): anatomy, enzymes, phenolics and lipids

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Original article Fruits, vol. 65 (4) 221 A set of data on green, ripening and senescent vanilla pod (Vanilla planifolia; Orchidaceae): anatomy, enzymes, phenolics and lipids. Abstract Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β- D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous β-glucosidase, liberates vanillin, the major aroma component of vanilla. Little is known on the spatial distribution of aroma-generating phenolics, and the enzymes responsible for their liberation (β-glucosidase) and oxidation (peroxidase). We report here quantitative data with respect to these three components in relation to the anatomy of the pod. Furthermore, the spatial progression of oxidation is shown. Materials and methods. Mature green vanilla pods were analyzed for their contents of phenolics (HPLC), and β-glucosidase and per- oxidase activities by spectrophotometric techniques using p-nitrophenyl glucoside and vanillin as sub- strates, respectively. Lipids were examined under fluorescence microscopy after Nile red staining. Oxidation development was observed on transverse slices of pods. Results and discussion. Phenolics, and β-glucosidase and peroxidase activities showed gradients of increasing-decreasing concentrations from the stem to the blossom end of pods. The β-glucosidase activity is distributed in between the pla- centae, mesocarp, and trichomes in a [7 / 2 / 1] proportion while that of peroxidase shows a [38 / 1] ratio in the mesocarp and placentae, and was absent from trichomes. Oxidation begins from the blossom end in the placentae, progressively invading the mesocarp and moving towards the stem end. Con- clusion. The green mature vanilla pod is spatially heterogeneous for its phenolics, and β-glucosidase and peroxidase activities, its placentae playing an important role in the liberation of vanillin and its sub- sequent oxidation. France / Vanilla planifolia / Orchidaceae / vanilla / β-glucosidase / peroxidase / glucovanillin / vanillin / lipids / oxidation / gradient / mesocarp / placentae / trichomes / xylem Ensemble de résultats sur la gousse de vanille verte, murissante et sénescente (Vanilla planifolia ; Orchidaceae) : anatomie, enzymes, composés phénoliques et lipides. Résumé –– Introduction. Les gousses de vanille vertes matures accumulent du 4-O-(3-méthoxy-ben- zaldéhyde)-β-D-glucoside (glucovanilline) qui, sous l’effet d’une hydrolyse par une β-glucosidase endo- gène, libère de la vanilline, le principal composé d’arôme de la vanille. Les répartitions spatiales des composés phénoliques générateurs d’arôme et des enzymes responsables de leur libération (β-gluco- sidase) et oxydation (peroxydase) sont mal connues. Nous donnons ici des résultats quantitatifs concer- nant ces trois composantes au regard de l’anatomie de la gousse. De plus, la progression spatiale de l’oxydation est illustrée. Matériel et méthodes. Des gousses de vanille vertes matures ont été analysées pour leurs teneurs en composés phénoliques, et activités β-glucosidase et peroxydase par des techniques spectrophotométriques en utilisant respectivement le glucoside de p-nitrophényle et la vanilline comme substrats. Les lipides ont été examinés en microscopie de fluorescence après coloration par le rouge de Nil. Le développement de l’oxydation a été observé sur des coupes transversales de gousses. Résultats et discussion. Les composés phénoliques et les activités β-glucosidase et peroxydase montrent des gra- dients de concentrations croissantes-décroissantes de l’extrémité pédonculaire vers l’extrémité florale de la gousse. L’activité β-glucosidase est distribuée entre placentas, mésocarpe et trichomes dans un rap- port [7 / 2 / 1] alors que l’activité peroxydase est répartie dans des proportions [38 / 1] entre le mésocarpe et les placentas ; elle est absente des trichomes. L’oxydation commence dans les placentas de la partie florale en envahissant progressivement le mésocarpe et se déplace vers la zone pédonculaire. Conclu- sion. La gousse de vanille verte mature est hétérogène d’un point de vue spatial pour ce qui concerne ses composés phénoliques et activités β-glucosidase et peroxydase, les placentas jouant un rôle important dans la libération de la vanilline et son oxydation consécutive. France / Vanilla planifolia / Orchidaceae / vanille / β-glucosidase / peroxydase / glucovanilline / vanilline / lipides / oxydation / gradient / mésocarpe / placentas / trichomes / xylème 1 CIRAD, Persyst, UMR QUALISUD, TA B-95/16, F-34398 Montpellier Cedex 5, France, [email protected] 2 UMR 5004, INRA-CNRS- UMII-SUPAGRO, and CIRAD, PHIV, TA/40/02, F-34398 Montpellier Cedex 5, France 3 UMR 1083, INRA, Univ. Montpellier I, F-34000 Montpellier Cedex, France A set of data on green, ripening and senescent vanilla pod (Vanilla planifolia; Orchidaceae): anatomy, enzymes, phenolics and lipids Jean-Marc BRILLOUET 1, 3 *, Eric ODOUX 1 , Geneviève CONEJERO 2 * Correspondence and reprints Received 6 January 2010 Accepted 05 February 2010 Fruits, 2010, vol. 65, p. 221–235 © 2010 Cirad/EDP Sciences All rights reserved DOI: 10.1051/fruits/2010018 www.fruits-journal.org RESUMEN ESPAÑOL, p. 235 Article published by EDP Sciences and available at http://www.fruits-journal.org or http://dx.doi.org/10.1051/fruits/2010018

