Nutritional Properties of Table Olives and Their Use in Cocktails

NUTRITIONAL PROPERTIES OF TABLE OLIVES AND THEIR USE IN COCKTAILS

14

Ambra Ariani⁎, Silvia Vincenzetti†, Paolo Polidori‡ ⁎

School of Advanced Studies, University of Camerino, Camerino, Italy, †School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy, ‡School of Pharmacy, University of Camerino, Camerino, Italy

14.1 Introduction The olive (Olea europaea L.) is a small tree, belonging to the family Oleaceae, native from tropical and warm temperate regions of the world. The tree, famous for its fruits, also named olive, is commercially important in the south of Europe because of the production of olive oil that is considered the best fat for human nutrition. There are around 2500 known varieties of olives; 250 are classified as commercial cultivars by the International Olive Oil Council (IOOC). The olive tree that is considered one of the oldest and more important domesticated crops raised by humans has received by the farmers a lot of attention during the centuries, with the consequent creation of several varieties. Olive cultivars are first and foremost divided into their land of origin; most names for cultivars come from place names. Secondarily, several cultivars have a double purpose: in some cases, olives may be preferred for olive oil production, other cultivars can be selected only for producing table olives. The tree is typically distributed in the coastal areas of the eastern Mediterranean Basin, the adjoining coastal areas of south-eastern Europe, western Asia and northern Africa as well as northern Iran and also near the Caspian Sea. Although olive is now cultivated in several regions of the world, the Mediterranean area is still a major production site, accounting for about 98% of the world’s olive cultivation. Spain, with a total cultivated area of 2,500,000 ha, is the biggest producer, followed by Italy (1,159,000 ha) and Greece (765,000 ha). The cultivation of olive tree dates back more than 7000 years, according to the archeological evidence. Olives were grown commer­ cially in Crete since 3000 BC, by the Minoan civilization. Ancient Greek literature describes the use of olive oil for body health. In the context of Nutrients in Beverages. https://doi.org/10.1016/B978-0-12-816842-4.00014-9 © 2019 Elsevier Inc. All rights reserved.

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religious importance, olive tree and its fruits are described several times in the Bible, both in the New and Old Testaments, and later also in the Quran. The olive tree has a long history of medicinal and nutritional values. Over the centuries, extracts from olive leaf have been used for promoting health and preservation. In Egypt, in fact, olive leaves were used to mummify Pharaohs. Similarly, they have been used as a popular remedy to treat fever and some tropical diseases such as malaria. Considering the economic value, olive is an important food because it is possible to obtain a nutritious edible oil with potential health functions. In fact, table olives are considered as a complete food from a nutritional point of view. It is a drupe consisting primarily of water, fat, carbohydrates, protein, fiber, pectin, biophenols, vitamins, organic acids, and mineral elements. The nutritional properties of this product are related to the combined effect of various factors, such as the suitability of raw materials, the processing technologies, the chemical composition and also the sensory properties. The processing technologies are necessary: olives are rarely used in their natural form because of the severe bitterness; they are consumed in one of the two forms, oil or table olives (Fig. 14.1). Oleuropein, a bitter-tasting secoiridoids glycoside present in olive leaves and fruits, is the main phenolic compound in olive fruits; its content greatly decreases during the course of fruit ripening and processing. Although the majority of the polyphenols found in table olives

Fig. 14.1  Olives in Puglia Region, Italy.

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are derivatives from its hydrolysis, in most cases, oleuropein concentration in table olives and olive oil is very low. The main purpose of table olives processing is the removal of bitterness related to oleuropein. This aim is obtained through different processes mainly based on alkaline or enzymatic hydrolysis in order to decrease oleuropein content and to increase hydroxytyrosol content. The knowledge of the nutritional and chemical characteristics of oleuropein for table olive processing is important in order to produce high-quality table olives and to develop innovative methods for table olives production. Considering the nutritional properties, olive fruit is a drupe, constituted by three distinct anatomical zones: epicarp (skin), mesocarp (pulp), and endocarp (stone) containing the seed (Montaño et  al., 2010). All these anatomical zones influence the quality of the final product. The epicarp and mesocarp constitute the edible part of the olive fruit that is around 70%–85%. The energy value contained in 100 g of edible portion (e.p.) of Italian olives is around 200–250 kcal, but the total range is very wide, being the higher value represented by 455 kcal for Majatica olives and the lower value of 190 kcal for Intosso olives (see Table 14.1).

Table 14.1  Nutritional Characteristics of Some Italian Table Olives Nutrients (100 g e.p.)

Sevillan Green Olives Intosso

Ferrandina Black Olives Majatica

Natural Black Olives Taggiasca

Natural Green Olives Itrana

Natural Black Olives Itrana

Energy (kcal) Proteins (g) Carbohydrates (g) Sugars (g) Fats (g) SFA (g) MUFA (g) PUFA (g) Fiber (g) Sodium (g) Calcium (mg) Polyphenols (mg)

190 1.0 2.8 tr 17.5 2.7 13.6 1.2 2.6 1.3 33.6 168

455 2.2 n.d. 4.4 46.9 6.3 36.7 4.0 3.4 0.9 168.1 263

226 1.5 8.9 tr 19.9 3.7 15.2 0.9 2.6 1.8 92.7 206

193 1.5 5.0 0.6 17.7 2.8 14.0 0.9 3.6 1.2 21.9 109

235 1.4 6.5 0.3 21.7 2.7 17.7 1.3 4.0 1.5 28.9 211

n.d., not detected; tr, traces. Modified from Lanza, B., 2012. Nutritional and sensory quality of table olives. In: Muzzalupo, I. (Ed.), Olive Germplasm—The Olive Cultivation, Table Olive and Olive Oil Industry in Italy. InTech, Rjieka, pp. 343–370.

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The protein content is low (1.0–2.2 g/100 g), but the biological value is high for the presence of essential amino acids for adults, threonine, valine, leucine, isoleucine, phenylalanine, and lysine, and for children, arginine, histidine, and tyrosine (see Table  14.2). Aspartic and glutamic acids are the most representative amino acids, however in some preparations threonine, valine, leucine and arginine contents can be higher than 100 mg/100 g (Lanza, 2012). The carbohydrate content in the olive fruit is lower than any other edible fruit. The table olives have lower proportions of this compound because during the fermentation process or brine storage the microorganisms present in brines cause fermentation of carbohydrates. The drupe is a rich source of dietary fiber that is also characterized by a high digestibility rate. Most varieties of olives have a content of fiber ≥3 g/100 g of e.p. that represents an interesting amount of dietary fiber (see Table 14.1). Table olives mineral content must be carefully analyzed. Interesting calcium content was found (168.1 mg/100 g for Majatica and 92.7 mg/100 g for Taggiasca),

Table 14.2  Amino Acid Pattern of Some Italian Table Olives Amino Acid (mg/100 g e.p.)

Treated Green Olives Intosso

Ferrandina Black Olives Majatica

Natural Black Olives Peranzana

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Phenylalanine + Tyrosine Lysine Histidine Arginine Other amino acids

150 70 80 150 50 70 80 60 100 140 100 10 30 70 tr

214 129 124 226 tr 115 115 104 86 173 168 18 26 120 tr

131 80 74 128 tr 66 66 63 50 98 117 tr 28 68 tr

tr., traces. Modified from López-López, A., Montaño, A., Garrido-Fernández, A., 2010. Nutrient profiles of commercial table olives: proteins and vitamins. In: Preedy, W. (Eds.), Olives and Olive Oil in Health and Disease Prevention. Elsevier, San Diego, pp. 705–714.

