Mean values and comparison of mean separation analysis (ANOVA) of the fatty acids relative to the 1108 monovarietal olive oil samples.
\r\n\t
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Chapoval",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10324.jpg",keywords:"Th2 Response, Th2 Cytokines, Asthma Classification, Differences in Diagnostics, FDA-Approved Treatments, Occupational Asthma, Exercise-Provoked Asthma, Medication-Induced Asthma, Th2-Independent Asthma, Asthma and COPD, Future Perspectives, Asthma Research",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"October 26th 2020",dateEndSecondStepPublish:"December 22nd 2020",dateEndThirdStepPublish:"February 26th 2021",dateEndFourthStepPublish:"May 17th 2021",dateEndFifthStepPublish:"July 16th 2021",remainingDaysToSecondStep:"2 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Assistant Professor at the University of Maryland School of Medicine, well-recognized for her work on HLA Class II-restricted allergen T cell epitopes, VEGF-induced lung DC modifications, and her recent discoveries on neuroimmune semaphorins 4A and 4D contributions to allergic airway inflammation and to Treg cell phenotype and function. 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She joined the Center for Vascular and Inflammatory Diseases and the Program in Oncology of the University of Maryland Marlene and Stewart Greenebaum Cancer Center at University of Maryland School of Medicine in 2006 as an Assistant Professor. Dr. Chapoval’s research is focused on cellular and molecular mechanisms of lung chronic inflammatory diseases, asthma in particular, and novel molecules for disease immunotherapy. She is well-recognized for her work on HLA Class II-restricted allergen T cell epitopes, VEGF-induced lung DC modifications, and her recent discoveries on neuroimmune semaphorins 4A and 4D contributions to allergic airway inflammation and to Treg cell phenotype and function. Dr. Chapoval has served and continue to serve as a reviewer for 20+ peer-reviewed scientific journals. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"39064",title:"Italian National Database of Monovarietal Extra Virgin Olive Oils",doi:"10.5772/51772",slug:"italian-national-database-of-monovarietal-extra-virgin-olive-oils",body:'\n\t\tThe abundance of indigenous Italian olive germplasm, numbering over 800 cultivars [1] and rising, guarantees the ongoing production of high quality extra virgin olive oils, thus contributing to the preservation of much of the ancient genetic biodiversity of the olive.
\n\t\t\tThe Olea Europea species has maintained much of its genetic diversity as a result of limited genetic erosion. This is due to breeding programs of this species having begun relatively recently compared to those of other fruit species.
\n\t\t\tKnowledge and development of the characteristics of Italian monovarietal extra virgin olive oils will also lead to an improvement in knowledge of the areas where these oils are produced, in turn developing tourism, a crucial sector for the Italian economy.
\n\t\t\tIn Italy, new regulation was recently introduced forcing virgin and extra virgin olive oil producers to indicate the location of both olive harvest and oil production. More recently the European Commission has established compulsory standards for the labelling of origin for extra virgin and virgin olive oils (Reg EC n.182/2009). The significant increase in demand for extra virgin olive oils is due not only to the health benefits it offers, but also to its organoleptic properties; the large number of Italian olive cultivars allows for the production of different monovarietal oils marked out by a wide range of pleasant flavours.
\n\t\t\tAs the genotype of origin affects the chemical and sensory characteristics of extra virgin olive oil deeply, the preservation and characterization of authocthonous cultivars and clones play a key role in the marketing of high quality olive oils.
\n\t\t\tConservation of genetic resources for olives has important implications for both adaptation of the cultivars to their local environment and their agronomical performance under specific conditions. This also implies that every initiative to promote olive cultivation ought to take into consideration the local varieties and also that every region should preserve its own plant material to safeguard olive adaptation and productivity and to maintain the intrinsic characteristics of its olive oil which represent a deep connection with the territory of origin.
\n\t\t\tIn the EU olive oils can be linked to the cultivar of origin and in turn to its area of production under the rules of the Protected Denominations of Origin (PDO) or of the European Protected Geographical Indication (PGI).
\n\t\t\tSeveral typical Italian extra virgin olive oils have qualified for PDO and PGI status, as many as 42 PDO and 1 PGI. Generally, these products are blends of different varieties according to the different cultivar percentage reported in the Product specification; some Italian PDO oils are obtained from the transformation of a single cultivar (monovarietal oils) for example the PDO Nostrana di Brisighella.
\n\t\t\tItalian olive cultivation is marked out by its extremely rich and varied varietal heritage. An important objective being pursued by every region is the protection and preservation of autochthonous Italian olive cultivars. This can be seen in the spread of regional varietal catalogs and also in the ongoing rise in the number of monovarietal olive oils taking part in the Italian National Review of Monovarietal olive oils as organized by ASSAM Marche [2].
\n\t\t\tIn Italy this review serves to characterize monovarietal oils in terms of both chemical and sensory profiles. The organization of events, courses and forums involving olive farmers, crushers, consumers and catering operators has contributed to an improvement in the visibility of the market for Italian monovarietal olive oils. Studies into the quality of monovarietal oils increase the value of the product while showcasing the region of origin and educating the consumer about their nutritional and organoleptic value.
\n\t\t\tIn Italy the market for monovarietal and organic oils is growing due to consumers paying greater attention to both flavour and health benefits of the product.
\n\t\tASSAM and IBIMET-CNR have created and are managing a database of chemical and sensory profiles of extra virgin oils participating in the Italian National Review of Monovarietal olive oils. This dynamic database includes a large number of observations for each monovarietal oil, and can allow for ongoing updates every year, thus providing more accurate chemical and sensory average data for the oils. For each monocultivar oil, chemical and sensory profiles were calculated and described, including a large number of oil samples from different regions.
\n\t\t\tThe Review reached its ninth edition as of 2004, and the large number of oil samples has led to improvements in results, in turn diminishing the effect of the main variables which have a significant influence on the quality of the oil: seasonality, ripening and different milling technologies.
\n\t\t\tSensory analysis was laid out by the “ASSAM – Marche Panel” as recognized by the IOOC (International Olive Oil Council) and the Italian Ministry for Agriculture, Food and Forestry Policy under the conditions described in EC Reg. 640/2008.
\n\t\t\tThe 150 samples collected during the first and the second year (edition 2004-2005) were used to identify the specific descriptors for the sensory analyses of monovarietal extra virgin olive oil and to set up the relative profile sheet [3].
\n\t\t\tEach panellist smelled and tasted the oil, in order to analyse olfactory, gustatory, tactile and kinaesthetic characteristics. Thirteen attributes were evaluated: 9 during the olfactory phase (olive fruity, olive fresh leaf, grass, fresh almond, artichoke, tomato, apple, berries and aromatic herbs) and 4 during the gustatory phase (olive fruity, bitter, pungent and fluidity). Attributes were assessed on an oriented 10-cm line scale and quantified measuring the location of the mark from the origin. Data obtained for the 13 descriptors were used to define the sensory profile of each sample using the median values [4].
\n\t\t\tFatty acid composition, determined according to Reg. EC Reg.796/2002 methodology [5], and total phenolic content determined according to the Folin-Ciocalteu spectrophotometric method expressed as milligrams of gallic acid per kilogram of oil, were determined by Centro Agrochimico ASSAM, Jesi (AN).
\n\t\t\tChemical and sensory data were processed using SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA). Explorative analysis and descriptive statistics were performed for each set of data in order to identify outliers, extreme observations and to obtain distributional properties of the data. Descriptive measures (moments, basic measures of location and variability, confidence intervals for the mean, standard deviation, and variance) of chemical and sensory variables were calculated for each monovarietal oil.
\n\t\t\tCurrently, the database includes 2092 oils produced from 130 different cultivars from 18 Italian regions. Nutritional properties, expressed as fatty acid and total phenol content, and the sensory profiles of each Italian monovarietal oil were published in the Catalogue of Italian Monovarietal oils [6] and at
Below are listed the average sensory profiles of the 16 most represented monovarietal oils. The number of samples belonging to each cultivar are indicated in brackets: Ascolana Tenera (36 samples), Bianchera (34), Biancolilla (28), Bosana (133), Casaliva (39), Coratina (80), Frantoio (122), Itrana (102), Leccino (105), Mignola (38), Moraiolo (83), Nocellara del Belice (49), Peranzana (47), Piantone di Mogliano (57), Raggia (54), Ravece (101).
\n\t\t\tAscolana Tenera – Marche region. Sensory profile: intense olive fruity, strongly grassy with hints of tomato and artichoke; balanced in taste, with medium intensity of bitter and pungent notes.
Bosana – Sardegna region. Sensory profile: medium olive fruity, grassy with prevalent scent of thistle and artichoke and hints of almond and tomato. Medium intensity of bitter and pungent notes.
Bianchera – Friuli Venezia Giulia region. Sensory profile: medium-intense olive fruity, with grassy scent, artichoke, almond and tomato; medium bitter and pungent flavours.
Casaliva – Lago di Garda area. Sensory profile: medium-intense olive fruity, with marked almond scent and light flavour of grass and artichoke; well balanced taste with medium intensity of bitter and pungent notes.
Biancolilla – Sicilia region. Sensory profile: medium-intense olive fruity, with a marked grass scent and light hint of almond, artichoke and tomato; bitter and pungent flavours are of medium-light intensity.
