen.
44
44
5
4.5
4
0.5 o 2 4 6 8
Time (days) 10
· · · .. ·· 0%: 40 e ·· ·. · · 0%: 200e - -...- 10%: 200 e - . • . - 10%: 200 e
50%: 40 e
- . ._ . 50%: 200 e
12 14
A. Dissolved oxygen changes of electro-reduced milk samples at 4V during storage
5
4.5
1.5
0.5 --- ._._ .. ~-_.
o 2
· ·· .. ·· 0%: 40 e · · ·. - · · 0%: 200 e
- -... - 10%: 40 e - .• . - 10%: 200 e -0-- 50%: 40 e - -__ - 50%: 200 e
. = ~. "::.. ":".-::.. -:- .-- -: .-- -:: -:--- . -: -- . .• . -: ~ .-: - - - - - - - - - - - - - - - - - - - -
::-: ::: ::-. ~ ::" ~':!:_'---" .. -.. -.-.-' - . - . - . ~ - -- ._-. -_ ._._ ---- -- -- ---- -- -_._._._.-
- ::::~ : ~::: ~~: ~:::: ~:: ~: ~:~ ~: ~::: _::: ::_- -- ---
4 6 8 10 12 Time (days)
B. Dissolved oxygen changes of electro-reduced milk samples at OV during
storage.
14
45
45
5.0
4 .5 ~
4.0
3.5
E S
3 .0
8 2.5
2.0 ··· .. · - 0%: 40C ···. ··- 0%: 200C
1.5 - -.- - 10% : 40C - .• . - 10%: 200 e
1.0 -0-50%: 40C _ . ..- . 50%: 200 e
0.5 -
0 2 4 6 8 la 12 14 Time (days )
c. Dissolved oxygen changes of control milk samples during storage.
Fig 2.9) Changes in the dissolved oxygen value during storage of milk samples. Where 4°C and 20°C= temperature of storage, 0%, 10%, 50%= headspace of sample jars
46
46
3.2.3 Effect ofsto~age on pH
The repeated measure analysis of variance of the data showed that the anode/cathode
voltage difference has no effect on the pH (P>0.1063) while time (P<O.OOOl) had a
significant effect on the pH evolution during electro-reduction treatment.
The mean initial pH value of the pasteurized milk used in ~his study was 6.63 which is in
agreement with pH 6.6-6.8 reported in other articles (Amiot et al, 2002, White and Davies,
1958). The slight variations seen in the results (Figure 2.10) were similar to those observed
by Schreyer et al., ( 2008, IFSET) where the variations of pH ranged from -0.10 to +0.05
pH unit. These slight variations in pH level are indicative of the absence of bacterial
contamination in the stored milk products. However, after 4 days of storage a significant
d~crease was seen in the pH" value of electro-reduced samples and control samples which
may be due to the growth of bacteria in the milk sample~ during storage.
The contaminations might have been introduced during the preparation of the emulsions or
by contact to the electro-reduction unit and/or from ambient air. Perhaps the sodium azide
added in this study to the milk emulsions was not sufficient to eliminate bacteria
throughout the storage period of 14 days.
47
47
:a
:a
6.7
!II 6.65 .. ~ .
6.6 . ~.
~:
6.55
6.5
6.45
6.4
6.35
6.3
6.25 0
." .
2 4 6 8 Time (days)
- - .• - - 0%: 40C
- - -. - - 0%: 200C
-.....- - 10%: 40C
- ' 0 - - 10%: 20°C
50%: 40C
- -._ . 50%: 20°C
10 12
A. pH changes of electro-reduced milk samples at 4V during storage
6.7
"'. -' 0%: 4°C 6.65
···. ·- 0%: 20°C
6.6 -.....-- 10% : 4°C
6.55 --;;·- 10% : 20°C
6.5 --9-- 50% : 4°C
_ . .- . 50% : 20°C 6.45
6.4
6.35
6.3
6.25 ---
0 2 4 6 8 Time (days)
10 12
B. pH changes of electro-reduced milk samples at OV during storage.
48
14
14
48
49
6.7 • . ' , \ . , ' . " 0%: 40C
6.65 ~ ' .'\ " \ , , ... ,. 0%: 20°C
6.6 - -.- - 10%: 4°C
6.55 - .• ' - 10% : 20°C
6.5 50%: 4°C :a 6.45 , 50%: 20°C
6.4 _ .. :-.. ..:.. '" -=- ... ~~ a-: _ _ .. -r:"~:;-""' '''' .''' ''' '''
6.35 .. _ ... - .. _._ ... .-:..:. ~. :_:..: ::-... .. - .:..- - - - .:.. -- -~':.~: ~#'---_ .. -
6.3
6 .25 -0 2 4 6 8 10 12 14
Time (days)
C. pH changes of control milk samples during storage
Fig 2.10. Changes in pH during storage of milk samples. Where 4°C and 20°C= temperature of storage, 0%, 10%, 50%= headspace of sample jars
49
Conclusion
The electro-reduction treatment reduced the redox potential quickly and decreased the
dissolved oxygen concentration of the oil/water emulsion samples studied, without causing
any major changes in their pH. Although samples electro-reduced at 4V maintained their t
negative redox values throughout the 14 day storage period a significant increase was seen
at the end of storage period. The increase in the redox value may be due to the buffering
capacity of dairy products which causes the changes in the redox state of the milk species .
during electro-reduction (Jacob, 1970). The rise in ORP during storage confirms the
instability and reversibility of the electro-active species of milk which may be the result of
the re-oxidation of electro-reduced species of milk. The decline of DO during treatment is
due to electro-reduction of oxygen at the cathode. The decrease of the DO for aU milk . .
. samples during storage shows the consumption of the dissolved oxygen concentration in
the samples which can be a result of the oxidation of polyunsaturated fatty acids.in milk, re
oxidation of electro-reduced species and/or growth ofaerobic micro-organisms.
During storage, the storage factors of headspace and temperature showed to have an
important impact on the ORP value as weU a~ the DO value of aIl samples whether electro
reduced or not The· factor : of headspace in the electro-reduced samples at 4V showed that
when the amount of oxygen present is higher (headspace of 50%), the ORP value niaches
higher values more quickly than when the amount of oxygen present is lower (headspace of
10% and 0%). The dual effect of storage temperature and headspace had an important
effect on the decrease in the DO value as weIL
These results indicate that when electro-reduced samples are kept in optimal storag~
conditions in regards to storage temperature and the absence of oxygen (or when oxygen is
limited), the low values of ORP may be maintained for a longer period. This matter may be
helpful for preventing or controlling many of the unwanted oxidation -reduction .reactions
that take place in milk especially lipid oxidation reactions.
50
50
Chapter 3
Lipid stability of electro-reduced omega-3 enriched commercial milk during storage
51
51
Abstract
Consumer demand for specific nutritional qualities is encouraging the dairy industry to
develop products supplemented in omega-3 fatty acids .- Although these fatty acids are
known to have many health benefits, they . are extremely susceptible to oxidative
deterioration which causes difficulties during their storage. Methods to prevent or reduce
the oxidation phenomena in milk are continuously being put forward. In this study an
. electro-reduction process was performed to modify the redox state of omega-3 enriched
milk. A 4V electro-reduction treatment was applied ' for 1 hour on pasteurized omega-3
enriched milk, at room temperature. Parameters such as redox potential, dissolved oxygen,
pH and conductivity were measured during the course of treatment. Significant decrease of
redox potential and dissolved oxygen was observed. The electro-reduced and control
samples were stored at room temperature for up to 3 weeks in the presence of fluorescent
light and in dark. The analysis showed that while the dissolved oxygen continued to
decrease for aU samples throughout storage, the low values of redox potential for electro
reduced milk were not stable, and reached positive values after two weeks. During the
storage perjod the composition of the fatty acids of electro ... reduced milk was measured at
intervals of one week by Gas Chromatography. The results showed that a significant
change was not seen in the fatty acid composition of the samples placed in dark, which
iridicates that the decrease of their DO was not only due to lipid oxidation. On the contrary,
storage under fluorescent light involved a degradation of the fatty-acids , whereas the
electro-reduction treatment slowed down the oxidative degradation of electro-reduced
samples in comparison to untreated milk samples. Results of this study show that the
electro-reduction treatment can be a potential method of enhancing the shelf-life of
products containing unsaturated fatty acids.
