in vivo 30: 291-302 (2016)

Assessment of Fatty Acid Allocation in Plasma and Tissues in Piglets, Using Feed Supplemented with Byproducts from Processed Mill Wastewater

KONSTANTINOS GERASOPOULOS1,2, DIMITRIOS STAGOS1, ALEXANDROS KROUEZAS1, CHRISTINA KARAVELI1, CHRISTINA BARDA1, HELEN GKIKA1, DIMITRIOS MITSIOU1, KONSTANTINOS PETROTOS2, PANAGIOTIS GOULAS2 and DEMETRIOS KOURETAS1

1Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece; 2Department of Biosystem Engineering, Technical Education Institute of Thessaly, Larissa, Greece

Abstract. Background/Aim: A previous study revealed the Fatty acids (Fas), both free and as complex lipids, are improvement of redox status in blood and tissues of young playing key roles in the metabolism (e.g. for storage and piglets (ablactation period), that consumed feed containing transportation of energy), as essential components of all polyphenolic additives from byproducts of processed olive mill membranes and as gene modulators, usually, produced from wastewater (OMWW). The polyphenolic additives strengthened triglycerides or phospholipids. Hydrolysis of triglycerides the defense of the piglets. Herein we analyzed the performed by lipases, results in releasing of energy. Through fatty acid (FA) composition of these animals in various tissues. the action of lipases glycerol and fatty acids are produced. Materials and Methods: The steps followed during the analysis When not linked to other molecules, they are known as were: Preparation and isolation of byproducts containing "free" fatty acids (FFA). These FAs are important energy polyphenolic compounds from OMWW processing, silage and sources, because when metabolized, they produce large piglet feed preparation, blood and tissue collection, fatty acid quantities of ATP (nucleoside triphosphate) carrying methyl esters synthesis and GC/MS analysis. Results: The chemical energy within cells for metabolism. The ratio young piglets, that consumed feed containing polyphenolic phosphate/oxygen (P/O), depends on the ratio of H+ additives from byproducts of processed OMWW, were found to transported away from the mitochondrial matrix by 2e– have a decreased ω6/ω3 ratio, compared to samples of the passing NADH to O2 in the electron transport chain and the control group. For example, in the quadriceps tissue the control number of H+ traveling through ATP synthase, to synthesize group has a ω6/ω3 ratio of 10.1, while in the polyphenolic a ATP (1, 2). A molecule of palmitic acid is produced after group this ratio was decreased to 2.93. Regarding the ratio of the complete oxidation at 106 ATP molecules. The fatty acids UFA/SFA, no significant differences were observed. Finally, the are cleaved to acetyl-CoA by oxidation. Also the beta- polyphenolic group exhibited almost in all tissues lower values oxidation generates NADH. The beta-oxidation of fatty acids of the ratio of PUFA/MUFA than the control group. is the major route for energy release from lipids. The beta- oxidation enzymes are located in the mitochondria in animals and in the peroxisome in plants. At the end of beta-oxidation, generated acetyl-CoA enters in the citric acid cycle or the Abbreviations: ATP, Adenosine triphosphate; FA, fatty acid; FAME, glycosylation cycle. Many cell types can use either glucose fatty acid methyl ester; GC-MS, gas chromatography-mass or fatty acids for this purpose. More particularly, in the heart spectrometry; OMWW, olive mill waste waters; MDA, malondialdehyde; NADH, nicotinamide adenine dinucleotide; NIST, and skeletal muscle fatty acids are preferred. Despite claims national institute of standards and technology; PUFA, polyunsaturated to the contrary, other than glucose and ketone bodies, fatty fatty acids; SFA, saturated fatty acids; TFA, trans fatty acid. acids are used as energy source for brain cells, at least in some rodents (3, 4). Fats are decomposed in the digestive Correspondence to: Demetrios Kouretas, Department of tract to glycerol and FAs by the enzymes secreted therein. Biochemistry and Biotechnology, University of Thessaly, Ploutonos FAs are the largest and most important part of fat. Full 26 & Aiolou, Larissa 41221, Greece. Tel: +30 2410565277, Fax: hydrolysis is performed at a rate of 30-45%, while a similar +30 2410565293, e-mail: [email protected] percentage of monoglyceride is hydrolyzed and the rest Key Words: Olive mill wastewater, antioxidant feed, fatty acid, remain as lipids. By their absorption in the intestinal villi piglet, ω6/ω3 ratio. they are completely hydrolyzed, and both the glycerol and

