UNIVERSITA’ DI PISA
DIPARTIMENENTO DI SCIENZE AGRARIE, ALIMENTARI E
AGROAMBIENTALI
TESI DI LAUREA MAGISTRALE IN
BIOSICUREZZA E QUALITA’ DEGLI ALIMENTI
EFFECTS OF PRE-SLAUGHTER DIETS ON THE VITAMIN E AND COLOUR STABILITY OF LAMB MEAT
RELATORE CANDIDATO
PROF. ANDREA SERRA ANNA VALENTINA LUPARELLI
RELATORE
PROF. FRANK MONAHAN
CORRELATORE
PROF.SSA ANNA MARIA RANIERI
Ai miei nonni , le mie radici.
“A volte mi mancate cosi tanto che credo di non farcela.
Poi ce la faccio, però mi mancate lo stesso.”
Charles Bukowski
"No single feature of man's past equals in importance
his attempt to understand the forces of Nature and himself."
Herbert Mc Lean Evans
Summary
1 INTRODUCTION ... 4
2 VITAMIN E ... 7
2.1 Nomenclature, definition, and classification of vitamins ... 7
2.2 History of vitamin E ... 9
2.3 Nomenclature and structure of vitamin E ... 10
2.3.1 Alpha and Gamma tocopherols ... 12
2.3.2 Antioxidant mechanism... 14
3 VITAMIN E IN LAMB METABOLISM ... 20
3.1 Vitamins in rumen ... 20
3.2 Lipid Digestion ... 20
3.2.1 Vitamin E absorption and transport ... 22
3.3 Food sources of Vitamin E ... 25
3.4 Animal intake of vitamin E ... 27
3.5 Human Intake of Vitamin E ... 28
4 VITAMIN E IN MEAT... 30
4.1 Role of vitamin E in meat ... 30
4.2 Meat shelf life ... 31
4.3 Meat colour ... 31
4.4 Reduction of weight by drip loss ... 34
5 LAMB MEAT ... 35
5.1 Nutritional values present in lamb ... 35
5.2 Irish lamb ... 38
6 THE AIM OF THE THESIS ... 39
7 MATERIALS AND METHODS ... 41
7.1 Breeding and slaughtering of animals... 41
7.2 Sample preparation ... 42
7.3 Analyzed parameters ... 42
7.5 Vitamin E measurement in meat ... 43
7.5.1 HPLC analysis ... 46
7.6 Recovery (meat samples) ... 48
7.7 Vitamin E measurement in feeds ... 49
7.7.1 HPLC analysis ... 51
7.8 Recovery (feeds samples) ... 51
8 RESULTS AND DISCUSSION ... 53
8.1 Vitamin E in lamb meat as affected by diet ... 53
8.2 Vitamin E in lamb meat as affected by storage ... 56
8.3 Vitamin E in lamb meat as affected by diet and storage... 59
8.4 Meat colour descriptors ... 62
9 CONCLUSION ... 65
10 BIBLIOGRAPHY ... 66
1 ACKNOWLEDGEMENTS
Ed eccomi qui a, scrivere l’ultima pagina di un percorso che ha avuto inizio otto anni fa. Sembra ieri che, ancora piccola e ingenua, ho ascoltato la mia prima lezione universitaria a Roma, convinta di voler diventare una dietista. Ed invece oggi sono qui, non più convinta di niente ma speranzosa di poter fare tutto.
Pisa è stata uno splendido imprevisto, in un percorso già più o meno stabilito, che mi ha fatto capire come nella vita tutto possa cambiare in un batter d’occhio, e quello che prima ritenevi essere una certezza per il tuo futuro diventa tutto a un tratto il tuo passato. La cosa più bella di questa storia è che è stato il fato, o in qualunque altro modo voglia essere chiamato, a guidarmi in questo viaggio pieno di imprevisti, dolori e sacrifici, ma allo stesso tempo pieno di gioie, soddisfazioni e sorrisi ,che mi ha portato proprio dove dovevo essere.
E in questo strano gioco del destino ci sono stati molti complici. Infatti, se sono qui oggi non è certo solo merito mio ed è per questo che voglio e devo ringraziare molte persone a me care che hanno reso possibile tutto questo e ad oggi, come FINE del mio percorso universitario, voglio farlo nel miglior modo possibile.
Innanzi tutto mi sembra doveroso ringraziare il mio relatore, il professor Andrea Serra per avermi permesso di vivere una delle esperienze più belle della mia vita, quella dell’erasmus. Ricordo ancora quando andai nel suo ufficio e, senza avere idea di quello che avrei potuto fare o di dove sarei potuta andare, lui, più entusiasta di me, in meno di cinque minuti mi trovò quel tirocinio che poi si rivelò essere fatto su misura per me. Professore, la (ti) ringrazio per l’entusiasmo con cui mi è stato accanto e per la fiducia che ha riposto in me.
Vorrei poi ringraziare le persone che mi sono state più vicine a Dublino e che mi hanno accompagnato nel percorso di tesi nella maniera migliore possibile ovvero il professor Frank Monahan e la dottoressa Rufielyn Gravador. Grazie per la vostra gentilezza, disponibilità e interesse nei miei confronti, non avrei potuto incontrare persone migliori di voi. Un grandissimo grazie va soprattutto a te, Rufi, sei una delle persone più gentili e dolci che io abbia mai conosciuto. Sei stata fondamentale in quei sei mesi e ti ricorderò sempre con grande affetto, sperando di non perderci mai di vista. Un grazie anche a
2 tutte le persone conosciute li a Dublino, soprattutto a Elisabetta e Dora, che mi hanno fatto sentire a casa.
Un grazie speciale va alla mia mamma, una donna che ha sempre pensato di non essere forte e che invece ho capito esserlo molto più di altri, non arrendendosi mai, proteggendoci e sostenendoci sempre, non avendo paura di annullare se stessa per noi. Cara mamma, non ho mai conosciuto una forza e un amore più grandi.
Al mio papà, che ha sempre creduto in me e che mi ha amata incondizionatamente, che mi ha insegnato l’umiltà e a guardare avanti senza avere mai rimpianti, che ha sempre fatto di tutto per rendermi felice e accontentare ogni mio capriccio. Spero di averti reso fiero di me. Una bacio forte anche a Paolo che, nonostante tutto, sarà sempre il mio fratellino. Grazie perché so che sarete sempre al mio fianco.
Un grazie amorevole a zia Mimmi che mi ama come una madre e che mi è sempre stata vicina. Grazie per aver creduto in me e non aver mai dubitato del mio potenziale. Un grazie speciale anche a tutti gli altri zii, anche quelli acquisiti, che mi hanno sempre amata incondizionatamente come una figlia e sempre mi sono stati accanto.
Un grazie affettuoso ai miei nonni, che, anche se non ci sono più, so che da lassù mi hanno sempre protetta e accompagnata, senza mai allontanarsi. Nei momenti più importanti ho sempre sentito la vostra presenza vicino a me. Un pensiero in particolare, oggi, va a mia nonna: il mio più grande rimpianto è che tu oggi non possa essere qui a vedermi superare questo traguardo che non vedevi l’ora che raggiungessi per poter stare più con te. Mi piace pensare che l’ultima spinta me l’abbia data tu da lassù. Grazie perché è anche per te se oggi sono qui. Mi manchi tanto.