1. Introduction
Consumer demand for food that is natural and has higher nutritional values has encouraged
the food industries to produce products with such qualifications. Functional foods that
contain such needs promise to deliver health and wellness to consumers while having a
tasty formulation as weIl. Milk is a natural, multi-:component, nutrient-rich beverage. With
these characteristics, milk is an ideal delivery system for newly discovered food ingredients
targeting modem lifestyle diseases. Interest in the omega-3 fatty acids as health-promoting
nutrients has expanded dramatically in the recent. years. The health benefits of these fatty
acids 'are known for . the human body and their nutritional and therapeutical effects for the
prevention of diseases such as cardiovascular disease, inflammatory disease, brain function
and mental health have been shown. Dietary sources of omega-3 fatty acids include plants
(particularly flax, canola, walnuts and hemp) and fish (particularly ocean fish such as
sardines, anchovies, salmon and mackerel). Plants contain the parent omega-3 , alpha
linolenic acid (ALA), which can be converted into eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). About 58% of the total acid composition of flaxseed oil
contains alpha-linolenic acid (ALA; C 18:3). Many assessments have shown that most
western . populations' consumption of omega-3 fatty acids is much less than the amount
. considered nutritionally desirable. Efforts to supplement foods with omega-3 fatty acids has
been made and these fatty acids have been incorporated into a range of food products which
one of the most favorable and most consumed has been in milk products
(Sharma et al., 2005). The problem with omega-3 fatty acids is that they are
polyunsaturated fatty acids (PUF A) which makes them extremely susceptible to oxidative
deterioration durihg their process (heating) as weIl as during their storage in the presence of
light and oxygene
Oxidative degradation of polyunsaturated fatty acids (PUF A) takes place under 2 main
mechanisms; the auto-oxidation and the photo-oxidation. The auto oxidation of lipids in
milk is usually initiated by a removal of hydrogen from the methylene group attached to
double -bonds. The auto-oxidation of unsaturated fatty acids causes chain effects which
result in the decomposition of hydro peroxides and consequently a wide range of carbonyl
products are produced. These products contribute to the off flavors in dairy products.
53
53
54
Milk is .a complex biological system containing many factors, which may act as
antioxidants and/or pro-oxidants. The relative amounts of these factors in milk as weIl as
processing and storaging conditions have been shown to influence the rate and extent of
lipid oxidation in milk. These factors include oxygen, light, storage temperature, metals,
enzymes and water activity (O 'Conner et al, 2006).
Several techniques and . methods have been used to prevent oxidative deterioration of milk
and other dairy products, such as selection of packaging materials with low oxygen
permeability and light transmis'sion (Moyssiadi et al. , 2004); vacuum qr inert gas packaging
to reduce oxygen concentration (Mortensen et al. , 2004) and . application of natural
antioxidants for stabilizing polyunsaturated fatty acids (Boyd, 2001). An electrochemical
method--electro-reduction-has also been put forward as a mean .of inhibiting and/or
reducing oxidation phenomena in milk (Swanson et Sonvner, 1940b; Inoue & Kato, 2003 ;
Bolduc et al. , 2006a) This process uses electrolysis (supply of electrons from the cathode)
to reduce electro-chemically active species, causing a decrease in the redox potential of the
product treated.
The objective of this study was to verify if the decrease in the redox potential due to the
electro-reduction treatment . improves the oxidative stability of the lipids in omega-3
enriched milk during storage by determining the factors of storage which affect the
oxidative stability of the lipids ofmilk, especially the omega-3 fattyacid.
2. Materials and Methods
2.1 Material
Milk enriched with omega-3 fatty acids
The electro-reduction treatmentswere carried out on co,nmercially available pasteurized
partly skim milk enriched with omega-3 fatty acids (1 % fat milk enriched with flaxseed oil,
Natrel, Québec, Canada).
54
2.2 Electro-reduction system
H 20. ~02 +2H+ +2e
Electrolyte
•
+ Eleclrical Source -
Treated Milk
•
li °2 +2H+ ~ H 20
H 20+2e- ~20H- + H 2
Milk to be treated •
Figure 3.1 Simplified diagram of the electrolysis cell. (Source: Bolduc et al. , 2006a)
55
The system used was a Microflow type electrodialysis cell with a membrane electrolysis
configuration (ElectroCell AB, Karlskoga, Sweden). The cell was separated in two different
compartments by a cationic membrane (CMX-SB, Tokuyama Soda Corp, Tokyo, Japan).
" In each compartment, one polypropylene spacer (2.02 mm thick)" was placed to allow the
" liquid to flow through and to have contact with a 10 cm2 surface of both the membrane and
the corresponding electrode. On one side of the membrane, the milk was in contact with a
food-grade stainless steel cathode and on the other side of the membrane, the electrolyte
(0.1 H2S04 solution) was in contact with a dimensionally stable electrode (DSA-02). The
assembly was made watertight with rubber gaskets (1.23 mm thick), placed next to e"ach of
the electrodes, spacers and membrane. Each cell compartment was connected to their own
external tanks (300 mL for the milk and .309 mL for the acid solution) to allow a continuous
circulation during each treatment. Both solutions were circulated by two centrifugaI pumps
(Iwaki Magnet Pump, Iwaki Co, Ltd., Tokyo, Japan), and their flows controlled by flow
55
56
meters were kept ai' 300 mL/min (Aalborg Instruments and Controls, Inc., Orangeburg,
USA). This arrangement has the same configuration as that used by Bolduc et al. , 2006a.
A DC current of 4V between the two electrodes was supplied by an electrical power supply
(Model · HPD 30-10, Xantrex, Bumaby, Canada). The treatment was caITied out in
triplicates. During each 60 min treatment, oxidation-reduction potential (ORP), dissolved
oxygen (DO), pH and conductivity of milk were recorded as well as the CUITent intensity at
intervals of 1 min during the first 10 minutes and at intervals of 5 minutes thereafter.
2.3 Storage
Part one:
The electro-reduction treatment was done in three repetitions and after each electro
reduction treatment, samples of 35 mL of ORP modulated milk were rapidly poured in
tinted glass jars of 70 mL volume (50% headspace in each sample) and stored at 20°C for 3
weeks in darkness. While other samples were placed in the presence of fluorescent light
(fluorescent lamp Coolwhite, 60 watts, #F48T12WWHO) at 20°C for the same storage
time. These samples were placed in glass jars that were hot tinted.
At intervals of one week the oxidation-reduction potential (ORP), dissolved oxygen (DO),
pH and the fatty acid composition of aIl milk samples were recorded whereas for each
storage time, analyses were performed on different jars of samples in order to pre vent
sample and headspace contamination.
Pari Iwo:
After each electro-reduction treatment, samples of 70 mL of ORP modulated milk were
rapidly poured in tinted glass jars of the same volume and stored at 20°C for 6 ·weeks in
paraIlel with control non electro-reduced milk. The headspace in each jar was minimized
by overfilling the containers. Tinted glass jars were used in this study to limit the influence
of light on oxidation reactions in stored samples. Sodium azide wasadded to aIl modulated
samples in order to prevent microbial growth in the samples during stprage.
56
2.4. Analytical methods
2.4.1 Oxidation-reduction potential measurement:
The ORP .was measured using a VWR Symphony platinum electrode CyWR Scientific
Products, West Chester, PA, USA) with an internaI. Ag/AgCI reference electrode and filled
with the recommended solution containing KCI and AgCl. · This electrode was connected to
a VWR Symphony portable SP20 pH/ISE meter. The ele~trode reading was verified with a
solution of potassium ferrocyanide and potassium ferricyanide having an ORP of +234m V.
2.4.2 Dissolved oxygen (DO) measurement:
The DO was measured using a VWR Symphony electrode (VWR Scientific Products)
mounted with the specified membrane and fiJled with the supplied DO electrolyte solution.