0258-851X/2016 $2.00+.40 291 in vivo 30: 291-302 (2016) the FAs are used in the villi for reconstruction of body fat Table I. Final piglet feed. Ingredients and nutrient composition of while others, especially the FAs of carbon atoms less than experimental diets. 12 (C12: 0 and below) move directly to the liver where Ingredients Composition (% w/w) metabolized without being used to form lipids (5). Out of the FAs in food technology those with a straight aliphatic chain, Corn 46.5a an even number of carbon atoms and a single carboxyl group Soybean 21.0 are the most interesting. Milk powder 20.0 Lipid oxidation is influenced by many factors: the Pig grower concentrate 10.0 Balancer (piglet corn) 2.5 medium, oxygen concentration, temperature, light, degree of unsaturation, and metal ions among others. In the presence aCorn contained 60% solids and 40% liquid in control feed; 56% solids, of oxygen, oxidation cannot be entirely prevented nor can it 4% OMWW retentate and 40% liquid in feed supplemented with be reversed, but it can be inhibited, delaying the buildup of OMWW . oxidized products to unacceptable levels. Anti-oxidants can interact with several steps of free-radical or photo oxidation. Their performance is medium and concentration – dependent Buxtehude, Germany,) was used for the lactic fermentation of corn. and requires care as they can also act as prooxidants under The lactic bacteria had been dissolved in water (10% w/v) by stirring certain conditions (6). The most widely used are and warmed at 40˚C in order to be activated prior to mixing with free radical scavengers that remove reactive radicals formed corn. After activation, lactic bacteria were mixed with corn (1 g of in the initiation and propagation steps of auto-oxidation. bacteria with 100 kg of corn). For producing the silage, the mixture of Many fatty foods are susceptible to auto-oxidation, involving lactic bacteria and corn was placed in special airtight-seal plastic bags the oxidation of lipid molecules to produce malodorous and was fermented for 3-4 weeks. To prevent the bags from rupturing due to the inflation caused by the carbon dioxide production during ketones, alcohols and acids that adversely affect the texture, fermentation, the material was repackaged in new plastic bags every flavour and taste of food. two to three days. Finally, the resulting silages were mixed with other The polyphenols that have been identified in OMWW ingredients to make the final piglet feed (Table I). include and tyrosol as their major components, as well as p-coumaric acid, homovanillic acid, caffeic acid, Blood and tissue collection. At 50 days post birth (i.e. after piglets protocatechuic acid, 3.4-dihydroxymandelic acid, vanillic acid had been fed with ration for 30 days), 7 blood samples were and ferulic acid (7). It should be noted that polyphenols in obtained from each group. For blood collection, piglets were restrained manually and 4 ml of blood was collected from the OMWW contain about 50% of the total polyphenols found in anterior vena cava and placed in tetraacetic acid olive fruit (8). Despite the toxic effects exhibited at high Vacuette® K3 EDTA tubes (VWR, Radnor, PA, USA). Blood concentrations, these polyphenols also possess antioxidant samples were centrifuged immediately at 1,370 × g for 10 min at activity (7). The findings of the study (9) suggest that feed 4˚C and the plasma was collected and then stored at −80˚C until gas containing OMWW byproducts could be used for enhancing chromatography (GC) analysis. the redox status of piglets by reducing the oxidative damage of For tissue collection, the piglets were sacrificed in a fully biological molecules (i.e. protein oxidation, lipid peroxidation) automated slaughter complex (Slaughterhouses of Larissa S.A., Girtoni, Greece). All relevant procedures (e.g. CO stunning, and increasing antioxidant mechanisms. Furthermore, in the 2 slaughter, bleeding, skin removal, gutting, viscera separation and present study, by means of gas chromatography-mass washing) were executed by special machines and specialized staff. spectrometry, the FAs allocation in pigs (plasma and in nine Tissues were quickly removed and snap-frozen in liquid nitrogen. different tissues; brain, cardiac, kidney, liver, lung, pancreas, In preparation for tissue biochemical analysis, mortar and pestle stomach, spleen and quadriceps muscle) was assessed, at the were used for crushing and grinding the samples with the assistance age of fifty days after their birth. of liquid nitrogen. One part of tissue powder was then homogenized with two parts (weight/volume) of 0.01 M phosphate buffered saline Materials and Methods pH 7.4 (138 mM NaCl, 2.7 mM KCl, and 1 mM EDTA) and a cocktail of protease inhibitor tablet (complete mini inhibitor Preparation and isolation of byproducts containing polyphenolic protease cocktail tablet; Roche, Munich, Germany) was added. The compounds from OMWW processing. The byproducts isolation homogenate was vigorously vortexed and a brief sonication containing polyphenolic compounds from OMWW processing was treatment on ice was applied. The homogenate was then centrifuged based on a patented OMWW powder production scheme at 12,000 × g for 30 min at 4˚C and the supernatant was collected. that has been previously described (9). Tissues were then stored at −80˚C until GC analysis. Fatty acid methyl esters (FAME) synthesis. The method of FAME Silage and piglet feed preparation. OMWW retentate was used for synthesis (10) was applied as follows: 0.5 ml of sample was making silage corn. For this purpose, corn was mixed with OMWW homogenized in PBS with addition of proteases inhibitors, by means retentate at a ratio of 24:1. Thus, silage corn was made that contained of a special microspatoula placed in heat resistance glass tubing 56% solids, 4% OMWW retentate and 40% liquid. Then, standard length 16 cm, diameter 1.6 cm that had a teflon screw cap for commercial formulation of lactic bacteria (11CFT Pioneer, hermetic seal. Then, 1 ml methanolic solution of tridecanoid acid