Un grazie a Nigel, che in questi quattro anni, anche se a volte a modo suo, mi è sempre stato vicino. Anche se siamo molto diversi, riusciamo a completarci in un modo che non avrei creduto possibile. Sei tranquillità nella mia ansia, sei sostegno nella mia tristezza, sei un trampolino nei miei infiniti dubbi.
Un grazie gigantesco, infine, a Pisa, la mia seconda casa, che mi ha accolta e mi ha permesso di scoprirmi, conoscermi e crescere. Grazie agli aperitivi in vettovaglie, alle birre in cavalieri, alle vasche in corso Italia, al “ci vediamo in piazza Garibaldi”, alle nuove e vecchie amicizie, alle serate quelle belle e alle serate senza senso. Grazie a tutti coloro che mi sono stati accanto in questi anni, e che hanno reso indimenticabile questa esperienza.
3 Grazie soprattutto alla mia Vale che ha reso speciale ogni momento. Grazie per le chiacchierate, per i pianti e le risate, per le nottate al telefono, per le sfogate, per gli abbracci, per i consigli; perché ci sei sempre stata e perché so che ci sarai sempre. A tutti voi che siete qui oggi a rendere speciale questo giorno…per ora non posso immaginare una gioia più grande di quella che sto provando adesso ed è bellissimo poterla condividere con tutti voi.
4
1 INTRODUCTION
Animal nutrition is one of the most important and varied topic in the animal sciences. This discipline is interested in development and improvement of animal feeding systems and ranges from the chemistry of nutrients, through their digestive physiology, to the modelling of nutrient requirements, with considerations of the characteristic level of food intake. Food intake is important to check the effects of the food on the animal health and final nutritional quality of animal products like meat, milk etc. These products, in fact, depending on how the animals were fed, will have different chemical and nutritional features. The diets of the various animal species are composed of mixture of nutrients with a more or less complex molecular structure and are represented by: proteins, lipids, carbohydrates, vitamins, minerals, water which cannot be absorbed as such. The set of mechanical, enzymatic, and microbiological processes that determines the conversion of nutritive principles contained in food in small diffusible and assimilable molecules, is called digestion.
As far as nutrition is concerned, there are a lot of different types of animals. Based on their digestive characteristics and on the main types of food they usually eat, they can be grouped into four classes. There are mainly carnivores (which eat meat and have a mainly enzymatic digestion and a poor microbial digestion), omnivores (which eat meat and vegetables and exhibit a mainly enzymatic digestion, even if microbial fermentation processes occur in their large intestine) and herbivores, the group we are interested in for this work, which eat food of vegetable origin. They are mainly divided into polygastric herbivores (cattle, sheep, goats) which have an intense microbial fermentation process before undergoing the action of digestive enzymes, which starts in the abomasum and continues in the small intestine, and monogastric herbivores (horse and rabbit) in which the microbial fermentative processes are considerable, but are carried out in the back of the digestive tract (blind and colon), when the food has already undergone enzymatic attack in the stomach and in the small intestine.
One of the most important classification of herbivores is by Hofmann and Stewart (1972), who grouped these animals into grazers (which eat mainly grass), concentrate selectors (which eat sugar, protein and fat- rich food) and intermediate feeders, animals which select their diet according to availability.
5 Hofmann (1989), in his classification, described many differences between ruminant and non-ruminant herbivores; The ruminant’s digestible tract is distinguishable from monogastric one by the presence of three different communicant pre-stomachs, rumen, reticulum and omasum before the glandular stomach (abomasum). Among these, the abomasum is morphologically and physiologically similar among all mammal. The pre-stomachs are colonized by a set of micro-organisms which, through the development of anaerobic fermentation process, allows the animals using food which cannot be used by monogastrics. He concluded that grazer have a smaller liver and a more complex and bigger rumen than concentrate selectors.
This grazer’s developed ruminant digestive tract is very important because allows the animal to maximize the extraction of digestible constituents from vegetative plant material. The vegetal cell is characterized by the cell wall, a fibrous material, made up by both digestible (pectin) and indigestible matter (cellulose, hemicellulose and lignin). Both ruminant and non-ruminant species, but especially the intermediate feeders and grazers, are able to utilize the cellulose and hemicellulose, thanks to the microbial population throughout the rumen fermentation of the fermentable matter, being hemicellulose faster fermentable than cellulose., can use these food components. On the other hand, the monogastric mammals don’t have endogenous enzyme suitable for this digestive process. Ruminants have also a big malleability in food intake and digestion; Among them, especially sheep, can increase the size of their rumen and the retention time of undigested food, depending on the type of food (more or less mature, fibrous etc.), in order to release the single nutrients by digestion from their combination in food and absorb them from the digestive tract.
Nutrients are food constituents and they are used for a lot of metabolic functions. Some of them can be named “essential” because they are required for the proper functioning of the animal body and cannot be synthetized by the animal or at least cannot be made in sufficient amount to meet the animal’s requirement (Dryden, 2008).
These essential nutrients include some amino acids (about 10), some fatty acids (linoleic and linolenic), minerals and vitamins, known to be very important for the animal and the final product nutritional value. Some vitamins are also important for the quality of the final animal product, such as meat, because they play an important role of antioxidants and can improve the shelf life of the product during storage.
6 The most important vitamin with this role is vitamin E (tocopherols). It is a biological antioxidant and in association with other vitamins and several enzymes (glutathione peroxidase etc.), it protects cells against free radicals and oxidative damage, which can negatively affect the cell membranes functionality, cell tissues and cell nuclear material. Antioxidant, such as vitamin E, are of vital importance in maintaining the health of the living animal and in protecting the stability of the animal final product during the storage. Vitamin E in the meat, fights in preventing oxidation of polyunsaturated fatty acids, which are constituents of cellular membranes, offsets endogenous muscle pro-oxidants and product preservation time increases.
In this work, the effect of finished diets with different vitamin E content on final vitamin E concentration of lamb meat were investigated.
The objective of this study was to assess the vitamin E content and colour stability during a 14 days long storage period under modified atmosphere packaging (MAP) composed by 80% O2 : 20% CO2 at 4°C. It was hypothesized that each diet would have
7
2 VITAMIN E
2.1 Nomenclature, definition, and classification of vitamins
The term Vitamin indicates a certain number of organic substances essential for the life. In terms of chemical structure, they are very different each other’s, showing a well specific function, which cannot be replaced by other nutrients. Some of these are involved in the regulation of gene expression (such as vitamin A, D, vitamin B12 and thiamine), others are enzyme cofactors (vitamins of B group and vitamin K), some of them regulate the chemical reactions of the cells, or ensure vital functions in the tissues, or participate in the production of energy, or protect visual function (vitamin A), or improve the defense mechanisms of the immune system, or play the important role of antioxidants against free radicals etc.
Vitamins are nutrients that animals cannot synthesize themselves. Through the evolution, in fact, animals lost the ability to synthesize these substances or, at least, synthetize them in adequate amounts. However, their functions in animal survival are so important that it is therefore necessary to ensure the correct daily intake of these components in another way. They must be assumed inevitably through the diet and for this reason they are defined as essential.
Animals, in fact, need these important factors in their diet in addition to basic nutrients (proteins, fats and carbohydrates), for their importance in preventing diseases and maintaining the right physiological state of the organism. A no-enough amount in the tissues (due both to the diet and to failure in absorption from the diet) causes a specific deficiency syndrome, well recognized since ancient times. For instance there is an accurate description of scurvy, a disease caused by a deficiency of vitamin C, characterized by swollen bleeding gums and the opening of previously healed wounds, which particularly affected poorly nourished sailors until the end of the 18th century, by Hippocrates (c. 420 B.C.) and in the Eber papyrus in c. 1150 B.C..
