Phenolic Compounds in Maple Syrup - ACS Symposium Series (ACS

Chapter 15

Phenolic Compounds in Maple Syrup Thomas L. Potter and Irving S. Fagerson

Downloaded by CORNELL UNIV on June 13, 2012 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0506.ch015

Mass Spectrometry Facility, College of Food and Natural Resources, Chenoweth Laboratory, University of Massachusetts, Amherst, MA 01003

" A c i d / n e u t r a l " dichloromethane e x t r a c t s o f two grade A medium amber maple syrups were prepared and analyzed by gas chromatography/mass spectrometry. In excess o f 133 compounds were detected. P h e n o l i c s accounted f o r 41 o f these compounds and over 70 percent of the t o t a l mass o f compounds detected. The composition o f the syrup e x t r a c t s was qualitatively s i m i l a r but e x h i b i t e d s u b s t a n t i a l q u a n t i t a t i v e d i f f e r e n c e s . The data i n d i c a t e t h a t d u r i n g syrup production o x i d a t i o n o f p h e n o l i c l i g n i n monomers in maple sap i s l i m i t e d . I m p l i c a t i o n s f o r product f l a v o r and q u a l i t y are d i s c u s s e d . Maple syrup i s d e r i v e d from evaporative c o n c e n t r a t i o n of the s p r i n g sap flow o f the sugar maple (Acer saccharum March). I t i s unique i n t h a t i t i s the only commercially a v a i l a b l e food d e r i v e d s o l e l y from the sap of deciduous t r e e s . Under c u r r e n t c l a s s i f i c a t i o n s , t h e U.S. Department o f A g r i c u l t u r e (USDA) recognizes two syrup grades, A and B, and, w i t h i n grade A., l i g h t , medium and dark amber. The syrup i s comprised p r i m a r i l y o f sucrose with s m a l l e r amounts o f glucose and f r u c t o s e ( 2 ) . Key f l a v o r c o n s t i t u e n t s i n c l u d e v a n i l l i n , 3-methyl-2-hydroxycyclopenten-2-one ( c y c l o t e n e ) , 2,5-dimethyl-4-hydroxy 2(2H)-furanone ( f u r a n e o l ) and r e l a t e d compounds (2,3). These compounds are not present i n a p p r e c i a b l e quant i t i e s i n sap and are presumed d e r i v e d from the sugars and other sap c o n s t i t u e n t s d u r i n g evaporative c o n c e n t r a t i o n (3). I n v e s t i g a t i o n s with model systems have served t o demonstrate t h a t formation o f f l a v o r compounds d u r i n g maple syrup production f o l l o w s two pathways. One 0097-6156/92/0506-0192$06.00/0 © 1992 American Chemical Society In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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Phenolic Compounds in Maple Syrup 193

i n v o l v e s the thermal decomposition of sugars under a l k a l i n e c o n d i t i o n s t o form c y c l o t e n e , f u r a n e o l and r e l a t e d compounds. Shaw e t a l . (5,6) have reported t h a t these compounds are formed upon h e a t i n g a l k a l i n e sucrose and f r u c t o s e s o l u t i o n s . Given the reported d e t e c t i o n of t r a c e q u a n t i t i e s of f r e e amino a c i d s i n maple saps (2), non-enzymatic browning r e a c t i o n s may a l s o c o n t r i b u t e (7). The second f l a v o r production pathway i n v o l v e s a d i f f e r e n t s e t of precursor compounds, namely, l i g n i n monomers present i n sap. O x i d a t i o n and a l k a l i n e h y d r o l y s i s of these p h e n o l i c compounds are presumed r e s p o n s i b l e f o r v a n i l l i n and syringaldehyde formation. F i d d l e r e t a l . (8) have reported t h a t v a n i l l i n i s a major product of the thermal decomposition of f e r u l i c a c i d i n a i r . V a n i l l i n i s a l s o found i n c o n i f e r y l a l c o h o l p r e p a r a t i o n s l e f t exposed t o a i r ( P o t t e r and Fagerson unpublished r e s u l t s ) . The s i g n i f i c a n c e of o x i d a t i o n has a l s o been i l l u s t r a t e d by Underwood and F i l i p i c , ( 3 ) . They showed t h a t a l k a l i n e nitrobenzene o x i d a t i o n of sap e x t r a c t s , "lignaceous m a t e r i a l " i s o l a t e d from sap, and chloroform e x t r a c t s of maple syrup g r e a t l y i n c r e a s e d v a n i l l i n and t o a l e s s e r extent syringaldehyde content. The l a t t e r observation i n d i c a t e s t h a t there i s a s u b s t a n t i a l q u a n t i t y of unoxidized p h e n o l i c m a t e r i a l s i n maple syrups and l e d i n p a r t to our i n v e s t i g a t i o n . In the c u r r e n t work, we r e p o r t i d e n t i f i c a t i o n of p h e n o l i c and r e l a t e d f l a v o r compounds i n dichloromethane e x t r a c t s of two grade A medium amber Massachusetts syrups. The data has confirmed hypotheses t h a t sap l i g n i n monomers are present i n r e l a t i v e l y high c o n c e n t r a t i o n i n maple syrup. I m p l i c a t i o n s f o r improving syrup f l a v o r and q u a l i t y are discussed. M a t e r i a l s and Methods M a t e r i a l s . Syrup A was purchased a t a l o c a l market. Label information provided on i t s c o n t a i n e r l i s t e d the product as Grade A medium amber. Syrup B, a l s o l a b e l e d as grade A medium amber, was obtained d i r e c t l y from a l o c a l producer. The syrups were produced i n the 1990 syrup season. Sample P r e p a r a t i o n . One hundred grams of syrup were d i l u t e d t o 500 ml with d i s t i l l e d - d e i o n i z e d water and spiked with 25 ug each of naphthalene-d8 and phenol-d6 u s i n g 15 u l of acetone t o a i d d i s s o l u t i o n . S o l u t i o n pH was then adjusted t o 2 with 6N s u l f u r i c a c i d , followed by s e r i a l e x t r a c t i o n i n a separatory funnel with 3 X 50 ml a l i q u o t s of " d i s t i l l e d i n g l a s s " dichloromethane. The r e s u l t i n g " a c i d / n e u t r a l " e x t r a c t was concentrated