292 Gerasopoulos et al: Feed Supplemented with Byproducts of OMWW in Piglets

(C13:0) was added at a concentration of 600 μg/ml, as an internal (ω-6) (Figure 1A). Even though, there were no statistically standard. Subsequently, 10 N 0.4 ml KOH (potassium hydroxide) and significant differences between the polyphenolic and control 2.7 ml of pure methanol were added. For proper hydrolysis of group, the ω6/ω3 ratio in the polyphenolic group was 8.71 samples, the tubes by careful secure-screwing were placed in a water bath at 55˚C for a period of 1.5 and every 20 min vigorous stirring and in the control group was 9.12 (Table II). The FAs followed. Cooling with tap water was followed for 15 min. For the allocation in the control group was: SFA 34.5%, MUFA correct composition of fatty acid methyl esters, 0.3 ml 24 N H2SO4 26.4% and PUFA 39.1%, while in the polyphenolic group it were added, hermetic screw and the tubes were placed in a water was: SFA 32.3%, MUFA 29.1% and PUFA 38.6%. bath at 55˚C for a period of 1.5 h, followed by vigorous stirring every 20 min. Cooling with tap water was followed for 15 min. Assessment of lipid oxides in tissues Finally, 3 ml hexane were added as solvent and the samples were Brain. Seven FAs were detected in the control group: stirred at vortex for 3 min. Then, placed in the centrifuge 6.000 × g, 15 min at room temperature and the supernatant (the hexane layer palmitic, palmitoelaidic, stearic, oleic (ω-9), linoleic (ω-6), containing the FAME), placed in GC vials of 2 ml and stored at - linolenic (ω-3) and arachidonic acid (ω-6). In the 20˚C until GS/mass spectrometry (MS) analysis. polyphenolic group two more FAs were detected: palmitoleic and eicosepentaenoic (timnodonic) acid (ω-3), a PUFA that GC-MS analysis. The fatty acids profile assessment in plasma and nine acts as a precursor for prostaglandin-3 (that inhibits platelet different tissues (brain, heart, kidney, liver, lung, quadriceps, pancreas, aggregation), thromboxane-3 and leukotriene-5 eicosanoids. spleen and stomach) of young pigs (50 days old), was determined using the methodology FAME final optimal. A paired calculation was carried In most brain FAs, there were statistically significant out, using the following assay protocol; The assay of concentrations was differences between the polyphenolic and control group made using the peak areas after correction with the internal standard and (Figure 1B). Even if the ω6/ω3 ratio in the control group is using standard 37 fatty acids supplied by SUPELCO (Sigma-Aldrich, as low as 2.60, in the polyphenolic group it is getting lower Munich, Germany) known as 37 Component FAME Mix Standard (this and reaches 1.28 (Table II). The FAs allocation in the control certified reference material may be used to identify and quantify key group was: SFA 37.8%, MUFA 36.8% and PUFA 25.4%, fatty acid methyl esters). To identify the various esters of lipid oxides, while in polyphenolic group it was: SFA 40.9%, MUFA the ability of the MS to perform recognition/identification of peaks in conjunction with the number of spectral data available from the NIST 25.4% and PUFA 33.7%. (National Institute of Standards and Technology) chromatograph was used. The GC-MS Agilent 7890A GC chromatography apparatus Heart. Sixteen FAs were detected in the control group: (Agilent, Frankfurt, Germany) was used. GC columns were located myristic, pentadecanoic, palmitic, palmitoleic, margaric, inside a temperature-controlled oven. Generally, one end of the column palmitoelaidic (ω-9), stearic, oleic (ω-9), linoleic (ω-6), is attached to the inlet, while the other end is attached to the detector. linolenic (ω-3), eicosenoic (gondoic) (ω-9), dihomo-γ- Columns vary in length, diameter, and internal coating. Each column is linolenic (ω-6), arachidonic (ω-6), eicosepentaenoic designed for use with different compounds. The purpose of the column and the oven is to separate the injected sample into individual (timnodonic) (ω-3), tetracosenoic (nervonic) (ω-9) and compounds as it travels through the column. To aid this process, the GC ducosahexuenoic acid (cervonic) (ω-3). In the polyphenolic oven can be programmed to speed the sample flow through the column. group one more FA was detected and this was the arachidic The silica column Agilent J&W 112-88A7: 804.11246 HP-88 250˚C: acid in a low percentage (Figure 2A). In six (6) heart FAs, 100 m × 250 μm × 0.25 μm (Agilent,) was used. The gas carrying the there were statistically significant differences between the substances to be separated should be inert and not react with the polyphenolic and control group, especially in the ω-3FAs. stationary phase or the analytes. The gas is driven to the column from The ω6/ω3 ratio in the control group was 9.53 and in the the high pressure cylinder through the flow regulator. The entire sample is introduced to the importer of the gas chromatograph directly or with polyphenolic group it drastically dropped to 2.24 (Table II). an auto sampler, entrained by the carrier gas, along the column in which The FAs allocation in the control group was: SFA 39.4%, the separation of the analytes occurs. Fractions of components are led MUFA 27.8% and PUFA 32.8%, while in the polyphenolic to a computer. Helium gas with 45.2 ml/min flow was used. group it was: SFA 40.0%, MUFA 29.8% and PUFA 30.2%.

