new 13 - aracne · 2017. 9. 20. · expression: when odorants are forced out of the natural source...

Determination of Volatile Organic Compounds

Application of SPME on NaturalMatrices and in Forensic Science

Maurizio D’Auria

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I edizione: aprile 2006

Contents

1. The importance of volatile organic compounds (VOC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 7

Flavors and fragrances . . . . . . . . . . . . . . . . . . . . . . . p. 8Isolation from a natural matrix . . . . . . . . . . . . . . . . p. 8Perfume ingredients . . . . . . . . . . . . . . . . . . . . . . . . . p. 16Problems related to the use . . . . . . . . . . . . . . . . . . . p. 22

2. Use of SPME in the determination of VOCs in naturalmatrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 29Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 29Truffles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 53Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 68Honey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 80Saffron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 87Thyme, oregano, lavender . . . . . . . . . . . . . . . . . . . . p. 111Horseradish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 154Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 157

3. Experimental details . . . . . . . . . . . . . . . . . . . . . . . p. 164Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 164Analysis of olive oil . . . . . . . . . . . . . . . . . . . . . . . . . p. 166Analysis of truffles . . . . . . . . . . . . . . . . . . . . . . . . . . p. 167Analysis of wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 168Photochemical reaction of ethyl hexanoate in thepresence of riboflavin . . . . . . . . . . . . . . . . . . . . . . . p. 169Analysis of honey . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 169Analysis of saffron . . . . . . . . . . . . . . . . . . . . . . . . . . p. 170Analysis of thyme, oregano, and lavender . . . . . . . . p. 171Analysis of horseradish . . . . . . . . . . . . . . . . . . . . . . . p. 172Analysis of cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 173

4. Forensic applications of SPME . . . . . . . . . . . . . . . p. 175The accelerants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 175Fire of a pre-fabricated wood house . . . . . . . . . . . . . p. 180Fire of a warehouse . . . . . . . . . . . . . . . . . . . . . . . . . . p. 185Attempt to set fire to a woman . . . . . . . . . . . . . . . . . . p. 188Fire in a house . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 193

5

Fire in a school . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 198Burned boats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 201Crude oil in the soil . . . . . . . . . . . . . . . . . . . . . . . . . . p. 206

5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 209

6

The importance of Volatile Organic Compounds (VOC)

Smell and taste are the oldest of our senses. They probably developed in very primitive organisms as a means of obtaining information about chemical changes in the organism’s environment. Animals use smell and taste to find food and to assess its quality. The smell of food has a powerful effect on animals.

Living organisms use the chemical sense as a means of communication. If the communication is between different parts of the same organism, the messenger is referred to as a hormone. Chemicals used to carry signals from one organism to another are known as semiochemicals, which can be grouped into two main classes: pheromones and allelochemicals. If the signal is between to members of the same species, the messenger is called pheromone. Sex pheromones are amongst the most widespread. The social insects also use alarm, aggregation, dispersal and social pheromones to warn of danger and to control group behavior. Chemicals that carry messages between members of different species are known as allelochemicals. Kairomones benefit the receiver of the signal, allelomones its sender and with synomones both the sender and the receiver benefit.

Some plants produce compounds known as antifeedants, the taste of which insect find repulsive.

Many odorous chemicals in nature are produced for properties other than their odor. Many plants, when damaged, exude resinous materials as a defense mechanism: the shrub Commiphora abyssinicaproduces a resin that contains a number of antibacterial and antifungal compounds. The resin has a pleasant odor and so was put to use by man as a perfume ingredient: it is known as myrrh. As a result of its antimicrobial properties, myrrh was also used as an antiseptic and preservative material, for instance, in the imbalsaming the corps.

Table 1 illustrates a near ten billion dollar industry in the provision of flavor composition, whilst Table 2 details the fifteen billion dollar market for cosmetic and fragrance on a global basis. Their products, essentially fragrance and flavor concentrates are provided in 50 kilo,

7

Chapter 18

100 kilo and 200 kilo drum and occasionally in 1 ton isocontainer.However, the multinational fragrance houses do not sell to general public. Their marketplace is the large multinational companies thatmanufacture the well known branded products, or indeed, the multipleretail chains in any domestic economy that produce own brand products.

Table 1. Estimated world consumption of flavor and fragrance products in 1994 (millions of dollars)

UnitedStates

WesternEurope

Japan Rest of World

Total Total(%)

Fragrancecomposition

660 1190 250 720 2820 29.2

Essentialoils/naturalextracts

450 768 158 320 1716 17.5

Aroma chemicals 464 582 182 206 1434 14.8Flavorcomposition

820 1060 977 860 3717 38.5

Total 2304 3600 1567 2106 9687 100.0Total (%) 24.7 37.2 16.4 21.7 100.0

Flavors and Fragrances

The four categories of secondary metabolites, in decreasing order of importance as sources of perfume ingredients, are terpenoids, shikimic acid derivatives, polyketides and alkaloids. Terpenes are the most important (Table 3).

Isolation from a natural matrix

Importance of VOCs 9

Table 2. World consumption of cosmetics and perfumes in 1994

Billions of dollars PercentCosmeticsNorth America 7.4 35Japan 6.0 29Western Europe 4.5 22Rest of the World 2.9 14

Total 20.8 100

PerfumesWestern Europe 7.1 48North America 5.4 37Japan and rest of the World 1.4 10Duty free shops 0.8 5

Total 14.7 100

Table 3. Classifications of terpenes

Name Number of isopreneunits

Number of carbonatoms

Hemiterpenes 1 5Monoterpenes 2 10Sesquiterpenes 3 15Diterpenes 4 20Sesterterpenes 5 25Triterpenes 6 30Carotenes 8 40Steroids

Chapter 110

The method used to extract perfume ingredients from their natural sources fall into three basic classes: expression, distillation and solvent extraction (Table 4).

Table 4. Some of the more important natural fragrance materials

Oil Type ofprocess

Plant part extracted

Approximate annual production (tones)

Typicalcountry of origin

Ambrette S seed 0.5 China,Colombia

Angelica S root 1 BulgariaAnise S seed 1200 China,

VietnamArtemisia S aerial

parts16 Morocco,

Tunisia.India

Basil S Floweringtops

15 Reunion

Bay S leaf 20 Dominica,Puerto Rico

Bergamot E leaf 120 ItalyBenzoin C exudate 3 Thailand.

IndonesiaBirch tar D wood 50 Austria.,

Germany,Russia

Cabrueva S wood 10 Brazil,Paraguay

Cade D wood 12 Portugal,Yugoslavia

Cajeput S Leaves &twigs

50 Indonesia

Calamus S rhizome 10 N. Korea,India

Camphor S wood 250 China


Table 4. Continued

Oil Type ofprocess




Cananga S flowers 45 Indonesia,ComorosIslands,Reunion

Caraway S seeds 10 Bulgaria,Egypt,Australia

Cassia S leaves 160 ChinaCedarwood

S wood 2200 China, USA

Cedar leaf S leaf 25 USA,Canada

Celery S seed 25 Bulgaria,India

Chamo-mile

S flowers 10 Morocco,France

Cinna-mon bark

S bark 5 Sri Lanka

Cinna-mon leaf

S leaf 100 Sri Lanka,India,Seychelles

Citronella S leaves 2300 Sri Lanka,Indonesia

Clarysage

A, S Flowers/leaves

45 Russia,USA,Bulgaria,France

Clove bud S Flowerbud

70 Indonesia,Madagascar

Clove leaf S leaf 2000 Indonesia,Madagascar

Chapter 112

Table 4. Continued

Oil Type ofprocess




Copaibabalsam

U exudate 40 Brazil

Coriander S Seeds 100 RussiaCornmint S Aerial

parts3000 China,

BrazilCumin A, S seeds 10 IndiaDill S Aerial

parts140 USA,

Hungary,Bulgaria

Elemi C, S exudate 10 PhilippinesEucalyp-tus

S Leaves

E.citriodora

800 Brazil, S.Africa, India

E. dives 50 AustraliaE.globolus

1600 Spain,Portugal

E. staige-riana

50 Australia,Brazil, S. Africa

Fennel S seeds 80 SpainFir needle S leaves 55 Canada,

USA,Russia

Galba-num

C, S exudate 10 Iran,Lebanon,Turkey

Geranium A, S Leaves/stems

150 Reunion,Egypt

Ginger S root 55 China,Jamaica


Table 4. Continued

Oil Type ofprocess




Grape-fruit

E fruit 250 Israel,Brazil, USA

Gauaiacwood

S wood 60 Paraguay

Ho S Leaf &wood

30 China

Jasmine A, C flower 12 Egypt,Morocco

Juniper S fruit 12 Yugoslavia,Italy

Labda-num

A, U, S exudate 20 Spain

Lavender S Aerialparts

1000 France,Spain,Tasmania

Lemon E fruit 2500 USA, Italy,Argentina,Brazil

Lime E fruit 1200 Mexico,Haiti

Litseacubeba

S fruit 900 China

Mandarin E fruit 120 Italy, ChinaMarjoram S Leaves &

flowers30 Morocco

Neroli S flowers 3 TunisiaNutmeg S Fruit 200 Indonesia,

Sri Lanka Oakmoss A, C Aerial

parts100 Yugoslavia,

Italy, France

Chapter 114

Table 4. Continued

Oil Type ofprocess




Olibanum C exudate 10 Ethiopia,Yemen

Orange E fruit 15000 USA,Brazil,Israel, Italy

Origanum S Aerialparts

10 Spain,France

Orris C, S rhizome 5 Italy,France,Morocco

Palma-rosa

S leaves 55 India, Brazil

Patchouli S leaf 800 IndonesiaPenny-royal

S Aerialparts

10 Morocco,Spain

Pepper-mint

S Aerialparts

2200 USA

Petitgrain S leaves 280 ParaguayPerubalsam

C, S exudate 45 SanSalvador,Brazil

Pimento S fruit 50 JamaicaPine oil S wood 1000 USA,

Mexico,Finland,Russia

Rosemary D Aerialparts

250 Spain,Morocco,Tunisia


Table 4. Continued

Oil Type ofprocess




Rose C, S flower 20 Bulgaria,Turkey,Morocco

Rose-wood

S wood 250 Brazil, Peru,Mexico

Sage S Aerialparts

45 Yugoslavia,Spain,Greece

Sandal-wood

S wood 250 Indonesia,India

Sassafras S roots 750 BrazilSpearmint S Aerial

parts1400 USA, China,

BrazilStyrax C, S exudate 25 Turkey,

HondurasTangerine E fruit 300 BrazilTarragon S Aerial

parts10 Italy,

MoroccoThyme S Aerial

parts25 Spain

Ti tree S leaves 10 AustraliaVanilla C, T fruit 2500 Reunion,

MadagascarVetiver S root 260 Reunion,

Haiti,Indonesia

Ylang-ylang

A, C, S Flower 90 Comores,Madagascar

A = absolutes; C = concretes and resinoids; D = dry distilled oils; E = expressedoils; S = steam-distilled oil; T = tincture; U = untreated.

