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A Comprehensive Review on Vanilla Flavor: Extraction, Isolation and Quantification of Vanillin and Others Constituents

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Vanilla, being the world's most popular flavoring materials, finds extensive applications in food, beverages, perfumery and pharmaceutical industry. With the high demand and limited supply of vanilla pods and the continuing increase in their cost, numerous efforts of blending and adulteration in natural vanilla extracts have been reported. Thus, to ensure the quality of vanilla extracts and vanilla-containing products, it is important to develop techniques to verify their authenticity. Quantitatively, vanillin is the major compound present in the vanilla pods and the determination of vanillin is a vital consideration in natural vanilla extracts. This paper provides a comprehensive account of different extraction processes and chromatographic techniques applied for the separation, identification and determination of chemical constituents of vanilla. The review also provides an account of different methods applied for the quantification and the authentification of chemical constituents of vanilla extract. As the various properties of vanilla are attributed to its main constituent vanillin, its physico-chemical and bioactive properties have also been outlined.
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A comprehensive review on vanilla flavor: Extraction,
isolation and quantification of vanillin and others
constituents
Arun K. Sinha
a
; Upendra K. Sharma
a
; Nandini Sharma
a
a
Natural Plant Products Division, Institute of Himalayan Bioresource Technology,
Palampur, Himachal Pradesh, India
First Published on: 18 September 2007
To cite this Article: Sinha, Arun K., Sharma, Upendra K. and Sharma, Nandini
(2007) 'A comprehensive review on vanilla flavor: Extraction, isolation and
quantification of vanillin and others constituents', International Journal of Food
Sciences and Nutrition, 59:4, 299 - 326
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A comprehensive review on vanilla flavor: Extraction,
isolation and quantification of vanillin and others
constituents
ARUN K. SINHA, UPENDRA K. SHARMA, & NANDINI SHARMA
Natural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur,
Himachal Pradesh, India
Abstract
Vanilla, being the world’s most popular flavoring materials, finds extensive applications in food,
beverages, perfumery and pharmaceutical industry. With the high demand and limited supply of
vanilla pods and the continuing increase in their cost, numerous efforts of blending and
adulteration in natural vanilla extracts have been reported. Thus, to ensure the quality of vanilla
extracts and vanilla-containing products, it is important to develop techniques to verify their
authenticity. Quantitatively, vanillin is the major compound present in the vanilla pods and the
determination of vanillin is a vital consideration in natural vanilla extracts. This paper provides a
comprehensive account of different extraction processes and chromatographic techniques
applied for the separation, identification and determination of chemical constituents of vanilla.
The review also provides an account of different methods applied for the quantification and the
authentification of chemical constituents of vanilla extract. As the various properties of vanilla
are attributed to its main constituent vanillin, its physico-chemical and bioactive properties have
also been outlined.
Keywords: Comprehensive review, gas chromatography, high-performance liquid chromato-
graphy, vanilla flavor, Vanilla planifolia, vanillin
Introduction
In recent years, there has been growing interest in natural and healthy foods, especially
with regards to the ingredients such as flavoring agents and preservatives. Amongst the
variety of natural flavors in use today, vanilla occupies a prominent market place and
has been in use for the preparation of ice creams, chocolates, cakes, soft drinks,
pharmaceuticals, liquors, perfumery and in nutraceuticals (Ranadive 1994). Natural
vanilla is a complex mixture of flavor components extracted from the cured pods of
different species of plant genus Vanilla: Vanillus planifolia and Vanillus tahitensis (Rao
and Ravishankar 2000). However, V. planifolia is valued most because of its pod
quality and yield. The fruity, floral fragrance of cured vanilla pods, combined with a
deep, aromatic body, makes it a widely accepted flavoring agent.
The history of vanilla begins with its discovery in Mesoamerica during the 1300s.
The Aztecs (natives of Mexico) are more often cited as the first culture to use and
Correspondence: Arun K. Sinha, Natural Plant Products Division, Institute of Himalayan Bioresource
Technology, IHBT Communication no.006, Post Box No. 6, Palampur 176061, Himachal Pradesh, India.
Tel: 91 1894 230426. Fax: 91 1894 230433. E-mail: aksinha08@rediffmail.com
ISSN 0963-7486 print/ISSN 1465-3478 online # 2008 Informa UK Ltd
DOI: 10.1080/09687630701539350
International Journal of Food Sciences and Nutrition,
June 2008; 59(4): 299326
Downloaded By: [CSIR eJournals Consortium] At: 13:10 28 April 2008
domesticate vanilla to flavor their drinks. Thereafter, the Spanish conquest of Aztec
culture brought it to Europe. Efforts were made to cultivate vanilla outside Mexico
but, owing to the absence of a natural pollinator, the crop was a failure*until the
botanist Charles Morren (1836) discovered the secret of vanilla’s reluctance to bear
fruit outside Mexico, which has led to the discovery of artificial pollination of vanilla
flowers (Reineccius 1997). From the time of the Aztecs, vanilla has been considered
an aphrodisiac, carminative and a stimulant. Venezuelans commonly use the pods for
treatment of fever and spasm. It is used as an antispasmodic and aphrodisiac in
Argentina. In Palau, vanilla is used for curing dysmenorrhea, fever and hysteria (Duke
et al. 2003). There have been reports whereby vanilla is known to protect the skin
against free radicals (Sophie and Francois 2003). Thus, owing to its medicinal
properties, besides being a highly valued flavoring agent, vanilla has tremendous
potential to be used as a food preservative and health food agent.
The active constituents of vanilla are responsible for its various biological and
therapeutic activities. The flavor profile of vanilla contains more than 200 compo-
nents, of which only 26 occur in concentrations greater than 1 mg/kg. The aroma and
flavor of vanilla extract is attributed mainly due to presence of vanillin (4-hydroxy-3-
methoxybenzaldehyde; Figure 1), which occurs in a concentration of 1.02.0% w/w in
cured vanilla pods (Westcott et al. 1994; Bettazzi et al. 2006; Sharma et al. 2006).
True vanilla pods possess a pure delicate spicy flavor that cannot be duplicated exactly
by synthetic products. For this reason, and because of limited supply, natural vanilla is
able to command a premium price, leading to numerous efforts of its blending and
adulteration. Also, the flavor quality of vanilla extracts vary considerably, depending
upon the origin, curing technique used, storage conditions, extraction methods, and
age of the vanilla extract itself. Thus, the availability of effective analytical techniques
for monitoring the quality and, as far as possible, maintaining uniformity of quality
over time is imperative (Poole et al. 1995). As a consequence, reliable and practical
analytical methods for the identification and determination of chemical constituents in
vanilla pods are of considerable interest.
Keeping the above in view, the present review is focused on various methodologies
available for the extraction, separation and quantification of chemical constituents of
vanilla flavor. In addition, various physico-chemical and bioactive properties of its
main constituent (i.e. vanillin) and methods applied for its authentification for quality
assurance have also been reviewed.
Figure 1. Structure of vanillin.
300 A. K. Sinha et al.
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Plant sources of vanilla
Vanilla is obtained from different species of plant genus Vanilla (family: Orchidaceae),
a tropical climbing orchid. Out of the total 110 species, only three species find
commercial importance. These are V. planifolia Andrews or Vanillus fragrans (Salis-
bury) Ames, V. tahitensis JW Moore and Vanillus pompona Scheide. However,
commercial vanilla is obtained from V. planifolia. V. pompona, also called vanillon,is
of lowest grade and is often used as an adulterant or by perfumers or tobacco
manufacturers (Arditti 1992). Although pods obtained from V. pompona are not
valued due to their low quality, the plant has some important traits such as growth
under adverse conditions and resistance to root-rot disease. These traits make it an
ideal candidate for use in cross-breeding programs and thus improving the
commercial source of vanilla (Havkin-Frenkel and Dorn 1996). Green vanilla pods
possess no flavor. The characteristic flavor and aroma of vanilla pods develops during
the curing process in which enzymatic changes occur. The action of naturally induced
b-glycosidases on the glycosides releases various vanilla flavor components. Curing
process consists of four steps: scalding/killing, sunning/sweating, drying and con-
ditioning/aging (Karas et al. 1972; Havkin-Frenkel and Dorn 1996; Dignum et al.
2002).
Chemistry of vanilla
Since the last century, identification of the chemical components of vanilla has
attracted considerable attention and more than 200 compounds have been identified
(Klimes and Lamparsky 1976; Galetto and Hoffman 1978; Adedeji et al. 1993;
Ramaroson-Raonizafinimanana et al. 1997; Wekhhoff and Guntert 1997; Pe´rez-Silva
et al. 2006). The characteristic aroma of the vanilla flavor is due to the presence of a
large number of compounds in the vanilla extract. Various non-volatile constituents
that impart the characteristic flavor to vanilla include tannins, polyphenols, free amino
acids and resins (Rao and Ravishankar 2000). An extract containing large amounts of
resins will retain aromatic compounds far longer than one that has smaller quantity of
them (Reineccius 1997).
Volatile constituents that are responsible for the aroma and flavor of vanilla are
acids, ethers, alcohols, acetals, heterocyclics, phenolics, hydrocarbons, esters and
carbonyls (Klimes and Lamparsky 1976). A comprehensive account of various
chemical constituents present in green and cured vanilla pods has been reviewed
earlier (Dignum et al. 2001). Among the various volatile compounds reported, vanillin
is the single most characteristic component of flavor. The chemical identity and
physico-chemical properties of vanillin are summarized and presented in Table I.
Bioactive properties
Because of advancements in chemistry and pharmacology, most of the earlier uses of
vanilla have given way to functional uses of vanillin, vanilla’s main constituent. In
recent years, researchers have been exploring vanillin’s properties as an antioxidant,
antimicrobial, anticarcinogenic and antisickling agent. Also, in the food industry there
is a growing interest in naturally occurring flavor compounds that exhibit antioxidant
and antimicrobial activity and therefore provide a potential source of novel
Comprehensive review on vanilla flavor 301
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preservatives. In this regard, some of the reported bioactive properties of vanillin are
presented below.
