Caporali Silvia tesi

Caporali Silvia tesi
Alma Mater Studiorum – Università di Bologna
Università degli Studi di Perugia (Sede consorziata)
DOTTORATO DI RICERCA IN
SCIENZE E BIOTECNOLOGIE DEGLI ALIMENTI
Ciclo XXIII
Settore Concorsuale di afferenza: 07/F1
Settore Scientifico disciplinare: AGR/15
TITOLO TESI
VALORIZATION OF POMACES FROM THE
MECHANICAL EXTRACTION OF VIRGIN OLIVE OILS IN
DAIRY ANIMAL FEEDING
Presentata da:
Dott.ssa Silvia Caporali
Coordinatore Dottorato
Relatore
Prof. Claudio Cavani
Prof. Maurizio Servili
Esame finale anno 2013
Ai miei figli
Gaia e Francesco
Table of Contents
1. INTRODUCTION
1
1.1. Olive oil and the Mediterranean diet
1
1.2. Extra-virgin olive oil: chemical composition
3
1.2.1. Phenolic compounds in olive fruit and in virgin olive oil
4
1.3. Health benefits of phenolic compounds
13
1.3.1. Antioxidant activity
14
1.3.2. Neuroprotective effects
17
1.3.3. Cardiovascular protection
17
1.3.4. Anticancer activity
18
1.3.5. Anti-inflammatory activity
18
1.3.6. Microbial activity
19
1.3.7. Bone health
19
1.3.8. Antiviral activity
20
1.4. Beneficial effect of virgin olive oil monounsaturated fatty acids
20
1.5. Olive by-products
21
1.5.1. Vegetation water
23
1.5.2. Pomace
26
1.5.3. Olive mill waste: regulations and management
28
1.5.3.1. Waste or by-product: regulations
31
1.6. Environmental problems posed by olive-mill waste
33
1.7. Possible uses of olive-mill waste
36
1.7.1. Vegetation water
37
1.7.2. Pomace
38
1.8. Animal feed supplements
39
1.9. Olive pomace in livestock feeding
41
1.9.1. Digestibility
42
1.9.2. Effect of olive pomace supplementation on fatty acid composition of
milk and meat
43
1.9.3. Effects of olive pomace supplementation on animal’s health
44
1.9.4. Storage
45
1.10. Unsaturated fatty acid sources as supplements in animal feeding
46
2. PURPOSE OF THE THESIS
47
3. MATERIALS AND METHODS
49
3.1. Research 1: olive pomace supplementation in buffalo feeding
49
3.1.1. Production of stoned olive pomace
49
3.1.2. Animals and diets
50
3.1.3. Chemical analyses of animal feeds
51
3.1.4. Buffalo milk and mozzarella cheese analyses
53
3.1.5. Analysis of the phenolic compounds of milk
54
3.1.6. Statistical analysis
55
3.2. Research 2: olive paté supplementation in sheep feeding
55
3.2.1. Production of olive paté
55
3.2.2. Preparation of feedstuff
56
3.2.3. Animals and diets
56
3.2.4. Chemical analyses of animal feeds
58
3.2.5. Ewes’ milk and cheese analyses
58
3.2.6. Analysis of the phenolic compounds of milk
60
3.2.7. Statistical analysis
60
4. RESULTS AND DISCUSSION
61
Research 1
4.1. Characterization of dried pomace and feedstuff
61
4.2. Effects of pomace supplementation in buffalo
65
4.2.1. Production and quality of buffalo milk
65
4.2.2. Buffalo milk fatty acid composition
66
4.2.3. Antioxidants and oxidative status of buffalo milk
71
4.2.4. Fatty acid composition of buffalo mozzarella cheese
74
4.2.5. Oxidative status of buffalo mozzarella cheese
75
Research 2
4.3. Phenolic characterization of fresh paté
79
4.4. Preservation of fresh paté over time
79
4.5. Paté pre-drying treatment
84
4.6. Characterization of dried paté and feedstuff
84
4.7. Effects of paté dietary supplementation in Comisana ewes
90
4.7.1. Production and quality of ewes’ milk
90
4.7.2. Ewes’ milk fatty acid composition
91
4.7.3. Antioxidants and oxidative stability of ewes’ milk
93
4.7.4. Oxidative status of ewes’ cheese
97
4.9. Milk phenols: extraction method
99
5. CONCLUSIONS
100
6. REFERENCES
102
RINGRAZIAMENTI
131
1. INTRODUCTION
The olive (Olea europaea L.) is one of the earliest fruit trees to be cultivated and
certainly one of the most important fruit trees in history. The evaluation of archeological
remains and the examination of wild relatives suggests that olive tree was most likely
domesticated in the Near East (Palestine, Israel, Jordan) in the third and fourth millennia
B.C. (Galili et al., 1988; Zohary and Spiegel-Roy, 1975), from the wild oleaster through
nine domestication times (Breton et al., 2009). Wild olives are distributed over the
entire Mediterranean basin and have a small fruit size and low oil content. The main
changes in olives under domestication include the increase of fruit production and
growth ring enlargement; this development involves the fleshy and oil containing
mesocarp (Terral, 1996). Olive production developed along the coastal and subcoastal
areas of the eastern Mediterranean sea, including southern European and northern
African countries, spreading later with the Romans to the northern areas of Italy, Spain,
France and the Balkans (Blázquez Martínez, 1996). Olive production began in Italy in
the first millennium B.C. thanks to, first, the Phoenicians, and later, the Greeks. The
passage of the olive to the Roman world (Acerbo, 1937; Smartt and Simmonds, 1988) is
reported, to be from Sicily, around the sixth century B.C., probably through Etruria
(Boardman, 1977).Olives are long-living with a life expectancy of over 500 years. In the
past seedling olive trees required many years before producing the first crop of fruit,
hence, the olive tree is synonymous with peaceful endeavors in a stable society,
important requirements for population growth. Therefore, since the beginning, the olive
has always been considered a symbol of peace and abundance in the historical and in
religious events which evolved over the centuries.
1.1. Olive oil and the Mediterranean diet
The olive is the most extensively cultivated fruit crop in the world counting 9.49 million
hectares of harvested area in 2010 (FAOSTAT, 2012) and its cultivation area has tripled
in the past 50 years. Italy ranks second in the world, after Spain, with 24% of the world
1
production of olive oil and 1,190,800.000 million hectares of harvested area in 2010
(FAOSTAT, 2012). The olive in Italy is very important for both economic and
environmental purposes. Olive-groves contribute to prevent soil erosion in hilly areas,
provide landscape related environmental benefits, and tourism related economic aspects.
Italian olive biodiversity is a peculiar element of the Italian landscape with over 500
varieties, approximately 42% of world heritage, even though the Italian olive growing is
based on approximately only 50 varieties (Santilli et al., 2011). The presence on the
national territory of a large number of cultivars provides the peculiarity of the sensory
profiles of Italian extra-virgin olive oils, as it consists of different characteristics
depending on the regions, resulting from the interaction between genotype and territory.
Virgin olive oil plays an important and essential role in the Mediterranean diet. The
term “Mediterranean diet” reflects food patterns typical of Crete, much of the rest of
Greece and southern Italy in the early 1960s and is based mainly on the fact that in these
areas the life expectancy of the populations is among the highest in the world and there
is a lower incidence of coronary heart disease than that seen throughout the rest of the
world (Willett, 1995). Keys et al., (1984; 1986) conducted the ‘Seven Countries Study’
on this subject; this provided evidence that the reduced risk of coronary heart disease
(CHD), degenerative diseases, cancer of the breast and colon were associated with the
Mediterranean diet. In particular, it emerged that the number of deaths due to coronary
heart disease was low in the trial groups using olive oil as the main fat (Keys et al.,
1986). This study inspired much research on the combination pattern of the
“Mediterranean diet”, analysing the individual foods of the diet in relation to the health
status of the population (Hu, 2002). Higher adherence to a Mediterranean diet,
associated with other lifestyle factors, predicts increased longevity especially when
consuming wholegrain cereals, foods rich in polyunsaturated fatty acids. Tognon et al.,
(2011) showed the beneficial role of this diet on the incidence of Parkinson’s disease
and Alzheimer’s disease (Sofi et al., 2008).
The Mediterranean diet of the early 1960s was characterized by high intake of a large
amounts of plant food such as vegetables, legumes, fruits, nuts and seeds, cereals
(unrefined in the past), minimally processed, seasonally fresh and locally grown. A
moderately high intake of fish, a moderate intake of dairy products (mostly cheese and
2
yogurt), a low intake of meat and meat products, a regular and moderate intake of
ethanol in the form of wine and generally during meals, and a high intake of olive oil as
the principal source of fat (Kromhout et al., 1989; Willett et al., 1995).
1.2. Extra-virgin olive oil: chemical composition
Extra virgin olive oil (EVOO) is obtained exclusively by mechanical extraction from
the olive fruit and can be consumed crude without any further physical-chemical
treatments of refining. The chemical composition of EVOO is strongly affected by the
agronomic and technological conditions of its production. Several parameters can
modify its composition such as geographical and genetic origin, cultivar, fruit ripening,
pedoclimatic conditions of production, some agronomic techniques, processing system
and storage of the oil.
The chemical composition of VOO is characterized by the presence of two main groups:
the major components (98-99% of the total oil weight), and the minor components (12% of the total oil weight). The major components represents more than 98% of the
total oil weight include mainly triacylglycerols (Gunstone et al., 1994) and small
concentrations of diacylglycerols, monoacylglycerols and some free fatty acids. The key
aspect of this fatty fraction is the low concentration of saturated fatty acids (SFAs) and
the high concentration of monounsaturated (MUFAs) and polyunsatured (PUFAs) fatty
acids. Virgin olive oil is the main source of MUFAs in Mediterranean countries; the
area of production is one of the parameters which affect the fatty acid composition of
olive oil. The more concentrated fatty acids in olive oil are oleic acid (C18:1) a
monounsaturated ω-9 fatty acid (Table 1), followed, in order of concentration, by
linoleic acid, a polyunsaturated ω-6 fatty acid, palmitic acid a saturated fatty acid of
olive oil, stearic acid (C18:0) a saturated fatty acid of olive oil and alpha-linolenic acid
(C18:3), a polyunsaturated ω-3 fatty acid. Virgin olive oil is the main source of MUFAs
in Mediterranean countries; the area of production is one of the parameters which affect
the fatty acid composition of olive oil. The unsaponifiable fraction is low, about 2% of
oil weight and includes more than 230 chemical compounds such as aliphatic and
3
triterpenic alcohols, sterols, hydrocarbons, volatile compounds and antioxidants (Servili
et al., 2004).
Table 1. Fatty acid composition
(%) of virgin olive oil.
Fatty acid
Myristic acid
Palmitic acid
Palmitoleic acid
Heptadecanoic acid
Staric acid
Oleic acid
Linoleic acid
Linolenic acid
Arachidic acid
Gadoleic acid
Behenic acid
Lignoceric acid
IOOC (a)
<0,05
7.5-20.0
0.3-3.5
<0.3
0.5-5.0
55.0-83.0
3.5-21.0
<1.0
<0.6
<0.4
<0.2
<0.2
(a: IOOC-2011)
1.2.1. Phenolic compounds in olive fruit and in virgin olive oil
The healthy properties of olive oil in the past have been ascribed to its high oleic acid
content as a source of monounsaturated fatty acid. However, in recent years converging
evidence, reporting biological activities in vitro, in vivo and in clinical assays of
phenolic compounds present in virgin olive oil (VOO), clarified that many of the
beneficial effects of virgin olive oil intake, are mainly due to its minor compounds.
Polyphenols contribute to the organoleptic properties of olive oil; in particular the
phenolic constituents confer a bitter and pungent taste to the oil, both positive attributes
(Gutiérrez-Rosales et al., 1992; Montedoro et al., 1992a). Moreover, acting as free
radical scavengers, they are chiefly responsible for the defence against the autooxidation of unsaturated fatty acids, increasing the shelf life of the oil (Baldioli et al.,
1996). Phenolic compounds are present at levels between 200 and 1500 mg/kg,
4
depending on the olive tree variety, climatic and agronomic conditions, degree of
maturation at harvest, and the manufacturing process (Servili et al., 2004) and up to
now the methods commonly used to evaluate the phenolic content of extra virgin olive
oil are the Folin-Ciocalteau colorimetric assay (Visioli et al., 1995b) and the HPLC
(Montedoro et al., 1992b).
Tyrosol
[(p-hydroxyphenyl)ethanol]
or
p-HPEA
and
hydroxytyrosol
[(3,4-
dihydroxyphenyl)ethanol] or 3,4-DHPEA are the main phenolic alcohols in the olive
fruit and in virgin olive oil (Montedoro et al., 1992b). It has been proven that the latter
is the most interesting phenolic alcohol, because of its pharmacological and antioxidant
activity. The secoiridoids, are present exclusively in the Oleaceae family that includes
Olea europea (Servili et al., 2004) and are characterized by the presence of either
elenolic acid or elenolic acid derivatives in their molecular structure (Montedoro et al.,
1992c, 1993), in VOO are present in aglycon forms and are derivatives of oleuropein,
demethyloleuropein and ligstrostide, the seicoiridoid glucosides contained in all the
constitutive part of olive fruit (Figure 1). Another seicoridoid, detected in the olive seed
is the nüzhenide (Servili et al., 1999).
5
Figure 1. Chemical structures of the secoiridoids glucosides in the olive fruit.
The olive fruit contains a large amount of phenols, between 1% and 3% of the fresh
pulp weight. In addition to secoiridoids, the main phenolic compounds in the olive fruit
are phenolic acids, phenolic alcohols, and flavonoids (Table 2).
6
Table 2. Phenolic compounds in olive fruit.
Phenolic acid
Chlorogenic acid
Caffeic acid
P-Hydroxybenzoic acid
Protocatechuic acid
Vanillic acid
Syringic acid
p -Coumaric acid
o -Coumaric acid
Ferulic acid
Sinaptic acid
Benzoic acid
Cinnamic acid
Gallic acid
Seicoridoids
Oleuropein
Demethyloleuropein
Ligstroside
Nuzhenide
Phenolic alcohols
(3,4-Dihydroxyphenyl) ethanol (3,4-DHPEA)
(p -Hydroxyphenyl) ethanol (p -HPEA)
Flavonols
Quercitin-3-rutiniside
Flavones
Luteolin-7-glucoside
Luteolin-5-glucoside
Apigenin-7-glucoside
Antocyanins
Cyanidin-3-glucoside
Cyanidin-3-rutinoside
Cyanidin-3-caffeygucoside
Cyanidin-3-caffeylatinoside
Delphinidin 3-rhamosylglucoside-7-xyloside
Hydroxycinnamic acid derivatives
Verbascoside
Oleuropein and demethyloleuropein are the main secoiridoids of the olive fruit, which
also contains verbascoside, a derivative of hydroxicinnamic acid (Figure 2).
Figure 2. Chemical structure of verbascoside.
7
The most important antioxidants in virgin olive oil are polyphenols that include
lipophilic and hydrophilic phenols. The first group includes tocotrienols and
tocopherols, among which, α-tocopherol is the most abundant in virgin olive oil, about
90% of total concentration. The second group is shown in Table 3 and consists of
hydrophilic
phenols:
phenolic
acids,
phenolic
alcohols,
hydroxy-isocromans,
flavonoids, secoiridoids and lignans.
Table 3. Phenolic composition of virgin olive oil (VOO).
Phenolic acids and derivatives
Vanillic acid
Syringic acid
p -Coumaric acid
o -Coumaric acid
Gallic acid
Caffeic acid
Protocatechuic acid
p -Hydroxybenzoic acid
Ferulic acid
Cinnamic acid
4-(Acetoxyethyl)-1,2-dihydroxybenzene
Benzoic acid
Hydroxy-isocromans
Phenolic alcohols
(3,4-Dihydroxyphenyl) ethanol (3,4-DHPEA)
(p -Hydroxyphenyl) ethanol (p -HPEA)
(3,4-Dihydroxyphenyl) ethanol-glucoside
Lignans
(+)-1-Acetoxypinoresinol
(+)-Pinoresinol
Flavones
Apigenin
Luteolin
Hydroxy-isochromans
1-(3'-methoxy-4'-hydroxyphenyl)-6,7-dihydroxyisochroman
1-phenyl-6,7-dihydroxyisochroman
Secoiridoids
Dialdehydic form of decarboxymethyl elenolic acid linked to 3,4-DHPEA (3,4-DHPEA-EDA)
Dialdehydic form of decarboxymethyl elenolic acid linked to p -HPEA (p-HPEA-EDA)
Oleuropein aglycon (3,4-DHPEA-EA)
Ligstroside aglycon
Oleuropein
p -HPEA-derivative
Dialdehydic form of oleuropein aglycon
Dialdehydic form of ligstroside aglycon
8
The phenolic acids (Figure 3) were the first group of phenols described in virgin olive
oil (Mannino et al., 1993; Montedoro et al., 1992b; Servili et al., 2004; Tsimidou et al.,
1996).
Figure 3. Chemical structure of the main phenolic acids of VOO: benzoic acid [I], phydroxybenzoic acid [II], vanillic acid [III], protocatechuic acid [IV], syringic acid [V], gallic
acid [VI], cinnamic acid [VII], p-coumaric acid [VIII], o-coumaric acid [IX], caffeic acid [X],
ferulic acid [XI].
9
These compounds are present in small amounts in virgin olive oil, together with phenylalcohols, hydroxy-isocromans and flavones. Hydroxy-isochromans were detected in
VOO by Bianco et al., (2001), and are formed by the reaction between hydroxytyrosol
and vanillin [1-(3'-methoxy-4'-hydroxyphenyl)-6,7-dihydroxyisochroman] and between
hydroxytyrosol and benzaldehyde [1- phenyl-6,7-dihydroxyisochroman].
Secoiridoids and lignans, are the most abundant hydrophilic phenols in virgin olive oil;
in particular Owen et al., (2000a) reported 27.72 mg/kg of total seicoridoids and 41.53
mg/kg of total lignans in virgin olive oil. The lignans were isolated and characterized by
Owen et al., (2000b) who indentified (+)-1-Acetoxypinoresinol and (+)-Pinoresinol as
the most concentrated in virgin olive oil (Figure 4).
Figure 4. Chemical structures of the lignans occurring in VOO.
10
The aglyconic form of secoridoids (Figure 5) in VOO arises from the hydrolytic
process, catalyzed by endogenous β-glucosidases, according to the proposed mechanism
reported in Figure 6 (Servili et al., 2004). In this way the secoiridoids derivatives are
released into the oil and in by-products.
Figure 5. Chemical structures of the secoiridoids derivatives and phenolic alcohols of VOO.
11
Figure 6. Biochemical mechanism of secoiridoids derivatives formation: (I) R = H: ligstroside;
R = OH: oleuropein; (II) R = H: ligstroside aglycon; (III) R = OH: 3,4-DHPEA-EA; (IV) R = H:
dialdehydic form of ligstroside aglycon; R = OH: dialdehydic form of oleuropein aglycon; (V)
R = H: p-HPEA-EDA; R = OH: 3,4-DHPEA-EDA.
12
1.3. Health benefits of phenolic compounds
In Figure 7, the biological activities of phenolic compounds on health are summarized.
Antioxidant
activity
Antiviral
activity
Bone health
Health properties
of virgin olive oil
hydrophilic phenols
Microbial
activity
Neurological effects
Cardiovascular
protection
Anticancer
activity
Anti-inflammatory
activity
Figure 7. Biological activities of phenolic compounds.
Assessing the olive oil phenols bioavailability was an important step in demonstrating
in vivo effects on humans. Up to now the research on the bioavailability of olive oil
phenolic compounds, was mainly focused on hydroxytyrosol, tyrosol and oleuropein
(Cicerale et al., 2010). In particular it was found that hydroxytyrosol and tyrosol,
absorbed dose-dependently from olive oil after oral ingestion in humans, rise quickly in
plasma, reaching a peak at around 1h (Weinbrenner et al., 2004; Miró-Casas et al.,
2003) and 0-2 h in urine (Miró-Casas et al., 2001). The excretion of hydroxytyrosol and
tyrosol in humans is between 30-60% and 20-22% of the total amount ingested,
respectively (Visioli et al., 2000b). The different polarities of these compounds were
supposed to play an important role in the absorption process; hydroxytyrosol and
tyrosol are polar and permeate cell membranes of human intestinal cells via a passive
diffusion mechanism (Manna et al., 2000). Hydroxytyrosol and tyrosol are present in
plasma and urine in their glucoronide conjugated forms, (Caruso et al., 2001) after
13
virgin olive oil ingestion; an extensive first pass intestinal/hepatic metabolism of the
ingested primary forms has been proposed (Miró-Casas et al., 2003). Bonanome and coauthors (2000) showed that the olive phenolic compounds are incorporated in human
lipoproteins after ingestion and it has been proven that hydroxytyrosol preserves its
antioxidant properties following ingestion (Visioli et al., 2005). Miró-Casas et al.,
(2003) developed a method to quantify absorption of hydroxytyrosol and its
metabolites, and also found that absorption of hydroxytyrosol is nearly complete and its
plasma half-life is 2.43 hours. The high bioavailability represents a starting point to
confirm its beneficial effects on health.