Coratina – Puglia region. Sensory profile: medium olive fruity, with a marked fresh almond scent together with notes of grass and artichoke; bitter and pungent flavours are of medium-high intensity.
Frantoio – Central-North Italy. Sensory profile: medium-high olive fruity, with a marked fresh almond and and light flavour of grass and artichoke; bitter and pungent flavours are of medium intensity.
Itrana – Lazio region. Sensory profile: high olive fruity intensity, with grass, tomato and artichoke scent and light almond flavor; well balanced taste with a bitter and pungent medium-light intensity.
Leccino – North-central Italy. Sensory profile: medium olive fruity intensity, with almond scent and light grass and artichoke flavor; medium intensity of pungency and bitter taste
Mignola – Marche region. Sensory profile: medium olive fruity intensity, with a peculiar flavor of soft fruits; medium intensity of pungency notes and marked bitter taste.
Nocellara del Belice – Sicilia region. Sensory profile: medium-high olive fruity intensity, with grassy and tomato notes and light scent of artichoke and almond; well balanced taste with medium intensity of bitter and pungency notes.
Piantone di Mogliano – Marche region. Sensory profile: medium olive fruity intensity, with almond scent; medium-light intensity of pungency and bitter taste.
Moraiolo – Central Italy. Sensory profile: medium olive fruity intensity, with scents of grass, almond and artichoke; medium intensity of pungency and bitter taste.
Peranzana – Puglia region. Sensory profile: medium olive fruity intensity, with scent of grass, artichoke, almond and tomato; medium intensity of pungency and bitter taste.
Raggia – Marche region. Sensory profile: medium olive fruity intensity, with strong green almond scent and light grass and artichoke flavor; well balanced to taste with medium intensity of pungency and bitter taste.
Ravece – Campania region. Sensory profile: medium-high olive fruity intensity, with grass, tomato and artichoke scent along with light almond scent; medium intensity of pungency and bitter taste.
The availability of this monovarietal oil database allows for a statistic elaboration of the data in order to meet different aims of the research into olive cultivation and olive oil quality. Some studies will be carried out considering the cultivars Frantoio and Leccino, which are widespread along the Italian peninsula, in order to evaluate the effect of the environment (climate, altitude and latitude) on the chemical and sensory profiles of monovarietal olive oils.
\n\t\t\tMoreover the quality and typicality of extra virgin olive oil are primarily determined by genetic, agronomical, environmental factors, and by technological parameters of oil processing [7,8,9]. Genetic matrix (cultivar) plays a key role in the chemical and sensory quality of the oil [10].
\n\t\t\tIt is important to underline that a smaller number of studies has considered the seasonal effect on the chemical and sensory profile of olive oil. The seasonality, which is deeply related to the different climate events of the crop year, may influence the ripening process of olives, thus affecting the oil composition and the resulting quality of olive oil. This thesis is supported by a study carried out by IBIMET-CNR and ASSAM on the evaluation of the influence of the cultivar and seasonality, as well as their interaction on monovarietal oil composition.
\n\t\t\tThe study was performed on 1108 monovarietal oils from the 16 most representative Italian cultivars.
\n\t\t\tNutritional properties, expressed as fatty acid and total phenols contents, and the sensory profiles were considered.
\n\t\t\tThe procedure was based on the analysis of variance (ANOVA) by a complete factorial design in order to examine treatment interdependencies (variety and crop year). A Principal Components Analysis (PCA) was also performed on chemical and sensory data separately, using mean values of each crop year of each cultivar collected.
\n\t\t\tFatty acids with the highest index of variability (heptadecenoic, linoleic, oleic, stearic and palmitic) were selected according to their p-level and F-values and submitted to PCA.
\n\t\t\t\n\t\t\t\tTable 1 reports mean values and comparison of mean separation analysis of the fatty acids belonging to the 1108 monovarietal olive oil samples. Regarding oleic acid, the contents of which as reported to EC Official Reg. EC Reg.702/2007 [11] range from 55 to 83%, the range of oleic acid of oils considered in this study varied from 71% to 77% showing their high nutritional level, some cultivars such as Coratina and Itrana are characterized by the production with the highest amount of acid oleic (above 77%).
\n\t\t\t\n\t\t\tConsidering organoleptic quality, Table 2 shows mean values and comparison of mean separation analysis of the sensory attributes of the monovarietal oils. All monovarietal oils have presented significant intensities of grass attributes with the highest levels of 3.2 noted in Ascolana Tenera and Biancolilla. Considering the peculiar attributes, which are heavily cultivar–dependent, such as fresh almond, artichoke, tomato, aromatic herbs and berries [12, 13], oils produced by Coratina, Frantoio, Leccino, Moraiolo and Piantone di Mogliano are distinguished for their high intensity of fresh almond, a typical pleasant flavour which characterized these cultivars.
\n\t\t\t\n\t\t\t\t\t\t | \n\t\t\t\t\t\tHeptadecenoic | \n\t\t\t\t\t\tLinoleic | \n\t\t\t\t\t\tOleic | \n\t\t\t\t\t\tPalmitic | \n\t\t\t\t\t\tStearic | \n\t\t\t\t\t\tTotal phenols | \n\t\t\t\t\t||||||
ASCOLANA T. | \n\t\t\t\t\t\t0.20 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t6.10 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t75.57 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t13.42 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t1.98 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t394 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t
BIANCHERA | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t5.98 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t76.26 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t12.70 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t2.52 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t646 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t
BIANCOLILLA | \n\t\t\t\t\t\t0.25 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t9.38 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t71.92 | \n\t\t\t\t\t\ti | \n\t\t\t\t\t\t13.69 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t2.22 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t327 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t
BOSANA | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t10.07 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t72.83 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t12.71 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t2.09 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t440 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t
CASALIVA | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t6.74 | \n\t\t\t\t\t\te | \n\t\t\t\t\t\t76.84 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t12.38 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t1.70 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t411 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t
CORATINA | \n\t\t\t\t\t\t0.08 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t7.07 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t77.74 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t11.24 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t1.80 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t588 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t
FRANTOIO | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t6.99 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t76.09 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t12.77 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t1.81 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t495 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t
ITRANA | \n\t\t\t\t\t\t0.09 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t6.17 | \n\t\t\t\t\t\tfgh | \n\t\t\t\t\t\t77.66 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t12.06 | \n\t\t\t\t\t\tg | \n\t\t\t\t\t\t1.80 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t329 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t
LECCINO | \n\t\t\t\t\t\t0.11 | \n\t\t\t\t\t\td | \n\t\t\t\t\t\t6.69 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t75.00 | \n\t\t\t\t\t\ted | \n\t\t\t\t\t\t14.01 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t1.74 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t414 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t
MIGNOLA | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t8.71 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t71.42 | \n\t\t\t\t\t\ti | \n\t\t\t\t\t\t14.63 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t1.84 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t503 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t
MORAIOLO | \n\t\t\t\t\t\t0.09 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t7.41 | \n\t\t\t\t\t\td | \n\t\t\t\t\t\t75.59 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t13.00 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t1.68 | \n\t\t\t\t\t\tg | \n\t\t\t\t\t\t504 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t
NOCELLARA B. | \n\t\t\t\t\t\t0.11 | \n\t\t\t\t\t\td | \n\t\t\t\t\t\t8.20 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t73.73 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t12.85 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t2.63 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t358 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t
PERANZANA | \n\t\t\t\t\t\t0.08 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t9.41 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t73.30 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t13.00 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t1.87 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t375 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t
PIANTONE M. | \n\t\t\t\t\t\t0.22 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t6.55 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t76.58 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t12.14 | \n\t\t\t\t\t\tg | \n\t\t\t\t\t\t1.97 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t395 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t
RAGGIA | \n\t\t\t\t\t\t0.10 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t7.49 | \n\t\t\t\t\t\td | \n\t\t\t\t\t\t74.45 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t13.40 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t1.89 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t414 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t
RAVECE | \n\t\t\t\t\t\t0.08 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t9.23 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t73.28 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t12.51 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t2.78 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t473 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t
Mean values and comparison of mean separation analysis (ANOVA) of the fatty acids relative to the 1108 monovarietal olive oil samples.
All monovarietal oils considered in this study presented a significant intensity of artichoke flavours.
\n\t\t\tBosana, Peranzana, Itrana and Ravece oils exhibited the highest intensity, while in Piantone di Mogliano and Leccino oils, slight artichoke attributes were noted. With regard to this last attribute, oils of Ravece, Ascolana Tenera and Itrana are distinguished also for their high intensity of tomato flavour. Berries flavour characterized the oil produced by the Mignola cultivar.
\n\t\t\tAll monovarietal extra virgin olive oil considered in this study were characterized by a significant level of bitterness showing a range from 3.9 to 5.3. In particular Piantone di Mogliano, and Biancolilla oils presented the lowest intensity of bitterness. It is interesting to underline that the same oils were also characterized by a lower total phenol content (see tab. 1). By contrast, the monovarietal oils of Coratina, Bianchera and Mignola which exhibited the highest intensity of bitterness, also showed the highest phenolic content.