Antimicrobial activity
Vanillin and its direct structural analogues exhibit varying degrees of antifungal
activity against the different moulds and yeasts that cause food spoilage (Fitzgerald
et al. 2005). Vanillin has been reported to inhibit the growth of moulds and yeasts in
fruit purees, fruit-based agar systems (Lo´pez-Malo and Alzamora 1995; Cerrutti and
Alzamora 1996; Cerrutti et al. 1997; Lo´pez-Malo et al. 1998) and in soft drinks
(Fitzgerald et al. 2004a). The antifungal activity of vanillin against the medicinally
important yeasts Candida albicans and Cryptococcus neoformans has also been
documented (Boonchird and Flegel 1982). The aldehyde moiety of vanillin has
been shown to play a key role in the antifungal activity. However, the side-group
position on the benzene ring seems to be important structural feature that can
contribute to these effects (Fitzgerald et al. 2005).
The role of vanillin as an antibacterial agent against Escherichia coli, Lactobacillus
plantarum and Listeria innocua appears to be promising (Fitzgerald et al. 2004b).
Schiff bases derived from vanillin were evaluated for their potential as antibacterial
agents against some Gram-positive and Gram-negative bacterial strains such as
Pseudomonas pseudoalcaligenes, Proteus vulgaris, Citrobacter freundii, Enterobacter aero-
genes, Staphylococcus subfava and Bacillus megaterium (Vaghasiya et al. 2004). However,
further research needs to be conducted to determine the minimum concentration at
which vanillin act as an antimicrobial agent or a natural preservative.
Antioxidant activity
Vanillin has been recognized for its antioxidant potential (Burri et al. 1989a; Teissedre
and Waterhouse 2000) as well as its free radical scavenging activity (Sawa et al. 1999;
Mahal et al. 2001; Kumar et al. 2002, 2004). The presence of vanillin in micro
quantity enhanced the protection of food against oxidation (Burri et al. 1989b, 1991).
The hydroalcoholic extract of vanilla also exhibited antioxidant properties (Toshio
Table I. Chemical identity and physico-chemical properties of vanillin.
Property Data
Molecular formula C
8
H
8
O
3
CAS number 121-33-5
Chemical structure (CH
3
O)C
6
H
3
(OH) CHO
Physical state White or slightly yellow needles
Molecular weight 152.15
Melting point 80818C
Boiling point 2858C
Water solubility 1 g/100 ml
Density 1.056 g/ml
Vapor density (air1) 5.2
Vapor pressure 2.210
3
mmHg at 258C
Flash point 1478C
Dissociation constant pKa
1
7.40, pKa
2
11.4 (258C)
Adapted from http://cira.ornl.gov/documents/vanillin.pdf
302 A. K. Sinha et al.
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et al. 2000), thus acting as a food preservative (Aruoma 1999). Vanillin has also been
reported to inhibit the auto-oxidation of milk fat (Mykolaiivna and Petrivna 2005).
Using rat liver mitochondria as model systems, Kamat et al. (2000) have examined the
ability of vanillin to protect the cell membrane against oxidative damage induced by
photosensitization.
Anticarcinogenic and antimutagenic activity
Earlier studies suggested that vanillin has antimutagenic and anticarcinogenic activity
(Imanishi et al. 1990; Ferguson 1994). It has been shown to decrease the number of
colon tumors induced by multiple agents in rat models (Akagi et al. 1995). Vanillin
has been reported to suppress chromosomal aberrations caused by ultraviolet (UV)
and X-rays (Sasaki et al. 1990; Keshava et al. 1998), to inhibit mutation at the CD59
locus on human chromosome (Gustafson et al. 2000) and also to act as a DNA-PK
inhibitor, thus proving its utility in DNA strand repair (Durant and Karran 2003).
Hypolipidemic activity
Vanillin, as a food additive, is also used for preventing the development of pathological
conditions like hyperlipidemia. The pharmacological action of vanillin as a
hypolipidemichypotriglyceridemic agent over a wide range of concentration in the
treatment of diabetes (type 2), cardiovascular disturbances and obesity has been
recognized (Mokshagundam and Mokshagundam 2003).
Antisickling activity
Vanillin, in vitro, demonstrated antisickling effect through covalent bonding of the
aldehyde group with the hemoglobin in the red blood cells (Abraham et al. 1991).
However, orally administered vanillin has no therapeutic effect because of its rapid
decomposition in the upper digestive tract. To overcome this problem, a vanillin
prodrug, MX-1520, has been synthesized, which is biotransformed to vanillin in vivo.
The bioavailability of this drug was found to be 30 times higher than that of vanillin.
At the same time, this prodrug was also found to be five times more effective in
reducing sickling than natural vanilla (Zhang et al. 2004).
Other activities
Sun et al. (2001) found the alcoholic extracts of leaves of V. fragrans toxic to mosquito
larvae. The volatile oils derived from turmeric, citronella grass and hairy basil,
especially with 5% vanillin, were very effective against the mosquito species (Tawatsin
et al. 2001). A significant enhancement of repellence towards black fly species
(Silulium venstum Say and Prosimulium hirtipes Fries) was observed when vanillin,
along with toluamide, was used as a spray (Retnakaran 1984).
Other important activities shown by vanilla are anti-aggregant, anti-hepatotoxic,
anti-inflammatory, antiviral, analgesic, anesthetic, antiseptic, and so on (Duke et al.
2003). However, keeping the low bioavailability of vanillin in focus, additional in vivo
studies would be helpful to amplify its medicinal use and to make it an effective part of
prevention diet.
Comprehensive review on vanilla flavor 303
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Extraction methods
Extraction or sample preparation is the most important step in the development of
methods for isolation of target analytes or for the analysis of botanical and herbal
preparations. It is regarded as a foremost step in the qualitative and quantitative
analysis of any plant constituents. Ideally, an extraction technique should be
exhaustive with respect to constituents to be analyzed, rapid, simple and inexpensive.
Since vanilla is traded in international market as an ethanolic extract, rapid,
convenient and safe extraction methods will play an important role in ensuring a
high-quality product for consumers worldwide.
Commercial extraction of natural vanilla flavor
In some countries, notably France and Germany, the pods themselves are frequently
demanded by consumers. In America, where convenience is on a par with quality,
homemakers prefer extract, which is prepared mainly by percolating or macerating
chopped vanilla pods with ethyl alcohol and water, instead of distillation that destroys
the gentle fragrance of aromatic compounds (Reineccius 1997). According to US
FDA regulations, vanilla extract must contain at least the sapid and odorous principles
extracted from one unit weight (13.35 oz pods, maximum moisture content 25% by
weight per gallon of solvent) of vanilla pods by an aqueous alcohol solution of not less
than 35% ethyl alcohol (Bartnick et al. 2005). The concentration of extract is noted by
its ‘fold’. A single fold of vanilla extract contains the extractable material from 13.35
oz vanilla pods per gallon of solvent or 100 g extractable material per liter.
Commercially, natural vanilla extract is sold as a dilute ethanolic extract containing
about 1.0 g/l vanillin. Commercial vanilla extraction may fall under two categories: the
percolation method and the oleoresin method. The percolation method consists of a
circulating mixture of ethanol and water containing 3550% alcohol for 4872 h,
which results in a four-fold strength vanilla extract. The oleoresin method consists of
pulverizing whole pods and then circulating ethanol over the pods under vacuum at
about 458C. The excess ethanol is removed by evaporation. This process takes about
89 days. Using the oleoresin process, approximately 10-fold strength vanilla extract
can be prepared (Rao and Ravishankar 2000). The color of vanilla extract is
influenced by the quality of vanilla pods, the strength of alcoholic menstrum, the
duration of extraction, and the presence of glycerin that is added to retard the
evaporation and to retain the flavor in the extract (Reineccius 1997).
Other conventional methods
Other conventional methods employed for the extraction and analysis of natural
vanilla include soxhlet, heat treatment, homogenization, maceration (Voisine et al.
1995) and liquidliquid and liquidsolid extraction using a special reciprocating plate
extraction contactor (Zhu and Zhou 2002). Voisine et al. (1995) compared the
different extraction techniques for the simultaneous extraction of vanillin and
glucovanillin from Java and Bourbon vanilla pods with methanol and ethanol as the
extraction solvent. The highest yield for glucovanillin was obtained with 24-h soxhlet
extraction in 47.5% ethanol, and for vanillin with 24-h extraction by maceration in
47.5% ethanol or 80% methanol. In another study, customary soxhlet apparatus, the
Buchi 810 soxhlet extractor and maceration/percolation has been compared for the
304 A. K. Sinha et al.
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extraction of vanilla pods using isopropanol, ethanol, methanol, and a mixture of
ethanol/water. The results were compared on the basis of vanillin content and several
ratios of other principal flavor components. The highest extraction yield for vanillin
was obtained using a Buchi 810 soxhlet apparatus with methanol as the extraction
solvent and an extraction time of 24 h (Ehlers et al. 1999). However, the conventional
extraction procedures suffer from a number of drawbacks which include low
extraction yields, large extraction time, higher solvent consumption, and so forth.
Supercritical fluid extraction
Due to increasing stringent environmental regulations, supercritical fluid extraction
(SCFE) has gained wide importance as an alternative to conventional solvent
extraction for the separation of organic compounds in many industrial and analytical
processes. In SCFE, the solvation power of fluid can be manipulated by changing
pressure and/or temperature resulting in high selectivity. Due to their low viscosity and
relatively high diffusivity, supercritical fluids can penetrate through porous solid
material more effectively than liquid solvents*the outcome of which is high extraction
yields. The extracts are produced under gentle conditions, virtually free of solvent,
and contain only the plant ingredients. Because of its low cost, little toxicity, and
favorable critical parameters (T
c
31.18C, P
c
74.8 atm), CO
2
is one of the most
commonly used extracting agents. A mixture of CO
2
with modifiers (polar organic
solvents) is generally used for the extraction of polar substances (Lang and Wai 2001).
SCFE has been applied successfully for the extraction of vanillin using supercritical
CO
2
as an extraction solvent (Ehlers and Bartholomae 1993; Fang et al. 2002; Fu et
al. 2002). Extraction conditions varied from 35 MPa pressure at 458C for 150 min
(Fu et al. 2002) to 350 bar at 458C for 140 min yielding 20.05 mg vanillin from 1 g
vanilla pods (Fang et al. 2002). Since carbon dioxide is a lipophilic solvent, extraction
is much more selective towards the flavor ingredients, leaving behind colors, sugars
and other polar components of the alcoholic extraction process that do not contribute
to the vanilla flavor. Purity of vanillin was found to be higher with SCFE than with
conventional aqueous ethanol extraction (Nguyen et al. 1991). However, the ratio of
main constituents of vanilla flavor (namely, vanillin, p-hydroxybenzaldehyde, p-
hydroxybenzoic acid, vanillic acid) was found to be different from that present in
conventional alcoholic extraction procedures. This could lead to erroneous results in
regards to authenticity analysis, since the ratio of these compounds is used to evaluate
the authenticity of vanilla extracts (Ehlers and Bartholomae 1993). Hence, serious
efforts in this direction ought to be put in before ascertaining SCFE as an effective
alternate to conventional methods.