Chapter 116

Expression: when odorants are forced out of the natural source by physical pressure; the process is referred to as expression and the product is called an expressed oil.

Distillation: there are three ways to perform it: Dry (high temperature: oils of highest boiling point). Steam distillation: water or steam is added to the still pot andthe oils are codistilled with the steam (less degradation than in dry distillation). In the hydrodiffusion the steam is introduced at the top of the pot and the water and oil taken of as liquid at the bottom. Perfume materials are referred to as essential oils. Solvent extraction: ethanolic extraction is not used very muchbecause the high proportion of water. The traditional solventfor extraction was benzene, but this has been superseded by other solvents because of concern over the possible toxic effects of benzene. Petroleum ether, acetone, hexane and ethylacetate are typical solvents. Recently, there has been a great deal of interest in the use of carbon dioxide as an extraction solvent. Carbon dioxide has the advantage that it is easily removed and there are no concerns about residual solvent levels. The products of such extraction is called a concrete or resinoid.

Obtaining an extract can be a very laborious work. It takes about 3000000 jasmine flowers to produce 1 kg of oil. The flowers have to pick by hand (no one has yet devised a mechanical method of harvesting jasmine) in the first few hours of the day when their oil content is at its highest.

Perfume ingredients

Terpenes form the largest group of natural odorants (Table 5). The most important members of the terpene family are the oxygenatedmonoterpenes.


Table 5. Some of the more important terpene fragrance materialsMaterial Odor Approx. usage

(tones/annum)Amberlyn®/Ambrox®/Ambroxan®

Ambergris 6

Carvone Spearmint 600Citronellol and esters Rose 6000Dihydromyrcenol Citrus, floral 2000Geraniol-nerol and esters

Rose 6000

Hydroxycitronellal Muguet 1000Borneol/isoborneol and acetate

Pine 2000

Linalool Floral, wood 4000Linalyl acetate Fruit, floral 3000Menthol Mint, coolant 5000(Methyl)ionones Violet 2000

-Terpineol and acetate Pine 3000Acetylated cedarwood Cedar 500

The terpene hydrocarbons generally have weaker odors and are used mainly ad feedstock. The higher molecular weights of the sesquiterpenes result in their having lower vapor pressures than theirmonoterpenes counterparts. Thus, sesquiterpenes are present at lowerconcentrations in the air above a perfume than are monoterpenes, with the result that to be detected they must have a greater effect on thereceptor of nose. Hence, a lower percentage of sesquiterpenes have useful odors than monoterpenes. Sesqui- and higher terpenes that do have odors are very tenacious because their lower volatility meansthey are lost more slowly from perfumes. Such materials form the base of perfumes and serve also to fix the more volatile components.

Geraniol-nerol, linalool, citronellal, citronellol and citral are five ofthe most important terpenes as far as the perfume industry is concerned (Figure 1).

Chapter 118

OH

linalool

O

citral

O

OH

geraniol - nerol

OH

citronellal citronellol

Figure 1. The most important terpenes in perfume industry

L-Menthol occurs in a number of mint oils and is used not only for the minty odor, but also, and more importantly, for its physiological cooling effect. When applied to skin or mucus membranes, L-mentholcreates the sensation of cooling independent of the actual temperature of the tissue concerned. It is used in toothpaste and other oral-careproducts, in confectionery and tobacco, and in some cosmeticproducts. L-Carvone is the principal odor component of spearmint oil. The chirality of carvone is crucial to the odor.

Monocyclic terpene hydrocarbons occur in many essential oils. They have relatively weak odors, although some add dryness and reen notes to the oilsg

oil. Terpinolene is thcontaining them. D-Limonene occurs in citrus e dehydration product of -terpineol. -

Phellandrene occurs in eucalyptus oil. The phenols carvacrol andthymol are important in some herbal odor types. Piperitone and


pulegone are strong mintly odorants, the latter being the majorcomponent of pennyroyal oil. 1,8-Cineole is the major component of some eucalyptus oils.

Camphor, isoborneol and isobornyl acetate are all used for theirwoody odors. Nonyl acetate has a sweet-fruity odor.

Nerolidol, farnesol and bisabolol have some perfumery use, although their odors are weak. Bisabolol is used mostly of its anti-inflammatory and antibacterial properties. The sesquiterpenesresponsible for the odor of vetiver and patchouli oils have complexstructures (Figure 2).

O O

vetivone vetivone

HO HO

Patchouli alcohol Norpatchoulenol

love oil.ongifolene is present in Indian turpentine, which is obtained from the

species Pinus longifolia.The major components of the Juniperus wood oil are cedrol,

cedrene an illation ofthe parasiti f the oilsre santalols (Figure 3).

Figure 2. Main components of vetiver and patchouli oils

Caryophyllene is the main hydrocarbon component of cL

d thujopsane. Sandalwood oil is obtained by distc tree Santalum album. The major components o

a

Chapter 120

Very few diterpenes are sufficiently volatile to possess an odor. One diterpene is used in perfumery because it and the derivativesconcerned are odorless. They are used as solvents. These solvents also have fixative properties. Abietic acid is the major component of tall oil. Esterification and hydrogenation produces two solvents.

HOOH

-Santalol -Santalol

Figure 3. Major components in sandalwood oil.

The sperm whale produces, in its intestinal tract, a triterpene calledambreine. It is not known exactly why the whole produces ambreine:it is probably in response to some irritation. Lumps of ambreine,which can weight up to 100 kg, are excreted into the sea. Then, in the presence of salt water, air and sunlight, the ambreine undergoes a variety of degradation reactions to produce a complex mixture ofbreakdown products. The mixture is known as ambergris, from the French “amber gris”. Perhydronaphthofuran possesses thecharacteristic animalic note of ambergris.

Ionones possess odors which are reminiscent of violet, sometimesalso with woody notes. The damascones are components of rose oils and have very intense fruity-floral odors.

The original musk components of perfumes were extracted fromanimal source. They were extracted from the anal glands of the muskdeer and civet cat. Muscone and civetone are the most important odor components of musk and civet respectively. Ambrettolide is a plant product, occurring in the seeds of the ambrette plant which iscultivated in Madagascar, the Seychelles, Columbia and Ecuador.

2-Phenylethanol is a major component of rose oils and it is one of the most important of all perfumery ingredients. Many esters of 2-


phenylethanol are used in perfumery, the acetate, isobutyrate and phenyl acetate in particular.

The hydrocynnamic aldehyde is another family of materials derived from benzene and which possess fresh, white-floral notes reminiscentof muguet and cyclamen.

Benzyl acetate is the major component of jasmine oils.Cinnamaldehyde is used as starting material for the corresponding alcohol, cinnamyl alcohol. This is an important component of spixy perfumes in which a cinnamon note is required. Its esters, the acetatein particular, are also used for their odor. Much more important are the higher members of the series, amylcinnamic aldehyde andhexylcinnamic aldehyde. They possess odors reminiscent of the fatty ba

ate to perfumery is amyl, hexyl and benzyl de

xcellent blenders and fix

nctional products, where the pH is not neutral, ey undergo a variety of reactions leaguing to discoloration. For

example, inclusion of vanillin in a white soap, after a matter of days,produces a color close to that of chocolate. Protection of the phenolic group of vanillin through the isobutyrate ester gives Isobutavan®. Reduction of the aldehyde group of ethylvanillin to a methyl group gives Ultravanil®. Both of these compounds provide vanilic notes, bur are much more stable in use than vanillin itself.

Clove oil is available at moderate cost and in moderate quantityfrom several tropical countries. Similarly, sassafras oil is available from Brazil. The major component of clove oils eugenol, while that of sassafras oil is safrole.

Methyl anthranilate occurs naturally in many flowers and has a very characteristic and intense, sweet smell.

A large number of aliphatic fragrance ingredients are used. They are mostly aldehydes, nitriles and lactones, the majority of which have

ckground note of jasmine.The esters of salicylic acid are important to the fragrance industry.

Methyl salicylate is the major component of oil of wintergreen. The most important salicyl

rivatives, which are used in very significant quantities. These have persistent, floral, herbaceous odors and make e

atives for floral perfumes.Vanillin and ethylvanillin are not particularly stable chemically.

This is not surprising since they possess both and aldehyde and aphenolic group. In futh

Chapter 122

very intense odors t incorporated into afragrance (Figure 4).

hat limit the amount that can be

O O O OO

O-Decalactone hexyl acetate allyl heptanoate

O CN

O

OMe

Aldehyde MNA Frutonitrile Beauvertate

Figure 4. Aliphatic fragrances

Problems related to the use

Compounds that are prepared by chemical means but that areidentical in structure to those found in nature are known as nature-identical materials. The synthesis of compounds closely related to those nature-identical materials has led to a discovery of ingredients that are similar in odor, but which are much easier and cheaper to make than their natural counterparts.

When inserted in a white soap, to prevent discoloration on storage, vanillin may be substituted by ethyl vanillin which, because it is moreintense, can be dosed at about one-third the vanillin level. Smallquantities of Ultravanillin® are also incorporated to boost the vanilla effect without causing discoloration. Indole is replaced by Indolal® at a slightly higher level, as it gives less intense, animalic jasminecharacter in soap.

In shampoo we have some other problems: molecules with a low relative molecular mass, for example ethyl hexanoate and limonene,


diffuse most speedily on opening the cap of the shampoo bottle.Molecules such as limonene with relative high log P (4.46) diffuse more readily than more polar molecules of similar mass, such as 2-phenylethanol, whose log P is 1.52. Another consideration is the active detergent level of the shampoo, which affects both theappreciation of the fragrance in the headspace above the shampoo andthe

e perfume dosage in a shower gel or bath product is do

ther chemicals onto the surface, which can int fragrance. Lastly, the skin is less porous in

ble fort molecules. I c ed for the bath, thandiffusion from the bath water becomes important.

Most fragrance ingredients are classed as mild or moderated skin i dilutedc nlikel o be ce of irr ion.Flavors and fragrances can induce skin sensitization and phototoxicity. In Table 6 we h a c e restriction in t ance proposed by the International Fragrance A

ows the relativ rgan of a ra ofmuguet ingredients in an aerosol antip ther the ofthe fragrance ingredient has virtually disappeared to leave only the odor of the product base or the sam ped und bleolfactory notes, the fragrance redie as having poororganoleptic stability.

solubility of the perfume oil in the detergent system. A system with low concentration of active detergent has a smaller reservoir of micelles in which to solubilize the perfume mixture. Then, moreperfume is available for the headspace because less can be “dissolved”in the shampoo base.