\n\t\t\t\n\t\t\t\t\t\t | \n\t\t\t\t\t\tOlive fruity | \n\t\t\t\t\t\tGrass | \n\t\t\t\t\t\tFresh almond | \n\t\t\t\t\t\tArtichoke | \n\t\t\t\t\t\tTomato | \n\t\t\t\t\t|||||
ASCOLANA T. | \n\t\t\t\t\t\t5.9 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t3.2 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t0.9 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tbcde | \n\t\t\t\t\t\t2.7 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t
BIANCHERA | \n\t\t\t\t\t\t5.3 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t\t2.2 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t2.1 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t1.6 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\t\t1.0 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t
BIANCOLILLA | \n\t\t\t\t\t\t5.4 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t3.2 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tbcde | \n\t\t\t\t\t\t1.0 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t
BOSANA | \n\t\t\t\t\t\t5.3 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t2.6 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\t1.7 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t2.3 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t0.8 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t
CASALIVA | \n\t\t\t\t\t\t5.4 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t2.4 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t3.3 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t1.5 | \n\t\t\t\t\t\tdefg | \n\t\t\t\t\t\t0.2 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
CORATINA | \n\t\t\t\t\t\t5.1 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t1.9 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t2.5 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t\t1.7 | \n\t\t\t\t\t\tbcde | \n\t\t\t\t\t\t0.3 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
FRANTOIO | \n\t\t\t\t\t\t5.2 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t2.3 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t2.9 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t1.7 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\t\t0.3 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
ITRANA | \n\t\t\t\t\t\t5.7 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t3.1 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t1.3 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t2.2 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t2.3 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t
LECCINO | \n\t\t\t\t\t\t4.8 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tfgh | \n\t\t\t\t\t\t2.6 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\t1.1 | \n\t\t\t\t\t\tg | \n\t\t\t\t\t\t0.2 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
MIGNOLA | \n\t\t\t\t\t\t5.0 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t1.4 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t1.2 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t0.6 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
MORAIOLO | \n\t\t\t\t\t\t5.2 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t2.3 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t2.4 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t2.0 | \n\t\t\t\t\t\tabcd | \n\t\t\t\t\t\t0.4 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t
NOCELLARA B. | \n\t\t\t\t\t\t5.6 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t2.9 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t1.3 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tbcde | \n\t\t\t\t\t\t2.5 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t
PERANZANA | \n\t\t\t\t\t\t5.2 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t2.8 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t2.3 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t1.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t
PIANTONE M. | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\th | \n\t\t\t\t\t\t1.7 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t2.2 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t\t1.2 | \n\t\t\t\t\t\tfg | \n\t\t\t\t\t\t0.4 | \n\t\t\t\t\t\tde | \n\t\t\t\t\t
RAGGIA | \n\t\t\t\t\t\t4.8 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t1.7 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t3.0 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t1.4 | \n\t\t\t\t\t\tefg | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\te | \n\t\t\t\t\t
RAVECE | \n\t\t\t\t\t\t5.7 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t2.8 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t1.3 | \n\t\t\t\t\t\tgh | \n\t\t\t\t\t\t2.1 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t2.5 | \n\t\t\t\t\t\tAb | \n\t\t\t\t\t
\n\t\t\t\t\t\t | Berries | \n\t\t\t\t\t\tAromatic herbs | \n\t\t\t\t\t\tBitter | \n\t\t\t\t\t\tPungent | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t | ||||
ASCOLANA T. | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.4 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tcd | \n\t\t\t\t\t\t5.0 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
BIANCHERA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t5.1 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t5.2 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
BIANCOLILLA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.0 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.5 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
BOSANA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.8 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
CASALIVA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.3 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\t4.5 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
CORATINA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t5.3 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t5.0 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
FRANTOIO | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
ITRANA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.3 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t4.2 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.2 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
LECCINO | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.3 | \n\t\t\t\t\t\tef | \n\t\t\t\t\t\t4.4 | \n\t\t\t\t\t\tdef | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
MIGNOLA | \n\t\t\t\t\t\t1.8 | \n\t\t\t\t\t\ta | \n\t\t\t\t\t\t0.3 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t5.1 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
MORAIOLO | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t5.0 | \n\t\t\t\t\t\tabc | \n\t\t\t\t\t\t4.8 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
NOCELLARA B. | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.2 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t4.1 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.5 | \n\t\t\t\t\t\tcde | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
PERANZANA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.2 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.1 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
PIANTONE M. | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t3.9 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.4 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
RAGGIA | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.1 | \n\t\t\t\t\t\tc | \n\t\t\t\t\t\t4.1 | \n\t\t\t\t\t\tf | \n\t\t\t\t\t\t4.5 | \n\t\t\t\t\t\tcdef | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
RAVECE | \n\t\t\t\t\t\t0.0 | \n\t\t\t\t\t\tb | \n\t\t\t\t\t\t0.2 | \n\t\t\t\t\t\tbc | \n\t\t\t\t\t\t4.7 | \n\t\t\t\t\t\tbcd | \n\t\t\t\t\t\t4.9 | \n\t\t\t\t\t\tab | \n\t\t\t\t\t\n\t\t\t\t\t | \n\t\t\t\t\t |
Mean values and comparison of mean separation analysis (ANOVA) of the sensory attributes relative to the 1108 monovarietal olive oil samples.
Regarding the influence of the genetic matrix and the crop year in Table 3 we see that in all fatty acids analysed, the effects of the cultivar and crop year are highly significant. The effect of the interaction between the two factors is also highly significant on the content of fatty acid, with the exception of palmitoleic, heptadecenoic and heptadecanoic acids. Also the total phenolic contents are heavily influenced by both the cultivar and the year, as well as the interaction between the two factors. It is interesting to underline that both factors (cultivar and crop year) have similarly significant influence on the contents of the most important fatty acids as linoleic, linolenic, oleic, palmitic and palmitoleic.
\n\t\t\tFor oleic and palmitoleic the main factor was, however, the cultivar, in fact ANOVA procedure explains the 68.30% and 70.32% of its variation respectively. The cultivar did not represent a great source of variability for linolenic acid: only 7.88%, while the crop year shows a variation of 85.95%.
\n\t\t\t\n\t\t\t\t\t\tParametr | \n\t\t\t\t\t\tCultivar | \n\t\t\t\t\t\tCrop year | \n\t\t\t\t\t\tCultivar x crop year | \n\t\t\t\t\t
Eicosanoic | \n\t\t\t\t\t\t24.34 *** | \n\t\t\t\t\t\t10.49 *** | \n\t\t\t\t\t\t65.17 *** | \n\t\t\t\t\t
Eicosenoic | \n\t\t\t\t\t\t10.98 *** | \n\t\t\t\t\t\t80.78 *** | \n\t\t\t\t\t\t8.24 *** | \n\t\t\t\t\t
Heptadecanoic | \n\t\t\t\t\t\t56.17 *** | \n\t\t\t\t\t\t20.08 *** | \n\t\t\t\t\t\t23.75 * | \n\t\t\t\t\t
Heptadecenoic | \n\t\t\t\t\t\t82.95 *** | \n\t\t\t\t\t\t8.11 *** | \n\t\t\t\t\t\t8.94 ns | \n\t\t\t\t\t
Linoleic | \n\t\t\t\t\t\t69.61 *** | \n\t\t\t\t\t\t20.07 *** | \n\t\t\t\t\t\t10.32 *** | \n\t\t\t\t\t
Linolenic | \n\t\t\t\t\t\t7.88 *** | \n\t\t\t\t\t\t85.95 *** | \n\t\t\t\t\t\t6.17 *** | \n\t\t\t\t\t
Oleic | \n\t\t\t\t\t\t68.30 *** | \n\t\t\t\t\t\t17.58 *** | \n\t\t\t\t\t\t14.12 *** | \n\t\t\t\t\t
Palmitic | \n\t\t\t\t\t\t55.37 *** | \n\t\t\t\t\t\t27.72 *** | \n\t\t\t\t\t\t16.91 *** | \n\t\t\t\t\t
Palmitoleic | \n\t\t\t\t\t\t70.32 *** | \n\t\t\t\t\t\t9.44 *** | \n\t\t\t\t\t\t20.24 * | \n\t\t\t\t\t
Stearic | \n\t\t\t\t\t\t52.43 *** | \n\t\t\t\t\t\t39.64 *** | \n\t\t\t\t\t\t7.93 *** | \n\t\t\t\t\t
Total phenols | \n\t\t\t\t\t\t51.32 *** | \n\t\t\t\t\t\t26.64 *** | \n\t\t\t\t\t\t22.04 *** | \n\t\t\t\t\t
Variability expressed as percent of the Total Sum of the Squares for fatty acid composition and total phenols. *, **, *** Significant F-values the 0.05 (*), 0.01 (**) or 0.001 (***) level, respectively; ns = nonsignificant.
The sensory profiles of the 1108 oil samples were submitted to the ANOVA procedure by a complete factorial design. The effect of the cultivar factor is highly significant on the sensory attributes. Olive fruity, grass, fresh almond, tomato and berries were strongly influenced by the cultivar. For these attributes the variability, expressed as percentage of the total sum of the squares, the cultivar factor is characterized by a range from 71.77% to 90.75%. In general the crop year factor for all the sensory attributes remains limited and the interaction with the cultivar is not significant with the exception of berries, bitter and pungent (table 4).