Microwave-assisted extraction and ultrasound-assisted extraction
In the past 10 years, there has been increased interest in using techniques involving
microwave-assisted extraction and ultrasound-assisted extraction as these are found to
be simpler and more effective alternatives to conventional extraction methods. The
main advantages of these techniques are a reduced extraction time and minimized
solvent consumption, rendering them rapid, safe and cheap (Hroma´dkova´ et al. 2002;
Pan et al. 2002). A focused microwave has been employed for extraction of vanillin and
p-hydroxybenzaldehyde from vanilla pods whereby the extraction time decreased up to
62 times with 4050% higher vanillin and p-hydroxybenzaldehyde concentrations
Comprehensive review on vanilla flavor 305
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when compared with the official Mexican extraction method (Longares-Patro´n and
Canizares-Macı´as 2006). Recently, Sharma et al. (2006) have compared microwave-
assisted extraction and ultrasound-assisted extraction methodology for the extraction
of vanillin from vanilla pods, wherein microwave-assisted extraction proved to be the
better of the two. The results showed that using microwave-assisted extraction and
ultrasound-assisted extraction the extraction time decreased between 50 and 70 times
with respect to the conventional cold percolation method, and between six and eight
times with respect to Soxhlet extraction. The maximum yield of vanilla extract
(29.81%) was obtained with a mixture of ethanol/water (40:60, v/v), while dehydrated
ethanolic extract showed the highest amount of vanillin (1.8%). Thus these
approaches provide a simple, rapid and environment friendly tool for the extraction
as well as quantitative analysis of chemical constituents of vanilla flavor.
Enzymatic extraction
The use of enzymes with glycosidase activity has been applied successfully for
producing natural vanilla extract (Bartnick et al. 2005). The use of enzymes is quite
helpful to improve the yield without affecting the flavor quality. Waliszewski et al.
(2007b) studied the effect of enzymatic pretreatment of vanilla pods using different
cellulytic enzymes and found that as much as one-half of the amount of vanillin
trapped in the cellulose structure of cured vanilla pods in free form or in glucovanillin
form can be extracted and liberated by enzymatic pretreatment.
In another report, the amount of vanillin transformed from green vanilla pods using
viscozyme and celluclast enzymes was higher than that in conventional methods. Use
of these two enzymes for extraction of vanillin may increase the yield 3.13 times more
than that obtained with the Soxhlet method. Thus, enzymes are useful not only in the
conversion of precursor glucovanillin to vanillin, but also in extraction from the pods,
avoiding the fermentationextraction process (Ruiz-Teran et al. 2001).
Solid-phase extraction
Solid phase extraction (SPE) is an effective alternative to liquidliquid extraction. SPE
involves absorbing the analyte from the sample onto a modified solid support. The
analyte is then desorbed either by thermal means or by using a solvent. The primary
advantage of SPE is the reduced consumption of high-purity solvents, thereby
reducing laboratory costs and diminishing the need for solvent disposal (Arthur and
Pawliszyn 1990). The glucosides in the vanilla extract have been isolated using SPE on
a 35-ml Oasis HLB cartridge, whereby these were collected by elution with methanol/
water (1:1, v/v; 60 ml) (Dignum et al. 2004).
More recent development in this field is solid phase microextraction (SPME),
wherein the polymeric phase is immobilized onto a silica fiber. SPME eliminates the
problems associated with SPE while retaining the advantages; solvents are completely
eliminated, blanks are greatly reduced, and the extraction time can be reduced to a
few minutes. In this procedure a small diameter fiber coated with a stationary phase is
placed in an aqueous sample. The analytes partition into the stationary phase and are
then thermally desorbed, on-column, in the injector of a gas chromatograph (Arthur
and Pawliszyn 1990). Sostaric et al. (2000) have developed a SPME method for
analysis of volatiles in vanilla extract using optimum conditions such as polyacrylate
fiber, a 40-min sampling time at room temperature and a 2-min desorption time.
306 A. K. Sinha et al.
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Biphasic sonoelectroanalysis
Biphasic sonoelectroanalysis involves the use of ultrasonic emulsification and
voltammetric measurements in biphasic systems. The use of ultrasound to form
emulsions ensures that, regardless of the relative densities of the two liquids, both
remain in constant contact with the electrode surface during voltammetric analysis.
Biphasic sonoelectroanalysis removes the need for sample degradation or a separation
step, which would lengthen and complicate the analytical protocol. Thus, biphasic
sonoelectroanalysis may be used as an alternative extraction technique, which
demonstrates the possibilities of simultaneous sono-extraction with an organic phase
sonoemulsified with the target medium (Banks and Compton 2003). Biphasic
sonoelectroanalysis has been employed for the simultaneous extraction and determi-
nation of vanillin in food flavorings using ethyl acetoacetate as an electrochemical and
sonoelectrochemical solvent with quantitation efficiency comparable with the high-
performance liquid chromatography (HPLC)UV method (Hardcastle et al. 2001).
Qualitative and quantitative determination of chemical constituents
Owing to the limited supply and high price of vanilla pods, the creation of imitation
vanilla flavors to replace the natural extracts has now reached a very high level.
Artificial vanilla flavorings usually contain synthetically produced vanillin, ethyl
vanillin and coumarin (banned in several countries) in order to increase the vanilla
flavor perception. In this perspective, to determine the variation in the quality of
vanilla extracts as well as for the separation, identification and quantitative
determination of various chemical constituents, chromatography seems to be a
powerful analytical tool. Various chromatographic techniques such as thin layer
chromatography (TLC), gas chromatography (GC), HPLC, capillary electrophoresis
(CE) and micellar electrokinetic chromatography (MECK) offer very useful informa-
tion, in terms of identification and quantitation, furnishing excellent resolution and
selective retention times. Recently, techniques such as GCmass spectrometry (MS)
and HPLCMS have come up for the identification of new compounds on the basis of
their mass fragmentation pattern while avoiding unnecessary isolation of common
compounds of minor interest. In the present review, the analytical methods currently
available for the analysis of chemical constituents of vanilla flavor are summarized.
Thin layer chromatography
The separation and identification of various natural compounds including vanillin
and its derivatives have been performed using paper chromatography (Anwar 1963)
and TLC (Ramaroson-Raonizafinimanana et al. 1997). TLC has some advantages,
such as rapidity and ease of handling, besides being economical (Stahl 1969).
Analysis of vanilla flavor metabolites obtained from biotransformation has been
achieved successfully on silica gel plates with benzene as the solvent system (Rao
and Ravishankar 1999). Gerasimov et al. (2003) applied TLC for the determination
of vanillin and its homologue ethyl vanillin in food flavorings using hexane/ethyl
acetate (9:1) as the solvent system. A mixture of heptanone, ethanol and sulfuric
acid (4:5:1, v/v/v) was used as developing agent. Recently, TLC has given way to its
modern counterparts: high-performance TLC (HPTLC) and automated multiple
development TLC (AMD-TLC).
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HPTLC is a modern, instrumentalized, quantitative method allowing greater
separation efficiency and improved detection limits as compared with conventional
TLC. HPTLC using silica gel or cellulose layers combined with selective spray
reagents has been used to identify the adulterated vanilla extracts (Association of
Official Analytical Chemists 1990; Poole et al. 1995) and detection of principal flavor
components*namely, vanillin, vanillic acid, p-hydroxybenzaldehyde and p-hydro-
xybenzoic acid*in vanilla extract (Lavoine et al. 1998). Kiridena et al. (1994) have
applied HPTLC to quantify 5-(hydroxymethyl)-2-furfural, one of the important
constituents in vanilla extract, using chloroform/ethyl acetate/1-propanol (94:2:4, v/v)
as the mobile phase.
Introduction of AMD-TLC has simplified the separation of complex mixtures by
TLC. The AMD-TLC technique employs incremental multiple development in
combination with a stepwise solvent gradient that allows optimization of the
separation selectivity through the chromatogram for mixtures of a wide polarity
range. Belay and Poole (1993) applied the AMD-TLC technique for determination of
vanillin and related flavor compounds in natural vanilla extract. They were able to
separate nine phenolic compounds in a standard mixture with the detection of five
phenolic compounds in vanilla extract. In this method, a mixture of chloroform, ethyl
acetate and 1-propanol was used as the developing solvent while acetic acid was added
to minimize tailing of polar compounds.
TLC and its modern counterparts hold ground for routine quality control
applications over other techniques due to their simplicity, minimum clean-up
procedures, less solvent consumption and the possibility of analyzing several samples
in less time.
Gas chromatography
GC is one of the most popular chromatographic techniques for separating volatile
mixtures. It has certain advantages such as high efficiency and better resolution, and
can also be used for quantitative analysis. Gasliquid chromatography and gassolid
chromatography have proven of value in both differentiating and quantitating vanillin
and ethyl vanillin, as reported in earlier works (Martin et al. 1973). The presence of
vanillin in various citrus fruits has been identified and confirmed using high-resolution
GC retention index values and aroma quality (Makkar and Beeker 1994).
Identification based on GC retention data alone is not sufficient; therefore, the
recent trend is to complement GC with MS in the electron impact mode for better
quantitation and analysis of vanilla volatiles. This technique offers the possibility to
gain additional information by mass spectra. GCMS has proven of worth for the
analysis of vanilla constituents; more than 150 compounds have been identified in
different extracts. Identification of some benzyl ethers in commercially prepared
pentane extract of vanilla has been reported using GCMS (Galetto and Hoffman
1978). The above technique has been applied for the identification of 54 hydro-
carbons from three species of Vanilla (Ramaroson-Raonizafinimanana et al. 1997).
Pe´rez-Silva et al. (2006) identified 65 volatile constituents of vanilla in pentane/ether
extract by GCMS analysis, which include 25 acids, 15 phenolic compounds,
10 alcohols, four aldehydes, four heterocyclic compounds, four esters, two hydro-
carbons and one ketone, of which 26 compounds were found to be odor active by GC
308 A. K. Sinha et al.
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olfactometry analysis. The GC conditions used for the above mentioned studies are
presented in Table II.