1.3.1. Antioxidant activity
Elevated levels of high density lipoprotein cholesterol (HDL-C) have protective and
anti-inflammatory properties (Chrysohoou et al., 2006). However, on the other hand
elevated levels of total cholesterol and low density lipoprotein cholesterol (LDL-C)
have been established as a critical factor in the development of atherosclerosis, which is
the primary cause of cardiovascular disease (Gonzalez-Santiago et al., 2010; VázquezVelasco et al., 2011). Coronary events are reduced by 2-3% for every 1% decrease in
LDL-C, as reported in the Helsinki Heart Study (Manninen et al., 1988) and are reduced
by 3% for every 1% increase in HDL-C (Feldman, 2002).
There is evidence that oxidative modification of low density lipoprotein (LDL) plays a
key role in the development of atherosclerosis (Witztum et al., 1994a). The formation of
oxidized LDL depends on its antioxidant content, such as vitamin E and phenolic
compounds, present in LDL (Reaven et al., 1994). In vitro the antioxidant effect is
evaluated through the assessment of various markers of LDL oxidation such as reduced
formation of short-chain aldehydes and lipid peroxides, high vitamin E content in the
residual LDL, protection of the apoprotein layer (Visioli et al., 1995a). Hydroxytyrosol
reduces oxidation of the low-density lipoproteins carrying cholesterol (LDL-C); it also
has a potential protective effect against oxidative stress induced by tertbutyl
hydroperoxide (Goya et al., 2007). Both hydoxythyrosol and oleuropein inhibit the
14
copper sulfate-induced oxidation of LDL (Visioli and Galli, 1994) which indicate that
these molecules are potent free radical scavengers.
Up to now in humans it has been proven that phenolic compounds linked to human LDL
increases in a dose dependent manner with the phenolic content of the olive oil
administered (Covas et al., 2006) (Figure 8), hence, the phenolic content of the olive oil
protects the LDL phenolic content from degradation (Fitò et al., 2000).
Figure 8. Relationship between the change in the total phenolic content (PC) of the LDL and
plasma hydroxytyrosol concentrations 30 minutes after ingestion of 40 ml of a high phenolic
content (366mg/kg) of olive oil. R=0.780, P=0.009, Spearman’s correlation coefficient. Adapted
from Covas et al., (2006), by Fitò et al., (2007).
Oxidative stress produced by reactive oxygen species (ROS) has been linked to several
diseases such as atherosclerosis, certain cancers and neurodegenerative diseases
(Witztum et al., 1994b). Manna and co-workers (2002) found that the phenolic fraction
of extra virgin olive oils plays a protective role against ROS-mediated (reactive oxygen
species) oxidative injuries in erythrocytes and human intestinal cells (Caco-2). It has
15
also been reported that total plasma antioxidant activity increases in humans after the
ingestion of olive oil phenolic compounds (Visioli et al., 2005).
The abundance of polyunsaturated fatty acids makes the kidney an organ particularly
vulnerable to the lipid peroxidation process (Kubo et al., 1997): lipid peroxidation is
considered to be crucial in the mechanisms involved in a wide range of renal diseases,
such as tubulointerstitial alterations (Martin-Mateo et al., 1999). A recent study has
shown that a pre-treatment with hydroxytyrosol inhibits H2O2 induced oxidative
damage in a porcine kidney epithelial cell line (LLCPK1), preserving the level of
membrane lipids with no significant detection of oxidation products (Deiana et al.,
2008).
The olive oil polyphenols contribute to the protection of human blood lipids from
oxidative stress; the new European regulation about the “health claims in foods” fixed
for the phenolic compounds of EVOO a minimum level of 5 mg of hydroxytirosol and
its derivatives (e.g. oleuropein complex and tyrosol) in 20 g of oil (Reg. EU 432/2012).
Oxidative stress is involved in age-related degeneration of retinal pigment epithelium
and the photoreceptors in the macular area of the retina; Liu et al., (2007), have shown
that hydroxytyrosol protects human retinal pigment epithelial cells (ARPE-19) from
oxidative damage induced by an environmental toxin (acrolein), endogenous end
product of lipid oxidation, that occurs at increased levels in age-related macular
degeneration lesions. A further study on the same disease showed that hydroxytyrosol is
an important inducer of phase II detoxifying enzymes and an enhancer of mitochondrial
biogenesis (Zhu et al., 2010). Several studies reported the protective rule on DNA
damage of the phenolic compounds of virgin olive oil, in vitro (Fabiani et al., 2008)
and in animals.
A study conducted, by Salvini and co-workers (2006) reports that in humans the
ingestion of phenol rich virgin olive oil decreases oxidative DNA damage by up to 30%,
compared to a low phenolic content virgin olive oil. A further study demonstrated that
after consumption of phenol rich virgin olive oil there was decreased urinary excretion
of 8-oxo-deoxygyuanosine a systemic marker of DNA oxidation (Cooke et al., 2003).
16
1.3.2. Neuroprotective effects
Aging is the result of the oxidative injury, mainly to mitochondria. Some of the
oxidative damage leads to cellular dysfunctions during an entire lifetime. Mitochondrial
membranes are very sensitive to free radical attacks because of the presence of double
bond carbon-carbon in the lipid tails of its phospholipids, which can lead to cognitive
and neurodegenerative diseases (Omar, 2010). In a study Bazoti et al., (2006) reported
that oleuropein decreases or even prevents β-amyloid peptide aggregation, this peptide
is the major proteinaceous component of senile plaques formed in Alzheimer's disease
brain. An animal study has shown the protective role of virgin olive oil phenols on lipid
peroxidation: mice fed from middle age to senescence with extra-virgin olive oil (10%
wt/wt dry diet) rich in phenols (total polyphenol dose/day, 6 mg/kg) improved
contextual memory to levels similar to young animals and prevented age-related
impairment in motor coordination. This effect appeared to be correlated with reduced
lipid peroxidation in the cerebellum (Pitozzi et al., 2012).
Another study showed that administration of virgin olive oil in mice improves learning
and memory, increasing brain glutathione levels, suggesting reduced oxidative stress as
a possible mechanism (Farr et al., 2012).
1.3.3. Cardiovascular protection
It is recognized that platelets play a key role in the development of thrombosis, (Osler,
1886) and it is widely accepted that platelets play a central role in the development of
cardiovascular diseases (Bhatt and Topol, 2003).
Hydroxytyrosol has been proven to inhibit platelet aggregation completely in human
blood (in vitro) in the range of 100-400 µM (Petroni et al., 1995). A more recent study
shows that a number of olive oil phenolic compounds such as oleuropein aglycone and
luteolin are also potent inhibitors of platelet aggregation (Dell’Agli et al., 2008).
Leukocyte adhesion to vascular endothelial represents a key step in the formation of
17
atherosclerotic, plaque; the ability of phenols to inhibit these kinds of cells was also
proven (Carluccio et al., 2003).
1.3.4. Anticancer activity
Several epidemiologic studies have demonstrated the association between olive oil
consumption and a reduced risk of cancer in different sites such as the breast (La
Vecchia et al., 1995; Martin-Moreno et al., 1994; Trichopoulou et al., 1995), the
prostate (Hodge et al., 2004), the lung (Fortes et al., 2003), the larynx (Bosetti et al.,
2002a), the ovary (Bosetti et al., 2002b) and the colon (Stoneham et al., 2000). Cell
proliferation is the factor chiefly responsible for tumour formation and progression. It
has been proven that hydroxytyrosol shows a pro-apoptotic effect by modulating the
expression of genes involved in tumour cell proliferation of promyelocytes (HL60 cells)
(Fabiani et al., 2006, 2008, 2009, 2011). Moreover, it has been proven that
hydroxytyrosol inhibits proliferation of human MCF-7 breast cancer cells (Bulotta et al.
2011; Sirianni et al., 2010), human HT29 colon carcinoma cells (Guichard et al., 2006),
human M14 melanoma cells (D’Angelo et al., 2005) and human PC3 prostate cells
(Quiles et al., 2002). Hashim and co-workers (2008) demonstrated a dose-related
inhibition of colon cancer cell invasion exerted by olive oil phenolic compounds.
1.3.5. Anti-inflammatory activity
Recent studies on the mechanisms involved in atherosclerosis disease has focused on
inflammatory cytokines that are responsible for vascular inflammation, stimulating the
generation of endothelial adhesion molecules which may enter the circulation in a
soluble form. Elevated concentrations of inflammation markers in serum are associated
with increased cardiovascular risk (Packard and Libby, 2008). Plasma thromboxane B2
has the ability to increase blood platelet aggregation and leukotriene B4 has a
chemostactic effect on neutrophils, directing the cells to damaged tissue, and both are
known proinflammatory agents (Bogani et al., 2007). Polyphenols have been shown to
18
decrease the production of inflammatory markers, such as leukotriene B4, in several
systems (Biesalski, 2007).
Recently, it has been established that HT-20, an olive oil extract containing about 20%
of hydroxytyrosol, inhibits inflammatory swelling and hyperalgesia, and suppresses
proinflammatory cytokine in a rat inflammation model (Gong et al., 2009).
1.3.6. Microbial activity
Antimicrobial activity of oleuropein, tyrosol and hydroxytyrosol has been studied in
vitro against bacteria, viruses and protozoa (Bisignano et al., 1999). Hydroxytyrosol and
oleuropein have been shown to have antibacterial properties in particular cytotoxic
against many bacterial strains, mainly against bacteria responsible for intestinal and
respiratory infections (Medina et al., 2006). Also, the dialdehydic form of
decarboxymethyl ligstroside is not hydrolyzed in the stomach and plays an important
role in the inhibition of the growth of the Helicobacter pylori bacteria (Romero et al.,
2007), chiefly responsible for the development of gastric cancer and peptic ulcers.
1.3.7. Bone health
Tyrosol and hydroxytyrosol seem to be involved in increased bone strengthening in rats
(Puel et al., 2008). In particular these compounds could represent effective remedies in
the treatment of osteoporosis symptoms; Hagiwara and co-authors (2011) evaluated the
effects of oleuropein, hydroxytyrosol and tyrosol on bone formation using cultured
osteoblasts and osteoclasts. The results showed that oleuropein and hydroxytyrosol
inhibited the loss of bone density, stimulating the deposit of calcium in a dosedependent manner. Both compounds, also, suppressed the loss of bone density of
trabecular bone in femurs of ovariectomized mice.
19
1.3.8. Antiviral activity
Hydroxytyrosol and oleuropein have been identified as a unique class of HIV-1
inhibitors that prevent HIV from entering into the host cell and binding the catalytic site
of the HIV-1 integrase (Fernández-Bolaños et al., 2012). Oleuropein and
hydroxytyrosol were identified as HIV inhibitors at both the fusion and integration
stages (Lee-Huang et al., 2007a, 2007b) in a dose-dependent manner. Hydroxytyrosol
was also found to be useful as a microbicide for preventing HIV infection, in fact,
thanks to this property, it has recently been patented as a product for topical use
(Gómez-Acebo et al., 2011). Furthermore, it has been reported in another study that
hydroxytyrosol inactivated influenza A viruses, including H1N1, H3N2, H5N1, and
H9N2 subtypes. Electron microscopic analysis revealed morphological abnormalities in
the hydroxytyrosol-treated H9N2 virus, this suggested that the structure of the H9N2
virus could be disrupted by hydroxytyrosol (Yamada et al., 2009).
1.4. Beneficial effect of virgin olive oil monounsaturated fatty acids
Monounsaturated fatty acids intake leads to enhanced resistance of LDL oxidation
(Bonanome et al., 1992), it also increases the levels of the protective high-density
lipoprotein more than polyunsaturated fatty acids when these two classes of fatty acids
replace carbohydrates in the diet (Mensink et al., 2003). Another effect of dietary
MUFA, is lower endothelial activation (Massaro et al., 2002; Massaro and De Caterina,
2002). The intake of monounsaturated fatty acid appeared to be associated with a
reduced risk of age-related cognitive decline, mainly due to the role these fats play in
maintaining the structural integrity of neuronal membranes (Panza et al., 2004a;
Solfrizzi et al., 1999). Moreover, high ingestion of monounsaturated fatty acids appears
to exert a contrasting action on Alzheimer’s disease (Panza et al., 2004b).
20
1.5. Olive by-products
The virgin olive oil (VOO) extraction system and related processing parameters can
strongly affect the qualitative and the quantitative composition in phenols of VOO and
their main by-products. In particular, the concentration in the final product and byproducts of those compounds, is largely affected by agronomic and technological
conditions of VOO production. Cultivar, ripening stage, geographic origin of olives and
olive trees irrigation are the main agronomic aspects that can modify the VOO
secoiridoid composition as well as some operative conditions applied during crushing,
malaxation and VOO separation (Servili et al., 2004, 2009). So far, different extraction
technologies, such as pressure and centrifugation and selective filtration (i.e. "surface
tension" or "percolation"), are used to enable the separation of oily must from the olive
paste (Boskou, 1996; Di Giovacchino et al., 1994), however, the majority of VOO in
the Mediterranean area is currently extracted by centrifugation; in Italy this technique
represents more than 80% of total production (Roig et al., 2006).
In particular two main different systems have been used, up to now, using
centrifugation: the three-phases and the two-phases extraction systems (Sequi et al.,
2001). However innovative technologies in recent years have improved, from both
environmental and qualitative point of view, the existing extraction systems; for
example the new three-phases decanters, and a new two-phases decanter which
produces a new kind of pomace, more easily reusable (i.e. paté).
The three-phase system, introduced in the 1970s to improve extraction yield, produces
olive oil, vegetation water (VWs), which consists of the water plus organic matter
incorporated into drupes and the water added during the process, pomace made up of
pulp bulk and pit; the dilution of the malaxed pastes produces 50-90 l of VWs/100 kg
of olive pastes (Servili et al., 2011). The traditional three-phases decanters, featuring
water addition ranging from 0.5 to 1 m3/ton, while the “new three-phases decanters”,
works at low water consumption (ranging from 0.2 to 0.3 m3/ton) (Servili et al., 2012).
The two-phase centrifugation system was introduced in the 1990s in Spain as an
ecological approach for olive oil production and, thanks to this, there was a significant
decrease in water consumption during the process producing two fractions: a semisolid
21
sludge called by various names (“two-phases olive mill waste”, “alperujo”, “olive wet
husk”, or “wet pomace”) and olive oil (Roig et al., 2006). From an environmental point
of view, VWs from the three-phase system is considered the worst waste in both
quantity and quality terms; the two-phases decanters, which can operate without the
addition of water do not produce vegetation water as a by-product of the extraction oil
process (Servili et al., 2012). Figure 9 reports the comparison of the two main different
olive oil extraction systems.
Figure 9. Comparison of the three-phases and two-phases centrifugation systems for olive oil
extraction (Alburquerque et al., 2004).
Lesage-Messen and co-workers (2001) studied the phenolic composition of by-products
obtained from three-phase and two-phase extraction systems. The study reported that
the phenolic composition, identified by HPLC, after acid extraction with ethyl acetate,
was similar in VWs and pomace from the two different extraction systems, with
hydroxytyrosol, approximately 1% dry residue, and tyrosol being the main compounds
22
detected. Nevertheless, the contents of individual compounds (hydroxytyrosol, tyrosol,
caffeic acid, ferulic acid and p-coumaric acid), with the exception of vanillic acid, were
higher for the two-phase system.
The phenolic composition of virgin olive oil and by-products is strongly affected by the
enzymatic reactions such as β-glucosidase, polyphenoloxidase (PPO), peroxidase
(POD), occurring during the different phases of the mechanical extraction process of the
oil (Di Giovacchino et al., 1994; Servili et al., 2004).
The first ones cause the release of phenols in the oil and in the water, while the latter
promote their oxidation. In recent years the understanding of those phenomena,
acknowledged by the industry in some innovations in the mechanical extraction process,
has led to the production of a VOO significantly richer in phenolic compounds and at
the same time an increase in those compounds in the VWs.
1.5.1. Vegetation water
The vegetation waters (VWs) is characterized by the following special features and
components (Lòpez, 1993; Vásquez-Roncero et al., 1974):
•
intense violet-dark brown up to black colour;
•
strong specific olive oil smell;
•
high degree of organic pollution (COD values up to 220 g/l);
•
pH between 3 and 6 (slightly acid);
•
high electrical conductivity;
•
high content of polyphenols (0.5-24 g/l);
•
high content of solid matter.
The composition of the VWs varies according to cultivar, degree of maturity,
cultivation soil, harvesting time, use of pesticides and fertilizers, olive’s water content
and climatic conditions. The VWs pH value ranges from 4.5 to 6 and the VWs contain
an average of 3-16% of organic compounds, in which 1-8% of sugars, 1.2-2.4% of
nitrogen containing compounds and 0.34-1.13% of phenols (Niaounakis and
Halvadakis, 2004). VWs contain various amounts of sugars depending on olive cultivar,
23
climatic conditions during growth and the extraction system. The sugars constitute up to
60% of the dry substance, and are, in order of concentration, fructose, mannose,
glucose, saccharose, and traces of sucrose, and some pentose. The olive oil phenolic
compounds are amphiphilic in nature and are more soluble in the water than in the oil
phase, consequently, a large quantity of antioxidants is lost with the wastewater during
processing. These compounds in the VWs reach concentrations ranging from 0.5 to 24
g/L and are strictly dependent on the processing system used for olive oil production
(Niaounakis et al., 2006).
The hydrophilic phenols identified and quantified in VWs include phenolic alcohols,
phenolic acids, phenyl alcohols, secoiridoids, flavonoids, and lignans. Up to now, more
than 30 phenolic compounds have been identified in VWs. The main phenolic
compounds detected in VWs are:
•
Derivatives from cinnamic acid: (cinnamic acid), o-and p-coumaric acid (4hydroxycinnamic acid), caffeic acid (3,4-dihydroxycinnamic acid), and ferulic
acid (4-hydroxy-3-methoxycinnamic acid).
•
Derivatives from benzoic acid: (acid benzoic), protocatechuic acid, and b-3,4dihydroxyphenyl ethanol derivatives. A large amount of phenolic compounds
detected in vegetation water are represented by secoiridoids, in particular pHPEA
(p-hydroxyphenylethanol
or
tyrosol),
3,4-DHPEA
(3,4-
dihydroxyphenylethanol or hydroxytyrosol), and p-HPEA-EDA and the 3,4
DHPEA-EDA (dialdehydic form of decarboxymethil elenolic acid linked to
tyrosol and hydroxytyrosol respectively) and also verbascoside (Servili et al.,
1999).
•
catechol, 4-methylcatechol, p-cresol, and resorcinol (Capasso et al., 1992a,
1992b; Vinciguerra et al., 1993).
•
VWs can contain, under certain conditions, small amounts of oleuropein,
demethyloleuropein and verbascoside (Servili et al., 1999).
•
VWs also contains relatively high concentrations of flavonoids: the main
flavonoids detected in VWs are anthocyanins (cyanidin-3-glucoside, cyanidin-3rutinoside), flavones (luteolin, luteolin-7-glucoside, apigenin, apigenin-7-
24
glucoside), and flavonols (quercetin, rutin) (Duràn Barrantes, 1990; Servili et
al., 1999).
A specific complex that has an active biological and specific activity, consisting of
hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and the dialdehydic form of
decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl) ethanol (3,4DHPEA-EDA) and accounting for 6% of the total phenolic content of the VWs, has
recently been identified in VWs (Angelino et al., 2011).
The chemical composition of VWs as reported by Morillo et al., 2009, is shown in
Table 4.
Table 4. Chemical composition of VWs from two-phase
olive mill extraction system.