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as indicated in Table  14.1. The high sodium content of some traditional preparations (≥1.5 g) is the consequence of fermentations and packing brines. Table olives also contain natural antioxidants such as vitamins; B group vitamins content is low, as well as liposoluble vitamins such as provitamin A and vitamin E, that are characterized by remarkable antioxidant effects. The vitamin C content is low (<1 mg/kg of e.p.), as determined by Lanza (2012). Many green olives preparations have been enriched with ascorbic acid, an antioxidant ingredient, with the consequent increase of vitamin C content in the food. This compound may be progressively lost during shelf life, but if the olives are consumed close to the production date, this food may eventually represent an interesting source of vitamin C. Other antioxidant source in table olives is polyphenols that can have functional effects on human health. The phenolic compounds present in table olives ranges between 100 and 350 mg/100 g of e.p., and their variability depends on agronomic and/or technological processes (Lanza, 2012). Recent study has focused on a phenolic compound of nutraceutical value, oleocanthal (dialdehydic form of deacetoxy-ligstroside aglycon) as nonsteroidal anti-inflammatory (NSAI)-like drugs, with Ibuprofenelike activity. Organic acids (oxalic, succinic, malic, citric, and lactic) have been determined in small contents, giving to the olive pulp a total acidity ranging between 4 and 10 g/kg (expressed as citric acid) and a pH value ranging between 3.8 and 5.0. The content of oxalic and malic acids decreases during the fruit maturation while citric acid content increases; on the other hand, succinic acid seems to remain constant. In addition, the ratio citric acid/malic acid decreases during the fruit maturation to reach, at the moment of maximum oil accumulation (inolition), values close to 1. Evaluating olive fatty acid composition, oleic acid (C18:1 ω9) is the predominant one (see Table 14.3), followed by palmitic acid (C16:0), linoleic acid (C18:2 ω6), and stearic acid (C18:0). In human intestine, mucosal cells utilize dietary oleic acid as a substrate to produce the lipid messenger oleoylethanolamide (OEA) that plays an important role in the regulation of animal food intake and body weight in human physiological and pathophysiological conditions. Monounsaturated fatty acids (MUFA) are the most represented fatty acids (66.8%–82.1%), while saturated fatty acids (SFA) represent less than 22.4% and polyunsaturated fatty acids (PUFA) range between 4.9% and 14.2%. The trans fatty acids (TFA) are present in very small amount (less than 0.02%). The intake of α-linolenic acid (C18:3 ω3), precursor for the synthesis of long-chain 3 fatty acids, is appreciable but the ratio ω6:ω3 is greatly influenced by the evident greater amount of ω6 fatty acids, even if each different cultivar shows different ratio (see Table 14.4).

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Table 14.3  Fatty Acid Composition of Some Italian Table Olives (g/100 g Total Fatty Acids) Fatty Acid

Sevillan Green Olives Intosso

Ferrandina Black Olives Majatica

Natural Black Olives Peranzana

Natural Green Olives Itrana

C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 ω6 C18:3 ω3 C20:0 C20:1 C22:0 C24:0 Trans fatty acids

n.d. 11.8 0.6 0.1 0.3 2.9 76.4 6.3 0.5 0.8 0.3 0.1 n.d. 0.02

n.d. 9.8 1.1 n.d. 0.1 3.0 76.8 8.0 0.5 0.4 0.3 0.1 n.d. 0.02

n.d. 14.7 1.3 n.d. 0.1 1.7 70.2 10.7 0.7 0.3 0.2 0.1 n.d. tr

n.d. 13.6 1.4 0.1 0.1 1.8 76.5 5.5 0.5 0.3 0.3 0.1 0.1 0.01

n.d., not detected; tr, traces. Modified from Lanza, B., 2012. Nutritional and sensory quality of table olives. In: Muzzalupo, I. (Ed.), Olive Germplasm—The Olive Cultivation, Table Olive and Olive Oil Industry in Italy. InTech, Rjieka, pp. 343–370.

Table 14.4  Fatty Acid Categories in Italian Table Olives (g/100 g Total Fatty Acids) Fatty Acid Category

Sevillan Green Olives Intosso

Ferrandina Black Olives Majatica

Natural Black Olives Peranzana

Natural Green Olives Itrana

SFA MUFA PUFA MUFA/SFA PUFA/SFA C18:1/C16:0 ω6/ω3

15.7 77.6 6.8 4.9 0.4 6.5 12.6

13.3 78.3 8.5 5.9 0.6 7.8 16.0

16.8 71.8 11.4 4.3 0.7 4.8 15.3

16.0 78.3 6.0 4.9 0.4 5.6 5.6

Modified from Sousa, A., Casal, S., Bento, A., Malheiro, R., Oliveira, M. B. P. P., Pereira, J. A., 2011. Chemical characterization of “Alcaparra” stoned table olives from Northeast Portugal. Molecules 16, 9025–9040.

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The changes in sterol, fatty acids and triterpenic alcohol contents during processing have been investigated. The main phytosterols and phytostanols found in Ferrandina table olives are β-sitosterol (59.1%– 89.6%) and Δ5-avenasterol (1.5%–34.3%), followed by campesterol (1.8%–4.2%), Δ5,24-stigmastadienol (0.4%–1.2%), and chlerosterol (1.0%–1.5%), as illustrated in Table 14.5. The low content of β-sitosterol and high content of Δ5-avenasterol in Majatica olives is typical for this cultivar, but β-sitosterol (including Δ5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, Δ5-avenasterol, and Δ5,24-stigmastadienol) is higher than 93%, the limit fixed for extra virgin olive oil (EVOO) by the Commission Regulation (EEC) 2568/91 and its subsequent modifications (Lanza, 2012). Epidemiologic and experimental studies suggest that dietary phytosterols, and in particular β-sitosterol, can be considered as an help in preventing the most common cancers in the Western countries, such as colon, breast, and prostate cancer and can also be effective in reducing the cardiovascular disease risk. The consumption

Table 14.5  Sterolic Composition (%) of Some Italian Table Olives Sterol

Castelvetrano Green Olives Nocellara B.

Ferrandina Black Olives Majatica

Natural Black Olives Peranzana

Natural Green Olives Itrana

Cholesterol Brassicasterol 24-Methylen cholesterol Campesterol Campestanol Stigmasterol Δ7-Campesterol Δ5.23-Stigmastadienol Clerosterol β-Sytosterol Sitostanol Δ5-Avenasterol Δ5.24-Stigmastadienol Δ7-Stigmastenol Δ7-Avenasterol

1.4 0.3 0.2 4.2 0.1 1.8 0.2 n.d. 1.4 84.5 0.6 4.7 0.6 n.d. n.d.

0.4 0.1 0.5 1.8 0.1 0.5 0.1 0.1 1.0 59.1 0.4 34.3 1.2 0.3 0.1

0.5 n.d. 0.2 2.0 0.4 0.7 0.3 n.d. 1.2 77.4 0.6 15.9 0.5 0.2 0.2

0.5 0.1 0.1 2.4 n.d. 0.7 0.6 n.d. 1.0 88.7 0.7 4.3 0.6 0.3 0.2

n.d., not detected. Modified from Lanza, B., 2012. Nutritional and sensory quality of table olives. In: Muzzalupo, I. (Ed.), Olive Germplasm—The Olive Cultivation, Table Olive and Olive Oil Industry in Italy. InTech, Rjieka, pp. 343–370.

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of table olives, together with the consumption of olive oil, which are common foods in the Mediterranean diet, provide a large amount of nutraceutical compounds (polyphenols, phytosterols, and fatty acids) with antioxidant, anti-inflammatory, or hormone-like properties. Finally, table olives can be considered also as a functional food for inserting probiotic bacteria into the human intestine. Recently, the interest of food industries is in fact focusing on the so-called functional foods that are characterized by the property, being a part of the nor­ mal diet, of containing particular molecules, such as probiotics, that have positive effects to human health. The incorporation of health-­ promoting bacteria into table olives would add functional features to their current nutritional properties.

14.2  Main Preparation Techniques Table olives are a fermented product; in the past, the fermentation process was naturally performed by the microflora present on the fruit and in the environment. More recently, this process has been investigated and today the procedures used for the fermentation are carefully determined and subjected to accurate control. Table olives are defined by the International Olive Oil Council (2004) as the fruit obtained from specific varieties of the cultivated olive tree, harvested at an appropriate stage of ripeness and whose quality is such that they represent an edible product and ensure its good preservation as a food for the human consumption. Processing techniques in olives production can include the use of various spices as food additives to improve nutritional quality The IOOC and the Codex Alimentarius (FAO/WHO) have published the required standards on different aspects of table olive production, including technical description, size grading, chemical composition, quality properties, labeling, defects and tolerances, authorized food additives, residues of contaminants, and hygienic characteristics. The olives can be harvested at the beginning of ripening, when they are normally green colored, until the end of ripening, when they are fully mature and their color becomes black. In order to give a  tastier flavor to the fruit, it is important to eliminate the entire or part of the bitter phenolic glucoside oleuropein, which is present in all olives in different amounts. Procedures for removing oleuropein are related to many factors, such as the olive cultivar, fruit maturity, growth conditions, and consumer preference. In most of the procedures, the olives are subjected to a lactic fermentation in brine solution, which preserves them and increases palatability. The process is affected by several factors, including temperature, pH of the brine solution, concentration of the lye and brine solution, species of the

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Fig. 14.2  Olives from Ascoli Piceno Province, Italy.

