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Olive Biophenols as Food Supplements and Additives
Chapter 30
Olive Biophenols as Food Supplements and Additives Antonella De Leonardis and Vincenzo Macciola Department of Agricultural, Food, Environmental and Microbiological Science and Technology (DiSTAAM), Campobasso, Italy
30.1 Introduction Food, cosmetic and pharmaceutical industries are drawn together to promote new products, named ‘functional food’ (food with scientifically recognized health and therapeutic properties), food supplements, nutraceuticals or cosmeceuticals (Peschel et al., 2006). Functional food products meet the consumer’s demand for a healthy lifestyle. These foods are not intended only to satisfy hunger and provide humans with necessary nutrients, but also to prevent nutrition-related diseases and increase physical and mental well-being of consumers. Besides, the modern customers, especially those living in the industrial countries, demand ‘non-chemical’ ingredients in health products. The most important and dynamic market of the functional foods represent USA and Japan (Menrad, 2003). Also in Europe, the functional foods market is increasing, especially in Germany, France, the United Kingdom and the Netherlands. Since ancient times, in the Mediterranean countries, most of the plant parts of Olea europaea have been used as an effective medical treatment in traditional medicine and the principal common uses are listed in Table 30.1 (Yaseen Khan et al., 2007). In recent decades, the positive effects of some olive phenols on human health have been scientifically demonstrated numerous times. The olive phenols with biological activity are also called ‘biophenols’. Table olives and extra virgin olive oil, both typical ethnic products of the Mediterranean food culture, are certainly the principal food source of olive biophenols (Uccella, 2001). Nevertheless, the demand of olive biomolecules is quickly increasing across the world, including in countries far-off in the Mediterranean area. In fact, the American calibrated Food Guide Pyramid (USDA, 2000) also suggests eating foods containing olive biophenols, which combat dangerous free radicals and prevent the accumulation of cholesterol. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
Table 30.1 Traditional medical uses of plant parts of Olea europaea (Yaseen Khan et al., 2007). l Oil
with lemon juice to treat gallstones
l Leaves
taken orally for stomach and intestinal pathologies
l Hot
water extract of fresh leaves is taken orally to treat hypertension, for bronchial asthma and to induce diuresis
l Seed
oil is taken orally as a cholagogue and laxative; it is also applied externally as an emollient and pectoral and to prevent hair loss
l Decoction l Tincture
of dried leaves is taken orally for diabetes
of leaves is taken orally as a febrifuge
l Fruit
is applied externally to fractured limb and as a skin cleanser
l Hot
water extract of dried plant is taken
l Infusion
of the fresh leaf is taken orally as an anti-inflammatory
Nowadays, besides the traditional olive foods (table olives and olive oil), a lot of olive biophenol formulations are already available commercially. Some recent patents relative to the extraction techniques and potential applications of olive biophenols are listed in Table 30.2. In the industrial process, olive biophenols are extracted from different olive-based starting materials, including olives, olive pulps, olive oil, olive oil mill wastewaters and finally, olive leaves. Olive oil mill wastewaters, but also olive leaves, are certainly the most convenient and environmentally friendly
283
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
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Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 30.2 Some recent patents relative to the production and utilization of olive biophenols. Patent number
Title
US Patent 6358542/2002
Antioxidant compositions extracted from olives and olive byproducts
US Patent 5714150/1998
Method for producing extract of olive leaves
US Patent 6162480/2000
Fortification of a vegetable fat with antioxidants
EP1790234/2007
Natural antioxidant additive for feed and drinking water
WO/1999/038383
Method for producing extract of olive leaves and extract produced thereby
WO/1999/032589
Olive oil containing food composition
WO/1999/055349
Substance mixture for topical application comprising olive oil and honey
WO/2000/038541
Fortification of food products with olive fruit ingredients
WO/2001/076579
Treatment of skin damage using olive oil polyphenols
WO/2002/032387
Use of olive oil in the preparation of a product for oral hygiene for eliminating or reducing bacterial plaque and/or bacteria in the mouth
WO/2003/079794
Use of olive tree extracts in detergents, rinsing agents and cleaning agents
WO/2004/110171
Olive powder
WO/2004/005228
An hydroxytyrosol-rich composition from olive vegetation water and method of use thereof
WO/2005/123603
Process for recovering the components of olive mill wastewater with membrane technologies
WO/2006/020588
Olive compositions and methods for treating inflammatory conditions
WO/2006/043117
Processing of olive marc into alimentary meal
WO/2006/005986
Olive polyphenols concentrate
WO/2007/013032
Method of obtaining a natural hydroxytyrosol-rich concentrate from olive tree residues and subproducts using clean technologies
WO/2007/042742
Cured olive powder
WO/2007/074490
Process for producing triacetyl-hydroxytyrosol from olive oil mill wastewaters for use as stabilized antioxidant
WO/2007/096446
Use of olive extract as a pronutrient in animal feed
WO/2008/040550
Olive juice extracts for promoting muscle health
raw material to produce bio-technologically olive biophenols. In fact, owing to their potential health benefits, olive biophenols represent an opportunity to add value to the waste products that are an environmental problem in oliveproducing countries (Schieber et al., 2001). The principal ‘recovery-systems’ are based on the following techniques: extraction with solvents; resin chromatography; selective concentration by ultrafiltration and inverse osmosis; solid–liquid or liquid–liquid extraction;
supercritical fluid extraction (Vásquez et al., 1987; Capasso et al., 1999; Fernández-Bolanˇos et al., 2002; Agalias et al., 2007; Japón-Luján and de Castro, 2007). The olive biophenols, that are generally present in the commercial formulates, can be grouped as follows: oleuropeosides (oleuropein, demethyloleuropein, ligustroside, verbascoside) l flavonoids l