Data were calculated from eight independent studies reported in Roig
et al., (2006) and adapted by Morillo et al., 2009.
25
1.5.2. Pomace
Olive pomace, the other olive mill waste, is the solid phase resulting from the virgin
olive oil mechanical separation process. The chemical composition and nutritive value
of the olive pomaces are very different from those of pomaces coming from different
extraction systems, and depends on the proportion of the different physical components
such as skin, stone, pulp, water, year, geographic origin, contamination with soil and the
residual oil (Molina Alcaide and Yáñez-Ruiz, 2008). The pomace is made up by
residual oil (5-8%), vegetation waters (25-55%) while the remaining fraction is made up
by solid components. The pomace produced by the two-phase extraction system has a
moisture content in the range of 55-70%, while traditional pomaces have a moisture
content of around 20-25% in press systems and 40-45% in three-phase decanters (AlbaMendoza et al., 1990; Alburquerque et al., 2004). It also contains some residual olive
oil (2-3%) and 2% ash with 30% of potassium content. Tyrosol and hydroxytyrosol are
the most abundant phenolic compounds in pomaces (Fernández-Bolaños et al., 2002)
together with p-coumaric, caffeic (Lesage-Meessen et al., 2001), while vanillic acid is
found in lower amount. These phenolic compounds together with the lipid fraction have
been connected with the phytotoxic and antimicrobial effects attributed to olive-mill
wastes. The chemical composition is reported in Table 5. The recovery of the pomace
residual oil by means of solvent and its subsequent refining, will increase problems for
the placement of the product, considered as a low quality oil.
26
Table 5. Chemical composition of pomace from two-phase olive
mill extraction system.
Data were calculated from eight independent studies reported in Roig
et al. (2006) and adapted by Morillo et al., 2009.
a
(% w/w) of total organic matter.
The fibrous components vary depending largely on the proportion of stones in pomaces.
In the case of de-stoned pomaces the crude fibre value is lower (Sansoucy, 1985).
Analysis of fibres by the Van Soest (1975) method shows that pomace has high cell
wall constituents (NDF), ligno-cellulose (ADF) and lignin (ADL) contents (Table 6).
The crude fat (CF) and neutral detergent fiber (NDF) are the most variable components.
27
Table 6. Average chemical composition (g/kg D.M.)
of olive pomace.
Parameters
Dry matter (g/kg fresh matter)
Organic matter
Ether extract
Gross energy (MJ/kg DM)
Crude protein
Amino acid N (g/kg N)
Acid detergent insoluble
Neutral detergent fibre
Acid detergent fibre
Acid detergent lignin
Total extractable polyphenols
Total extractable tannins
Total extractable condensed tannins
Total condensed tannins
Free condensed tannins
Fibre bound condensed tannins
Protein bound condensed tannins
Pomace
805
901
54,5
19,7
72,6
846
10,7
676
544
289
13,9
9,78
0,81
12,4
1,64
4
5,87
S.D.
178
62
42.1
2
23,4
3,9
119
83
30
4,8
0,7
0,08
0,3
0,65
Lignin content is high and Crude protein content (CP) is generally low, and a substantial
part is linked to cell wall components, in fact a large proportion of the proteins (80 to
90%) is linked to the ligno-cellulose fraction (ADF-N) (Nefzaoui, 1983). Pomace fat is
high in unsaturated C:16 and C:18 fatty acids which constitute 96% of total fatty acids
(Chiofalo et al., 2002). Pomace is highly vulnerable to air oxygen which is the main
cause of spoilage of its organoleptic properties (Sansoucy, 1985).
1.5.3. Olive mill waste: regulations and management
In Italy land spreading of waste, wastewater and/or wet husk, is regulated by Law n°
574 dated 11/11/1996 regarding olive mill waste waters and olive pomace. “Nuove
norme in materia di utilizzazione agronomica delle acque di vegetazione e di scarichi
28
dei frantoi oleari” which in Art. 1, envisages that: “Le acque di vegetazione residuate
dalla lavorazione meccanica delle olive che non hanno subito alcun trattamento né
ricevuto alcun additivo ad eccezione delle acque per la diluizione delle paste ovvero per
la lavatura degli impianti possono essere oggetto di utilizzazione agronomica
attraverso lo spandimento controllato su terreni adibiti ad usi agricoli”. The provisions
of Italian Law n° 574 on land spreading of VWs and olive pomace, contain many points
and the main ones are summarized below.
Art.1. Agronomic use
1. Olive-mill wastewater: olive-mill wastewater without pre-treatment.
2. Olive pomace “…ai fini dell’applicazione della presente legge le sanse umide
provenienti dalla lavorazione delle olive e costituite dalle acque e dalla parte fibrosa di
frutto e dai frammenti di nocciolo possono essere utilizzate come ammendanti in deroga
alle caratteristiche stabilite dalla legge 19 ottobre 1984, n. 748, e successive
modificazioni.” (Replaced by Legislative Decree 217 dated 29 April 2006 “Revisione
della disciplina in materia di fertilizzanti”).
Art. 2. Limits of acceptability
1. Olive-mill wastewater: from traditional press at 50m3/ha/year or from centrifugation
at 80m3/ha/year.
2. The mayor can stop spreading operations if there is a chance of damage to the
environment or reduce the limits of acceptability.
Art. 3. Authorization
Spreading operations must be notified to the mayor 30 days before. Communication
must include: type of soil, spreading system, spreading time, hydrological condition.
Art. 4. Spreading systems
•
Distribution must be uniform and by-products must be ploughed in.
•
During spreading operation run off must be avoided.
29
Art 5. Prohibition
Spreading is forbidden, where:
•
Distance is less than 300m to the groundwater draining areas.
•
Distance is less than 200m to the built up areas.
•
Soil is used for growing vegetables.
•
Soil with a water table depth of less than 10 m.
•
Soil where percolation water could reach the water table.
Art. 6 Storage
As far as the storage is concerning:
•
Storage period max 30 days.
•
Storage must be in a water-proof container.
•
The mayor must be notified of storage location.
The following articles (Art 8. and Art. 9) refer to the sanctions and inspections,
respectively.
What is meant by agronomic utilization is defined in Legislativa Decree n. 152 dated 3
April 2006, entitled “Norme in materia ambientale” article 74 paragraph 1 letter p
envisages: “utilizzazione agronomica: la gestione di effluenti di allevamento, acque di
vegetazione residuate dalla lavorazione delle olive, acque reflue provenienti da aziende
agricole e piccole aziende agro-alimentari, dalla loro produzione fino all'applicazione
al terreno ovvero al loro utilizzo irriguo o fertirriguo, finalizzati all'utilizzo delle
sostanze nutritive e ammendanti nei medesimi contenute”
The norms on the olive mill waste, were later completed with the provisions enviseged
in the Ministerial Decree dated 06/07/2005 “Criteri e sulle norme tecniche generali per
la disciplina regionale dell’utilizzazione agronomica delle acque di vegetazione e degli
scarichi dei frantoi oleari” and with the relative regional resolution.
When the use of waste is not defined as “agronomic” use, the Cassation Court defined
the sphere of application “Al di fuori dell'applicazione agronomica per i residui oleari
non possono comunque trovare applicazione le disposizioni contenute nella L. n. 574
30
del 1996 ma vanno invece applicate le disposizioni generali in tema di inquinamento o
di rifiuti”. Cass., Sez. III, 27 Marzo 2007, N° 21773 In: Ambiente e sviluppo, 2007, 11
p.1024. e recentemente Cass., Sez. III, Sentenza del 24 Luglio 2012, N°30124.
1.5.3.1. Waste or by-product: regulations
Norms regarding waste, are established by the Legs. Decree dated 3 April 2006, n° 152
updated on 9 May 2012 with the “Modifiche al decreto legislativo 3 aprile 2006, n. 152,
e altre disposizioni in materia ambientale” establish by the Senate of the Repubblic.
The Legs. Decree n. 4 dated 2008 entitled "Ulteriori disposizioni correttive ed
integrative del decreto legislativo 3 aprile 2006, n. 152, recante norme in materia
ambientale" introduces important innovations in the definition of the by-product (art.
183, paragraph 1, lett. p), howevere in point 2 of lett. P it establishes that “ il loro
impiego sia certo, sin dalla fase della produzione, integrale e avvenga direttamente nel
corso del processo di produzione o di utilizzazione preventivamente individuato e
definito”. This definition has created doubts concerning the context in which the byproduct may be used, in particular if the production process or the use could be different
from the original one.
The “Directive 2008/98/EC of the European Parliament and of the Council of 19
November 2008 on waste and repealing certain Directives” proposes the necessity to
avoid “confusion between the various aspects of the waste definition, and appropriate
procedures should be applied, where necessary, to byproducts that are not waste, on the
one hand, or to waste that ceases to be waste, on the other hand”. In fact in Article 5
the by-product definition is given.
In Italy with Legs. Decree dated 3 December 2010, n. 205 the “Disposizioni di
attuazione della direttiva 2008/98/CE del Parlamento europeo e del Consiglio del 19
novembre 2008 relativa ai rifiuti e che abroga alcune direttive” have been defined.
In particular in Art. 12 “Sottoprodotto e cessazione della qualifica di rifiuto” after Art.
184 of Legs. Decree dated 3 April 2006, n. 152, Art. 184-bis is inserted; it defines the
“by-product”. In particular in point b of point 1 the context of use of the by-product is
31
defined; now it is separate from the initial production process. This article is cited
below:
“E' un sottoprodotto e non un rifiuto ai sensi dell'articolo 183, comma 1, lettera a),
qualsiasi sostanza od oggetto che soddisfa tutte le seguenti condizioni…
a) la sostanza o l'oggetto e' originato da un processo di produzione, di cui costituisce
parte integrante, e il cui scopo primario non e' la produzione di tale sostanza od
oggetto;
b) e' certo che la sostanza o l'oggetto sara' utilizzato, nel corso dello stesso o di un
successivo processo di produzione o di utilizzazione, da parte del produttore o di terzi;
c) la sostanza o l'oggetto puo' essere utilizzato direttamente senza alcun ulteriore
trattamento diverso dalla normale pratica industriale.
d) l'ulteriore utilizzo e' legale, ossia la sostanza o l'oggetto soddisfa, per l'utilizzo
specifico, tutti i requisiti pertinenti riguardanti i prodotti e la protezione della salute e
dell'ambiente e non portera' a impatti complessivi negativi sull'ambiente o la salute
umana.
2. Sulla base delle condizioni previste al comma 1, possono essere adottate misure per
stabilire criteri qualitativi o quantitativi da soddisfare affinche' specifiche tipologie di
sostanze o oggetti siano considerati sottoprodotti e non rifiuti. All'adozione di tali
criteri si provvede con uno o piu' decreti del Ministro dell'ambiente e della tutela del
territorio e del mare, ai sensi dell'articolo 17, comma 3, della legge 23 agosto 1988, n.
400, in conformita' a quanto previsto dalla disciplina comunitaria.”
In point 1 of Art. 184-ter the requisites necessary for the“Cessazione della qualifica di
rifiuto” is defined:
“Un rifiuto cessa di essere tale, quando e' stato sottoposto a un'operazione di recupero,
incluso il riciclaggio e la preparazione per il riutilizzo, e soddisfi i criteri specifici, da
adottare nel rispetto delle seguenti condizioni:
a) la sostanza o l'oggetto e' comunemente utilizzato per scopi specifici;
b) esiste un mercato o una domanda per tale sostanza od oggetto;
c) la sostanza o l'oggetto soddisfa i requisiti tecnici per gli scopi specifici e rispetta la
normativa e gli standard esistenti applicabili ai prodotti;
32
d) l'utilizzo della sostanza o dell'oggetto non porterà a impatti complessivi negativi
sull'ambiente o sulla salute umana.
At the end of point 5 of the above mentioned article it is established that: “La disciplina
in materia di gestione dei rifiuti si applica fino alla cessazione della qualifica di
rifiuto.”
As far as the use of pomace is concerned, until few years ago, not all types of olive
residues could be used for heating purposes. In fact the Italian decree dated 8 March
2002, mentioned that only olive pomace without physical or chemical treatment
processing could be used. However since 2006 with the D.Lgs n. 152 del 3/04/06 ed al
d.p.c.m. 8/10/04) all pomaces resulting from mechanical processing of agricultural
products, without chemical additives can be used and are considered a biomass fuel.
In Part 1, in the attachments of the fifth part, section 4 the “nuovo testo unico
dell’ambiente” defines the “Caratteristiche delle biomasse combustibili e relative
condizioni di utilizzo”. Point f of point 1, states that: ”Sansa di olive disoleata…ottenuta
dal trattamento delle sanse vergini con n-esano per l’estrazione con l’olio di sansa
destinato all’alimentazione umana, e da successivo trattamento termico, purchè i
predetti trattamenti siano effettuati all’interno del medesimo impianto; tali requisiti nel
caso di impiego del prodotto al di fuori dell’impianto stesso di produzione, devono
risultare da un sistema di identificazione conforme a quanto stabilito al punto 3”. The
point 3 is related to the “Norme per l’identificazione delle biomasse”. The normative
recognition of potential uses of these wastes represents a fundamental point for their
valorisation.
1.6. Environmental problems posed by olive-mill waste
Around 30 million m3 of by-products are produced annually in the Mediterranean area,
generating many environmental problems (Meksi et al., 2012) because of their polluting
effects on soil fertility and water (Piotrowska et al., 2011; Sierra et al., 2001) and its
potentially pathogenic consequences. Problems arise also from the fact that olive oil
production is seasonal; a large amount of waste is applied over a short period and
storage of such large amounts of liquid waste is difficult. Because of their
33
amphiphilicity a high fraction of phenols (>98%) is lost with the waste stream during
the mechanical extraction of olive oil (Rodis et al. 2002). In fact, the olive pulp is very
rich in phenolic compounds (Cardoso et al., 2005), but only 2.7% of the total phenolic
content of the olive fruit is released in the oil phase (Rodis et al., 2002) and the
remaining amount is lost in the VWs and in the pomace (Figure 10); the values shown
in Figure 10 refer to the pomace obtained from three-phases decanters, while the
percentage of phenolic compounds contained in the pomace obtained from two-phases
system are higher, because it consists also of the phenolic fraction generally present in
the vegetation waters.
Pomace
47.8%
Virgin olive oil
2.7%
Vegetation Waters
49.5%
Figure 10. Distribution of phenols in VOO and in by-products.
The phenolic components are the various phenolic acids such as caffeic, protocatechuic,
α-hydroxycinnamic, vanillic, then flavonoids, anthocyanes, and seleoprotein. The high
concentration of phenols provides a batteriostatic property to the VWs due to their
antimicrobial activity, so they become less biodegradable. Phenols are unstable and tend
to polymerise during storage into condensed high-molecular-weight polymers that are
very difficult to degrade (Crognale et al., 2006). The pollutant load of VWs is elevated
because of the maximum biochemical oxygen demand (BOD5) ranges between 15.000
and 120.000 mg/L and the chemical oxygen demand (COD) concentration of VWs
ranges between 40.000 and 220.000 mg/l. This high polluting power makes wastewaters
34
resistant to degradation and represents a severe environmental problem related to its
high organic content made up largely of simple phenolic compounds. The toxic load of
VWs in terms of phenolic compounds is estimated to be up to a thousand times greater
than that of domestic sewage (Niaounakis and Halvadakis 2004).
The antimicrobial and phytotoxic properties of olive mill wastes have been widely
investigated (Bonari et al., 1993; Capasso et al., 1992; Casa et al. 2003; Obied et al.,
2005). These phytotoxic effects are particularly evident during germination and seedling
development (Aliotta et al., 2000). Casa et al., (2003) and Asfi et al., (2006) showed
that phenolic compounds have the main phytotoxic properties using the durum wheat
and spinach (Spinacea oleracea L.), respectively, germinability as a biotest. El Hadrami
et al. (2004), reported that crude and undiluted VWs was lethal when applied to the
crops studied, i.e., maize (Zea mays L.), chickpea (Cicer arietinum L.), tomato and
wheat. Boz et al. (2003) studied the herbicidal effect of solid and liquid olive mill waste
on common weeds in wheat and maize crops, finding a high inhibition of the
germination of Portulaca oleracea species. However, Cayuela and co-workers (2008)
reports that inhibition of seed germination is species-specific and therefore the toxic
effect of these compounds should be investigated for each crop-weed system. Waste
spreading can be carried out without significant crop yield losses, at rates lower than 40
m3 ha-1, during the advanced growth stage of winter cereals (Bonari et al., 2001), on the
contrary spreading of higher rates before sowing caused phytotoxic effects and
decreases in yield, unless at least 60 days were allowed to elapse between spreading and
sowing (Bonari and Ceccarini, 1993).
The uncontrolled disposal of VWs can lead to an alteration in the balance of soil
(Moreno et al., 1987; Paredes et al., 1986) that results in anomalous fermentation of the
organic compounds, changing the environmental conditions for the microorganisms and
the reduction of soil fertility, also the sugar content of VWs leads to an alteration of the
invertebrate community (Cicolani et al., 1992). Moreover, the discharge of olive oil mill
waste into soils leads to the release of heavy metals retained in the waste, such as Pb,
Fe, Cu, Zn, Mn. The solubilisation tends to be a pH-dependent value for some of these
metals. The discharge of VWs can also affect the movement of pesticides in soil and in
streams. Furthermore, the olive mill waste is sometimes disposed in the latter and
35
nearby rivers with a considerable impact on the waters; as a result of these malpractices
many rivers in Spain and Italy become anoxic (Cabrera et al., 1984; Di Giovacchino et
al., 1976). The subsequent change in colour of these rivers, due to the oxidation and
polymerization of tannins giving darkly colored polyphenols, is difficult to be removed
(Hamdi, 1992).
Numerous studies have been carried out in order to solve the VWs disposal problem and
at the same time valorise it, through physico-chemical or biological pre-treatments to
reduce the pollutant load. Concerning this topic, treatments with fungi have been
successfully used to mitigate its phytotoxicity (D’Annibale et al., 1998; Kissi et al.,
2001; Sayadi and Ellouz, 1993) such as Pleurotus (Tsioulpas et al., 2002) and Lentinula
edodes (D’Annibale et al., 2004) whose cultures in VWs are able to reduce the total
phenol content by about 88% in 240 hours. This effect was preferentially exerted on
ortho-diphenolic structures, such as catechol, 4-methyl-cathechol and caffeic acid
(D’Annibale et al., 2004). Furthermore, some researchers have found that toxicity is
also found despite of the total removal of phenols, suggesting that other chemical
products contribute to the total amount of toxicity (Capasso et al., 1992; Greco et al.,
2006); it seems to be attributed to long-chain fatty acids and volatile acids (Linares et
al., 2003; Ouzounidou et al., 2008; Paixao et al., 1999). Several research works are
directed towards the exploitation of microorganisms isolated from VWs or from
substrates treated with it over long periods; an example is provided by Azotobacter
vinelandii strains originating from soil treated with VWs and used as an inoculum for
the aerobic biodegradation of the VWs; it was found to reduce the phytotoxicity
significantly by reducing COD (70% after three days) and phenolic compounds, up to
100% within seven days, with consequent enhancement of soil fertility (Ehaliotis et al.,
1999; Piperidou et al., 2000).
1.7. Possible uses of olive-mill waste
The growing interest in environmental protection has promoted studies on the
evaluation of the possible uses of the olive industry by-products.
36
1.7.1. Vegetation water
Numerous studies have suggested the use of vegetation waters as a substrate for the
production of methane (Fiestas Ros de Ursinos et al., 1982). Another use is the
production of pectic enzymes (Federici et al., 1988) through the use of the
microorganism Cryptococcus albidus var. albidus, strain IMAT-4735 grown on suitably
treated VWs. Vegetation water and in general all the olive industry by-products is also
used as a fertilizer (Garcia-Ortiz et al., 1999; Tejada and Gonzalez, 2004a), mixed with
irrigation water (Briccoli-Bati and Lombardo, 1990) and in animal rations (Martilotti,
1983). VWs is an organic ammendant, according to Italian law, so it is a soil fertilizer
and has been considered as an inexpensive method of disposal and recovery of the
mineral and organic components present and a valid method of improving soil fertility
(Di Giovacchino et al., 1990, 2000). The greatest benefits of the effective use of the
plant nutrients of the waste, mainly K, N, P, and Mg the fact that they are a low cost
source, an important resource for irrigation and supply of organic matter for the soil,
which enhances microbial activity and improves the physical and chemical properties of
soil. However, this use as a fertilizer is controversial, mainly due to the presence of
phytotoxic compounds and their toxic effects, especially phenols, high mineral salt
content and low pH.