In shower and bath gel retentivity on the body must be considered. In general, th

uble that for shampoo, so the effect of any potentially discoloring ingredients is accentuated. The higher perfume level certainly helpsfragrance retentivity. However, substantivity on skin is usually lessthan that on hair for three reasons. Firstly, the skin is warm and hencethe rate of perfume vaporization is higher. Secondly, the skincontinually secretes o

eract with depositednature than hair, so potentially fewer bonding sites are availahe perfume f the produ ts destin

rritants when in an un form only, so the low levels found in onsumer products are u y t a serious sour itat

ave ollection of somhe use of fragrssociation.Figure 5 sh e o oleptic stability nge

erspirant. If ei odor

ple has develo esiraing nt is classified

Chapter 124

Table 6. Examples of fragrance gredients restricted by IFRA Rest ion

allowedon skin

inIngredients rict Reason %

Acetylated vetiver oil S Sensitization UAcetylethyltetramethyl-

e oil

ldehyde

t products P Sensitization 0

olarin

S Sensitization UP Phototoxicity 0

il sitizationTrans-Hept-2-enal P SensitizationHexahydrocoumarin P Sensitization

tetralinP Neurotoxicity 0

5-Acetyl-1,2,3,3,6-hexamethylindan

R Phototoxicity 2

Angelica root oil R Phototoxicity 0.78Bergamot oil R Phototoxicity 0.4Bitter orang R Phototoxicity 1.4p-t-butylphenol P Sensitization 0Cinnamic alcohol R Sensitization 0.8Cinnamic a Q Sensitization UCitralCostus roo

Q Sensitization U

Cumin oil R Phototoxicity 0.4Cyclamen alcoh P Sensitization 0Dihydrocoum P Sensitization 0FarnesolFig leaf absolute Grapefruit o R Photosen 4.0

00

Trans-Hex-2-enal R Sensitization 0.002Hydroxycitronellal R Sensitization 1.0Isoeugenol R Sensitization 0.02Lemon oil cold pressed R Phototoxicity 2.0Lome oil cold pressed R Phototoxicity 0.7Limonene S Sensitization U6- and 7-methyl-coumarin

P Photosensitization 0

Methyloctine carbonate R Sensitization 0.01Musk ambrette P Neurotoxicity 0Nookatone S Sensitization U


T tinued

Rest ionallowedon skin

able 6. Con

Ingredients rict Reason %

Trans-Hex-2-enal 0.002R SensitizationHydroxycitronellal R Sensitization 1.0Isoeugenol R Sensitization 0.02Lemon oil cold pressed R Phototoxicity 2.0

e oil cold pressed LomLim

R Phototoxicity 0.7onene S Sensitization U

6-

0.78Safrole P Chronic toxicity 0St

and 7-methyl-coumarin

P Photosensitization 0

Methyloctine carbonate R Sensitization 0.01Musk ambrette P Neurotoxicity 0Nookatone S Sensitization UOppoponax R Sensitization 0.60Phenylacetaldehyde Q Sensitization UPseudoionone P Sensitization 0Rue oil R Phototoxicity

yrax R Sensitization 0.6R =source or method of production = quenching, this material can only be used in conjunction with an agent that prevents sensitization; U =unrestricted.

Antiperspirant formulations are acidic because of partial hydrolysis of the active antiperspirant agents, such as aluminum chlorohydrate. Florosa (Figure 6) performs well in antiperspirants it is chemicallymore stable than the aldehydes. After 4 weeks storage at 37°C it remains unchanged. In contrast, Lilial remaining is only 15%. This occurs via an auto oxidation process. Autoxidation is defined as the reaction of organic compounds with oxygen under mild conditions.Although Florosa is more stable than the aldehydic muguet materials, it cannot be used as a simple replacement in a formulation.

restricted; P = prohibited; S = specification, there is a defined grade, botanicalfor this material; Q

Chapter 126

0

2

4

6

8

10

Organolepticstability

Hyd

roxy

Mouguet ingredient

c C

Flitron

ella

l

ycla

men

alde

hyde

Lilia

l

Lyra

l

oros

a

Bour

geon

alFigure 5. Organoleptic stability of some muguet ingredient in an

antiperspirant.

O

OH

O O

HO

CHO

O

OHO

Hydroxycitronellal Cyclamen aldehyde Lilial

Lyral Florosa Bourgeonal

Figure 6. Muguet ingredients


Its floral odor is rosier and it lacks both the impact and the green and watery notes associated with materials such as Lilial andBourgeonal.

Use of SPME in the determination of VOCs in natural

matrices

Oil (Bentivenga et al., 2001; Bentivenga et al., 2002a; Bentivenga et al., 2002b)

Flavor has a role in the definition of preference for virgin olive oil (Monteleone et al., 1997a,b). Recently some efforts have been performed in order to determine the aroma of the olive oil (Guth and Grosch, 1991; Morchio et al., 1994; Guinda et al., 1996; Monteleone et al., 1996; Morales et al, 1997; Angerosa et al., 1997; Aparicio and Morales, 1998; Lerker et al., 1999; Del Signore, 1999). Most of these works have been carried out by using gas-chromatographic analyses of the oils (Bocci et al., 1992; Morales et al., 1994; Raghavan et al., 1994; Servili et al., 1997).

everal compounds coSn

ntributing to the aroma of olive oil have been e tified. Aldehydes from C2 to C18 have been determined in the

ritsakis, 1998). re the

ma om he olivro extra ) have

a i logy t e a s(Zhang and Pawliszyn, 1993). This method has been applied to the analysis of flavors (Yang and Peppard, 1994).

ase m action ( ME) is a sample preparation technique based on sorption, which is useful for extraction and concentration ana submersion in a liquid phase or by exposure to a gaseous phase. Following exposure of the fiber to the sample, sorbed analytes can be thermally desorbed in a conventional gas chromatography injection port. SPME has been used in a range of fields including studies of flavors and taints, especially for quick screening of the h been applied , g et t al., 1998), vegetable oils (Yang and Peppard, 1994; Field et al., 1996), offee (Yang and Peppard, 1994; Deibler et al., 1999), wine (Garcia et

idolive oil (Flath et al., 1973; Montedoro et al., 1978; KiHexanal, trans-2-hexenal, 1-hexanol and 3-methylbutanol a

jor volatile cRecently, solid phase m

pounds of t e oil. ction (SPMEic

ve methodoallowed us to nalysis of vola new non-invas o perform th tile

Solid ph icroextr SP

lyses either by

volatile compositioto fruits (Penton

n of a wide range1996; Son

of products. It al., 1997; Jia eas

c

29

Chapter 230

al., 1996; Garcia et al., 1997; Garcia et al., 1998; Wada and Shib 97 1998; Hayasaka and Bartowsky, 1999; Ong and Acree, 1999), cork (Fischer and Fischer, 1997; Ong and A ree, 1999), bee al., 19 eat (Ruiz et al., 1998), milk(Marsili, 1999), and biological fluids (Nishikawa et al., 1997; Lee et al., 1997; Namera et al., 1999; Cardinali et al., 2000).

SPME provid t es over conventional samplep paration techn E echnique is simple to use, takes less than one hour to complete, is less expensive, does not require solvent extraction ha of the headspace inc tact with the e od obviates e classical steamdistillation, which to modify unstable constituents.

Montedoro (1978) reported that the headspace analysis of the oliveoil

l, -terpineol, and lovandulol (Flath etl., 1978; Fedeli, 1977).

T C ide n o adsClass of

Compoumpound of

C unds

amoto, 19 ; Vas et al.,

c r (Jelen et 98), m

es many advan agre iques. The SPM t

and allows c racterizationon sample. This m

is liableth th

allowed the identification of several compounds. They are reported in Table 7. In the olive oil aroma some terpenes has been identified: inparticular, 1,8-cineole, linaloa

able 7 – ompoundCo

ntified i live oil heClass

pace.Compound

nds ompoHy carb talddro ons n-Hexane Aldehydes Ace ehyde

tl an

Alcohols Ethanol ana 1-Penten-3-ol -Pe 3-Methylbutan- s-2 l

1-Pentanol na 1-Hexanol ana

- Benzaldehyde

s-3l

Ketones 3-Pentenone

n-Oc ane 2-MethylbutanalEthy benzene Pent al

Hexcis-2

lntenal

1-oltran -Hexena

Octa lNon l

cis-3 Hexen-1-oltran

o-Hexen-

1-

SPME in natural matrices 31

Ta

CompoundsClass of

CompoundsCompound

ble 7. ContinuedClass of Compound

1-Octanol 4-Methyl-3-penten-2-one

Esters Ethyl acetate Isobutyl acetate 3-Methylbutyl

acetate Hexyl acetate

cis-3-Hexenylacetate

In Table 8 we have collected some data useful to characterize theolive oil we used. All the olive oils, with the exception of sample 2, were extra virgin olive oils.

Table 8 – Characterization of olive oils Sample Place of

originSpecie Acidity

degreeAcid

NumberK

1 Armento primitive 0.22 0.044 0.01 2 Mon

scaglioso 0.01 te- coratina 1.22 0.24

3 Armento primitive 0.31 0.063 0.01 4 Monte-

scagliosocoratina 0.44 0.087 0.01

5 Corleto Perticara

coratina 0.21 0.042 0.01

6 Armento primitive 0.36 0.073 0.01 7 Corleto primitive 0.34 0.067 0.01

Perticara8 Corleto

Perticaramaiatica 0.17 0.034 0.01

9 Monte-scaglioso

coratina 0.26 0.051 0.01

Chapter 232

Sample 2 can be considered virgin olive oil. A typical UV spectrumof used olive oil is reported in Fig. 7.

Figure 7 – UV spectrum of olive oil.

At retention time 6.69 min. the chromatogram showed a signal whose mass spectrum had peaks at m/z (relative abundance) 136 (7%), 121 (16), 105 (17), 93 (100), 92 (22), 91 (44), 80 (34), 79 (39), 67 (11), 53 (11), and 41 (21).

The analysis of sample 1 (Fig. 8) showed the presence of ethanol, ethyl acetate, (E)-2-hexenal, and 2-hexanol. Furthermore, we found acompound with a retention time 6.56 min. It showed peaks in the massspectrum at m/z (relative abundance) 136 (22%), 121 (23), 107 (26), 94 (31), 93 (73), 91 (26), 79 (40), 68 (100), 67 (74), and 53 (26).

This spectrum is identical to that reported for dl-limonene (1)(Scheme 1).