Chapter | 30 Olive Biophenols as Food Supplements and Additives
simple phenols (tyrosol, hydroxytyrosol, vanilic, p-coumaric, ferulic and caffeic acids, catechol) l others (elenolic acid). l
The composition of commercial formulates is extremely variable in relation to the olive-based starting materials and the extraction techniques. Specific technologies, e.g. supercritical fluid extraction, nanofiltration and reverse osmosis, are used to concentrate singular phenols, especially oleuropein and hydroxytyrosol. The olive biophenol extracts can be used in liquid form or in powder manufactured by spray drying or selective absorption. Olive biophenols can be formulated into dietary supplements, food, beverages, cosmetics and pharmaceutical products and health fortificant for feed. One qualitative system to discriminate the olive biophenol commercial products could be the percentage and the purity grade of the free hydroxytyrosol (HT). In nature, native HT is rarely found free in common foods, with the exception of ripened olives, in which it is present in quantities ranging from 1.0 to 3.0 g per 100 g of dried weight. On the contrary, free HT is present in considerable amounts in olive mill solid–liquid wastes from two-phase olive oil processing (Fernández-Bolanˇos et al., 2002), as well as in olive-mill waste waters from traditional and industrial threephase plants (Capasso et al., 1999; Allouche et al., 2004). The natural mechanism that occurs when the olive tree forms free HT is enzymatic hydrolysis, and specific native -glycosidase and esterase are implicated. On the contrary, in the industrial processes, acid or alkaline hydrolysis is generally the mechanism used to produce free HT. Finally, with the aim to produce a more stable and effective antioxidant ingredient, HT can also be transformed into triacetylhydroxytyrosol (Capasso et al., 2006). The principal health-promoting activities that are claimed for olive biophenol commercial products (supported on the bases of a copious scientific literature) are listed in Table 30.3. In particular, the antioxidative, antimicrobial and antivirus activities are certainly the most important properties of olive biophenols that are advertised throughout the marketing strategy.
30.2 Olive biophenols from olive leaves The most popular olive phenol dietary supplements are those produced from olive leaves. Commercial formulates of olive leaves are as follows: dried olive leaves olive leaf extracts as gel capsule or tablets with phenol standardized composition l liquid olive leaf extracts. l l
The dried olive leaves are used to make a beverage similar to tea.
285
Table 30.3 The principal health-promoting activities that are claimed for olive biophenol commercial products. Protection of human erythrocytes against oxidative damage Cardioprotective and antiatherogenic Scavenges and reduces superoxide anion production in human promonocyte cells Inhibition of the proliferation of tumor cells Antimicrobial human pathogens Anti-inflammatory prostaglandin sparing and antioxidant Inhibition of leukocytes leukotriene B4 Radical scavenging activity within biomembrane Inhibition of LDL oxidation and platelets aggregation Hypoglycemic activity Antihypertensive vasodilator Antimicrobial and antiviral activity Anti-HIV activity
The olive leaf extracts contain several different types of phytochemicals, but generally oleuropein is the most abundant, comprising 20–40% of total components (De Leonardis et al., 2008).
30.3 Olive phenols from olive-oil mill byproducts During the process of olive oil extraction, the native olive phenols migrate into the fat phase or aqueous phase in function of their lipophilic/hydrophilic nature. At the end of the process, only about 200 mg kg1 of total phenols could be found in the virgin olive oil, 5000 mg kg1 in the wastewaters and about 10 000 mg kg1 in the solid waste (Rodis et al., 2002). The phenol substances found in olive oil mill waste waters (OMWW) are different from those of olive fruits. In fact, olives are very rich in secoiridoid glucosides, while OMWW actually contain secoiridoid derivates, especially hydroxytyrosol and dialdehydic form of decarboxymethyl oleuropein aglycon (Macciola and De Leonardis, 2006; De Marco et al., 2007). The phenol composition of raw material could vary with the cultivars, ripening status and quality of the olives and with the industrial ‘oil-extraction’ technology, but also with the storage conditions of the same wastes (Feki et al., 2005).
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Section | I Lipids, Phenolics and Other Organics and Volatiles
Consequently, characteristics of the raw material and extraction techniques have a significant effect on the composition of the final extracts, and so in the industrial process, the olive phenol extract composition must be standardized. Finally, the oil-mill byproducts phenol extracts are generally available in the market as powder, gel capsules or tablets.
30.4 Olive biophenols directly from olives Sometimes, a fine dried powder can be directly obtained from olives. Generally, green olives are used because they are richer in phenol substances. The solid matter obtained from olive fruits has variable particle sizes, ranging from 0.1 m to 5.0 mm. Olive fruit powder can not be de-bittered or can be de-bittered, by a treatment with sodium hydroxide solution, before it is used as an ingredient in other foods.
30.5 Olive biophenols as food antioxidant additive It is well known that olive phenols have antioxidant properties, so they could be useful in improving the oxidative stability of different edible fats as an alternative to synthetic compounds. However, not all the olive phenol substances are effective as food antioxidants and especially, concentration of free HT is demonstrated to be fundamental in improving the antioxidant effectiveness of olive extracts. The graph of Figure 30.1 shows the results of laboratory tests in which the antioxidant effectiveness of four
Induction time (minutes)
4
3
3.5
IT control IT fortified PF 2.6 2.2
2
1.8
1
0 E1. (TP: 7.0 g/L; HT/TP: 1.1%)
E2. (TP: 2.5 g/L; HT/TP: 4.0%)
E3. (TP: 1.5 g/L; HT/TP: 8.4%)
E4. (TP: 0.8 g/L; HT/TP: 18.8%)
Phenol extracts from olive oil mill waste waters
Figure 30.1 Antioxidant effectiveness of different olive oil mill wastewater extracts measured on lard by Rancimat test (under 120°C temperature and 20 L h1 air flow). IT, induction time; PF, protection factor; TP, total phenols; HT/TP, free hydroxytyrosol on total phenols.