Transcript of ): anatomy, enzymes, phenolics and lipids

Page 1: ): anatomy, enzymes, phenolics and lipids

Original article

Fruits, vol. 65 (4) 221

A set of data on green, ripening and senescent vanilla pod (Vanilla planifolia;Orchidaceae): anatomy, enzymes, phenolics and lipids.Abstract — Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β-D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous β-glucosidase, liberates vanillin,the major aroma component of vanilla. Little is known on the spatial distribution of aroma-generatingphenolics, and the enzymes responsible for their liberation (β-glucosidase) and oxidation (peroxidase).We report here quantitative data with respect to these three components in relation to the anatomy ofthe pod. Furthermore, the spatial progression of oxidation is shown. Materials and methods. Maturegreen vanilla pods were analyzed for their contents of phenolics (HPLC), and β-glucosidase and per-oxidase activities by spectrophotometric techniques using p-nitrophenyl glucoside and vanillin as sub-strates, respectively. Lipids were examined under fluorescence microscopy after Nile red staining.Oxidation development was observed on transverse slices of pods. Results and discussion. Phenolics,and β-glucosidase and peroxidase activities showed gradients of increasing-decreasing concentrationsfrom the stem to the blossom end of pods. The β-glucosidase activity is distributed in between the pla-centae, mesocarp, and trichomes in a [7 / 2 / 1] proportion while that of peroxidase shows a [38 / 1]ratio in the mesocarp and placentae, and was absent from trichomes. Oxidation begins from the blossomend in the placentae, progressively invading the mesocarp and moving towards the stem end. Con-clusion. The green mature vanilla pod is spatially heterogeneous for its phenolics, and β-glucosidaseand peroxidase activities, its placentae playing an important role in the liberation of vanillin and its sub-sequent oxidation.France / Vanilla planifolia / Orchidaceae / vanilla / β-glucosidase / peroxidase /glucovanillin / vanillin / lipids / oxidation / gradient / mesocarp / placentae /trichomes / xylem

Ensemble de résultats sur la gousse de vanille verte, murissante etsénescente (Vanilla planifolia ; Orchidaceae) : anatomie, enzymes, composésphénoliques et lipides.Résumé –– Introduction. Les gousses de vanille vertes matures accumulent du 4-O-(3-méthoxy-ben-zaldéhyde)-β-D-glucoside (glucovanilline) qui, sous l’effet d’une hydrolyse par une β-glucosidase endo-gène, libère de la vanilline, le principal composé d’arôme de la vanille. Les répartitions spatiales descomposés phénoliques générateurs d’arôme et des enzymes responsables de leur libération (β-gluco-sidase) et oxydation (peroxydase) sont mal connues. Nous donnons ici des résultats quantitatifs concer-nant ces trois composantes au regard de l’anatomie de la gousse. De plus, la progression spatiale del’oxydation est illustrée. Matériel et méthodes. Des gousses de vanille vertes matures ont été analyséespour leurs teneurs en composés phénoliques, et activités β-glucosidase et peroxydase par des techniquesspectrophotométriques en utilisant respectivement le glucoside de p-nitrophényle et la vanilline commesubstrats. Les lipides ont été examinés en microscopie de fluorescence après coloration par le rougede Nil. Le développement de l’oxydation a été observé sur des coupes transversales de gousses. Résultatset discussion. Les composés phénoliques et les activités β-glucosidase et peroxydase montrent des gra-dients de concentrations croissantes-décroissantes de l’extrémité pédonculaire vers l’extrémité floralede la gousse. L’activité β-glucosidase est distribuée entre placentas, mésocarpe et trichomes dans un rap-port [7 / 2 / 1] alors que l’activité peroxydase est répartie dans des proportions [38 / 1] entre le mésocarpeet les placentas ; elle est absente des trichomes. L’oxydation commence dans les placentas de la partieflorale en envahissant progressivement le mésocarpe et se déplace vers la zone pédonculaire. Conclu-sion. La gousse de vanille verte mature est hétérogène d’un point de vue spatial pour ce qui concerneses composés phénoliques et activités β-glucosidase et peroxydase, les placentas jouant un rôle importantdans la libération de la vanilline et son oxydation consécutive.

France / Vanilla planifolia / Orchidaceae / vanille / β-glucosidase / peroxydase /glucovanilline / vanilline / lipides / oxydation / gradient / mésocarpe / placentas /trichomes / xylème

1 CIRAD, Persyst, UMRQUALISUD, TA B-95/16,F-34398 Montpellier Cedex 5,France, [email protected]

2 UMR 5004, INRA-CNRS-UMII-SUPAGRO, and CIRAD,PHIV, TA/40/02, F-34398Montpellier Cedex 5, France

3 UMR 1083, INRA, Univ.Montpellier I, F-34000Montpellier Cedex, France

A set of data on green, ripening and senescent vanilla pod (Vanilla planifolia; Orchidaceae): anatomy, enzymes, phenolics and lipidsJean-Marc BRILLOUET1, 3*, Eric ODOUX1, Geneviève CONEJERO2

* Correspondence and reprints

Received 6 January 2010Accepted 05 February 2010

Fruits, 2010, vol. 65, p. 221–235© 2010 Cirad/EDP SciencesAll rights reservedDOI: 10.1051/fruits/2010018www.fruits-journal.org

RESUMEN ESPAÑOL, p. 235

Article published by EDP Sciences and available at http://www.fruits-journal.org or http://dx.doi.org/10.1051/fruits/2010018

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1. Introduction

Vanilla pod, the fruit of Vanilla sp. vine,exhales an excellent aroma, making it a highadded-value product. When it is greenmature at the time of harvest (8–9 monthsafter pollination), it does not smell; it is onlyafter a lengthy treatment that it becomes acommercial product. Up until the presentday, preparation of vanilla comprises sev-eral steps [1–4], “killing” in a hot water bath,“sweating” in a cloth-lined close woodenbox, drying on racks in the morning sun,and conditioning in a wax paper-linedwooden trunk, the number and length of thevarious steps depending on the area of pro-duction. Basically, the aims and conse-quences of these steps are:

– “killing”, to stop the vegetative life of thepod to prevent dehiscence [3]; decompart-mentation of the inner volumes of the cells(vacuole, cytoplasm, etc.) [5], outset ofhydrolysis of glucovanillin by the vanilla β-glucosidase [6] and of oxidation with pro-duction of brown pigments; no variation inthe water content [3],

– “sweating”, to take benefit of the innerheat generated by the “killing” to go on withhydrolysis of glucovanillin by the vanilla β-glucosidase [7] and continue oxidation; novariation in the water content,– “drying”, a lengthy step (several months)aiming at bringing pods to 30–50% dry mat-ter content,– conditioning, to further increase the drymatter content to 50–60%, depending on itsduration.