­ icroorganisms, carbohydrates content of the olives, and exposure to m light and temperature (Fig. 14.2). Most olive fermentations are anaerobic, but also aerobic fermentations can be performed. The main organisms used in olive fermentation are lactic acid bacteria (LAB), able to produce a fermentation of sugars (glucose and fructose) from the olive pulp, producing lactic and other organic acids that reduce brine pH, a requirement to ensure food safety. Because of the anaerobic conditions created during fermentations, molds do not grow well; however, if enough air space is available on top of the brine surface, mold will grow on the surface. To prevent their growth, considering that most of the yeasts and all molds are strict aerobes, it is important to remove air by applying a vacuum, the same strategy of vacuum packing, or replacing it with gas (such as carbon dioxide or nitrogen). However, it is important to consider that anaerobic organisms such as Clostridium botulinum can still reproduce themselves in the absence of oxygen. The temperature affects the growth and activity of microorganisms; the best conditions for an optimal fermentation are in the range between 15°C and 30°C with 25°C being very favorable. Below 15°C, fermentation is very slow and above 30°C, the growth of undesirable food contaminants is very frequent. The best temperature for a very good efficacy for most lactic acid is in the range between 18°C and 22°C, including Leuconostoc species that initiate fermentation. Temperatures above 22°C favor the Lactobacillus species. The acidity in brine is determined using pH value that represents a measure of the hydrogen ion (acidic) concentration in solution, in this case fermentation brine or packing solution. The actual amount of acid produced

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during fermentation or added to brine for adjustment is generally calculated as grams of lactic acid per 100 mL equivalent; this procedure is very important for the preservation of table olives. Foods with a pH value of 4.5 or less are considered as high-acid foods; when acidity reaches these levels, it is not possible a growth of bacterial spores associated with food poisoning. Food with pH values greater than 4.5 is susceptible to spoilage due to the growth of bacterial spores. Effective fermentations of table olives achieve brine pH values between 4.3 and 4.5 or less. If these pH values are not achieved, organic acids, for example, lactic or acetic acids, can be used to reduce pH value in brine, obtaining as a result of this technique safe olives resistant to bacterial spoilage. Lactobacillus spp. and Streptococcus spp. are acid tolerant bacteria. Yeasts can grow when pH values are in the range between 4.0 and 4.5, therefore, in spontaneous fermentations yeasts are often present, associated with fermentative bacteria. Even if molds prefer acid environments, they can grow over a wide range of pH values, ranging from 2.0 till 8.5. Fermentation is a natural process by which organic substrates, such as carbohydrates as glucose and fructose, undergo biochemical changes provoked by microorganisms or enzymes, with the final production of organic acids, ethanol, carbon dioxide, and other metabolites, so its use permits the production of “natural” olives. Controlled fermentation process that occurs in the brine is generally an efficient technique requiring low-energy inputs and increasing the safety and shelf life of olives. Fermentable substrates must get out of the olive pulp into the brine and fermentation products (lactic and acetic acids) and salt must get in the olives. When processed correctly, the olives are efficiently preserved thanks to the effects of salt, pH, and the organic acids, therefore, the olives will not need heat treatment to ensure safety and stability. However, with the aim to obtain the best result in terms of precaution, many fermented table olive products are packed and pasteurized, and sometimes preservatives such of sorbic or benzoic acids are added, too. It is essential with olive fermentation to ensure that only the desired bacteria or yeasts start to multiply and grow in the brine, not the undesirable pathogenic and spoilage microorganisms, which fortunately cannot survive the salt/acidic environment. During fermentation, organic acids such as lactic and acetic acid are produced, with a consequent increase of the acidity level of the brine and a decrease of pH value. Alcohol is also produced during some types of fermentation. During the fermentation process, it is very important to perform several controls in order to reduce the risk of overgrowth of undesirable or harmful bacteria that can cause a food deterioration or food poisoning. Process control involves checking the salt and acid levels adding, if necessary, sodium chloride and food acids, respectively.

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A simple debittering process for any olive, green ripe, turning color, or black ripe, can be performed putting the olives in 8%–10% w/v salt brine solution for a certain time. Olive’s pulp is affected by some textural changes, too. The process of fermentation involves the degradation of organic molecules by microbial enzymes into simpler substances, for example, carbohydrates are converted into lactic acid, acetic acid, and alcohol. The natural microflora of raw olives shows: Gram-negative bacteria; homofermentative and heterofermentative LAB and/or yeasts; oxidative yeasts and molds; and other bacteria such as Clostridia, Propionibacteria, and Bacillus spp. Some of these microorganisms are involved in the fermentation process, while others, if not controlled, can eventually lead to soft and not palatable olives. Olives are generally fermented in brine (8%–10% w/v salt brine). The initial brine has a pH value ranging between 6.5 and 7.5, possibly higher if the olives are pretreated with lye. These bacteria produce large amounts of carbon dioxide, which, after dissolving in the brine, produces carbonic acid. Some organic acids released from raw olives can also contribute to the initial acidity, while oxygen is also consumed. The result is a moderate increase of acidity in the brine, with a fall in pH value close to 5, which creates anaerobic conditions in the brine for fermentation. For completing this process generally are necessary 3–4 days. If the pH value does not fall, then Gram-negative bacteria persist and the olives can produce gas pockets, with soft olives as a final result. The addition of organic acid, such as lactic acid, causes the fall of pH value close to 5 in the brine, reducing and/or avoiding this problem. The levels in the brine should be constantly maintained around 8% w/v during processing. The fermentation containers (barrels and tins) must be kept full of brine continuously. During the period of active fermentation (4–5 days) when gas production causes excessive frothing and bubbling, care must be taken to replace all lost brine. When gas is produced, it is very important to keep the closures or lids tightened firmly, in order to avoid air penetration and keep oxidative yeast and mold growth at the surface to a minimum. If the olives being fermented are low in carbohydrates, sugar (dextrose, sucrose, or corn syrup) can be added after 3–4 days from the beginning of the fermentation process. Depending upon the final product, homofermentative and heterofermentative LAB and/or yeasts are able to proliferate under these anaerobic conditions and lowered brine pH value. Usually, faster growing heterofermentative bacteria dominate at this stage, utilizing sugars and other fermentable substrates released into the brine from the olive pulp to produce carbon dioxide, lactic acid, acetic acid, and ethanol. Using this strategy, a further decrease of brine pH value occurs and anaerobic conditions are maintained, preventing in this way further proliferation of Gram-negative bacteria. Maintaining the strict

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anaerobic conditions during the fermentation process, the growth of surface molds and yeasts is inhibited. Otherwise, these organisms would consume acids produced during fermentation, resulting in an increase in brine pH, with a consequent reduction in the olives stability and an increase of the risk of spoilage. When brine acidity increases, heterofermentative species are replaced by homofermentative LAB, for example, Lactobacillus pentosus (formerly Lactobacillus plantarum), with lactic acid as the main final products. Heterofermentative bacteria do not produce as much acid as the homofermentative bacteria. If the brine sugars are low or depleted, insufficient acid is produced and preservation problems can occur. During natural fermentation, yeasts are often present with the LAB, namely mixed flora. In Greek-style olives, yeasts predominate. If the process has been carefully checked, the final products have desirable sensorial qualities. If poorly controlled, the olives soften, change color, and become gassy or fritzy. At the end of the fermentation process, if the salt content is close to 10% w/v or more, the olives can be stored in the same brine for up to 2 years. In this paragraph, it was described only the processing of the olive fruit, while the use of food additives such as herbs, spices, and other flavorings to the product either during or after processing have not been considered. Fruit color, processing method, or processing style are the three most common criteria used for table olives classification; obviously, the three criteria are strictly correlated. There are three main types of table olives based on color, mainly influenced by the degree of maturity at harvest time. The International Olive Oil Council (2004) defines three main categories: green, turning color, and black. Green olives are harvested when the fruit has reached final size, with a color described from green to straw yellow. Olives classified as turning color are rose, wine-rose, blush, or brown-colored fruit, the harvest takes place before complete ripeness. Black olives are harvested fully ripe or just before complete ripeness: in this case, the olive color varies from reddish black, violet black, deep violet, greenish black, or deep chestnut. Black olives Californian style are blackened by oxidation or dyeing. As regards the processing methods, the classification of table olives is based on the procedure used to remove the oleuropein, and are indicated as treated or natural (Kailis and Harris, 2007) (Fig. 14.3).