In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

194

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I


by r o t a r y evaporation and taken t o i t s f i n a l volume (0.3 ml) under a stream of dry n i t r o g e n . Emulsions formed during e x t r a c t i o n were broken by f r e e z i n g . Solvents were obtained from Burdick and Jackson (Muskegeon, MI) and deuterated standards from A l d r i c h Chemical Co. (Milwaukee, WI). I d e n t i c a l e x t r a c t i o n c o n d i t i o n s were a p p l i e d t o a reagent blank c o n s i s t i n g of 500 ml of d i s t i l l e d - d e i o n i z e d water. A l l e x t r a c t s were s t o r e d a t -20°C p r i o r t o GC/MS a n a l y s i s . GC/MS A n a l y s i s . GC/MS a n a l y s i s was performed with a Hewlett-Packard 5985B system equipped with a 30 m X 0.33 mm I.D. DB-WAX ( J + W S c i e n t i f i c , Folsom, CA) fused s i l i c a c a p i l l a r y column (0.25 micron f i l m ) . Helium i n l e t pressure on the column was f i x e d a t 104 kPa and i n j e c t i o n was i n the Grob s p l i t l e s s mode a t 250°C. The oven temperature p r o f i l e was as f o l l o w s : 60°C(hold 1 minute) t o 240°C a t 2°C/min (hold 29 minutes). The column was i n t e r f a c e d t o the mass spectrometer through an o p e n - s p l i t i n t e r f a c e ( S c i e n t i f i c Glass and Engineering, A u s t i n , TX). Mass s p e c t r a were obtained a t 70eV over 33 t o 350 amu a t a mass f i l t e r scan r a t e of 300 amu/sec. The instrument was tuned t o meet standard p e r f l u o r o t r i b u t y l a m i n e criteria. Reference Compounds. The corresponding a l c o h o l s and a l dehydes o f f e r u l i c , coumaric, s y r i n g i c , s i n a p i c , v a n i l l i c , h o m o v a n i l l i c , and homoveratric a c i d s were prepared by L i A l H r e d u c t i o n i n e t h e r . Products were i s o l a t e d by dichloromethane e x t r a c t i o n a f t e r quenching with water. T y p i c a l l y , the a l c o h o l , aldehyde, d i h y d r o a l c o h o l , and i n some cases the a l l y l phenols were obtained. A l l reagents and other r e f e r e n c e compounds were obtained from A l d r i c h Chemical Co. (Milwaukee, WI). 4

R e s u l t s and D i s c u s s i o n T o t a l i o n c u r r e n t chromatograms obtained from the a n a l y s i s of the syrup e x t r a c t s are shown i n F i g u r e 1. In excess of 133 compounds were d e t e c t e d . E i g h t y - f o u r were assigned s t r u c t u r e s with 48 confirmed by a n a l y s i s of r e f e r e n c e compounds. P h e n o l i c s accounted f o r 41 o f the compounds detected. O v e r a l l , r e s u l t s were i n q u a l i t a t i v e agreement with p u b l i s h e d s t u d i e s o f maple syrup v o l a t i l e s composition (3,4). The exception i s i n the much l a r g e r number o f com-pounds detected i n the c u r r e n t work. A minor d i f f e r e n c e noted i s t h a t K a l l i o e t a l . (10) reported h o m o v a n i l l i c a c i d t o be among the major v o l a t i l e c o n s t i t u e n t s o f maple syrup but d i d not r e p o r t d e t e c t i o n o f d i h y d r o c o n i f e r y l a l c o h o l . We detected only t r a c e amounts o f the a c i d and r e l a t i v e l y high



30

\

surrogate standards

\

Time

60

vanillin

(minutes)

ι

.