Figure 8 - Typical gaschromatogram obtained using SPME GCMS. 1: 1,4-hexadiene, 2: 1-hexanol, 3: octamethylcyclotetrasiloxane, 4: dl-limonene, 5: -trans-ocimene, 6: decamethylcyclopentasiloxane, 7: decamethylcyclohexasiloxane, 8: -copaene, 9: tetradecamethylcycloheptasiloxane, 10: E,E- -farnesene, 11: muurolene, 12: ethyl phthalate, 13: diisobutylphthalate.

This spectrum can be assigned without doubt to trans- -ocimene(2) (Scheme 1). At retention time 9.37 min. we had a new productshowing in the mass spectrum peaks at m/z (relative abundance) 204 (20%), 162 (13), 161 (100), 133 (14), 121 (12), 120 (24), 119 (93), 107 (13), 106 (12), 105 (81), 93 (41), 92 (23), 91 (34), 81 (23), 79 (12), 77 (18), 55 (11), and 41 (29). This spectrum can be identified as

-copaene (3) (Scheme 1) (Fig. 9).

Chapter 234

Scheme 1

1 23

4

5

Figure 9 – Mass spectrum of -copaene

6


The chromatogram showed also a peak with retention time 10.10mi

3), 81 (27), 80 (21), 79 (42), 77 (30), 69 (56), and 55 (41). The above described spectrum is identical to that of (E,E)- -farnesene (4) (Scheme 1).

Finally, with a retention time 10.17 min. we detected a new c(9), 161 (61), 149 (11), 147 (14), 133 (22), 121 (12), 119 (28), 107

94 (28), 93 (32), 9 1 (21), 79 (20),77 (23), and 55 (11). This spectrum is compatible with the

n as -muurolen ) e 1). E)-2-hexenal, 1-hexanol, trans

ene E,E - rn en Fur er re, e f ndt of oi lav r w h ten n e 10.13 mectru we pe s re ive bu n 2

147 (70), 143 (48), 123 (20), 121 ), 109 0 30 10 7 10 (2 10 (10 ), 9, 93 (75 (51 , 81 (50), 79 (55), 77 (28), 69 (15), 67

om u ca be den ied asme

e pr n of ethanol, ethyl acetate, (E)-anol, o e n - a . sa le 4 we

ol, ethy te E)-2-hexenal, 1-hexanol, d im etrans- ne, (E ar en an -muurolene.

e, at ret time 11.10 min. we found a compound s in th sp ctru a 38, 25, 11, 7, , 6 ,

pound can be identified as 8-heshowe vo co )-2-hexe al, 4-

ol, -ocime , a d op ne. n th sa ple 6 ol, m ac ate 3-h en- ol, E)-

hexenal, trans- -ocimene, and -copaene. Similar results wereobtained in the analysis of the sample 7: in this case we found ethanol, methyl acetate, ethyl acetate, (E)-2-hexenal, 1-hexanol, trans- -

n. The mass spectrum of this component of olive oil flavor hadpeaks at m/z 161 (9%), 147 (6), 135 (6), 133 (8), 123 (35), 119 (42), 107 (50), 105 (24), 94 (12), 93 (100), 91 (4

ompound showing peaks at m/z (relative abundance) 204 (38%), 189

(15), 106 (18), 105 (100), 1 (34), 8

identificatio e (5 (SchemIn sample 2 we found ethanol, (

ocimene, -copa- -

, and ( )- fa eits e

re. th mo w ou a

new componenwhose mass sp

olive l fd

o tiolat

tim i .04n

m sho ak at m/z ( a nda ce)(30%), 189 (30), 161 (21), 148 (33), (32), 119 (36(40), 94 (21)

(17), 1 8 ( ), 7 ( 7), 6 2), 5 0 5), 91 )

(33), 65 (12), and 55 (35). Thisguaiene (6) (S

c po nd n i tif -che

Sample 3 show1).

d the ese ce 2-hexenal, 1-hex dl-lim

l acetanen, (

, a d cop ene In mpl-lfound ethan on ne,

-copaene,Furthermor

-ocime ,E)- -f nes e, dentione massshowing peak

55, 43, 41, and 28. This come m t m/z 2 1 1 9 83 9

ptadecene.Sample 5 d as fla r mponents ethanol, (E n

methyl-1-pentanwe found ethan

trans- ne n -c ae I e methyl et , ethyl acetate, ex 1- ( 1-

Chapter 236

oc

S analysis.

imene, and -copaene. Sample 8 contained ethanol, (E)-2-hexenal,-copaene, and 2,4-di-t-butylphenol.

Finally in the sample 9 we found ethanol, (E)-2-hexenal,acetophenone, -copaene, and (E,E)- -farnesene. In the Table 9 we have collected all the compounds we found.

Table 9 – Components of olive oil flavor by SPME-GCM

Compound r.t. [min.]

Sample

1 2 3 4 5 6 7 8 9ethanol 1.83 x x x x x x x x xmethyl acetate 2.02 x xethyl acetate 2.41 x x x x(E)-2-hexenal 4.74 x x x x x x x x3-hexen-1-ol 4.77 x1-hexanol 4.89 x x x x x x4-mp

ethyl-1- 4.92 xentanol

dl e 7 x-lim enon 6.5 x xtrans- ocim 9 x x x- ene 6.6 x x xacetophenone 6.95 x

-copaene 7 x x x x x9.3 x x x x-

farnesene0 x x10.1 x x

2,4-di-t-butylphenol

310.1 x

guaiene 310.1 x-m lene .17 x x uuro 10

8-he ecen x x ptad e 11.09

In conclusion, we have shown that SPME-GCMS analysis of virgin olive oil gave quite diff c h p ious works. We found ethanol and h l, ethyl ace E)-2-hexenal, 3-hexen-1-ol, but we found also the presence of terpenes never detected before. In particular, we found that e is p in all th amples we tested. Also (E,E)- -farnesene and -ocime re pre most of mples we e ed.

These results could indicate that olive oil flavor is due to more omplex mixture of component as described previously.

rvi ostu re he in o oil obtained using different procedure (high pressure squeezing, spinning, milling witho

The identification of flavor components has been performed by sing SPME technique. In Table 10-12 we have collected some data

us

erent results in omparison wit revexano tate, (

some-copaen resent

e s trans-ne a sent in the sa xamin

cConsidering that flavor has a role in the definition of preference fo

rgin olive oil (Monteleone et al., 1997a and 1997b), we decided tdy the p sence of t above described terpenes in virg live

ut hazel).

ueful to characterize the olive oils we used.

Table 10 – Characterization of olive oils from oil-mill at high pressure

Sample Cultivar Acidity degree Acid Number K 1 primitive 0.34 0.067

Chapter 238

Table 11 – Characterization of olive oils from oil-mill using spinning Sample Cultivar Acidity degree Acid Number K

14 locali 0.08 0.41


Figu – h m tog a of sa plere 10 C ro a r m m 1

Figure 11 – Mass spectrum of trans- -ocimene.

Chapter 240

Table 13 – Flavor components of olive oil from oil-mill at highpressure

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13Compoundethanol x x x x x x x x x x x x xpropanone x(E)-2-hexenal x x x x x x x x x(Z)-3-hexen-1-ol x x x x x x2-hexen-1-ol x1-hexanol x x x x x x x x x x x x xcycloisosativene x x xtrans- -ocimene x x x x x x x x

-copaene x x x x x x x x x x x x xlimonene x x x x x

-muurolene x x x

x

x x x x xundecanal xdodecanal x x x x x x xtetradecanal x(E,E)- -farnesene x xisopentanol x1,1-dodecanedioldiacetate

x

Thus, samples 3, 9, and 12 showed the presence of trans- -ocimene, -copaene, and -muurolene. Sample 4 contained only -copaene. Sample 5 showed the presence of limonene, trans- -ocimene, cycloisosativene, -copaene, E,E- -farnesene (r.t. 16.39 min.), and -muurolene. In the samples 6 and 8 we found limoneneand -copaene, while, in sample 7, limonene, trans- -ocimene, and

copaene were present. Samples 10 and 13 were found to contain -copaene and -muurolene. Finally, in sample 11 we found trans- -ocimene, -copaene, E,E- -farnesene, and -muurolene.

The analysis of flavor of olive oils obtained by spinning (Table 11) showed a very different behavior. In sample 14 we found ethanol, E-2-hexenal, -copaene, tetradecanal (r.t. 17.28 min.), and 1,1-dodecanediol diacetate (r.t. 17.29 min.).

Chapter 242


Sample 15 showed the presence of only ethanol, E-2-hexenal, 1,1-dodecanediol diacetate, and tridecanal (r.t. 17.30 min.). Sample 16 contained ethanol and E-2-hexenal while in sample 17 we found only ethanol, tetradecanal, and tridecanal. Finally, the head-space analysisof the sample 18 led to the identification of ethanol, E-2-hexanal, andtridecanal.

The analysis of these samples showed that, in this case, the terpenes identified in the flavor are only rarely present.

We studied also a sample deriving from milling without hazels(Table 12). This sample showed the presence of ethanol, E-2-hexanal,

-copaene, dodecanol, -muurolene, 1,1-didecanediol diacetate, and tridecanal.

In conclusion, we have shown that a. the presence of somecomplex terpenes in the flavor of the olive oil has been confirmed; b. these compounds are not present when spinning oil mill has been usedto obtain olive oil; c. the use of olives without hazel in the millingprocess does not inhibit the presence of terpenes in the flavor.

After this work we decided to analyze a larger number of samplesthan in our previous work and on commercial samples of extra virginolive oils.Table 14 collects the results of the chemical characterization of the olive oils. On the basis of these data samples 2, 3, 5, 7, and 26 can not be considered extra virgin olive oils.

The SPME analysis of these samples showed the same trendobserved in the previous set of data. For example, the analysis of sample 1 (Table 15) showed the presence of ethanol, pentanal, hexanal, (Z)-3-hexen-1-ol, 1-hexanol, dl-limonene, trans- -ocimene,

-copaene, and -farnesene. In sample 2 (Table 15) we found ethanol, ethyl acetate, 3-methyl-1-butanol, 1-hexanol, octanal, and dl-limonene. In the aroma of sample 3 we found ethanol, ethyl acetate, 3-methyl-1-butanol, (Z)-3-hexen-1-ol, 1-hexanol, and -copaene (Table15). Ethanol, ethyl acetate, 3-methyl-1-butanol, (E)-2-hexenal, (E)-2-hexen-1-ol, 1-hexanol, and -copaene were found in sample 4 (Table 15). Sample 5 (Table 15) contained ethanol, (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, 1-hexanol, trans- -ocimene, nonanal,

-copaene, and -farnesene. On the contrary, in sample 6 (Table 15)

Chapter 244

we found only ethanol and ethyl acetate. The analysis of sample 7 (Table 15) showed the presence of ethanol, acetic acid, ethyl acetate,3-methylbutanol, 2-methylbutanol, hexanal, (E)-2-hexenal, (Z)-3-hexen-1-ol, 1-hexanol, and trans- -ocimene.