The electrode was connected to a VWR Symphony SP50D portable DO meter. Th,e
electrode was calibrated every 2 h as described in the supplier' s manual.
2.4.3Conductivity measurement:
The conductivity was measured with an immersible YSI probe (model 3417, K) 1 cm-l ,
Yellow Springs Instrument, Yellow Springs, OH) connected to an YSI 3232 adaptor to
aUow readings on the YSI 3100 conductivity meter of the same manufacturer.
Since the conductivity varied proportionally with temperature and the values were not
automaticaUy compensated by the conductivity meter, aU readings were corrected to 25 oC
using the method described by Bazinet et al., (2004).
2.4.4 pH measurement:
The pH was measured using aVWR Symphony electrode (VWR Scientific Products)
equipped with an automatic temperature compensation device and connected to a yWR
Symphony SR601C benchtop pH meter.
57
57
2.4.5 Current intensity measurement:
The CUITent passing through the electrodes was read from a Mastercraft numerical
multimeter (Model 52-0060-2, Mastercraft, Toronto, Canada).
2.4.6 Fatty acid composition measurement:
2.4.6.1 Milk fat extraction
Milk fat extraction was done according to Wolff using hexane/isopropanol (3 :2, vol/vol)
with sorne minor modifications. A representive sample (lOg) was dispersed in isoproponol
(50mL). and after addition of hexane (75mL); a second dispersion was carried OUt. The ·
suspens"ion was filtered, transferred into a separatory funnel, and washed with an aqueous
solution of sodium chloride (5% wt/vol, 2>< 100mL). The organic phase was dried over
anhydrous sodium sulfate, filtered, and the solvents were in a rotary evaporator at 45°C
under vacuum. The lipid extract was stored at -18°C until removed for use. This method
was the same as the method used by Destaillats et al., (2003).
2.4.6.2 Fame methylation
Methylation of milk fatty acids (20mg in 2rnL hexane) was carried out in a sealed tube, with
0.4N sodium methoxide (0.5mL). After hornogenization, the mixture was held at 40°C for
15 min, cooled to room temperature, washed with water (lmL; vortex for 5s ,allowed to
stand 1min), and fatty acid methyl esters (FAME) were extracted with hexane
(3 x 1 mL). The organic extracts were combined, dri'ed over anhydrous sulfate, filtered and
kept under N2 in closed vials at -18°c ~ntil use.
2.4.6.3 Fatty acid analysis
Analysis of the methyl esters were performed with agas chrornatograph (Model 5890
Series II; Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector. 1.0
Jll of sarnples were injected on a DB-225 capillary column (J&W, Folsom, CA; 30m x
0.25 mm x 0.25 Jlm). The carrier gas was hydrogen, with a linear velocity of 35.5 cm/s at
50°C. The injector and detector temperatures were maintained, respectively, at 215 and 230
oC. The initial oven temperature was 35 oC and increase~ at a rate of 10°C/min to 190 oC
58
58
and held for 3 minutes, and then programmed at a rate of 3 oC/min to 200 oC and held for
13 minutes, for a total mn time of 30 minutes. Correction factors were determined-by
analysis of a standard mixture of fatty acid methyl esters (GLC Reference Standards,
Catalogue No. GLC-60 and GLC-506; Nu-Chek-Prep, Elysian, MN).
2.5. Statistical analysis
Data obtained during treatments and storage was subjected to multivariate analysis of
variance using JMP iN software (Version 5.1, SAS Institute inc., Cary, NC).
3. Results and Discussion
3.1 Electro-reduction treatment of omega-3 enriched milk
3.1.1 Effect on redox potential (ORP):
>' .s Cü :t: c: Cl) .... 0 a.. >< 0 "C Q)
0:::
300
200
100
0
-100
-200
-300
-400
-500
0 1 2 3 4 5 6 7 8 9 10 15 20 25 30 3540 45 50 55 60
Time (min)
Fig 3.2. Changes in the redox potential (ORP) during treatment of omega-3 enriched milk at 4V.
In this experiment, the mean initial ORP value for the pasteurized omega-3 enriched milk 1
was + 198m V. A similar mean value was observed (+ 130m V) for pasteurized skim milk in a
recent study (Bazinet et al., 2009). AlI treatments performed on milk samples-resultèd in a
decrease (P < 0.0001) in their ORP values as can be seen on Figure 3.2.
- 59
59
1t should also be noted that the decrease- in the ORP during electro-reduction is inversely
exponential: 88% of the decrease occurs in the first 10 mip.utes, after vyhich the ORP tends
to stabilize (Figure 3.2).
This decline in the ORP can be explained by the fact that the operating principle of electro
reduction is to generate the electrons needed to reduce electro-active species. This is ~
emphasized by using a potential difference between the electrodes, which -speeds up the
transfer of electrons' between electrodes (Tallec, 1985). Therefore, the ORP tends to ..
decline, since it represents the relationship between a standard redox potential and the
concentration ratio of oxidizers and reducers (Morris, 2000). Aiso the denaturation of sorne
whey proteins after pasteurization rnay have sorne effect on the rnilk ORP value during electro
redu~tion (Amiot et al., 2002).
3.1.2 Effect on Dissolved oxygen (DO)
8 ~-----------------------------------------~
7
'[ 6 a. c:: 5 <1> Cl
~ 4 o -g 3 > g 2 fi)
C 1
o 1 2 3 4 . 5 6 7 8 9 10 15 20 25 30 35 40 45 50 55 60 lime (min)
Fig 3.3. Changes in the dissolved oxygen concentration during treatment of omega-3 enriched milk at 4V.
Figure 3.3 shows the evolution of DO in milk during the electro-reduction treatment
performed. The mean initial value of DO in the milk used in this experiment was 7.1 ppm.
However, the DO of milk is linked to the handling of the milk: agitation during the
different processing and distribution stages incorporates air, increasing the dissolved
oxygen concentration. The initial value was brought down to 1.6 ppm during the course of
60
60
the electro-reduction treatments at 4V. Therefore, the treatment caused a significant linear
decrease in DO of the milk samples as shown in Figure 3.3.
The decrease in the concentration of oxygen is due to its reduction" that takes place at the
cathode (Yz02 + 2H+ + 2e- ~ H20). Electrons are transferred from the electric circuit to
the cathode and then to electro-active species in milk, which one of the most active is
oxygen (Tallec, 1985).As electrons are transferred throughout the treatment, the
concentration of oxygen decreases. It is difficult t6 establish a link between ORP and DO;
however since oxygen is an electrochemically active compound its reduction should
contribute 'to a decrease in the ORP.
3.1.3 Effeet on pH:
Figure 3.4 presents the evolution of pH during the course of treatment. The mean initial pH
value of the pasteurized milk used in this study was 6.73. When milk was treated at 4 V, the
pH did not show a significant decrease and only decreased 0.04 pH units to reach 6.69 at
the end of the treatment.
The very slight decrease of pH duringthe electro-reduction process can be explained by the
migration of protons towards the cathode compartment. The cationic membrane which is
permeable to protons, allows them to 'migrate through the membrane toward the cathode
compartment from the aqueous anolyte H2S04 whereas simultaneously OH- ions are
produced. Consequently, the migration of H+ cations was responsible for the slight
decrease of pH during the treatment since there were more H+ ions crossing the membr"ane
than OH- ions being produced at the cathode (Figure 3.4).
61
61
6.8 ,.--------------~-------__,
6.75
6.7
:a 6.65
6.6
6.55
-0 1 2 3 4 5 6 7 8· 9 10 15 20 25 30 35 40 45 50 55 60
lime (min)
Fig 3.4. Changes in the pH during treatment of omega-3 enriched milk at 4V.