90

F i g u r e 1. T o t a l i o n c u r r e n t chromatograms o f maple syrup dichloromethane e x t r a c t s

SYRUP A



196


c o n c e n t r a t i o n s o f the a l c o h o l . We a l s o obtained n e a r l y i d e n t i c a l mass s p e c t r a f o r these compounds under e l e c t r o n impact c o n d i t i o n s and observed t h a t t h e i r chromatographic s e p a r a t i o n c o u l d not be achieved u s i n g c a p i l l a r y columns coated with non-polar l i q u i d phases. Thus, peak mis-assignment by K a l l i o e t a l . (20) may provide a p o s s i b l e e x p l a n a t i o n . The Table I data show t h a t t h e estimated t o t a l c o n c e n t r a t i o n o f compounds i n t h e syrup e x t r a c t s ranged from 5.8 t o 14.7 ug/g. Concentration estimates were based on the assumption o f u n i t t o t a l i o n c u r r e n t response t o naphthalene-d8. Recovery o f t h e surrogate standards, naphthalene-d8 and phenol-d6, ranged from 41 to 69 percent. Given t h i s , c o n c e n t r a t i o n values r e p o r t e d are expected t o represent minimum v a l u e s . The Table I data a l s o show t h a t " p h e n o l i c s " accounted f o r 72 t o 78 percent o f t h e t o t a l mass o f compounds detected.

Table I . C l a s s e s o f compounds detected i n maple syrups Concentration (ug/g) a

A 13

sucrose p y r o l y s i s p r o d u c t s fatty acids phenolics^ unknowns miscellaneous p l a s t i c i z e r s total 5.79 14.7 c

e

Β 0.44 0.49 10.6 2.28 0.91

0. 24 0. 23 4. 52 0. 68 0. 11

a

Concentration based on assumed u n i t t o t a l i o n c u r r e n t to the surrogate standard, naphthalene-d8; Compounds r e p o r t e d as sugar degradation products i n c l u d e v a r i o u s l a c t o n e s , furanones, cylcopentenones and r e l a t e d compounds; f r e e f a t t y a c i d s ; ° compounds with a hydroxy-phenyl moiety and/or aromatic c a r b o x y l i c a c i d s , a l c o h o l s and aldehydes; miscellaneous phthalate and adipate e s t e r p l a s t i c i z e r s . b

c

e

The c o n c e n t r a t i o n s of major syrup p h e n o l i c s a r e summarized i n Table I I . As i n d i c a t e d , v a n i l l i n and syringaldehyde, p h e n o l i c o x i d a t i o n products o f and major c o n t r i b u t o r s t o syrup f l a v o r were detected a t much lower c o n c e n t r a t i o n s than t h e i r presumed p r e c u r s o r s , c o n i f e r y l , d i h y d r o c o n i f e r y l and d i h y d r o s i n a p y l a l c o h o l . T h i s o b s e r v a t i o n provides q u a n t i t a t i v e support t o Underwood and F i l i p i c ' s


15.


POTTER AND FAGERSON

experimental r e s u l t s (3). In s h o r t , there i s a s i g n i f i c a n t p o r t i o n of the lignaceous m a t e r i a l i n maple sap which i s not o x i d i z e d d u r i n g evaporation. Table I I . P r i n c i p a l p h e n o l i c and r e l a t e d compounds i n maple syrups

flavor Concentration (ug/g) b


Compound

a

phenethyl a l c o h o l eugenol isoeugenol isomer isoeugenol isomer vanillin syringaldehyde dihydroconiferyl alcohol c o n i f e r y l aldehyde syringyl alcohol coniferyl alcohol dihydrosinapyl alcohol v a n i l l i c acid s i n a p y l aldehyde + h o m o v a n i l l i c a c i d 4-allyl-3,5-dimethoxytoluene syringic acid

A

Β

0,.03 0,.01 0,.09 0,.34 1,.04 0,.09 0,.15 1,.27 0,.48 0..03 0,.04 0,.31 0..06

0,.07 0,.08 0,.09 0..90 0,.93 3,.30 0,.40 0..76 1,.31 0,.86 0,.12 0,.21 0,.12 0,.22

a

a l l compounds confirmed by a n a l y s i s of r e f e r e n c e compound except 4-allyl-3,5-dimethoxytoluene; c o n c e n t r a t i o n based on assumed u n i t response t o the surrogate standard, naphthalene-d8. b