\n\t\t\t\n\t\t\t\t\t\tParametr | \n\t\t\t\t\t\tCultivar | \n\t\t\t\t\t\tCrop year | \n\t\t\t\t\t\tCultivar x crop year | \n\t\t\t\t\t
Olive fruity | \n\t\t\t\t\t\t71.77 *** | \n\t\t\t\t\t\t5.93 *** | \n\t\t\t\t\t\t22.30 ns | \n\t\t\t\t\t
Grass | \n\t\t\t\t\t\t75.92 *** | \n\t\t\t\t\t\t2.54 ns | \n\t\t\t\t\t\t21.54 ns | \n\t\t\t\t\t
Fresh almond | \n\t\t\t\t\t\t73.59 *** | \n\t\t\t\t\t\t9.81 *** | \n\t\t\t\t\t\t16.60 ns | \n\t\t\t\t\t
Artichoke | \n\t\t\t\t\t\t56.96 ** | \n\t\t\t\t\t\t15.38 *** | \n\t\t\t\t\t\t27.66 ns | \n\t\t\t\t\t
Tomato | \n\t\t\t\t\t\t90.75 *** | \n\t\t\t\t\t\t1.48 * | \n\t\t\t\t\t\t7.77 ns | \n\t\t\t\t\t
Berries | \n\t\t\t\t\t\t75.71 *** | \n\t\t\t\t\t\t0.99 ** | \n\t\t\t\t\t\t23.30 *** | \n\t\t\t\t\t
Aromatic herbs | \n\t\t\t\t\t\t26.86 ** | \n\t\t\t\t\t\t10.17 * | \n\t\t\t\t\t\t62.97 ns | \n\t\t\t\t\t
Bitter | \n\t\t\t\t\t\t49.02 *** | \n\t\t\t\t\t\t17.04 *** | \n\t\t\t\t\t\t33.94 *** | \n\t\t\t\t\t
Pungent | \n\t\t\t\t\t\t26.35 *** | \n\t\t\t\t\t\t42.63 *** | \n\t\t\t\t\t\t31.02 *** | \n\t\t\t\t\t
Variability expressed as percent of the Total Sum of the Squares for sensory attributes. *, **, *** Significant F-values the 0.05 (*), 0.01 (**) or 0.001 (***) level, respectively; ns =not significant.
The decision to carry out a study of a large number of labelled commercial extra virgin olive oils was taken in order to provide the consumer with information about the chemical and sensory properties of extra virgin olive oils which are currently available on the Italian market. The commercial potential of the monovarietal oils can be exploited either in terms of purity, relying on the specific characteristics of the single cultivar, or mixing the monovarietal oils from each cultivars as a "blend" based on the different typologies of Italian olive oil.
\n\t\t\tFor this purpose these oil typologies were assessed by clustering the collected olive oil data according to different sensory profiles. Descriptive analysis and hierarchical cluster analysis of sensory characters were performed. Monovarietal oils were clustered in six different sensory typologies emphasising the variability and the depth of aromas characterising Italian Monovarietal oils.
\n\t\t\tSuch classification of monovarietal oils typologies may help the consumer in making an informed choice, and in matching more easily with the wide range of flavours found in Italian cuisine.
\n\t\t\t\n\t\t\tThese monovarietal oils were classified as belonging to typology 1:
\n\t\t\tCaninese, Carboncella, Carpellese, Cornetta, Dolce Agogia, Dolce di Rossano, Dritta, Gentile di Chieti, Gentile di Larino, Leccino, Limoncella, Nebbio, Ogliarola, Ogliarola del Bradano, Paesana Bianca, Piantone di Mogliano, Raggia, Raggiola, Rajo, Razzola, Rosciola, Salviana, Sargano di Fermo, Taggiasca.
\n\t\t\t\n\t\t\t\n\t\t\tTypology 1 Sensory profile: medium olive fruity intensity, with prevalent almond scent and light notes of grass/leaf and artichoke; pungent and bitter taste of medium-light intensity.
These monovarietal oils were classified as belonging to typology 2:
\n\t\t\tCasaliva, Coratina, Correggiolo, Frantoio, Moraiolo, Ogliarola Garganica, Oliva Nera di Colletorto, Olivastra Seggianese, Pendolino, Raggiolo, Razzo, San Felice, Sargano di Ascoli.
\n\t\t\tTypology 2 : Sensory profile: medium olive fruity intensity, with prevalent almond scent and light notes of grass/leaf and artichoke; pungent and bitter taste of medium intensity.
Typology 3 Sensory profile: medium olive fruity intensity, with peculiar soft fruits scent; pungency and bitter taste of medium intensity.
These monovarietal oils were classified as belonging to typology 3: Cellina di Nardò, Mignola, Ogliarola Salentina
\n\t\t\t\n\t\t\tThese monovarietal oils were classified as belonging to typology 4: Biancolilla, Bosana, Carolea, Coroncina, I77, Majatica di Ferrandina, Maurino, Orbetana, Peranzana, Prempesa, Salella, Semidana, Tonda del Matese.
\n\t\t\t\n\t\t\tTypology 4 Sensory profile: medium olive fruity intensity, with scent of grass, artichoke fresh almond and tomato; pungency and bitter taste of medium light intensity.
Typology 5 Sensory profile: medium high olive fruity with grassy notes, tomato and artichoke scent and light flavour of fresh almond; pungency and bitter taste of medium intensity.
These monovarietal oils were classified as belonging to typology 5. Ascolana Tenera, Cerasuola, Ghiacciolo, Itrana, Nera di Oliena, Nocellara del Belice, Nocellara Etnea, Nocellara Messinese, Ortice, Ravece, Tonda Iblea
\n\t\t\tTypology 6 Sensory profile: medium high olive fruity with grass/leaf and artichoke notes, light flavor of fresh almond and tomato, medium intensity of bitter and pungency taste.
These monovarietal oils were classified as belonging to typology 6: Bianchera, FS17, Intosso, Lantesca, Leccio del Corno, Nostrana di Brisighella, Piantone di Falerone, Picholene
\n\t\t\n\t\tAt a national level the varietal biodiversity culture is being promoted ever more heavily, resulting in increasing diversification of production of extra virgin olive oil which constitutes the necessary basis for creating blends and PDOs which appeal to consumers.
\n\t\t\tVarious Italian regions are conducting research into the promotion of the genetic heritage of the olive cultures, drawing on social and cultural elements of olive culture. For a strong and healthy olive culture, the cultivation process should not only fulfil the demands of intensification and optimization of production, but also balance this with respect for the ancient traditions and heritage- traditions and heritage which we see throughout Italy in the form of monumental trees, archaeological exhibits, ancient tools and gastronomic traditions which have extra virgin olive oil at their very heart.
\n\t\t\tDevelopment of olive production with care being taken to respect biodiversity and cultural traditions- and, as a consequence, the different autochthonous genotypes- is key to ensuring sustainable and environmentally friendly olive production processes.
\n\t\t\tThe will to proceed with recovery and exploitation of Italian germplasm, encourages the development of marginal areas, but also allows for the protection of biodiversity and ecological systems in specific areas where the olive tree plays an important role for buoyancy and hydro-geological protection for the characterization of the landscape.
\n\t\t\tThe exploitation of monovarietal oil results in the propagation of many native Italian varieties involving research institutes, University and nurseries called upon to halt the erosion of genetic heritage of Italian olive.
\n\t\t\tThe unique Italian monovarietal heritage plays a key role in the "diversification" culture that should guide Italian production in order to avoid the standardization of products recently observed in the GDO market. The promotion of the "diversification" culture has to be reached both by increasing the cultivated land – and also discouraging the substitution of pre-existing cultivar or the implantation of new "universal" ones with the sole aim of greater production - and by the reinforcement of the elements that characterize GDO territoriality: organoleptic and sensorial diversification.
\n\t\t\tThe quality oil market is expanding and Italy is still the reference point at an international level. The operators of the production chain and national institutions have the task of developing appropriate strategies to strengthen the position of production, sales and marketing.
\n\t\t\tAs has happened in the case of wine, the varieties typical to these regions may become a symbol of high quality product and find a better place in the market.
\n\t\t\tThis approach can be a first step toward traceability and authenticity of these particular productions in order to protect the interest of both consumer and producer.
\n\t\t\tThe authors are aware of the numerous variables: mill typology, olive ripening index and agronomic practice, which influence the overall olive oil quality. These variables are not usually known for commercial oils.
\n\t\t\tKnowledge of the chemical and sensory profiles of the Italian monovarietal olive oils could potentially start a certification process for these oils, thus leading to greater guarantees of origin and consequently greater guarantees of quality for the consumer.
\n\t\tExtracellular vesicles (EVs) are a collective term for tiny vesicles with a phospholipid bilayer structure that are actively secreted by cells. Almost all known cell types can be secreted. The two main categories of EVs are exosomes and microvesicles (Table 1). Exosomes (30-150 nm in diameter) are intraluminal vesicles, formed by the invagination of the multivesicular endosome membrane, and are released into the extracellular space after the multivesicular endosomes fuse with the cell membrane [1]. Microvesicles (50–1,000 nm in diameter) are a group of highly heterogeneous EVs characterized in that their origin and secretion are budding through the plasma membrane [1]. Considering the complexity of identifying its biogenesis, the size of the vesicle is the most widely used parameter for classifying EV types, and on this basis they are described as small EVs or medium and large EVs. In this article, unless otherwise specified, the term "EVs" generally refers to small EVs.