Statistical analysis. Data were analyzed by one-way ANOVA Kidney. Seventeen FAs were detected in the control group: followed by Dunnett’s test for pairwise comparisons. The level of statistical significance was set at p<0.05. All results are expressed as pentadecanoic, palmitic, palmitoleic, margaric, mean±SEM. Data were analysed using SPSS, version 13.0 (SPSS palmitoelaidic (ω-9), stearic, oleic (ω-9), linoelaidic (ω-6), Inc., Chicago, IL, USA). linoleic (ω-6), arachidic, linolenic (ω-3), eicosenoic (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), arachidonic (ω- Results 6), eicosepentaenoic (timnodonic) (ω-3), tetracosenoic (nervonic) (ω-9) and ducosahexuenoic acid (cervonic) (ω-3). Assessment of lipid oxides in plasma. Seven FAs were In the polyphenolic group one more FA was detected and detected in the plasma in both groups. According to the this was the myristic acid (Figure 2B). In ten FAs, there were retention time these were: palmitic, palmitoleic, stearic, oleic statistically significant differences between the polyphenolic (ω-9), linoleic (ω-6), linolenic (ω-3) and arachidonic acid and control group, especially in the ω-3FAs. The ω6/ω3

293 in vivo 30: 291-302 (2016)

Figure 1. Fatty acids allocation, in plasma (A) and brain tissue (B). *Significantly different from values of control group at the same sampling time (p<0.05). Results are presented as the mean±SEM.

ratio in the control group was 4.79 and in the polyphenolic MUFA 23.7% and PUFA 34.5%, while in polyphenolic group was decreased to 2.25 (Table II). The FAs allocation in group it was: SFA 37.6%, MUFA 33.3% and PUFA 29.1%. the control group was: SFA 38.3%, MUFA 26.8% and PUFA 34.9%, while in polyphenolic group it was: SFA 38.4%, Lung. Fourteen FAs were detected in the control group: MUFA 33.2% and PUFA 28.4%. myristic, pentadecanoic, palmitic, palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω-9), linoleic (ω-6), Liver. Seventeen FAs were detected, both in the control and arachidic, dihomo-γ-linolenic (ω-6), arachidonic (ω-6), polyphenolic groups: myristic, pentadecanoic, palmitic, eicosepentaenoic (timnodonic) (ω-3), and ducosahexuenoic palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω- acid (cervonic) (ω-3). In the polyphenolic group one more 9), linoleic (ω-6), arachidic, linolenic (ω-3), eicosenoic FA was detected and this was the tetracosenoic (nervonic) (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), arachidonic (ω- (ω-9) acid (Figure 3B). The ω6/ω3 ratio in the control 6), eicosepentaenoic (timnodonic) (ω-3), tetracosenoic group was 3.14 and in the polyphenolic group was 3.00 (nervonic) (ω-9) and ducosahexuenoic acid (cervonic) (ω-3) (Table II). The FAs allocation in the control group was: (Figure 3A). The ω6/ω3 ratio in the control group was 2.79 SFA 50.9%, MUFA 27.9% and PUFA 21.2%, while in and in the polyphenolic group it dropped to 2.72 (Table II). polyphenolic group it was: SFA 50.6%, MUFA 29.4% and The FAs allocation in the control group was: SFA 41.8%, PUFA 20.0%.