Nevertheless, high concentration of antioxidants provides nutraceutic properties to VWs
and the interest of the pharmaceutical industries in natural antioxidants is growing
constantly. VWs could represent an important source of polyphenolic compounds; in
fact as the phenolic compounds in virgin olive oil, the waste water extracts have shown
powerful antioxidant activity in vitro (Visioli et al., 1999). Visioli and co-workers
(2000a) have shown that in rats the intake of a hydroxytyrosol-rich VWs extract (10
mg/kg) was associated with an increase in the antioxidant capacity in plasma. Another
human study tested the antioxidant effect of a hdroxytyrosol-rich phenolic extract from
VWs, administered during breakfast to patients with uncomplicated type I diabetes; the
main result was the plasma enhanced antioxidant capacity, hence, an antiaggregating
platelet action (Leger et al., 2005). Concerning the molecules responsible for the
protective effect, Angelino and co-workers 2011, found in VWs a bioactive a
37
phytocomplex made up of hydroxytyrosol (3,4 DHPEA), tyrosol (p-HPEA) and the
dialdehydic form of decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl)
ethanol (3,4-DHPEA-EDA); this is able to permeate the cell membrane, exhibiting
antioxidant activity inside the red blood cells.
One of the methods used for recovering hydrophilic phenols and reduce polluting power
from VWs is a process consisting of three consecutive membrane-filtration steps with
decreasing cut-off values: microfiltration, ultra filtration and reverse osmosis, facilitated
by previous enzymatic treatment (Ghanbari et al., 2012; Paraskeva et al., 2007; Servili
et al., 2011). The products obtained is a crude phenolic concentrated that has been
proven to improve VOO phenolic content when is re-utilised in a virgin olive oil
extraction process (Servili et al., 2011) and a permeate without any phenolic
compounds and organic residue. Another extraction method for phenolic compounds is
super critical fluid extraction with carbon dioxide.
1.7.2. Pomace
Traditionally this by-product is sent for further treatment to obtain an olive pomace oil.
The recovery is carried out by extraction with solvent, and by subsequent
deacidification and bleaching, in order to make the oil edible. After refining, olive
pomace oil is mixed with virgin olive oil and then it is eligible for the commercial class
of olive pomace oil as defined by European Community (EC) Regulation No. 356/92.
The refining process generates a final solid waste, whose main use is as a fuel resource,
in drying ovens or steam boilers because of its thermal capacity, even though,
environmental problems associated with smoke emission from burning have led to
restrictions on this practice. The use of olive pit for the production of bio-energy is very
important, mainly from an environmental point of view; to confirm this in a recent study
Russo et al., 2008 applied Life Cycle Assessment (LCA) methodology to compare the
environmental performance of the recovery of by-products from the olive-oil sector
with that of wood-pellet production, and the results showed that the recovery of olive pit
offers environmental advantages with respect to other alternative fuels, mainly due to
38
the higher net calorific value and also the simple recovery method. Therefore, with the
continued production of these by-products the need for utilization strategies represent an
important step. Further possible uses of pomace are gasification (Ollero et al. 2003) and
recycling of pomace as a soil amendment (Brunetti et al., 2005; López-Piñeiro et al.,
2006).
Another possible use of olive pomace is in the production of "compost" that is an
aerobic exothermic process implemented by micro-organisms that make a partial
biodegradation and bioconversion of organic matter that provides a final product
fertilizer for agricultural use. This shows significant improvements of the physicalchemical characteristics of soils due to the nitrogen, phosphorus and organic carbon
deposited. Many studies looked into the utilization of olive cake for composting
purposes (Alburquerque et al., 2004; Giannoutsou et al., 2004). Pomace contains a large
amount of organic matter (90%), and valuable nutrients, especially potassium, and this
makes pomace a valuable resource. Pomace has also been successfully tested as a foliar
fertilizer (Tejada and Gonzalez, 2004b) and as a soil-less substrate in combination with
peat (García-Gómez et al., 2002).
Several studies showed the high potential of olive mill wastes as biobased pesticides
against several species of fungi, weeds and nematodes. In particular Cayuela et al.,
(2008) reports the nematicidal potential of pomace and pomace compost which is able
to pass through the nematode egg.
Compared to traditional uses, new uses of pomace, such as the use of virgin pomace in
agriculture and in floro-vivaistic productions, and the use of olive stoned pomace as
supplements in animal feeding in the livestock sector are being evaluated
1.8. Animal feed supplements
The valorization of by-products from various food industries, is nowadays an important
area of research. Concerning this topic, in 2007 the FAO conducted a study entitled
"Feed supplementation blocks", on the use of solidified blend of agro-industrial byproducts as supplement in ruminants feeding, carried out in areas of the world where the
39
farming conditions is difficult, such as India, Bangladesh, China, Thailand, Sri Lanka,
Vietnam, Venezuela, Pakistan, Sudan, Malaysia, and parts of tropical Africa are
collected. Ruminants in arid, semi-arid or mountainous areas rely almost exclusively on
pasture as a major component of their diet, however a wide range of secondary
compounds, particularly tannins, have been implicated as anti-nutritional components of
shrubs and trees, and these limit the nutritional potential, reducing the absorptionprocess. This is mainly due to the ability of tannins to form complexes with proteins,
carbohydrates, amino acids and minerals.
However, it is now recognized that these phenolic compounds can be toxic, innocuous
or beneficial depending on the type, chemical structure, amount administrated and
animal species (Makkar, 2003; Mueller-Harvey, 2006; Toral et al., 2011).
The negative effects of tannins include inhibition of digestive enzymes, hence, lower
digestibility, loss of endogenous protein and systemic effects as a result of the
absorption of hydrolysable tannin degradation products from the digestive tract.
Furthermore tannins limit N availability (Mangan, 1988).
Vermeire and Wester, (2001) highlighted the fact that animal performance is reduced
when tannins exceeded 5% of forage weight, and Hegarty et al., (1985) assessed that
both condensed and hydrolysable tannins can exert detrimental effects, however tannins
can also exert a preventive action against gastrointestinal parasitism (Paolini et al.,
2002). Several methods have been tested to de-activate tannins and reduce the negative
effect of these in animal nutrition; the use of Polyethylene glycol (PEG) is the only
approach that is proven to reduce the negative effect of tannins by forming complexes
with them and seems to be the more effective way to increase the nutritive value of tree
foliage and shrubs as found in goats (Ben Salem et al., 2003; Decandia et al., 2000;
Titus et al., 2001), and in sheep (Villalba and Provenza, 2002). PEG was usually
incorporated into feed blocks that are an efficient supplement for increasing intake,
rumen fermentation, digestibility and daily weight gain. The use of solid feed
supplementation blocks provides nitrogen, the minerals and vitamins missing in fibrous
feeds; other advantages are ease of transport and storage.
Furthermore feed blocks can be used as a carrier for some additives such as minerals, to
increase reproductive performance or anthelmintic drugs for the control of
40
gastrointestinal parasites. Formulation of this supplement includes one or more binders
such as lime, cement or bentonite, common preservatives such as salt, urea as a source
of non-proteic nitrogen, molasses and other ingredients to ensure an adequate supply of
energy, nitrogen and minerals. "Molasses-free" blocks have demonstrated equivalent
benefits both in terms of degree of acceptance by ruminants and the nutritional value of
low quality roughage. The development of "mini-blocks" without urea, has facilitated
integrated feeding for rabbits (Ramchurn and Ragoo, 2000). Most research on feedblocks has been published over the last three decades (Sansoucy, 1995, 1996) however
Cordier (1947) highlighted that the use of these feed supplements was already
implemented in Tunisia in 1930. In recent years, about 25 formulations have been used
(Ben Salem and Nefzaoui, 2003) and at least 60 countries have adopted this type of
dietary supplement for ruminants in difficult conditions, especially cows, sheep and
goats. The widespread use of these blocks in the world reveals the importance of this
supplement in the livestock sector and the improvement in revenue for farmers and
ranchers. Their using in fact, not only provides a continuous and balanced supply of
energy and nutrients such as nitrogen, minerals and vitamins a low cost, but it is also
represents an effective method for the enhancement of agro-industrial by-products with
a high moisture content which are not always eco-compatible and otherwise expensive
to dispose of.
1.9. Olive pomace in livestock feeding
In the last 50 years olive growing has undergone an expansion due to strong demand;
furthermore the increasing interest concerning sources of fats with a healthy fatty acid
profile has led to an increase in production, moreover the use of by-products as sources
of nutrients for animals can improve the economy and the efficiency of agricultural,
industrial and livestock production (Molina-Alcaide, 1996). In Italy from the early
decades of the 1900s, several studies were conducted to assess the effect of olive
pomace feeding on animals (Gugnoni, 1920; Maymone and Giustozzi, 1935) and the
41
results showed the positive effect of pomace intake as a feed integrator on the end
products.
The main types of pomace that are subjected to the drying process are virgin olive
pomaces, pomaces destoned in pre-extraction and pomace destoned in post-extraction,
among these, only the last two are the more suitable for a zootechnical use. The use of
virgin olive pomace can, however, be evaluated, but only after its enzymatic
stabilization. This process is involved in the degradation of the polysaccharide fraction
of the pomace, which consists mostly of cellulose, pectins and hemicelluloses,
increasing the amount of soluble fiber and consequently its dietary value as animal feed
(Servili et al., 2007b).
1.9.1. Digestibility
The low degradable fraction of dry matter, crude protein and NDF of olive by-products
are the most important limitation factors in ruminant nutrition. In fact, crude fibre is
mainly constituted of lignin, which limits the feed value of olive cake; furthermore a
large proportion of protein is linked to the ligno-cellulose fraction decreasing the biodigestion of pomace. This evidence induced many studies to improve the nutritive value
of olive pomace. However, among domestic ruminants, goats seem to be the ones most
suitable for utilizing these high lignin-cellulose and low protein forages (Beede et al.,
1986; Molina-Alcaide et al., 1997) while the other ruminants require an adequate
supply of protein nitrogen for the ruminal microorganisms in addition to specific deactivating tannin compounds (Molina-Alcaide et al., 2003).
Alkali treatments have been the most studied procedures, treatments with NaOH seems
to improve in vitro digestibility (Abdouli, 1979). Another trial study was carried out by
Nefrazoui (1986), ensiling the pomace with different levels of sodium hydroxide and
ammonia on a laboratory scale and assessing the digestibility of the pomace as feed for
sheep; the main results were that the ammonia treatment increased the organic matter
digestibility by 4 % units and the nitrogen retention by 6%.
42
The partial destoning is considered a valid economic possibility to improve the nutritive
value of this by-product and increase its digestible content. Sadeghi et al., (2009) in a
recent study evaluated the effect after feeding ewes with four different olive pomaces:
crude, exhausted, partly destoned and partly destoned exhausted pomace. These were
included in experimental rations of ewes and tested the effect on fattening performance.
The results of this trial show that destoned olive pomace has a higher nutritive value
than the other pomaces and improves body weight gain and growth rate.
1.9.2. Effect of olive pomace supplementation on fatty acid composition of milk and
meat
Several studies have shown positive effects arising from the use of virgin olive pomaces
as animal feed supplements such as the increase in the amount of unsaturated fatty acids
from the meat and milk of ruminants (Molina-Alcaide et al., 2008), increased oxidation
stability of both milk and cheese products, and an increase in vitamin E (α-tocopherol)
in milk fat (Pauselli et al., 2007; Servili et al., 2007). The intake of SFAs is strongly
related to an atherogenic and thrombogenic risk (Givens, 2005). At this regard, it has
been estimated that for every 1% reduction in dietary SFA intake, there is an associated
3% decrease in cardiovascular risk (Minihane, 2006). Therefore olive pomace with its
high amount of oleic acid has been considered in dairy ewe nutrition to modify the fatty
acid composition of milk (Chiofalo et al., 2004) increasing the unsaturated-saturated
fatty acid ratio, improving oleic acid content and the oxidative stability of milk due to
its high content of antioxidants (Chiofalo et al., 2004; Pauselli et al., 2007).
Another study conducted by Vera and co-workers (2009) evaluated lamb carcass quality
and fat composition providing a destoned dry pomace-based ration, instead of a
conventional ration or pasture feeding. The results showed a decrease of the amount of
saturated fatty acid and an increase in monounsaturated fatty acid with a high increase
in oleic acid. Pork fed with olive pomace (10% inclusion rate) was significantly lower
in saturated fatty acids, polyunsaturated fat and ω-6 fatty acids (p<.05) compared to the
control diet, evaluated on the Longissimus muscle (Doyle et al., 2006).
43
1.9.3. Effects of olive pomace supplementation on animal’s health
Olive stone removal before the crushing can reduce the phenolic oxidative degradation,
during processing providing by-products having an increased content of phenols. In fact
as previously shown, about 48% of the total phenolic content of the olive fruit, is
transferred into the olive pomaces (Figure 10), consequently the virgin pomaces
destoned in pre-extraction are more richer in natural antioxidants, with a residual oil
content ranging from 8% to 15% (Dal Bosco et al., 2007; Pauselli et al., 2007; Servili et
al., 2007a, 2007b). Stoned pomaces have a fatty acid composition similar to that of
virgin olive oil and also show a high concentration of bio-active phenolic compounds
such as lignans and secoiridoids. For this reason, the virgin destoned pomace is
considered as a good supplement animal feed in the livestock sector and an important
source of monounsaturated fatty acids and antioxidants.
The use of dried destoned pomace could be considered interesting in dairy production to
increase the oxidative stability of milk and cheeses. Several studies have shown that the
phenolic compounds of pomace are able to prevent lipid oxidation with a potent free
radical scavenging activity (Amro et al., 2002).
The effect of pomace antioxidant and radical scavenging activity, such as
hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and their secoiridoid derivatives
(dialdheydic form of decarboxymethyl elenolic acid, 3,4-DHPEA-EDA or p-HPEAEDA) as well as verbascoside, is proven by several studies.
Dal Bosco et al., (2012) shows the effects on a rabbit diet of including (5%) pomace
from different olive cultivars characterized by different phenolic concentrations; this
feed integration leads to an increase in oxidative stability and nutritional value, as
revealed by the low concentration of lipid peroxidation and the high nutritional indexes,
the greater the increase, the greater the phenol content. This shows how pomaces with
high polyphenols content are able to prevent the oxidation of unsaturated lipids,
contributing to the preservation of the dietetic-nutritional value of the meat, moreover,
also stone removal contributed to reducing the oxidative degradation of phenols,
considering that seed has the highest peroxidative activity (Dal Bosco et al., 2007).
44
1.9.4. Storage
The use of pomace as a supplement animal feed in the livestock sector has been taken
into consideration even if the production is seasonal and therefore requires adequate
techniques for storage (Molina-Alcaide and Yáñez-Ruiz, 2008). Concerning this topic
the main problem is related to the high water content and the large amount of oil
retained, which quickly becomes rancid when exposed to air. It has been estimated that
pomace obtained by centrifugation deteriorates after 4-5 days, whereas pomace obtained
by pressure deteriorates after about 15 days (Sansoucy, 1985).
Silage has been reported to be an efficient procedure to preserve pomace, alone
(Hadjipanayiotou, 1999), or mixed with other feed (Hadjipanayiotou, 1994) or with urea
(Al-Jassim et al., 1997) or with alkali. In particular the ensiling technique can be used
for long term storage of pomaces, and can partially replace conventional roughage in
diets of mature, growing and lactating ruminants (Hadjipanayiotou, 2000). Often
molasses is added in order to supply water soluble carbohydrates necessary for the
ensiling fermentation. In a recent study it was concluded that molasses added at 3%
could improve the ensiling fermentation (Weinberg et al., 2008) of pomaces without
substantial losses, while with a higher application of molasses more yeasts developed
and ensiling losses increased. However results from a recent study conducted by
Rowghani and Zamiri (2007), indicated that treating pomaces before ensiling with 8%
molasses, 0.4% formic acid and 0.5% urea could provide a good and economical source
of a non-conventional feed and help to improve the diet formulation for ruminants.
Vera et al., (2009) evaluate the stability of destoned, unexhausted olive cake,
originating from the two-phase process dry pomace over time, the results showed that
dehydrated pomace can be preserved for several months with no detrimental effects on
its lipid composition and quality.
45
1.10. Unsaturated fatty acid sources as supplements in animal feeding
Dairy products are the main source of 12:0 and 14:0 in the human diet and make a
significant contribution to 16:0 and trans fatty acids intake (Shingfield, 2008a). To
improve the quality of milk from a nutritional point of view is one of the most important
purposes of the milk industry.
The reduction of saturated fatty acids, in favour of polyunsaturated fatty acids,
increasing the content of n-3 series, including α-linolenic acid (ALA), has been proven
to have beneficial effects. In particular ALA is recognized as minimizing the risk of
cardiovascular disease, modulation of the inflammatory response, and shows a positive
impact on both central nervous system function and behaviour (Stark et al., 2008). Also
the increase in concentration of conjugated linoleic acid (CLA) has been reported to
decrease tumorigenesis in animals both in vitro and in vivo (Deker, 1995; Ip et al.,
1994; Parodi, 1997). CLA refers to a group of polyunsaturated fatty acids that exist as
positional and stereo-isomers of conjugated dienoic octadecadienoate (18:2) and the
predominant geometric isomer in foods is the c9, t11-18:2, also called “rumenic acid”
(Kramer et al., 1998). The diet influences milk composition and fatty acid profile,
hence, the use of vegetable lipid sources aimed at increasing the level of MUFA, PUFA
and CLA in milk has been tested in numerous studies both in cattle and in sheep (Castro
et al., 2009; Gómez-Cortés et al., 2011; Mele et al., 2007; Zhang et al., 2006a, 2006b).
However, is well known the importance of ingestion of grass grazing directly, on the
level CLA in milk and its derivatives both in cattle (Bargo et al., 2006) and in the sheep
(Addis et al., 2005); in fact the highest concentrations of CLA in milk fat and
unsaturated fatty acids (UFA) are mostly found in milk from animal diet based on long
periods of pasture feeding. Studies on grazing cows shows high proportions of UFA and
more CLA content than cows fed just on silage, which on the contrary shows an
increased proportion of saturated fatty acids (Elgersma et al., 2004; GonzalezRodríguez et al., 2010).
The two main sources of linolenic acid are grass, both fresh and ensiled (Boufaied et al.,
2003), and linseed (Doreau et al., 2009). Linseed is particularly rich in ALA and it has
been proven that its intake can lead a significantly increase in linolenic acid content in
46
both milk (Loor et al., 2005) and meat (Normand et al., 2005). These benefits are
independent of the form of administration; whole seed (Ward et al., 2002), ground seed
(Collomb et al., 2004), extruded seed (Schori et al., 2006), or oil (Dhiman et al., 2000).
Oilseeds can be either provided as whole seeds or processed by different techniques, the
most common is the extrusion. The animal products, after linseed intake are enriched in
rumenic acid, which has been proven to have positive effect on human health,
decreasing the incidence of cancer (Shingfield et al., 2008b). The moderate amount of
linseed added to the diet has no detrimental effects on organic matter or fiber digestion
(Ueda et al., 2003). Concerning this topic, Doreau et al., (2009), investigates the
consequences of a linseed supply on the changes in organic matter and fiber digestion
comparing three linseed forms; rolled, extruded and oil. They found that linseed
supplementation increases the duodenal flow of unsaturated intermediates of
biohydrogenation, and this effect is more evident using extruded seeds. The ability of
extruded linseed in modifying fatty acids composition and conjugated linoleic acid
concentration, by dietary supplementation, were assess in several studies, in ewes (Mele
et al., 2007) in goats (Nudda et al., 2006) and cows (Pezzi et al., 2007). Hurtaud et al.,
(2010) found that increasing the amount of extruded linseed dietary ratio in dairy cow
feeding leads a linearly decrease of milk fat content and globule size and at the same
time a linearly increase the percentage of milk unsaturated fatty acid, specifically αlinolenic acid and trans fatty acid. A similar result was obtained by Oeffner et al.,
(2013) whose have been shown that the increase of supplementation rates of extruded
linseed improved the fatty acid profile of milk, butter, and cheese gradually with a
simultaneous decrease in saturated fatty acids in serum in Holstein cows.