3.1.4 Effeet on Conduetivity:
62
According to the repeated rneasure analysis of variance, there was not a significant effect of
time (P>0.4741) on the evolution of conductivity rneasurernents during the electro
reduction treatment. Figure 3.5 shows the evolution of conductivity measurements during
the electro-reduction treatrnent. The conductivity of milk did not change throughout the
treatment at 4V. Since electrical conductivity is known to be a function of the nature and (
concentration of the different electrolytes that are present in a solution, this stable value
during the treatrnent suggests that the electro-reduction treatrnent did not change the
electrolyte composition in ornega-3 enriched rnilk. This is sirnilar to the results obtained by
Schreyer, (2007 ,PhD thesis) concerning that the electro-reduction treatment haS only an
impact on the redox state ofelectro-active species of milk without causing modifications in
the physico-chemical composition of pasteurized skim milk.
62
63
4550
4500
Z' 4450 ">
~ (.)
::l '0 c: 4400 0
~ .. - ~ - ____ -a.~
u
4350
4300
o 2 4 6 8 10 14 18 22 26 30. 34 38 42 · 46 50 54 58 60
Time (min)
Fig 3.5) Changes in the conductivity of omega-3 enriched milk at 4V during treatment
3.1.5 Effect on Current Intensity:
During electrolysis, monitoring of CUITent intensity, makes it possible to determine the
progress of the reaction (Gardais, 1990). The intensity measured at the start of electro
reduction treatment reflects the amount of electro-active substances present in the milk that
are likely to become reduced during electro-reduction in response to the electrolysis
potential applied. The flow ofelectrons transferring to electro-active species in milk
increases as a function of the voltage applied between the electrodes. Mean values · of
.cUITent intensity measured during milk electro-reduction varied between 130 and 100 mA
(Figure 3.6). The values measured for milk are in accordance with those of Bazinet et al. ,
(2007). This value remained almost constant during the treatment. Bolduc et al., (2006a)
also concluded that since the voltage applied between both electrodes remains constant, the
increase in the global resistance of the system might have also cause the decrease of CUITent
intensity (U = RI). The rise in resistance can be caused by a slight fouling on the membrane
which is due to prote in coagulation on the surface of the membrane. Bazinet et al., (2007)
concluded that the acidic pH at the surface of the membrane causes the coagulation Qf the
pro teins present in milk. This phenomenon could be minimized by increasing the flow rate
of the milk within the electric cell throughout the electro-reduction treatment.
63
140~--------------------------------------~
130 ~""'-~~""'-'~-6--6-___ '"
120
;( 110 E - 100 ~ ~ 90
~ 80
70
60
50+-~~~~~~~~~~~~~~~~~--~~
o 1 2 3 4 5 6 7 8 9 10 15 20 25 30 35 40 45 50 55 60
Time(min)
Fig 3.6) Changes in the current intensity of omega-3 enriched milk at 4V during treatment
3.2 Storage of electro-reduc.ed omega-3 enriched milk
64
After the treatment of the omega-3 enriched milk by electro-reduction, the objective was to
verify. the effect of different storage conditions such as temperature, light and/or dark and
headspace of sample on the negative ORP value, the low DO and the stable pH during the
storage period of 3 weeks as weIl as the effect of electro-reduction treatment on the fatty
acid composition.
3.2.1 Effect of storage on redox potential (ORP)
Figure 3.7 represents the evolution of ORP in treated and control milk samples during
3 weeks of storage at 20°C in dark and in the presence of fluorescent light.
This figure shows that theORP value of aIl the treated samples increased significantly
(P<O.OOOl) during storage while the ORP in non treated control samples remained stable
and higher than treated samples throughout the storage period. A significant difference
(P<O.0011) was seen between the ORP values of electro-reduced samples in comparison to
untreated control samples. Although the storage conditions of headspace (P<O.6368) an.d
dark/light (P<O.1489) did nothave an important impact on the ORP values.
The increase in the ORP of aU the electro-reduced milk samples reflects the instability and ·
reversibility of the changes in redox state of sorne milk species during electro-reduction.
64
65
Reoxidation may occur because the compounds that are electro-reduced during electrolysis
consume thedissolved oxygen still present in the milk. According to Jacob (1970), dairy
products could profit from a capacity to buffering their ORP. This capacity is known as the
poising effect of milk species which indicates that milk can easily counteract the changes in
its redox state, for example the changes caused by the electro-reduction treatment.
250
150
> g 50 1e ~
-50 Cl)
& )(
.g -150 Cl)
0:::
-250
-350
0
- ~ --- :,: -: : -~ -. - ----. --- '--- -.- ~ - ._,:_~- = _-:-:: _:_ ~ -:;' ~ _ .. ;:_ :,:,:::-; -:---=-'- : -~- ~-=:-:=: -:-=- ~:- :::: -:-: -::::: -:- : -:-:- :-
lime (weeks) 2
o c ligtt 50% ---- .. ---- c dark50%
• t dark50% ~ tight50% - - 0- _·c darkO% -~-tdarkO% · .
3
Fig 3.7) Changes in the redox potential (ORP) of omega-3 enriched milk saniples during storage. c= control samples, t= treated sa~ples, O%=10% =50% =headspace of sample jars
In previous studies (Bolduc et al., 2006a and Bazinet et al., 2007) where electro-reduced
milk samples were stored in polypropylene jars the. ORP values of the treated milk
increased during storage to reach their initial values after less than 7 days of storage.
Although in this current research the treated samples maintained their negative ORP values
up ta 2 weeks. Maintaining the ' negative ORP values for a longer period of time in this
research may be due to many factors. One factor may be due ta the usage of glass sample
jars under aerobic conditions. This hypothesis was confirmed in a study by Bazinet et al. ,
2007 where the ORP of electro-reduced milk increased of 50% in polypropylene jars after a
24 h storage period, but remained constant in tinted glas~ jars like those used in this study.
Another hypothesis is that in the previous studies of Bolduc et al., (2006a), the milk
samples were opened daily for taking measurements while in this study analyses were
65
66
performed on different jars of samples in order to prevent the exposition to oxygen which
could be responsible for the increase in ORP values of milk samples.
AIso, in the previous studies, stored samples did not contain antibacterial agent like sodium
azide. In a study, Schreyer et al. (2007)observed an important increase in ORP values in
milk containing sodium azide within 24 h storage in polypropylene tubes. This fast rise in
the ORP value of milk could be due to the oxygen perm~ability of the polypropylene tubes.
Figure 3.7 shows that despite the increase of ORP values of treated samples placed in dark
the electro-reduced milk samples stored in the presence of fluorescent light reached the
positive ORP values more rapidly. Therefore; the storage factors applied on the electro
reduced milk samples ·seem to have an important i~pact on the rate of reaching the positive
values. Another storage factor that seems to have. an impact oh the increase of ORP values
even when stored in dark, is the headspace of the sample glass jars. As seen in Figure 3.7,
the increase of the O~ values of electro-reduced milk samples with 50% headspace
(consuming half the volume of glass jar) was faster than the electro-reduced milk samples
with 0% headspace (consuming an the volume of the glass jar). In order to verify the effect
. of headspace on electro-reduced milk, the samples with 0% headspace were placed in dark
for a period of 6 weeks. These samples reached values similar to the untreated milk
samples after 6 weeks storage.
On the contrary, the results showed (Fig 3.7) that neither the storage condition of light nor
dark had an effect on the ORP value of non treated control samples whether placed in glass
jars with 0% headspace or in glass jars with 50% headspace.
66
3.2.2 Effect on dissolved oxygen (DO):
-0- c light 50% --- c dark 50% -<>-- t light 50% --+-- t dark 50%
4
E ~ 3 t: (1) Cl
~ 2 0
"C (1)
> (5 1 .~ Cl
0
0 2 3
Time (weeks)
Fig 3.8) Changes in the dissolved oxygen concentration (DO) of omega-3 milk samples during storage with 50% headspace.
Figure 3.8 illustrates the evolutionof DO in. the ORP modulated milks during storage of
3 weeks in dark and light with 50% headspace. In both the electro-reduced milk samples
and the untreated control samples a decrease of the dissolved oxygen concentration was
observed which was significant for the treated samples (P<0.0005). Whereas, at the end of
the storage period a significant difference was not seen between treated and control milk
samples (P<0.0034) as well as between samples placed in dark or light (P<0.1489).