different olive oil mill wastewater phenol extracts (E1., E2., E3., E4.) are measured on lard by using a Rancimat apparatus, under operative conditions of 120°C temperature and 20 L h1 air flow (De Leonardis et al., 2009). Antioxidant effectiveness has been calculated on the protection factor (PF) that is the percentage ratio of the fat induction times, with and without each OMWW phenol extract. Specifically, the OMWW extracts were different for content and composition of phenols. Total phenols were measured by the colorimetric Folin-Ciocalteu methods (Gutfinger, 1981), while phenol composition was determined by HPLC analysis. It has clearly emerged that antioxidant effectiveness is scarcely correlated to the values of total phenols, while it is strongly correlated with the percentage of free HT (Figure 30.1). In conclusion, antioxidant effectiveness of an olive phenol extract is affected more from its phenol composition, than from its total phenol contents. Without doubt, Folin-Ciocalteu’s reagent reacts also with several other compounds, especially condensed phenols, that have no antioxidant properties. HT is the best antioxidant component among the OMWW olive phenols. Nevertheless, native content of HT into the OMWW could be extremely variable; in addition, it is only partially in free form. Therefore, adequate and standardized processes are needed to produce a good antioxidant extract, preferably enriched with major amounts of free hydroxytyrosol. In Table 30.4, the antioxidant effectiveness of an aqueous HT-rich olive leaf extract (at least 92% of HT on total phenols) on different edible lipids is shown (De Leonardis et al., 2008). In particular, the antioxidant effect has been evaluated on butter, lard and cod liver oil, by using the Rancimat apparatus and a dosage of 50 mg kg1 of HT on fat. For each lipid sample, HT effectiveness has been measured on the protection factor (PF). In the above-mentioned experiment, HT confirmed to be a good antioxidant for food lipids and in fact, there has been a significant increase in the induction time in all the lipid samples fortified with the HT extract, if compared with the relative control. More specifically, at 120°C, the induction times were 10.7 and 3.6 times higher than those of the control in lard and in butter, respectively; for the cod liver oil at 100°C the induction time was 1.7 times than that of the control. In the specific case of the lard, the HT-rich extract can delay the oxidative stability by at least 7–15 times, when under low and high temperature (De Leonardis et al., 2007). Such treatments comprising crushing, freezing and thawing, sonication, heat shock, drying and high-pressure shock, can simplify the fortification of the edible fats with the hydrophilic HT. Lard or other edible fats enriched with olive phenols, especially HT, have the following advantages: improved oxidative stability and increased nutritional value.
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Chapter | 30 Olive Biophenols as Food Supplements and Additives
Table 30.4 Oxidation stability, measured by Rancimat test, of different food lipids fortified with and without hydroxytyrosol-rich olive leaf extract. Operative temperature °C
120
120
100
Edible lipids
Lard
Butter
Cod liver oil
Determinations
IT
Control
0.41
Proof with 50 mg kg1 of HT extract
4.37
PF
IT
PF
5.02 10.7
18.04
IT
PF
1.80 3.6
3.07
1.7
IT: induction time (minutes); PF: protection factor.
30.6 Olive biophenols as functional food Nutritional value of a food product, particularly vegetable oils, spreads, mayonnaises, salad dressings and sauces, can be enhanced by the addition of olive biophenol ingredients. In recent years, new food formulations, derived from the combination of nutraceutical compounds and probiotic microorganisms, are setting quite a trend. Indeed, co-cultures of Streptococcus thermophilus and Lactobacillus delbruechii ssp. bulgaricus are generally used in yogurt and similar fermented milk. The effects of olive leaf extracts at high content of free HT (at least 92% on total phenols) has been observed on the growth of co-cultures of Streptococcus thermophilus and Lactobacillus delbruechii ssp. bulgaricus, at very variable concentrations, ranging from 25 to 3200 g mL1 (as HT) (De Leonardis et al., 2008). At all the tested concentrations, the antimicrobial activities of olive leaf extract showed no growth inhibition and minimum antimicrobial activity (MIC) was a value 3200 g mL1 of HT. In conclusion, olive phenols, especially HT, can be added as an integrator or antioxidant to fermented milk, to increase both the quality and the nutritional value of the final milk product, without inducing any negative effects on the viability of the lactic acid bacteria.
30.7 Health fortificant for feed A recent return to ‘natural medicine’ to treat animals is also an increasing trend (Viegi et al., 2003). The use of antibiotic or chemotherapeutic substances, referred to as ‘growth promoters’, in the production of livestock is well known. In recent years, attention has increasingly focused on problems related to the widespread use of antibiotics or chemotherapeutics as ‘growth promoters’. As a consequence, in various countries a complete cessation of the use of classical growth promoters in swine production
has been imposed. New alternative methods are sought to ensure the livestock’s growth conditions, free of both disease and use of antibiotics. Olive biophenols, especially those extracted from leaves, can be used as an antioxidant into products for feed for domestic animals. Olive phenols in feed (5% in weight) improve the productivity and health status in animals by having antimicrobial and antiviral properties, improving feed conversion, the utilization of nutrients and the health status of the animals. Olive biophenols can be used for monogastric animals and ruminants but also for fish, crustaceans, and pets.