Likewise, beriberi, a disease causing inflammation of the nerves and heart failure, caused by a deficiency of vitamin B1 (thiamin) was described in Chinese herbals, dating back around 2600 B.C..
8 Although it was clear the link between certain diseases and the lack of vitamins, this connection was not considered until the 20th century, when, in 1886, Christian Eijkman was sent in Indonesia to discover the cause of beriberi, which was initially associated with a bacterium. He realized quickly that there isn’t a bacterial cause but the problem stemmed from a food shortage. In fact, he noticed that some chickens kept in the laboratory manifested a disease similar to beriberi, until the disease suddenly disappeared. He immediately associated this with a change in diet for the chickens. He confirmed the link between the disease and the lack of some nutrients and understood that polished rice causes the disease, whole meal wheat prevented it.
Subsequently Casimir Funk, relying on this model, came close to isolating the anti-beriberi factor. So, the first vitamin, thiamin, was discovered by Funk (1911, 1913). He characterized it as an amine and, because of its essential role, gave it this name, vitamin or “Vital amine”.
After his discover, he observed: “I must admit that when I chose the word vitamin, I
was well aware that these substances might later prove to not all be of an amine nature. However, it was necessary for me to use a name that would sound well and serve as a catch word”.
In the following decades, the different vitamins were gradually identified. Vitamins are bio-regulators of decisive importance because they preside, together with hormones, the performance of all physiological processes both directly and indirectly through enzymatic mechanisms. Vitamins have very different chemical structures each other’s and are mainly classified in two groups based on solubility: water-soluble and fat-soluble. Currently, four fat-soluble vitamins (Vitamin A: retinol; Vitamin D: calciferol; Vitamin E: tocopherol; Vitamin K: phylloquinone and menachinone) and 13 water-soluble vitamins (Vitamin B1: thiamin; Vitamin B2: riboflavin; Vitamin B3: niacin; Vitamin B5: pantothenic acid; Vitamin B6: pyridoxine; Vitamin B8: biotin; Vitamin B9: folic acid; Vitamin B12: cyanocobalamin; Vitamin C: ascorbic acid; Choline, Lipoic acid; myo- inositol; p-aminobenzoic acid (AINCN, 1987)) are recognized.
9 2.2 History of vitamin E
Vitamin E was discovered more than 80 years ago, exactly in 1922 at H.M. Evans and his assistant, K.S. Bishop, during an experiment on rats. They realized that rodents fed a rancid lard diet, vitamin E deficient, were mostly sterile or gave birth to completely sterile animals. So, they determined that this new nutritional lipophilic factor, initially called factor X, was a compound necessary to sustain reproductive capacity in rodents and for the prevention of fetal animal death, which, with this nutrient deficiency, occurred despite a normal ovarian function of the animal (Evans e Bishop,
1922). The experiment consisted of the evaluation of two groups of rats fed same diet. The only difference was that the diet of the second group also included vegetables (salad, sprouted wheat), therefore a source of this factor X.
The two researchers observed that the rats subjected to a rancid lard diet grew well but could not reproduce because the embryos were reabsorbed before the end of gestation. Only when vegetables such as fresh lettuce green leaves were integrated into the diet we see a relatively quick restoration of fertility in sterile rats. The same conclusion was formed by Barnett Sure who performed similar dietary experiments and coined the term vitamin E since vitamins A, B, C and D were already identified.
Subsequently, thanks to further investigations on vitamin E, the isolation of α-tocopherol (one of the most important form of vitamin E) from wheat germ and β- and γ- tocopherol from vegetable oils was reached, even if these last two compounds have only about 45 and 13 % of the activity of the α form, respectively.
If initially vitamin E has been identified as a vital substance for reproduction, today it is considered an essential nutrient, involved in numerous biological systems, including mechanisms against oxidation, the cardiovascular and neuromuscular system, as well as the regulation of cell growth and it is known that the lack of vitamin E causes anomalies, including dangerous motor dysfunction in humans. Though most of the
10 molecular causes of these anomalies remain unknown, an association with the lack of vitamin E is clear, since a reintegration into the diet of this fat-soluble factor removes them.
2.3 Nomenclature and structure of vitamin E
Actually, the alphabetic designation of the vitamin E is not sufficiently comprehensive of the all characteristics of this factor, so is it better to define it more specifically.
Vitamin E is a collective term to describe a group of derivatives of 6-hydroxychroman carrying a phytol side chain. This term refers to all structurally similar tocotrienols and tocopherols (termed “Tocochromanols” or the “Vitamers” of vitamin E) and to all their derivatives that have the biological activity of α-tocopherol in prevention of rat fetal resorption. The term tocopherol derives from the Greek language and is a combination of the words childbirth (tokos), -to bring forth (phero) and alcohol (ol) that explains the role of vitamin E in animal reproduction.
Basically, there are four tocopherols and four tocotrienols in nature. Chemically all these eight lipophilic compounds derive from 6-chromanol, which presents in position 2 a methyl group and a side chain with 16 carbon atoms. They differ in the number and position of methyl groups on the phenol ring of the chromanol head group.
The side chain can be saturated or unsaturated. This is the difference between tocopherols (with fully saturated side chain) and tocotrienols (with three double bonds at the 3’, 7’ and 11’ positions). For each series, four homologues exist, α, β, γ and δ, based on the number and position of methyl groups present on the chromanol head.
11 Fig. structure of tocopherols and tocotrienols
For example, when the chromanol head is fully methylated and the phytol tail is saturated, than this “vitamer” is called α-tocopherol (2,5,7,8,-tetramethyl-2R-( 4’R,8’R,12 trimethyltridecyl)-6-chromanol).
All the compounds are found in nature but only α-, β, and γ-tocopherols and α- and β-tocotrienols are widespread.
A- and γ-Tocopherol are the most abundant forms of vitamin E found biologically and in the diet.
12 2.3.1 Alpha and Gamma tocopherols
The α-tocopherol is chemically the 5,7,8- trimethyl-tocolo, a hydroxy derivative of the chromane, formed in turn by a benzene nucleus condensed with a pyranic core, and carrying an isocetyl chain in position 2.
Thus, the tocopherols can be considered tocolo derivatives bound to a saturated isoprenoid chain with three chiral centers.
The α-tocopherol molecule has three chiral centers, so stereoisomers can occur. The naturally occurring molecule is the D-α-tocopherol (or RRR-α-tocopherol) configuration; this compound shows the highest biological activity.
Synthetic DL-α-tocopherol acetate is used as a vitamin E supplement and comprises all eight possible stereoisomers; only one molecule out of eight is in RRR form. The vitamin activity of the four stereoisomers in the L forms is considerably lower than the four that make up the D forms; in the latter, the RRR form is the predominant form of vitamin E, especially in plasma and tissues of animals, and the most active. In addition to α-tocopherol antioxidant ability, this form of vitamin E has a contributory role in cell signaling. α-tocopherol may inhibit a protein involved in cell proliferation and differentiation in smooth muscle cells, monocytes and platelets, protein kinase C. A-tocopherol can also alter other proteins like VCAM-1 and ICAM-1, important factors in cardiovascular disease risk since they cause the fixation of some blood cellular components to the vascular endothelium.