Commercial podswill have variable vanil-lin contents depending on origin (country,areas of production, and, for the sameorigin,variability of the pod batches cured),agropedoclimatic conditions, cultural prac-tices, curing conditions, etc. [1, 8–10].

Although a great amount of work hasbeen done on vanilla for a century, there arestill zones of uncertainty concerning thesequence of events leading to the develop-ment of the vanilla flavor. The enzymaticrelease of vanillin from its non-aromatic pre-cursor, glucovanillin, has been establishedfor a long time [1], but it is only recently thatthe vanilla enzyme responsible was charac-

terized [6]; although Arana [1] demonstratedearlier that glucovanillin was unequally dis-tributed along the length of the pod, no fineanatomical picture of its distribution in thedifferent compartments of the pod (meso-carp, placentae, trichomes) was availableuntil recently [11–13]; it is the same in thecase of the β-glucosidase [11].

Oxidative alteration of vanillin has alsobeen known for a long time [1] and is exem-plifiedbydrastic color changesoccurringdur-ing the curing process, the green vanilla podturning black in the end. Since then severalauthors have measured the peroxidase activ-ity of vanilla [14–16], and a peroxidase wasrecentlypurifiedandcharacterized [17].How-ever, it is only recently that Gatfield et al. [18]showed the presence in some vanilla extractsof a taste-active compound, divanillin, anoxi-dation product of vanillin by a peroxidase,and no anatomical picture of the distributionof peroxidase activity in the vanilla pod is, toour knowledge, available today.

Thus, the aim of this publication is togather apparently dispersed data, all of themconverging for a better comprehension ofthe formation of the vanilla aroma. Moreprecisely, objectives were:

– to analyze the variability in populationsof green vanilla pods with regard to glucov-anillin and β-glucosidase contents,

– to examine in detail the existence of lon-gitudinal and radial gradients in the pod forβ-glucosidase, peroxidase, phenolics andsugars,

– to describe the anatomical distribution ofβ-glucosidase, peroxidase and phenolics inthe pod,

– to describe the longitudinal develop-ment of phenol oxidation in the pod,

2. Materials and methods

2.1. Plant materials

Two large batches of green mature vanillapods (~8–9 months after pollination) werefrom Réunion Island (France) and Madagas-car; other minor batches were from Tamil

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Nadu (India) and Papantla (Vera Cruz, Mex-ico). Their mass ranges were 15–20 g. Theirwater content was determined by dryingovernight at 105 °C.

2.2. Extraction and measurementof the β-glucosidase activity

Immediately after dissection [13], portions ofmesocarp, placentae and trichomes (~30–50 mg each) were dilacerated with a Micro-Turrax at ambient temperature in 10 mL of0.1 M phosphate buffer (pH 7.0). After cen-trifugation (14 000 g, 5 min, 20 °C), the fil-trate was diluted 10 × with the same buffer(20 °C).

Activity was measured as described byOdoux et al. [6]: briefly, an aliquot of enzymeextract was added to 200 µL of 4 mM p-nitro-phenyl β-D-glucopyranoside in the abovephosphate buffer and the total volume wasmade to 400 µL with phosphate buffer. After20 min of incubation at 40 °C, 1 mL of 0.5 MNaOH was added and the optical densitywas read at λ = 400 nm. 1 nanokatal (nKat)is defined as the amount of enzyme hydro-lyzing 1 nanomole of substrate per secondin the above conditions.

Undissected representative portions ofpods (~50–250 mg) were also submitted tothe same extraction-determination protocol.

2.3. Extraction and measurementof the peroxidase activity

Immediately after dissection, portions ofmesocarp, placentae and trichomes (~50–100 mg each) were dilacerated with a Micro-Turrax at ambient temperature in 1.8 mL of0.1 M acetate buffer (pH 4.5) containing1 M NaCl, and the slurry was stirred for30 min at ambient temperature. After cen-trifugation (14 000 g, 5 min, 20 °C), thesupernatant (400 µL) was mixed with thesame volume of buffer, and 200 µL of 40 mMvanillin in buffer was added, then 10 µL of30.9 % hydrogen peroxide; the increase inabsorbance was followed at λ = 480 nmbetween (1 and 3) min at 20 °C. A unit (U)of peroxidase activity was defined as theamount of enzyme giving a 0.001 absorb-ance unit increase per minute.

Undissected representative portions ofpods (~50–250 mg) were also submitted tothe same extraction-determination protocol.

2.4. Extraction and determination ofglucovanillin and related phenolics

When mentioned, the word “phenolics” willinclude aglycons and their glucosylatedforms. Analyses were performed accordingto Odoux and Brillouet [13]: briefly, portionsof mesocarp, placentae and trichomes (~50–100 mg each) were extracted with [metha-nol:water] (50:50, v/v); the supernatant wasthen submitted to quantitative analysis byHPLC separation of phenolics and their gly-coconjugates on a (250 × 4.6) mm Modulo-cart QSLichrospher 5-µm ODS2 columnoperated at 0.5 mL·min–1 and 30 °C. Themobilephase consistedof [water:formic acid](98:2, v/v) (eluant A) and [water:acetoni-trile:formic acid] (18:80:2, v/v/v) (eluant B).The elution program was as follows: 8–13%eluant B for 0–10 min; 13–20% eluant B for10–30 min; 20–8% eluant B for 30–35 min.Duplicate samples were injected at a level of20 µL. The column effluent was monitoredat 280 nm. Triplicate samples were injectedat a level of 10 µL. External standardizationwas achieved with pure glucosides, andaglycones. Data were expressed as % perfresh weight.