In most of the samples we found dl-limonene, trans- -ocimene, -copaene, and (E,E)- -farnesene. In this set of samples we did not find

-guaiene and -muurolene.Table 16 contains the physical data of some commercial olive oils

tested in this work.All the oils we used were extra virgin olive oils. We have to note

the higher peroxide values than in the previous set of samples.The SPME-GC-MS analysis of these samples gave the results

showed in Table 17. In our samples we did not find dl-limonene and trans- -ocimene. Furthermore, -farnesene is present in only two samples, while we found -copaene only in one sample.

In conclusion, we have shown that SPME-GCMS analysis of virgin olive oil gave quite different results in comparison with previousworks. We found ethanol and hexanol, ethyl acetate, (E)-2-hexenal,(Z)-3-hexen-1-ol, but we found also the presence of some terpenes never detected before. In particular, we found that -copaene, (E,E)-

-farnesene and trans- -ocimene are present in most of the sampleswe examined. The year of cultivation does not affect this result. Theseresults could indicate that olive oil flavor is due to more complexmixture of components as described previously. Surprisingly, wefound a poor presence of these terpenes in commercial extra virgin olive oils.

In our previous works in the olive oil characterization, we showed that SPME could be used as analytical method for the determinationof olive oil flavor.

Furthermore, GC-MS analysis of methanol-water extracts of oliveoils showed the presence of several components: the main class was alcohols, but also acids, esters, aldehydes, alkanes and alkenes were found.

Chapter 246

Table 17 – Components of olive oil flavor by SPME-GCMS analysis

Sampleon samples 1-5 of commercial olive oils.

Compound r.t [

4 5

Ethanol 1.88 6 0.8 0.7 1.0min.]

1 2 3

0. 0.8Acetic acid 2.28 2 0.9 0.3Ethyl acetate 2.51 0.3Hexanal 5.05 1.6 1.6 2.8(E)-2-Hexenal 6.20 2.4 0.2 2.1(Z)-3-Hexen-1-o 6.28 1(E)-2-Hexen-1-ol 6.50 3 0.8 0.61-Hexanol 6.55 0.2 0.2 (E)-2-Heptenal 8.53 0.8 0.45(

4. 3.70.3

2.3l 0.

0. 0.6

Z)-3-Hexen-1-olacetate

9.65 0.5 0.5

1-Octanol 11.00 0.3Nonanal 11.71 0.8 2.2 1.5 1.1 2.0(E)-2-Decenal 14.72 0.4

-Copaene 16.87 0.4(E,E)- -Farnesene 18.83 0.4 0.5

The most diffused components were 4-hydroxybenzeneethanol, lauric acid and 1-hexadecene, followed by palmitic acid, (E)-2-(9-octadecenyloxy)ethanol, and oleic acid (D’Auria et al., 2002).

Recently the use of 1H NMR in order to characterize olive oilreceived a certain interest (Sacchi et al., 1996; Mannina et al., 1999). The use of 1H NMR allows to determine fatty acid composition, the presence of volatile organic compounds, and the presence ofaldehydes.

Our analyses were performed on six samples of virgin olive oils deriving from the cultivations of Corleto Perticara (Potenza, Southern Italy). The cultivars used were maiatica, olearia, frantoiana, primitiva,


and leccina. Some chemical properties of the olive oils we used areollected in Table 18.

cteri ive o n

Sample Ac[mg KOH/g]

c

Table 18 – Chara zation of ol

Acidity degree[% oleic acid]

ils used in NMR a

id Number

alyses

1 0.28 0.062 0.34 0.07

0.100.080.090.110.11

3 0.50 4 0.38 5 0.45 6 0.53 7 0.33

the spectra wererec

e oil contains a high amount of oleic acid, a low lev

estimated by referring to the allylic protons at 2.02 ppm to all fatty acid chains measurable from the intensity of C2 protons at 2.33 ppm.

1H NMR analyses of our samples were performed indeuterochloroform. In previous work in this field

orded on a solution containing dimethylsulfoxide-d6, in order to achieve to a complete dissolution of the samples. The presence of thisco-solvent did not show the same importance with our samples.

NMR analysis can give some information on fatty acid compositionin the oil. Virgin oliv

el of linoleic acid, and less than 1% of linolenic acid. A higher level of linolenic acid is considered one of the addition of seed oil addition. We can obtain a direct measurement of the linolenic acid level from a characteristic signal at 0.99 ppm. The obtained values are collected in Table 19. Two samples showed a value higher than 1%.

Using the methyl group at 0.90 ppm, we can obtain an indication of the global amount of saturated, monounsaturated fatty acids andlinoleic acid. Table 19 contains these data. These data confirm that this way we can consider most of the fatty acids present in the oil.

The amount of monounsaturated fatty acids and linoleic acid can be

Chapter 248

Trange 49-74% and e of 66%.

T le 1 N ar tio att on in o

Sa le L oleniacid [%

Sa d,nou rated li fatt

acids [%]

onou saturand lin leic fa

aci [%]

atatty acid

[

he obtained values are reported in Table 19: the values are in themost of the samples showed a valu

ab 9 – 1H MR ch acteriza n of f y acid c tents liveoil.

mp in c] mo

turatensatu d, a

an noleic y

M n ted So tty fds

urated

%]

1 0.85 98 65 332 1.70 97 66 343 1.04 99 66 334 0.74 98 74 245 0.86 99 66 336 1.06 99 49 507 0.84 98 72 25

Finally, saturated fatty acids (mainly palmitic and stearic acid) canbe

nce of aldehydic signals in theran

signal at 1.150 ppm (2), unk

evaluated, giving the results showed in Table 19. Some other minor components can be evaluated by using 1H NMR

spectra. We examined a signal of a C18 methyl group of -sitosterol,the presence of volatile organic compounds responsible of signals inthe range 4.5-5.0 ppm, and the prese

ge 8.0-10 ppm.The intensities of the selected resonances were compared with that

at 1.553 ppm, due to methylenes, normalized to 1000; this procedure gives an index proportional to the molar ratio between each compoundand the total amount of fatty chains. We selected a singlet at 0.685 ppm (1 in Table 20) due to -sitosterol, a

nown methyl, the signals at 4.597 (3), 4.667 (4), and 4.719 ppm (5) due to volatile organic compounds. In literature are reported also a signal at 4.886 ppm we did not find. We also considered the signal at 8.068 ppm, identified with a hemiacetalic group, while we did not find signals in the range 9.45-9.70 ppm, identified with aldehydic protons.


Table 20 – Normalized intensities of selected resonances in the 1HNMR spectra of virgin olive oils.

Signal Sample Sample1 2

Sample3

Sample4

Sample5

Sample6

Sample7

1 11.21 5.48 11.03 20.8 17.43 3.92 5.792 20.86 10 19.12 8.43 .85 3.064 0.92 .02 3.075 .81 0 7.6 5.13 3.09 6 0.22 3.48 2.7

.68 20.71.25 1.20 7.4 4

0.86 7.6 50 .83

In two samples we were not able to detect the presence of volatilerganic compounds. In the other four samples the amount of volatile

owork. Furthermore, it is notewo re not able to identifieda c unds vo e ooff-flavor. yd c n in onpresence of tive th

In conclusion we have shown that alysis of lucan virginolive oils can give s sting data for the evaluation of thequality of this oil. We showed that in this oil we have a low content oflinolenic acid, a high amount of volatil ganic co unds,low ntent ehydic

In 2002 we performed oint analys live oi ng bNMR and SPME techniques. Our analyses were performed on five amples of virgin olive oils deriving from the cultivations of Corleto erticara (Potenza). The cultivars used were maiatica, olearia,ra

de

f linoleic acid, and less than 1% of linolenic

organic compounds is remarkable higher than in previous reported

rthy that we aldehydic ompo

Aldehoxida

, whose flaic compounds

r is responsiblan give a

e olive oils.1

of both ardicati

ma andof the

processes in

ome intereH NMR an

e or mpo and aco of ald compounds.

a j is of o ls usi oth 1H

sPf ntoiana, primitiva, and leccina. Some chemical properties of the olive oils we used are collected in Table 21.

1H NMR analyses of our samples were performed in uterochloroform. NMR analysis can give some information on fatty

acid composition in the oil. Virgin olive oil contains a high amount of oleic acid, a low level o

Chapter 250

aci

-0.72 %.

r NMR and SPME

d. A higher level of linolenic acid is considered one of the indices of seed oil addition. We can obtain a direct measurement of the linolenic acid level from a characteristic signal at 0.99 ppm. The obtained values are collected in Table 22. Two samples showed values in the range 0.43

Table 21 – Characterization of olive oils used foanalyses.

Sample Acidity degree[% oleic acid]

Acid Number[mg KOH/g]

1 0.28 0.062 0.34 0.073 0.50 0.104 0.38 0.085 0.45 0.09

Table 22 – 1H NMR characterization of fatty acid contents in oliveoil.

Sample Lino-lenicacid[%]

Saturated,monounsatu-

rated, and linoleic fatty

acids [%]

Monounsatu-rated and

linoleic fattyacids [%]

Saturatedfatty

acids [%]

Syto-sterol[%]

1 0.72 99 87 12 0.172 0.50 99 87 12 0.093 0.43 99.6 89 10.6 0.204 0.50 99 84.6 14.4 0.135 0.62 98.8 88 10.8 0.18

Using the methyl group at 0.90 ppm, we can obtain an indication of the global amount of saturated, monounsaturated fatty acids and linoleic acid. Table 22 contains these data.

These data confirm that this way we can consider most of the fattyacids present in the oil.


The amount of monounsaturated fatty acids and linoleic acid can beestimated by referring to the allylic protons at 2.02 ppm to all fatty cid chains measurable from the intensity of C2 protons at 2.33 ppm.

The obta in the%.

Saturated fatty acids (m inly pa itic ste ac, giving the resu wed b

er minor com ts ca ev ed sin Nxamined a s of a e gr f os

ce o tile o ic pounds responsible of .5-5. , and the presence of aldehydic signals pm.

cted a singlet a 5 ppm due to -sy rol, a signal atm, unknown methyl, the signals at 4.597, 4.667, and 4.719

atile organ pounds. In literature are reported also m we d t find also considered the signal at

ied with iacetalic group, and the signals in the .70 ppm, iden with hy rot

ples we cted pre e la rganics. Furthermor ree les (1, 2, and 5) we detected

of aldehydi oun n t amp s (1 and 5) we nce of the at 8.068 ppm

pec nd t anti tive e luat f th ecan be diffi sually the sitie of eleere compared with that at 1.553 ppm, due to methylenes,

gives an index pro ar ratio between each compound and the total amount of fatty

ch

xen-1--ocimene, nonanal, and -

ained values are reported in Table 16: the values are

range 85-89a lm and aric id) can be

evaluated lts sho in Tan e

le 22.tSome oth ponen b alua b uy g 1H MR

spectra. We e C18 m thyl oup o -syt terolignal(Table 22), the presen f vola rgan comsignals in the range 4 0 ppmin the range 8.0-10 p

We sele t 0.68 toste1.150 ppppm, due to vol ic coma signal at 4.886 pp id no . We8.068 ppm, identif a hemrange 9.45-9 tified alde dic p ons.