Recently, GCMS has been used in conjugation with SPME to discriminate
between different types of vanilla extracts and flavorings (Sostaric et al. 2000).
Bourbon, Tahitian and Indonesian vanilla extracts were analyzed as a part of these
studies (Figure 2).
High-performance liquid chromatography
To date, HPLC is the most powerful and preferred technique for the quantification of
organic molecules due to its simplicity, sensitivity, precision and selectivity. HPLC
methods can be adapted according to the problem at hand using various strategies
such as different types of stationary phases, mobile phases and wide range of selective
detectors. This makes HPLC suitable for analysis of active components in the natural
extracts.
Various HPLC methods have been reported on separation and quantitative
determination of vanillin and other related phenolics in natural vanilla extract
(Ranadive 1992; Lamprecht et al. 1994; Voisine et al. 1995; Negishi and Ozawa
1996; Dignum et al. 2004). For the separation of various components of vanilla flavor,
the chromatographic conditions of the HPLC generally include the use of a reverse-
phase C
18
column. Sachan et al. (2004) have developed a HPLC method for the
analysis of phenolic flavor compounds using various C
18
columns (Prodigy
TM
ODS2,
Synergi
TM
Hydro-RP, Lichrosorb
and Columbus
TM
). Six phenolics have been
separated within 21 min on a reverse-phase C
18
column (Synergi
TM
Hydro-RP).
Recently, Sinha et al. (2007) have developed a HPLC method using a Purospher
-
Star RP-18e column, resulting in separation of 21 phenolics in a standard mixture
including adulterants and quantification of 10 phenolics (namely, 4-hydroxybenzyl
alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic
acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid and piperonal) in
crude extract of vanilla pods cultivated in India (Figures 3 and 4).
In addition to choice of column, the efficiency of HPLC analysis is also dependent
upon the selection of mobile phase. Most of the HPLC methods (see Table III) have
employed acidified water mixed with a polar organic solvent such as methanol or
acetonitrile as the mobile phase. All of the reported HPLC protocols show vanillin
elution time varying from 7 min (Ehlers 1999) to 36 min (Voisine et al. 1995) at
detection wavelengths ranging between 254 and 340 nm (Table III), but recently a
rapid HPLC technique for vanillin determination has been developed where the
elution time of vanillin is as low as 2.2 min at 231 nm (Waliszewski et al. 2007b)*
however, the method does not report the quantification of other phenolic compounds.
The role of HPLC has been highlighted by various authors (Ehlers and
Bartholomae 1993; Voisine et al. 1995; Ruiz-Teran et al. 2001; Sharma et al. 2006)
for comparison of different vanilla extraction protocols. Besides this, HPLC has also
been applied successfully for composition analysis of different species of genus Vanilla
(Ehlers et al. 1994; Ehlers and Pfister 1997) and to detect possible adulteration in
vanilla flavorings (Ehlers 1999; Jagerdeo et al. 2000; Scharrer and Mosandi 2001).
HPLC analysis is not only confined to quantification of vanillin in vanilla extract,
but has also been applied extensively for determination of vanillin and other related
phenolics produced through biotransformation pathway (Rao and Ravishankar 1999;
Comprehensive review on vanilla flavor 309
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Table II. GC/GCMS analysis of vanilla extract/other matrices.
Analysis/matrix Sample preparation Column Conditions Reference
GC/vanilla extract Extraction of pods with
pentane
Glass column packed with 5%
Carbowax 20M on Chromosorb
W.A.W. 8 ft0.25 in
Helium: 60 ml/min, with column
temp. 802308Cat48C/min, TCD
Galetto and Hoffman (1978)
GC/vanilla extract Soxhlet extraction with
pentaneH
2
O; extraction
with diisopropyl ether;
organic layer washed, dried
and evaporated; column
chromatography of
unsaponifiable extracted
over Al
2
O
3
gel with hexane
OV-I glass capillary column,
25 m 0.31 mm i.d., 0.15 mm
Hydrogen: 3 ml/min, split 60 ml/min,
column temp. 702208Cat38C/min,
FID
Ramaroson-Raonizafinimanana
et al. (1997)
GCMS/vanilla extract OV-1701 fused capillary column,
50 m 0.32 mm i.d., 0.30 mm
Helium: 4 ml/min, split 80 ml/min,
column temp. 1002808C; 38C/min,
i.s. 2708C, i.v. 70 eV
Ramaroson-Raonizafinimanana
et al. (1997)
GCMS/vanilla extract SPME DB-5 glass capillary column,
30 m 0.2 mm i.d., 0.25 mm
Helium: 1.0 ml/min, 2008Cat88C/
min, then 2508Cat508C/min
Sostaric et al. (2000)
GC/fruit juices Extraction with 50:50 mix-
ture of pentane and diethyl
ethercentrifuge
DB-5 glass capillary column,
30 m 0.25 mm i.d., 0.5 mm
Helium: 1.55 ml/min, 352758Cat
68C/min, FID
Goodner et al. (2000)
GCMS/fruit juices RTX5-MS column, 30 m
0.25 mm, 0.25 mm
352218Cat48C/min to 2758Cat
108C/min, i.s. 1708C, i.v. 70 eV
Goodner et al. (2000)
GC/vanilla extract Extraction with pentane/
ether
DB-Wax fused silica capillary
column, 30 m0.32 mm i.d.,
0.25 mm
Hydrogen: 2 ml/min, column temp.
402458Cat38C/min, FID
Pe´rez-Silva et al. (2006)
GCMS/vanilla extract DB-Wax fused silica capillary
column, 30 m0.32 mm i.d.,
0.25 mm
Helium: 1.1 ml/min, column temp.
402458Cat38C/min, i.s. 2308C, i.v.
70 eV
Pe´rez-Silva et al. (2006)
310 A. K. Sinha et al.
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Tripathi et al. 2002; Suresh et al. 2003; Li et al. 2004; Velanker and Heble 2004) as
well as to quantify vanillin in several other matrices (Anklam et al. 1997; Farthing
et al. 1999; Sobolev 2001). The HPLC conditions used for the above-mentioned
studies are presented in Table III.
More recently, hyphenated techniques coupling HPLC with MS detection have
been developed, allowing the online detection and identification of chemical
Figure 2. GC profile of headspace volatile components sampled by solid-phase microextraction at room
temperature from (a) Tahitian natural vanilla extract, (b) Indonesian natural vanilla extract and (c) Bourbon
natural vanilla extract (for GC conditions see Table II). Peak identification: (1) ethyl hexanoate, (2)
p-methoxybenzaldehyde, (3) 5-propenyl-1,3-benzodioxole, (4) ethyl nonanoate, (5) unidentified compo-
nent, (6) p-methoxybenzoic acid methyl ester, (7) 3-phenyl-2-propenoic acid methyl ester, (8) ethyl
decanoate, and (9) vanillin. Adapted from Sostaric et al. (2000). # 2000 American Chemical Society.
Comprehensive review on vanilla flavor 311
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AU
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
1
2
3
4
5
6
11
7
8
12
13
9
14
15, 16
10
18, 19
20
21
17
Figure 3. HPLC chromatogram of a standard mixture of phenolic compounds (121) (for HPLC
conditions see Table III). Peak identification: (1) 4-Hydroxy benzyl alcohol, (2) Vanillyl alcohol, (3) 3,
4-Dihydroxybenzaldehyde, (4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzaldehyde, (7)
vanillin, (8) p-coumaric acid, (9) ferulic acid, (10) piperonal, (11) isovanillin, (12) syringaldehyde, (13)
acetovanillone, (14) m-coumaric acid, (15) vanillin methyl ester, (16) ethyl vanillin, (17) 2,4-dihydroxy
acetophenone, (18) o-vanillin, (19) coumarin, (20) p-anisaldehyde, and (21) eugenol. Adapted from Sinha
et al. (2007). # 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
UA
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
Minutes
5.00 10.00 15.00 20.00 25.00 30.00
1
2
3
4
5
6
7
8
9
10
Figure 4. HPLC chromatogram of ethanolic extract of pods of V. planifolia (for HPLC conditions see Table
III). Peak identification: (1) 4-hydroxy benzyl alcohol, (2) vanillyl alcohol, (3) 3,4-dihydroxybenzaldehyde,
(4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzaldehyde, (7) vanillin, (8) p-coumaric acid,
(9) ferulic acid, and (10) piperonal. Adapted from Sinha et al. (2007). # 2007 WILEY-VCH Verlag GmbH
& Co. KGaA, Weinheim.
312 A. K. Sinha et al.
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Table III. HPLC conditions for analysis of vanillin and related flavor compounds in vanilla extract/other matrices.
Analysis/matrix Sample preparation Stationary phase (column) Mobile phase Detector Reference
MPLC/vanilla extract Extraction with 44% EtOH,
filtered
MPLC RP-8 spheri-5
column, 1004.6 mm,
5 mm
Methanol/acidified water
(10:90), 1.5 ml/min
UV at 254 nm Ranadive
(1992)
Prep. HPLC/vanilla extract Crude vanilla extract diluted with
water/methanol 1:1, extract with
ether, added ethanol 30%,
concentration
Lichrospher 100 RP-18,
251 cm i.d., 5 mm
(A) H
2
O acidified with HCl,
pH 2.8; (B) methanol.
Isocratic elution at 0% 7A
and 30% B, 2.7 ml/min.
DAD at 340 nm Lamprecht et al.
(1994)
HPLC/vanilla extract Vanilla extract diluted with
methanol/water 1:1
LiChroCart Superspher
100 RP-18, 504 mm i.d.,
4 mm, guard: RP-18, 354
mm, 5 mm
Aqueous sol. 0.01 M
CH
3
COONa, pH 4.0 with
(A) HCl and (B) methanol;
gradient: 15% B85% B,
025 min; 100% B, 2530
min; 0.8 ml/min
DAD at 340 nm Lamprecht et al.
(1994)
Prep. HPLC/ vanilla extract Extraction and filtration m Bondapak RP C18, 25
100 mm i.d., 10 mm, guard:
2510 mm
Water acidified with 1.25%
CH
3
COOH and methanol
in 90:10, isocratic elution, 8
ml/min
UV at 270 nm Voisine et al.