294 Gerasopoulos et al: Feed Supplemented with Byproducts of OMWW in Piglets

Table II. ω6/ω3, UFA/SFA, PUFA/MUFA ratios and percentage of SFA–MUFA–PUFA.

ω6/ω3 Ratio UFA/SFA Ratio PUFA/MUFA Ratio SFA – MUFA – PUFA %

Plasma 9.12a 1.90a 1.48a 34.5 – 26.4 – 39.1a 8.71b 2.10b 1.33b 32.3 – 29.1 – 38.6b Brain 2.60a 1.64a 0.69a 37.8 – 36.8 – 25.4a 1.28b 1.44b 1.33b 40.9 – 25.4 – 33.7b Heart 9.53a 1.54a 1.18a 39.4 – 27.8 – 32.8a 2.24b 1.50b 1.02b 40.0 – 29.8 – 30.2b Kidney 4.79a 1.87a 1.33a 38.3 – 26.8 – 34.9a 2.25b 1.60b 0.85b 38.4 – 33.2 – 28.4b Liver 2.79a 1.40a 1.46a 41.8 – 23.7 – 34.5a 2.72b 1.66b 0.88b 37.6 – 33.3 – 29.1b Lung 3.14a 0.96a 0.76a 50.9 – 27.9 – 21.2a 3.00b 0.97b 0.68b 50.6 – 29.4 – 20.0b Quadriceps 10.2a 2.88a 0.46a 26.3 – 50.4 – 23.3a 2.91b 1.91b 0.54b 34.3 – 42.7 – 23.0b Pancreas 10.5a 1.47a 0.42a 40.4 – 42.0 – 17.6a 4.40b 1.21b 0.79b 45.3 – 30.5 – 24.2b Spleen 2.65a 1.07a 1.46a 48.3 – 21.0 – 30.7a 2.17b 1.26b 0.88b 44.3 – 29.6 – 26.1b Stomach 7.45a 1.56a 0.76a 39.2 – 34.5 – 26.3a 2.54b 1.58b 0.80b 38.7 – 34.0 – 27.3b aControl group, bpolyphenolic group.

Quadriceps. Seventeen FAs were detected, both in the control 9), linoleic (ω-6), arachidic, linolenic (ω-3), eicosenoic and polyphenolic groups: myristic, pentadecanoic, palmitic, (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), arachidonic (ω- palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω- 6), eicosepentaenoic (timnodonic) (ω-3), tetracosenoic 9), linoleic (ω-6), arachidic, linolenic (ω-3), eicosenoic (nervonic) (ω-9) and ducosahexuenoic acid (cervonic) (ω-3) (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), arachidonic (ω-6), (Figure 5A). The ω6/ω3 ratio in the control group was 2.65 eicosepentaenoic (timnodonic) (ω-3), tetracosenoic (nervonic) and in the polyphenolic group was 2.17 (Table II). The FAs (ω-9) and ducosahexuenoic acid (cervonic) (ω-3) (Figure 4A). allocation in the control group was: SFA 48.3%, MUFA The ω6/ω3 ratio in the control group was 10.15 and in the 21.0% and PUFA 30.7%, while in the polyphenolic group it polyphenolic group drastically dropped to 2.91 (Table II). The was: SFA 44.3%, MUFA 29.6% and PUFA 26.1%. FAs allocation in the control group was: SFA 26.3%, MUFA 50.4% and PUFA 23.3%, while in the polyphenolic group it Stomach. Seventeen FAs were detected, both in the control was: SFA 34.3%, MUFA 42.7% and PUFA 23.0%. and polyphenolic groups: myristic, pentadecanoic, palmitic, palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω- Pancreas. Seventeen FAs were detected, both in the control and 9), linoelaidic (ω-6), linoleic (ω-6), arachidic, linolenic (ω- polyphenolic groups: myristic, pentadecanoic, palmitic, 3), eicosenoic (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω- arachidonic (ω-6), eicosepentaenoic (timnodonic) (ω-3) and 9), linoleic (ω-6), arachidic, linolenic (ω-3), eicosenoic tetracosenoic (nervonic) (ω-9) (Figure 5B). The ω6/ω3 ratio (gondoic) (ω-9), dihomo-γ-linolenic (ω-6), arachidonic (ω-6), in the control group was 7.45 and in the polyphenolic group eicosepentaenoic (timnodonic) (ω-3), tetracosenoic (nervonic) drastically dropped to 2.54 (Table II). The FAs allocation in (ω-9) and ducosahexuenoic acid (cervonic) (ω-3) (Figure 4B). the control group was: SFA 39.2%, MUFA 34.5% and PUFA The ω6/ω3 ratio in the control group is 10.51 and in the 26.3%, while in the polyphenolic group it was: SFA 38.7%, polyphenolic group drastically dropped to 4.40 (Table II). The MUFA 34.0% and PUFA 27.3%. FAs allocation in the control group was: SFA 40.4%, MUFA 42.0% and PUFA 17.6%, while in the polyphenolic group it Discussion was: SFA 45.3%, MUFA 30.5% and PUFA 24.2%. Industrialized societies are characterized by an increase in energy Spleen. Seventeen FAs were detected, both in the control and intake and decrease in energy expenditure, an increase in SFA, polyphenolic groups: myristic, pentadecanoic, palmitic, ω-6 FAs and TFAs and a decrease in ω-3 FAs intake (11). The palmitoleic, margaric, palmitoelaidic (ω-9), stearic, oleic (ω- beneficial health effects of ω-3 fatty acids, eicosapentaenoic acid