14.3  Treated Olives The “Trade Standard Applying to Table Olives” (International Olive Oil Council, 2004) defined “treated olives” as: “Green olives, olives turning color or black olives that have undergone alkaline treatment, then packed in brine in which they undergo complete or partial fermentation, and preserved or not by the addition of acidifying agents.” Below are listed the main styles used to prepare table olives.

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Fig. 14.3  Black olives from Greece.

14.3.1 Treated Green Olives (Sevillan or Spanish-Style) Sevillan or Spanish style is the most used method for the production of Italian green table olives. In this case, the fruits are debittered in NaOH ranging from 2.0% to 3.5%, depending on the olive variety and ripening. The alkaline treatment performs the function of hydrolyzing the compound principally responsible for the bitter taste (oleuropein). This solution covers completely the olives which remain in this alkaline solution until it has entered between 2/3 and 3/4 of the distance between the skin and the pit. To verify a correct treatment, olives are cut with a razor blade or scalpel near the pit, checking the surface after air exposition. After the alkaline treatment, the olives are washed with potable water. The sequence of washings is: 1. the first washing is strong, with potable water, using a shower for 15–20 min in order to eliminate the lye attached to the fruit surface; the olives washed are left at the end in the final washing water; 2. after 2–3 h a second faster and simpler washing takes place using a filling-emptying procedure; and 3. in the next 24–48 h are performed three to four other washings, using always a filling-emptying procedure. After water washings to eliminate the residual lye, olives are covered with a sodium chloride solution (brine) and left to develop a spontaneous lactic fermentation. Initial brine concentration is 8%–10% NaCl but, after a short period of time, decreases to 5% because of the exit of water from the olives. A spontaneous fermentation starts as soon as the olives are placed in brine. After the alkaline treatment, the pH

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value of olive pulp reaches the value of 11.0–13.0 down to the value of 8.0–9.0 due to the frequent washes. It has to be considered that the liquid obtained is a sort of culture broth in which some microorganisms are able to grow. Reducing sugars and glucosides, the basic sources of carbon needed in the development of lactobacilli and other microorganisms pass from olive pulp to the brine, where they are used by heterofermentative or homofermentative microorganisms to produce lactic acid. In the first phase of fermentation, when Gram-negative bacteria prevail, the pH ranges from 8.0–9.0 to about 6.0. At this pH value, LAB can grow easily, since their optimal growth is between pH 5.5 and 5.8. When the lactic fermentation is done, the pH reaches values <4.0 and acidity increases, ensuring thus the preservation of the product. The lactic fermentation ends with the exhaustion of available carbohydrates (glucose from glucosides and reducing sugars). Several studies have been carried out to evaluate the technological functionality of Enterococcus casseliflavus and Lactobacillus pentosus during the Spanish-style green olive processing (De Castro et al., 2002; Sánchez et al., 2001). E. casseliflavus and Lactobacillus pentosus have been proposed as starter cultures to accelerate lactic acid formation at pH 9 (immediately after washings). In this case, obviously, the strain used as starter is not necessarily oleuropeinolytic because lye has just demolished the bitter glucoside. The aim of this type of starter is to reduce the lag phase and the risk of spoilage (Bevilacqua et al., 2010; Leal-Sanchez et  al., 2003). As a result of this treatment, important changes in structural and nutritional molecules located in the tissue of the food can be caused (Lanza, 2012).

14.3.2  Californian/Spanish-Style Black Olives This method/style of olive, inappropriately called “black-ripe olives,” was originally developed in California. It has also been adopted in Spain and in some countries of North Africa where table is produced. Fresh green olives or turning color olives are soaked in several caustic soda (lye) solutions at different concentration, in order to ­allow the lye penetration through the pulp until it gets to the stone. After this treatment, the lye is removed and replaced with potable water and the air passed through tanks. The olives turn a brown/black color through the oxidation/polymerization of polyphenols (hydroxytyrosol and caffeic acid). It is possible to acidify the water with HCl or carbon dioxide, in this case few washes are necessary. After this step, the olives are put in a solution containing 0.1% w/v of ferrous gluconate, which stabilizes the color. Iron in excess is removed washing the olives that are later packed in a 2%–3% w/v food-grade sodium chloride solution and sterilized. The IOOC Table Olive Standards (2004) declares that residual iron in the olives should not exceed 150 mg/kg.

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No fermentation takes place during this process and the processed olives are preserved by sterilization. The equipment necessary for this method of olive processing is expensive, the cost of inputs (especially water) must be considered, and the light level of technical expertise is required. In big food factories, raw olives are processed in large horizontal tanks, for example, 10 ton stainless steel, or polyester and fiberglass tanks, where water, lye solution, brine, and compressed air can be inserted through different inlets for water and can be drained easily during processing. Specifically designed rotating tanks that facilitate even processing are used in some centers. Processing is undertaken at around 20°C. The choice of lye used is based on the olives variety, the maturation state, the fruit size, and the possible previous storage in brine before lye treatment. In some centers, prebrined olives are purchased from other sources, often from different regions or even different countries. This method produces sterilized olives, therefore, the final packing is based on the principles of good manufacturing practice. Typically, the pH ranges between 5.8 and 8.0 and sodium chloride is 1%–5% w/v (depending on the commercial product). The maximum iron level can be up to 0.15 g/kg of fruits. This method is basically a chemical process and no fermentation occurs on olives. The two main advantages associated with this method are that the olives retain the firmness of green-ripe/turning color olives and, because only a few days are necessary for this process, they are ready for the market 1 or maximum 2 weeks after harvest. The main disadvantages are the large volumes of water necessary for lye treatments and washing, and the disposal of the resulting wastewater at the end of the process. California/Spanish-style black olives require sterilization to ensure safety (Kailis and Harris, 2007).

14.4  Natural Olives According to the “Trade Standard Applying to Table Olives” (IOC, 2004), the so-called “natural olives” are defined as: “green olives, olives turning color or black olives placed directly in brine in which they undergo complete or partial fermentation, preserved or not by the addition of acidifying agents.” The most important industrial preparation for natural black olives takes the name “Greek-style” because it is traditionally practiced in Greece utilizing Conservolea cultivar.

14.4.1  Natural Turning Color Olives The so-called “natural turning color olives” are table olives produced in Italy. In this type of processing, the olives are brined in 8%–10% of sodium chloride which, stimulating the fermentation process, can reduce the bitterness due to the presence of oleuropein. It is a

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time-consuming process, since in this case there is not the presence of an alkaline solution which facilitates the diffusion of the soluble components through the pulp. There is a variety of microorganisms in the brine, but the most abundant are yeasts which are present throughout the process. The first 7–15 days are mainly present Enterobacteriaceae, but as soon as the composition of brine changes, they decrease. LAB content depends on the salt concentration of the brine and on the polyphenol content, the latter is a characteristic of the olive variety. Although traditionally the brining procedure is performed under anaerobic conditions, an aerobic method, based on the presence of a central column in the fermenter by which air is bubbled, can also be used. Using this method, a final product with better quality can be obtained, since the ratio between fermentative and oxidative yeasts changes (Garrido Fernández et  al., 1997). During the process, the olives lose their color but this problem can be overcome by aerating olives for 2/3 days or by treating them with 0.1% ferrous gluconate or lactate to turn their color into deep black. Finally, olives are selected and packed in barrels or cans filled with 8% of fresh brine, the fruit can be commercialized when is sufficiently debittered. To reduce the debittering phase, several researches have evaluated the use of selected oleuropeinolytic LAB as starter cultures in Greek-style olive processing. The following bacteria: Lactobacillus plantarum (Marsilio et al., 2005; Panagou et al., 2008) and Lactobacillus pentosus (Panagou et al., 2003, 2008; Servili et  al., 2006) are characterized by a fast growth, a good acidifying ability and furthermore are able to grow even in the presence of high salt concentration and in the presence of phenolic compounds (known for their antimicrobial action) and, thanks to the marked oleuropeinolytic activity, degraded the oleuropein in no-­ bitter compounds, reducing thus considerably the time of debittering (Lanza, 2012).