(1995)
HPLC/gluco-vanillin and
vanillin
_ ODS 2 Spherisorb, RP
C18, 25 0.46 cm, 10 mm
Solvent A, water acidified
with 1.25% CH
3
COOH;
Solvent B, methanol;
gradient: 9590%, 0.5 min,
hold for 5 min; 65% A, 535
min, hold for 10 min, 1 ml/
min
DAD at 270 nm Voisine et al.
(1995)
HPLC/vanilla extract Vanilla beans extracted with
MeOH20 ml H
2
O, extracted
with pentane then ether, aqueous
passed through XAD-2 and eluted
by H
2
O and MeOH
C18 TSK-gel, ODS 80Ts,
1504.6 mm i.d., 5 mm
Solvent system I,
H
2
O:MeOH:AcOH:Et
3
N,
A97:3:0.3:0.3. pH 4.6;
B20:80:0.3:0.3. Solvent
system II, 1 mM H
3
PO
4
,
pH3.1:MeOH, A97:3,
B20:80, 0.8 ml/min
UV at 280 nm Negishi and
Ozawa (1996)
Comprehensive review on vanilla flavor 313
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Table III (Continued)
Analysis/matrix Sample preparation Stationary phase (column) Mobile phase Detector Reference
HPLC/plasma and red
blood cell (RBC) analysis
for vanillin
150 ml plasma150 ml ACN,
vortex 15 sec, 13,000g, 10 min,
100 ml RBCs100 ml deionized
H
2
O, vortex 15 sec200 ml ACN,
13,000g, 10 min, 40 ml urine460
ml deionized H
2
O, vortex 10 sec
ODS, 150 mm3.2 mm
i.d., 3 mm guard: C18, 30
mm4.6 mm i.d., 4050
mm
For plasma and RBC
analysis: aq. CH
3
COOH
(1%)/ACN (85:15), pH 2.9,
for urine analysis: aq.
CH
3
COOH (1%)/ACN;
gradient: 90:10 for 10 min;
70:30 at 10 min and hold 5
min, 0.5 ml/min for plasma,
0.6 ml/min for urine and
RBCs
UV at 280 nm Farthing et al.
(1999)
HPLC/vanilla flavorings Lichrospher RP H
3
PO
4
in H
2
O (1:10,000)/
ACN (14:86), 1 ml/min
UV at 278 nm Ehlers (1999)
HPLC/vanilla flavorings Nova-Pak C18 Aqueous AcOH (0.05%)/
MeOH/THF (70:30:0.2), 1
ml/min
UV at 275 nm Jagerdeo et al.
(2000)
HPLC/green vanilla beans Enzymatic extraction
(viscoenzyme and celluclast)
with 47.5% aq. EtOH
Beckman C18 column 4.66
mm25 cm i.d.
MeOH/H
2
O acidified with
AcOH, pH 4; gradient:
acidified H
2
O/MeOH,
6040% for 3 min. at 0.8 ml/
min, 6535% for 9 min at
1.0 ml/min, 6040% at 0.8
ml/min
UV at 278 nm Ruiz-Teran et
al. (2001)
HPLC/boiled peanuts Extraction with ACN, purified
over Al
2
O
3
Zorbax Rx-SIL column,
250 mm4.6 mm i.d.,
5 mm
Isocratic elution with
n-hexane/2-propanol/H
2
O/
AcOH (2,100/540/37/2, v/
v), 1.5 ml/min
DAD at 220450 nm Sobolev (2001)
HPLC/vanilla beans Lichrospher 60 Aqueous H
3
PO
4
(1%)/
ACN/MeOH (95:2:3), 1 ml/
min
UV at 275 nm Scharrer and
Mosandi (2001)
314 A. K. Sinha et al.
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Table III (Continued)
Analysis/matrix Sample preparation Stationary phase (column) Mobile phase Detector Reference
HPLC/biotransformation
(vanilla flavor metabolites)
Capsicum frutescens cell
culture
Medium was extracted with
EtOAc (3 40 ml), dried and
concentrated
Shim-pack ODS column,
4.6 mm 150 mm i.d., 5
mm
Solvent A, 0.1% HCOOH
aq. sol. Solvent B, ACN.
Solvent B started at 20%,
hold 5 min, 40% at 5 min,
80% at 6.5 min, hold 1.5
min, return to 20% at 10
min, re-equilibrated for 5
min, 1.0 ml/min
UV at 280 nm Tripathi et al.
(2002)
HPLC/vanilla extract Commercial extract diluted with
H
2
O, filtered; for reference
extract, beans extracted with H
2
O/
MeOH by maceration and filtered
RP-18-5, 2504 mm Aqueous CH
3
COONa (10
mM/l) pH 4.0, with conc.
HCl (compound A) and
methanol (compound B);
gradient: A/B 85:15, in 35
min to A/B 15:85, in 5
min to A/B85:15, 0.8 ml/
min
DAD at 210360 nm Pyell et al.
(2002)
HPLC/biotransformation
(vanilla flavor metabolites)
Capsicum frutescens root cul-
ture
Roots were extracted with EtOAc
(three times), dried and
concentrated
Shim-pack ODS column,
4.6 mm 150 mm i.d.,
5 mm
Isocratic elution with
MeOH/AcOH/H
2
O
(20:5:75) 1.0 ml/min
UV at 280 nm Suresh et al.
(2003)
HPLC/vanilla glucosides Extraction with 0.1 M acetate
buffer, pH 5, then SPE
Phenomenex
ODS-Hypersil C18,
254.6 mm, 5 mm,
guard: C18 as precolumn
(A) H
2
O/ACN/
CH
3
COOH95:5:0.75,
(B) H
2
O/ACN/
CH
3
COOH5:95:0.75;
gradient: 0% B20% B,
030min; 80% B, 3037.5
min, 2.5 ml/min
DAD at 254275 nm Dignum et al.
(2004)
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Table III (Continued)
Analysis/matrix Sample preparation Stationary phase (column) Mobile phase Detector Reference
Semi Prep HPLC/glucosides Bondapak C18, 3007.8
mm i.d.
Solvent A, H
2
O/ACN/
CH
3
COOH95:5:0.75.
Solvent B, H
2
O/ACN/
CH
3
COOH5:95:0.75;
gradient: 010% B in 30
min, 80% B in 7.5 min,
100% B for 1.5 min, 2.5ml/
min
DAD at 254275 nm Dignum et al.
(2004)
HPLC/bioconversion broth 20 g/l isoeugenol as substrate,
bioconversion with enzyme at
288C, 180 rpm, pH 9, for 3 days
Lichrospher 100 RP-18-5,
2504 mm i.d., 5 mm
Solvent A, 0.01% AcOH aq.
sol. Solvent B, methanol;
gradient: 60% B for 5 min,
50% B for 7 min, 100% B
for 18 min, 60% B for 5 min,
1 ml/min
UV at 270 nm Li et al. (2004)
HPLC/microbial plant
sources
Synergi
TM
Hydro-RP C
18
Isocratic elution with aq.
CF
3
COOH (1 mM)/
methanol (17:8), 1.0 ml/min
Sachan et al.
(2004)
HPLC/vanilla extract Extraction with EtOH RP-C18 ODS column, 4.6
mm i.d. 250 mm, 5 mm
Solvent A, ACN. Solvent B,
H
2
O/H
3
PO
4
, 99.999:0.001,
pH 6.0; gradient: 010 min,
1040% A, 1020 min,
4080% A, 2025 min,
80100% A, 1.4 ml/min
PDA at 254 nm Sharma et al.
(2006)
HPLC/vanilla extract Extraction with EtOH Nucleosil C18 H
2
O/MeOH (40:60), 05
min. H
2
O/MeOH (40:60),
510 min. MeOH (100), 10
15 min. H
2
O/MeOH
(40:60), 1 ml/min
UV at 231 nm Waliszewski
et al. (2007)
Note: AcOH, acetic acid; ACN, acetonitrile; MeOH, methanol; CF
3
COOH, trifluoroacetic acid; EtOH, ethanol; THF, tetrahydrofuran.
316 A. K. Sinha et al.
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constituents. de Jager et al. (2007) have applied liquid chromatographyelectrospray
ionizationmass spectrometry for the determination of vanillin, coumarin, and ethyl
vanillin in vanilla products. All MS data were acquired in the positive ionization mode.
Also, comparison made between liquid chromatographyMS and liquid
chromatographyUV in terms of precision and accuracy found liquid
chromatographyMS to be more accurate due to its higher level of specificity.
Capillary electrophoresis
Although HPLC remains the most dominating technique for the analysis of chemical
constituents of vanilla, CE is gaining popularity as it has several unique advantages
over HPLC because of its speed, efficiency, reproducibility, small sample volume, low
consumption of solvent and ease of removal of contaminants. A high-performance CE
method has been developed for analysis of vanillin, syringaldehyde, coniferaldehyde
and sinapaldehyde in brandy and wine. The optimized conditions selected for the
purpose of monitoring were a capillary of length 53.5 cm, borate buffer of pH 9.3
(50 mM) as a mobile phase at temperature 208C, voltage at 30 kV and UV absorptions
at 348, 362, 404, and 422 nm. Recoveries ranged between 99.9% and 107.7% for all
of the compounds tested. The repeatability of the method was found to be high
(Panossian et al. 2001). In addition to CE, more recent capillary techniques such as
micellar electrokinetic capillary chromatography (MEKC) and mixed micellar
electrokinetic capillary chromatography are also being investigated for component
analysis.
MEKC, a separation mode of CE, was introduced by Terabe et al. (2001). It is one
of the latest chromatographic techniques incorporating many unique features and is
highly competitive with HPLC in the field of food analysis. Because of the absence of a
stationary phase and solubilization of the sample by the micellar phase, direct sample
injection is possible in many cases. Other advantages are the high peak capacity and
short run times. MEKC has been used as a rapid screening method for the analysis of
vanilla flavorings and vanilla extracts. Under optimized conditions, the separation of
nine vanilla constituents and three probable adulterants was possible within 9 min
(Bu
¨
tehorn and Pyell 1996).
There have been attempts to study the comparative advantages of MEKC vis-a-vis
HPLC for the component analysis of vanilla extracts and vanilla-containing
commercial preparations (Pyell et al. 2002). The results showed that MEKC could
be regarded as a competitive alternative to HPLC, since investigation can be
concluded in much shorter analysis time with high resolution and efficiency (Pyell
et al. 2002) (Figure 5). This study used a capillary electrophoresis system equipped
with an UV absorbance detector and fused-silica capillaries (75 mm i.d., 375 mm o.d.).