Vesicle | Size (nm) | Density (g/mL) | Origin | Markers |
---|---|---|---|---|
Exosomes | 30-150 | 1.13-1.18 | Endosomes | Tetraspanins, Alix, TSG101 |
Microvesicles | 50-1000 | 1.16-1.19 | Plasma membrane | Intergrins, Selectins, CD40 |
In recent years, people’s understanding of the biogenesis, composition, function and mechanism of EVs has continued to deepen [3, 4, 5]. Their application in clinical treatment has also attracted more and more attention. One of the most useful properties of EVs is their ability to cross barriers, such as the plasma membrane and blood/brain barrier. This makes them very suitable for delivering therapeutic molecules. With their natural source material transport properties, inherent long-term blood circulation capabilities and excellent biocompatibility, EVs can deliver a variety of chemical drugs, proteins, nucleic acids, gene drugs and other drugs. They have great potential in the field of drug carriers. CD47 is the ligand for signal regulatory protein alpha (SIRPα), and CD47-SIRPα binding initiates the ‘don’t eat me’ signal that inhibits phagocytosis. Therefore, CD47 on EVs prevents them from being engulfed by immune cells [6]. EVs are more efficient than their synthetic analog liposomes. The application of EVs as drug delivery carriers is like putting a "stealth coat" on the drug molecules, which can maximize the stability of the drugs, reduce the immune system’s clearance of them, and make "precise delivery" possible. Therefore, EVs can be described as "stealth transport aircrafts" for drugs. EVs therapy has shown great application prospects from oncology to regenerative medicine.
A number of studies have shown that EVs derived from mesenchymal stem cells (MSCs) can be used for stem cell replacement therapy [7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]. In most cases, it is not clear which component of the unmodified EVs exerts curative effects. The researchers’ operations are only the separation and purification of EVs produced by therapeutic cells. The curative effects are based on the biological functions of the donor cells, such as the regulation of the immune environment, the repair of damaged cells and the promotion of angiogenesis.
At present, the most extensive research is the attempt to use stem cell-derived EVs for disease treatment. The main application ranges are to repair and regenerate tissues and organs. Such researches involve central nervous system diseases [7, 8], cardiovascular diseases [9, 10, 11, 12] and other organ damage repair and regeneration [13, 14, 15, 16, 17, 18, 19, 20, 21].
In the treatment of central nervous system disease, there is a blood-brain barrier, which often results in that drugs can not reach the diseased site and work well. Stem cells have been gradually used in the treatment of central nervous system diseases in recent years. A large number of research results have been obtained [22, 23]. However, there are still potential risks faced by direct stem cell transplantation, such as tumorigenicity, infection, transplant failure, graft versus host disease, hemorrhagic cystitis, and long-term complications [24].
The application of stem cell EVs avoids a variety of potential risks of direct stem cell transplantation. EVs have low immunogenicity and are easy to preserve and transport, showing unique advantages as a "cell-free stem cell therapy technology". Spinal cord injury (SCI) is one of the deadliest diseases. The complex inhibitory microenvironment needs to be fully mitigated. EVs derived from MSCs have the function of microenvironmental regulation. Studies have established innovative implantation strategies using human MSC-derived EVs immobilized in peptide-modified adhesive hydrogels (Exo-pGel) [7]. Exo-pGel plays an important role in nerve recovery and urinary tissue protection by effectively reducing inflammation and oxidation [7]. In addition, small extracellular vesiclesderived from embryonic stem cells (ESC-sEVs) can significantly reduce the time-related aging of hippocampal neural stem cells (H-NSCs) through intravenous injection into vascular dementia (VD) rats. ESC-sEVs can restore the damaged proliferation and neuronal differentiation ability, and reverse cognitive impairment. The application of ESC-sEVs may be a new cell-free treatment tool for VD and other diseases related to aging [8].
Stem cells can be induced to differentiate into cardiomyocytes. Early studies believed that the transplanted stem cells can differentiate into heart cells and necrotic cells in the body to repair damaged myocardium and maintain heart function [25]. At present, a large number of preclinical studies have found that EVs derived from transplanted stem cells also have the function of myocardial repair [26, 27]. EVs mainly promote myocardial regeneration by activating cardiac precursor cells, promoting the survival and proliferation of cardiomyocytes, inhibiting their apoptosis, promoting cardiac angiogenesis, reducing infarct size and tissue fibrosis, and regulating inflammation. Extracellular vesicles secreted by cardiovascular precursor cells (hCVPC-EVs) derived from human pluripotent stem cells (hPSCs) play a role in protecting the heart in myocardial infarction by improving cardiomyocyte survival and angiogenesis [9]. Mouse ESC-derived EVs promote angiogenesis, cardiomyocyte survival and proliferation, reduce cardiac fibrosis, and improve cardiac function by carrying miR-294-3p [10]. IPSC-derived EVs inhibit cardiomyocyte apoptosis through miR-21 and miR-210 loaded, and also have a cardioprotective effect [11]. Exosomes produced by immature bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a. They can reduce the necrotic lesions of atherosclerosis [12].
With the continuous discovery of the repair and regeneration effects of stem cell EVs in brain tissue and cardiovascular tissues and organs, the application of stem cell EVs in the repair and regeneration of other tissues has also made a lot of progresses.
MSC-derived EVs reduce radiation-induced lung injury through miRNA-214-3p [13]. Replacing autologous cells with EVs derived from hair follicle papillary cell spheres can promote hair growth [14]. Human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exo) can inhibit pyrolysis and repair muscle ischemic injury by releasing circular RNA circHIPK3 [15]. Hertwig’s EVs derived from epithelial root sheath cells promote the regeneration of dentin plasma tissue [16]. Exosomes from neural progenitor cells retain photoreceptor cells during retinal degeneration (RD) by inactivating microglia. This suggests that NPC-exos and its contents may be the mechanism of stem cell therapy to treat RD [17].
Aging is the process of cell and tissue dysfunction. Small extracellular vesicles (sEVs) isolated from primary fibroblasts from young human donors can improve certain biomarkers of cellular senescence from elderly and Hutchinson-Gilford progeria donors. Studies have shown that sEVs have GST activity to improve aging-related tissue damage [18]. In obesity diseases, EVs derived from adipocytes, as new adipokines, are related to the body’s metabolic homeostasis. EVs released from brown adipose tissue or adipose stem cells can help control the remodeling of white adipose tissue, making it brown and maintaining metabolic homeostasis. EVs have been considered as new regulators of diseases such as insulin resistance, diabetes and non-alcoholic fatty liver. The results provide new treatment strategies for obesity and metabolic diseases [19].
In addition, some reports suggest that some EVs derived from mesenchymal stem cells contain some tumor suppressor molecules. For example, it has been reported that miR-206 in exosomes derived from bone marrow mesenchymal stem cells could inhibit the progression of osteosarcoma by targeting TRA2B [20]. The exosomes derived from human umbilical cord mesenchymal stem cells deliver miRNA-375 to delay the progression of esophageal squamous cell carcinoma [21]. However, although EVs contain these small RNAs that have been reported to exert anti-cancer effects, they also contain a large number of growth factors and pro-angiogenesis factors. When these substances are transported to tumor cells by EVs, can EVs derived from MSCs still exert a tumor suppressor effect? This needs more research to prove.
At present, cell replacement therapy based on the characteristics of donor cells has been studied earlier and more frequently in the field of EVs. There is also a clearer understanding of the components that play a major role. With the continuous increase of clinical needs, people began to try to modify the surfaces and contents of EVs to adapt to more disease treatments.
Although natural EVs have been used for cell replacement therapy based on their source and achieved good results, their therapeutic range is far from meeting the current treatment needs. One of the most important therapeutic areas is the treatment of malignant tumors. The secretion ability of EVs in malignant tumor itself is enhanced and contributes to tumor progression. Considering that MSC-derived EVs generally contain high levels of growth factors and pro-angiogenic factors, most natural EVs are not suitable for tumor therapy, except that EVs derived from antigen-presenting cells can be used as tumor vaccines to activate anti-tumor immune responses [28]. Based on the biological characteristics of EVs, it has become the focus of researchers and biopharmaceutical companies to transform EVs as carriers of multiple drugs.
Most diseases have characteristic down-regulation of small RNA expression. Small RNA is the main content of extracellular vesicles, the most abundant and the most easily carried component. Therefore, EVs can be used to carry and deliver small RNA and other gene therapy systems. This section will discuss the progress of engineered EVs to deliver nucleic acid drugs and the strategies of drug loading and targeting.
There are three main problems in the development of nucleic acid drugs: the instability of nucleic acid molecules in the body, potential side effects and difficulties in drug delivery systems. The most important one is the development of delivery systems. Because a good drug delivery system can improve drug stability and target cell absorption efficiency, and can reduce its side effects. At present, the commonly used delivery vehicles in the field of nucleic acid drugs are mainly adeno-associated virus (AAV) and liposomal nanoparticles (LNPs). A small number of companies also use lentivirus (LV) and exosomes as delivery vehicles.