30.8 Final safety consideration The claimed effects of olive biophenols are often studied in vitro, while in the same way they are not adequately welldocumented in humans. Indeed, several questions are still open about the correct use of olive biophenol (Pokorný, 2007). The increased use of fortified foods and functional supplements has increased the intake of nutrient substances around the world. In turn, there has been growing interest on an international basis for determining the levels of intake that may pose risk. ‘Habitual intake’ is the long-term average daily intake of the nutrient substance. The upper level of intake is the maximum level of ‘habitual intake’ from all sources of a nutrient or related substance, judged to be unlikely to lead to adverse health effects in humans. It is approximately estimated that the average daily intake of native antioxidants present in the diet consisting of normal food is totally about 1000 mg, of which about 50–200 mg are polyphenols (Murkovic, 2003). In the case of olive biophenols, the safety and physiological limits are mostly not known. Theoretically, an adequate intake of olive phenol supplements should be determined on the quantity of extra virgin olive oil eaten usually by Mediterranean people. By considering a typical
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Section | I Lipids, Phenolics and Other Organics and Volatiles
Mediterranean diet, the daily quantity eaten of extra virgin olive oil, containing presumably 200 mg kg1 of olive phenols, is about 30 g, so the calculated daily intake of olive phenols should be about 6 mg. Another nutritional doubt is the very different composition between the olive oil phenols and that of the commercial olive biophenol formulates. The greatest part of natural phenols often shows, in vitro, a strong inhibitor effect on cell proliferation. It is known that the degree of toxicity of a phenol varies in relation to its concentration but also with the chemical structure. In human cells, the cytotoxicity of several phenols typically present in virgin olive oil, is in the following order of toxicity: oleuropein aglycone oleuropein glycoside caffeic acid o-coumaric acid cinnamic acid tyrosol and syringic protocatechuic and vanillic acids (Babich and Visioli, 2003). Specific studies on the cytotoxicity of free HT are not numerous. However, it is known that at high concentrations (higher than 1 mM) olive phenols exert a cytotoxic effect; nevertheless, at lower concentrations increased cell proliferation has been observed more than once (Manna et al., 1997; Owen et al., 2000). Mouse fibroblasts NIH/3T3 and human umbilical vein endothelial cells (HUVEC) have been exposed to an olive phenol extract at the concentrations of 0.01, 0.16, 0.32, 0.64 and 1.28 mM (as hydroxytyrosol) for 12, 24 and 48 h (De Leonardis et al., 2008). The tested cell types gave similar results; cytotoxicity varied with olive phenol extract concentration, and at constant concentration it varied with the stimulation time. At concentrations higher than 0.32 mM, the cytotoxic effect of olive phenol extract emerged on both cell samples in the first hours. At 0.32 mM, the number of stimulated cells equalled those of the control. Finally, at concentrations lower than 0.32 mM there was little antiproliferative activity and during the first 12 h, the cell numbers were higher than in the control in both cases. Therefore, at concentrations less than 0.32 mM, an olive phenol extract can be considered non-cytotoxic. In conclusion, the information on safety and health advantages of olive biophenols is still insufficient. Major specific toxicity tests and nutritional studies are needed to regulate an appropriate use of olive biophenols as antioxidant and health-promoting supplements, ingredients and additives.
Summary points Since ancient times, in the Mediterranean countries, most of the plant parts of Olea europaea have been used as effective medical treatments in traditional medicine. l Olive phenols with biological activity (also called ‘biophenols’) are sold in several formulations, in liquid form or in powder. l
In the industrial process, olive biophenols are extracted from different olive-based starting materials, including olives, olive pulps, olive oil, olive oil mill wastewaters, and finally, olive leaves. l Composition of the commercial formulates are extremely variable in relation to the olive-based starting materials and the extraction techniques. l Olive ‘biophenols’ can also be used as a functional ingredient and antioxidant additive for food and feed. l
References Agalias, A., Magiatis, P., Skaltsounis, A.L., Mikros, E., Tsarbopoulos, A., Gikas, E., Spanos, I., Manios, T., 2007. A new process for the management of olive oil mill waste water and recovery of natural antioxidants. J. Agric. Food Chem. 55, 2671–2676. Allouche, N., Fki, I., Sayadi, S., 2004. Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters. J. Agric. Food Chem. 52, 267–273. Babich, H., Visioli, F., 2003. In vitro cytotoxicity to human cells in culture of some phenols from olive oil. Il Farmaco 58, 403–407. Capasso, R., Evidente, A., Avolio, S., Solla, F., 1999. A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste waters. J. Agric. Food Chem. 47, 1745–1748. Capasso, R., Sannino, F., De Martino, A., Manna, C., 2006. Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bio-antioxidant. J. Agric. Food Chem. 54, 9063–9070. De Leonardis, A., Macciola, V., Lembo, G., Aretini, A., Nag, A., 2007. Studies on oxidative stabilisation of lard by natural antioxidants recovered from olive-oil mill wastewater. Food Chem. 100, 998–1004. De Leonardis, A., Aretini, A., Alfano, G., Macciola, V., Ranalli, G., 2008. Isolation of a hydroxytyrosol-rich extract from olive leaves (Olea Europaea L.) and evaluation of its antioxidant properties and bioactivity. Eur. Food Res. Technol. 226, 653–659. De Leonardis, A., Macciola, V., Nag, A., 2009. Antioxidant activity of different phenol extracts from olive-oil mill wastewaters. Acta Alim. 38, 77–86. De Marco, E., Savarese, M., Paduano, A., Sacchi, R., 2007. Characterization and fraction of phenolic compounds extracted from olive-oil mill wastewaters. Food Chem. 104, 858–867. Fernández-Bolanˇos, J., Rodríguez, G., Rodríguez, R., Heredia, A., Guillén, R., Jiménez, A., 2002. Production in large quantities of highly purified hydroxytyrosol from liquid–solid waste of two-phase olive oil processing or ‘‘Alperujio’’. J. Agric. Food Chem. 50, 6804–6811. Feki, M., Allouche, N., Bouaziz, M., Gargoubi, A., Sayadi, S., 2005. Effect of storage of olive mill wastewaters on hydroxytyrosol concentration. Eur. J. Lipid Sci. Technol. 107, 497–504. Gutfinger, T., 1981. Phenols in olive oil. J. Am. Oil Chem. Soc. 11, 966–968. Japón-Luján, R., de Castro, M.D.L., 2007. Static-dynamic superheated liquid extraction of hydroxytyrosol and other biophenols from alperujo (a semisolid residue of the olive oil industry). J. Agric. Food Chem. 55, 3629–3643. Macciola, V., De Leonardis, A., 2006. Natura e proprietà antiossidanti di fenoli estratti da un olio extra-vergine d’oliva e suoi sottoprodotti. Ingredienti Alimentari 5, 6–14. Manna, C., Galletti, P., Cucciolla, V., Moltedo, O., Leone, A., Zappia, V., 1997. The protective effect of the olive oil polyphenols (3-4-dyhydroxyphenyl)-ethanol counteracts reactive oxygen metabolite induced cytoxicity in caco-2-cells. J. Nutrit. 127, 286–292.