The detection was performed at 230 nm. The separation buffer was an aqueous
solution of sodium dodecyl sulfate (80 mmol/l), disodium tetraborate (10 mmol/l),
boric acid (10 mmol/l) and urea (1.5 mol/l) having pH 9.14.
Recently, a mixed micellar electrokinetic capillary chromatography method for the
qualitative and quantitative determination of key components of vanilla flavor,
including vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid and
3-methoxybenzaldehyde, has been developed (Boyce et al. 2003). The separations
were performed using a fused silica capillary (52 cm effective length 75 mm i.d.) at
258C with an applied voltage of 18 kV. The detection wavelength was 214 nm. Buffer
Comprehensive review on vanilla flavor 317
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consisted of 10 mM sodium tetraborate, 10 mM boric acid, 100 mM sodium dodecyl
sulphate and 40 mM sodium cholate at pH 7.0.
Production of vanillin
Vanillin, being the most characteristic component of vanilla flavor, has an annual
consumption in the world flavor market touching 12,000 t per annum. The limited
supply and consequently high price of natural vanillin (i.e. US$4,000/kg) (Rao and
Ravishankar 2000) has led to the usage of a large amount of synthetic equivalents. The
production of vanillin, from its earliest days to the present, is a fascinating story of the
progress made in chemistry and chemical engineering, which is reflected by the
gradual lowering of price up to $12/kg. At present about 97% of vanillin sold in the
market comes mainly from the synthetic sources using coniferin, eugenol, safrole,
guaiacol (Bedoukian 1986) and lignin (Hearon and Lo 1980; Wu et al. 1994; Sande
and Sears 1996; Hocking 1997; Bjørsvik 1999; Qiang and Zhonghao 2001; Kozlov
and Gogotov 2001). Although vanillin produced by these means is able to meet the
global annual demand, it suffers from serious drawbacks. For one, the aroma of
synthetically produced vanillin is not comparable with that of natural vanillin.
Secondly, chemical synthesis involves use of hazardous chemicals (and hence under
current US and European legislations cannot be used in natural flavors), resulting in
decreased consumer appeal the world over.
However, the production and isolation of vanillin from natural sources present an
altogether different scenario. The reason behind it is the huge disparity in efforts put
in and the yield per hectare. The cultivation of vanilla is a time-consuming and labor-
intensive process, yet the yield is not very high (Rao and Ravishankar 2000). Very few
attempts have been reported for the isolation of natural vanillin from vanilla extract. In
one process, extract containing about 0.10.2 g vanillin was extracted three times with
ether. The combined ether layers were concentrated and the residue was taken up in
Figure 5. Electropherogram (MEKC) of commercial vanilla extract. Peak identification: (2) 4-hydro-
xybenzyl alcohol, (3) vanillyl alcohol, (4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzalde-
hyde, (7) vanillin, and (8) syringaldehyde. Adapted from Pyell et al. (2002). # 2000 WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim.
318 A. K. Sinha et al.
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methylene chloride, removing any water, and dried (Na
2
SO
4
). This solution was
concentrated to solid, from which approximately a 10% solution in ether was
prepared. Pure vanillin was then isolated by preparative GC of the solution (Hoffman
and Salb 1979). In another report, Krueger and Krueger (1985) adopted the
procedure suggested by Dr Warren Wong of Givaudan Cooperation for the isolation
of natural vanillin. To a 100 ml portion of one-fold extract (or an equivalent amount of
multi-fold extract diluted to 100 ml with 35% alcohol) taken in a beaker, 200 ml
methylene chloride was added and the solution was stirred magnetically overnight (it
was found that extraction in a separatory funnel yielded intractable emulsions). The
methylene chloride layer was separated, dried over Na
2
SO
4
and evaporated in vacuo to
dryness. The residue in flask was extracted with 50 ml boiling pentane whereby
vanillin crystallized out on cooling in an ice bath.
Recently, there has been a huge upsurge in the exploration of more eco-friendly
biosynthetic procedures for the production of natural flavors as the products derived
from these have already been identified in plants or other natural sources, and hence
in principle defined as ‘natural’. Biotechnological approaches for the production of
vanillin have been based on bioconversion of certain natural precursors such as lignin,
eugenol, isoeugenol, ferulic acid and phenolic stilbenes and on de novo biosynthesis
applying fungi, bacteria, plant cells, or genetically engineered microorganisms
(Priefert et al. 2001; Rao and Ravishankar 2000). However, the yields from most of
the precursors are very low since the toxicity of both the precursor and the vanillin
formed presents a major obstacle. Ferulic acid, being least toxic of all the above, is a
suitable candidate for these microbial conversions (Benz and Muheim 1996), thereby
providing a unique biocatalytic pathway for commercial production. Another notable
feature about ferulic acid is that it is a cheap raw material and can be easily obtained
from agricultural waste residues such as cereal bran and sugar beet pulp (Zheng et al.
2007). Several reviews are already documented on the biotechnological production of
vanillin (Rao and Ravishankar 2000; Priefert et al. 2001; Walton et al. 2003).
Although the consumers’ preference for ‘natural’, ‘bio’ or ‘organic’ products is
undoubtedly an important market pull in the food sector (Cheetham 1997), the
industrial interest lies in the cost-effective production of flavor molecules, which are
otherwise difficult to obtain in the proportions present in natural extracts. In this
context, research exploiting the advantages of biocatalysis is certainly to overplay
chemical approaches in the near future.
Authenticity of vanilla extract for quality assurance
A huge market demand together with a high price of authentic vanilla extract has
provided an economic incentive to pretend inferior and synthetic products as natural
vanilla. Often adulterants like vanillin isolated from lignin, ethyl vanillin, coumarin or
piperonal are heavily used to mimic the flavor. Also, the flavor of extracts varies
significantly with environmental, geographical and storage conditions. Thus, deter-
mination of the origin of vanilla is a significant problem nowadays. Several methods
have been developed in an attempt to identify the primary components responsible for
the delicate and unique flavor and to verify the authenticity of vanilla extract,
including AOAC methods. Adulteration can be detected chromatographically by an
abnormal excess of vanillin relative to the profile of minor components in a vanilla
preparation, but again the possibility is there to manipulate this profile artificially
Comprehensive review on vanilla flavor 319
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(Lamprecht et al. 1994; Benz and Muheim 1996). Three basic approaches for
resolving this problem are: ash analysis (Martin et al. 1981), compositional profiling
(Kauzinger et al. 1997; Dennis 1998) and, most importantly, isotopic profiling
(Martin et al. 1981; Toulemonde et al. 1983; Maubert et al. 1988; Caer et al. 1991;
Remaud et al. 1997; Dennis 1998; Dennis et al. 1998; Bensaid et al. 2002; Asche et al.
2003; John and Jamin 2004).
The potential of isotopic profiling as a tool for determination of origin is based on the
fact that the distribution of isotopes on different sites of the molecule is not statistical,
but rather depends on the origin of the precursor and on the type of process to which the
precursor has been subjected (Schmidt 1986; Schmidt et al. 2003). Vanillin contains
three elements (hydrogen, carbon, oxygen) that can be used for isotopic discrimination.
Thus, d
18
O values obtained by isotope ratio MS have been used to distinguish natural
vanillin from guaiacol and lignin-derived vanillin (Fronza et al. 2001), although this
parameter is susceptible to chemical exchange for the aldehyde position during
laboratory or industrial procedures (Heck et al. 1997; Bensaid et al. 2002).
Quantitative deuterium nuclear magnetic resonance provides another powerful tool
for distinguishing between natural and synthetic origins of the aromatic ring
(Toulemonde et al. 1983) but is unable to differentiate between different ways by
which the chain shortening has been achieved (Bensaid et al. 2002). Also, aromatic
hydrogen exchange under certain conditions cannot be ruled out (Tenailleau et al.
2004a). The carbon skeleton, however, is not susceptible to the same restrictions and
all eight atoms of carbon present should contain
13
C/
12
C ratios that reflect their
origin. Thus, the use of
13
C isotopic distribution as an efficient means to determine
the origin of vanillin has been explored by different workers (Krueger and Krueger
1983, 1985; Breas et al. 1994; Tenailleau et al. 2004b).
13
C-nuclear magnetic resonance has the advantage that it not only efficiently detects
the
13
C content at both the carbonyl (C1) and methyl (C8) positions, but also
simultaneously provides additional information on the six aromatic positions. Sample
preparation is simple and chemical treatment that could introduce fractionation is
avoided. Moreover, it readily measures the relative molar fractions between these three
parts of the molecule, making it much easier to detect fraudulent enrichment due to
the addition of either methyl-
13
Corcarbonyl-
13
C vanillin (Tenailleau et al. 2004b).
Vanillin from vanilla beans exhibits a very characteristic d
PDB
13
C value, which is the
ratio between
12
C and
13
C in the sample with Pee Dee Belemnite (PDB), a calcareous
Cretaceous fossil, as a standard. The PDB value for natural vanillin lies between 19%
and 20% (Table IV) resulting from a unique metabolic pathway of vanillin formation
within orchid, thus providing an effective way to determine the origin and authenticity
of vanillin in vanilla extract.
Conclusion
Vanilla is one of the most widely used flavoring agents, with wide-ranging applications
in food, confectionary and pharmaceutical industries. Besides, it has also been used as
a traditional medicine. Antimicrobial, antioxidant, antimutagenic and antisickling
effects are some of the biological activities that have been reported.
Both the traditional and modern methods of extraction have been used for the
isolation of chemical constituents of vanilla. A variety of chromatographic methods
have also been reported for the quantification of active principles. HPLC with Diode
320 A. K. Sinha et al.
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Array Detector DADUV detection is the method of choice for quantification of active
principle. Recent techniques such as capillary electrophoresis and MEKC are
particularly useful, with advantages such as shorter analysis time, small sample
requirement and low running cost.
The future holds promise for more extensive utilization of this precious natural
resource. But some pressing problems need to be addressed if the above goal is to be
realized. An urgent area of concern is the wide disparity between the global demand
and the actual supply of natural vanillin. There is a need to have a multipronged
strategy in this direction. While on one hand studies to increase the yield of the plant
through modern biotechnological approaches should be encouraged, at the same time
the need of the hour is the development of newer and more efficient extraction
methodologies to supplement the biosynthetic efforts. The widespread adulteration of
natural vanillin is another area of concern, so studies aimed at devising effective
authentification tools ought to be taken up.