14.5  Use of LAB LAB are Gram-positive, anaerobic bacteria which produce energy thanks to the fermentative metabolism which permits to ferment a sugar (such as glucose) into lactic acid. Among LAB family are the rod-shaped bacteria such as lactobacilli and the cocci: Streptococci, Lactococci, Enterococci, Pediococci, or Leuconostoc. LAB can grow in the presence of several growth factors which are present naturally in foods or in the mammal’s digestive tract. LAB are used for so long in dairy, meat, fish, and vegetable fermentations: in particular, among vegetable fermentation, are used in the production of sauerkraut, cucumbers, and table olives. Among the main commercialized table olives such as treated green olives (Spanish style), naturally black ­olives (Greek style), and alkaline-treated olives (Californian style), only

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treated and natural olives are subjected to the fermentation process. In particular, in the case of treated olives, LAB are involved in this process together with yeast, competing among them, but, in some cases, yeasts are the only one responsible for fermentation (Aponte et  al., 2010). Independently of their color, green, turning color, or black, all olives can be processed as treated or natural. The fermentation process can be influenced by a number of factors such as the cultivar itself, the agricultural, and industrial practices. As regards on the cultivar is a good practice to prepare each cultivar following a single procedure and the well-established local experiences. Generally, these processing methods have as principal purpose to conserve the olives thanks to the action of LAB and to ensure a good de-bitterness of the fruit, with the final aim to preserve the olives and improve the final product (Panagou and Tassou, 2006; Sánchez-Gómez et al., 2006). As shown in Table 14.6, Lactobacillus is the most representative genus in fermentation, followed by Enterococcus, Pediococcus, Leuconostoc, and Lactococcus which are present but at lower extent. In particular, Lactobacillus plantarum and Lactobacillus pentosus are predominant in most fermentations, however, other lactobacilli or

Table 14.6  Lactic Acid Bacteria (LAB) Species Identified in Table-Olive Fermentations of Different Cultivars Identified Species

Olive Cultivar

L. plantarum L. plantarum, L. paracasei, L. pentosus, L. pentosaceus L. plantarum, Enterococcus sp. L. plantarum, Pediococcus sp. L. plantarum L. lactis, L. plantarum, E. faecalis L. plantarum L. casei, L. rhamnosus, L. brevis, E. faecium L. plantarum, L. collinoides L. pentosus L. pentosus, L. coryniformis L. coryniformis, L. paracasei, L. plantarum, L. pentosus L. plantarum, L. pentosus, L. brevis, Pediococcus pentosaceus L. pentosus, L. plantarum, L. vaccinostercus, L. suebicus L. plantarum, Pediococcus pentosaceus

Treated green olive (Spain) Galega natural green olive (Portugal) Treated green olive (Spain) Edincik and Gemik natural black olives (Turkey) Picholine treated green olives (Morocco) Natural green olives (Algeria) Oblica natural olives (Croatia) Natural green olives (Italy) Natural olives (Tunisia) Conservolea natural black olives (Greece) Nocellara del Belice treated green olives (Italy) Bella di Cerignola treatd green olives (Italy) Picholine treated green olives (Morocco) Aloreña natural green olives (Spain) Lecino natural black olives (Italy)

Modified from Hurtado, A., Reguant, C., Bordons, A., Rozès, N., 2012. Lactic acid bacteria from fermented table olives. Food Microbiol. 31, 1–8.

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genera can play an important role or can be present as predominant species and this fact can depend on the cultivar, geographic origin, and processing methods. In general, in natural processed olives, there is a larger LAB variety with respect to the alkaline-treated olives. The LAB reactions produce, in terms of the number of substrates generated and metabolic pathways, are even more complex than the name of the bacterial group suggests. Despite its metabolic complexity, the lactic fermentation of the table olive is based on the ability of the microorganisms, LAB, to produce acid by lowering the pH and increasing free acidity. The lactic acid produced is effective at ­inhibiting the growth of other bacteria which can decompose and spoil the olives. While Streptococcus and Leuconostoc are the least acid-producing species, the members of the Lactobacillus group are homofermentative and produce the highest amounts of acid. Pediococcus are somewhere between the two. Correct fermentation requires the presence and the fast growth of LAB. Although initial conditions have to be considered, it is established that LAB grow spontaneously in treated olives but they can be substituted by yeasts in natural olives. LAB convert carbohydrates into CO2, lactic acid, and other organic acids without the need for o ­ xygen in the medium. In these conditions, the changes that take place do not modify greatly the olives composition. It is possible to divide treated olives fermentation into three phases, which are differentiated by the microbial species associated with each of them and the physical and chemical changes that occur both in brine and olives. The first phase starts when the olives are placed in the brine and finishes with the appearance of the first LAB. It is characterized by high numbers of Enterobacteriaceae. Thanks to these processes, the growth of the spoiling microorganism is inhibited, while is favored the growth of LAB. The second phase starts when the pH of the medium reaches the value of 6.0 and is characterized by the presence of Pediococcus and Leuconostoc. At the end of this phase, Enterobacteriaceae can no longer be found in the brine. The last phase is characterized by the fast growth of Lactobacillus and the pH tends to reach the value of 4.0. LAB populations tend to decrease once the fermentable substrates are exhausted. During the process, the number of yeasts also varies (104–106 CFU/mL) and they have a wide variety of functions during the fermentation (Arroyo-López et al., 2008). Although several authors recommend using inocula to ferment treated olives, the practice is not widespread and most processes are spontaneous. The fermentation ecosystem of natural olives, like that of treated olives, consists of a complex mixture of Gram-negative bacteria, LAB, and yeasts. The process can also be divided into different phases but the beginning and the end of each phase are not as well defined as they are in treated olives. Gram-negative bacteria are very important

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(107–108 CFU/mL) during the first steps of the process, in fact they reach their larger amount 2 days after the olives have been placed in the brine. Meantime, LAB or yeasts, or both, whichever are responsible for the fermentation, start to emerge. Gram-negative bacteria disappear in a few weeks (1–4), that means at the end of the second step of the process. In optimal conditions, the pH will reach the value 4, at this point the third step will start and the LAB or yeasts, sometimes both, will consume the nutrients of the medium (Sánchez-Gómez et al., 2006; Hurtado et al., 2008). The factors that influence the correct development of fermentation and the growth of LAB are several: the pH of the pulp; the amount of residual NaOH in treated olives; the washing process of the olives; the NaCl concentration in the brine; the temperature, the nutrient availability, and their diffusion through olive skin; and the polyphenol content of the fruit, the aeration, and the size of the vats where the fermentations take place (Chammen et al., 2005; Hurtado et al., 2009). Total and free acidity are normally checked to evaluate the progress of fermentation. In natural olives, organic acids can be added to adjust the initial pH, in order to favor the LAB development (International Olive Oil Council, 2004; Panagou and Tassou, 2006). Furthermore, it is very important to always check NaCl concentration in the brine, to ensure a correct fermentation from the beginning of the process and also to keep the concentration constant during the process, if necessary the salt must be renewed. Table olives from different cultivars are fermented in brines containing NaCl levels ranging from 4% to 15% (w/v). In general, low-NaCl brines favor the LAB development while higher concentrations stimulate yeast growth (Hurtado et  al., 2009). Lactobacillus pentosus can grow in up to 82 g L−1 of NaCl (BautistaGallego et al., 2008). The growth of 100 Lactobacillus isolates from table olives in a basal medium with NaCl concentrations of between 8% and 12% were assayed (Hurtado et al., 2011), showing that salt tolerance is a strain-dependent factor. Another very important factor is temperature, but it is hard to check without adapted fermentations (Hurtado et al., 2008). Temperature is greatly affected by the size of the fermentation vats. Small volumes have less inertia to microbiological changes and in a group of fermentations, the LAB growth and olive quality are very irregular. Olive cultivar is a crucial factor, too, especially in natural olives. Depending on the local habits, each cultivar is harvested at a different stage of maturation, so the amount of sugar, polyphenol content, and cell wall permeability will be affected. Natural olives are not subjected to lye treatment. In this case, there is no additional influence on the permeability of the skin or polyphenol hydrolysis. Despite the fact that most of the strains can degrade oleuropein and some strains can metabolize specific compounds, in