Chapter | 30 Olive Biophenols as Food Supplements and Additives
Menrad, K., 2003. Market and marketing of functional food. J. Food Eng. 56, 181–188. Murkovic, M., 2003. Phenolic compounds. In: Caballero, B., Trugo, C., Finglas, P.M. (eds) Encyclopedia of Food Sciences and Nutrition, 2nd edn. Academic Press, Amsterdam (The Netherlands), pp. 4507–4514. Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Spiegelhalder, B., Bartsch, H., 2000. The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur. J. Cancer 36, 1235–1247. Peschel, W., Sànchez-Rabaneda, F., Diekmann, W., Plescher, A., Gartzìa, I., Jimènez, D., Lamuela-Raventçò, R., Buxaderas, S., Codina, C., 2006. An industrial approach in the search of natural antioxidants from vegetable and fruit wastes. Food Chem. 97, 137–150. Pokorný, J., 2007. Are natural antioxidants better-and safer-than synthetic antioxidants? Eur. J. Lipid Sci. Technol. 109, 629–642. Rodis, P.S., Karathanos, V.T., Mantzavinos, A., 2002. Partitioning of olive oil antioxidants between oil and water phases. J. Agric. Food Chem. 50, 596–601.
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Schieber, A., Stintzing, F.C., Carle, R., 2001. By-products of plant food processing as a source of functional compounds–recent developments. Trend Food Sci. Tech. 12, 401–413. Yaseen Khan, M.D., Panchal, S., Vyas, N., Butani, A., Kumar, V., 2007. Olea europaea: a phyto-pharmacological review. Pharmacognosy Reviews 1, 114–118. Uccella, N., 2001. Olive biophenols: novel ethnic and technological approach. Trend Food Sci. Tech. 11, 328–339. USDA, 2000. ‘The Food Guide Pyramid’ in Center for Nutrition Policy and Promotion. US Department of Agriculture, Washington, DC. Vásquez, R., Maestro Durán, R., Graciani, C., 1987. Phenols components of olive oil mill waste water. Grasas y Aceites 25, 341–345. Viegi, L., Pieroni, A., Guarrera, P.M., Vangelisti, R., 2003. A review of plants used in folk veterinary medicine in Italy as basis for a databank. J. Ethnopharmacol. 89, 221–244.
Olive Biophenols as Food Supplements and Additives Antonella De Leonardis and Vincenzo Macciola Department of Agricultural, Food, Environmental and Microbiological Science and Technology (DiSTAAM), Campobasso, Italy
30.1 Introduction Food, cosmetic and pharmaceutical industries are drawn together to promote new products, named ‘functional food’ (food with scientifically recognized health and therapeutic properties), food supplements, nutraceuticals or cosmeceuticals (Peschel et al., 2006). Functional food products meet the consumer’s demand for a healthy lifestyle. These foods are not intended only to satisfy hunger and provide humans with necessary nutrients, but also to prevent nutrition-related diseases and increase physical and mental well-being of consumers. Besides, the modern customers, especially those living in the industrial countries, demand ‘non-chemical’ ingredients in health products. The most important and dynamic market of the functional foods represent USA and Japan (Menrad, 2003). Also in Europe, the functional foods market is increasing, especially in Germany, France, the United Kingdom and the Netherlands. Since ancient times, in the Mediterranean countries, most of the plant parts of Olea europaea have been used as an effective medical treatment in traditional medicine and the principal common uses are listed in Table 30.1 (Yaseen Khan et al., 2007). In recent decades, the positive effects of some olive phenols on human health have been scientifically demonstrated numerous times. The olive phenols with biological activity are also called ‘biophenols’. Table olives and extra virgin olive oil, both typical ethnic products of the Mediterranean food culture, are certainly the principal food source of olive biophenols (Uccella, 2001). Nevertheless, the demand of olive biomolecules is quickly increasing across the world, including in countries far-off in the Mediterranean area. In fact, the American calibrated Food Guide Pyramid (USDA, 2000) also suggests eating foods containing olive biophenols, which combat dangerous free radicals and prevent the accumulation of cholesterol. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
Table 30.1 Traditional medical uses of plant parts of Olea europaea (Yaseen Khan et al., 2007). l Oil
with lemon juice to treat gallstones
l Leaves
taken orally for stomach and intestinal pathologies
l Hot
water extract of fresh leaves is taken orally to treat hypertension, for bronchial asthma and to induce diuresis
l Seed
oil is taken orally as a cholagogue and laxative; it is also applied externally as an emollient and pectoral and to prevent hair loss
l Decoction l Tincture
of dried leaves is taken orally for diabetes
of leaves is taken orally as a febrifuge
l Fruit
is applied externally to fractured limb and as a skin cleanser
l Hot
water extract of dried plant is taken
l Infusion
of the fresh leaf is taken orally as an anti-inflammatory
Nowadays, besides the traditional olive foods (table olives and olive oil), a lot of olive biophenol formulations are already available commercially. Some recent patents relative to the extraction techniques and potential applications of olive biophenols are listed in Table 30.2. In the industrial process, olive biophenols are extracted from different olive-based starting materials, including olives, olive pulps, olive oil, olive oil mill wastewaters and finally, olive leaves. Olive oil mill wastewaters, but also olive leaves, are certainly the most convenient and environmentally friendly
283
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
284
Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 30.2 Some recent patents relative to the production and utilization of olive biophenols. Patent number
Title
US Patent 6358542/2002
Antioxidant compositions extracted from olives and olive byproducts
US Patent 5714150/1998
Method for producing extract of olive leaves
US Patent 6162480/2000
Fortification of a vegetable fat with antioxidants