Mele and co-workers (2010) studied the milk fatty acid composition in Sarda ewes,
particularly evaluating the change in rumenic acid content, in dietary supplementation
with whole extruded linseed over a long period (70 days). The results showed that
concentrations of cis-9, trans-11, and vaccenic acid (VA), reached the highest levels of
enrichment after 7-8 weeks (3.06, 7.31 g/100 g milk fat for RA and VA, respectively),
furthermore, the milk from the ewes fed with linseed showed a significant reduction:
(-17%) in saturated fatty acid concentration in the milk, improving its quality and
providing it of important transferable properties.
47
2. PURPOSE OF THE THESIS
This research project is aimed at the valorisation of two types of pomace obtained from
the extra virgin olive oil mechanical extraction process such as olive pomace and a new
by-product named ‘paté’. For this purpose, these by-products have been used in the
livestock sector as important sources of antioxidants and unsaturated fatty acids. This
work project includes two parallel researches:
Research 1. The suitability of dried stoned olive pomace as a dietary supplement for
dairy buffaloes was evaluated. The effectiveness of this utilization in modifying fatty
acid composition and improving the oxidative stability of buffalo milk and mozzarella
cheese was assessed by means of the analysis of qualitative and quantitative parameters;
Research 2. The use of paté as a new by-product in dietary feed supplementation for
dairy ewes, already fed with a source of unsaturated fatty acids such as extruded linseed,
was studied in order to assess the effect of this combination on the dairy products
obtained. The characterization of paté as a new by-product was also carried out,
studying the optimal conditions of its stabilization and preservation at the same time.
The transfer of bioactive compounds and the improvement of the quality of milk fat
could positively interact in the prevention of some human cardiovascular diseases and
some tumours.
48
3. MATERIALS AND METHODS
3.1. Research 1: olive pomace supplementation in buffalo feeding
This research project was carried out in 2008; however in the following year (2009) a
partial repetition of the trial was carried out, in order to evaluate how the processing
system to obtain mozzarella cheese could affect the difference in the fatty acid
composition of the lipid fraction, due to dietary treatment. The phenolic and acidic
composition of the pomace, the fatty acid composition of the milk obtained, mozzarella
fatty acid composition and its oxidative stability were evaluated for the year 2009. The
experimental plan implemented in the second year and the feed used were the same as
those of the previous year, except for the amount of the daily pomace ration that was
slightly higher: 1.05 kg DM/d of pomace per head in 2008 vs. 1.2 kg DM/d of pomace
per head in 2009.
3.1.1. Production of stoned olive pomace
The fresh pomaces were obtained, in 2008 and 2009, from virgin olive oil mechanical
extraction using the following operative conditions: the olives were stoned and malaxed
for 40 min at 25°C, the oil extraction was performed using an RCM Rapanelli three
phase decanter mod. 400 eco. The stoned pomaces were stored at room temperature for
a maximum period of 36 hours before drying, then were dried using a fluid-bed dryer,
with a capacity of 20 kg/hour of evaporated water, following the process defined by
Servili et al., (2007a); the initial temperature of the flow of drying air was 120°C and
the maximum temperature of the pomaces during the drying process was 45°C. After
drying, the dried stoned olive pomaces (DSOP) were stored at room temperature.
49
3.1.2. Animals and diets
Sixteen pluriparous Mediterranean buffaloes were used, divided at the beginning of the
trial into two uniform groups: control and experimental. The two groups were not
statistically different for the following parameters: milk production (2192 and 2102 kg)
and duration of the lactation (254 and 252 d) of the previous year; distance from calving
(51 and 43 d), milk production (9.71 and 10.18 kg/d), body condition score (BCS) (6.44
and 6.31) and weight (617 and 653 kg) at the beginning of the trial. The trial lasted 40
days. The animals were weighed at the beginning and the end of the trial and the
nutritional status was determined using the BCS, utilizing the scale of Wagner et al.
(1988) modified, for the buffalo species, by Campanile et al. (1998); this method
provides for the use of a score from 0 to 9. The feedstuffs used were second cut alfalfa
hay, corn silage and two concentrates especially formulated using the same feed
materials (Table 7), the experimental concentrate contained DSOP (15.50% as fed). The
control and experimental diets were isoenergetic and isoproteic. Both the diets had the
following formulation dry matter basis: second cut alfalfa hay 20%, maize silage 42%,
and concentrate 38%. The feeding was by group and the feed was given as a mixed
ration once a day; 17 kg DM/d was administered to each animal and each buffalo in the
experimental group received 1.05 kg DM/d (2008) and 1.2 kg DM/d (2009) of DSOP
which represented respectively 6.17% and 7.05% of the diet. The ingestion of 17 kg
DM/d per head for each group was constantly maintained for the duration of the trial; in
this phase of lactation, this quantity represents the maximum ingestion capacity
(Bartocci et al., 2006).
50
Table 7. Formulation of the two concentrates (% as fed).
Items
Control
Bran flour
Maize flour
Soya extract flour
Flour from distillery residues
Flour of dehydrated alfalfa
Flour from sunflower extract
Flour from beet pulp
Flour from stoned olive pomace
Molasses
Vitamin-mineral supplementation
27.00
21.00
12.00
10.00
10.00
9.00
3.50
3.00
4.50
Experimental
Concentrates
20.00
25.00
15.00
5.00
12.00
15.50
3.00
4.50
3.1.3. Chemical analyses of animal feeds
The following analytical determinations were frequently performed on samples of the
feedstuffs used: dry matter (DM), crude protein (CP), crude fibre (CF), ether extract
(EE) and ash (AOAC, 1995); neutral detergent fibre (NDF), acid detergent fibre (ADF)
and acid detergent lignin (ADL) according to the method reported by Goering and Van
Soest (1970), the non-structural carbohydrates were calculated according to the method
reported by Van Soest et al. (1991).
The phenolic compounds of DSOP were extracted following the procedure described by
Servili et al. (2011), using 10 g of the sample and reported below:
Preparation of the aqueous extract: 10 gr of the sample was homogenized with 100 ml
of a mixture of methanol and water (80:20, v/v) containing 20 mg/L of sodium
diethyldithiocarbamate (DIECA); the extraction was performed in triplicate. After
methanol removal, using rotavapor, the aqueous extract was used for SPE phenol
separation.
Solid phase extraction (SPE): the cartridge was activated with 10 ml methanol + 10 ml
of distilled water and then dried with N2. Than the SPE procedure was applied by
loading 1 ml of the aqueous extract into an Extraclean High-load C18 cartridge (Alltech
51
Italia S.r.l.) and eluting with methanol (50 ml). After solvent removal under vacuum at
30°C, the phenolic extract was resolubilized with 5 ml of methanol and after sovent
removal, re-dissolved in methanol (1 ml).
Sample preparation for HPLC analysis: the phenolic extract was recovered with 1 ml of
methanol, filtered with 0.2 m PVDF filter and recovered in a vial.
The HPLC-DAD/FLD analyses of the phenolic extracts were conducted according to
Servili et al. (2011) with an Agilent Technologies system, model 1100, composed of a
vacuum degasser, a quaternary pump, an autosampler, a thermostatted column
compartment, a DAD and a fluorescence detector (FLD). The C18 column used was a
Spherisorb ODS-1 measuring 250 mm X 4.6 mm with a particle size of 5 µm (Phase
Separation Ltd., Deeside, United Kingdom); the injected sample volume was 20 µl.
To extract the residual oil from the DSOP, 250 ml of hexane was added to 100 g of
DSOP. The mixture was homogenized with the Ultra-Turrax T50 (IKA-Labortechnik,
Staufen, Germany) at 6000 rpm for 10 minutes at 20°C and then filtered with filter
paper. The extraction was performed twice. The filtered homogenate was evaporated
under vacuum in a nitrogen flow at 35°C. In the residual oil, the acidic composition was
determined according to the 1989/03 Commission Regulation (2003).
Feed fatty acid composition was ascertained by gas chromatography, as methyl ester
derivatives after transesterification with sulphuric acid. A MEGAL FISONS gas
chromatographic apparatus was equipped with a detector and a capillary column (D-B
Wax 30 m in length, 0.25 mm in thickness and 0.25 µm in thickness of the internal film)
with helium as the carrier gas. The temperature was programmed at 130°C for 5 min
followed by an increase to 230°C at a rate of 20°C/min. The temperatures of the injector
and detector were 270°C and 260°C, respectively. The peak areas of the individual fatty
acids were identified by means of previous fatty acid standard injection (Sigma-Aldrich)
and quantified as a percent of total fatty acids.
The extraction of tocopherols, tocotrienols and retinol from the feeds was carried out
according to the method of Havemose et al. (2004), by means of chromatography.
52
3.1.4. Buffalo milk and mozzarella cheese analyses
Milk production, representative of two milkings at intervals of 12 h, was determined at
the beginning of the trial and after 15, 30 and 40 days. The individual milk samples,
representative of the two milkings, taken at each control, underwent the following
analytical determinations: fat, protein (Nx6.38), lactose, urea and pH (ASPA, 1995).
The parameters for milk coagulation were determined: rennet clotting time (r), curd
firming time (k20), curd firmness (a30) by means of the thromboelastograph Formagraph
as reported by Zannoni and Annibaldi (1981).
During the last two weeks of the trial, the bulk milk of the two experimental groups was
processed according to the following artisanal method: a pilot plant consisting of
thermoregulated (37°C) vats and the natural whey culture (“cizza”) were used. The
natural starter was added to the vat milk to bring the acidity of the mixture to 10°SH.
Eight cubic centimeters (1:20,000) of liquid rennet was added to obtain milk clotting
within 20 min, as required by the traditional method of cheese making. After one hour,
the curd was cut and left to rest for approximately five hours under serum. Then, the
curd was stretched in hot tap water (95ºC), and afterward cheese pieces of about 70 g
were docked. The cheese pieces were then cooled in cold water for 1 h and then kept at
room temperature in skim water resulting from the stretching, diluted with skim whey
from previous manufacturing up to 5-6 °SH (pH 3.842) and 1% NaCl (referred to in this
study as “governing liquid”).
After each processing, the mozzarella and governing liquid samples were collected
according to the following procedure; Mozzarella was divided into two halves and was
subsequently homogenized with an Omnimixer homogenizer at low speed without other
solutions. The governing liquid filtered with Whatman N°1 filters.
The milk and mozzarella fatty acid composition were evaluated by gas chromatographic
analyses as were the methyl ester derivatives after transesterification with sulphuric acid
following the procedure reported above for the animal feeds samples. To evaluate the
risk of coronary disease, atherogenic and thrombogenic indexes were calculated as
suggested by Ulbricht and Southgate (1991). Tocopherol, tocotrienol and retinol
extraction was conducted according to the method proposed by Havemose et al. (2004).
53
The evaluation of the oxidative stability was carried out by the determination of
thiobarbituric acid reactive substances, expressed as mg/L of malondialdehyde
according to the method proposed by Fenaille et al. (2001).
3.1.5. Analysis of the phenolic compounds of milk
A specific method has been developed for the extraction and purification of the phenolic
fraction from milk, and it is reported at the end of “Results and Discussion” section.
For LC-MS/MS analysis, 20 µl aliquots of purified phenolic fraction, were injected into
an HPLC system consisting of two 218 pumps and a Varian 1200L triple-quadrupole
mass spectrometer equipped with an electrospray ionization interface (Varian, Palo
Alto, CA). A Varian C18 Intertsil ODS-3 column (250 x 4.6 mm, 5 µm) was used as the
HPLC column. Elution was performed at a flow rate of 1.0 ml/min using a mixture of
water/acetic acid (99.8:0.2 v/v) (solvent A) and methanol (solvent B) as mobile phases.
The gradient was changed as follows: to 95% solvent A for 2 min, to 75% solvent A in
8 min, to 60% solvent A in 10 min, maintained for 2 min, to 100% solvent B in 4 min,
maintained for 14 min, and returned to initial conditions in 7 min. The total run time
was 47 min. Data were acquired using the Varian MS Workstation data system. A
splitting valve allowed a solvent flow of 0.2 ml/min from the HPLC to the electrospray
source. LC MS/MS analysis was performed in negative multiple reaction monitoring
(MRM) mode. The mass spectra were obtained under the following conditions: a spray
voltage of 4500 V, shield voltage of 600 V, capillary voltage of 60 V, nebulizing gas
pressure of 50 psi, drying gas pressure of 18 psi, temperature of 230°C and electron
multiplier voltage of 1500 V. The formation of product ions in the MS/MS experiments
was induced by collision (CID) of the selected precursor (3,4-DHPEA m/z 153) with
argon.
54
3.1.6. Statistical analysis
The differences between the two groups were tested by means of the GLM procedure
(SAS, 2001) using the monofactorial model:
Yij = µ + αi+ εij
where µ = general mean; αi = diet (i = 1, 2); εij = error of model.
3.2. Research 2: olive paté supplementation in sheep feeding
3.2.1. Production of olive paté
The paté is a new by-product obtained by virgin olive oil mechanical extraction, by
separation from the innovative two-phases Pieralisi decanter, named DMF.
This decanter, even though is a two-phases, and does not require the use of added water,
produces three types of products: oil, a fairly dry pomace (like that of a three-phase
extraction system, but contains all the hardest parts of the olive, such as epicarp, stone,
seed as well as a small amount of pulp) and the paté (consisting of pulp and olives
water). The estimated amounts of this products for 100 kg of processed olives are: 1520% oil, 42-43% of the remaining percentage is pomace and 42-43% is paté.
It represents, as well as the virgin olive pomace, the solid fraction separated from the
horizontal centrifuge, but without the woody fraction and epicarp of the drupe. The paté
used for the tests of storage in silos was obtained from olives harvested in three
successive batches of product, during the month of December 2008, in advanced state of
maturation. The third batch was also used for testing storage in silos; samples of this
batch were analyzed in the following months (January, February, March and April
2009), in order to assess the stability of the ensiling by-product over time. The paté used
for the supplementation in ewes’ feed was obtained from olives harvested during the
month of November 2009. The paté, both in year 2008 and in year 2009, was dried
using a fluid-bed dryer, following the procedure mentioned above for pomace.
55
3.2.2. Preparation of feedstuff
Concerning the three batches of fresh paté a part of the first two, and a part of the third
batch related to the trial on freshly ensiled paté, was analyzed fresh, and the following
samples were dried before analysis:
-pate fresh as it is;
-pate fresh + 20% alfalfa;
-pate fresh + 20% soybean meal;
-pate fresh + mixture of soybean meal 10% and alfalfa meal 10 %.
Only the mixing of the paté with vegetable flours at a rate of 20% was evaluated for the
second batch.
3.2.3. Animals and diets
Fifteen multiparous Comisana ewes an average body weight of 63 ± 4.5 kg and 161 ±
14 days of lactation were kept at the Experimental Section of the Department of Applied
Biology, University of Perugia. The animals were allotted into 3 experimental groups,
corresponding to three different dietary treatments. The experiment was conducted in
2010 and lasted 28 days after 14 days of acclimatization. The animals were grazed each
day from 6:00 p.m to 7:00 a.m, on a natural pasture characterized by the botanical
composition reported in Table 8, following the strip-grazing technique. Grazing periods
lasted 3 days each by the displacement of the electrified fence, yielding approximately
40 m2/ewe/day, water was always available.
Treatments were: 1) 150 g/head/d rolled linseed (L), 2) 162 g/head/d dried paté (mixed
with 20% of dehydrated alfalfa hay) (OP) and 24 g/head/d olive oil and 3) 75 g/head/d
rolled linseed, 81 g/head/d dried paté (mixed with 20% of dehydrated alfalfa hay) and
12 g/head/day olive oil (LOP).
The percentage composition of the three test concentrations is shown in Table 9. The
experimental concentrates (600 g/head/d) were offered after the morning and the
afternoon milking, while a 100 g/head of rolled barley were offered during milking. The
ewes were milked daily at 07:30 AM and 17:30 PM, and daily milk yield recorded.
56
Table 8. Pasture grasses (%).
Bromus sterilis L.
Medicago arabica (L.) Hudson
Bellis perennis L.
Bromus hordeaceus L.
Hordeum murinum L.
Avena barbata Potter
Calamintha nepeta (L.) Savi
Dactylis glomerata L.
Geranium dissectum L.
Orchis purpurea Hudson
Poa pratensis L.
Trifolium incarnatum L.
Vicia sativa L.
40
30
5
5
5
5
+
+
+
+
+
+
+
Table 9. Dietary treatments composition (%).
Feeds
Wheat middlings
Soybean meal
Corn
Dried Beet Pulp
Corn Gluten Meal
Barley
Rolled Linseed
olive paté mixed with alfalfa meal
Olive oil
Molasses
Calcium Carbonate
Sodium Chloride
Sodium di-carbonate
Di-calcium Phosphate
Mineral-Vitamins Premix
Experimental
concentrates
L
P
PL
13.9
4.5
9.0
1.0
6.0
3.4
21.0
22.0
22.0
20.0
1.9
11.0
2.0
2.0
2.0
10.0
10.0
10.0
25.0
12.5
44.5
22.0
4.0
2.0
4.0
2.0
3.0
1.0
1.0
1.0
0.5
0.5
0.4
0.5
0.5
0.5
0.5
0.5
0.5
0.6
0.6
0.6
57
3.2.4. Chemical analyses of animal feeds
The analytical determinations were frequently performed on samples of the feedstuffs
used as above reported with pomace. The phenolic compounds of feed were extracted
and analysed by HPLC-DAD/FLD following the procedure previously described by
Servili et al. (2011), using 10 g of product (20 g for the phenolic extraction from fresh
paté). The feed fatty acid composition was evaluated by gas chromatographic analyses
as reported above for the buffalo feeds and milk samples. The extraction of residual oil
from paté was carried out following the method above reported for residual oil from
pomace. In the residual oil, the acidic composition was determined according to the
1989/03 Commission Regulation (2003). The extraction of tocopherols, tocotrienols and
retinol from the feeds was carried out according to the method of Havemose et al.
(2004), by means of chromatography.
3.2.5. Ewes’ milk and cheese analyses
Production was recorded daily and milk samples were collected weekly to assess
chemical, physical parameters (following the method reported above for buffalo milk).
The collected milk was promptly place to freezing and stored at -80° C until the time of
analysis while another part was processed into cheese.
The milk of each group was processed into cheese using a polyvalent tank, built in
stainless steel with a maximum capacity of about 50 l.
The starter used was obtained by inoculating ewes’ milk treated at 90 ° C x 7 minutes,
allowed to cool and maintained at 42° C in an oven, with a lyophilized culture of
various selected strains of Lactobacillus delbrurckii (ssp. Bulgaricus and Streptococcus
thermophilus). Then the inoculum was fractionated and frozen. The amount of rennet
used was 30g/100L of milk, diluted in a ratio of 1:10 with water at 25° C. After the the
frozen inoculum was added, the milk, stirred continuously, was brought, to a
temperature of 35° C in the polyvalent. After about 6-7 minutes, with a milk
temperature of 37.5° C the diluted rennet paste was added; after 15-17 minutes from the
uniform distribution of the rennet, a first break was carried out with the help of the
58
cutter and a knife to obtain a checkerboard with squares approximately 2.5 cm from the
side. After 5 minutes, the breaking of the curd was carried out with a simultaneous
heating of the mass until a temperature of 38-40° C was reached. The gradual increase
of the speed of agitation allowed the curd to refine in about 15 minutes. Subsequently,
with the aid of perforated molds, the curd was extracted from polyvalent and the serum
recovered for the subsequent production of ricotta. The shapes, slightly pressed initially,
were then turned over every 30 minutes for the first two hours in order to facilitate the
elimination of the serum still retained by the cheese. The cheese was then maintained at
a temperature of about 40-45° C for about 3h to ensure the multiplication of the
inoculum. After about 15-20 hours from the production, the shapes were placed in brine
(saturated NaCl solution) for 6-7 hours. The process ended with the aging at a
temperature of 10-13 °C with RH 80%.
After each processing, the cheese samples were were grated at low temperature to to
make it more homogeneous and then homogenized. The milk fatty acid composition
was evaluated by gas chromatographic analyses as were the methyl ester derivatives
after transesterification with sulphuric acid following the procedure reported above for
buffalo milk. To evaluate the risk of coronary disease, atherogenic and thrombogenic
indexes were calculated as suggested by Ulbricht and Southgate (1991). Tocopherol,
tocotrienol and retinol extraction was conducted according to the method proposed by
Havemose et al. (2004). The reactive substances with tiobarbituric acid (TBARs),
expressed as µg MDA/g of fat, (Fenaille et al., 2001) per ml of milk measured at 0, 24
and 72 hours of exposure to light at 4°C, were determined.