Undissected representative portions ofpods (~100–200 mg) were also submitted tothe same extraction-determination protocol.

Estimation of “total” vanillin was achievedaccording to Odoux [3]: “total” vanillin con-tent is that obtained after full hydrolysis ofglucovanillin with almond β-glucosidaseplus free vanillin, the latter being generallylow in the total. Thus “total” vanillin is anacceptable approximation of the glucova-nillin level.

2.5. Extraction and determinationof sugars

Undissected representative portions ofpods (~40–250mg)wereextractedby1.5 mLof 0.01 N sulfuric acid for 2 h at ambient tem-perature, then centrifuged (14 000 g, 5 min).HPLC analyses were performed using a

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Spectra-SERIES separation system P100(Thermo Separation Products, USA) includ-ing a quaternary pump and controlled byChemstation A.10.02 software. Separationswere achieved using a (300 × 7.8 mm i.d.)AMINEX® Ion HPX-87H (7 µm, BioRad) col-umn with a guard column operated at 25 °C.The mobile phase consisted of 0.01 N sul-furic acid at 0.6 mL·min–1.Duplicate sampleswere injected at a level of 20 µL. The columneffluent was monitored with a Shimadzu dif-ferential refractometer. Quantification wasachieved by injection of solutions of knownconcentrations of sucrose, glucose and fruc-tose; data were expressed as % per freshweight.

2.6. Fluorescence microscopy

Fresh cross-sections (100 µm) wereobtained from pods using a MicromHM650V vibratome, stained for few min-utes with Nile red (0.5 mg·mL–1 acetone,then 1/500 dilution in water), thenobserved with a Leica DM6000 epifluores-cence microscope (filter cube A, excitation450–500 nm, emission > 528 nm) (LeicaMicrosystems, Rueil-Malmaison, France).

2.7. Chemicals

Vanillin and p-hydroxybenzaldehyde werefrom Fluka (Buchs, Switzerland). Glucova-nillin was from ChromaDex (Irvine, CA,USA), and glucoside of p-hydroxybenzalde-hyde was synthesized according to Dignumet al. [19].

3. Results

3.1. Variability in the total vanillincontent and β-glucosidase activityamongst populations of maturegreen vanilla pods

Two large batches of mature green vanillapods were analyzed for their “total” vanillincontent and β-glucosidase activity (fig-ure 1). One must note that the mature greenstage was chosen by local producers accord-ing to visual estimation of the pods. As seen,this population exhibits a wide variabilityfrom 0.7% to more than 5.2% of the pod dryweight, with the majority of pods having4.5% of vanillin, i.e., 9.2% as glucovanillin.The same remark may be applied to meas-urement of the β-glucosidase activity:indeed, it varied in a second populationfrom 110 nKat·g–1 fresh weight to more than1680 nKat·g–1 fresh weight.

3.2. Longitudinal distributionof mass, water, β-glucosidase,peroxidase, sugars and phenolicsin mature green vanilla pods

After removal of the corky lignified zonesof attachment to the stem and residual blos-som pieces (3 mm length), vanilla podswere cut into 16 slices of equal thicknessesfrom their stem to their blossom end andanalyzed (figure 2).

Mass percents and contents of water anddry matter along the length of a maturegreen vanilla pod show that a pronouncedwater gradient develops along this axis fromthe highly hydrated stem end (slices n°1, 2,~89.2%), descending to ~84.5% in the drier~2/3-length zone (slice n°12); then, water

Figure 1.Distribution of the « total »vanillin content andβ-glucosidase activity amongsttwo populations of greenmature vanilla pods (n = 70 andn = 95, respectively).

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content increases up to the blossom end(slice n°16, ∼ 87.8%) (figure 2A). All calcu-lations done, slice n°14, i.e., close to theblossom end, is the most representative ofthe entire pod water content. The mass dis-tribution follows an almost symmetrical pat-tern: indeed, the pod shows a tronconicshape from slices n°1 to 6, then adopts apseudo-cylindrical structure (n°7 to 15), andfinally terminates as a cone (n°16).

The β-glucosidase activity exhibits a pat-tern of variation similar to that of the mass(figure 2B): actually, it is also somewhatsuperimposable on the variation in dry mat-ter (figure 2A). Its distribution in nkatals by1/3-length portion (slices n°1 to 6, n°7 to 11,n°12 to 16) was: 22 / 40 / 38. Peroxidase isdifferently distributed: from slices n°6 to 14,its level was constant while it increased gen-tly up to the stem end; on the other end ofthe pod (blossom), it increased tremen-dously up to the terminal slice n°16. Its dis-tribution in units by 1/3-length portion was31 / 27 / 42. Sugars (i.e., sucrose, glucoseand fructose in a constant weight ratio of0.47 / 0.26 / 0.27) decreased from the stemend to slice n°13, then they increased mod-erately up to the floral end (figure 2B).