A decrease in DO concentrations in control milk and treated milk from a mean initial value
of 3.3 ± 0.3 mg/L to 0.8 ± 0.2 and 1.5 ±0.3 mg/L to 0.5± 0.1 respectively was observed
after 3 weeks of storage.
The decreas~ in dissolved oxygen concentrations in milk control samples was also observed
by Bazinet et al., (2007). However, the variations in DO concentrations during storage of
electro-reduced samples was smaller than the values shown in this study, with a mean DO
. concentrations decrease of 0.9 mg/L in comparison to a mean DO concentrations decrease
of 3.2 mg/L. Similar results to this study concerning the decrease in the DO concentrations
of electro-reduced milk samples during storage has been observed (Bolduc et al. , 2006a;
Schreyer et al., 2007, PhD thesis; Bazinet et al., 2009).
67
67
The DO percentage decrease of the control samples observed in this study was
approximately 81 % and 75% for samples placed in light and dark respectively. Whereas,
the DO percentage decrease of the electro-reduced samples was approximately 73% and
40% for samples placed in light and dark respectively.
There are several possible hypotheses or a combination of aU to explain the decrease of the
dissolved oxygen concentration of milk during storage of 3 weeks.
A) One hypothesis for the decrease of the oxygen concentration especially in milk samples
stored in light is due to lipid oxidation. The hydrogens at the a-position of the double bonds
(allylic hydrogens) in the fatty acid chain react with oxygen to form free radicals and
hydroperoxides (Collomb et al., 1996). Light, oxygen and transition metals such as iron or
copper are well-known catalysts of these reactibns. In the presence of light and a photo
s'ensitizer,. singlet oxygen is formed from triplet state oxygen. Pigments such as chlorophyll
and Fiboflavin and heavy metals which aIl naturally occur as minor components in food
have be shown as sensitizers. In these reactions the singlet oxygen generated from triplet
oxygen . is highly reactive and can directly react with unsaturated fatty acids and form
hydroperoxides. In this study, comparing the samples placed in light and dark showed the
effect of light in the rate of the consumption of dissolved oxygen .. These results showed that
the consumption of DO is at the most when samples were placed in the presence of light.
The DO percentage decrease of the control samples observed in this study was
approximately 81 % and 75% for samples placed in light and dark respectively. Whereas,
the DO percentage decrease of the electro-reduced samples was approximately 73 % and
40% for samples placed in light and dark respectively.
B) The significant increase of the ORP value during storage for all electrà-reduced milk
samples caused the assumption that reoxidation takes place on the electro-reduced species
by consuming the dissolved oxygen still present in the milk. Riboflavin is an electro
reducible specie of milkwhich has been converted to its reduced form during electro
reduction at the cathode compartment. During storage riboflavin may . consume the
dissolved oxygen still present in the milk and reoxidise. Therefore, with the oxidation of
riboflavin, a weIl known photo sensitizer, lipid oxidation may happen more easily with a
faster rate in samples in the presence of light compared to milk samples placed in dark.
68
68 ·
69
Comparison of the samples placed in light and dark in this study shows the effect of light in
the rate of lipid oxidation.
C) Another hypothesis for the decrease of dissolved oxygen concentration involves the
growth of psychrotrophic bacteria. In a study done by Allen et al., (1983) on, the oxygen
consumption of pasteurized milk during storage in dar~ for 6 days, they concluded that
most of the oxygen concentration is consumed by bacteria in approximately 1.48 mg/L
compared to the total consumption of 4.10 mg/L. Aiso in other previous studies it was
shown that the electro-reduction treatment did not have a significant effect on the total
psychrotrophic bacteria population in milk (Bolduc et al., 2006b). Therefore, it may be
assumed that these microorganisms cou,ld have consumed the oxygen present in 'the milk
during their growth, decreasing its concentration to values near zero ppm during storage.
Although standard plate counts of the pasteurized milk was not done on the day of its
purchase but since the milk used for this study was pasteurized, it can be assumed that the
contamination might have been introduced frqm contact of the milk with the electro
reduction unit and/or from ambient air and materials during measurements. Although
sodium azide was used to eliminate bacteria in this study the enzymes released during the
destruction of microflora remained active and they could have' had a role in the
consumption of remaining oxygen. Bolduc et al., 2006a observed the same trend of
decrease in DO in pasteurized skim milk when oxygen concentration reached to 0.1 ppm
after 6 days of storage at 4oC.
D) Heat treatments cause numerous chemical reactions, such as Maillard reactions,
hydrolysis and vitamin degradation, that affect the organoleptic and nutritional properties
of milk (van Boekel, 1998; Walstra & Jenness, 1984). Nevertheless, it has been shown that
heat treatment could enhance the antioxidant activity of milk (Taylor and Richardson,
1980; Alamed et al., 2006). The increased antioxidative capacity which is obtained by
increased heat treatment has been referred to protein unfolding. Protein unfolding that
occurs in the globular proteins especially in b-Iactoglobulin, causes the exposure of the
thiol groups (Walstra and Jenness, 1984). Due to the low binding energy of the S-H bond,
the thiols (R -SH) can, act as hydrogen atom donors and inactivate reactive oxygen species
such as lipid alkoxy and peroxy radicals, while the resulting thiyl radicals (RS') yield
inactive disulphides in chain-terminating reactions (Huxtable, 1986). The antioxidant effect 69
70
of sorne advanced Maillard reaction products have also been shown that may cause a
decrease in the dissolved oxygen concentration (van Boekel, 1998).
The relationship between ORP valu~s and DO level is not well-understood. On one hand, a
decrease in the DO causes a decrease in the ORP (during treatment). However, during
storage the opposite occurs: the ORP increases as the DO of all milk samples reaches low
values.
It seems that an oxygen levelas low as 1.5 ppm in electro-reduced milk caused an increase
in redox potential during storage, but on the other hand, a decrease in oxygen level by
either of the hypothesis mentioned or a combination of them, 'did not 'result in a redox
potential decrease. Even after 6 weeks of storage the DO decreased but still did not reach
zero. It could be possible that a DO level of about 0 ppm would be necessary to slow the
ORP increase as described by Giroux et al., 2007.
3.2.3 Effeet on pH:
, 7.00
6.50 !
J: 6.00 a.
5.50
5.00
wO . w1
--+- control dark
• - • 6- - • controllight
Time (weeks)
w2 w3
---- treated dark
- - ~~ - - treated Iight
Fig 3.9) Changes in the pH of omega-3 milk samples during storage.
The repeated measure analysis of variance showed a significant effect of time (P<O.OOOl)
on the pH evolution of electro-reduced milk samples during storage. However, the control
milk samples showed a different pH evolution in comparison with theelectro-reduced milk
70
samples stored under the same conditions. Figure 3.9 shows the evolution of the pH of
modulated milk during storage at room temperature placed in dark and light. The storage
condition of light or dark did not have a significant effect on the evolution of the pH of
electro-reduced milk samples. However, pH value of the electro-reduced milk sa1p.ples
changed more significantly in time compared to control milk samples. The electro-reduced
milk samples that were placed in clark or light had, a significant decrease after 3 weeks of
storage. In the same condition of storage, control milk presented a higher initial value,
which was constant during the 3 weeks of storage. The pH evolution. during storage for the
control milk samples was in accordance with the evolution described by Bolduc et al. ,
(2006a) which they mentioned that there was not a significant change of the pH during their
storage period of 7 days. The authors suggested that the contaminating microorganisms
were nonacid-producing microorganisms therefore a significant decrease in pH for electro
reduced milk samples was not seen in storage of 7 days.
Although sodium azide was added to aH milk samples in order to prevent microbial growth
during storage, the significant decrease of the pH of electro-reduced milk may be due to
contamination from contact of the milk with theelectro-reduction unit and/or from ambient
air and materials during measurements.