Nature seems to have enriched vanilla with a wide range of useful properties. The
sustainable exploitation of this natural resource calls for integrating information on
various aspects of vanilla science and their holistic application on the part of the users.
Acknowledgements
The authors are highly grateful to the Council of Scientific & Industrial Research,
New Delhi, India (Mission mode project COR-0010) for its financial support. The
authors are also thankful to the Director of IHBT, Palampur for providing the
necessary facilities during the course of the project.
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... Native to Mexico, named Xanath and valued by Totonac culture, early reviews date from years 1427-1440 period during which Aztecs conquered that empire, receiving vanilla as tribute named "tlil-xochitl" in nahuatl dialect that means black flower. Aztecs used it as flavoring for a drink made from cocoa which was demanded and consumed only by nobility (Mayorga, 2002;Bythrow, 2005;Sinha et al., 2008;Palama et al., 2011;IMPI, 2016;Havkin-Frenkel and Belanger, 2018) (Fig. 1). ...
... V. planifolia is the most appreciated variety because of its aroma and flavor qualities whereby it has widely grown internationally. Due to plant conditions as creeper or climber plant, the use of stakes is required as support further providing the required shadow and also organic matter (Damiron-Velázquez, 2004;Sinha et al., 2008;Palama et al., 2011;Anuradha et al., 2013;Khoyrattya et al., 2018;He et al., 2019). ...
... with information (Mayorga, 2002;IMPI, 2016). global commercial production, further being the most valued because of its aromatic qualities; it is highly appreciated in food industry while constituting the most complete profile among all extracts (Mayorga, 2002;Sinha et al., 2008;Palama et al., 2011;Khoyrattya et al., 2018). ...
Article
Vanilla (Vanilla planifolia) native to Mexico, is a plant that belongs to the orchid family. It is formed by the cutting, orchids and their fruits (beans). Since its discovery by the Totonacas until today, it has been used to flavor many beverages and foods, due to its sensorial properties developed during a traditional curing process, which has given Mexican vanilla beans an important international demand for their exquisite flavor and for having the most complete aromatic profile among all the species marketed, enabling to hold a denomination of origin facilitating its commercialization and ensuring its quality. The green vanilla bean lacks aroma and flavor, so to develop these characteristics it must be subjected to a traditional curing process, which is carried out in an artisanal way and consists of 4 stages (killing, sweating, sun drying and conditioning), which allow the development of phenolic and aromatic compounds, fatty and organic acids that contribute to the development of its aromatic profile, which may be related to morphological, structural, chemical changes, endophytic microorganisms, which together interact giving their aromatic profile so appreciated. However, this has only been studied during the bean physiological maturity and at the end of the curing process. According to the above, this review consists of the integration of knowledge about the vanilla generalities, current regulations, composition, endophyte microorganisms and microstructural changes that develop in the vanilla bean during the traditional curing process; as well as their interrelation between each of them; in addition to a possible evaluation of the waste use generated after the extraction systems of the compounds responsible for the aroma. In order to provide information and scientific knowledge on the integral behavior of the vanilla bean during the curing process, related to the development of molecules responsible for the aromatic profile, as well as technological and commercial of importance, which together promote a better understanding of the structure-property-functionality relationship of green vanilla beans and during their curing process, allowing the use of technological processes that contribute to the development of value-added products and vanilla integral use
... On the other hand, in recent years, there has been a worldwide interest in natural, healthy, and minimally processed foods, especially with regards to ingredients such as flavorings [7]. Additionally, the industry must ensure the use of technologies with minimal environmental impact. ...
Article
Full-text available
The curing process (CP) of Vanilla planifolia pods, which is a long and tedious process, is necessary to obtain the natural vanilla extract. This research evaluated the application of microwave (M) and ultrasound (U) during the “killing” stage of the CP and its effect on vanillin content and β-glucosidase activity. The pods were immersed in a container with water or with moistened samples for the M treatments. In U treatments, the pods were immersed in an ultrasonic bath. After this stage, the samples were subjected to an additional U treatment. The results show that the application of these technologies significantly improves vanillin yield (p < 0.05) and the curing time is reduced to 20 days. U treatments subjected to additional sonication at 38 °C obtain more than double the yield of vanillin regarding control. The effect of M and U on cell structure damage increases with additional sonication, but at 15 min, β-glucosidase inactivation decreases the final yield. Disposition of samples in M also affects the final vanillin content. There is no significant correlation between β-glucosidase and vanillin in the different treatments. The application of M and U with the appropriate parameters reduces the CP time without affecting the compounds of interest.
... Additionally, it has been employed as a conventional medication. Biological actions that have been documented include those with antimicrobial, antioxidant, antimutagenic, and antisickling properties (Sinha et al., 2008). Indian processing businesses purchase green vanilla beans from farmers, process them, and then export them to overseas customers who extract the vanilla essence. ...
... It has biological activity effects such as antioxidant, anti-inflammatory, anti-diabetic, nephroprotective, gastroprotective, neuroprotective and hepatoprotective [41,42]. In both leaf and flower parts of the plants, other phenolic compounds such as caffeic acid having various bioactivity, which is present in many food sources including blueberry, coffee drinks, apple and cider [43,44] and vanillin the main component of natural vanilla, which is common used as an aroma and flavor enhancer in foods [45], and quercetin, which is a powerful antioxidant that protects the plant against biotic and abiotic stress factors were also detected. p-coumaric acid and vanillin in both flower and leaf ethanolic extracts of L. mucronatum subsp. ...
Article
Plants include compounds having high antioxidant activity such as flavonoids, phenolics, and carotenoids. Antioxidant defense mechanisms play an important role in the prevention and treatment of oxidative stress diseases in humans. In the present study, the flower and leaf parts of Linum mucronatum subsp. armenum were extracted in five different solvents. The antioxidant activities of the extracts were determine using six antioxidant activity determination assays (Iron (III) reducing/antioxidant power (FRAP), DPPH radical scavenging activity, copper (II) reducing antioxidant activity (CUPRAC), ABTS radical scavenging capacity, total flavonoid content and total phenolic content). While, the methanol extract showed the highest activity for the flower part, ethanol extracts of leaf part showed the highest antioxidant activity in the DPPH, FRAP and CUPRAC tests. The highest activity values in both flower and leaf parts was measured in acetone extract with SC50=0.287 mg/mL and (SC50=0.163 mg/mL in ABTS test, respectively. Lowest activity values of solvent extracts were measured in hexane extracts in all tests. Phenolic compounds of the plant were identified using LC-MS/MS. These phenolics are kaempferol, vanillin, protecatechuic acid, caffeic acid, p-coumaric acid, p-OH benzoic acid, salicylic acid, quercetin and rutin. The leaf and flower parts have α-glucosidase enzyme inhibitor effect. It was determined that the leaf part of the plant (IC50=4.533 mg/mL) have higher enzyme inhibition than in the flower (IC50=6.096 mg/mL). As a result, it was determined that the plant showed the biological activity. The results will contribute to the studies on the biological activity of the other plant.
... El 95 % de la vainilla comercializada en el mundo corresponde a V. planifolia (Bory et al., 2008). V. pompona es de menor uso, casi restringido a la perfumería, pero cuenta con la característica de ser una de las especies del género más resistente a patógenos y cambios ambientales (Soto-Arenas y Solano-Gómez, 2007;Sinha et al., 2008). Entre 2019 y 2020, Madagascar, Indonesia y México generaron el 77.2 % (5739 t) de la vainilla que se produjo en el mundo (FAO, 2022). ...
Article
Full-text available
Vanilla planifolia es la principal fuente vegetal de vainillina, saborizante de amplia importancia comercial. Fusarium oxysporum f. sp. vanillae, principal patógeno de V. planifolia, ha devastado cultivos enteros a nivel mundial. Vanilla pompona posee resistencia a patógenos comunes del género. Va-riaciones climáticas extremas repercuten en las interacciones planta-patógeno. A partir del supuesto de que temperaturas superiores a 28 ºC intensifican la infectividad de F. oxysporum en vainilla, se determinó la influencia del incremento de la temperatura en la infectividad de F. oxysporum f. sp. vanillae (cepa M21C5) en V. planifolia y dos híbridos de V. planifolia x V. pompona. Se inocularon raíces de esquejes de V. planifolia y de los híbridos con suspensiones de esporas del hongo. Se midió el avance de la enfermedad durante 60 días a 25, 30 y 35 ºC. Se emplearon cinco réplicas por tratamiento, incluyen-do un lote testigo. Se utilizó ANOVA post hoc Tukey (P ≤ 0.05) para analizar los datos. V. planifolia fue susceptible a 35 °C y altamente susceptible a 25 y 30 °C. Ambos híbridos mostraron resistencia al patógeno en las temperaturas evaluadas. Por la resistencia mostrada, los híbridos de V. planifolia x V. pompona son una alternativa viable ante el patógeno.
... Vanilla fruits produce up to 200 aromatic compounds, and it is well established that vanillinperhaps the most widely used natural aroma and flavoring used in human societies-is the most abundant compound in the fruits, playing a key role in their sweet, agreeable aroma. 34 We show that Euglossini and Meliponini bees displace or collect the seeds of dehiscent V. odorata and V. planifolia fruits through fragrance and pulp collection behaviors on the inner part of the pod. This coincides with the site of vanillin glucoside biosynthesis, where the compound accumulates within the mesocarp and placental laminae. ...
Article
Identifying the mechanisms for seed dispersal and persistence of species is a central aim of ecology. Seed dispersal by animals is an essential form of dissemination in many plant communities, including seeds of over 66% of neotropical canopy tree species. 1 ,2 Besides physical dispersal, animals influence seed germination probabilities through scarification, breaking dormancy, and preventing rotting, so plants often invest important resources in attracting them. Orchids are predominantly adapted to wind dispersal, having dust-like seeds that are easily uplifted. Exceptions include bird-, 3 ,4 cricket-, 5 ,6 and mammal-dispersed 7 species, featuring fleshy fruits with hard seeds that germinate after passing the animal’s digestive system. Given the similarity in fruit and seed morphology, zoochory has also been suggested in Vanilla, 8 ,9 ,10 ,11 ,12 ,13 ,14 ,15 a pantropical genus of 118 species with vine-like growth. 16 ,17 ,18 We test this prediction through in situ and ex situ experimentation using fruits of Vanilla planifolia, and wild relatives, from which vanillin—a widely used natural aroma and flavoring—is obtained. Seeds from dehiscent fruits are removed by male Euglossini collecting fragrances, a unique case in plants, and female Meliponini bees gathering nest-building materials, a first among monocots. By contrast, mammals, mostly rodents, consume the nutritious indehiscent fruits, passing the seeds up to 18 h after consumption. Protocorm formation in digested and undigested seeds proves that scarification in the gut is not strictly required for germination. Multimodal seed dispersal mechanisms are proven for the first time in Orchidaceae, with ectozoochory and endozoochory playing crucial roles in the unusually broad distribution of Vanilla.