A-tocopherol is also an important antitumor factor because is able to increase the production of vasodilator prostanoids (prostaglandin I2 and prostaglandin E2).
In addition, this form of vitamin E increases the expression of phospholipase A2 and the activity of COX-1, but at the same time decreases C- reactive protein and monocyte interleukin-6 concentrations and inhibits the TNF-α and the NF-κB (a redox- sensitive transcription factor) binding activity by 5-lipoxygenase inhibition (Robert E.C.Wildman, 1964).
This last effect is observed for α-tocopherol succinate and not for α-tocopherol acetate even if in an animal investigation it was observed that α-tocopherol acetate decreased liver NF-κB activity without affecting other endogenous antioxidant systems.
13 Because of the RRR-α-tocopherol superior potency in bioassays for prevention of deficiency symptoms, a lot of supplements contain only α-tocopherol. For this, recently, α-tocopherol has been studied in an isolated form, not considering the positive characteristics of other forms of vitamin E.
In fact, a lot of recent studies suggest that other form of vitamin E can be biologically active (Hensley et al.,2004; Jiang et al., 2001), like γ-tocopherol.
There are some differences in the chemical reactivity of α- and tocopherol. γ-Tocopherol more effectively inhibits lipid peroxidation, LDL oxidation and peroxide generation. Similarly, it stimulates the expression and activity of superoxide dismutase more effectively (T.P.Coultate, 2002).
The physiological metabolite of γ-tocopherol is γ-CEHC (γ-carboxy-ethyl-hydroxy-chroman) which has anti- inflammatory activity. This γ- form of vitamin E inhibits COX activity in lipopolysaccharide-stimulated macrophages and interleukin-1β-stimulated epithelial cells.
Γ-tocopherol supplementation also inhibites protein nitration and saves vitamin C against inflammation (Robert E.C.Wildman, 1964).
The 5 position of γ-tocopherol is highly nucleophilic and reactive toward electrophiles such as NO• and NO2•. During peroxynitrite-induced lipid peroxidation studies,
α-tocopherol was converted to α-tocopherilquinone, whereas γ-α-tocopherol, in several models of inflammation and nitrosative stress, can be nitrated by reactive nitrogen species and converted to 5-NO2-γ-tocopherol (a biomarker of nitrosative stress)
(Christen et al., 1997). α-Tocopherol doesn’t have this ability, because it is methylated at C-5 on the phenol ring. As γ-tocopherol has an unsubstitued position on the chromanol head, it can scavenge nitrogen oxides. γ -tocopherol may also protect other biomolecules, such as tyrosine, from nitration.
Thanks to a lot of research, it is possible to see that supplementation with γ-tocopherol inhibites protein nitration and ascorbate oxidation in rats with inflammation. (Jiang et al.,2002)
Several studies on rats demonstrated that a γ-tocopherol supplementation reduces inflammation by suppressing the synthesis of various pro inflammatory substances like prostaglandin E2 and leukotriene B4 and results in a greater reduction in blood clots than in alpha-tocopherol.
14 Even if the role of γ-tocopherol on rats is clear, its role in humans is still uncertain. It seems to be positive against coronary artery diseases, it can protect cells from the mutagenic and carcinogenic effects of very dangerous reactive molecules. It can be a protective molecule against cancer and against Alzheimer.
In conclusion, although the α-tocopherol has long been recognized as one of the most active forms of vitamin E, above all for its important antioxidant function, research has shown that the full range of vitamin E is much more effective. The different forms of vitamin E exert complementary effects against free radicals. Together they can fight a much wider spectrum of free radicals than individually taken alpha-tocopherol. The alpha-tocopherol, in fact, with other forms such as γ-tocopherol, is able to immobilize and remove highly toxic free radicals such as nitrogen peroxide.
2.3.2 Antioxidant mechanism
Fig. phases of the antioxidant mechanism
Antioxidant are important substances necessary to protect the animal’s cell and tissues from damage due to the presence of free radicals, highly reactive products containing one or more unpaired electrons. Formed during cellular metabolism, free radicals are highly unstable molecules and their reactivity comes from the need to lose or accept an electron. Because of their instability, free radicals are capable to react with numerous
15 biological molecules, creating modifications in structures, damaging cell membranes, enzymes and cell nuclear material, bringing often irreversible damages. All classes of biological molecules are vulnerable to free radical damage, especially proteins, DNA and lipids, which are the most susceptible.
The oxidative process is a mechanism that requires a trigger, such as the interaction of certain substances with light radiation, high temperatures, metals, etc. but, above all, it is caused by the interaction of some molecules, such as PUFAs, with reactive oxygen species (ROS) ore reductant metals, which lead to the formation of alkyl radical, which continue the oxidative process. Once this process started, it is difficult to control and decelerate it. The oxidative process, in fact, becomes a real chain reaction which can be extremely damaging especially in the destruction of polyunsaturated fatty acids and, in absence of protective molecules, proceeds undisturbed.
Oxidative stress, especially lipid peroxidation, which perturbs the structural integrity of the membrane lipid bilayer, is a non-enzymatic process initiated when a free radical attacks the methylene carbon, especially when it is present between a pair of double bonds. This mechanism may result in the formation of carbon-centered radicals that can react with oxygen to form peroxyl radicals. Lipid peroxidation is continually propagated through the regeneration of a carbon-centered radical, becoming an autocatalytic process.
Fig. oxidation phases
16 To contrast this process, break the chain reaction and maintain cell integrity, the animal’s cell requires protection mechanism, an antioxidant system, which is provided by molecules with particular characteristics, able to oppose the free radicals and convert them into less reactive substances. These antioxidants include some enzymes such as superoxide dismutase (containing copper, it eliminates superoxide radicals formed in the cell), glutathione peroxidases (containing selenium, able to detoxifies lipid hydroperoxides) and catalase (which breaks down hydrogen peroxide), and a group of vitamins such as vitamin A, vitamin C and vitamin E, which is the main antioxidant. It is located in the mitochondria and endothelial reticulum of the mammalian cell.
It donates a hydrogen atom to the free radical to form a stable molecule, stopping the oxidative reaction.
This protection is particularly important in preventing oxidation of polyunsaturated fatty acids, which are the primary constituents of subcellular membranes and precursor of prostaglandins. Because many tissues contain substantial quantities of essential fatty acids, it is important that they also contain an adequate supply of antioxidant system to protect against lipid peroxidation.
2.3.2.1 Vitamin E Antioxidant activity
The tocopherols and tocotrienols have always been considered to be the most effective free radical quencher in the nature. Vitamin E is the major lipid-soluble antioxidant found in tissues, red blood cells and plasma. α-tocopherol shows highest antioxidant activity; in fact, it contains three methyl groups on the chromanol head functioning to stabilize phenoxyl radicals, whereas the other tocopherols lack one or more methyl groups). It’s well know biological function is to be a peroxyl radical scavenger, which stops lipid auto-oxidation, prevents the propagation of lipid peroxidation and maintains the integrity of biological membranes (Burton and Traber, 1990).
It is known that a single vitamin E molecule is able to protect about 1000 lipid molecules from the second step of oxidation. Vitamin E, in fact, has a greater affinity to react with peroxyl radicals, compared to PUFAs, so it is able to protect them, even because the tocopherol radical is relatively unreactive.
17 itself and forming a tocopheroxyl radical.