EP1790234/2007
Natural antioxidant additive for feed and drinking water
WO/1999/038383
Method for producing extract of olive leaves and extract produced thereby
WO/1999/032589
Olive oil containing food composition
WO/1999/055349
Substance mixture for topical application comprising olive oil and honey
WO/2000/038541
Fortification of food products with olive fruit ingredients
WO/2001/076579
Treatment of skin damage using olive oil polyphenols
WO/2002/032387
Use of olive oil in the preparation of a product for oral hygiene for eliminating or reducing bacterial plaque and/or bacteria in the mouth
WO/2003/079794
Use of olive tree extracts in detergents, rinsing agents and cleaning agents
WO/2004/110171
Olive powder
WO/2004/005228
An hydroxytyrosol-rich composition from olive vegetation water and method of use thereof
WO/2005/123603
Process for recovering the components of olive mill wastewater with membrane technologies
WO/2006/020588
Olive compositions and methods for treating inflammatory conditions
WO/2006/043117
Processing of olive marc into alimentary meal
WO/2006/005986
Olive polyphenols concentrate
WO/2007/013032
Method of obtaining a natural hydroxytyrosol-rich concentrate from olive tree residues and subproducts using clean technologies
WO/2007/042742
Cured olive powder
WO/2007/074490
Process for producing triacetyl-hydroxytyrosol from olive oil mill wastewaters for use as stabilized antioxidant
WO/2007/096446
Use of olive extract as a pronutrient in animal feed
WO/2008/040550
Olive juice extracts for promoting muscle health
raw material to produce bio-technologically olive biophenols. In fact, owing to their potential health benefits, olive biophenols represent an opportunity to add value to the waste products that are an environmental problem in oliveproducing countries (Schieber et al., 2001). The principal ‘recovery-systems’ are based on the following techniques: extraction with solvents; resin chromatography; selective concentration by ultrafiltration and inverse osmosis; solid–liquid or liquid–liquid extraction;
supercritical fluid extraction (Vásquez et al., 1987; Capasso et al., 1999; Fernández-Bolanˇos et al., 2002; Agalias et al., 2007; Japón-Luján and de Castro, 2007). The olive biophenols, that are generally present in the commercial formulates, can be grouped as follows: oleuropeosides (oleuropein, demethyloleuropein, ligustroside, verbascoside) l flavonoids l
Chapter | 30 Olive Biophenols as Food Supplements and Additives
simple phenols (tyrosol, hydroxytyrosol, vanilic, p-coumaric, ferulic and caffeic acids, catechol) l others (elenolic acid). l
The composition of commercial formulates is extremely variable in relation to the olive-based starting materials and the extraction techniques. Specific technologies, e.g. supercritical fluid extraction, nanofiltration and reverse osmosis, are used to concentrate singular phenols, especially oleuropein and hydroxytyrosol. The olive biophenol extracts can be used in liquid form or in powder manufactured by spray drying or selective absorption. Olive biophenols can be formulated into dietary supplements, food, beverages, cosmetics and pharmaceutical products and health fortificant for feed. One qualitative system to discriminate the olive biophenol commercial products could be the percentage and the purity grade of the free hydroxytyrosol (HT). In nature, native HT is rarely found free in common foods, with the exception of ripened olives, in which it is present in quantities ranging from 1.0 to 3.0 g per 100 g of dried weight. On the contrary, free HT is present in considerable amounts in olive mill solid–liquid wastes from two-phase olive oil processing (Fernández-Bolanˇos et al., 2002), as well as in olive-mill waste waters from traditional and industrial threephase plants (Capasso et al., 1999; Allouche et al., 2004). The natural mechanism that occurs when the olive tree forms free HT is enzymatic hydrolysis, and specific native -glycosidase and esterase are implicated. On the contrary, in the industrial processes, acid or alkaline hydrolysis is generally the mechanism used to produce free HT. Finally, with the aim to produce a more stable and effective antioxidant ingredient, HT can also be transformed into triacetylhydroxytyrosol (Capasso et al., 2006). The principal health-promoting activities that are claimed for olive biophenol commercial products (supported on the bases of a copious scientific literature) are listed in Table 30.3. In particular, the antioxidative, antimicrobial and antivirus activities are certainly the most important properties of olive biophenols that are advertised throughout the marketing strategy.
30.2 Olive biophenols from olive leaves The most popular olive phenol dietary supplements are those produced from olive leaves. Commercial formulates of olive leaves are as follows: dried olive leaves olive leaf extracts as gel capsule or tablets with phenol standardized composition l liquid olive leaf extracts. l l
The dried olive leaves are used to make a beverage similar to tea.
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Table 30.3 The principal health-promoting activities that are claimed for olive biophenol commercial products. Protection of human erythrocytes against oxidative damage Cardioprotective and antiatherogenic Scavenges and reduces superoxide anion production in human promonocyte cells Inhibition of the proliferation of tumor cells Antimicrobial human pathogens Anti-inflammatory prostaglandin sparing and antioxidant Inhibition of leukocytes leukotriene B4 Radical scavenging activity within biomembrane Inhibition of LDL oxidation and platelets aggregation Hypoglycemic activity Antihypertensive vasodilator Antimicrobial and antiviral activity Anti-HIV activity
The olive leaf extracts contain several different types of phytochemicals, but generally oleuropein is the most abundant, comprising 20–40% of total components (De Leonardis et al., 2008).