The packaging capacity of AAV is small (≤5kb). AAV will be used more than once in patients for therapeutic purposes and the second use will cause the body to produce a strong immune response. The safety of LNPs is relatively high, and the carrier capacity and delivery efficiency can meet the current demand for drug carriers. However, the organ selectivity of LNPs is still relatively limited. The main delivery area is concentrated in the liver, and the delivery efficiency to other tissues and organs is relatively low.
EVs are now candidate carriers for nucleic acid drugs by virtue of their advantages. The red blood cell extracellular vesicles (RBCEVs) have a large loading capacity (≤11kb), can be loaded with many types (including DNA, mRNA, antisense oligonucleotides, siRNA and other nucleic acid types), and contain very little nucleic acid. The advantages make them high-quality natural blank nucleic acid carriers. RBCEVs can be delivered to many different organs and tissues. In mouse experiments, the delivery effects of lung, liver, kidney, bone tissue, immune cells, etc. are all obvious [29]. Moreover, the raw materials used to produce RBCEVs are mainly blood from type O blood donors. This means large quantities of raw materials are readily available, and costs are controllable. Carmine Therapeutics focuses on the research and development of nucleic acid delivery technology using RBCEVs as carriers.
In addition, researchers are also committed to modifying the surfaces of EVs to improve their targeting. Many results show that this strategy can indeed improve the therapeutic effect of engineered EVs [30, 31, 32, 33].
The researchers combined ligand-coupled superparamagnetic nanoparticles with specific membrane proteins of blood exosomes to achieve the separation, purification and tumor targeting of exosomes [30]. The chemotherapy drug doxorubicin (Dox) and the cholesterol-modified single-stranded miRNA-21 inhibitor (chol-miR21i) were co-loaded onto the exosomes. Then the cationic endolysin peptide was absorbed on the negatively charged membrane surface of exosomes to promote the cytoplasmic release of the packaged cargo (Figure 1). The research results showed that these effectively released drugs and RNA simultaneously interfered with nuclear DNA activity and down-regulated the expression of oncogenes, thereby significantly inhibiting tumor growth and reducing side effects [30].
Schematic representation of engineered blood exosomes for effective gene/chemo combined antitumor therapy [30].
Chimeric antigen receptors (CAR) are cell surface receptors that recognize specific proteins (antigens). Tumor cells express their specific antigens. Modification of EVs surfaces with CAR targeting tumor antigens enables EVs to target tumors for drug delivery. Modified EVs with CAR can serve as a biosafety delivery platform for the CRISPR/Cas9 system to improve their tumor selectivity. Compared with unmodified EVs, CAR-EVs accumulate rapidly in tumors and effectively release the CRISPR/Cas9 system targeting MYC oncogenes in vitro and in vivo [31].
Rabies virus glycoprotein (RVG) is neurogenic. At present, it has become the most active guide molecule for brain targeted drugs. Lysosomal-associated membrane glycoprotein 2b (Lamp2b) is the membrane surface protein of EVs. RVG fused with Lamp2b is located on the surface of the EV to achieve brain targeting. Engineered Lamp2b-RVG-circSCMH1-extracellular vesicles (Lamp2b-RVG-circSCMH1-EVs) can selectively deliver circSCMH1 to the brain. The treatment can improve the functional recovery of mice and monkeys after stroke [32].
In addition, EVs without modification for targeting have also shown certain curative effects. The miR-214 inhibitor was transfected into HEK293T cells. Their exosomes Exo-anti-214 can reverse the resistance of gastric cancer to DDP [33]. HEK293T cells were transfected with HGF siRNA and their exosomes were harvested. In vivo and in vitro experiments have shown that exosomes loaded with HGF siRNA can inhibit the proliferation and migration of cancer cells and vascular cells [33].
Methods of loading nucleic acids into EVs include: chemical reagent transfection, electroporation transfection, transfection of donor cells, protein and characteristic sequence targeting methods. The application scope and advantages and disadvantages of different methods are shown in Table 2.
Methods | Application scope | Merit and demerit | References |
---|---|---|---|
Chemical reagent transfection | Broad-spectrum. | Easy to operate, but EVs should be purified before and after transfection. | [34] |
Electroporation transfection | The most commonly used method, but not for miRNA, shRNA, mRNA containing chemical modification. | Easy to operate, but EVs should be purified before and after transfection. | [35] |
Transfection of donor cells | Broad spectrum, but not for biotoxic molecules. | Purify EVs after transfection, but the effect of the transfected molecule on the donor cell should be taken into account (e.g. biotoxicity). | [33, 36, 37] |
Protein and characteristic sequence targeting | mRNA and miRNA. | High specificity of loading, but the therapeutic molecules will be modified. Whether this will affect the efficacy remains to be determined. | [38, 39] |
Methods of loading nucleic acid drugs into engineered EVs.
The use of proteins that can bind to specific RNA sequences (Figure 2) or the conservative sequences of Exosome-enriched RNAs (eRNAs) to achieve active packaging is a promising direction. The researchers used the specificity of protein binding to the RNA sequence to develop EXOtic devices for mRNA delivery [38]. Archaeal ribosomal protein L7Ae specifically binds to the C/Dbox RNA structure [40, 41, 42]. Based on this, L7Ae was conjugated to the C-terminus of CD63. C/D box was inserted into the 3′-untranslated region (3′-UTR) of the reporter gene. Therefore, the mRNA encoding the reporter protein could be well incorporated into exosomes via the interaction between L7Ae and the C/D box in the 3′-UTR. Exosomes containing the RNA packaging device (CD63-L7Ae), targeting module (RVG-Lamp2b to target CHRNA7), cytosolic delivery helper (Cx43 S368A) and mRNA (nluc-C/Dbox) were efficiently produced from exosome producer cells by the exosome production booster. The engineered exosomes were delivered to target cells and the mRNA was delivered into the target cell cytosol with the help of the cytosolic delivery helper. Finally, protein encoded in the mRNA was expressed in the target cells [38] (Figure 2). In the future, researchers need to obtain more specific RNA sequence binding proteins and conserved sequences of eRNAs through bioinformatics analysis.
EXOtic devices for mRNA delivery. A schematic illustration of the EXOtic devices [38].
The lack of protein and malfunction are important causes of many diseases. For example, the occurrence of malignant tumors is related to the lack of certain tumor suppressor factors and malfunctions. Therefore, increasing the corresponding protein level is one of the ways to treat diseases. Considering the risk of genome changes, researchers aim to deliver therapeutic protein molecules to the lesion through effective drug delivery vehicles. This section will introduce the use of EVs to transport protein molecules for the prevention and treatment of tumors, immune diseases, cardiovascular diseases, atherosclerosis, myocardial infarction and other diseases.
Compared with the previous small molecule compound drugs, protein drugs have the characteristics of high activity, strong specificity, low toxicity, clear biological functions, and are beneficial to clinical application. However, protein drugs are unstable in the internal and external environments, and may undergo a variety of complex chemical degradation and physical changes, such as aggregation, precipitation, racemization, hydrolysis, and deamidation. Protein drugs have short half-life, high clearance rate, large molecular weight, poor permeability, susceptibility to the destruction of enzymes, bacteria and body fluids in the receptor, and low bioavailability of non-injection administration. These problems greatly limit the use of protein drugs. Although researchers have improved the stability and absorption efficiency of protein drugs through methods such as PEG modification, microsphere sustained release, and liposome embedding, they still look forward to the emergence of better drug carriers. The application of EVs has brought dawn to this field.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. Delivery of TRAIL through EVs can efficiently induce cancer cell apoptosis. When combined with dinaciclib, they inhibit the growth of drug-resistant tumors [43]. Immunosuppressive drugs must be taken after organ transplantation, but long-term use of these drugs increases the risk of infection and other serious diseases. Using bioengineering methods, researchers prepared exosome-like nanovesicles (NV) displaying the dual target cargo of PD-L1/CTLA-4. These NVs enhanced the PD-L1/PD-1 and CTLA-4/CD80 immunosuppressive pathways and could be used as prospective immunosuppressive agents in organ transplantation [44]. Using extracellular nanovesicles to package CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping can avoid off-target mutagenesis and immunogenicity. And this method can achieve effective genome editing in a variety of cell types that are difficult to transfect, including human induced pluripotent stem cells (iPS), neurons and myoblasts [45]. Catalase could be loaded into exosomes by incubating at room temperature, saponins penetrating the membrane, repeated freezing and thawing and mechanical extrusion for the treatment of Parkinson’s disease (PD) [46].