Then, this molecule can turn into different ways: it can lose a second electron, becoming further oxidized to form a tocopherol quinone; it can react with other radical forming a nonreactive product; it can turn back into a tocopherol thanks to another antioxidant such as vitamin C; it could potentially reinitiate lipid peroxidation through a particular process called tocopherol-mediated peroxidation.
.Fig. tocopherol-mediated peroxidation
Although the oxidation of tocopherol to α-tocopheroxyl radical is reversible, oxidation of
the α-tocopheroxyl radical to α-
tocopherilquinone is not reversible.
The 6-hidroxyl group of the chroman ring is the reactive portion of the tocochromanols. The phenolic hydrogen atom is donated to a free radical, neutralizing it and forming the aforementioned tocopheroxyl radical.
18 Many studies have been done regarding the antioxidant activity of vitamin E and has been highlighted that the antioxidant activity of tocopherols varies considerably depending on the experimental conditions of the assessment method employed (Kamal-Eldin and Appelqvist,1996).
Vitamin E is not the only piece of means by which cells are protected from oxidative damage. However, vitamin E occupies a unique position in the overall antioxidant picture owing to its localization in cell membranes and its efficacy at remarkably dilute concentrations. Cell membranes typically contain only one molecule of α-tocopherol per several hundred phospholipid molecules.
19
2.3.2.2 Other functions of vitamin E
Although the fundamental task of vitamin E in the living animal cell is to represent the physiological antioxidant that protects vitamin A and saturated and unsaturated fatty acids from possible oxidation process, vitamin E in general is involved in many other functions. For example, it has great importance for the male and female reproductive sphere. It appears to have a preventive action against premature abortion and curative action in respect of some forms of non-anatomical infertility.
Moreover, it participates in synthesis and metabolic processes. It is endowed with surfactant properties and it is indispensable for the genesis of many enzymes and coenzymes, for the synthesis of ascorbic acid and of nucleic acids. It increases the body's tolerance to toxic substances.
Vitamin E may also play an important role in the regulation of cell signaling and gene expression, in the development of the immune system and in preventing chronic disease such as heart disease and cancer. Alpha-tocopherol, in fact, inhibits the activity of protein kinase C, an enzyme involved in cell proliferation and differentiation in smooth muscle cells, platelets, and monocytes.
Due to vitamin E repletion endothelial cells lining the interior surface of blood vessels are better able to resist blood-cell components adhering to this surface. Vitamin E also increases the expression of two enzymes that suppress arachidonic acid metabolism, increasing the release of prostacyclin from the endothelium, which, in turn, dilates blood vessels and inhibits platelet aggregation.
Concluding with animal sphere, in according with the National Research Council, it is known that vitamin E is able to reduce the incidence of mastitis, retention of placenta and hypofertility, especially in cows and sheep. In several studies, it was observed that supplementation of animal diets with the vitamin provide some protection against heart disorders, muscular dystrophy, infection with pathogenic organism.
It is known that, exactly like vitamin A, the transfer of vitamin E across the placenta is limited so the neonate need colostrum to meet its requirements. Nonetheless, in sheep, the passage through the placenta occur, so that there is a good concentration in muscle and brain in lambs, even if colostrum remain a very important source of vitamin E for the new born in general.
20
3 VITAMIN E IN LAMB METABOLISM
3.1 Vitamins in rumen
Vitamins are essential and irreplaceable nutritional elements for animals and ruminants in general. They play the role of organic catalysts and must be regularly employed to guarantee health and productivity at all times in the life of the animal. Speaking more specifically about lambs, they are unable to synthesize water-soluble vitamins and vitamin K until the rumen has completely developed. The vitamin requirement is almost completely satisfied when the lamb is fed with breast milk. In adult ruminants, rumen microflora is able to synthesize both the water-soluble vitamins (group B) and the vitamin K. For this reason, it was believed that an animal with functioning rumen was almost entirely self-sufficient in maintaining adequate levels of water-soluble vitamins but instead, it needed the integration of the diet with liposoluble vitamins (A, D, E). In fact, these are not synthesized in the rumen. Today we know that, in order to guarantee high production levels, not only the right requirements of fat-soluble vitamins but also of B vitamins must be provided. In fact the use of large quantities of concentrates or other by-products modifies significantly ruminal pH and composition and the activity of rumen bacterial flora, reducing endogenous production of vitamins. Sometimes it is appropriate to supplement the diet of dairy and meat animals with complete supplements of fat-soluble vitamins and with vitamins belonging to group B.
In dairy animals, it is now known the improvement effect on fertility allowed by high levels of vitamins A, β-carotene, but above all E.
3.2 Lipid Digestion
As we said, vitamins A, D and E must be mandatorily supplied to the animal through the diet as the body is unable to synthesize them. Since these vitamins are fat-soluble, their digestion and absorption within the animal organism will be very similar to the other lipid substances in the diet.
21 Lipid supplementation is used to cover energy requirement and/or to supply some fatty acid able in improving the milk nutritional quality. Lipid can be provided as oil (rumen protected or not), or by protein-oleaginous concentrate (soy, sunflower, safflower, canola, linseed, etc.). These lipids are mainly represented by triglycerides and, depending to their chemical structure, can have a double fate: can be “intercepted” by the rumen bacteria, or can bay-pass prosecuting thought digestive tract. When rumen bacteria pick triglycerides up, unsaturated fatty acids are bio-hydrogenated, giving rise saturated fatty acids. During rumen bio-hydrogenation process, some important fatty acids such as rumenic acid (cis9,trans11 C18:2) and trans vaccenic acid (trans11 C18:1) are produced.
If the triglycerides bypass rumen, they are digested and adsorbed in a same way of a monogastric specie, thus occurs in the small intestine. This can happen also for the vitamin E.
Degradation of vitamin E in the rumen (contribution 20.000 U.I.)
grain corn medical hay raw cellulose
(%SS) Degradation (%) 20 80 24,1 8,4 50 60 18,9 22,2 60 40 11,9 25,0 80 20 3,8 42,5
22 3.2.1 Vitamin E absorption and transport
Because of its insolubility in water, vitamin E requires special transport mechanism during its absorption. It is absorbed with other lipids components of the diet, which are incorporated into particular molecules named chylomicrons, and eventually transported by other circulating lipoproteins.
Tocopheryl esters, if present, are hydrolyzed to free tocopherol by pancreatic esterases in the proximal lumen of the small intestine. In addition to pancreatic esterases that are required to cleave fatty acids from triglycerides, bile acid secretion is equally important, as both are necessary for the formation of mixed micelles which makes vitamin E absorption possible.
After this, tocopherols and tocotrienols are absorbed into enterocytes lining the small intestine. When they are inside the enterocytes, tocochromanols are incorporated, along with the other lipid components, inside the so-called chylomicrons, lipoproteins with lower density and greater diameter, responsible for collecting triglycerides, cholesterol, phospholipids and other liposoluble components, allowing then the transport mechanism of these compounds in the aqueous milieu of the blood.
Fig. vitamin E absorption
23 This transport is possible thanks to the hydro-solubility conferred by the proteins, which increases the level of solubility of the chylomicron in the aqueous medium.
The chylomicrons, secreted into the intercellular space, after exiting the enterocyte with a mechanism of exocytosis, pass into the interstitial fluid, from here to the lymphatic vessels, and then into the bloodstream.