In all the sam dete the senc of vo tile ocompound e, in th sampthe presence c comp ds. I wo s lefound the prese signal . However, these signals were very low in the s trum, a he qu ta va ion o escomponents cult. U , inten s the s ctedresonances wormalized to 1000; this procedure n

moportional to the

lains. Considering both that the error in this evaluation could be high

and that we were able to identify volatile organic components andaldehydes in the oil, we preferred to use SPME analysis of the samples in order to obtain more detailed information.

This type of analysis allows us to characterize the volatile organiccomponents in the olive oil. In all the samples we found relevant amounts of ethanol (Table 23). In the flavor we found ethyl acetate, 2-

ethyl-1-propanol, 3- and 2-methyl-1-butanol, octane, (Z)-3-hemol, (E)-2-hexen-1-ol, 1-hexanol, trans-

Chapter 252

farnesene. In most of the samples we also found (E)-2-hexenal, 1-nonanol, and -copaene.

Table 23 – Volatile organic compounds in olive oils. Compound r.t. [min.] Sample

1 2 3 4 5Ethanol 1.62 15.6 16.0 14.3 3.3 16.4Acetic acid 1.92 0.7 0.6Ethyl acetate 2.08 0.5 0.6 0.9 0.4 0.82-Methyl-1-propanol 2.14 0.7 0.7 1.1 0.5 0.73-Methyl-1-butanol 3.25 0.2 0.8 0.6 0.3 0.72-Methyl-1-butanol 3.72 0.1 0.5 0.4 0.3 0.41-Octene 4.14 0.2Octane 4.28 1.7 0.7 1.7 0.6 1.0(E)-2-Hexenal 5.42 0.1 0.3 0.6 0.3(Z)-3-Hexen-1-ol 6.47 (-Hexanol 5.72 1.7 1.4 2.0 3.3 2.3ans- -ocimene 9.67 0.5 1.1 0.7 0.6 0.8

No

0.2 0.1 0.5 0.8 0.2E)-2-Hexen-1-ol 5.68 1.6 0.5 1.4 3.8 0.8

1tr

nanal 10.81 1.5 1.2 1.2 0.7 1.11-Nonanol 12.11 0.1 0.2 0.1(E)-2-Decenal 13.80 0.3Endo Bornyl acetate 14.33 0.2 0.3

-Copaene 15.88 0.1 0.1 0.4-Farnesene 17.87 0.5 0.7 0.4 1.1 0.9

It is noteworthy that in 1H NMR analysis we found aldehydic protons only in three samples while we found nonanal in all the

mples. Furthermore, (E)-2-hexenal was found in four samples and )-2-decenal in one sample. Sample five contains three aldehydes

the 1H NMR spectrum (Figure 2). Then, 1H NMR analysis does not represent a valid method for the

de

sa(Eand we observed only a week signal in1

termination of the amounts of aldehydes in a virgin olive oil.


Figure 12 – Aldehydic signal 1in the H NMR spectrum of sample 5.

In conclusion, we have shown that fatty acids of the virgin olive oil from Basilicata are mainly due to the presence of saturated,monounsaturated, and linoleic fatty acids, those monounsaturated fatty acids constitutes the most important component in this oil, and that the amount of saturated fatty acids is in the range 11-14%. Furthermore,we have shown that 1H NMR analysis of olive oils is not able to givean exact description of the presence of volatile organic compoundsand aldehydes. More detailed information can be obtained from theuse of SPME-GC-MS. Finally, our data confirms the presence of terpenes as components of the flavor of virgin olive oils.

Truffles (Mauriello et al., 2001, 2004)

uring the last thirty years the volatile organic compounds (VOCs)in truffle aroma hav methods (Fiecchiet al., 1967; Ney an 1; Balestreri et al.,

De been analyzed by using severald Frietag, 1980; Claus et al., 198

Chapter 254

19

tensively studied and several compounds have been identified in its flavor.

On the contrary, bis(methylthio)methane was identified as the maincomponent of white truffle (Tuber magnatum Pico) aroma (Fiecchi et al., 1967). VOCs have been determined also in the mycelium of Tuberborchii Vitt. few years ago (Tirillini et al., 2000; Bellesia et al., 2001).

SPME was applied to the analysis of flavors (Boyd-Boland et al., 1994; Yang and Peppard, 1994; Chin et al., 1996; Matich et al., 1996;Clark and Bunch, 1997; Elmore et al., 1997; Song et al., 1997; Steffen and Pawliszyn, 1997). The use of SPME in the determination ofsulphur components of black and white truffle flavor has been also reported (Pelusio et al., 1995).

Figure 13. Tuber mesentericum

86; Talou et al, 1987; Bellina Agostinone et al., 1987; Angeletti et al., 1988; Fiecchi, 1988; Balestreri et al., 1988; Hanssen and Kühne, 1988; Flament et al., 1990; Pacioni et al., 1990; Talou et al., 1990 ;Bellesia et al., 1996a ; Bellesia et al., 1996b ; Bellesia et al., 1998a; Bellesia et al., 1998b). Black truffle (Tuber melanosporum Vitt.) hasbeen ex


We analyzed samples of Tuber mesentericum Vitt., Tuber borchiiVitt., Tuber aestivum Vitt. and its form uncinatum (Chatin), Tubermagnatum Pico, Tuber brumale Vitt. and its form moschatum (Ferry),Tuber excavatum Vitt., Tuber melanosporum Vitt., Tuberoligospermum (Tul. & C. Tul.) Trappe, and Tuber panniferum Tul. & C. Tul.

Chapter 256

Twenty-seven VOCs were identified in samples of T. mesentericu(Fig. 13, T

mable 24).

The same table contains the per cent area for each identifiedmponent. This is the first report

uffle species. The compounds found with high frequency were ethylsulphide (found on 100% of the sam , 3% uene (57%) ethylbutanal (43%), and nO (Fig. 14) are listed in Table 25. High lt bserved in th avor anal sam les.e e rese in thr the rc h VOCs ere fo only al ctanol, ethanol, acetic acid, and decane,

re were encountered in single carpophores.

co dealing with the aroma of this trdim ples) -methylanisole(79 ), 2,5-dimethoxytol , 3-mbuta one (36%).

T. excavatumV Cs found inresu variability was o e fl

e pof yzed p

Dimasco

thylsulphide and 3-octanone warps subjected to analysis. Ot

rer

ntw

ee ofu

fouinnd

coup e of samples, i.e. 3-owhe as the remaining ones

Figure 14. Tuber excavatum


Table 25 –VOCs identified in T. excavatum and corresponding per centarea.

Compound Sample1 2 3 4

2 ethanol 38.9 2.37 dimethylsulphide 2.4 3.4 2.215 2-methyl-1-propanol 4.416 acetic acid 4.5 0.418 3-methylbutanal 4.821 cis-methylpropenylsulphide 1.5 22 3-methyl-1-butanol 5.124 2-methyl-1-butanol 2.826 toluene 0.334 1,3-dimethylbenzene 0.137 styrene 0.339 3-octanone 6.4 5.2 17.440 benzaldehyde 7.244 1-octen-3-ol 48.545 2-pentylfuran 0.747 3-octanol 6.2 31.359 decane 1.1 1.363 benzo[c]thiophene 0.2

Samples of T. borchii (Fig. 15) also showed a high variability in thearoma composition (Table 26). It is noteworthy that all of them were lacking dimethylsulphide. Two VOCs (2-methyl-1,3-butadiene and 1,2-pentadiene) were found with high per cent area in two ascocarps which also contained 5-11 other volatile substances. VOC number persample varied from 6 to 16. Sometime 1,2-pentadiene was present in lower percentage but was accompanied by discrete amounts of 1-methylpropyl formate, tetradecanal, and tetradecane, or by 3-octanoneand very little amounts of other 15 VOCs.

Chapter 258

Figure 15. Tuber borchii

Tir coworkers analyze y lium . b rchiill 0). We have to note that there is not superposition e ts and those repo in hat w In , o y 3-

n ne, and ethynylbenze e were found in both the studies. n es of T ber a um (Fig. 16) and T.

iv atum gave th o ed in ble 27. e pound found with the highest frequency

5% of the samples). Butanone and 3-methylbutanal were also ens, respectively. Other VOCs

fou

illini and d VOCs in m ce of T o(Tiri ini et al., 200betw en our resul rted t ork. fact nlocta one, deca n

A alyses of carpophor u estivaest um f. uncin e results sh w TaDim thysulphide was the com(8identified in 60% and 55% of specim

nd in discrete amounts in some samples were dimethylsulphide, 1-methoxy-3-methylbenzene, butanone, ethanol, and ethyl acetate. It is noteworthy that in T. aestivum f. uncinatum the amounts ofdimethylsuphide were always lower than in T. aestivum.


Table 26 –VOCs identified in T. borchii and corresponding per cent area.

Compound Sample1 2 3 4 5

2 ethanol 0.7 0.84 1,2-pentadiene 63.1 5.7 6.45 2-methyl-1,3-butadiene 42.7 2.3 11 2-butanol 0.1 14 2-methylfuran 0.2 2.5 0.215 2-methyl-1-propanol 0.2 17 2-methylbutanal 0.7 0.6 0.318 3-methylbutanal 0.1 2.819 pentanal 1.0 20 1-methylpropyl formate 8.622 3-methyl-1-bu .3tanol 224 2-methyl-1-butanol 1.8 0.6 0.526 toluene 0.3 0.1 29 3-methylthiophene 0.3 0.3 0.334 1,3-dimethylbenzene 0.2 0.1 37 styrene 0.2 0.1 39 3-octanone 0.6 6.648 -ocimene 0.7 0.3 1.249 decane 1.7 0.5 0.963 benzo[c]thiophene 0.2 0.4 68 tetradecane 23.2 0.1 69 tetradecanal 17.2 1.2

The above results are in substantial agreement with those of Bellina Agostinone (1987) who found same main VOCs in both the truffle entities. On the contrary, other VOCs (i.e. 3- and 2-methylbutanal, 2-phenylethanol, 3- an by Bellesia et al.(1998a) in T. uncinatum were not detected here.

d 2-methyl-1-butanol) found

Chapter 260

Figure 17. Tuber magnatum

Figure 16. Tuber aestivum

Chapter 262

SPME-GC-MS analysis of white truffle samples (T. magnatum)showed that only seven VOCs were present (Fig. 17, Table 28).

imethylsulphide was present in all the examined samples in p rce ing from 16.6%. With the exception of 2- and 3-meth butanal (found in sam s), only sulphur compounds were p se ent with th resu sr or nd ne 87) B ia 96 sd io did not find 1,2,4-trithiolane, m th thio)methyldisulphide, a tris thy o)m anr r io (199

Cs iden in agn tum co pond g pcent area

p

De ntage vary 0.4 to

yl two plere nt. The most abundant VOC, in agreem e ltep ted by Hansen a

pentane. WeKüh (19 a dn elles (19 ), wa 2,4-

ithe yl(methyl nd (me lthi eth e as

epo ted by Pelus 5).