295 in vivo 30: 291-302 (2016)

Figure 2. Fatty acids allocation, in heart (A) and kidney tissue (B). *Significantly different from values of control group at the same sampling p<0.05. Results are presented as mean±SEM.

(EPA) and docosahexaenoic acid (DHA) were first described in In recent years it has become apparent that the oxidation Greenland Eskimos who consumed a seafood diet and had low of lipids, or lipid peroxidation, is a crucial step in the rates of coronary heart disease, asthma, type 1 diabetes mellitus, pathogenesis of several disease states in adult and infant and multiple sclerosis. Since that observation, the beneficial patients. Lipid peroxidation is a process that occurs naturally health effects of ω-3 FAs have been extended to include benefits in small amounts in the body, mainly due to the effect of related to cancer, inflammatory bowel disease, rheumatoid several reactive oxygen species (e.g. hydroxyl radical, arthritis, and psoriasis (12). hydrogen peroxide etc.). It can also be caused by the activity

296 Gerasopoulos et al: Feed Supplemented with Byproducts of OMWW in Piglets

Figure 3. Fatty acids allocation, (A) in liver tissue and (B) in lung tissue. *Significantly different from values of control group at the same sampling time p<0.05. Results are presented as mean±SEM.

of phagocytes. These reactive oxygen species readily attack on fats and FAs in nutrition, November 10-14, 2008, WHO the polyunsaturated fatty acids of the fatty acid membrane, HQ, Geneva, there exists convincing evidence that: replacing initiating a self-propagating chain reaction (13). SFA (C12:0–C16:0) with PUFA decreases LDL cholesterol According to the Joint FAO/WHO (Food and Agriculture concentration and the total/HDL cholesterol ratio. A similar Organization/World Health Organisation) expert consultation but smaller effect is achieved by replacing these SFA with

297 in vivo 30: 291-302 (2016)

Figure 4. Fatty acids allocation, (A) in quadriceps tissue and (B) in pancreas tissue. *Significantly different from values of control group at the same sampling time p<0.05. Results are presented as mean±SEM.

MUFA. Replacing SFA (C12:0–C16:0) with trans-fatty acids of CHD. Therefore, it is recommended that SFA should be (TFA) decreases HDL cholesterol and increases the total replaced with PUFA (ω-3 and ω-6) in the diet and the total /HDL cholesterol ratio. Based on coronary heart disease intake of SFA does not exceed 10% E (energy percentage). (CHD) morbidity and mortality data from epidemiological Based on both the scientific evidence and conceptual studies and controlled clinical trials (using CHD events and limitations, there is no compelling scientific rationale for the death), it was also agreed that: there exists convincing recommendation of a specific ratio of ω-6 to ω-3 FAs or LA evidence that replacing SFA with PUFA decreases the risk (linoleic acid) to ALA (α linolenic acid). The ω-3 and ω-6

298 Gerasopoulos et al: Feed Supplemented with Byproducts of OMWW in Piglets

Figure 5. Fatty acids allocation, (A) in spleen tissue and (B) in stomach tissue. *Significantly different from values of control group at the same sampling time p<0.05. Results are presented as mean±SEM.