3.2.4 Effec! on fatty acid composition:
Table 3.1 Fatty acid composition of omega-3 enriched milk
Fatty acid 0/0 C4:0 5.8 ± 0.1 C6:0 2.7 ±O.2 C8:0 1.5 ± 0.2
C10:0 2.9 ± 0.3 C12:0 3.3 ± 0.2 C14:0 9.7 ± 0.4 C16:0 25.4 ± 0.3 C18:0 7.3 ± 0.3 C18:1 16.8 ± 0.3 C18:2 4.5 ± 0.3 C18:3 Il.1 ± 0.1
71
71
Us~ng the Gas chromatography (G.C) method the fatty acid composition of omega-3
enriched milk was measured and as seen in table 1 the profile contains a high level of
saturated fatty acids (about 65%), and a moderate level of unsaturated (about 36%) fatty
acids. Considering that the objective is to determine the effect of electro-reduction
treatment on the oxidative stability of the lipids of omega-3 enriched milk, C16:0, C18:0,
CI8:1 , C18:2 and the omega-3 fatty acid C18:3 were observed during the storage period of
3 weeks. C 16:0 was used as an internaI standard of the GC and its value for the first week
was maintained throughout the weeks which the values of the other four fatty acids were
compared to this reference in order to determine the changes of the unsaturated fatty acids
during storage.
35
30 ~ c: 25 g iii 0 c.. 20 E 0 0
"C u 15 lU >. t: lU U. 10
5
0
• treated dark
~::::~:::.'-'FH.:: :t----------------------i [] control dark
0
~ N ("")
~ ~ ~ o ....- ' N ("")
~ ~ ~ ~ o ....- N ("")
~ ~ ~ ~ ~ ~ ~ ~ o ....- N ("")
~ ~ ~ ~ C16:0 C18:0 C18:1 C18:2 C18:3
Fig 3.10) Changes in the fatty acid composition of electro-reduced and untreated (control) milk during storage in dark for 3 weeks.
As shown in figure 3.10 during storage in darkness, the decrease of the unsaturated fatty
acids, C 18: 1, C 18:2 and C 18:3, was not significant showing that lipid oxidation was not
taking place as what had been seen for the samples placed in the presence of light Fig 3.11.
It appears that the conditions to continue lipid oxidation was not favourable since the
primary reaction rate could not be maintained by the autoxidation process. In order to
maintain the rate of free radical reactions, external energy from heat, light, radiation and
catalysts such as metal ions is required. These findings sho~ed that the storage condition of
72
72
dark was not favourable for lipid oxidation to further take place. In fact when the samples
were placed in dark with 0% headspace inthe samples (indicating that the glass jars were
full) no significant decrease was seen in the fatty acid composition of the unsaturated fatty
acids up to 6 weeks ·of storage. A slight change can be seen after the third week of storage
in dark for the polyunsaturated fatty acid C18:3 (ALA). Lipid oxidation of the omega-3
fatty acid (ALA) was approximately 17% less in samples placed in dark when compared to
the samples placed in the presence of fluorescent light during 3 weeks of storage. Although
a decrease is seen in the composition of the omega-3 fatty ·acid for both the control samples
and the electro-reduced milk samples, indicating that lipid oxidation has occurred; the
amount of omega-3 decrease in the electro-reduced milk is less· than in the control milk
sample. This shows that the electro-reduction ·treatment has slowed down the rate of lipid
oxidation in the polyunsaturated fatty acid by 6% compared to the control milk when
placed in the same conditions of storage.
35
E 30
· .! 25 '5 • 20 • • 1,1 ...
15 '~ • Z 10 • ..
5
0 0
~ N ct) 0
~ ~ ~ ~ ~ ~ ct) 0
~ N
~ ~ ~ ct) 0
~ ~ ~ N
~ ('t')
~
• treated light
D controllight
Fig 3.11 Changes in the fatty acid composition of electro-reduced and untreated (control) milk during storage in Iight for 3 weeks.
As shown in Fig. 3.11 an important difference was observed in t~e fatty acid composition
of unsaturated fatty acids, especially the omega-3 fatty acid (ALA) in milk samples placed
in the presence of fluorescent light. The reaction seemed to be photo-catalyzed and required
a constant source of energy in order to further take place as which had been concluded by
73
73
Mehta and Bassette (1979). Lipid deterioration reactions are seen both in electro-reduced
and control milksamples. For electro-reduced milk samples it is in accordance with the
hypothesis that riboflavin, a well known photo sensitizer, is re-oxidized during storage
which increases theORP value. When riboflavin is once more availabl~ (in comparison to
when it has been reduced) in electro-reduced milk and the samples have been stored in
light, photo-oxidation reactions may occur by the formation of singlet oxygen from triplet
state oxygen.
o treated dark Il control dark 20 ~-------------------------------------
< ~ 15 't-o è: o
:;:;
.~ 10 c. E o o
"'C
.~ 5 ~ --ta
LL
~treated light Dcontrollight
WO . W1 Time (weeks) ';N2 W3
Fig 3.12 Changes in the fatty acid composition of ALA in electro-reduced and untreated (control) milk during storage for 3 weeks.
Fig. 3.12 shows the fatty acid composition of the omega-3 fatty acid ALA;C18:3 in both
treated and untreated milk 'samples during 3 weeks storage placed in light and dark. The
most important change was seen when samples were untreated and were placed in the
presence of fleurescent light, where about 37% of ALA was degraded. On the contrary the
least degradation of ALA was observed when the sample was electro-reduced and placed in
the absence of light (only 10% degradation of ALA).
74
74
Conclusion
The electro-reduction treatment reduced the redox potential value of omega-3 enriched
milk samples quickly and decreased their dissolved oxygen concentration as weU, without
causing any major changes in their pH. The significant decrease in the ORP of milk during
treatment is explained by the fact that the operating principle of electro-reduction is to
generate the electrons needed to reduce electro-active species. The decline of the OD of
milk during treatment is due to the electro-reduction of oxygen, an important electro-active
species of milk, at the cathode. The rise in ORP during storage confirms t~e instability and .
reversibility of the . electro-active species of milk which may be the result of the re
oxidation of electro-reduced species such as Riblofl~vine. The decline of OD for aU milk
samples during storage shows the consumption of the dissolved oxygen concentration in
the samples. This may be due to many reactions such as; lipid oxidation (especially for the
samples stored in light), re-oxidation of Riboflavin (an electro-active species of milk),
oxidation of thiol groups (proteins) and/or growth of aerobic micro-organisms.
The electro-reduction treatment does not seem to have a direct affect on the lipid oxidative
stability of omega-3 enriched milk although this treatment caused a retardation of lipid
oxidation by about (6%) in electro-reduced samples compared to control untreated milk
samples. During storage the factor of light had a more significant effect on the degradatiori
of the fatty acid composition. of milk compared to the factor of dark which concluded that
the reactions ~eem to be photo-catalyzed and require a constant source of energy such as
light in order to further take place.
Results of this study show that the electro-reduction treatment can be a potential method of
enhancing the shelf-life of products containing unsaturated fatty acids. In future work,
natural electrochemically active compounds can be added to the milk samples to further
decrease the ORP value. AIso, the selection of the appropriate storage conditions to
maintain the reductive environment throughout storage would be useful.
75
75
76
Bibliographie
Alais C., (1984). Propriétés physicochimiques du lait. Science du lait. Paris, p264-268.
Alamed J. , McClements J. D., Decker E. A., (2006). Influence of heat processing and calcium ions on the ability of EDTA to inhibit "lipid oxidation in oil-in-water "emulsions containing omega-3 fattyacids. Journal of Food Chemistry. 95, p585-590
Ami6t 1., Fournier S., Lebeuf Y., Paquin P., Simpson R., (2002). Chapitre 1 : Composition, propriétés physicochimiques, valeur nutritive, qualité -technologique et techniques d'analyse du lait. Science et technologie du lait: Transformation du lait. Presses international Polytechnique, p 1-73. "
Anese M., Calligaris S., Nicoli M.C., Massini R., (2003). Influence of t otal solids concentration and temperature on the changes in redox potential of tomato pastes. International Journal of Food Science and Technology, 38, p55-61.