It is thought that this obligatory path is linked to the permeability of the chylomicrons, which by virtue of their important dimensions, would encounter many difficulties in crossing the blood capillaries inside the villus.
The chylomicrons, carried by the blood, bind to sites on the capillary wall.
Thanks to this bond, the chylomicron yields part of the triglycerides to the tissues (above all to the muscular and adipose tissues), reducing its lipid load.
Subsequently, the chylomicrons (remnant chylomicrons), poor in triglycerides, reach the liver and penetrates inside. The hepatocytes digest the outer envelope of a protein nature, freeing their lipid content (residual triglycerides, cholesterol, phospholipids and fat-soluble vitamins). This mechanism allows absorption, transport and use of lipids by the body. Estimates of the absorption efficiency of tocopherol vary widely, but it appears that roughly half of the tocopherols consumed in the foods is absorbed, with the remainder excreted in the feces. It appears to be no major differences in the rates of intestinal absorption of the various forms of vitamin E (Kayden and Traber, 1993). It is also important to remember that fat-soluble vitamins share absorption mechanisms and compete with each other. Thus, an excess of vitamin A, for example, increases the demands of vitamin E, D and K (Goncalves, Aurélie, et al., 2015).
But the vitamin E journey does not end here. Its fate continues in the liver. The recently absorbed vitamin E is taken from the cells of the hepatic parenchyma and is incorporated into nascent low-density lipoproteins named VLDLs. Linked to these structures, vitamin E is secreted back into the bloodstream or, more rarely, metabolized in the liver to a water-soluble metabolite. The process of secretion of α-tocopherol from the liver via VLDLs is extremely important to maintain normal plasma vitamin E levels. Endogenous vitamin E in other tissues appears to be released to high-density lipoproteins (HDLs) and reported to the liver or other tissues.
24 Vitamin E can be transported by different lipoproteins and can be exchanged between lipoproteins and tissues or between the only lipoproteins, facilitated by a phospholipid transfer protein found in plasma (Kostner et al., 1995).
The alpha tocopherol transfer protein (α-TTP), a protein that binds tocopherols, plays a fundamental role in vitamin transport. This α-TTP is one of two proteins known to bind vitamin E with high affinity. It is found in the liver and it mainly binds RRR-α-tocopherol (Manor and Morley, 2008). It can transfer α-RRR-α-tocopherol from one membrane to another in a process involving direct membrane interaction (Morley et al., 2008). The other protein with high affinity with the vitamin is cytochrome P450-4F2.
This tocopherol-binding protein result in a preferential enrichment of LDLs and HDLs with α-tocopherol compared with the other forms of vitamin E.
In general tocopherols, especially α-tocopherol, are equally distributed between LDLs and HDLs. The tocopherols contained in the lipoproteins are released into the tissues; this is mediated by some receptors. There are several mechanisms of cell uptake and they are all different, such as the selective uptake from HDLs via scavenger receptors, as in the case of cholesterol, or part of the vitamin E, in association with chylomicrons and VLDLs may be transferred to peripheral cell during the lipolysis of these triacylglycerol-rich lipoproteins, by lipoproteins lipase.
A lot of studies demonstrated enhanced vitamin E uptake by skeletal muscle but not by adipose tissue or brain (Sattler et al., 1996).
25 3.3 Food sources of Vitamin E
Vitamin E, unlike vitamin A, is not stored in large quantities in the animal body, so it must be taken regularly with the diet.
Fortunately, different kinds of food contain vitamin E. It is constantly present in vegetable and animal food; cereal germs, related oils and shoots are particularly rich of it. A significant amount of vitamin E is also available in green parts of all plants. This is very important for animal nutrition as green fodders are good sources of α- tocopherol. Generally young grass is a better source of vitamin E than mature herbage. The leaves contain 30 times as much vitamin E as the stems. Losses during haymaking can be as high as 90 per cent, while losses during ensilage or artificial drying are much lower. It is contained in green leafy vegetables and in untreated hay, too. Cereal grains are also good sources of the vitamin, but the amount of it depends on species. Wheat and barley are similar to grass in content of α-tocopherol and maize, instead, contains a significant amount of γ-tocopherol as well. During the storage of moist grain in silos, the vitamin E activity can drastically decline. A recent study said that the reduction in the concentration of the vitamin can be from 9 to 1 mg/kg in moist barley stored for 12 weeks. Nuts and seeds in general and vegetable oils are among the best sources of alpha-tocopherol.
Even for humans the main source of vitamin E is linked to cereals, vegetables and oils. In products of animal origin, in fact vitamin E is contained in small doses (except for the placenta and for the hypophysis which are particularly rich of it). This happens although the amount present is related to the level of vitamin E in the diet. But, even if we speak more of α- tocopherol, according to some studies, the majority of the tocopherols consumed in the world by humans, especially in United States, are not α-tocopherols but γ-α-tocopherols.
This form of vitamin E is particularly present in soybean oil and corn oil and represents more than half of the estimated total tocopherol intake (Chow et al., 1967).
The α-tocopherol, instead, is mainly present in sunflower oil, safflower oil and olive oil. - and δ- tocopherol are present in other aliments and oil but their importance is lower than the α- and γ-form.
26 At present, the vitamin E values of food are stated in terms of international units; this unit represents a measure of biological activity rather than quantity. One IU of vitamin E being defined as the specific activity of 1 mg of synthetic all-racemic α-tocopherol acetate. Naturally sourced vitamin E is called RRR-alpha-tocopherol (commonly labeled as d-alpha-tocopherol). It is generally accepted that 1 mg of RRR-α-tocopherol is equivalent to 1.49 IU vitamin E and 1 mg RRR-α-tocopherol acetate is equivalent to 1.36 IU vitamin E. 1 IU of the synthetic form is equivalent to 0.45 mg of alpha-tocopherol. However, recent evidence suggests that the equivalence of all-racemic to RRR forms is related to species, age and the criteria used to assess them and that it may be as high as 2:1.
Food sources of vitamin E
α-Tocopherol (mg) Total tocopherols (mg)
Vegetable Oils (1 tbsp, 14 g)
Soybean oil 1 13
Olive oil 2 2
Corn, canola oils 2 6
Meat and Fish (3 oz, 85g)
Beef 0,3 0,3 Chicken 0,3 0,3 Salmon 0,7 0,7 Nuts (1/2 cup, 60 g) Walnuts 1 20 Pecans 1 14 Pistachios 1 15
Vegetables, Fruits, Legumes (1/2 cup)
Red pepper 75 g 2 3 Broccoli 45 g 1 1 Spinach 15 g 1 1 Tomato 90g 1 1 Apple 55 g 0,1 0,1 Kidney beans 125 g 0,2 2
Data from U.S. department of Agriculture, Agricultural research Service. (2010). USDA National Nutrient Database for Standard Reference, Release 23. Retrieved from www.ars.usda.gov/ba/bhnrc/ndl
27 3.4 Animal intake of vitamin E
Vitamins, in general, are required by animals in very small amounts compared with other nutrients. For example, vitamin B1, thiamin, requirement of a 50-kg weight animal, is only about 3 mg/day. Also, the vitamin E demand for the animal is not very high and it is generally calculated based on the amount of unsaturated fatty acids present in the diet (for 1 g of PUFAs, 3 mg/kg of food of vitamin E; for 1% of added fat, 5 mg/kg of food of vitamin E) because the main function of vitamin E is to protect against oxidation the unsaturated fatty acids and membrane lipid components. According to Muggli (1994), the intake of vitamin E by the animals must be increased by 0.13 - 2.70 mg per gram of unsaturated fatty acid in the food, depending on the number of double bonds. A continuous lack of this nutrient in the diet has a great impact on the meat oxidation processes and causes metabolism disorders and several diseases in the animal. Also, the conditions in which the food supplied to the animals is presented, might affect the presence of vitamin E in the tissues. The results of experimental tests, following the administration of mixtures of concentrates containing rancid substances, show that, if there is a great amount of rancid food in the ration, many of the vitamin reserves present in the animal are destroyed by oxidation and this can cause symptoms of vitamin E secondary deficiency and a worse shelf life of the final product of animal origin.