30.3 Olive phenols from olive-oil mill byproducts During the process of olive oil extraction, the native olive phenols migrate into the fat phase or aqueous phase in function of their lipophilic/hydrophilic nature. At the end of the process, only about 200 mg kg1 of total phenols could be found in the virgin olive oil, 5000 mg kg1 in the wastewaters and about 10 000 mg kg1 in the solid waste (Rodis et al., 2002). The phenol substances found in olive oil mill waste waters (OMWW) are different from those of olive fruits. In fact, olives are very rich in secoiridoid glucosides, while OMWW actually contain secoiridoid derivates, especially hydroxytyrosol and dialdehydic form of decarboxymethyl oleuropein aglycon (Macciola and De Leonardis, 2006; De Marco et al., 2007). The phenol composition of raw material could vary with the cultivars, ripening status and quality of the olives and with the industrial ‘oil-extraction’ technology, but also with the storage conditions of the same wastes (Feki et al., 2005).
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Consequently, characteristics of the raw material and extraction techniques have a significant effect on the composition of the final extracts, and so in the industrial process, the olive phenol extract composition must be standardized. Finally, the oil-mill byproducts phenol extracts are generally available in the market as powder, gel capsules or tablets.
30.4 Olive biophenols directly from olives Sometimes, a fine dried powder can be directly obtained from olives. Generally, green olives are used because they are richer in phenol substances. The solid matter obtained from olive fruits has variable particle sizes, ranging from 0.1 m to 5.0 mm. Olive fruit powder can not be de-bittered or can be de-bittered, by a treatment with sodium hydroxide solution, before it is used as an ingredient in other foods.
30.5 Olive biophenols as food antioxidant additive It is well known that olive phenols have antioxidant properties, so they could be useful in improving the oxidative stability of different edible fats as an alternative to synthetic compounds. However, not all the olive phenol substances are effective as food antioxidants and especially, concentration of free HT is demonstrated to be fundamental in improving the antioxidant effectiveness of olive extracts. The graph of Figure 30.1 shows the results of laboratory tests in which the antioxidant effectiveness of four
Induction time (minutes)
4
3
3.5
IT control IT fortified PF 2.6 2.2
2
1.8
1
0 E1. (TP: 7.0 g/L; HT/TP: 1.1%)
E2. (TP: 2.5 g/L; HT/TP: 4.0%)
E3. (TP: 1.5 g/L; HT/TP: 8.4%)
E4. (TP: 0.8 g/L; HT/TP: 18.8%)
Phenol extracts from olive oil mill waste waters
Figure 30.1 Antioxidant effectiveness of different olive oil mill wastewater extracts measured on lard by Rancimat test (under 120°C temperature and 20 L h1 air flow). IT, induction time; PF, protection factor; TP, total phenols; HT/TP, free hydroxytyrosol on total phenols.
different olive oil mill wastewater phenol extracts (E1., E2., E3., E4.) are measured on lard by using a Rancimat apparatus, under operative conditions of 120°C temperature and 20 L h1 air flow (De Leonardis et al., 2009). Antioxidant effectiveness has been calculated on the protection factor (PF) that is the percentage ratio of the fat induction times, with and without each OMWW phenol extract. Specifically, the OMWW extracts were different for content and composition of phenols. Total phenols were measured by the colorimetric Folin-Ciocalteu methods (Gutfinger, 1981), while phenol composition was determined by HPLC analysis. It has clearly emerged that antioxidant effectiveness is scarcely correlated to the values of total phenols, while it is strongly correlated with the percentage of free HT (Figure 30.1). In conclusion, antioxidant effectiveness of an olive phenol extract is affected more from its phenol composition, than from its total phenol contents. Without doubt, Folin-Ciocalteu’s reagent reacts also with several other compounds, especially condensed phenols, that have no antioxidant properties. HT is the best antioxidant component among the OMWW olive phenols. Nevertheless, native content of HT into the OMWW could be extremely variable; in addition, it is only partially in free form. Therefore, adequate and standardized processes are needed to produce a good antioxidant extract, preferably enriched with major amounts of free hydroxytyrosol. In Table 30.4, the antioxidant effectiveness of an aqueous HT-rich olive leaf extract (at least 92% of HT on total phenols) on different edible lipids is shown (De Leonardis et al., 2008). In particular, the antioxidant effect has been evaluated on butter, lard and cod liver oil, by using the Rancimat apparatus and a dosage of 50 mg kg1 of HT on fat. For each lipid sample, HT effectiveness has been measured on the protection factor (PF). In the above-mentioned experiment, HT confirmed to be a good antioxidant for food lipids and in fact, there has been a significant increase in the induction time in all the lipid samples fortified with the HT extract, if compared with the relative control. More specifically, at 120°C, the induction times were 10.7 and 3.6 times higher than those of the control in lard and in butter, respectively; for the cod liver oil at 100°C the induction time was 1.7 times than that of the control. In the specific case of the lard, the HT-rich extract can delay the oxidative stability by at least 7–15 times, when under low and high temperature (De Leonardis et al., 2007). Such treatments comprising crushing, freezing and thawing, sonication, heat shock, drying and high-pressure shock, can simplify the fortification of the edible fats with the hydrophilic HT. Lard or other edible fats enriched with olive phenols, especially HT, have the following advantages: improved oxidative stability and increased nutritional value.
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Table 30.4 Oxidation stability, measured by Rancimat test, of different food lipids fortified with and without hydroxytyrosol-rich olive leaf extract. Operative temperature °C
120
120
100
Edible lipids
Lard
Butter
Cod liver oil
Determinations
IT
Control
0.41
Proof with 50 mg kg1 of HT extract
4.37
PF
IT
PF
5.02 10.7
18.04
IT
PF
1.80 3.6
3.07
1.7
IT: induction time (minutes); PF: protection factor.