Article
In this work, nanostructured pectin aerogels were prepared via a sol-gel process and subsequent drying under supercritical conditions. To this end, three commercially available citrus pectins and an in-house produced and enzymatically modified watermelon rind pectin (WRP) were compared. Then, the effect of pectin's structure and composition on the aerogel properties were analysed and its potential application as a delivery system was explored by impregnating them with vanillin. Results showed that the molecular weight, degree of esterification and branching degree of the pectin samples played a main role in the production of hydrogels and subsequent aerogels. The developed aerogel particles showed high specific surface areas (468–584 m2/g) and low bulk density (0.025–0.10 g/cm3). The shrinkage effect during aerogel formation was significantly affected by the pectin concentration and structure, while vanillin loading in aerogels and its release profile was also seen to be influenced by the affinity between pectin and vanillin. Furthermore, the results highlight the interest of WRP as a carrier of active compounds which might have potential application in food and biomedical areas, among others.
Article
Vanillin (VLN) is generally used in bakery food products as an aroma enhancer and flavoring agent. Hence it is important to develop a powerful and rapid tool to monitor the concentration of VLN levels concerning food safety. In this paper, a simple sensor was proposed by electropolymerized Aspartic acid (ASP) modified on Graphene and Graphite composite paste electrode (PASPMGN/CPE) for the detection of VLN by differential pulse voltammetry (DPV). PASPMGN/CPE ranked to have high electrocatalytic kinetics towards electro‐oxidation of VLN, in optimized conditions. The electrochemical process of VLN is portrayed to be irreversible and adsorption‐controlled kinetics by the cyclic voltammetry (CV) method. The surface features were studied using Field Emission Scanning Electron Microscopy (FE‐SEM), Energy Dispersive X‐ray Spectroscopy (EDX), Electrochemical Impedance Spectroscopy (EIS) and CV. From DPV results, the oxidative peak current showed linear growth toward the concentration of VLN ranging from 1.0 μM to 15.0 μM with a limit of detection and quantification of 4.85 μM and 16.2 μM. The modified sensor attained to be highly selective with common possible interferents. PASPMGN/CPE was efficient in detecting VLN in vanilla pods, essence and bakery product samples. The electrode based on electropolymerized Aspartic acid (ASP) modified on Graphene and Graphite composite paste electrode (PASPMGN/CPE) applied for the detection of VLN using differential pulse voltammetry (DPV). PASPMGN/CPE was highly conductive and validated for the electrochemical oxidation of Vanillin (VLN) using DPV technique. The quantified one peak revealed to be irreversible carrying two electrons and two protons transfer. The application of PASPMGN/CPE was succeeded in validating in the samples of vanilla pods, essence and bakery products. PASPMGN/CPE presented with reproducible surface and sensitive with low limit of detection 4.85 μM.
Article
Purpose This study aims to synthesize two different benzoxazines (Bz) monomers using bio-based and petroleum-based primary amines, respectively, and they have been compared to study their thermal and mechanical performances. Design/methodology/approach A bio-based bisphenol, Divanillin (DiVa), was formed by reacting two moles of vanillin with one mole of ethylenediamine (EDA) which was then reacted firstly with paraformaldehyde and EDA to form the benzoxazine DiVa-EDA-Bz, and secondly with paraformaldehyde and furfuryl amine (FFA) to form the benzoxazine DiVa-FFA-Bz. The molecular structure and thermal properties of the benzoxazines were characterized by fourier transform infrared spectroscopy and nuclear magnetic resonance (1H,13C) spectroscopies, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), respectively. The benzoxazines were further coated on mild steel panels to evaluate their mechanical properties and chemical resistance. Findings The DSC results of DiVa-FFA-Bz showed two exothermic peaks related to crosslinking compared to the one in DiVa-EDA-Bz. The DiVa-FFA-Bz also showed a higher heat of polymerization than DiVa-EDA-Bz. The TGA results showed that DiVa-FFA-Bz exhibited higher thermal stability with a residual char of 54.10% than 43.24% for DiVa-EDA-Bz. The chemical resistance test results showed that DiVa-FFA-Bz showed better chemical resistance and mechanical properties due to its higher crosslinking density. Originality/value This study shows the use of bio-based materials, vanillin and FFA, for synthesizing a benzoxazine resin and its application at high temperatures.
A method is described for isolating vanillin from vanilla extract, followed by stable isotope ratio analysis to determine the amount of natural vanillin contained in adulterated vanilla extracts. After the potassium content is determined, the percent Madagascar and/or Java vanilla beans incorporated into the extract may then be approximated from the vanillin/ potassium ratio.
Article
Vanillin, a food additive, has been evaluated as a potential agent to treat sickle cell anemia. Earlier studies indicated that vanillin had moderate antisickling activity when compared with other aldehydes. We have determined by high performance liquid chromatography that vanillin reacts covalently with sickle hemoglobin (HbS) both in solution and in intact red blood cells. Hemoscan oxygen equilibrium curves show a dose- dependent left shift, particularly at low oxygen tensions. Rheologic evaluation (pO2 scan Ektacytometry) of vanillin-reacted HbS erythrocytes shows a dose-dependent inhibition of deoxygenation-induced cell sickling. Ektacytometry also suggests that vanillin may have a direct inhibitory effect on HbS polymer formation. Vanillin has no adverse effects on cell ion or water content. X-ray crystallographic studies with deoxyhemoglobin (HbA)-vanillin demonstrate that vanillin binds near His 103 alpha, Cys 104 alpha, and Gln 131 beta in the central water cavity. A secondary binding site is located between His 116 beta and His 117 beta. His 116 beta has been implicated as a polymer contact residue. Oxygen equilibrium, ektacytometry, and x-ray studies indicate that vanillin may be acting to decrease HbS polymerization by a dual mechanism of action; allosteric modulation to a high-affinity HbS molecule and by stereospecific inhibition of T state HbS polymerization. Because vanillin is a food additive on the GRAS (generally regarded as safe) list, and because it has little or no adverse effects at high dosages in animals, vanillin is a candidate for further evaluation as an agent for the treatment of sickle cell disease.
Book
"Let food be your medicine, medicine your food." -Hippocrates, 2400 B.C. When the "Father of Medicine" uttered those famous words, spices were as important for medicine, embalming, preserving food, and masking bad odors as they were for more mundane culinary matters. Author James A. Duke predicts that spices such as capsicum, cinnamon, garlic, ginger, onion, and turmeric will assume relatively more medicinal importance again, as the economic costs and knowledge of the side-effects of prescription pharmaceuticals increase. After all, each spice contains thousands of useful phytochemicals. Pharmaceuticals usually contain only one or two. Discover the Science behind the Folklore Spices are important medicines that have withstood the empirical tests of millennia. Nearly 5,000 years ago Charak, the father of Ayurvedic medicine, claimed that garlic lightens the blood, reduces tumors, and is an aphrodisiac tonic. Today scientists say it thins the blood, prevents cancer, and increases libido. For centuries people worldwide have used spices to cure a myriad of ailments and to preserve foods. Now science is proving that these spices may preserve us with their antioxidant and antiseptic activities. Organized by scientific name, the CRC Handbook of Medicinal Spices provides the science behind the folklore of over 60 popular spices. For each spice, it lists: Scientific name Common name Medicinal activities and indications Multiple activities Other uses, especially culinary Cultivation Chemistry Important phytochemical constituents and their activities The handbook also includes market and import data, culinary uses, ecology and cultural information, and discusses at length the use of spices as antiseptics and antioxidants.
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Flavor is unquestionably one of the most extremely secretive one-reluctant to dis­ close anything that might be of value to a important attributes of the food we eat. competitor. Thus, little information about Man does not eat simply to live but even the activities of the flavor industry itself is more so lives to eat. Take away the pleasure offood and life becomes relatively mundane. available to the public. There now is a substantial body of liter­ The goal of the original Source Book of ature dealing with food flavor. The "golden Flavors, written by Henry Heath, was to years" of flavor research in the United States bring together in one volume as much of the were the 1960s and 70s. Numerous academic worldwide data and facts and as many flavor­ and government institutions had strong related subjects (e. g. , food colors) as was flavor programs and money was readily possible. Henry Heath added a wealth of available for flavor research. In the 1980s personal information on how the industry and 90s, research funding has become diffi­ accomplishes its various activities, which cult to obtain, particularly in an esthetic had never been published in any other liter­ area such as food flavor. The number of ature. It has been the intent of this author to research groups focusing on food flavor has update and build upon the original work of declined in the United States. Fortunately, Henry Heath.
Article
The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l(-1) and alpha-naphthalene acetic acid (NAA) 2 mg l(-1) were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.
Article
Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.
Article
This is a large and expensive book. Free copies are not available for distribution. Please do not ask. It can be purchased from Zip Publishing (info@zippublishing.com). This illustrated reference work provided a detailed scientific approach to orchid biology. There are 15 chapters: history (in Asia, Africa, Europe, New Guinea and Australia), including the history of the discovery of orchid reproduction; classification and naming of orchids; evolution of the Orchidaceae, and of plant parts individually; cytology; physiology; phytochemistry; morphology; anatomy; mycorrhiza (including orchid-fungus specificity, seed germination and root characteristics); pollination (with attention to attractants and pollinators); embryology; reproduction (including reproduction through seeds, germination, and sexual and asexual propagation); heredity and breeding; ecology (with an account of the habitats in which orchids exist, as well as notes on climate, carbon fixation, seed dispersal and conservation); and commercial and ethnobotanical uses. Each chapter has a bibliography. -J.W.Cooper This a book. The author cannot send copies. It is available for purchase at www.amazon.com. -Joseph Arditti