Surface modification of EVs carrying protein drugs can greatly improve their targeting. In the study of stroke, nerve growth factor (NGF) exerts various neuroprotective functions after ischemia. NGF was loaded into EVs with RVG peptide modification on the surface. Through systemic administration, NGF was effectively delivered to the ischemic cortex. The delivered NGF reduced inflammation by remodeling microglia polarization, promoted cell survival, and increased the number of neuroblast marker doublecortin-positive cells. The results of the study indicated the potential therapeutic effect of NGF@Exo (RVG) on stroke [47]. In addition, integrin αVβ5 exhibits tropism for the liver while integrin α6Vβ4 and integrin α6β1 target lung [48, 49]. The iRGD specifically recognizes αV integrins on the surface of tumor cells [50]. RVG and c(RGDyK) peptides target brain tissue [51]. Klotho protein has the property of binding to circulating endothelial progenitor cells (EPCs) [52]. And chimeric antigen receptor (CAR) targeting specific tumor antigens and so on. These guiding molecules are utilized either by fusion with EVs membrane surface proteins (such as Lamp2b, VSVG, CD63, and other transmembrane proteins, etc.), or by chemical cross-linking on the surface of EVs to achieve the EVs targeting modification. Liu et al. summarized the surface modification strategies to improve the targeting of EVs (Figure 3) [53]. In addition, EVs derived from antigen-presenting cells with tumor antigens can be used as tumor vaccines to activate anti-tumor immune responses.
Design strategies for therapeutic exosome targeting [53].
How to load protein drugs into EVs? There are currently the following strategies:
Transfect donor cells with plasmids carrying the gene of interest. The cell will synthesize the target protein. These proteins are then secreted into EVs through a natural packaging process. Subsequent separation and purification of EVs in the cell culture supernatant is sufficient [54]. Although this method seems simple and easy to implement, cytotoxicity, mixed interactions and impaired biological responses will provide great obstacles to the production of EVs. And the loading efficiency of the target protein is relatively low. Therefore, researchers have carried out various attempts to specifically load target proteins into EVs. For example, the fusion of therapeutic proteins with the constituent proteins of EVs and the specific modification of therapeutic proteins.
The therapeutic proteins are fused with the constituent proteins of EVs. Then they will be distributed into EVs mediated by the constituent proteins. This method can improve the specificity of protein loading into EVs. The fused constituent proteins of EVs that have been tried include: CD63, Nef [55], vesicular stomatitis virus glycoprotein (VSVG) [56], apolipoprotein E (ApoE) [57], lysosome-associated membrane glycoprotein 2 (LAMP2B) [58], etc.
In addition, based on the idea of fusion proteins, researchers have developed a conditional loading method called "exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs)" [59]. The principle is to couple the exosomal membrane protein CD9 with CIBN, and CRY 2 with the therapeutic protein. After light excitation, CIBN and CRY2 interact, and the therapeutic protein can be loaded into EVs through "photoreversible protein-protein interaction" [59].
All in all, the fusion expression of therapeutic proteins with the constituent proteins of EVs can indeed increase the enrichment level of therapeutic proteins in EVs. However, whether the fusion protein affects the uptake and function of the therapeutic protein by the recipient cells needs to be verified. Therefore, exploring the fusion of peptides that can play a sorting role with therapeutic proteins and minimize the impact on protein functions will become one of the research hotspots in the field of engineered EVs.
Currently, known protein modifications that can target therapeutic proteins into EVs mainly include two types. One is ubiquitination modification. The fusion of ubiquitin to the C-terminus of therapeutic protein can make the concentration of the fused therapeutic protein in EVs increased by nearly 10 times [60]. The other is to fuse the N-terminus of the therapeutic protein with a palmitoylated or myristoylated peptide, which can further increase the therapeutic protein in EVs [61]. However, it is still unknown whether the modification of proteins, especially ubiquitination, will cause the degradation of the therapeutic protein by the proteasome and affect its function in the recipient cell.
Expression of therapeutic protein in donor cells, combined with mechanical methods that pass through different pores, can produce small vesicles containing the therapeutic proteins [46, 62]. In addition, there are methods such as incubation at room temperature, permeabilization with saponin, freeze-thaw cycles and sonication, [46]. There are two main problems with engineered EVs obtained by mechanical methods. One is that the technical requirements for the separation and purification of EVs are relatively high. The second is the maintenance of the integrity and biological activity of EVs. The composition of EVs actively produced by cells is different from the composition of mechanically produced EVs. The difference may affect the efficacy of engineered EVs. In the future application research of EVs, these two problems need to be solved and proved urgently.
So, what are the possible development directions for the existing cytotoxicity and the interaction of biological functions? The expression of tumor suppressor protein molecules may cause cytotoxicity to donor cells, which is not conducive to the production of EVs. If an inducible expression system is established, the coding DNA containing the inducible promoter is introduced into the donor cell to make the donor cell produce EVs containing the coding DNA, which will avoid cytotoxicity to the donor cell. Then prepare EVs containing small molecules that induce DNA expression. The two types of EVs can be used in combination to express tumor suppressor molecules in target cells. It can play a therapeutic role without affecting the production efficiency of EVs. The dual targeting of the two EVs will greatly reduce the impact of engineered EVs on non-targeted tissues. Because single-component EVs are randomly engulfed by cells and will not affect the cells. This may become one of the follow-up development directions in this field.
Chemotherapeutics and traditional Chinese medicine ingredients with anticancer effects are often used in the clinical treatment of a variety of malignant tumors. However, their toxic, side effects and short half-life limit their efficacy. The packaging and transportation with EVs will improve the targeting of chemotherapeutic drugs, increase the uptake efficiency of tumor cells, promote drug stability, reduce the use concentration, and reduce toxic side effects on other organs and normal tissues [63].
The hydrophobic drug curcumin could be packaged into exosomes by direct mixing for tumor treatment [64]. Paclitaxel (PTX) was loaded into EVs secreted by macrophages by different methods such as room temperature incubation, electroporation and sonication. Studies have found that ultrasound treatment increases the load of EVs on drug molecules and the sustained release [65]. Small compounds can also be naturally secreted into EVs by incubating with donor cells. Incubation with paclitaxel make mesenchymal stromal cells produce microvesicles containing paclitaxel [66]. Injecting methotrexate-containing plasma membrane microvesicles (MTX-TMP) from apoptotic human tumor cells into the bile duct lumen of extrahepatic CCA patients could mobilize and activate neutrophils, and relieve the bile duct obstruction in 25% of patients, almost no adverse reactions [67].
At present, small molecule drugs are often loaded into EVs by passive loading methods, such as direct mixing, incubation, ultrasonic treatment, vortexing, saponin permeation, repeated freezing and thawing, and mechanical extrusion. The disadvantages of these methods have always existed, that is, the loss and quality reduction of EVs caused by multiple purifications. In addition, long-term in vitro processing and the physical and chemical properties of drug molecules will also affect the biological activity and stability of EVs. Therefore, before EVs can be widely used in treatment, the storage methods and stability factors of EVs are also worthy of research.
Why are EVs a “stealth cap” for drugs? Because we know viruses to use them exactly like this. In nature, viruses "hijack" EVs to secrete and infect other cells. This method helps to provide a "cover" for the virus to prevent the virus from being cleared by the immune system or neutralized by antibodies, such as the infection process of HAV, HBV and HCV.
In gene therapy, currently widely used adeno-associated virus (AAV), oncolytic adenovirus (OAV) and lentivirus (LV) mediated gene therapy can cause the body’s immune response. After the same kind of AAV is used once, the body will produce a strong immune response, making the second injection ineffective. If EVs encapsulate viral particles to mediate their delivery, perhaps the therapeutic effect will be better.
Studies have shown that AAV isolated from conditioned media could bind to exosomes (exo-AAV) [68]. Compared with conventional AAV, exo-AAV was more resistant to neutralizing antibodies. After systemic injection in mice, compared with conventional AAV, exo-AAV delivered genes to the brain more efficiently at low vector doses. Importantly, no cytotoxicity was found in exo-AAV transduced cells. This may make exo-AAV widely used as a neuroscience research tool [68]. Compared with non-targeted modified EV-AAV, the expression of brain-targeting peptides on the surfaces of EVs can significantly enhance transduction [69].
In gene therapy of ophthalmic diseases, transferring genes to the retina is challenging. Because it requires a carrier system to overcome physical and biochemical barriers to enter and spread throughout the retinal tissue. After the exo-AAV was injected into the vitreous cavity (IVT), it was found that the expression of exo-AAV was better than the traditional AAV. Exo-AAV exhibited a deeper penetration in the retina, effectively reaching the inner core and outer plexus, and to a lesser extent the outer nuclear layer. Exo-AAV is a reliable mouse retina gene delivery tool. Its simplicity of production and isolation makes it widely used in basic eye research [70].
Due to the low efficiency of gene delivery to the inner ear sensory hair cells. AAV is not so advanced in the field of gene therapy for hearing impairment. Studies have shown that Exo-AAV1-GFP is more effective than traditional AAV1-GFP, whether injected in mouse cochlear explants in vitro or directly injected into the cochlea in vivo. Exo-AAV was not toxic in the body. Exo-AAV1 gene therapy partially rescued the hearing in a mouse model of hereditary deafness. It was a powerful hair cell gene delivery system that could be used for gene therapy of deafness [71].
Oncolytic viruses show unique anti-cancer mechanisms in cancer treatment. Chemotherapeutic drugs combined with oncolytic viruses showed stronger cytotoxicity and oncolytic effects. Someone has studied the systemic delivery of oncolytic adenovirus and paclitaxel encapsulated by EVs. The results have shown that this combination therapy enhanced anticancer effects in lung cancer models both in vitro and in vivo. EVs play a key role in the effective transmission of oncolytic viruses and chemotherapeutic drugs [72].