In this case, to avoid a sub-optimal intake of vitamin E, it is good not only to increase the daily intake of vitamin E but also to prevent the rancidity phenomena and check the state of the food, before their administration to the animals in order to guarantee, then, a better preservation of the final product. To avoid vitamin loss, it is also essential to check the conditions under which the animal origin food is stored, because these will affect the final vitamin potency.
Increased vitamin E requirement caused by unsaturated fatty acids in the diet. (Muggli, 1994) Fatty acid formula vitamin E requirement (UI/g)
Oleic acid 18:1 0,13 Linoleic acid 18:2 0,90 Linolenic acid 18.3 1,35 Arachidonic acid 20:4 1,80 Eicosapentaenoic acid 20:5 2,25 Docosahexaenoic acid 22:6 2,70
28 3.5 Human Intake of Vitamin E
The right intake of vitamin E is also essential for humans, who can satisfy their daily needs through the diet. This is necessary to allow the vitamin making the most of all its functions. Humans, through food, must reach the recommended daily intake allowance (RDA).
Although in the foods from animal, compared to the products of plant origin, the vitamin E is present in small concentrations, these are however important to allow the achievement of the right daily intake.
The vitamin E content in lamb meat, the object of our study, is related to the animal diet, and allows both preserving the meat and helping in achieving the RDA.
Estimates, based on the 1999-2000 data from the National Health and Nutrition Examination Survey (NHANES) place the median dietary intake of α-tocopherol alone at 7.6 mg/day for man and 5.8mg/day for woman aged 19 years or older (Moshfegh et al., 2005). The ranges of α-tocopherol intake are 4.1 to 14.2 mg/day for man and 3.1 to 11.0 mg/day for woman.
RDAs for vitamin E are provided in milligrams (mg), however, for the manufacture and addition of vitamin E to dietary supplements and nourishments, as well as for labeling the vitamin E content, the U.S. Food and Drug Administration (FDA) states that the older conversion factors published by the FNB in 1968 have to be used: 1 IU = 0.67 mg for d-alpha-tocopherol = 0.90 mg for dl-alpha-tocopherol. Under FDA’s new labeling regulations for foods and dietary supplements that take effect by July 26, 2018 (for companies with annual sales of $10 million or more) or July 26, 2019 (for smaller companies), vitamin E will be listed only in mg and not IUs (Food and Drug Administration. Food labeling: Revision of the Nutrition and Supplement Facts labels. Federal Register 2016;81:33741-999).
29
Recommended Dietary Allowances (RDAs) for Vitamin E (Alpha-Tocopherol) (Jacob, Robert A. et al.,
1996)
Age Males Females Pregnancy Lactation 0–6 months* 4 mg (6 IU) 4 mg (6 IU)
7–12 months* 5 mg (7.5 IU) 5 mg (7.5 IU)
1–3 years 6 mg (9 IU) 6 mg (9 IU)
4–8 years 7 mg (10.4 IU) 7 mg (10.4 IU)
9–13 years 11 mg (16.4 IU) 11 mg (16.4 IU)
14+ years 15 mg (22.4 IU) 15 mg (22.4 IU) 15 mg (22.4 IU) 19 mg (28.4 IU)
*Adequate Intake (AI) Table lists the RDAs for alpha-tocopherol in both mg and IU of the natural form; for example, 15 mg x 1.49 IU/mg = 22.4 IU. The corresponding value for synthetic alpha-tocopherol would be 33.3 IU (15 mg x 2.22 IU/mg).
30
4 VITAMIN E IN MEAT
4.1 Role of vitamin E in meat
An adequate intake of vitamin E through diet is important for many functions through animal life, such as the improvement in fertility, prevention of placental retention, improvement of immune performance, reduction of the incidence of mastitis, and after the animal slaughtering as well, in protecting meat against lipid oxidation, and thus, contributing in increasing meat shelf life.
The presence of vitamin E in livestock food must therefore satisfy the physiological needs of the animals and also take into consideration future aspects linked to the quality of food of animal origin.
Numerous studies have shown that there is a relationship between the administration of vitamin E through grass and feed and certain parameters of meat quality.
In fact, after slaughter, most of the mechanisms that counteract the oxidative processes are deactivated and within a few days considerable alterations in the properties of the meat can occur.
Numerous experiments have shown that the content of α-tocopherol in the tissues is greatly affected by vitamin E in the diet. It is known that vitamin E accumulates mainly in dorsal fat and in the liver and, secondly, in the heart and muscles of the back (Peter Hoppe, 1993). The accumulation in organs and tissues depends significantly by the species of the animals, the composition of the feed, the origin and dosage of the vitamin. An adequate content of vitamin E in the muscle is essential to ensure proper protection against oxidation. Some authors (Augustini and Freudenreich, 1996) argue that the optimal vitamin content is 3.0 - 3.5 mg/kg of muscle tissue. Other authors, instead, recommend slightly higher concentrations of vitamin E in meat, to optimize the stability of fats.
Concluding, vitamin E guarantees an improvement in the oxidation stability of meat and meat products and moreover a softer consistency, a more intense and stable colour of meat combined with lower losses due to dripping and a greater preservability (Giuseppe Succi, 1976).
31 4.2 Meat shelf life
The shelf life coincides with the commercial life of the product which is the period of time in which a food can be kept under certain storage conditions and maintain its optimal quality and safety. The shelf life starts from the food production and depends on many factors, such as the production process itself, the type of packaging, the storage conditions and the presence of particular compounds such as vitamin E.
An optimal combination of all these factors allows obtaining a greater shelf life time of the meat. The permanence of vitamin E in tissues, therefore, thanks to all its numerous protective functions, guarantees a longer commercial life of the product and contributes to a better management of food safety.
4.3 Meat colour
The meat colour depends on the concentration of and on kind of myoglobin in muscle fiber, which, on turn is affected by animal species, sex, age and type of food. Myoglobin is a very complex protein which transports oxygen in muscle cells and allows them contracting.
The Myoglobin is composed by two different portions: a protein and a no-protein. The first one is a 16,900 kD weight. This, which is an anomalous protein as, due to lack in cysteine, has no disulfide bonds, is composed by 8 alfa-helices (the biggest one is 23 amino acids) and by a total of 153 amino acids.
The protein portion of myoglobin is uncolored but some colour differences among different species are due to some differences of the primary structure of the protein. The no-protein portion is composed by heme-group which contains iron. Four of the six valence electron of the iron allow iron linking pyrrolic ring, the fifth is used to link heme-group to protein portion (by the histidine 93), and the last is used to link oxygen. The meat is red thanks to the heme-group and, particularly, to the great numbers of conjugated double bounds of the four pyrrolic groups. The colour variation of the meat
32 is related both to the oxidation status of the iron of the heme group of the relationship with oxygen as listed in following table.