FAs are important components of cell membranes and a ALA and LA compete for their metabolism by the delta-6- precursor of many substances in the body, such as substances desaturase enzyme. The above fact is considered important involved in regulating blood pressure and inflammatory to health, since high uptake LA could reduce the amount of response of the body. There is evidence that ω-3 FAs help enzyme available for the metabolism of ALA, thereby protect against heart disease and are known for their anti- increasing the risk for cardiovascular diseases. The foregoing inflammatory effects, which play an important role in the theory was based on data showing that the last 150 years, the above, but also in many other diseases (14). In the body the ω-6 intakes were increased, whereas the intake of ω-3

299 in vivo 30: 291-302 (2016) decreased alongside increasing heart disease. Consequently, Conflicts of Ιnterest an idea of having an "ideal" ratio of ω-6 to ω-3 FAs was developed (15). The Authors declare that there are no conflicts of interest. Administration of feed supplemented with OMWW retentate reduced lipid peroxidation as demonstrated by reduction in Acknowledgements TBARS levels in plasma and all tissues (1). Inhibition of lipid peroxidation is important, since it has been reported to affect pig The work was funded by the ‘Biotechnology-Nutrition & productivity and health (16). Moreover, lipid peroxidation is one Environment’ and ‘Molecular Biology and Genetics Applications’ MSc programmes in the Department of Biochemistry & of the primary causes leading to meat’s quality deterioration, Biotechnology at the University of Thessaly. while it may also result in production of toxic compounds (17). It is also believed that lipid peroxidation and protein oxidation, References especially when occurring in meat products, are interrelated processes at which the former probably takes place faster and 1 Gerasopoulos K, Stagos D, Kokkas S, Petrotos K, Kantas D, subsequently induces the latter (18). Due to the increased Goulas P and Kouretas D: Feed supplemented with polyphenolic amounts of ω-6 FAs in the Western diet, the eicosanoid byproduct from olive mill wastewater processing improves the metabolic products from AA (arachidonic acid), specifically redox status in blood and tissues of piglets. Food and Chem prostaglandins, thromboxanes, leukotrienes and lipoxins, are Toxicol 86: 319-327, 2015. formed in larger quantities than those formed from ω-3 FAs, 2 Garrett RH and Grisham CM: Biochemistry 4th Edition. 2010. specifically EPA (19). Furthermore, in the present study, the GS 3 Ebert D, Haller RG and Walton ME: Energy contribution of octanoate to intact rat brain metabolism measured by 13C analysis in plasma and all tissues showed that the ω-6/ω-3 ratio nuclear magnetic resonance spectroscopy. J Neurosci 23(13): is getting lower in the polyphenolic group compared to the 5928-5935, 2003. control group. The quadriceps tissue in the polyphenolic group 4 Marin-Valencia I, Good LB, Qian M, Malloy CR and Pascual has a low ratio 2.91 compared with 10.2 of the control group and JM: Heptanoate as a neural fuel: energetic and neurotransmitter that is because in the polyphenolic group the presence of EPA is precursors in normal and glucose transporter I-deficient (G1D) more than five times higher compared to that of the control brain. J Cereb Blood Flow Metab 33(2): 175-182, 2012. group. The EPA acts as a precursor for the biosynthesis of 5 Zerfiridis G K: Human Nutrition. 4th Edition, Giachoudi, Thessaloniki. 1998. eicosanoid groups: prostaglandin-3 (which inhibits platelet 6 Kumar De U, Mukherjee R, Nandi S, Patel BH, Dimri U, aggregation), thromboxane-3 (antithrombotic and vasodilator Ravishankar C and Verma AK: Alterations in oxidant/antioxidant activity) and leukotriene-5 (cellular communication). In the brain balance, high-mobility group box 1 protein and acute phase tissue although the ratio ω-6/ω-3 in the control group is low response in cross-bred suckling piglets suffering from rotaviral 2.60, in the polyphenolic group further descends and reaches enteritis. Trop Anim Health Prod 46(7): 1127-1133, 2014. 1.28. Regarding the ratio of UFA/SFA, no significant differences 7 Frankel E, Bakhouche A, Lozano-Sánchez J, Segura-Carretero are observed. The polyphenolic group almost entirely shows A and Fernández-Gutiérrez A: Literature review on production process to obtain extra virgin enriched in bioactive lower values than the control group in the ratio of PUFA/MUFA. compounds. Potential use of byproducts as alternative sources of Oleic (18:1 ω-9), linoleic (18:2 ω-6), and palmitic (C16:0) acids polyphenols. J Agric Food Chem 61(22): 5179-5188, 2013. accounted for the greatest proportion of the total peak area of 8 Rodis PS, Karathanos VT and Mantzavinou A: Partitioning of monounsaturated, polyunsaturated, and saturated fatty acids, olive oil antioxidants between oil and water phases. J Agric Food respectively. Interestingly, it has been reported that ω-3 fatty Chem 50: 596-601, 2002. acids exerted a significant influence on subcutaneous and 9 Gerasopoulos K, Stagos D, Kokkas S, Petrotos K, Kantas D, intramuscular fat and on meat quality (colour and tenderness) Goulas P and Kouretas D: Feed supplemented with byproducts (20). Moreover, the reduction of the dietary ω6/ω3 resulted in from olive oil mill wastewater processing increases antioxidant capacity in broiler chickens. Food and Chem Toxicol 82: 42-49, nutritionally-enriched meat with higher content of desirable 2015. PUFA, especially AA, EPA and DPA (20). 10 O'Fallon JV, Busboom JR, Nelson ML and Gaskins CT: A direct This finding is important, since as mentioned above piglets’ method for fatty acid methyl ester synthesis: Application to wet weaning is a stressful event that may cause oxidative stress meat tissues, oils, and feedstuffs. J Anim Sci 85: 1511-1521, 2007. and subsequently manifestation of pathological conditions (21- 11 Eaton SB and Konner M: Paleolithic nutrition. A consideration 23). The use of byproducts from OMWW processing for of its nature and current implications. N Engl J Med 312: 283- making animal feeds is also interesting, since OMWW cause 289, 1985. 12 Simopoulos AP: Omega-3 fatty acids in inflammation an serious environmental problems. It is of particular interest that autoimmune diseases. J Am Coll Nutr 21: 495-505, 2002. polyphenols contained in the feed of pigs, from byproducts 13. Mylonas C and Kouretas D: Lipid peroxidation and tissue from OMWW, are beneficial for lipid ratio in the meat of pigs damage. In Vivo 13(3): 295-309, 1999. (decrease of ω-6/ω-3 ratio). Study of the specific mechanism 14 Lunn J and Theobald H: The health effects of dietary unsaturated is one of our aims in the near future. fatty acids. Nutrition Bulletin 31: 178-224, 2006.