The same method was used to asses the oxidative stability of cheese at the time of
sampling, after 3 and 7 days of storage in the dark and at a temperature of 4 ° C in a
cell. On cheese (40 d ripening) stored at 4°C and dark TBARs after 0, 3 and 7 d was
evaluate. Also, yellow index was evaluated on cheese following the method proposed
by Giangiacomo and Messina (1988).
59
3.2.6. Analysis of the phenolic compounds of milk
A specific method has been developed for the extraction and purification of the phenolic
fraction from milk, and it is reported at the end of “Results and Discussion” section.
The analysis of these compounds was carried out following the procedure reported
above for buffalo milk
3.2.7. Statistical analysis
Data were performed according to the following model:
Yijkl = µ + αi + β(α)ij + dimijk + εijkl
where:
Yijk = experimental parameters
µ = general mean
αi = fixed effect due to treatment (Linseed, Olive Paté, Linseed+Olive Paté);
βij = random effect due to ewe within tratment;
dimijk = covariate for days in milking;
εijk = residual errror.
60
4. RESULTS AND DISCUSSION
Research 1
The results reported below refer to the first year of research (2008).
In addition the ripetition of the analysis of the phenolic and acid composition of the
pomace and the fatty acid composition of milk carried out in the following year (2009),
are reported below. During this second year mozzarella was made and then the fatty
acid composition and the oxidative stability were evaluated.
4.1. Characterization of dried pomace and feedstuff
The chemical characteristics of the stoned olive pomace (DSOP) are reported in Table
10, and the protein content is similar to that reported by Malossini et al. (1983). The
fibrous component was lower than the values reported by Molina-Alcaide and YañezRuiz (2008) for olive cakes. The destoning process determines a reduction in ADL
content (207.8 g/Kg), this value represents a further reduction of the values obtained by
Chiofalo et al. (2004) in partially stoned virgin pomace (308.0 g/kg DM). The DSOP
shows a high level of ether extract (206.6 g/kg DM).
The fatty acids profile is characterized by a high amount of C18:1cis9 (75.5%) (Table
11), this value is consistent with that reported by Chiofalo et al. (2002); the reduction in
lignin associated with the high content of ether extract determine an improvement in the
dry matter digestibility (Sadeghi et al., 2009).
61
Table 10. Dried Stoned Olive Pomace (DSOP) chemical
characteristics (% D.M.).
Dry Matter
95.67
Crude Protein
10.08
Ether Extract
20.66
Neutral Detergent Fiber
42.16
Acid Detergent Fiber
32.92
Acid Detergent Lignin
20.78
Non Structural Carbohydrates
19.53
Ash
7.56
Table 11. DSOP fatty acids profile
(% FAME).
Fatty acids
2008
2009
C14:0
0.1
0.1
C16:0
12.3
12.7
C16:1
0.9
0.7
C17:0
0.1
0.1
C18:0
1.9
2.0
C18:1n9
75.5
76.5
C18:2n6
8.3
6.9
C18:3n3
0.6
0.7
C20:0
0.2
0.1
C20:1n9
0.3
0.2
The phenolic composition of the DSOP (2008 and 2009) shows high amounts of
secoiridoids, such as 3,4-DHPEA (1.2 g/kg DM), 3,4-DHPEA-EDA (12.6 g/kg DM), p-
62
HPEA-EDA (5.6 g/kg DM) and lignans, including 1-acetoxypinoresinol (Table 12). For
the year 2009 also the phenolic composition of fresh pomace is shown (Table 22), in
comparison with paté.
Table 12. Phenolic compounds of the dried stoned olive pomace
(g/Kg D.M.) (± Rmse).
Phenolic compounds
2008
2009
3,4-DHPEA
3.5
1.2 ± 0.8
p-HPEA
0.9 ± 0.07 2.8
8.0
Verbascoside
10 ± 0.5
9.4
3,4-DHPEA-EDA
12.6 ± 0.7
3.8
p-HPEA-EDA
5.6 ± 0.4
0.5
(+)-1-Acetoxypinoresinol 0.2 ± 0.002
Sum of phenols
30.4 ± 1.2 27.9
±
±
±
±
±
±
±
0.1
0.1
0.9
1.1
0.5
0.1
1.5
Several phenolic compounds occurring in the DSOP, such as 3,4-DHPEA, 3,4-DHPEAEDA, p-HPEA-EDA, are now considered to be the main bio-active phenols of extravirgin olive involved in the prevention of cardiovascular disease and cancer in humans
(Servili et al., 2009; Covas, 2008; EFSA, 2011).
The chemical characteristics of the feedstuff are shown in Table 13.
Table 14 shows the tocopherols and tocotrienol content and the acidic composition of
the lipid fraction of the feedstuffs. The experimental concentrate shows a higher amount
of α-T (P<0.05), of γ e δ-T (P<0.01) and consequently of the total tocopherols (62.88 vs
55.87 µg/g, (P<0.05) compared to the control group, however a good level of α-T is
present in the maize silage.
The DSOP supplementation improved acidic composition of the lipid fraction of the
experimental concentrate with a significant decrease, with the exception of C18:0, of all
the saturated fatty acids reported.
There is significant increase (P<0.01) both in C18:1ω9 and for C22:6ω3, on the
contrary there is a decrease (P<0.01) both of C18:2ω6 and of C18:3ω3 and also
C20:5ω3 was lower (P<0.05) compared to the value of the control group.
63
Table 13. Dry matter (g/kg as fed) and chemical composition (g/kg D.M.) of the
feedstuffs.
Items
DM
CP
CF
EE
NSC
Ash
NDF
ADF
ADL
Alfalfa hay
879.6
179.9
339.2
27.1
207.2
85.3
500.5
385.1
87.6
Maize silage
334.2
83.9
216.7
24.8
327.9
46.9
516.5
259.0
44.9
Control
concentrate
Experimental
concentrate
Control diet
907.3
189.6
124.4
29.7
366.0
99.8
314.9
175.3
65.5
912.5
198.3
124.7
47.5
348.2
84.8
321.2
168.6
78.2
661.1
143.3
206.1
27.1
318.2
74.7
436.7
252.4
61.3
663.1
146.6
206.2
33.9
311.4
69.0
439.1
249.9
66.1
Experimental
diet
Table 14. Tocopherols, tocotrienol (µg/g) and fatty acids (%) of the feedstuffs.
Items
α-tocopherol
γ-tocopherol
δ-tocopherol
γ-tocotrienol
Toc. totals
Fatty acid profile
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1ω9
C18:2ω6
C18:3ω3
C20:4ω6
C20:5ω3
C22:6ω3
Control
Experimental
Alfalfa Maize
Rmse
hay
silage concentrate concentrate
2.36
43.32
45.64b
50.26a
1.33
B
0.16
2.79
1.59
2.72A
0.19
1.10
1.43B
1.73A
0.14
7.94
7.21*
8.18*
0.47
2.52
55.15
55.87b
62.88a
2.17
0.24
1.04
1.76
29.01
4.83
5.51
16.85
22.30
1.03
1.40
1.37
0.06
0.39
0.57
17.20
2.02
20.67
45.92
7.03
0.23
0.27
0.17
0.20A
1.34A
0.71A
17.13a
3.00
27.53B
42.18A
4.02A
0.05
0.11a
0.10B
0.07B
0.56B
0.49B
15.01b
2.86
42.05A
32.76B
2.95B
0.06
0.07b
0.24A
0.01
0.03
0.02
0.40
0.07
0.89
0.94
0.09
0.01
0.01
0.01
A, B: P < 0.01; a, b: P < 0.05; *: P = 6.28%
64
4.2. Effects of pomace supplemetation in buffalo
4.2.1. Production and quality of buffalo milk
As can be seen from the comparison with the control group, shown in Table 15, the use
of DSOP as supplemented feed does not affect the BCS (body condition score), ADG
(average daily gain) the fat, protein, lactose and urea content of the milk. These results
demonstrate, as found by Chiofalo et al. (2004) and Pauselli et al. (2007), that the
experimental diet did not alter the level of the parameters considered and did not lead to
any detrimental effect on the activity of the ruminal bacteria due to the long-chain
unsaturated fatty acids, as has already been proved by Nefzaoui and Vanbelle (1986)
and by Chiofalo et al. (2004). Moreover it is important to note that water buffalo has a
better fibre utilization than the dairy cow (Batista et al., 1982; Bartocci et al., 2002).
The use of the dried stoned olive pomace does not affect the milk production, this
confirms what was reported by Hadjipanyiotou (1999) on dairy cows and Chiofalo et al.
(2004) and Pauselli et al. (2007) on milk of ewes. No difference was found between the
milk produced by the control group and the experimental group.
Table 15. Live weight, body condition score (BCS ), milk yield and quality.
The
Rmse
17.00
DSOP
group
17.00
Live weight (kg)
625.93
662.50
90.16
ADG (g/d)
421.88
437.50
391.47
BCS (1÷9)
6.41
6.53
0.45
Milk production (kg/d)
9.69
10.08
2.53
Fat (%)
7.16
7.36
1.07
Protein (%)
4.51
4.45
0.33
Lactose (%)
4.88
4.90
0.19
Urea (mg/100ml)
32.58
33.13
3.22
Items
Control group
Ingestion of DM (kg/d)
-
quantity
65
of crude protein administered per head in the DSOP animal group was 110.60 g/d
(4.42% of the total protein). The pH, the milk coagulation parameters of the two groups
are shown in Table 16. No significant differences were found for the pH values, for the
rennet clotting time (20.34 and 22.35 min), for the curd firming time (2.30 and 2.66
min) and for the curd firmness (44.40 and 35.25 mm). These data are similar to those
obtained by Bartocci et al. (2006) and Tripaldi et al. (2010).
Table 16. Acidity, thromboelastographic parameters, of the milk
of the two groups.
Items
Control group
DSOP group
Rmse
pH
r (min)
k20 (min)
a30 (min)
6.78
20.34
2.30
44.40
6.80
22.35
2.66
35.25
0.08
4.15
1.28
14.22
4.2.2. Buffalo milk fatty acid composition
Table 17, shows the fatty acid composition of the lipid fraction of buffalo milk, refer to
the first year of research (2008).
A significant difference was found in the DSOP group in C18:0, C18:3ω6 and C18:1ω7
milk content and also a large amount of C18:1ω9, although not significant.
The use of non-rumen-protected, vegetable C:18 unsaturated fatty-acid sources resulted
in an increase in rumen of the C18:0 synthesis due to bacterial biohydrogenation
activity (Mosley et al., 2002), which could be partially converted to C18:1ω9 in the
mammary gland; in fact the increase of C18:1ω9, could in part be attributed to the
activity of desaturation of the mammary gland both for C18:1ω7 and C18:3ω6
(Chilliard and Ferlay, 2004). In dairy cows, 40% of the C18:1ω9 is formed in the
mammary gland by ∆9-desaturase activity (Lock and Gainsworth, 2003). The fatty acid
composition of the milk of the experimental group has been proven to be similar to that
observed by Selner and Schultz (1980) in cattle fed with rumen unprotected oleic acid
66
sources. The DSOP intake led to an increase in the MUFA represented principally by
C18:1ω9 and an increase in the PUFA represented principally by C18:2ω6. The
consequent reduction of SFA led to a reduction of the saturated/unsaturated ratio (2.75
and 3.04), of the atherogenic (3.60 and 3.84) and thrombogenic indices (3.75 and 3.87)
of the milk produced by the animal of the experimental group. In the milk of the
experimental group there was a slight reduction in content of the short-chain (C6:0C10:0) and medium-chain fatty acids (C11:0-C16:3ω4) and a slight increase in the longchain fatty acids (C17:0-C24:1).
67
Table 17. Fatty acid composition (%) of the milk fat of the
two groups (1st year 2008).
Fatty acid
composition
Control
group
DSOP
group
Rmse
C6:0
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1ω9
C18:1ω7
C18:2ω6
C18:3ω3
C18:3ω6
C18:4ω3
C20:4ω6
C20:5ω3
C22:6ω3
SFA
MUFA
PUFA
SFA/UFA
ω3
ω6
ω6/ω3
Atherogenic index
Thrombogenic
index
5.20
3.17
4.08
4.31
14.43
34.10
7.34b
16.83
0.96*
2.15
0.43
0.06b
0.45
0.18
0.07
0.04
74.52
21.08
4.40
3.04
1.47
3.47
2.40
3.84
4.72
2.74
3.72
3.95
14.30
33.53
8.60a
17.81
1.27*
2.29
0.46
0.09a
0.52
0.20
0.06
0.03
73.30
22.15
4.55
2.75
1.45
3.64
2.51
3.60
1.00
0.79
1.01
0.56
1.02
2.91
0.93
2.58
0.33
0.39
0.08
0.02
0.11
0.08
0.03
0.03
2.97
2.69
0.74
0.41
0.42
0.46
0.26
0.57
3.87
3.75
0.51
SCFA
MCFA
LCFA
12.35
57.56
30.09
10.93
56.17
32.90
2.53
3.11
3.69
a, b: P < 0.05; *: P = 8.67%
68
The fatty acid composition of milk from buffalo cows supplemented with DSOP
relative to the year 2009, is reported in Table 18. The milk of the DSOP group shows a
significant higher proportion in C18:1 n9 content (P<0,001), with respect to the content
of the control group, that consequently leads to an important increase in LCFA, UFA
and MUFA milk content. The simultaneous reduction in the SAT amount in the milk
from the pomace-feeding group resulted also in a reduction of the atherogenic and
thrombogenic index; similar results were obtained on the fatty acid composition of the
milk fat of sheep and on the indices by Chiofalo et al. (2004). These results are similar
to those found by Molina Alcaide et al. (2008) in milk from ewes fed with olive cake
and by Pauselli et al. (2007) in ewes fed with a concentrate of stoned olive pomace.
Also a significant increase in of the long-chain fatty acids (LCFA) emerged; this could
be determined by the protective effect from oxidation exerted by the vitamin E on the
long-chain fatty acids (Chiofalo et al., 2004).
The differences between the pomace group and the control group appear to be more
pronounced in the second year of experimentation; this is due to a higher amount of
pomace used in the diet, 1.2 kg DM/d in year 2009 compared to 1.05 kg DM/d in year
2008.
69
Table 18. Fatty acid composition (%) of the milk fat of the
two groups (2nd year 2009).
Fatty acid composition
C6:0
C8:0
C10:0
C12:0
C14:0
C14:1n6
C16:0
C16:1n7
C18:0
C18:1n9
C18:2n6
C18:3n3
C20:4n6
C20:5n3
C22:6n3
SCFA
MCFA
LCFA
SAT
INS
MUFA
PUFA
n6
n3
n6/n3
Atherogenic Index
Thrombogenic index
Control
group
3.55
2.52
5.15
0.10
17.37
1.23
41.49
1.84
5.90
13.49B
1.50
0.35
0.08a
0.06
0.0009
11.46
61.21
26.82b
78.39a
21.61b
18.37b
3.23
3.09
0.74
4.26
5.06a
3.78
DSOP
group
3.28
2.19
4.26
0.09
17.39
1.34
39.77
3.00
6.36
16.67A
1.52
0.36
0.07b
0.08
0.0004
9.98
59.87
30.25a
75.33b
24.67a
21.44a
3.22
3.20
0.81
4.00
4.37b
3.41
Rmse
1.37
0.58
0.73
0.02
2.94
0.24
1.81
0.42
0.59
1.54
0.14
0.05
0.01
0.03
0.00
2.51
2.34
2.08
1.89
1.90
1.73
0.30
0.30
0.10
0.48
0.74
0.29
a-b: P< 0.05; A-B: P < 0.01
70
4.2.3. Antioxidants and oxidative status of buffalo milk
The occurrence of tocopherols, retinol and TBARs values of the milk of DSOP-group
and control group was evaluated for the first year (2008) and is reported in Table 19.
Among tocopherols only α-T, γ-T, δ-T and γ-T3 were present in appreciable quantities
in the milk of both groups. Alpha-T was present in a high concentration (79.42%),
followed by y-T (15.93%) and δ-T (1.98%) in the milk of the control group. The DSOP
dietary supplementation resulted in a significant increase in all the aforementioned
tocopherols, except for δ-T, in the milk of the treated group, without altering their
distribution. As a consequence, the total amount of vitamin E in the milk reached 8.6
and 10.45 µg/g fat (P<0.01) for the control and the treated group respectively.
Table 19. Tocopherols, tocotrienol, retinol (µg/g fat ) and oxidative
stability (µg MDA/g fat) of the lipid fraction of the milk of the
two groups (1st year 2008).
Experimental
group
8.19a
1.70A
Rmse
α-tocopherol
γ-tocopherol
Control
group
6.83b
1.37B
δ-tocopherol
0.17
0.19
0.03
Items
γ-tocotrienol
Total (Vitamin E)
Retinol
TBARs
0.23
B
8.60
B
2.54
B
15.05
A
0.37
A
10.45
3.17
A
A
12.09
B
1.00
0.21
0.05
1.12
0.28
1.79
A, B: P < 0.01; a, b: P < 0.05
These results may be ascribed, in part, to the occurrence of a higher amount of vitamin
E in the experimental concentrate (62.88 vs. 55.87 µg/g, P<0.05). These findings are
consistent with those reported by Weiss et al. (2003), who showed evidence of the
relationship between dietary α-T content and its concentration in milk independently
from the tested fat sources. The high amount of phenols characterized by strong
antioxidant activity, such as 3, 4-DHPEA, 3, 4-DHPEA-EDA, found in the DSOP,
71
might also be implicated in the increase in the amount of α-T in the milk. In fact, the
DSOP phenolic antioxidants can protect tocopherols from oxidation thereby increasing
the total amount absorbed through digestion and thus their concentration in the milk.
In this regard, one of the main results of this research work was the detection and partial
characterization of hydrophilic phenols in the milk. Figure 11 shows the LC-MS/MS
chromatogram and the mass spectra of the occurrence of 3,4-DHPEA in the milk of
water buffaloes fed with DSOP. The other phenols, such as 3,4-DHPEA-EDA, pHPEA-EDA and lignans, which are the most concentrated compounds of DSOP, were
not found in the milk of DSOP-fed animals.
These results suggest that 3,4-DHPEA-EDA, very abundant in DSOP, could be
probably hydrolyzed during digestion, releasing the 3,4-DHPEA that is transferred and
accumulated in the milk. In terms of the absolute concentration of 3,4-DHPEA found in
the milk of water buffaloes treated with DSOP, the average value was 36.0 µg/l, but the
disparity due to subject variability was very large, ranging between a minimum of 8.9
µg/l and a maximum of 56.2 µg/l.
72
kCounts
50
1
40
30
20
10
10
20
30
40
50
60
minutes
Figure 11. HPLC-ESI-MS/MS chromatogram obtained from a milk sample produced by a
DSOP-fed buffalo cow. [1] 3,4-DHPEA (hydroxytyrosol).
The milk from DSOP-fed animals also revealed a higher retinol level (3.17 vs. 2.54
µg/g fat, P<0.01) in comparison to the milk from the control group (Table 19). The
higher amount of tocopherols and retinol might have contributed to reducing the level of
TBARs (12.09 vs. 15.05 µg MDA/g fat, P<0.01) in the milk of the treated group and
consequently to a better oxidative status of this milk with respect to the milk derived
from the untreated animals. These results are similar to those reported by Pauselli et al.
(2007) in the milk of ewes fed with a concentrate enriched with stoned olive pomace.
The presence of a powerful natural antioxidant such as 3,4-DHPEA, in the milk of
DSOP-fed animals, an antioxidant that is normally present in virgin olive oil and its byproducts, might also contribute to the better oxidative status of milk fat from the DSOPtreated group, acting either directly or indirectly (synergism with vitamin E) against free
radicals (Owen et al., 2000; Mirò Casas et al., 2001; Servili et al., 2004). Among DSOP
phenols, ortho-diphenols, such as 3,4-DHPEA and 3,4-DHPEA-EDA, are known to
73
possess the highest antioxidant activity and are also effective radical scavengers
(Baldioli et al., 1996). In particular, 3,4-DHPEA scavenges aqueous peroxyl radicals
near the membrane surface, while oleuropein scavenges chain-propagating lipid peroxyl
radicals within membranes (Saija et al., 1998). Moreover, phenolic compounds have
been shown to play a role in vitamin E recycling and might have also accounted for the
higher milk tocopherol levels in the DSOP-treated group. A similar hypothesis has been
formulated for flavonoids on α-T recycling (Zhu et al., 1999; Pedrielli and Skibsted
2002; Pazos et al., 2002). However, further studies are required to better clarify the role
of each DSOP compound in the total milk antioxidant capacity.