EVs currently used for therapeutic research are mainly derived from the following sources: mesenchymal stem cells (MSCs), dendritic cells (DCs), tumor cells, red blood cells, macrophages and plants. EVs from different sources have different biological characteristics. Materials should be selected according to the purpose of treatment. The characteristics, advantages and disadvantages of EVs from different sources will be described below.
The MSCs involved in the study of EVs include adipose-derived MSCs, bone marrow MSCs, progenitor cells from different tissues, and so on. MSCs can be extracted from the patient’s bone marrow, fat, or other tissues. EVs derived from MSCs are very attractive. Because they have anti-inflammatory, anti-apoptotic and anti-microbial capability, and promote angiogenesis and the repair and regeneration of damaged tissues. As mentioned above, EVs derived from MSCs have been widely used in the treatment of central nervous system diseases, cardiovascular diseases, bone and cartilage damage repair and regeneration, wound repair, and other organ damage repair and regeneration [7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21].
One potential source of therapeutic EVs is immature dendritic cells (imDCs). EVs secreted by imDCs lack surface markers such as CD40, CD86, MHC-I and MHC-II. Therefore, they have low immunogenicity. They can be isolated from CD34+ cells from the patient’s peripheral blood. It is one of the preferred sources of therapeutic EVs.
The use of EVs derived from tumor cells to deliver drugs and vaccines for immunotherapy is very promising. Tumor EVs can deliver antigens to dendritic cells, thereby inducing T cell-mediated immune responses to tumor cells [73]. As tumor-derived EVs specifically express Tetraspanins, they can target different tissues. This makes it possible to use tumor-derived EVs for tumor-targeting and selective drug delivery [74]. However, tumor-derived EVs also have many potential risks. Due to the presence of Tetraspanins, Urokinase plasminogen activator, Cathepsin D, Vimentin and other molecules derived from the surface of tumor cells [75, 76], they may promote tumor proliferation and metastasis, and Immunosuppressive effect [77, 78, 79].
Blood EVs mainly secreted by reticulocytes (RTC) are a potential source of safe and sufficient EVs. Because they integrate various membrane proteins including Transferrin (Tf) receptors, but they do not have any immune and cancer stimulating activity [30]. Red blood cell EVs (RBCEV) also have the following advantages: large load; low self-nucleic acid content (red blood cells without nucleus and mitochondria); they can be delivered to a variety of different organs and tissues; large quantities of raw materials and easily available (the raw materials for producing RBCEVs are mainly O-type Blood of blood donors). Using blood EVs as carriers can efficiently target tumors to co-deliver chemotherapeutics and nucleic acid drugs. Significant tumor growth inhibitory effects were observed in tumor-bearing mice. There were no obvious side effects [30].
Macrophages are an important immune cell in the antigen-presenting cell family. EVs derived from immune cells can mimic immune cells to target tumor cells. Macrophage EVs can transfer miRNAs or proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, in the study of tumor treatment of EVs, in addition to using the targeting properties of macrophages-derived EVs, the influence of their contents must also be considered. It has been reported that the contents of EVs derived from macrophages can be removed. Then the EVs were used to carry chemotherapeutic drugs to achieve targeted therapy of triple-negative breast cancer [80].
Based on reliable sources and safety, fruits and plants have been used as alternative sources for the isolation of EVs for clinical use [81]. Plant-derived EVs have similar structural characteristics to animal cell-derived EVs. EVs from different plant sources have the physiological functions of the plant from which they are derived. For example, lemon-derived EVs have certain anti-cancer effects. Some researchers have tried to isolate lemon-derived EVs (LDEVs) for the treatment of gastric cancer. LDEVs caused s-phase arrest of gastric cancer cell cycle and induced cell apoptosis. LDEVs could be retained in the organs of the gastrointestinal tract and had strong anti-tumor activity against gastric cancer [82]. The isolated plant EVs can also be used after being engineered. Some researchers isolated EVs from grapefruit, modified the EVs in a targeted manner, and then loaded the anti-tumor drugs doxorubicin and curcumin. These modified EVs could target inflammatory tumors and have anti-inflammatory effects in mouse models [83].
Plant-derived EVs have a wide range of sources, are safe and non-toxic, have low immunogenicity, low cost, and are edible. They have great clinical application potential as edible chemotherapeutic drug carriers.
So far, no EVs drugs have entered the clinic. Codiak BioSciences, a leading company in the development of engineered EVs as a new type of biopharmaceutical, uses its proprietary engEx platform to engineer EVs with different characteristics, load them with various types of therapeutic molecules and change their orientation, so that they can reach specific cellular targets. Recently, Evox Therapeutics Ltd. and Eli Lilly and Co. reached a cooperation agreement to apply its exosome technology to the system to deliver RNA interference and antisense oligonucleotide drugs to the central nervous system, treating five unspecified Neurological diseases. Carmine Therapeutics is also a gene therapy company based on EVs, established in 2019. Carmine’s REGENT technology platform focuses on using red blood cell extracellular vesicles (RBCEV) as drug delivery vehicles. Mantra Bio also joined the emerging group of exosome drug development companies. With the deepening of research, more and more companies will join the field of EVs treatment.
The Severe Acute Respiratory Syndrome (which first appeared in December 2019) related to the new coronavirus (COVID-19) has rapidly developed into a pandemic, and the morbidity and mortality rates are increasing worldwide. COVID-19 respiratory tract infection is characterized by an imbalanced immune response, leading to an increased possibility of severe respiratory disease and multiple organ disease.
Because EVs derived from MSCs have anti-inflammatory, anti-apoptotic and anti-microbial capability, promote angiogenesis and the repair and regeneration of damaged tissues. In related lung disease models, including acute lung injury and sepsis, systemic administration of MSC-EVs preparations can modulate immune responses. In a mouse model of pneumonia induced by Escherichia coli, it was found that MSC-EVs administration could enhance the phagocytosis of bacteria. In the pig model, MSC-EVs could reduce influenza virus-induced acute lung injury by inhibiting influenza virus replication. In other disease models, the disease alleviation effect of MSC-EVs on the inflammatory immune response has also been observed. It is speculated that they may also have anti-COVID-19 efficacy. In cell therapy research for COVID-19, some registered clinical trials have turned their targets to EVs in the conditioned medium of MSCs. MSC-EVs can be administered intravenously (ChiCTR2000030484) or by inhalation (NCT04276987, ChiCTR2000030261).
However, before using MSC-EVs for COVID-19 patients, many other issues should be considered, such as the huge heterogeneity of MSC-EVs composition and source. In fact, comparing MSC-EVs harvested from the conditioned medium of bone marrow MSCs derived from different donors, there are significant differences in cytokine content and different therapeutic effects. In addition to immune regulation, MSC-EVs can also control other biological processes and may cause unpredictable side effects. For example, increasing the risk of thrombosis.
In short, in order to reduce the risk of potential life-threatening side effects, International Society for Extracellular Vesicles (ISEV) and International Society for Cell and Gene Therapy (ISCT) strongly require that the clinical data from reasonable clinical trial should be carefully weighed. The EV preparations with good characteristics and produced under strict GMP conditions and appropriate regulatory supervision could be used. Any application of EVs should be carefully evaluated [84].
The potential application of EVs in new diagnostic and therapeutic strategies has attracted increasing attention. However, due to the inherent complex biogenesis of EVs and their huge heterogeneity in size, composition and source, the research of EVs still faces huge challenges. It is necessary to establish a standardized method to solve the heterogeneity of EVs and the analysis of pre-processing and analysis of sources of variability in the study of EVs. The quality standards, extraction specifications and especially the stability of preparation conditions for therapeutic EVs also need to be clarified.
In addition, the diversity and uncertainty of EVs content are also issues that need to be considered in the application. Before metastasis, malignant tumor cells use EVs to modify the microenvironment of the organ targeted by cancer metastasis, making it a suitable "soil" for tumor cell growth. The contents of EVs secreted by most tumor cells play a role in promoting tumor metastasis and progression. As mentioned earlier in this article, macrophage EVs can transfer miRNAs and proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, if EVs from such sources are used as drug carriers, it is particularly important to first remove the adverse effects of their contents.
As an important medium of intercellular communication, EVs play an important physiological function and are also involved in the occurrence and development of a variety of diseases. In recent years, there have been numerous studies on the treatment of related diseases with EVs from different cell sources, and EV has shown its unique advantages in drug transportation. EVs are similar to natural liposomes, which can enhance the function of EVs to treat specific diseases through targeting modification and delivery of functional active substances and other technical modifications according to the characteristics of different diseases. EVs with improved function have shown obvious advantages in the treatment of tumors and difficult diseases of central nervous system. However, the clinical application of EVs technology is still in its infancy, and the challenges it faces are accompanied by the possibility of numerous new discoveries and new technologies. We expect that with the continuous in-depth research, EVs as a new drug carrier in the treatment of a variety of diseases will bring more and greater surprises.
This work was supported by the National Key R&D Program of China (2018YFA0900900), and the National Natural Science Foundation of China (81773251 and 81702735).
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