Variations in the concentrations of glu-covanillin and vanillin (% per FW) showprofiles somewhat symmetrical to the watercontent but slightly shifted (figure 2C).Their levels began from almost nil in thestem end, then progressively climbed up toslice n°14, and finally dramatically felldown to the blossom end; this is also trueon a dry matter basis, glucovanillin varyingfrom 0.08 (slice n°1) to a maximum of 9.43(slice n°12) % (DW), and vanillin from 0.01to 0.85 % (DW). The same trends wereobserved for p-hydroxy-benzaldehyde glu-coside and its aglycon; however, it must benoted that, while glucovanillin is more orless stable from slices n°8 to 15, p-hydroxy-benzaldehyde glucoside goes on increasingfrom slices n°10 to 14. Weight concentra-tion ratios between glucosides and theiraglycones were stable at: [glucovanillin /vanillin] = 11.2, [p-hydroxybenzaldehydeglucoside / p-hydroxybenzaldehyde] = 5.6.Distribution of total phenolics (FW) by 1/3-length portion (slices n°1 to 6, n°7 to 11,n°12 to 16) was: 17 / 43 / 40.

Similar variations were observed on sev-eral other green pods of different origins.

3.3. Radialdistributionofβ-glucosidaseand peroxidase in the differentcompartments of mature greenvanilla pods

Transverse slices (n°14) of vanilla pods werefinely dissected under a stereomicroscope,yielding chlorophyllous mesocarp, placen-tae and trichomes. The mesocarp portionwas further dissected into five concentriccoronae [(I) to (V)] and placentae weredivided into three layers [(VI) to (VIII)] fromtheir base to their top (figure 3).

Figure 2.Variations in mass, water anddry matter (A); β-glucosidase,peroxidase and sugars (B);glucovanillin,p-hydroxybenzaldehydeglucoside, vanillin andp-hydroxybenzaldehydecontents (C), along thelongitudinal axis of a maturegreen vanilla pod.

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Figure 3.Top: transverse slice of amaturegreenvanillapod (n°14)showing the diversecompartments analyzed:(I) epidermis, (II)–(III) outermesocarp, (IV)–(V) innermesocarp, (VI) placental base,(VII) lobes of the placentae,(VIII) funicles, (IX) trichomes.Bottom: anatomicaldistributions of the β-glucosidase (A), andperoxidase (B) activities, andglucovanillin (C) (from ref. [13]).Intensities of colors (clearto deep) are roughly indicativeof numerical values given intable I; white indicates nil.

Table I.Distribution of the β-glucosidase and peroxidase activities, and glucovanillin in the different compartments of atransverse slice (n°14) of a mature vanilla pod (n = 10).

Compartment Compartments N° β-Glucosidase activity(nKat·g–1 FW)

Peroxidase activity(U·g–1 FW)

Glucovanillin2

(% FW)

Mesocarp Epidermis (I) 268 ± 119

Outer mesocarp (II) 311 ± 192

(III) 294 ± 1057124 ± 1831 0.03 ± 0.021

Inner mesocarp (IV) 326 ± 177

(V) 210 ± 123

Placentae Placental base (VI) 1047 ± 305 9.63 ± 1.99

Lobes of the placentae (VII) 1377 ± 112186 ± 91 4.98 ± 1.271

Funicles (VIII) 1210 ± 152

Trichomes Trichomes (IX) 200 ± 89 Not detected 3.93 ± 1.28

1 Measured without sub-dissections of mesocarp and placentae.2 From Odoux and Brillouet [13]; (VII)–(VIII) are the placental lobes in this ref.

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The β-glucosidase activity was measuredonall these fractions including trichomes,while the peroxidase activity was deter-mined on whole mesocarp, placentae andtrichomes without sub-dissection (table I;figure 3). The β-glucosidase was mainly andrather uniformly located in the placentae;mesocarp was 3-4× poorer and no radial gra-dient was observed from its periphery to itsinner portion. Trichomes were the poorestcompartment (1.5× lower than mesocarp).

The peroxidase activity was differentlydistributed: it was absent from the trichomesand showed a [38 / 1] repartition betweenmesocarp and placentae (FW).

3.4. Longitudinal and radialdevelopments of oxidation inripening-senescent vanilla pods

A ripening-senescent pod clearly shows oxi-dation at the blossom end progressivelyinvading up to one-fourth of the lengthwhere green tissues are still present (fig-ure 4); oxidation is also visible at the stemend (∼ 10% of pod length). Transverse sliceswere obtained from different regions, start-ing from the green region and finishing atthe blossom end, from the green area (n°1)to greenish-brown (n°2), to brownish (n°3),to blackish (n°4), to deep black (n°5) ones.On the fresh green slice, one may see thechlorophyllous mesocarp, the diffuse whiteplacental zone, the glassy white V- and J-shaped trichome corners, and, on the frozenequivalent slice, a white area clearly demar-cating the placentae (see also ref. [11]); thefirst signs of oxidation resulting from thetransverse cutting are visible in the freshmesocarp portion as two brownish circles,vascular bundles. On fresh slice n°2, onesees oxidation superimposed on the placen-tal zone; on the frozen ring, three stages areobserved: an intact upper left white pla-centa, an upper right slightly brown one andbelow a deep brown one and there, oxida-tion invaded the mesocarp. On fresh slicen°3, a typical clear reddish-brown urchin-like shape radiating from the placentae out-wards half themesocarp thickness; one mustnote that no oxidation is visible in betweenthe mesocarpic vascular bundles. In this

slice, one may see residual whitish placentalzones and the trichomes stay white (see alsofigure 5). When moving towards the blos-som end, slice n°4 shows a more or less reg-ular annulus uniformly stained reddishwhile trichomes and funicles appear white;half of the mesocarp is still greenish. Finally,on slice n°5, one sees that the entire slice,except trichomes and funicles, turned deepchocolate brown.