4.2.4. Fatty acid composition of buffalo mozzarella cheese
Milk (year 2009), processing to obtain mozzarella cheese, led to an increase in the
difference in the fatty acid composition of the lipid fraction, due to dietary treatment
(Table 20). The percentages of C6:0, C8:0 and C10:0 were higher in the mozzarella
from the control group milk, while in the mozzarella from the milk of DSPO group, the
presence of C14:0 was lower (P<0.05) and the C18:1n9 content was higher (P<0.01).
Mozzarella of the DSPO group was also characterized by a higher MUFA content
(P<0.01), a reduced n6/n3 ratio (P<0.05) and lower atherogenic and thrombogenic
indexes.
74
Table 20. Effects of dietary treatment on mozzarella cheese
lipid fraction (% FAME) (2nd year 2009).
Fatty acid composition
C6:0
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1n9
C18:2n6
C18:3n3
C20:4n6
C20:5n3
C20:6n3
SCFA
MCFA
LCFA
SAT
INS
MUFA
PUFA
n6
n3
n6/n3
Atherogenic Index
Thrombogenic Index
C
3.89A
3.01A
4.12a
0.09
18.66a
39.00
6.82
14.06B
1.52
0.52
0.07
0.06
0.0009
11.00A
60.97a
27.92B
77.69A
22.31B
18.77B
3.53
3.33
0.74
3.76a
4.95A
3.68a
DSOP
2.44B
2.24B
3.60b
0.08
16.92b
36.07
8.05
20.90A
1.69
0.47
0.08
0.08
0.0005
8.39B
55.29b
36.31A
71.63B
28.37A
24.65A
3.71
2.76
0.81
2.73b
3.68B
3.14b
Rmse
0.38
0.21
0.23
0.02
1.00
2.00
0.92
1.57
0.32
0.53
0.01
0.03
0.00
0.59
2.31
2.31
1.79
1.79
1.61
0.75
0.30
0.10
0.54
0.44
0.30
a,b: P<0,0 5; A, B: P<0,01
4.2.5. Oxidative status of buffalo mozzarella cheese
No statistical differences due to dietary treatment were found in the concentration of
tocopherols, whose contents were similar to those found by Balestrieri et al. (2002) in
commercial mozzarella cheeses produced from water buffalo milk (Table 21).
75
Table 21. Effects of diet and storage on mozzarella tocopherol content
and oxidative status.
Dietary treatment
Items
Rmse
C
DSOP
α-tocopherol (µg /100 g)
γ-tocopherol (µg /100 g)
159.39
148.25
23.31
1.88
1.65
0.36
α-tocopherol (µg /g fat)
6.13
5.68
0.88
γ-tocopherol (µg /g fat)
0.07
0.06
0.01
Total tocopherols (µg /g fat)
6.20
5.75
0.88
TBARs (µg MDA/g fat)
2.690
2.569
0.6009
Governing liquid
TBARs (µg MDA/ml)
0.375
0.259
0.21
The storage period from 0 to 4 days resulted in a significant reduction in the αtocopherol content, which was slight higher in mozzarella from the control group
(Figure 12).
250
g/100g
200
C
150
DSOP
100
50
0
0
4
days of storage
Figure 12. Effect of storage on α-tocopherol content in mozzarella cheese obtained with the
milk from the two groups of buffaloes with different dietary treatments (C and DSOP).
76
In dairy products, tocopherols are scavengers of free radicals that catalyze the beginning
and propagation of chain reactions in lipid peroxidation (Burton and Ingold, 1986;
Madhavi et al. 1996). The reduction in the tocopherol content from milk to mozzarella
cheese is due to the strong oxidative conditions that occur during mozzarella
manufacturing, such as high temperature and oxygen exposure (Balestrieri et al., 2002),
taking in account also that buffalo milk has a lower content in ß-carotene than cow milk
(Addeo et al., 1995). Thiobarbituric acid-reactive substances in mozzarella cheese put in
evidence the consumption of antioxidants such as tocopherols during storage (Figure
µg MDA/g fat
13).
4
3.5
3
2.5
2
1.5
1
0.5
0
C
DSOP
0
4
days of storage
Figure 13. Effect of storage on the oxidative status (TBARs) of mozzarella cheese obtained
with the milk from the two groups of buffaloes with different dietary treatments (C and DSOP).
The slightly lower level of MDA content in the governing liquid from mozzarella of the
DSOP group (Table 21) could be due to the tranfer of hydrophilic polyphenols in such
liquid, and their consequent antioxidant action (Figure 14).
77
0.6
µg MDA/mL
0.5
0.4
C
DSOP
0.3
0.2
0.1
0
0
4
days of storage
Figure 14. Effect of storage on the oxidative status (TBARs) of the governing liquid of
mozzarella cheese obtained with the milk from the two groups of buffaloes with different
dietary treatments (C and DSOP).
78
Research 2
4.3. Phenolic characterization of fresh paté
From a comparison of the phenol content of the fresh olive paté with that of a fresh
pomace (Table 22), the patè shows a considerably high content in phenolic compounds
(76-96g/ kg), such as secoiridoids and verbascoside despite the late harvest of the olives
(December).
Table 22. Phenolic composition (g/kg D.M.) of fresh paté and fresh pomace.
Items
3,4-DHPEA
p-HPEA
3,4-DHPEA-EDA
Verbascoside
Sum of phenols
I batch
24.3
11.3
31.5
29.1
96.1
±
±
±
±
±
0.2
0.1
1.7
1.3
2.1
Fresh Paté
II batch
20.9
12.7
24.9
18.0
76.5
±
±
±
±
±
0.2
0.1
0.6
0.3
0.7
III batch
11.4
6.4
28.1
29.9
75.8
±
±
±
±
±
1.0
0.5
0.8
1.3
5.7
Fresh
Pomace
4.1
4.9
18.5
23.8
51.3
±
±
±
±
±
0.1
0.2
2.4
2.2
3.3
4.4. Preservation of fresh paté over time
The pre-drying storage conditions over time were evaluated on the third batch.
The silos were experimentally tested for the preservation of fresh paté. The evaluation
of storage stability of the by-product was carried out on paté samples after 30, 60 and
120 days of storage. It emerged from the results that the phenolic fraction shows a good
stability until day 30 of conservation, with a loss of just 10% compared to the initial
sample; then this percentage tends to increase to 48% after 60 days and to 76% after 120
days of conservation. Is important to consider that this trial was tested on the third
batch, which was carried out in late December and it is characterized not only by higher
initial moisture content (Table 23) but also by a lower concentration of phenolic
79
compounds (Table 24) comparing to the others two (Table 22). The results obtained
from the analysis of silage paté show that this type of storage could be an interesting
prospective.
Table 23. Moisture (%) of fresh pate, and paté blended with vegetable flours, stored in silos.
Items
Paté
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 %
+ soybean meal 10%
Initial point
(III batch)
78.8
± 3.6
63.3
± 3.4
64.1
± 3.9
77.5 ± 4.1 71.5 ± 3.8 78.2 ± 4.1
62.3 ± 3.1 56.3 ± 2.8 63.8 ± 3.2
63.4 ± 3.5 58.5 ± 3.3 65.2 ± 3.6
62.8
62.6 ± 3.7 56.8 ± 3.3 63.5 ± 3.7
±
4.3
30 days
60 days
120 days
Table 24. Phenolic composition (g/kg D.M.) of fresh paté during storage in silos.
Phenols
3,4-DHPEA
p-HPEA
Verbascoside
3,4-DHPEA-EDA
Total phenols
Initial point
(III batch)
11.4
6.4
28.1
29.9
75.8
±
±
±
±
±
1.0
0.5
0.8
1.3
5.7
30 days
11.7
6.1
24.0
25.8
67.6
±
±
±
±
±
0.5
0.6
0.7
0.9
5.2
60 days
7.3
6.3
12.7
15.9
42.2
±
±
±
±
±
0.6
0.5
0.4
0.2
2.8
120 days
3.2
4.3
6.4
8.1
22.0
±
±
±
±
±
0.2
0.5
0.1
0.2
1.7
The results on the characterization of dried paté after ensiling and after mixing with
vegetable flours have shown that the latter practice has had a stabilizing effect on
polyphenols in the drying phase from the thirtieth day. In fact from this moment on the
percentage of "recovery" of such substances on mixed paté was higher compared to
non-enriched paté (Table 25) despite the fact that the polyphenol content of the mixture
pate-vegetable flours was lower than that of the paté alone, due to a dilution effect.
This result could be due to an absorbent effect on the free water, exerted by vegetable
flour: during the fresh ensiling period of the paté, the vegetation water tended to
separate from the solid phase of the paté and was then absorbed by the vegetable meals
80
at the time of mixing, making the product more stable during drying, and consequently
more resistant to high temperatures.
The data relating to the fat fraction of the fresh paté stored in silos (Table 26), show also
a remarkable stability over time, both with respect to hydrolysis and oxidation; in fact
the differences in free acidity and peroxide value between the samples taken at different
storage times and then dried, are low.
81
Tabella 25. Phenolic composition (g/kg D.M.) and its recovery (%) in dried paté (after storage in silos) alone or mixed with vegetable flours.
3,4-DHPEA
p-HPEA
Initial point
30 days
60 days
120 days
5.8
5.9
4.2
1.1
±
±
±
±
0.5
0.3
0.3
0.1
6.0
6.0
5.5
3.3
Initial point
30 days
60 days
120 days
3.7
3.8
3.3
1.4
±
±
±
±
0.4
0.3
0.3
0.2
2.7
2.6
3.4
1.2
Initial point
30 days
60 days
120 days
3.5
3.5
3.4
1.7
±
±
±
±
0.4
0.2
0.2
0.1
2.9
2.5
2.5
1.5
Initial point
30 days
60 days
120 days
3.7
3.7
3.6
1.3
±
±
±
±
0.4
0.3
0.3
0.1
3.4
3.4
3.2
1.9
Verbascoside
3,4-DHPEA-EDA
Paté (III batch)
15.8 ± 0.8
14.8 ± 0.7
± 0.5
14.7 ± 0.6
11.5 ± 0.5
± 0.6
7.5 ± 0.4
5.5 ± 0.1
± 0.4
2.3 ± 0.1
3.3 ± 0.1
± 0.5
Paté + dehydrated alfalfa 20 %
0.1
7.7 ± 0.4
10.6 ± 0.5
±
7.2 ± 0.5
9.3 ± 0.6
± 0.2
5.2 ± 0.4
5.5 ± 0.1
± 0.3
2.8 ± 0.2
1.6 ± 0.3
± 0.1
Paté + soybean meal 20 %
6.8 ± 0.3
9.8 ± 0.5
± 0.1
6.6 ± 0.4
9.2 ± 0.4
± 0.1
5.4 ± 0.4
6.0 ± 0.4
± 0.1
2.6 ± 0.2
2.6 ± 0.1
± 0.1
Paté + dehydrated alfalfa 10 % + soybean meal 10%
7.1 ± 0.4
7.8 ± 0.4
± 0.2
6.9 ± 0.4
7.6 ± 0.4
± 0.2
5.5 ± 0.4
5.1 ± 0.4
± 0.2
2.8 ± 0.1
2.4 ± 0.1
± 0.1
*The results represent the average of two independent experiments ± standard deviation
Total phenols
Recovery
42.4
38.0
22.7
10.0
±
±
±
±
3.2
2.9
1.6
1.0
55.9
56.3
53.8
45.4
24.8
22.9
17.4
7.1
±
±
±
±
0.7
0.9
1.0
0.6
58.8
57.6
65.1
51.0
23.0
21.8
17.4
8.5
±
±
±
±
1.2
1.1
1.6
0.8
54.3
61.0
63.1
58.2
22.0
21.6
17.4
8.3
±
±
±
±
1.6
1.7
1.7
0.8
55.2
57.5
61.7
57.3
Tabella 26. Values of the stability parameters of the residual oil of fresh paté (after storage
in silos), alone or mixed with vegetable flours*.
Items
Paté (III batch)
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 % +
soybean meal 10%
Paté (III batch)
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 % +
soybean meal 10%
Paté (III batch)
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 % +
soybean meal 10%
Paté (III batch)
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 % +
soybean meal 10%
Acidity
Peroxide
(oleic acid g/100g
(meq O2/Kg oil)
oil)
Initial point
1.5 ± 0.2
3.7 ± 0.3
2.2 ± 0.2
5.5 ± 0.4
1.8 ± 0.1
5.7 ± 0.5
2.3
±
0.1
5.9
±
0.4
1.4
2.2
1.8
±
±
±
30 days
0.1
3.0
0.1
5.1
0.1
5.8
±
±
±
0.2
0.3
0.3
2.3
±
0.1
6.1
±
0.4
1.8
2.5
2.2
±
±
±
60 days
0.1
3.3
0.1
5.5
0.1
5.6
±
±
±
0.2
0.3
0.3
2.4
±
0.1
6.0
±
0.4
2.2
2.7
2.5
±
±
±
120 days
0.1
4.0
0.1
5.8
0.1
5.9
±
±
±
0.2
0.3
0.3
2.6
±
0.2
±
0.4
6.2
*The results represent the average of two independent experiments ± standard deviation
4.5. Paté pre-drying treatment
The high level of humidity of the fresh paté made direct drying difficult, using the normal
process applied in zoo-technical flour industry, which envisages a maximum level of
humidity of the row material at approximately 50%. Therefore the humidity of the paté
was reduced by pre-drying mixing with soya flour and alfalfa hay, usually used in zootechnical feed. It emerged from the tests that the addition of 20% vegetable flours, reduces
significantly the level of initial humidity of the fresh paté; in fact, in this way, the humidity
went down, as is shown in Table 27, to a value near of 50%, and consequently the dryingtime was significantly reduced.
Table 27. Moisture (%) and drying times, of fresh pate of the first two samples.
Moisture %
I batch II batch
Paté
68.8
68.7
Paté + dehydrated alfalfa 10 %
58.4
Paté + soybean meal 10 %
57.7
Paté + dehydrated alfalfa 5 % + soybean meal 5%
56.8
Paté + dehydrated alfalfa 20 %
53.6
53.1
Paté + soybean meal 20 %
54.64
54.7
Paté + dehydrated alfalfa 10 % + soybean meal 10% 53.84
53.7
Items
Drying time
I batch II batch
1 h 46'
1 h 40'
1 h 15'
1 h 10'
1 h 10'
35'
32'
39'
34'
35'
32'
4.6. Characterization of dried paté and feedstuff
The drying process was carried out following the same method above mentioned for the
pomace. This process brought the humidity of paté content down to values ranged between
5 and 8% (Table 28). The loss of polyphenols ranged between 30 to 40%, for the I and II
batch, and around 50% for the III, both for the dried paté alone and the ones containing
vegetable flours (Table 29). The greater loss in the third sampling is due to the fact that the
humidity was 79% while for the other two batches was 68%. These results are in line with
84
what had already been observed for stoned virgin pomace (Servili et al., 2007b). The
reduction in absolute value of the phenolic component is due essentially to the increase in
the content of vegetable flours, this led a dilution effect, however the antioxidant content
remains significant (between 20 and 40 g/kg), compatible with a concentrate to be used as
a feed integrator in the zoo-technical industry.
Table 28. Moisture content (%) of dry paté of the first two batches.
Items
I batch
II batch
Paté
7.9
7.2
Paté + dehydrated alfalfa 10 %
6.0
-
Paté + soybean meal 10 %
7.5
-
Paté + dehydrated alfalfa 5 % + soybean meal 5%
9.5
-
Paté + dehydrated alfalfa 20 %
5.9
5.5
Paté + soybean meal 20 %
6.8
6.3
Paté + dehydrated alfalfa 10 % + soybean meal 10%
7.0
5.8
85
Table 29. Phenolic composition* (g/kg DM) of dried paté as it is and mixed with vegetable flours (I batch).
Items
3,4-DHPEA
p-HPEA
Verbascoside
3,4-DHPEA-EDA
Total Phenols
Paté
13.9
±
0.3
10.3
±
1.0
18.7
±
1.0
24.8
±
0.4
67.6
±
1.5
Paté + dehydrated alfalfa 10 %
7.3
±
0.2
5.7
±
0.8
12.9
±
0.8
12.0
±
0.3
38.0
±
1.2
Paté + soybean meal 10 %
Paté + dehydrated alfalfa 5 %
+ soybean meal 5%
Paté + dehydrated alfalfa 20 %
8.1
±
0.2
6.4
±
0.1
13.8
±
0.1
12.8
±
0.1
41.1
±
0.3
8.8
±
0.1
7.9
±
0.1
12.6
±
0.1
13.0
±
0.3
42.4
±
0.3
5.2
±
0.1
3.9
±
0.4
9.6
±
0.4
10.4
±
0.1
29.1
±
0.6
6.2
±
0.3
3.1
±
0.4
8.7
±
0.4
10.3
±
0.6
28.3
±
0.8
5.5
±
0.3
4.0
±
0.0
9.6
±
0.0
10.9
±
0.2
30.0
±
0.4
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 %
+ soybean meal 10%
*The results represent the average of three independent experiments ± standard deviation.
86
Concerning the residual oil of the paté, the drying process did not cause negative effects on
the fat stability; in fact, both free acidity and peroxide number of all dried products were
low, corresponding to 1.9 and 2.2 g/100 of oleic acid, and 3.9 and 6.3 meq.O2/kg oil,
respectively (Table 30).
Table 30. Average values* of the first two samples, referred to the stability parameters of the
residual oil dried pate as it is and mixed with vegetable flours.
Items
Paté
Paté + dehydrated alfalfa 20 %
Paté + soybean meal 20 %
Paté + dehydrated alfalfa 10 % +
soybean meal 10%
Acidity (g of oleic
acid/100g of oil)
Peroxide value (meq
of O2/kg of oil)
1.9 ± 0.1
2.2 ± 0.1
2.0 ± 0.0
3.9 ± 0.1
5.7 ± 0.1
6.0 ± 0.1
2.2 ± 0.2
6.3 ± 0.2
*The results represent the average of two independent experiments ± standard deviation
The analysis of dried crude fiber of paté as it is and mixed with vegetables flours shows a
good content in NDF and ADF, levels that as expected increases with the addition of
vegetable flours, especially with alfalfa (Table 31). Similar values characterized the
experimental concentrates (Table 32).
Further interesting elements are the low lignin amount in the paté (Table 31), comparable
to the pomace de-stoned in pre-extraction and the crude protein content which values is
very similar to those found in the alfalfa flour. The protein content in paté is also low.
These characteristic are transferred into concentrates, which show similar values (Table
32).
87
Table 31. Content of crude fiber in the dried paté as it is and mixed with vegetables flours
% (DM).
Items
Paté
Dry Matter (%)
Ether Extract
Ash
Neutral Detergent Fiber
Acid Detergent Fiber
Lignin
Crude Protein
93.8
29.0
6.0
20.2
14.8
8.8
8.5
Paté +
dehydrated
alfalfa 20 %
94.1
18.5
8.0
36.2
25.2
7.9
8.6
Paté +
soybean
meal 20 %
93.6
18.4
6.7
28.0
12.4
5.1
22.3
Paté + dehydrated
alfalfa 10 % +
soybean meal 10%
94.4
19.5
7.5
29.9
12.4
5.1
16.0
Table 32. Chemical composition (%) of the pasture and the concentrates used.
Items
Rolled
Barley
Pasture
Dry Matter
88.66
23.03
Experimental Concentrates
L
P
LOP
87.75
89.03
88.35
Crude Protein
9.40
15.61
14.96
15.08
15.35
Fat
2.90
2.62
11.80
10.87
11.37
NDF
21.83
48.97
24.12
23.73
24.37
ADF
5.77
32.60
12.26
15.25
13.80
ADL
1.10
4.86
2.11
4.69
3.64
Ash
2.59
4.23
5.25
7.36
6.38
The addition of flour did not modify the fatty acid composition of the paté (Table 33). An
interesting point is that both the paté as it is and the other combination appear very rich in
C18:1n-9, consequently a similar amount characterized also the experimental concentrates
(Table 34) while, as expected, the group L was characterized by a high content of C18:2n6
and C18:3n3.