Another notable f e a t u r e of the Table I I data i s the observed q u a n t i t a t i v e d i f f e r e n c e i n the r e l a t i v e composition of the syrups e x t r a c t s . Syrup Β had a t e n f o l d g r e a t e r c o n c e n t r a t i o n of v a n i l l i n and t h r e e - f o l d g r e a t e r c o n c e n t r a t i o n s of syringaldehyde and d i h y d r o c o n i f e r y l a l c o h o l . These d i f f e r e n c e s demonstrate the q u a n t i t a t i v e v a r i a b i l i t y among syrups w i t h i n the same grade and produced i n the same season and geographic r e g i o n . In terms of sensory impact, i t i s i n t e r e s t i n g t o note t h a t the t a s t e d e t e c t i o n t h r e s h o l d f o r v a n i l l i n i n water has been reported t o be i n the 0.5 t o 1 p a r t s per m i l l i o n range (9). These observations i n d i c a t e t h a t v a n i l l i n might c o n t r i b u t e t o the t a s t e of syrup Β but not A. I m p l i c a t i o n s f o r syrup aroma can be i n f e r r e d , s i n c e K a l l i o e t a l . (4) found t h a t v a n i l l i n provided the most s i g n i f i c a n t a t t r i b u t e f o r maple syrups i n aroma panel t e s t s .



198


A p o s s i b l e explanation of the compositional d i f f e r e n c e s observed i s v a r i a t i o n i n o x i d a t i o n c o n d i t i o n s during sap evaporation. For example, processing v a r i a b l e s , such as foam c o n t r o l may have a s i g n i f i c a n t impact on the s u r f a c e area of contact with a i r . Thus, reduced foam c o n t r o l may promote p h e n o l i c s o x i d a t i o n and v a n i l l i n production. A n a l y s i s of a syrup produced i n a jacketed steam k e t t l e with no foam c o n t r o l showed t h a t v a n i l l i n and was the p r i n c i p a l phenolic c o n s t i t u e n t (Potter and Fagerson unpublished data). While s u b s t a n t i a l v a r i a t i o n was noted i n the " p h e n o l i c s " c o n c e n t r a t i o n i n the syrups, concentrations of key sugar degradation products detected were s i m i l a r . Cyclotene, f u r a n e o l , and i s o m a l t o l were detected a t 15 t o 27, 7 t o 9 and 2 t o 4 ng/g, r e s p e c t i v e l y . T h i s r e l a t i v e u n i f o r m i t y i n composition suggests t h a t these compounds c o n t r i b u t e t o the base syrup f l a v o r while v a n i l l i n and other compounds express s u b t l e d i f f e r e n c e s between products. Conclusions A wide array of n a t u r a l p h e n o l i c s and other compounds were i d e n t i f i e d i n the syrups s t u d i e d . Among the p h e n o l i c s , v a r i o u s l i g n i n monomers and t h e i r p a r t i a l o x i d a t i o n products were prominent. The l a t t e r compounds play a key r o l e i n maple syrup f l a v o r . Data obtained has provided q u a n t i t a t i v e support f o r observations t h a t o x i d a t i o n of sap p h e n o l i c c o n s t i t u e n t s does not approach completion during syrup production. T h i s presents the p o s s i b i l i t y t h a t , other t h i n g s being equal, d i f f e r e n c e s i n evaporator design and syrup production p r a c t i c e s may produce syrups of d i f f e r i n g sensory q u a l i t i e s . We conclude t h a t i n depth s t u d i e s of o x i d a t i o n processes during syrup production may a s s i s t i n the development of more uniform and higher q u a l i t y products. Another f e a t u r e of the study i s t h a t i t demonstrates the p o t e n t i a l f o r d e t e c t i n g syrup a d u l t e r a t i o n by gas chromatographic a n a l y s i s . Analyses focused on d e t e c t i n g c o n i f e r y l and d i h y d r o c o n i f e r y l a l c o h o l s and other n a t u r a l products provide a means f o r d i f f e r e n t i a t i n g between n a t u r a l and a d u l t e r a t e d products. Such analyses may r e p l a c e and/or supplement carbon isotope r a t i o measurements d e s c r i b e d by M o r s e l l i and Baggett (11). Acknowledgments Support f o r t h i s work was provided by the Massachusetts A g r i c u l t u r a l Experiment S t a t i o n . We thank W. C o l i f o r donation of a syrup sample. C o n t r i b u t i o n no. 3003 from the Massachusetts A g r i c u l t u r a l Experiment S t a t i o n .


15. POTTER AND FAGERSON


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November 20, 1991


Phenolic Compounds in Maple Syrup - ACS Symposium Series (ACS

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