Bazinet L., Lamarche F., Boulet M., Amiot J., (1997). Combinated effect of pH and temperature during électroréduction of whey proteins. Journal of Agricultural and Food Chemistry, 45-1, pI01-I07.
Bazinet L., Ippersiel D., Montpetit D., Mahdavi B., Amiot J., Lamarche F., (2000). Effect of membrane permselectivity on the fouling of cationic membranes during skim milk electroacidification. Journal of Membrane Science, 174, p97-110.
Bazinet L., Montpetit D., Ippersiel D., Amiot J., Lamarche F., (2001). Identification of skim milk electroacidification fouling : a microscopie approach. Journal of Colloid and Interface Science, 237, p62-69.
Bazinet L., Ippersiel D., Mahdavi B., (2004). Fractionation ofwhey proteins by bipolar membrane electroacidification. Food Sei. Technol. 5 (4), 17-25
Bazinet L., Araya-Farias M., (2005). Effect of calcium and carbonate concentrations on cationic membrane fouling during electrodialysis. Journal of C~olloid and Interface Science, 281, p188-196.
Bazinet L., Péricou J., Araya-Farias M., (2009). Effect of flow rate and " acid molarity on redox potential modulation during electroreduction of milk and simulated milk aqueous mineraI phase. Food chemistry,In press.
Bolduc M.P., (2005)." Effet de la modulation électrochimique du lait sur la ' flore psychrotrophe native" et sur la viabilité de cultures probiotiques ajoutés pendant l'entreposage sous réfrigération. Mémoire de maîtrise n023242, Université Laval, Québec.
76
77
Bolduc M.P., Bazinet L., Lessard l, Chapuzet lM., Vuillemard lC., (2006a) Electrochemical modification of the redox potential of pasteurized milk and its evolution during storage. Journal of Agricultural and Food Chemistry ,54-13, p4651-4657.
~olduc M.P., Raymond Y., . Fustier P., Champagne C.P., · Vui11emard J.C., (2006b). Sensitivity to bifidobacteria to oxygen and redox potential in non-fermented pasteurized milk. International of Dairy Journal, 16-9, pI 03 8-1 048.
Borie F., Sieber R., Bosset J.O., (2001). Photo-oxidation and photoprotection of foods with particular reference to dairy products - An update of a review article (1993-2000). Sciences des aliments, 21, p571-590.
Boyd L.C., (2001). Application of natural antioxidants in stabilizing polyunsaturated fatty acids in model systems and foods. Omega-3 Jatfy acids edited by Shahidi F. And FinleyJ.W ACS symposium 788
Bradley D.G. and MinD.B, (1992). Singlet 'oxygen oxidation of foods, Critical reviews. Food Science And Nutrition, 31, (3), p211-36
Britz T.J and Robinson K., (2008). Advanced dairy science and technology 1. ed.
Ca11igaris S., Manzocco L., A,nese M., Nicoli M.C., (2004). Effect of heat-treatment on the antioxidant and pro-oxidant activity of milk. International Dairy Journal, 14, p421-427. . -
Casimir C. Akoh., (2006). Handbook offunctionallipids IPublished by CRC Press, 2006
Cayot P., Lorient D., (1998). Structures natives des protéines majeures du lait de vache. Structures et technofonctions des protéines du lait. Éditions Lavoisier: Paris, p51-79.
Cesa, S. (2004). Malondialdehyde contents in infant milk formulas. Journal of Agricultural and Food Chemistry, 52, p2119-2122.
Cheftel J.C., (1984). Lait et produits. Introduction à la biochimie et à la technologie des aliments. Technique et documentation Lavoisier: Paris, p35-44.
Co 110mb M., Spahni M., (1996). Review of the methods for the determination oflipid oxidation products with special reference to milk lipids. Schweizerisch Milchwirtschaftliche F orschung, 25,3-24. .
Crowe L.K., Jenness R., Coulter S.T., (1948). The reducing -capacity of milk and milk products as measured by a modified ferricyanide method. Journal of Dairy Science, 31, p595-610.
77
78
Decker C.W., Ashworth U.S. , (1951). The keeping quality ofwhole milk powder - III. The use of antioxidants - ascorbic acid and sodium citrate. Journal of Dairy Science, 34, p633-640.
deI Alamo M., Nevares 1., Carcel L.M., (2006). Redox potential ' evolution during red wine aging in alternative systems. Analytica'Chimica Acta, 563, p223-228.
Frankel E.N., (1999). Lipid oxidation published by The Oily press
Gardais D. , (1990). Les procédés électriques de traitements des rejets .industriels. 487 Environnement et électricité. Avon: Dopee diffusion
Giroux H. 1., St-Amant 1. B., Fustier P. , Chapuzet 1., Britten M., (2007). Effect of electroreduction and heat treatments on oxidative degradation of a dairy beverage enriched with polyunsaturated fatty acids. Food Research International, doi: 10.10 16/j .foodres.2007.1 0.008
Granelli K., (1997). The oxidative stability of lipids in milk and milk powder in relation to sorne pro- and antioxidants. Dissertation Abstract International, 53, p860-933.
Greenbank G.~., (1940). Variation in the oxidation-reduction potential as a çause 'for the oxidized flavor in milk~ Journal of Dairy Science, 23, p725-744.
Greenbank R.G., Wright P.A., (1951). The 'deaeration of raw whole milk before heat treatment as a factor in retarding the development of the tallowy flavor in its dried product. Journal of Dairy Science; 34, p815-818.
Hamilton R.J., (1994). The chemistry of rancidity in foods. Rancidity in Foods, Blackie academic and professional
Harland H.A., Ashworth U.S., (1945). The use of thiamine disulfide for the estimation of reducing substances in processed milk. Journal of Dairy Science, 28, p15-23.
Harland H.A., Coulter S.T. , Jenness R., (1952). The interrer'ationships of processing treatments and oxidation-reduction systems as a factors affecting the keeping quality of dry whole milk. Journal of Dairy Science, 35, p643-654.
Hekal LM., (1983). Process for the preservation of color andflavour in liquid containing comestibles. United States Patent # 4.374.714.
Higginbottom C., Taylor M.M. , (1960). The oxidation-reduction potential of sterilized milk. Journal of Dairy Research, 27, p245-257. '
Huxtable ,R.J., (1986). IIi : Biochemistry ofSulfur, Plenum Press, New York
78
---------------- - - --- - ----- - -- - --- - - -
79
Inoue, T. T., and Kato, W. K. ,(2003). Powdery drinks and process for producing the same. International Patent W003053153.
Jacob H.E., (1970). Redox potential. Methods in Microbiology; Norris, 1. R. , Ribbons, D. W. , Eds.; Academic Press: London, England,; Vol. 2, p91-123.
Jacob H.E., (1979). Chapter 4 : Redox Potential. Methods in Microbiolgy - Volume 2. Morris J.R. , Ribbons D.W., Academic Press: London, p91
Janson H.V. , Lewis M.J. , (1994). Electrochemical coagulation of whey proteine Journal of the Society of Dairy Chemistry, 47-3, p87-90 ~
Jenness R. , Koops J., (1962). Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherland Milk and Dairy Journal, 16, p 153-164.
Jensen R.G.; Ferris A.M~ , Lammi-Keefe C.J., (1991). Symposium: Milk fat - composition, function and potential for change. Journal of Dairy Science, 74; p3228-3243.
Jensen·R.G. (2002). Invited Review: The Composition of Bovine Milk Lipids: January 1995 to Deceinber 2000 .. Journal of Dairy Science, 85, p295-350.
Karatapanis A.E., Badeka A.V., Riganakos K. A., Savvaidis I.N., Kontominas M.G. , (2006).Changes in flavour volatiles of whole pasteurized milk as affected by packaging material and storage time. Intl Dairy J. 16, p750-761
Kristott J., (2000). Fats and oils. The stability and shelf-life offood, Woodhead publishing limited. Edited by David Kilcast, Persis Subramaniam, Published by CRC Press
Labrecque R. , Théorèt A. , Lamarche F. and Boulet M., (1990). Application of Electrodialysis for the Extraction of Cytoplasmic Leaf Proteins from Alfalfa. Electrochemical techniques for a cleaner environment; N. Weinberg and D. Genders; The Electrosynthesis Company Inc.: East Amherst, USA
Lamontagne M, Champagne C.P., Reitz-Ausseur 1., Moineau S., GardnerN., Lamoureux M. , Jean J., Liss I., (2002). Science et technologie du lait: Transformation du lait. Fondation· de technologie laitière du Québec, Presses international Polytechnique :. Montréal, p75-151.