30.6 Olive biophenols as functional food Nutritional value of a food product, particularly vegetable oils, spreads, mayonnaises, salad dressings and sauces, can be enhanced by the addition of olive biophenol ingredients. In recent years, new food formulations, derived from the combination of nutraceutical compounds and probiotic microorganisms, are setting quite a trend. Indeed, co-cultures of Streptococcus thermophilus and Lactobacillus delbruechii ssp. bulgaricus are generally used in yogurt and similar fermented milk. The effects of olive leaf extracts at high content of free HT (at least 92% on total phenols) has been observed on the growth of co-cultures of Streptococcus thermophilus and Lactobacillus delbruechii ssp. bulgaricus, at very variable concentrations, ranging from 25 to 3200 g mL1 (as HT) (De Leonardis et al., 2008). At all the tested concentrations, the antimicrobial activities of olive leaf extract showed no growth inhibition and minimum antimicrobial activity (MIC) was a value 3200 g mL1 of HT. In conclusion, olive phenols, especially HT, can be added as an integrator or antioxidant to fermented milk, to increase both the quality and the nutritional value of the final milk product, without inducing any negative effects on the viability of the lactic acid bacteria.
30.7 Health fortificant for feed A recent return to ‘natural medicine’ to treat animals is also an increasing trend (Viegi et al., 2003). The use of antibiotic or chemotherapeutic substances, referred to as ‘growth promoters’, in the production of livestock is well known. In recent years, attention has increasingly focused on problems related to the widespread use of antibiotics or chemotherapeutics as ‘growth promoters’. As a consequence, in various countries a complete cessation of the use of classical growth promoters in swine production
has been imposed. New alternative methods are sought to ensure the livestock’s growth conditions, free of both disease and use of antibiotics. Olive biophenols, especially those extracted from leaves, can be used as an antioxidant into products for feed for domestic animals. Olive phenols in feed (5% in weight) improve the productivity and health status in animals by having antimicrobial and antiviral properties, improving feed conversion, the utilization of nutrients and the health status of the animals. Olive biophenols can be used for monogastric animals and ruminants but also for fish, crustaceans, and pets.
30.8 Final safety consideration The claimed effects of olive biophenols are often studied in vitro, while in the same way they are not adequately welldocumented in humans. Indeed, several questions are still open about the correct use of olive biophenol (Pokorný, 2007). The increased use of fortified foods and functional supplements has increased the intake of nutrient substances around the world. In turn, there has been growing interest on an international basis for determining the levels of intake that may pose risk. ‘Habitual intake’ is the long-term average daily intake of the nutrient substance. The upper level of intake is the maximum level of ‘habitual intake’ from all sources of a nutrient or related substance, judged to be unlikely to lead to adverse health effects in humans. It is approximately estimated that the average daily intake of native antioxidants present in the diet consisting of normal food is totally about 1000 mg, of which about 50–200 mg are polyphenols (Murkovic, 2003). In the case of olive biophenols, the safety and physiological limits are mostly not known. Theoretically, an adequate intake of olive phenol supplements should be determined on the quantity of extra virgin olive oil eaten usually by Mediterranean people. By considering a typical
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Mediterranean diet, the daily quantity eaten of extra virgin olive oil, containing presumably 200 mg kg1 of olive phenols, is about 30 g, so the calculated daily intake of olive phenols should be about 6 mg. Another nutritional doubt is the very different composition between the olive oil phenols and that of the commercial olive biophenol formulates. The greatest part of natural phenols often shows, in vitro, a strong inhibitor effect on cell proliferation. It is known that the degree of toxicity of a phenol varies in relation to its concentration but also with the chemical structure. In human cells, the cytotoxicity of several phenols typically present in virgin olive oil, is in the following order of toxicity: oleuropein aglycone oleuropein glycoside caffeic acid o-coumaric acid cinnamic acid tyrosol and syringic protocatechuic and vanillic acids (Babich and Visioli, 2003). Specific studies on the cytotoxicity of free HT are not numerous. However, it is known that at high concentrations (higher than 1 mM) olive phenols exert a cytotoxic effect; nevertheless, at lower concentrations increased cell proliferation has been observed more than once (Manna et al., 1997; Owen et al., 2000). Mouse fibroblasts NIH/3T3 and human umbilical vein endothelial cells (HUVEC) have been exposed to an olive phenol extract at the concentrations of 0.01, 0.16, 0.32, 0.64 and 1.28 mM (as hydroxytyrosol) for 12, 24 and 48 h (De Leonardis et al., 2008). The tested cell types gave similar results; cytotoxicity varied with olive phenol extract concentration, and at constant concentration it varied with the stimulation time. At concentrations higher than 0.32 mM, the cytotoxic effect of olive phenol extract emerged on both cell samples in the first hours. At 0.32 mM, the number of stimulated cells equalled those of the control. Finally, at concentrations lower than 0.32 mM there was little antiproliferative activity and during the first 12 h, the cell numbers were higher than in the control in both cases. Therefore, at concentrations less than 0.32 mM, an olive phenol extract can be considered non-cytotoxic. In conclusion, the information on safety and health advantages of olive biophenols is still insufficient. Major specific toxicity tests and nutritional studies are needed to regulate an appropriate use of olive biophenols as antioxidant and health-promoting supplements, ingredients and additives.
Summary points Since ancient times, in the Mediterranean countries, most of the plant parts of Olea europaea have been used as effective medical treatments in traditional medicine. l Olive phenols with biological activity (also called ‘biophenols’) are sold in several formulations, in liquid form or in powder. l
In the industrial process, olive biophenols are extracted from different olive-based starting materials, including olives, olive pulps, olive oil, olive oil mill wastewaters, and finally, olive leaves. l Composition of the commercial formulates are extremely variable in relation to the olive-based starting materials and the extraction techniques. l Olive ‘biophenols’ can also be used as a functional ingredient and antioxidant additive for food and feed. l
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