On the enlarged view of slice n°3 (fig-ure 5), one may see that the ends of raysfrom the radiating shape are actually vascu-lar bundles: three per triangle side, and atriplet at each corner in the outer mesocarparea made of twin little bundles and alarger one (see also figure 4, slice n°2, andrefs. [11, 20]). There, three small bundlesare also seen in the whitish upper right pla-centa. At this stage, one may see that oxi-dation draws upside-down funnel-likeshapes, their bottom being centered on themesocarpic vascular bundles; areas inbetween bundles remain yellowish-green.

3.5. Anatomical distribution of lipidsin mature green vanilla pods

In a partial transverse section of a nativemature green vanilla pod after lipid stainingwith Nile red (figure 6A), neutral lipid drop-lets intensely fluorescing in sulfur yellowwere seen in the single layer of endocarpcells lining the locule in between the tri-chomes and bordering the placental laminae(or blades); they were also seen in a singlecell layer beneath the trichomes. Whetherthese two single layers are of the sameontogenic origin remains uncertain, andthere is no apparent discontinuity in theirjunction area; the lipid droplets in the subtri-chomal layer, however, appear smaller thanin the properly so-called endocarpic layer.The trichomes also contain numerous smalllipid droplets. Differently from the two abovecompartments (endocarp and trichomes),deposits of an intensely golden orange fluo-rescing substance, not spherically structuredin droplets, were also seen in the terminalregion of the placental blades, i.e., the funi-cles. The autofluorescence of the upper partof the placental laminae (lobes) is apparentlyrestricted to the funicular ends (figure 6B).

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4. Discussion

Arana [1, 2] measured the glucovanillin con-tent in three portions of green mature pods(stem region, middle and blossom region;

presumably, 1/3 of the length each), andfound that glucovanillin mass distribution inthese three regions, taking into account theirweight proportions, was, in the pod, 20/40/40 of the total. We confirm more precisely

Figure 4.Transverse slices from the stemto the blossom end of aripening-senescent vanilla podshowing development andprogression of oxidation. Inpod slice n°2 mesocarpic andplacental vascular bundles areunderscored (black) (see alsorefs. [11, 22]).

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that pronounced gradients exist all along thepods for water, glucovanillin, vanillin, p-hydroxy-benzaldehyde glucoside, and itsaglycon; the shapes of these gradients muchresemble those of mass and dry matter dis-tributions, i.e., negligible levels of phenolicsat the stem end, progressively increasing upto pod slice n°14, then decreasing stronglyuntil the floral end. It is noteworthy that sug-ars followed a symmetrical trend. Comingback like Arana [1] to a 1/3 distribution ofthe three regions (i.e., stem end: fractionsn°1 to 6; middle fractions: n°7 to 11; andblossom end; fractions n°12 to 16; figure 2),we found, all calculations done, a 17/43/40distribution for the total phenolics (glucov-

anillin being largely dominant), a ratioalmost identical to Arana’s; this means thatthe two-thirds of the pod from its apex con-tain more than 80% of the total vanillin pre-cursor. It is noteworthy that the 22/40/38distribution for the β-glucosidase activity isquite similar to the phenolics one.

If one considers the glucovanillin levelsexpressed as mM in the water phase of thepod (i.e., 100 mM, dry matter basis), onemay calculate that its concentration fromslice n°8 to slice n°15 (blossom end) equals~50 mM, i.e., 10 × Km of the vanilla β-glu-cosidase in dilute solutions [6], or 2.2 × K0.5of the same enzyme working in heat-treatedpods [7]; thus, in the first comparison, this

Figure 5.Transverse fresh section (podslice n°3) from the middleregion of a ripening-senescentvanilla pod showingdistribution of oxidation.

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enzyme would work for a while at its Vmax,while, in the second case, hydrolysis wouldalso proceed at Vmax, but for a shorterperiod of time. As in addition to that, thelevel of the β-glucosidase activity shows agradient similar to that of glucovanillin, andalthough most of this activity is lost upon“killing” [7, 21, 22], the residual enzymewould, at the beginning, work at its maxi-mum speed in the middle-blossom endregions; then, its reaction rate woulddecrease progressively downwards to thestem end, as its activity level and glucova-nillin concentration do.

Both the above observations are thus tobe related to the preferential occurrence of

vanillin crystals on the epidermis of the mid-dle region and blossom end of cured vanillapods [1, 2]. It is also noticeable that a blackblossom end-split overripe pod was invadedby mold at the stem end only where vanillin,presumably toxic to some fungal species[23], is present at trace level [pers. com.].

Considering the peroxidase and β-gluco-sidase gradient shapes, one understandsnow why, as long as sufficient vanillin is lib-erated by the β-glucosidase at the blossomend where peroxidase is very high, oxida-tion starts in this portion by forming blos-som-end-yellow pods (figure 4) [1, 10]. Thedeterminism of this preferential oxidation atthe floral end must be searched for in thestructure and anatomy of the pod: vascularbundles, comprising xylem vessels, run itswhole length from the stem to the floralpieces. They are present in a constant num-ber of 27 (figures 4, 5 and ref. [20]) distrib-uted as follows: in the mesocarp – 3× 3(medium diameter; sides of the triangularsection) plus 3 (large diameter; corners) and3 twin (small diameter; corners); in the pla-cental bases – 3×3 (small diameter; sides ofthe triangular section). Since the volumeoccupied by these elements is constantalong the pod, and because both ends of thepod have much more reduced diametersthan the rest of it, then the volume (weight)proportions of xylem elements in these tis-sues increase there to the detriment ofparenchyma (number and/or size of cells);conversely, in the pseudo-cylindrical por-tion (slices n°7 to 5), the ratio (xylem/paren-chyma) is constant, and so is the peroxidaseactivity. Since cell wall-bound [24] and sol-uble peroxidases [25] are known to play arole in the lignification of secondary wallsof the xylem by converting the monolignols(coumaryl, coniferyl and synapyl alcohols)into free radicals which spontaneouslypolymerize to give lignin, one may hypoth-esize that xylem peroxidase(s) is somewhatinvolved in vanillin oxidation; however, theexistence of other non-vascular peroxidasescannot be ruled out (see below). Since glu-covanillin, and thus vanillin and β-glucosi-dase are very low in the very stem end, andalthough peroxidase is, as at the blossomend, higher than in the central core, oxida-tion does not develop there.