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general terms higher concentrations of polyphenols inhibit LAB. The beginning of the fermentation process is also influenced by the availability of nutrients (Sánchez-Gómez et al., 2006; Landete et al., 2008; Hurtado et  al., 2009; Rodríguez et  al., 2009; Ghabbour et  al., 2011). It has been demonstrated that an inappropriate alkaline treatment in treated green Manzanilla olives (low NaOH strength and insufficient alkali penetration) lead to the presence of several antimicrobial compounds in brines, inhibiting the growth of Lactobacillus pentosus (Medina et al., 2008). Yeasts, which have a minor role in treated olives fermentation, are supposed to have an important role in the development of table olive flavor and aromas and also in LAB growth (Arroyo-López et al., 2008; Sabatini et al., 2008). If yeasts are used as starter adjuncts, both undesirable contaminating yeasts and foodborne pathogens can be inhibited, which favors the imposition of LAB (Psani and Kotzekidou, 2006). It has been suggested that LAB starters compete with wild yeasts in Conservolea cultivar natural black olives (Panagou et al., 2008). It has been shown by Segovia-Bravo et al. (2007), that Lactobacillus pentosus shows a better growth when it is cocultured with Saccharomyces cerevisiae in green-table olive brine. Furthermore, other authors (Hurtado et al., 2010) show that the co-inoculation of Candida diddensiae and Lactobacillus pentosus led to a better microbial development profile than single inoculation. In the early stages of the fermentation, yeast metabolism can produce specific essential growth factors for the LAB development, particularly vitamins. Starter cultures are used to quicken and improve the fermentative processes and are composed of a variable number of microorganisms. Starter cultures not only decrease the risk of spoilage but also make acidification of the brine faster and more effective, and reduce the metabolic energy required during the process (Panagou and Tassou, 2006). The requirements for an ideal starter culture are: fast growth of the microbiota; ability to dominate the indigenous microbiota; homofermentative metabolism; ability to consume rapidly the fermentable substrates; high acidification rate; good medium tolerance for salt, organic acids, and polyphenols; ability to develop flavor and aromas; temperature range tolerance for growth; oleuropein-splitting capability; ability of bacteriocin production; minimum nutritional requirements; and the ability to resist freezing or lyophilization if required for the commercial purposes (Delgado et al., 2005; Hurtado et al., 2012). In particular, it is very important the dominance of the starter culture on the indigenous microbiota which can be exerted through its fast growth under the fermentation conditions in combination with its ability to produce antagonistic substances. Furthermore, it is necessary that the starter culture is able to resist to freezing and freeze-­ drying processes (for commercial purposes).

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Comparative studies between spontaneous and inoculated fermentations have been made since 1940s Lab starters are recommended by the majority of authors, and different starter cultures have been tested for each cultivar and fermentation process. Most of the studies on the use of starter cultures in fermented table olives production were focused on Lactobacillus pentosus and Lactobacillus plantarum, since they can be used for the fermentation of various olive table cultivars (Sánchez et al., 2001; Panagou et al., 2008), even if it has to be considered that the inoculation depends strictly on the cultivar and on the processing method (Panagou and Tassou, 2006; Hurtado et al., 2010, 2012). Other authors studied the use of other strains as starter cultures, inoculating them alone or together with another species such as Lactobacillus pentosus or Lactobacillus plantarum (De Bellis et al., 2010). In addition to the latter two microorganisms, only Lactobacillus paracasei (De Bellis et  al., 2010) and Leuconostoc cremoris (Kumral et al., 2009) have been able to ferment table olives when used as starter strains. Usually, the final concentration of the inoculum ranges from 106 to 107 CFU/mL of brine. In the case of for Lactobacillus paracasei, an inoculum of 109 CFU/mL can be used. Competitive microorganisms, which could grow during the fermentative process, can be effectively eliminated by the pasteurization of raw material, thus making olives more fermentable. Some authors in fact, during the pasteurized olive fermentation process in the presence of Lactobacillus plantarum strain as starter culture, found that the pH decreases less rapidly with respect to the olive not subjected to the pasteurization process, but final pH values and acid development were more pronounced (Chorianopoulos et al., 2005). As stated before, the LAB role, during olive fermentation, is very important: they can preserve olives by a progressive brine acidification, with a subsequent production of antimicrobial substances and bacteriocins, and can improve flavors and aromas of the final product (Marsilio et al., 2005). The majority of the authors acknowledged that the leader microorganisms in the fermentation processes are Lactobacillus plantarum (Chorianopoulos et al., 2005; Lamzira et al., 2005; Marsilio et al., 2005; Sabatini et al., 2008) and Lactobacillus pentosus (Panagou et al., 2008; Servili et al., 2006) since they are able to improve the microbiological control of the process, lead to an increase of lactic acid yield (therefore increase acidification), and finally lead to a high-quality table olive. Servili et al. (2006) used a particular strain of Lactobacillus pentosus (1MO) as a starter for the fermentation of black olives (from Itrana and Leccino cultivars) in brine at a temperature of 28°C, with a pH value of 6.0, 0.3% glucose and in the presence of growth factor (0.05% w/v yeast extract). The obtained result was that the olives were debittered in 8 days. Other authors used, as starter culture in olive fermentation, strains such as Lactobacillus plantarum LPCO10 able to

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produce bacteriocins which facilitate the dominance of the starter culture over the natural microbial population. The bacteriocins produced by Lactobacillus plantarum LPCO10 are named plantaricins S and T, and act against several natural competitors of Lactobacillus plantarum and against bacteria that cause the degradation of the olives (LealSanchez et al., 2003). Therefore, Lactobacillus plantarum LPCO10 is able to dominate the fermentation process, to induce a rapid pH decrease during the first fermentation steps, to induce a higher free total acidity and consequently can reduce olive spoilage. The effect of a mixed starter culture (Lactobacillus plantarum and a commercial preparation based on pure freeze-dried Lactobacillus pentosus) on fermentation of natural black olives (Conservolea cultivar) has been evaluated by Panagau and coworkers (2008). The fermentation brine contained 6% (w/v) NaCl, maintained constant by periodical addition of salt, and the temperature was 20°C, for a period of 30 days. As a result, the fermentation process was accelerated and the survival time of Gram-negative bacteria was reduced by 5 days when compared to the spontaneous process. Hurtado and coworkers found that the performance of Lactobacillus pentosus in the fermentation of table olive from Arbequina cultivar was better than Lactobacillus plantarum: in this case, the brine contained 8% NaCl (w/v) and the fermentation was performed at temperature of 20°C for 52 days. At the end of the fermentation process, the brine was replaced with a new sterile one containing 5% NaCl (w/v), 1% CH3COOH (v/v) and in order to inhibit any microbial growth, olives were stored at 4°C. During the fermentation process, the temperature exerts an important role since can influence the microbial metabolism and fermentation capacity. It has been shown that Lactobacillus plantarum and Lactobacillus pentosus require temperatures ranging from 20°C to 25°C. Finally, the starter culture should be able to growth also at low temperature. This could be important especially in winter when the low temperature could slow down the microbial activity. Recently, it was shown that fermented table olives can be used as a probiotic carrier: in this case, microorganism can have a dual role, acting as a starter and as a probiotic culture since they are able to control the fermentative process but contemporary it is possible to achieve a final probiotic product with probiotic characteristics. The obtained table olives could be ascribed as a “functional food.” In particular, Lavermicocca et al. (2005) used the probiotic strain Lactobacillus paracasei as starter for the olive fermentation process. These authors found that the olives treated with this strain had some additional ­benefit for health and nutrition: free-radical scavenger action, atherogenesis prevention; increasing of high-density lipoprotein (HDL) ­fraction; increasing the amount of vitamins A, B, and E; and consequently delaying of cellular aging. In the presence of probiotic strains, the fermentation process was performed under different conditions:

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4% and 8% (w/v) NaCl; at room temperature and at 4°C. At the end of the process, the probiotic colonized the olive surface dominating on the other LAB population and it has led to a decrease of the pH to values below 5 after 30 days until the end of fermentation. Furthermore, it was shown by the same authors that Lactobacillus paracasei can reduce the survival time of potential spoiling microorganisms. Recently, it was shown that the simultaneous inoculation of yeasts in the brine can increase the growth of LAB, because yeasts are able to produce vitamins B1 and B6, amino acids, and purines and can break down complexes carbohydrate in order to allow the growth of Lactobacillus spp. (Abbas, 2006). Some authors experimented the addition of S. cerevisiae during green olives fermentation, founding that the performances of Lactobacillus pentosus, used as starter culture, were significantly increased (Segovia-Bravo et al., 2007). Hurtado et  al. (2010) co-inoculated Lactobacillus pentosus and C. diddensiae in the fermentation of table olives (Arbequina cultivar) founding that the Enterobacteriaceae survival was reduced as well as the presence of contaminating yeasts and foodborne pathogens, the sensorial quality of the olives resulted increased and LAB activity was improved. Other yeasts were studied as candidates for co-inoculation in the fermentative process, such as Candida boidinii or P. membranifaciens (ArroyoLópez et  al., 2008). In conclusion, the use of starter cultures during the table olive fermentation process seems to be the most reasonable, since it is able to fully meet different requirements for obtaining high-quality healthy product thanks to the reduction of costs, fermentation time, risk of contaminations, and to the improvement of process control, sensory quality, and safety characteristics.

14.6  Different Strains in Table Olive Microbiota Olives microbiota differ among different cultivar and among the olive processing techniques. The microbiota present in the processed olives and in the brine includes members of Enterobacteriaceae, Clostridium, Pseudomonas, Staphylococcus, LAB, yeasts, and sometimes mold. Enterobacteriaceae are usually present at the beginning of the fermentation process (2.6–3.5 log CFU/mL in the brine obtained from cracked green table olives), but, during fermentation, they decrease until they disappear at the end of the fermentation (Alves et al., 2011; Randazzo et al., 2012). Other species can be found at the beginning of the fermentation process, such as Clostridium and Pseudomonas: Clostridium cannot survive longer because of the low pH value developed during the ­fermentative process (which is 4.1 when olives are preserved by its own physicochemical characteristics, or 4.3 when preserved by p ­ asteurization),

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Clostridium botulinum was isolated in heat-treated olives only if the pH value of the brine was up to 4.6 (Cawthorne et al., 2005). Other sources of contaminations may come from processing, packaging, and the transport of the final product (Jalava et al., 2011). However, if the abovementioned conditions are respected, Pseudomonas and Clostridium may not be a problem in fermented olives. As discussed before, LAB are the most important group of fermentative bacteria in the table olives production, they are divided in homofermentative bacteria (Lactobacillus, Streptococcus, and Pediococcus) and heterofermentative bacteria (Leuconostoc and some members of Lactobacillus) where the most important role is exerted by Lactobacillus spp. (Abriouel et al., 2011). Conversely, LAB were not revealed in some kind of natural green olives (Valencic et  al., 2010; Alves et  al., 2011; Aponte et  al., 2011). Bautista-Gallego et al. (2013) found that the population of LAB changes notably in Spanish-style green table olives; in one type of brined olives (Alorena cultivar) stored at 4°C, LAB were not detected; in another two types of treated olives (from Gordal and Manzanilla cultivar) LAB were detected (Bautista-Gallego et  al., 2013). More in particular it was found that in samples isolated from natural green olives of Spanish origin (Alorena cultivar) after 6 month of fermentation, the predominant bacteria were Lactobacillus pentosus (81.9%), followed by Leuconostoc pseudomesenteroides (10.4%) and Pediococcus parvulus (7.6%) (Abriouel et al., 2012). In natural black olives of Greek origin (Conservolea and Kalamata cultivar), was also observed the presence of Leuconostoc mesenteroides as dominant specie (Doulgeraki et  al., 2013). Lactobacillus paraplantarum and Leuconostoc pseudomesenteroides were found rarely in natural green and natural black olives, respectively (Bautista-Gallego et al., 2013; Doulgeraki et al., 2013). The microbiota of natural green olives from two Italian cultivars (Nocellara Etnea and Geracese) has been investigated (Randazzo et al., 2012). As a result, the olives from Geracese cultivar showed wide LAB population in fermented samples, in this case, olives were inoculated with Lactobacillus plantarum and L. casei and maintained for 180 days at the room temperature. During fermentation time were found some bacteria which resulted well adapted to the brine conditions: Lactobacillus brevis, Lactobacillus coryniformis, Lactobacillus plantarum, and Leuconostoc citreum, whereas L. paracollinoides, Lactobacillus paracasei, and Streptococcus thermophiles were detected also at the end of the fermentation process. As introduced before, yeast plays a fundamental role in fermented olive production especially when LAB are inhibited because of the presence of phenolic compounds in the brine (Arroyo-López et al., 2012a). Furthermore, it is important to remember that fermentative yeasts contribute to the organoleptic characteristics of table olives (Aponte et al., 2011). Yeasts population in table olives is shown in Table 14.7.

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Table 14.7  Yeast and Mold Species Isolated From Olives YEASTS

Type of Processing

Country of Origin

Candida apicola C. boldinii

Cracked directly brined green Ripe black Directly brined black Cracked directly brined green Directly brined green Cracked directly brined green Directly brined black Directly brined green Directly brined green Directly brined green Ripe black Directly brined green Directly brined black Directly brined green Cracked directly brined green Directly brined green

Spain Spain France Portugal Spain Portugal Greece Italy Spain Spain Spain Italy Greece Spain Spain, Portugal Spain

Natural fermented black Natural fermented black Natural fermented black Cracked directly brined green

Turkey Turkey Turkey Spain

C. oleophila C. olivae C. parapsilosis C. sorbosa C. tropicalis Pichia galeiformis P. guillermondii P. membranifaciens Saccharomyces cerevisiae MOLDS

Penicillium citrinum P. roqueforti P. brevicompactum Geotrichum candidum

Modified from Herpekan, D., 2013. Microbiota of table olive fermentations and criteria of selection for their use as starters. Front. Microbiol. 4, 143.

Candida, Pichia, and Saccharomyces are the yeast genera most frequently isolated in the brine from several olive varieties, but are present also in other genera such as Debaryomyces, Issatchenkia, Zygotorulaspora, and Wickerhamomyces (Arroyo López et  al., 2006; Coton et al., 2006; Nisiotou et al., 2010; Bautista-Gallego et al., 2011; Alves et al., 2011). Recently, Arroyo-López et al.(2012b) reported that some yeast could be used as starter culture (W. anomalous, S. cerevisiae, and P. membranifaciens), particularly, W. anomalous can grow easily under certain conditions such as low pH and high NaCl concentration and also show interesting technological properties (Bautista-Gallego et al., 2011). The population of yeasts increases during fermentation, since is about 4.9–5.0 log CFU/mL at the beginning of the process and then increase up to 6.0–6.5 log 10 CFU/mL at the end of the process (Alves et al., 2011). It is important, however, to maintain the oxidative

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yeast at a low level since they are able to oxidize lactic acid and therefore, can raise the pH value causing spoilage as observed by ArroyoLópez et al. (2012b). Furthermore, the production of high amounts of CO2 may cause blister formation. Finally, during the olive fermentation process, it is possible the development of some genera of mold: Aureobasidium, Geotrichum, and Penicillium were detected, but at low extent (see Table 14.7). Usually, Penicillium grow on the surface of the naturally fermented black olives, molds can cause softening of olive pulp, moldy taste, and can produce mycotoxins (Heperkan et al., 2006). Recently, it was found in the olive skin, processed according to the Spanish style, the coexistence of Lactobacillus pentosus (a LAB), Pichia galeiformis, Candida sorbosa (yeasts), and Geotrichum candidum (a mold) (Arroyo-López et al., 2012c; Heperkan, 2013). So, from what has been said, the success of the olive treatment depends on a lot of factors, first of all, the olive coming from different cultivars shows different fermentation behaviors when treated with brine, therefore, it is very important to know the chemical and physical characteristics of the olive cultivar used and its attitude to the treatment in order to obtain a final product of good quality. At this purpose, it is important to correlate each fermentation procedure to the relative chemical– physical composition of each cultivar. Another important aspect is the presence of pathogens that are detected during the olive treatments by several authors. At this purpose, severe controls on pH, good hygienic practices throughout the process are necessary, and also heat treatment of traditional products. All of these control and verification systems should be employed in order to guarantee a safe and hygienic product.