Name Oxidation status of iron Oxygen Colour
Deoxy myoglobin 2+ (reduced) No Purplish red
Oxy myoglobin 2+ (reduced) Yes Bright red
Met myoglobin 3+ (oxidized) No Brown
Iron is therefore the most important element involved in the final coloring of meat. as myoglobin role is carrying oxygen, and as oxygen requirement is higher in pasturing animals, the animal reared outdoor, need more myoglobin, thus meat is more red. nevertheless, as this protein, get oxidizing to metmyoglobin, will lead to the darker and brownish colour that is not desired.
Fig. Myogolobin Fig. metmyoglobin
Knowing the role of iron and myoglobin, it is therefore possible to control the variation in the colour of the meat, integrating animal nutrition with antioxidant substances such as Vitamin E, which reduces the oxidized form of myoglobin.
As an antioxidant, α-tocopherol is concentrated predominantly in cell membranes, making them more stable and reducing the risk of lipid peroxidation.
33 This results in a better final coloring of the meat, which remains stable for longer. There is a close correlation between lipid peroxidation and metmyoglobin content in meat (Faustman et al., 1989). When lipid peroxidation is high, the oxidation of myoglobin is also greater. When about 60% of myoglobin oxidizes producing met-myoglobin, the meat begins to take on a brown colour. Higher concentrations of vitamin E in meat and fat, due to the intake of greater quantities of vitamin E with diet, significantly limit the formation of met-myoglobin and the alteration of the colour of the meat.
The colour of the meat also depends on the structure of the muscle fiber. The meat with very tight muscle fibers typical of muscles that work over time and continuously, "red fibers", will have a redder colour because richer in myoglobin, while muscle fibers larger than those typical of muscles that work in quick shots in a short time , "White fibers", will be poorer than myoglobin.
34 4.4 Reduction of weight by drip loss
Numerous studies have shown that high amounts of vitamin E in animal feeds have positive effects on the quality of their meat. Asghar et al. (1991) observed that, after 10 days of refrigeration at 4 ºC under fluorescent light, samples of frozen meat that had consumed rations supplemented with 200 IU of α-tocopherol acetate per kg of feed, showed lower weight losses by dripping to those endured by samples from animals that had consumed elements integrated with 100 IU or 10 IU per kg.
35
5 LAMB MEAT
Lamb is the name given to the meat of young sheep, less than one year old. The meat of an older sheep is called hogget and later is known as mutton. It is possible to distinguish different cuts, including shoulder, rack, shank/breast, loin and leg.
Lamb is a delicious and really healthy meat. Some researchers believe that it is low in fat, an excellent source of vitamins and minerals and other essential nutrients for human health.
It has a unique taste and the final colour is directly related to the animal’s diet and the place where it is raised.
While, earlier, lamb was thought to be tough and stringy, today it has a much more tender consistency and it is more delicate and tasty due to modern breeding methods. Lamb meat is one of the most versatile meats in the world. It is consumed especially in Turkey, Greece, New Zealand, Australia, countries of the Middle East and is particularly famous in Ireland and the Anglo-Saxon countries.
5.1 Nutritional values present in lamb
Lamb is a rich source of easily digestible proteins. One hundred g of lamb meat yields about 22 grams of protein. The amino acids of lamb meat show a high biological value and are involved in the energy production mechanism.
The total fat content of lamb is very low; a lean portion of cooked lamb contains about 3 grams of fat. In addition, saturated fats represent only 35% of total fat. The remaining 65% is characterized, depending to rearing system and diet, by monounsaturated and polyunsaturated fats, positive for health.
Lamb meat is also one of the richest sources of vitamin A and the vitamins of group B such as B3, B6 and B12. The B-complex vitamins play an essential role in the metabolism of all the nutrients in the body and their deficiency in the diet can cause serious damage to health, especially in the case of vitamin B12. Vitamin B12, in fact, supports various metabolic processes in the body: it helps to produce red blood cells and reduce levels of harmful substances in the blood such as homocysteine. High levels of homocysteine can damage blood vessels, and cause cardiovascular disease, and increase
36 the risk of osteoporosis (Refsum, Helga, et al. 2006). Fortunately, lamb is one of the meat sources that provide high levels of vitamin B12.
The presence of vitamin E in lamb meat is equally important for all the functions that have been widely explained previously, including that of being a natural antioxidant. It depends mainly on the type of diet followed by the animal. The meat will, therefore, be richer in vitamin E if, during through life, the animal was fed a diet rich in foods high in tocopherols, such as foods of plant origin.
Lamb meat, furthermore, has a very varied content of minerals includes iron, phosphorous, calcium, selenium and zinc (trace minerals include manganese and copper). Iron is a vital mineral required for the formation of a fundamental protein, hemoglobin, which becomes part of the red blood cells. These transport oxygen throughout the body ensuring tissue oxygenation. Iron is also important in the support of the immune system. It is required, in fact, for the formation of white blood cells which act as antibodies. These fight infections and protect the body from diseases. One serving provides about 20 percent of the recommended daily value of iron.
Selenium shows antioxidant properties, able in helping to boost the body's immune system. It protects body cells from damage by free radicals. Selenium enhances thyroid functions and facilitates the activities of various essential enzymes. Selenium has also been found helpful in fighting viral infections. It promotes liver health and prevents some kind of cancers.
37 In conclusion lamb is a good source of easily absorbed zinc. This mineral is also important for various fundamental processes in the body. Together with iron, its crucial role is to support the immune system. It, supporting normal cell division and quick healing of wounds, also facilitates wound healing. Zinc is a mineral that is part of many enzymes and allows the performance of their biological functions. It, also, contributes to developing optimal odors and tastes in meat. A serving of the lamb meat provides 30 percent of the recommended daily value.
38 5.2 Irish lamb
Sheep breeding and the consequent production of sheep meat is one of the most important and profitable activities in Ireland, spread throughout the country. Unlike production in other European countries, where a large-scale intensive breeding and production is usually practiced, in Ireland sheep meat production remains mainly a traditional agricultural activity, carried out also by small companies, where producers are looking for maintaining high standards in food production. In terms of the total number of sheep, in the 80s there was an increase in livestock levels, from 1.5 million in 1980 to 4.75 million in 1992. Since 1992, however, numbers have decreased, with 3, 5 million sheep registered in 2005.
Irish lamb has a very high quality and is internationally recognized. Thanks to an optimal breeding method, in the uncontaminated countryside of Ireland, characterized by a temperate climate and guaranteeing an evergreen pasture, the Irish lamb meat, with its delicate flavor, has earned the respect and approval of consumers from all over Europe. Lambs raised in Ireland produce meat that is suitable for two different types of European market. The meat produced by lambs raised in the hills, fed with wild herbs and with wild plants is chosen mainly from the Mediterranean markets, while northern Europe prefers the meat of lambs raised in the Irish plain.
Today, the Irish sheep meat industry is trying to change the market, focusing more on the domestic market. The producers' desire is therefore to increase consumption by the Irish population of lamb and increase local sales, which have always been significantly lower (even if lately they are increasing in line with the increasing population) compared to those linked to exports, which exert a dominant influence on the returns of producers and processors and which generates a profit of around 240 million euros per year. The main European buyer of Irish lamb is France, which receives 60% of the product.