Table 28 –VO tified T. m a and rres in er.

Compound Sam le1 2 3 4 5

7 dimethylsulphide 16.6 0 4 .7 3.8. 9.4 017 anal 0 6 .12-methylbut . 018 thylbutanal 1.7 0.13-me23 thyldisulphi e .3dime d 0.6 033 pentane 7.0 2.1 6.42,4-dithio 541 hyltrisulph e .1dimet id 062 ethyltrisulp etrim hid 1.2

T. brumale ( Fig. 18) and T.b tum (T e 29

h entati T. brumale were d methylbutanal, 1-methylpropylform ropanal, and 1,4-dimethoxybenzene. In T. brumale f o dimethylsulphide reached lower amo nts th n inb main other VOCs found were 2-methylpropanal,butanone, 2-methylbutanal, and 1,4-dimethoxybenzene.

Twenty-eight VOCs were found inrum

Tale f. moschae most repres

a lbve com

).pounds found in

imethylsulphide, butanone, 2-ate, 2-methylp

. m schatum, u a T.rumale. The


Table 29 - VOCs identified in samples of Tuber brumale (*) and itsrm moschatum and corresponding per cent area.

ompound

fo

C Sample1 (*) 2 3 4 5 ) 76 (*

1 acetaldehyde 0.12 ethanol 0. 0.2 0.70.3 2 0.77 dimethylsulphide .3 0. 1. 3 0 6.715 3.4 6 2 .0 9.9 2-methyl-

propanal4.2 11.6 3.0 1.9 2.4 0.8 2.4

10 ylformate

4.3 0.8 1.1 5.7 10.7 4.3 1.1methyleth

12 butanone 2.5 11.8 7.3 0.8 1.3 1.5 2.713 ethyl acetate 0.3 0.2 0.3 0.1 0.2 1.115 2-methyl-1-

propanol0.8 1.9 0.8 3.3 1.3

17 2-methylbutanal 12.8 18.6 5.2 7.2 3.0 2.5 8.618 3-methylbutanal 0.7 2.2 0.4 0.5 0.3 0.4 0.420 1-methylpropyl

formate21.7 4.0 2.1 4.3 16.5 8.8 8.7

24 2-methyl-1-butanol

1.0 2.5 1.1 1.1 1.6

25 1-methylpropylacetate

0.1

26 toluene 0.134 1,3-dimethyl-

benzene0.1

37 styrene 0.238 anisole 0.1 0.8 0.3 0.2 0.3 0.339 3-octanone 0.144 1-octen-3-ol 0.14 1-methoxy-3-

methylbenzene2.7 0.4 0.2 2.66

49 decane 0.1 0.85 1-methoxy-4-

methylbenzene0.10

51 3-ethyl-5-methylphenol

0.2 0.2 0.3

52 limonene 0.0454 1-propynyl-

benzene0.1

Chapter 264

Table 29. Continued

Compound Sample 1 (*) 2 3 4 5 6

(*)7

60 1,4-dimethoxy-benzene

8.8 7.4 31.1 5.1 6.2 17.5

64 benzo[b]thio-phene

0.5

66 1,2,4-trimethoxy-benzene

1.3 3.5 4.5 9.0 0.5 5.6 0.3

Figure 18. Tuber brumale

ected, whereas 1-propanol, 3-octanol, 3-

It is interesting to note that our data fit the result reported by Bellesia (1996a). Furthermore, in this study six compounds never determined in the past (1-methoxy-3-methylbenzene, decane, anisole, 3-ethyl-5-methylphenol, 1,4-dimethoxybenzene, and 1,2,4-trimethoxybenzene) were det


m ,methylphenols, and 3-methy re absent.

Although the samples of T. melanosporum (Fig. 19) were only two, the results of the analyses were uniform. As showed in Table 30, little am ethysulphide were found. The main VOCs were 2-me lbu lpropanol, and 2-methyl-1-butanol. The following six esters, never detected before, were also found: ethyl 2-me lbu opyl 2-methylbutanoate, 2-methylbutyl2-m ylb lpropyl 2-methylpropanoate, and 3-me u ate. As it is well known, the organic este re avor. Therefore, these -mcom n ortant role in the definition of the aromaof t ru

Tuber melanosporu

he PME-GC-MS analyses of T. oligospermum ecollected in Table 31. Only propanone was detected in two samp s.The other VOCs were present with the former in mixture with

ren es.

ethyl-1-butanol, 5-hexen-2-ol, 3-nonanol, benzylic alcohollbutylamine we

ounts of dimthy tanal, 2-methy

thy tanoate, 2-methylpreth utanoate, 2-methy

lpropanothylb tyl 2-methyrs a strictly related to fl

pabove entioned

pou ds could play an imhis t ffle species.

Figure 19. m

T results of S arle

diffe t organic substanc

Chapter 266

Table 30 –VOCs identified in T. melanosporum and corresponding per cent area.

Compound Sample 1 2

1 acetaldehyde 2.0 1.1 2 ethanol 3.4 1.6 7 dimethyllsulphide 1.4 2.1 9 2-methylpropanal 3.2 4.7 12 butanone 1.3 1.3 15 2-methylpropanol 12.7 9.8 17 2-methylbutanal 9.6 18.4

1.3 1.1 18 3-methylbutanal0.3 0.8 20 1-methylpropyl formate0.922 3-methyl-1-butanol

1-butanol24 2-methyl- 24.9 25.5 27 hexanal 0.7 0.7 31 ethyl 2-methylbutanoate 0.2 0.3 36 2 0.3-methylpropyl 2-methylpropanoate38 anisole 0.1 42 2-methylpropyl 2-methylbutanoate 0.9 0.7 43 -m -methylpropano 0.2 0.5 2 ethylbutyl 2 ate53 -m thylbutyl 2-methylbutanoa 0.4 1.4 2 e te

Table V T. oligospermum co ondingent area.

C p le

31 – OCs identified in and rrespper c

om ound Samp 1 32

1 ac l 0.6eta dehyde3 p a 2.6rop none 0.917 2- tme hylbutanal 0.618 3- tme hylbutanal 2.0


Figure 20. Tuber panniferum

Table 32 - VOCs identified in T. panniferum and corresponding per cent area.

Compound Sample1 2 3

9 2-methylpropanal 1,0 1,7 1,812 butanone 16,7 3,817 2-methylbutanal 1,6 1,418 3-methylbutanal 1,3 1,423 dimethyldisulphide 0,3 0,533 2,4-dithiopentane 0,5 18,7 22,339 3-octanone 3,4 40 benzaldehyde 1,7 1,572 6(Z),9(E)-heptadecadiene 0,2 73 1-heptadecene 0,3 74 1-nonadecene 0,3

Chapter 268

Finally, Table 32 summarizes the results we obtained from theanalyses of T. panniferum (Fig. 20). It is noteworthy that in T.panniferum, as in T. magnatum, the main constituent of the aroma is 2,4-dithiopentane.

We have seen that SPME represents a useful non invasive methodto determine volatile organic compounds. SPME analysis is characterized by the absence of chemical manipulation of the samples.This way, when some truffle species have been analyzed in the past, we do not find some compounds. These results could be in agreementwith the hypothesis that they are artifacts.

Wine (D’Auria et al., 2003)

We tested the production of volatile organic compounds from different stocks of S. cerevisiae. In particular we verified the different capability of S. cerevisiae deriving from both Northern and Southern Italy to produce volatile organic compounds.

The results are reported in Table 33 and 34. S. cerevisiae stocks deriving from Southern Italy were able to produce more volatile organic compounds (59) than those from Northern Italy. The meanvalues were 19.5 ( 10.4) and 14.1 ( 5.7), respectively. The mainproducts obtained were ethanol, ethyl acetate, 3-methyl-1-butanol, 2-methyl-1-butanol, 2-phenylethanol, ethyl octanoate, ethyl 9-deceonoate, ethyl decanoate, ethyl dodecanoate, and ethyl hexadecanoate. Ethanol was produced in larger amount in Southern Italy stocks than in those deriving from Northern Italy [mean value 47.14% ( 18.1) vs 45.14% ( 14.0)]. Also the production of 2-phenylethanol, ethyl octanoate, and ethyl decanoate was larger from S.cerevisiae from Southern Italy [1.83% ( 1.3), 3.42 % ( 3.7). and 4.44% ( 4.4) vs 1.31% ( 1.5), 2.80% ( 2.4), and 3.60% ( 3.8), respectively]. On the contrary, the production of 3-methyl – and 2-methyl-1-butanol was higher by using S. cerevisiae deriving from Northern Italy [6.93% ( 4.0) and 2.47% ( 0.9) vs 7.98% ( 3.2) and 3.94% ( 0.9), respectively].

SPME in natural matrices 69SPME in natural matrices 69

Chapter 270

Chapter 272

Chapter 274

The production of the other main volatile organic compounds was almost the same for both the S. cerevisiae stocks.

Photodegradation can be responsible of off flavors in some ediblematerials. Some components of the flavor can be modified in thepresence of light. For example, the photoisomerization of humuloneint

ive (De Keukeleire, 2001). Gray et al. were first to show at thiols were involved in the development of an offending off-

flavor (Gray et al., 1947). In the early sixties, Kuroiwa et al. (1963) used model systems to establish that a photochemical reaction in the wavelength range of 350-500 nm, involving a flavin such as riboflavin, beer bitter agents (isohumulones), and sulfur-containing compounds, led to the so-called “lightstruck flavor”. Other drinks including champagne, wine, and milk are also sensitive to light; however, none produces the unique “skunky” odor and taste of light struck beer. Then, the need to avoid light irradiation is not restricted tobeer.

Champagne is one of the most famous sparkling wines in the world. It is obtained from three types of grapes, pinot noir, pinot meunier, and chardonnay, in a particular and well defined region in the North of France. With its northern geographical position at the limits of the vine’s cultural zone, the climate is harsh, softened only by an oceanic influence. The chalky sub-soil provides the vine with naturally constant irrigation. The vines’ position on the slopesprovides the best sunlight and the run-off of any excess water.