Figure 6.Top: cross-section of a vanillapod after staining with Nile red(V-shaped trichome corner ofthe pod; red fluorescingparticles are from thechlorophyllous mesocarp).Bottom: autofluorescence.

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The progressive longitudinal invasion ofvanillin oxidation from the blossom to thestem ends (see below) is also well explainedby these gradients: indeed, glucovanillin isnot prone to oxidation due to engagementof its vanillin phenoxy group in the β-glu-cosidic linkage, while free vanillin is; thus,action of the β-glucosidase is a prerequisiteto oxidation, and since its level decreasescontinuously from slice n°14 downwards tothe stem end, the amount of vanillin liber-ated per unit of time will also decrease, and,thus, the browning front will progressivelymove to the stem end, i.e., the length of timeto get from green to yellowish to chocolatebrown will increase.

The anatomical distributions of glucova-nillin and β-glucosidase were first shownby Odoux et al. [11]; the same authors latergave a more detailed analysis of the repar-tition of phenolics in the green maturepods [13]. We give here an even more com-plete anatomical picture and a quantitativeanalysis of the distributions of the β-glu-cosidase and peroxidase activities in a rep-resentative slice, the peroxidase repartitionhaving, to our knowledge, never been pub-lished (figure 3, table I). β-glucosidase, asformerly shown [13], is high in the placen-tae whatever the portion considered, andlower in the mesocarp and trichomes;again, one must point out the uselessnessof this enzyme, at least at the beginning ofhydrolysis, in the green glucovanillin-freemesocarp, contrary to the assertion ofArana [1]. Peroxidase is far higher in themesocarp than in placentae, and was notdetected in the trichome strips [13]; accord-ing to the fresh matter distribution of thetwo compartments [13], it is thus essentiallyfound in the mesocarp. This result is notsurprising since, as mentioned above, themesocarp contains most of the pod vascularbundles including xylem vessels. Ironically,the mesocarp does not contain oxidizablephenols (e.g., vanillin), but likely only thoseserving as substrates for lignin synthesis(e.g., p-coumaric acid) which are encoun-tered in xylem vessels [26]. Placentae alsoinclude vascular bundles of small diameterand, per placental pair, two of them arelocated at the base of each placental lam-ina, one of them in between (figures 4, 5

and ref. [20]). However, and since oxidationseems homogeneous in placental laminae(figure 4, frozen slice n°2, upper right), i.e.,there is no preferential oxidation surround-ing the bundles, one may imagine that pla-cental peroxidase(s) would not be onlyrestricted to the xylem, but would also beof parenchymatous origin. Indeed, plantperoxidases have been, for instance, alsoencountered in vacuoles [27]. Dignum et al.studied the stability of vanilla peroxidase incrude extracts [16], and Márquez et al.recently purified from whole pods with yel-low apices a cell wall-bound peroxidasefrom vanilla pod [17]. However, the finelocalization of peroxidases in the pod andtheir status (cell wall-bound, and possiblysoluble forms) is not yet known.

When examining the progression of oxi-dation along the longitudinal axis of pods,one may define successive steps:

– in the green parts, enzymatic hydrolysisof glucovanillin did not begin, and onlysome mesocarpic xylem vessels show, aftersection, signs of oxidation, likely of the phe-nyl-propanoids by the xylem peroxidase(s),

– when considering the blossom end, onemay see that oxidation, in addition to theabove observation, concerns the whole pla-cental laminae only, i.e., the mesocarpicparenchyma is not involved. Indeed, at thisstage, glucovanillin only present in the pla-centae and trichomes undergoes progres-sive hydrolysis with liberation of vanillinitself undergoing enzymatic oxidation in theplacentae; at that time, the incompletenessof hydrolysis is visible in residual whitishplacental areas,

– later on, oxidation progressively invadesthe inner mesocarp, drawing radiatingshapes of coalescence with the mesocarpicxylem oxidation zones,

– oxidation develops further into a con-centric annulus comprising half of the mes-ocarp surface, and the coloration intensityincreases, which indicates the completenessof glucovanillin hydrolysis and further sub-sequent oxidation of vanillin,

– finally, oxidation products spread allover the pod section, and the color turnsfrom reddish to chocolate brown.

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Thus, it is now possible to draw a hypo-thetical scheme of events leading to thebrown-blackish vanilla pod (figure 7):

– when ripening on the vine or during thetraditional curing process, a tissular decom-partmentation occurs [5], and phenolic β-glu-cosides and β-glucosidase become present,

– hydrolysis of glucovanillin and otherglucoconjugates by the β-glucosidasebegins in the placentae, and the presenceof this enzyme in the mesocarp serves, atthat time, no use; vanillin and other aglyconsare released, and the whitish zone slowlydisappears,

– rapidly or simultaneously, oxidation ofthese phenolics develops only in the pla-centae where substrates and peroxidase(s)are present; the mesocarp is not concerned,except its xylem vessels,

– reddish then brown oxidation productsdiffuse outwards into the inner mesocarp;however, the possibility that glucovanillinand/or vanillin migrate into the mesocarpand also undergo hydrolysis and oxidationthere cannot be ruled out; nevertheless, onemust recall that placentaehave by far enough

β-glucosidase for glucovanillin to be hydro-lyzed in situ without diffusing into anothercompartment [11, 13, this work]. At thatmoment, it is important to recall the com-ment of Arana [1] on the prerequisite for glu-covanillin to diffuse from the inner placentalparts of the pods outwards to the mesocarpfor being hydrolyzed by the β-glucosidase:actually, we show here that this sequence ofevents is not compulsory but may or may nothappen. It is presumably the oxidation prod-ucts accompanied or not by glucovanillinand vanillin which diffuse into the mesocarp.Indeed, the possibility that vanillin wouldalso be oxidized by the mesocarp xylem per-oxidase(s) cannot be ruled out. Until perox-idase activity is precisely localized in themesocarp (vascular bundles and paren-chyma?), this question remains open,

– finally, the whole pod turns deep brown,then blackens upon drying.