14.7  Olives and Cocktails Cocktail garnishes are decorative ornaments that add character or style to a mixed drink, most notably to cocktails. Alcoholic beverages, such as cocktails and highballs have been flavored by additions of flavoring agents in solid or liquid form. Fresh fruits, that is, citrus fruits such as lemons, limes and cherries, olives, and onions, are conventionally added to the alcoholic beverages for flavoring and visual effects. There are many different cocktail recipes which require a garnish of olives, all that are very tasty and elegant. The Martini cocktail is one of the best-known mixed alcoholic beverages. By 1922, the Martini reached its most recognizable form in which London dry gin and dry vermouth are combined at a ratio of 2:1, stirred in a mixing glass with ice cubes, with the optional addition of orange or aromatic bitters, then strained into a chilled cocktail glass (McElhone, 1922). Overtime the generally expected garnish became the drinker’s choice of a green olive or a twist of lemon peel. Martini the cocktail has become famous thanks to the Bond movies.

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The ­fictional spy James Bond also called 007 drinks only martini and likes his shaken not stirred! It is one of those iconic drinks that never goes out of style and has been endorsed by many a famous man down the ages including Ernest Hemingway. The cocktail that includes, first of all, the olive in the glass is the “Dirty Martini” which boasts important births, having been created by the 32nd President of the United States, Franklin Delano Roosevelt, and which takes its name from the presence of the brine of olives, which “dirty,” in fact, the compound. The original recipe includes 1/2 oz. of Martini Dry, 1 and 1/2 oz. of Gin (alternatively, the Vodka can also be used), 1 spray of brine, green olive to seal. After mixing everything, pour it into a cold cup, adding the olive as a final touch. One of the last creative cocktails of the “Martini” family is the “Martini Eyeball.” White Martini, enough to fill the glass, must be used together with radishes, green olives. It is extremely simple to prepare, providing only white Martini as a liqueur, but it is the addition of small radishes that makes the compound special. In fact, it is necessary to peel the radishes by making thin red stripes that look like an eye with veins. The final touch is to pierce the radish, applying a green olive to the center, and thus giving the idea of a spectral eye. The sweeter, earthier, more complex flavor of whiskey lends itself to a number of olive-garnished beverages. One of the most famous is the Manhattan, in which gin or vodka have been replaced with bourbon. If Scotch is the favorite spirit, a similar substitution produces the Rob Roy. A good whiskey can also serve as the base for a more elaborate drink, such as the Blarney stone, that needs eight parts Irish whiskey, with one part each of anise-flavored pastis and fruity orange Curacao. The shaker must be perfumed with a dash of maraschino liqueur and one of orange bitters, and garnish is provided by a strip of orange peel to bring up the fruit flavors, and an olive to provide them with a savory counterpoint. Finally, we can describe the trendiest cocktail of summer: it is based on EVOO and is called The Oliveto. The bar Artesian in London, who for the fourth consecutive year has won the first place in the ranking of the 50 best bars in the world, is located inside the Langham Hotel, in the chic district of Marylebone. This bar offers the highest quality and the best creative proposal according to a jury of over 400 experts coordinated by Drinks International, the highly regarded magazine in the beverage world. The barman created “The Oliveto” during the period when he was playing with the “fat-washing.” He explains that it is an alcohol flavoring technique using high-fat ingredients, such as butter or even bacon. After dissolving the fat in the alcohol, mixed and cooled for a certain period of time, the fat part is removed, which leaves its taste in the spirit. In this sense, he decided to use EVOO with gin, using, therefore, a high-quality product whose flavor was not too preponderant and seemed to blend well with the distillate. So, he realized that using an egg white in the cocktail he would not even need to

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do “fat-washing” with the gin, but he could have put the oil directly in the cocktail and the egg white would have emulsified! The final result was a perfect cocktail. This is the recipe of the Oliveto: 6 cL (abundant) Martin Miller’s Westbourne gin (or Gordon’s gin) 1 cL Licor 43 3 cL fresh lemon juice 1 cL sugar syrup 1.5 cL EVOO 1 egg white “dash” (a pinch, in jargon) of saline. Combine the ingredients in the shaker and stir them without ice. Add the ice and stir again for about 2 min. Strain into a glass of the “old fashioned” type previously filled with ice. With the different oils from all over the world, it is possible to have many different aromas and hints and experimentation with different products from different regions can be not only fun but also interesting (Fig. 14.4).

Fig. 14.4  Olives in a cocktail glass.

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14.8 Conclusions Table olives are a fermented product, and for most of their history, this has been a natural process driven by the microflora on the fruit and in the environment. More recently, this process has been investigated and subjected to more control. The olive contains a variety of compounds, including organic acids, tannins and the bitter glycoside, and oleuropein. They are all water soluble and their presence decreases as the olives ripen. Some bitterness is desirable, but depending on the ripeness some must be removed by processing methods. The current styles of table olive processing have evolved using standard cultivars that have been produced for many hundreds of years, and the details of protocol vary depending upon cultivar, region of production, and cultural preference. With the exception of California style and salt cured olives, all methods of curing involve a major fermentation involving bacteria and yeast that is of equal importance to the final table olive product. Of all the metabolites, lactic acid is the most important as it acts as a natural preservative lowering the pH of the solution to make the final product more stable against the growth of unwanted pathogenic species. The result is table olives which will store with or without refrigeration, and thus LAB dominated fermentations are generally considered as the most suitable method of curing olives. Yeast-dominated fermentations produce a different suite of metabolites which have fewer self-preservation characteristics and therefore, acid corrected, often with citric acid, in the final processing stage to achieve microbial stability. Finally, the concept that olive can be considered as a real “functional food” can be derived from several scientific studies, in which results obtained have shown that olives and their derivative, the olive oil, are healthful foods since they provide vitamins, minerals, and several other nutrients; there are also strong evidences that they can have a protective effect on the cardiovascular system, can reduce the risk of breast cancer and other disease. There are many different cocktail recipes which require to add a garnish of olives, all of them are very tasty and elegant. There are two different ways to use olives as a garnish. The easiest way is to use a cocktail stick through the olives, which will then rest in the glass. In this case, it is possible to have several olives instead of just the one. The second way will require a sharp knife and a steady hand. Simply make a small incision in the thickest part of the olive and squeeze. This should open up a little valley which is the perfect size to fit on the edge of your glass. This technique may take a bit of practice to get right, but looks fantastic at parties.

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Further Reading Ghanbari, R., Anwar, F., Alkharfy, K.M., Gilani, A.-H., Saari, N., 2012. Valuable nutrients and functional bioactives in different parts of olive (Olea europaea L.)—a review. Int. J. Mol. Sci. 13, 3291–3340. López-López, A., Montaño, A., Garrido-Fernández, A., 2010. Nutrient profiles of commercial table olives: proteins and vitamins. In: Preedy, W. (Ed.), Olives and Olive Oil in Health and Disease Prevention. Elsevier, San Diego, pp. 705–714. Ozdemir, Y., Guven, E., Ozturk, A., 2014. Understanding the characteristics of Oleuropein for table olive processing. J. Food Process. Technol. 5, 328. https://doi. org/10.4172/2157-7110.1000328. Pereira, A.P., Pereira, J.A., Bento, A., Estevinho, M.L., 2008. Microbiological characterization of table olives commercialized in Portugal in respect to safety aspects. Food Chem. Toxicol. 46, 2895–2902. Sousa, A., Casal, S., Bento, A., Malheiro, R., Oliveira, M.B.P.P., Pereira, J.A., 2011. Chemical characterization of “Alcaparra” stoned table olives from Northeast Portugal. Molecules 16, 9025–9040.