88
Table 33. Fatty acid composition (%) of dried paté as it is and mixed with vegetable flours.
Paté +
dehydrated
alfalfa 20 %
12.5
12.7
1.2
1.2
Fatty acid
Paté
composition
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1n9
C18:2n6
C18:n3
C20:0
C20:1n9
C22:0
0.1
0.1
2.4
72.7
9.7
0.6
0.4
0.3
0.2
Paté + soybean
meal 20 %
Paté + dehydrated alfalfa 10 %
+ soybean meal 10%
12.8
1.2
12.3
1.2
0.1
0.1
2.4
71.6
10.4
0.7
0.3
0.2
0.1
0.1
0.1
2.4
72.6
9.8
0.7
0.4
0.3
0.1
0.1
0.1
2.4
72.5
9.5
0.7
0.4
0.2
0.1
.
Table 34. Fatty acid composition (%) of the hay and the concentrates used.
Fatty acid
composition
C12:0
C14:0
C16:0
C16:1
C18:0
C18:1n9
C18:2n6
C18:3n3
SFA
MUFA
PUFA
Rolled
Barley
0.35
1.49
18.17
2.87
4.58
21.18
44.97
6.06
24.92
24.54
50.64
Pasture
0.12
1.90
16.32
0.36
4.20
11.82
22.18
37.50
24.38
12.91
62.71
Experimental Concentrates
L
OP
LOP
0.06
0.13
0.11
0.11
0.20
0.16
10.08
20.07
14.53
0.12
1.01
0.45
2.82
0.28
1.72
16.14
53.77
32.89
43.14
18.04
32.56
26.65
3.75
16.48
13.61
22.30
17.21
16.42
55.12
33.41
69.97
22.57
49.37
89
The addition of paté to the feed provided to it an high amount of phenolic compounds
(Table 35) equal to 10.3 g/kg (on D.M.). In the LOP group (paté mixed with linseed),
which has half paté content the concentration of phenolic compounds is halved (5.1 g/kg of
D.M). No trace of phenolic compounds was found in L group.
Table 35. Phenolic composition (g/kg DM) and fat-soluble vitamins (µ g/g D.M.)
of experimental concentrates used.
Items
Rolled Barley Pasture Experimental Concentrates
L
OP
LOP
Phenolic Compounds
3,4-DHPEA
p-HPEA
Verbascoside
3,4-DHPEA-EDA
Fat-soluble vitamins
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
4.72
1.12
2.58
3.26
2.81
0.57
1.35
1.47
γ-Tocotrienol
α-Tocotrienol
δ-Tocotrienol
γ-Tocopherol
α-Tocopherol
ΣTocotrienol
ΣTocopherol
Retinol
lutein + zeaxanthin
β-Carotene
ΣCarotenoids
7.21
17.12
1.97
3.24
22.59
24.35
27.82
Nd
Nd
0.61
1.65
76.13
Nd
78.39
0.39
0.39
104.95
51.22
156.17
5.16
7.14
0.51
7.97
113.64
12.30
122.13
251.96
nd
251.96
3.72
5.54
0.64
2.68
154.91
9.26
158.23
246.17
3.32
13.24
262.73
4.43
5.75
0.38
5.45
134.65
10.18
140.48
241.13
1.98
6.70
249.81
4.7. Effects of paté dietary supplemetation in Comisana ewes
4.7.1. Production and quality of ewes’ milk
The results reported in Table 36 show no significant differences between the values of the
parameters considered, with exception of the curd firmness value that resuls higher in L
group. Supplementing the diet with extruded linseed did not result in an increase in the fat
90
content of the milk, as observed Flowers et al. (2008) in grazing dairy cows fed with a
linseed oil supplementation feed. Milk protein and fat contents were not affected by the
supplementation, this is in line with that observed by Gómez-Cortés et al. (2009) in ewes
fed with different levels of extruded linseed and Nudda et al. (2006) in goats.
Table 36. Milk yield and quality.
Parameters
Milk yield
Fat
Lactose
Protein
r
k20
a30
g/d
%
%
%
min
min
mm
Dietary Treatments
L
OP
LOP
798.1
756.6
700.5
8.08
8.78
8.35
4.56
4.52
4.62
5.72
5.80
5.76
16’.23” 17’47” 16’18”
1’31”
1’32”
1’27”
58.78a 47.80b 50.96b
Rmse
55.3
0.58
0.08
0.37
2’56”
0’26”
6.93’
A. B: P<0.01; a. b: P<0.05.
4.7.2. Ewes’ milk fatty acids composition
The ewes’ milk fatty acids composition is shown in Table 37. The treatment with
laminated linseed has led a significantly higher content of ALA in the milk of L group
compared to OP group, while the levels of group LOP were slightly lower. The values
found in group L are slightly lower than those reported by Mele et al. (2007), this may be
related to the fact that in this research the amount of linseed used is lower. The
concentration of ALA in milk is closely related to the intake-amount derived from linseed.
as proven by Mughetti et al. (2012), who tested the dietary inclusion of extruded linseed at
different supplementation levels in dairy sheep feed.
The LOP combination appears to be interesting; even though it is based an availability of
ALA of about half, group L it still leads to a valid presence of it in the milk The
administration of linseed in extruded form compared to integral flax resulted in a
significantly higher increase in the ALA content in milk of ewes (Gomez-Cortes et al.
2009; Mele et al. 2007) and in dairy cows (Akraim et al. 2007).
91
The higher content of total n-3 fatty acids (ALA) in milk caused a linear decrease of the n6/n-3 fatty acid ratio in L group, with the same trend observed by Mughetti and co-workers
(2012).
As far as conjugated linoleic acid (CLA) is concerned, the level of RA is more than double
in the milk in groups L and LOP compared to that found in the milk of group OP with
values of 1.72 and 1.62 g/100 g of FAME. In the same way the level of VA is higher in the
milk of animals treated with linseed compared to those found in group P (P<0.05). These
results confirm the observation made by other authors (Gomez-Còrtes et al. 2009; Luna et
al. 2008) regarding the direct relationship between VA and RA in animals treated with
extruded linseed. Much of the RA in the milk originates from endogenous synthesis at the
level of the mammary gland from VA by the way of ∆9-desaturase. Since the
biohydrogenation of ALA does not generate RA as an intermediate, the secretion of high
concentrations of it in the milk can be obtained from the production of high levels of VA in
the rumen (Bauman et al. 2006). As for the ALA, the increase in the milk of RA can be
attributed both to the lamination process that the extrusion that can increase the availability
of ALA at rumen level, whose process of biohydrogenation generates a high amount of VA
that is subjected to the action of ∆9 desaturase at the level of the mammary gland.
The level of RA and VA were found to be higher in group P than observed by GómezCortés (2009) in ewes fed a diet enriched with olive oil and this is probably due to the high
concentration of ALA found in the pasture compared to the test conducted by these
authors.
92
Table 37. Fatty acid composition (%) of the ewes’ milk.
Fatty acid composition
C14:0
C16:0
C18:0
C18:1c9
C18:1t11
C18:2c9t11
C18:2c9c12
C18:3c9c12c15
C20:0
C20:4
C20:5
C22:5n3
C22:6n3
SFA
MUFA
PUFA
PUFA:SFA
PUFA n-6
PUFA n-3
n-6/n-3
AA/DHA
LA/LNA
C18:2c9t11/C18:1c9t11
TBARs (MDA mg/l)
L
6.89b
18.08b
15.20
20.09
5.14a
1.72a
2.21
1.65A
0.25
0.09B
0.06A
0.101
0.035
57.69
32.61
8.74A
0.15a
2.38
1.86A
1.29C
2.72
1.36C
25.43
0.857
Dietary Treatments
P
LP
8.01a
7.24ab
20.42a
19.14ab
13.30
13.70
23.85
22.48
1.96b
4.21a
0.87b
1.62a
2.24
2.33
0.62B
1.25A
0.28
0.28
0.13A
0.11AB
0.04B
0.05AB
0.091
0.093
0.047
0.034
62.31
57.74
32.41
34.18
4.94B
7.50A
0.08b
0.12a
2.47
2.52
0.81B
1.43A
3.06A
1.87B
3.10
3.30
3.63A
1.99B
30.30
27.51
0.706
0.587
Rmse
0.52
0.81
1.18
1.45
0.81
0.24
0.19
0.16
0.02
0.01
0.01
0.014
0.007
1.75
1.50
0.49
0.01
0.20
0.17
0.45
0.68
0.53
2.00
0.156
a.b: P<0.0 5; A. B: P<0.01
4.7.3. Antioxidants and oxidative stability of ewes’ milk
The most important result of this research is the detection of free-hydroxytyrosol in milk;
the chromatograms obtained by means of HPLC-ESI-MS/MS in the negative ion mode on
the precursor ions of the hydroxytyrosol and tyrosol with a ratio of m/z of 153 (1) and 137
(2) respectively, have been revealed the presence of these phenolic alcohols in the milk of
OL and LOP groups (Figure 15). Figure 16 shows the spectra of MS/MS of hydroxytyrosol
and tyrosol found in the extract of the phenolic obtained from ewes’ milk; data were
93
confirmed by retention time and from the spectrum of MS/MS of the respective standard
samples.
From a quantitative point of view there no proportional correlation between the amount of
phenolic substances in the paté used in feed and the concentration of hydroxytyrosol and
tyrosol in the milk. the latter was detected only in traces (Figure 15). However, the
detection of these compounds in milk is an important step in a better understanding of the
bioavailability of this substances; probably enzymatic hydrolysis of these compounds leads
to the liberation of the phenolic component with subsequent transfer into milk.
0
kCounts
100
L
75
50
1
25
2
0
kCounts
200
1
150
P+L
100
50
0
kCounts
1
400
P
300
200
100
2
0
10
20
30
40
50
60
minutes
Figure 15. Chromatograms HPLC-ESI-MS/MS from milk of ewes fed with linseed (L). paté and
linseed (P+L) and paté (P). 1. Hydroxythyrosol; 2. Tyrosol.
94
122.8
100%
105.8
100%
[M-H-CH2O]
75%
[M-H-CH2OH]
75%
118.9
[M-H-H2O]
50%
50%
25%
25%
0%
0%
95
100
105
110
115
120
95
m/z
A
100
105
110
115
120
m/z
B
Figure 16. ESI-MS/MS spectrum of hydroxytyrosol (A) and tyrosol (B).
95
As far as the oxidative status is concerned, the milk of L and OP groups is lower compared
to the LOP combination (Table 38); the level of TBARs remains higher in milk obtained
from animals fed with linseed alone, compared to the others. This differences are not
statistically significant. however is important to note that this reseach trial is about a dairy
product already partially protected by the large amount of antioxidants present in pasture
such as α-Tocopherol and β -Carotene (Table 35).
The concentration of tocopherols and retinol in the milk of the OL and LOP groups (Table
38), reflects the different concentration of hydroxytyrosol, this is probably due to the
protective action that the latter has on tocopherols protecting them from oxidation; in fact it
has been proven that in olive oil under thermal stress, hydroxythyrosol derivatives are the
first compounds to be oxidized, providing oxidative stability to the oil. while tocopherols
seems to be oxidized after a significant decrease on hydroxytyrosol derivatives
concentration (Nissiotis and Tasioula-Margari, 2001).
Also in this trial a high variability due to the animals emerged; the values of
hydroxytyrosol, ranged between a minimum of 2.0 µg/l and a maximum of 37.6 µg/l in the
OP group, and between 2.0 µg/l and 13.3 µg/l in the LOP group.
Table 38. Effect of dietary treatment on the hydroxytyrosol, tocopherols
and retinol content in ewes milk.
µg/l
µg/kg
µg/kg
µg/kg
Dietary Treatments
L
OP
LOP
0.02B
10.08A 7.09A
215
300
260
12
7
10
167
220
205
(MDA mg/l)
0.857
Items
3,4-DPEA-EA
α-Tocopherol
γ-Tocopherol
Retinol
TBARs
0.706
0.587
Rmse
1.57
160
5
97
0.156
A. B: P<0.01.
Further studies have also shown that hydroxytyrosol is a metabolite of dopamine and that
small concentrations within human fluids may be due to a combination of exogenous and
96
endogenous sources (Caruso et al. 2001; De la Torre, 2008). This could explain the
presence of traces of hydroxytyrosol in milk of L group (Figure 15).
4.7.4. Oxidative status of ewes’ cheese
The cheese made from the milk of animals of L group shows a more oxidizable lipid
fraction than that obtained from animals of the other two groups (Table 39). The MDA
expressed in mg/kg of fat, show good values in OP and LOP cheeses; these values are
significantly lower than those of L group cheeses, and about ¼ of those proposed by
Hamilton and Rossel (1986) as the value limit. These values are also lower than those
observed by Severini et al. (1998) in Parmesan cheese stored under vacuum.
The evolution of the oxidative status during storage of cheese, assessed at 3 and 7 days,
shows a constant trend in OP and LOP group cheeses (Figure 17), probably due to the
protective action that polyphenols exert against tocopherol oxidation protecting them from
heat treatment. These data are further confirmed by the "yellow index" of the cheese
(Figure 18); the color alteration which is linked to the oxidation process is lower in L
group than the others.
Table 39. Oxidation of the lipids of cheese as a function
of different dietary supplementation.
Items
mg MDA /kg
cheese
mg MDA /kg
of fat
Dietary Treatments
L
OP
LOP
Rmse
0.375a
0.138b
0.121b
0.073
1.291a
0.622b
0.493b
0.235
a.b: P<0.05.
97
L
350
OP
300
LOP
µg MDA/Kg cheese
.
400
250
200
150
100
50
0
0
3
7
Days
Figure 17. Evolution of the oxidative status of cheese during storage, assessed at 3 and 7 days.
L
60
Yellow index w
OP
50
LOP
40
30
20
10
0
0
3
7
Days
Figure 18. Effect of the dietary treatment on cheese yellow index.
98
4.9. Milk phenols: extraction method
A specific method for the extraction and purification of the phenolic compounds contained
in buffalo and ewes’ milk has been develop during this research work, because up to now,
there isn't enough information about a specific procedure in the literature.
The procedure is reported below.
The milk sample (80 ml) was skimmed by centrifuging at 5000 rpm for 1 min at room
temperature. A volume of 60 ml of skimmed milk was acidified with a solution of citric
acid (1.5 M) to pH 4.6 to precipitate the caseins. Then 120 ml of methanol was added to
the sample. and after 1 min of vortex mixing. the solution was centrifuged at 5000 rpm for
2 min. The supernatant was filtered with a fluted filter. The precipitate was washed with 80
ml of a mixture of methanol and water (80:20. v/v). and after 1 min of vortex mixing. the
solution was again centrifuged at 5000 rpm for 2 min. The supernatant was filtered and
collected with the previous fraction (this operation was repeated twice). The methanol was
removed with a rotary vacuum evaporator at 37°C. and the aqueous extract was passed
through an Extract-cleanTM SPE C18-HC column (5000 mg/25 ml) that was activated and
conditioned with 10 ml of methanol and 10 ml of water. respectively. The column
containing the analyte was washed with 10 ml of water. The phenolic compounds were
eluted with 100 ml of methanol. and the collected fraction was mixed with 40 ml of hexane
in a separatory funnel. After agitation. the methanolic fraction was transferred to the rotary
vacuum evaporator to eliminate the solvent. The residue was reconstituted in 5 ml of
methanol. filtered with a 0.2 µm PVDF filter (Alltech. Deerfield. IL). dried under a flow of
N2 gas and recovered with 1 ml of methanol for LC-MS/MS analysis.
99
5. CONCLUSIONS
This research project provides information on the effectiveness of the use of different types
of pomace, such as pomace and patè, obtained from the extra virgin olive oil mechanical
extraction process, in dairy animal feeding. The improvement of the quality of dairy
product fats could positively lead to beneficial effects in the prevention of some diseases in
humans.
The results obtained are summarized in the following points.
Research 1.
The results on virgin olive dried pomace supplementation in dairy water buffalo feeding,
confirm that its use does not have any detrimental effect on animals: in fact no changes due
to the different dietary treatment emerged from the analysis of the qualitative and
quantitative parameters of the milk from treated animals, despite the high amount of
phenolic compounds in the pomace. The elimination of the stone, also, determines a
significant reduction in the ADL content, improving the dry matter digestibility of the olive
pomace.
The milk from buffalo fed with pomace shows a higher proportion in C18:1 n9 content and
higher amount in LCFA, UFA and MUFA with respect to the control group, a significant
reduction in the SAT content and atherogenic index. Notable is the total amount of vitamin
E in milk, that increased from 8.605 µg/g of fat to 10.446 µg/g of fat in the milk of treated
buffalo with a consequent decrease of the TBARs value. The high amount of phenols,
characterized by strong antioxidant activity such as 3,4-DHPEA, 3,4-DHPEA-EDA, found
in the pomace may be involved in the increased amount of α- tocopherols in the milk; in
fact phenolic antioxidants can protect tocopherols from oxidation improving both their bioavailability during digestion and their concentration in the milk.
A lower level of SFA, a higher MUFA content, a reduced n6/n3 ratio and lower
atherogenic and trombogenic indexes were found in mozzarella from buffalo cows fed with
stoned pomace.
100
Research 2.
From a comparison of the nutritional-chemical characteristics of the olive paté with those
of the pomace, the patè showed better characteristics such as a lower content of lignin and
cellulose, and a higher concentration of the lipid fraction, crude fiber and high polyphenol
content.
The milk of LOP combination, even though it is based on an availability of ALA of about
half of that of the L group, is characterized only by a slightly lower presence of ALA in the
milk. The concentration of RA is higher in milk of the L and OP group; a similar trend is
followed by VA. The oxidative stability of the milk in diets L and OP is lower compared to
the LOP combination, the level of TBARs, even though, not statistically significant, is
lower in LOP group. These values, instead, become significant in cheese, where the
oxidation of the lipids is lower in the OP and LOP group and lead to a successive constant
evolution of the oxidative status during storage of cheese, assessed at 3 and 7 days, with a
significant improvement of the cheese shelf-life.
However the main results, common to both researches, have been the detection and the
characterization of hydrophilic phenols in the milk through a specific method for the
extraction and the purification of the phenolic fraction from milk developed in these years
of research. The analytical detection of hydroxytyrosol and tyrosol in the ewes’ milk fed
with the paté and hydroxytyrosol in buffalo fed with pomace showed for the first time the
presence in the milk of hydroxytyrosol, which is one of the most important bioactive
compounds of the oil industry products; the transfer of these antioxidants and the proven
improvement of the quality of milk fat could positively interact in the prevention of some
human cardiovascular diseases and some tumours, increasing in this manner the quality of
dairy products and also improving their shelf-life.
These results also provide important information on the bioavailability of these phenolic
compounds.
101
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RINGRAZIAMENTI
Desidero ringraziare tutto il Dipartimento di Scienze Economico-Estimative e degli
Alimenti, sezione di Biotecnologie degli Alimenti dell’Università degli Studi di
Perugia. In particolare un sentito ringraziamento va al mio tutor Prof. Maurizio
Servili che ha permesso lo svolgimento di questa attività di ricerca ospitandomi nel
suddetto Dipartimento e che, nonostante alcuni rallentamenti verificatesi durante lo
svolgimento di questa attività di ricerca, ha sempre mostrato comprensione e
disponibilità.
Un ringraziamento va al Dott. Stefano Terramoccia e al Dott. Settimio Bartocci che
hanno seguito la parte relativa all’integrazione della sansa di olive nella dieta delle
bufale.
Un sentito ringraziamento va al Prof. Mariano Pauselli, che ha seguito la parte
riguardante la prova di alimentazione delle pecore con il paté di olive e che ha
risposto sempre con molta disponibilità ad ogni mia richiesta.
Un ringraziamento va al C.R.A. - Consiglio per la Ricerca e la Sperimentazione in
Agricoltura, ente finanziatore della borsa di Dottorato ed al C.R.A.-OLI per avermi
ospitata per un periodo nelle loro strutture. In particolare un ringraziamento va al
Dott. Adolfo Rosati, al Dott. Giorgio Pannelli ed al Dott. Enzo Perri per il loro
sostegno ed incoraggiamento.
Ringrazio la mia famiglia per la serenità che mi ha trasmesso durante questo
percorso.
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