A particular point must be emphasized:throughout the ripening-ageing periodwhen pods turn from green to black, the tri-chomes and funicles, although sources ofglucovanillin and β-glucosidase (table I and

Figure 7.Sequence of someconsecutive enzymatic andchemical reactions occurringat the vanillin level in the vanillapod.

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refs. [11, 13]), do not show oxidation andremain whitish and glassy until late. This isto be related to the absence of peroxidaseand also to their richness in lipids (figure 3and refs.[13, 28]); as formerly mentioned[13], the absence of catechin-HCl staining ofvanillin-containing trichomes [12] was likelydue to the presence of high quantities oflipid reserves in this compartment. The con-stitution of trichome lipids is now partlydefined (γ-pyranones) [13], while the funic-ular ones remain unknown.

Finally, we also found that the “total”vanillin content, actually close to the glu-covanillin concentration [3], of green vanillapods was quite variable amongst a popula-tion: thus, to characterize a batch or followmaturation kinetics, one has to take intoaccount this variability and carry out meas-urements on a sufficient number of pods.The same observation was true for the β-glu-cosidase activity, one of the key enzymes inthe development of the vanilla aroma: it wasvariable amongst a population of pods ofthe same physiological age and origin. Thisis also likely true for a given set of vines fromone year to another, depending on the cli-matic conditions of culture. Again, one mustbe cautious and analyze numerous podsbefore giving an amount of activity per unitof fresh weight.

5. Conclusion

Green mature vanilla pods are heterogene-ous along their longitudinal axis, exhibitingpronounced gradients of decreasing-increasing concentrations of water, andincreasing-decreasing ones of β-glucosidaseand phenolics from the stem to the floralend; peroxidase activity is differently distrib-uted, stable in a major portion of the podand increasing at its two ends. These ele-ments are also heterogeneously distributedin the vanilla pod tissues, phenolics and β-glucosidase being preponderant in the pla-centae while peroxidase predominates inthe mesocarp. Finally, oxidation of phenolsdevelops from the placental region of theblossom end towards the stem end; then the

oxidation products (brown pigments) pro-gressively invade the mesocarp and, in theend, the whole pod.

Green mature vanilla pods are variablefor their glucovanillin and β-lucosidase con-tents, which must be taken into account instudies devoted to these components.

Acknowledgements

We thank M. Bourgois (Eurovanille, France)and Dr. K. Waliszewski (Inst. Tecnol., Ver-acruz, Mexico) for gifts of Indian and Mex-ican vanilla pods, respectively. Thanks arealso due to G. Morel (CIRAD-PERSYST) forhis helpful assistance in HPLC analyses.

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Conjunto de resultados sobre la vaina de vainilla verde, en proceso demaduración y sensescente (Vanilla planifolia; Orchidaceae): anatomía,encimas, compuestos fenólicos y lípidos.

Resumen — Introducción. Las vainas de vainilla verdes maduras acumulan 4-O-(3-metoxi-benzaldehído)-β-D-glucósido (glucovainilla) que, bajo el efecto de una hidrólisis medianteuna β-glucosidasa endógena, libera vainillina, el principal compuesto de aroma de la vainilla.Las reparticiones espaciales de los compuestos fenólicos generadores de aroma y de las enci-mas responsables de su liberación (β-glucosidasa) y oxidación (peroxidasa) son poco conoci-das. A continuación ofrecemos resultados cuantitativos de estos tres compuestos en relacióncon la anatomía de la vaina. Asimismo, se ilustrará la progresión espacial de la oxidación.Material y métodos. Se analizaron vainas de vainilla verdes maduras para su contenido encompuestos fenólicos y actividades β-glucosidasa y peroidasa mediante técnicas espectrofoto-métricas, gracias al empleo respectivo de glucósido de p-nitrofenilo y de vainilla como sustra-tos. Los lípidos se examinaron en microscopía fluorescente tras coloración roja de Nil. Eldesarrollo de la oxidación se observó en cortes transversales de vainas. Resultados y discu-sión. Los compuestos fenólicos, así como las actividades β-glucosidasa y peroxidasa mues-tran gradientes de concentraciones crecientes-decrecientes de la extremidad peduncular haciala extremidad floral de la vaina. La actividad β-glucosidasa se distribuye entre placentas,mesocarpio y tricomas con una relación [7 / 2 / 1], mientras que la actividad peroxidasa sereparte en proporciones [38 / 1] entre el mesocarpio y las placentas; está ausente de tricomas.La oxidación empieza en las placentas de la parte floral invadiendo progresivamente el meso-carpio y se desplaza hacia la zona peduncular. Conclusión. La vaina de vainilla verdemadura es heterogénea desde un punto de vista espacial en lo que se refiere a sus compues-tos fenólicos y actividades β-glucosidasa y peroxidasa, las placentas desempeñan un papelimportante en la liberación de la vainillina y en su oxidación consecutiva.

Francia / Vanilla planifolia / Orchidaceae / vainilla / β-glucosidasa / peroxídasas /glucovanillina / vanillina / lípidos / oxidación / mesocarpio / placentas / tricomas /xilema