300 Gerasopoulos et al: Feed Supplemented with Byproducts of OMWW in Piglets

15 Simopoulos AP: The importance of the omega-6/omega-3 fatty 21 Zhu LH, Zhao KL, Chen XL and Xu JX: Impact of weaning and acid ratio in cardiovascular disease and other chronic diseases. an antioxidant blend on intestinal barrier function and Exp Biol Med (Maywood) 233(6): 674-688, 2008. antioxidant status in pigs. J Anim Sci 90(8): 2581-2589, 2012. 16 Shurson GC, Kerr BJ and Hanson AR: Evaluating the quality of 22 Lykkesfeldt J and Svendsen O: Oxidants and antioxidants in feed fats and oils and their effects on pig growth performance. J disease: oxidative stress in farm animals. Vet J 173(3): 502-511, Anim Sci Biotechnol 21;6(1):10. doi: 10.1186/s 40104-015- 2007. 0005-4. e Collection, 2015. 23 Zhong RZ, Fang Y, Qin GX, Yang H and Zhou DW: Tea 17 Rey AI, Kerry JP, Lynch PB, López-Bote CJ, Buckley DJ and Protect Goat Skeletal Muscle against H2O2-Induced Morrissey PA: Effect of dietary oils and alpha- acetate Oxidative Stress by Modulating Expression of Phase 2 supplementation on lipid (TBARS) and cholesterol oxidation in Antioxidant Enzymes. J Agric Food Chem 63(36): 7921-7928, cooked pork. J Anim Sci 79(5): 1201-1208, 2001. 2015. 18 Zhou F, Zhao M, Zhao H, Sun W and Cui C: Effects of oxidative modification on gel properties of isolated porcine myofibrillar protein by peroxyl radicals. Meat Sci 96(4): 1432-1439, 2014. 19 Simopoulos AP: Omega-3 fatty acids in health and disease and in growth and development. Am J Clin Nutr 54: 438-463, 1991. 20 Qi KK, Chen JL, Zhao GP, Zheng MQ and Wen J: Effect of dietary omega6/omega3 on growth performance, carcass traits, Received December 4, 2015 meat quality and fatty acid profiles of Beijing-you chicken. J Revised January 20, 2016 Anim Physiol Anim Nutr 1;94(4): 474-485, 2010. Accepted February 2, 2016

301