French experts observed that the quality of champagne was

pposed to traditional liquor stores. Eventually, it was discovered that e

o trans- and cis-isohumulone in the beer has been studied (DeKeukeleire, 2001). The problem of a particular off-flavor in beer exposed to light was recognized as early as in 1875, and simple tests on the protective power of glass indicated that brown bottles weremost effectth

distinctly inferior when the bottles were sold in supermarkets asoth intense fluorescent lighting traditionally present in large retailstores produced that the struck favor (“goût de lumière”), triggered by photochemical transformations involving sulphur components, such as methionine and cisteine, which produce H2S, CH3SH, and (CH3)2S(Maujean and Seguin, 1983).


The “sunlight flavor” is reported to be produced easily in clear bottles of chardonnay and pinot gris wine with a riboflavin content of over 200 g l-1, when exposed to reflected light for two or three weeks, while a concentration below 100 g l-1 is considered safe for such wines (Pichler, 1996).

We analyzed the photochemical behavior of two samples of Piper-Heidsieck Champagne. In order to obtain valuable data on the modification of the flavor, we carried out the analysis of head space of

a pagne sample by using solid phase micro extraction technique ch m(Table 35)

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

Abs

orba

nce

5

263,8

269,8

275,8

281,8

287,8

293,8

299,8

305,8

311,8

317,8

323,8 33

836

839

842

8

wavelength [nm]

Figure 21. UV spectrum of champagne. Path length of the cell: 1 cm.

The sample we used showed the uv spectrum reported in Figure 21. It showed absorptions at 290 and 301 nm and a shoulder at 330 nm. We irradiated the first sample for 24 h with 15 W ultraviolet lampthrough Pyrex. To estimate modifications in the flavor compositionwe performed a calibration curve of one of the most abundant

Chapter 276

component after ethanol, 3-methyl-1-butanol. On the basis of this calibration curve we observed that in our sample 3-methyl-1-butanolwas contained in concentration of 155 mg l-1. This concentration did not change after the irradiation. On the basis of this result we report the concentration of the other components of champagne flavor as per cent referred to 3-methyl-1-butanol (100%). The result of this test is reported in Figure 22.

1-pr

opan

olet

hyl a

ceta

te2-

met

hylp

ropa

nol

3-m

ethy

lbut

anol

ethy

l but

anoa

tel 2

-hyd

roxy

prop

anoa

te1-

hexa

nol

yl-1

-but

anol

ace

tate

ethy

l hex

anoa

te2-

phen

ylet

hano

let

hyl b

utan

edio

ate

ethy

l oct

anoa

teet

hyl d

ecan

oate

6-di

-t-bu

tylp

heno

l

ethy

3-m

eth di 2,

S1

020

40

60

80

100

120

140

% referred to 3-methyl-1-butanol

Compound

.

Figure 22. Change in the composition of champagne flavor after 24 h irradiation. The concentration is reported as per cent referred to 3-methyl-1-butanol. Back: before irradiation; front: after irradiation.


We showed the presence of fourteen components in the flavor; some of these compounds were observed in very low concentration. We did not observe the formation of compounds containing sulfur. However, we observed some significant modifications in thecomposition of the flavor: 1-propanol and 1-hexanol contents did not change during the irradiation, the amount of 2-methylpropanolincreased, while 2,6-di-t-butylphenol disappeared after the irradiation.

3

4

5

6

d to

3-m

ethy

l-1-b

utan

o

7

l

1

2

% re

ferr

e

00 2 6

irradiation time

Figure 23. Evolution of the concentration of suitable components of champagne during irradiation. A: 1-propanol; C: 2-methyl-1-propanol;E: ethyl butanoate; G: 1-hexanol; H: 3-methyl-1-butanol acetate; J: 2-phenylethanol; L: diethyl butanedioate; O: 2,6-di-t-butylphenol.

Furthermore, the presence of esters in the wine after the irradiationwas completely modified. Ethyl acetate, ethyl butanoate, ethyl 2-

Chapter 278

hydroxypropanoate, 3-methyl-1-butanol acetate, ethyl hexanoate, and ethyl octanoate reduced their presence in the wine; ethyl decanoatedisappeared in the flavor after the irradiation. Furthermore, we did notfind the corresponding acids.

The second sample of champagne was irradiated for 2 and 6 hours. This way we could follow the evolution of the wine during the time:we follow this behavior in order to understand whether some other intermediates were formed. The results are collected in Figures 23 and 24.

0

20

40

60

80

100

160

120

140

-1-b

utan

ol

0 2 6

irradiation time (h)

% re

ferr

ed to

3-m

ethy

l

Figure 24. Evolution of the concentration of suitable components of champagne during irradiation. B: ethyl acetate; D: 3-methyl-1-butanol; F: ethyl 2-hydroxypropanoate; I: ethyl hexanoate; M: ethyl octanoate; N: ethyl decanoate.

The concentrations during irradiation of the minor components are collected in Figure 23: we can see that, while the contents of 2-mehyl-


1-p

thylexanoate, and ethyl decanoate decreased during the irradiation.

On the basis of the above reported results we can conclude that thet affect proteins in the wine with the

rmation of compounds containing sulfur. Furthermore, the morerel

-1) for wineslef

ac and Décor, 1967).

ited state and substrates. Thesem

ropanol,1-hexanol, and 2-phenylethanol did not undergo severemodifications, the concentrations of 1-propanol, ethyl butanoate, diethyl butanedioate, 3-methyl-1-butanol acetate, and 2,6-di-t-butylphenol decreased during irradiation. The same behavior was observed considering the main components (Figure 24): theconcentrations of ethyl octanoate, ethyl 2-hydroxypropanoate, eh

irradiation of champagne does nofo

evant change in the flavor is related to the decomposition of the ester contents in the wine.

As reported above, riboflavin was considered the compoundsresponsible for “goût de lumière”. The principal forms of riboflavin (vitamin B2) found in nature are flavin mononucleotide and flavin-adenine dinucleotide. Free riboflavin is also naturally present in raw and processed fruits (Goverd and Garr, 1974) and fermentedbeverages. Flavin mononucleotide and flavin-adenine dinucleotide can be converted to riboflavin prior to quantitation, in order to obtain the total riboflavin content.

The total riboflavin content was reported to be 50-70 g l-1 in grapeand in must, the content in wine rises to 110-250 g l-1 during fermentation and it can be further enriched (160-318 g l

t in contact with yeast for four to six days after fermentation is completed (Ournac, 1968; Ourn

The riboflavin absorption spectrum in aqueous medium exhibits four structure less peaks centered at 446, 375, 265 and 220 nm with high molar extinction coefficients (>104 M-1 cm-1) (Heelis, 1982).Riboflavin is particularly sensitive to uv and visible light and induces both Type I and II photosensitized oxidation mechanisms. The formerinvolves the formation of free radicals through hydrogen or electron transfer between riboflavin triplet exc

i oxidized substrate can undergo further oxidation in the presence of oxygen. The Type II process involves the formation of singlet oxygen by energy transfer from triplet excited riboflavin to molecular oxygen.

Chapter 280

We tested the capability of riboflavin to catalyze the decompositionof aliphatic esters.We verified whether riboflavin was able to induce decomposition of aliphatic esters. The reaction we carried out isdepicted in Scheme 2.

Scheme 2

OH

OH

O

O N

N

N

HN

HOCH2OH

O

O

+hv decomposition of the ester

One hour irradiation of 14 mg of ethyl hexanoate in the presence of riboflavin (2 mg) in ethanol water with a 125 W mercury arc through Pyrex induced 9% decomposition of the ester.

Riboflavin is able to induce decomposition of the esters: probably this reaction occurs through a Type I photosensitized mechanism. In fact, in our knowledge, singlet oxygen is not able to attack aliphatic esters.

In conclusion, we have shown that the “goût de lumière”, observed in champagne, can have a different origin from that described in previous reported articles in this field. We showed that irradiationinduces several modifications in flavor composition where esters are selectively decomposed. Furthermore, we showed that riboflavin is able to induce the same type of decomposition in ethyl hexanoate.

Honey (Bentivenga et al., 2004)

Honey aroma has been studied for years. The composition of thevolatile fraction is directed by floral origin, the foraging habits and physiology of the bees. Nowadays, more than 400 compounds have been identified and described as volatiles in honeys of different floral


types. However, a large number of new volatile compounds are expected to be identified, because there are still many honey types notyet studied. Considering only most recent work in this field, Radovic et al. (2001) showed the presence of 110 compounds in 43 authentic honey samples of different botanical and geographical origins. Four hundred compounds were separated by gas chromatography and massspectrometry in order to establish the presence of floral origin markers(Guyot et al., 1999). Seventy-two compounds were identified in Cashew and Marmeleiro honeys (Moreira et al., 2002).

SPME has been used in the determination of the flavor componentsin honeys (Perez et al., 2002).

The use of honey to determine the presence of pollutants such as benzopyrene has been proposed. In this work the determination offlavor components in honey has been performed on samples deriving from the zone of Corleto Perticara (Potenza, Basilicata, SouthernItaly): this land is interested to an intense oil extraction and in thiswork we want to verify if this activity induces the presence ofanthropogenic compounds in honeys.

We analyzed 13 samples of honey deriving from the same part ofBasilicata (Table 36 collects some characterization data about thesesamples). In all the samples (Table 37) we found a compound never detected before in honey, 2-ethyl-3-hydroxyhexyl 2-methylpropanoate. In eleven samples we found another 2-methylpropanoate ester, 2,2-dimethyl-1-(2-hydroxy-1-methylethyl)propyl 2-methylpropanoate (Table 37). This compoundhas not been recovered in honey until now. The first ester has been determined in apricot (Gomez et al., 1993), while the other ester was found in green tea (Shimoda et al., 1995). Also nonanal is present in eleven samples (Table 37). Decanal was found in nine samples, while 1-methylethyl dodecanoate in eight.

It is noteworthy the presence of 1-butylheptylbenzene in eightsamples, of 1-pentylheptylbenzene in nine samples, of 1-butyloctylbenzene in ten samples, of 1-propylnonylbenzene in eight samples, and of 1-pentyloctylbenzene in seven samples (Table 37). Furthermore, we found some other aromatic hydrocarbons in a lower number of samples.

Chapter 282

Chapter 284

Chapter 286

Table 38 – Volatile organic compounds from wax samples

Compound r.t. [min.] Area %Sample 1 Sample 2

Ethanol 1.62 0.5Limonene 9.29 0.2Nonanal 10.83 0.3 0.9Octanoic acid 12.08 0.2Dodecane 12.65 0.2De

new 13 - aracne · 2017. 9. 20. · expression: when odorants are forced out of the natural source...

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