Lanciotti R. , Anese M., Sinigaglia M., Severini C., Massini R., (1999). Effect of heated glucose-fructose-glutamic acid solutions on the growth of Bacillus stearothermophilus. Lebensmittel Wissenschaft and Technology, 32, p223':'230.
Let M. B., Jacobsen C., Frankel E. N., & Meyer A. S., (2003). Oxidative flavour deterioration of fish oil enriched milk. European Journal of Lipid Science and Technology, 105, p518-528.
- - - ------- - - - --- - - --------- - - - - - - - - - - --
79
80
Lingnert H.,(1993). Influence of food processing on lipid oxidation and flavor stability. Lipid oxidation in food. edited by St.Angelo Allen J., ACS symposium 500
Manas P., and Pagan R. (2005). Microbial inactivation by new technologies of food preservation. Journal of Applied Microbiology, 98, p1387-1399.
MatherLH., (2000). A review and proposed nomenclature for major proteins of the milkfat globule ·membrane. Journal of Dairy Science, 83 , p203-247.
Matthes H.D., Pastushenko V. , Heinrich H. , (2000). Redox potential of cow milk at three different months of the year. Milchwissenschaft 55 (4), p200-203 .
Matthes H. D. , Pastushenko V. , Heinrich H., (2001). Redox potential of cow .. milk at three different months of the year. Milchwissenschajt, 56-1, p 7 -10.
Mehta R. S., Bassette R. (1979). Volatile compounds in UHTsterilized milk during fluorescent light exposure and storage in the dark. Journal of Food Protection, 42, p256-258.
McPherson B.J., Kitchen J. , (1983). The bovine milk fat globule membrane~its formation, composition, structure and behaviour in milk. Journal of Dairy Research
Morales M. S., Palmquist D. L., and Weiss W P.,(2000). Milk fat composition of Holstein and Jersey cows with control or depleted copper status and fed whole soybeans or tallow. Journal of Dairy Sei. 83: 2112-211.
Morris J.G., (2000). Chapter Il : The effect of redox potential. The microbiological safety and quality offood - Volume 1. Lund B.M., Baird-Parker T.C., Gould G.W., Aspen ' Publishers Inc.: Maryland, p235-250.
Mortensen G., Bertelsen G.,Mortensen B. K., & Stapelfeldt H., (2004). Light-induced changes in packaged cheeses - a review. International Dairy Journal , 14, p85-102.
Mortensen G.,Sorensen J., Stapelfeldt H., (2002). Effect of light and oxygen transmission characteristics of packaging materials on photooxidative quality changes in Havarti cheeses. Packaging Technol. Sei. 15, p121-127.
Moyssiadi T., Badeka A. , Kondyli E., Vakirtzi T., Savvaidis L, & Kontominas M. G.(2004). Effect of light transmittance and oxygen permeability of various packaging materials on keeping quality of low fat pasteurized milk: chemical and sensorial aspects.International Dairy Journal, 14, p429-436.
MuirD.D and Banks J.M., (2000). Milk and milk products. The stability and shelf-life of
food, Woodhead publishing limited, Hannah Research Institute, Ayr,
O'Conner, T.P; O'Brien N.M.(2006). Lipids. Advanced Dairy Chemistry, vol2:,3rd ed. Springer,New York
80
L-________________________________________________________________________________ -- -
81
Pastushenko V., Matthes H.D., Heinrich H., (2000). Effect of fat content on the redox potential be~aviour of milk. Milchwissenschajt, 55, p547-549.
Pletcher D., Weinberg N. L., (1992). The green potential of electrochemistry: The applications. Journal ofChemical engineering, vol. 99, noll, pp. 132-141
(
Rosenthal 1., Rosert B., Bernstein S., (1993). Effects of milk fortification with ascorbic acid and iron. Milchwissenschajt, 48-12, p676-679.
Sharma R", (2005). Market trends and opportunities for functional dairy beverages. , Australian Journal of Dairy Technology. Melbourne:. Vol. 60, Iss. 2; pg. 195, P 4
Schreyer A., (2007). Régulation électrochimique du potentiel d'oxydoréduction du lait :Impact des composantes du lait et effet sur sa résitanee à l'oxydation après traitement. Ph.D. Thesis, Université Laval, Québée.
Schreyer A., Britten M., Chapuzet J.-M., Lessard J., Bazinet L., (2006).Electrochemical .modification of the redox potential ~f different milk products and its evolution during storage. Schreyer, A., Britten, M., Chapuzet, J.-M., Lessard, J., and Bazinet, L. (2008). Innovative Food Science and Emerging Technologies, 9(3), p 255-264. DOl: 10.10 16/j .ifset.2007 .07 .001 .
Sherbon, J. W., (1999) Physical properties of milk. Fundamentals of Dairy Chemistry, 3rd ed.; Wong, N. P., Ed.; Aspen Publishers: Gaithersburg, MD, p441-491.
Singh H., Fox P.F., (1987). Heat stability of milk : influence of modifying sulphydril. disulphide interactions on the heat coagulation time-pH profile. J~urnal of Dairy Research, 54, p347-359 .
. Small R.W.; (2002). Conservation grazing-a developing opportunity for rare breeds, the Ark Spring 2002, pp. 27-28.
Stapelfeldt H., Nielsen B. R., Skibsted L. H., (1997). Effeet of heat treatment, water activity and storage temperature on the oxidative stability of whole milk powder. International Dairy Journal, 7, p331-339.
Swanson A.M., Sommer H.H., (1940a). Oxidized flavor. in milk : 1. Effect of the development of oxidized flavor on the iodine number of the phospholipid fraction of milk. Journal of Dairy Science, 23, p201-208.
Swanson A.M., Sommer H.H., (1940b). Oxidized flavor in milk: II. The relation of oxidatioh-reduction potentials to its development. Journal of Dairy Science, 23, p597-614.
81
82
Tallec A. , (1985) Généralités sur l'électrochimie organique et les facteurs expérimentaux. Électrochimie organique: Synthèse et mécanisme, ed. Masson, Paris, 10-25
Taylor M.J., Richardson T., (1980). Antioxidant activity of skim milk : effect of heat and resultant sulfhydryl groups. Journal of Dairy Science , 63 , pI783-1795.
Vahcic N. , Palic A. , Ritz M., (1992). Mathematical evaluation of relationships between copper, iron, ascorbic acid' and redox potential. Milchwissenschajt, 47-4, p228-230.
van Boeckel M~A.J.S. , (1998). Effect of. heating on Maillard reactions in milk. Food chemistry, 62-4, p403-414.
Walstra P. , Jenness R. , (1984). Dairy Chemistry and Physics. Walstra P. , Jenness R. , Badings H. T., John Wiley & Sons: New-York, pI97-206.
Walstra P. , (1999). Dairy technology : principles of milk properties and pro cesses / New York M. Dekker.
Webb R.E., Hileman J.L., (1937). The relation of the oxidation-reduction potential of milk to oxidized flavor. Journal of Dairy Science, 20, p47-57.
,Webb B.H., (1965). Composition of milk. Fundamentals of dairy . chemistry. Webb B.H., Johnson A.H., The Avi Publishing company: Westport, p12.
Weinberg N. L., Pletcher D., (1992). The green potential of electrochemistry. II : The applications. Journal ofChemical engineering, vol. 99, noll, pp. 132-141
Wiking L., (2005). Milk Fat Globule Stability Lipolysis with Special Reference to Automatic Milking Systems. Doctoral thesis, Swedish University of Agricultur~l Sciences Uppsala .
82
