Kinetics of Formation of Alkylpyrazines - American Chemical Society

Chapter 7

Kinetics of Formation of Alkylpyrazines

Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: February 21, 1989 | doi: 10.1021/bk-1989-0388.ch007

Effect of Type of Amino Acid and Type of Sugar 1

2

M. M. Leahy and G. A. Reineccius 1

Ocean Spray Cranberries, Inc., R & D Building, Bridge Street, Middleboro, MA 02346 Department of Food Science, University of Minnesota, St. Paul, MN 55108

2

Pyrazines are heterocylic, nitrogen-containing compounds important to the flavor of many foods. Prior studies relating to the effects of type of amino acid and type of sugar on the formation of pyrazines have yielded contradictory results. This study investigates the effects of type of amino acid and type of sugar on the kinetics and distribution pattern of pyrazines formed. The amino acids, lysine and asparagine, and the sugars, glucose, fructose and ribose were chosen for this study. One-tenth molar sugar/amino acid solutions buffered at pH 9.0 were heat-processed. Samples were analyzed using a headspace concentration capillary gas chromatographic technique with nitrogen-selective detection. Rate of pyrazine formation fit pseudo zero order reaction kinetics. Effects of amino acid and sugar types on activation energies, yields and relative distributions of pyrazines are discussed. Pyrazines are heterocyclic, nitrogen-œntairiing compounds important t o the f l a v o r o f many foods. Alkylpyrazines have often been found i n heated foods and have been characterized as having roasted, toasted, nutty f l a v o r notes. Some excellent reviews have previously d e t a i l e d the presence o f pyrazines i n a great v a r i e t y of foods. Maga and S i z e r (1, 2) published the f i r s t comprehensive reviews on pyrazines i n foods. They reviewed the occurrences o f numerous pyrazines i n a wide v a r i e t y o f foods, pyrazine i s o l a t i o n , concentration, separation and i d e n t i f i c a t i o n techniques, pyrazine f l a v o r properties, pyrazine mechanisms o f formation and pyrazine synthesis techniques. Since then several others (3 - 10) have reviewed progress i n pyrazine research. Understanding both the e f f e c t s o f various parameters on the k i n e t i c s o f the formation o f pyrazines and the mechanism o f formation may allow f o r the β

0097-6156/89/0388-0076$06.00/0 1989 American Chemical Society

Teranishi et al.; Flavor Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


7. LEAHY AND REINECCIUS

Kinetics of Formation ofAlkylpyrazines

77

optimization o f pyrazine production i n both foods and i n r e a c t i o n flavors. Alkylpyrazines are most commonly found i n roasted and toasted foods and a r e believed t o form as a r e s u l t o f the M a i l l a r d browning r e a c t i o n (11). Several researchers have proposed mechanisms for alkylpyrazine formation in various cmbohydrate/amine systems (12 - 21). These pathways generally involve t h e formation o f aminocarbonyl fragments which condense, y i e l d i n g dihydropyrazines o r hydroxy aihydropyrazines. These i n t u r n y i e l d pyrazines through oxidation (17) o r dehydration reactions (18, 19). Aminocarbonyl fragments r e s u l t through various M a i l l a r d r e a c t i o n pathways. Some researchers postulate mechanisms i n which free ammonia formed as a r e s u l t o f amino a c i d decomposition reacts with sugars and sugar fragments y i e l d i n g alkylpyrazines (15, 16) · Others have proposed mechanisms by which sugars and amino acids condense through the generalized Hodge M a i l l a r d r e a c t i o n scheme (11, 12, 13) and Strecker degradation o f amino acids with cUcarbonyl fragments (17). Shibamoto and Bernhard (18) proposed the most d e t a i l e d scheme o f pyrazine formation pathways i n sugar^ammonia model systems, involving p^aminocarbonyl intermediates which condense to form alkylpyrazines. Various researchers have investigated factors a f f e c t i n g both y i e l d s o f i n d i v i d u a l pyrazines and t h e i r d i s t r i b u t i o n s , including source o f nitrogen and source o f carbon. Often these studies have y i e l d e d seemingly contradictory r e s u l t s . The e f f e c t o f source o f nitrogen on pyrazine formation was i n i t i a l l y investigated by Newell e t a l . (13). They reacted various amino acids with glucose and demonstrated t h a t q u a l i t a t i v e l y the same v o l a t i l e pyrazine compounds were produced regardless o f the amino a c i d employed as the nitrogen source. Van Praag e t a l . (15) obtained s i m i l a r r e s u l t s i n r e a c t i n g glycine, leucine, isoleucine, v a l i n e and alanine with D-fructose. Koehler e t a l . (16) observed formation of a s i m i l a r s e r i e s o f pyrazines when r e a c t i n g glucose with asparagine, glutzunine, glutamic a c i d and a s p a r t i c a c i d . Mostly a l k y l a t e d pyrazines were found, with only traces o f unsubstituted pyrazine. However, i n r e a c t i n g ammonium c h l o r i d e with glucose, unsubstituted pyrazine was the main pyrazine observed with only traces o f a l k y l a t e d pyrazines being found. Also, d i f f e r e n t pyrazine product d i s t r i b u t i o n s and t o t a l pyrazine concentrations were obtained f o r the d i f f e r e n t amino acids. Wang and O d e l l (14) reacted gycerol with alanine and glycine. Similar series of pyrazines r e s u l t e d , however t o t a l y i e l d s and d i s t r i b u t i o n s were different. Most recently, Wong and Bernhard (20) demonstrated t h a t t h e d i s t r i b u t i o n and y i e l d o f pyrazines formed i n the reactions o f glucose with ammonium hydroxide, ammonium formate, ammonium acetate, g l y c i n e and monosodium glutamate depended strongly on the nature o f nitrogen source. The e f f e c t o f source o f carbohydrate on pyrazine formation was f i r s t investigated by van Praag e t a l . (15). The same s e r i e s of pyrazine CGnpounds was i s o l a t e d from a r e a c t i o n mixture o f fructose o r glucose with ammonia. Koehler e t a l . (16) found t h a t the carbohydrate source a f f e c t e d the t o t a l y i e l d o f pyrazines formed. I n r e a c t i n g 10 irmoles each o f a sugar with ammonium c h l o r i d e , t o t a l y i e l d o f pyrazine was 59 jumoles with glucose and



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DEVELOPMENTS

195 jumoles with fructose. Koehler and Odell (22) monitored production o f methylpyrazine and diirethylpyrazine i n r e a c t i n g asparagine with glucose, fructose, sucrose and arabinose. They found the carbon source t o a f f e c t both t o t a l y i e l d and r e l a t i v e distribution. In a more comprehensive study, Shibamoto and Bernhard (18) reacted ammonia with several sugars. Reaction systems consisted o f a s o l u t i o n o f 8M ammonium hydroxide and 1M carbohydrate, heated a t 100°C f o r 2 h. They concluded t h a t pentoses g i v e greater t o t a l y i e l d s o f pyrazines than hexoses, and y i e l d s from mannose, glucose and fructose are about equal with galactose g i v i n g s l i g h t l y lower y i e l d s . D i s t r i b u t i o n patterns were e s s e n t i a l l y s i m i l a r f o r the pentoses and hexoses, except aldoses gave more unsubstituted pyrazine than the ketose, fructose. They concluded that epimers, diastereomers and enantiomers gave identical pyrazine pattern d i s t r i b u t i o n s , c o n t r a d i c t i n g the work o f Koehler and Odell (22). CXir study was conducted t o determine the e f f e c t s o f type o f amino a c i d and type o f sugar on both the k i n e t i c s and the d i s t r i b u t i o n p a t t e r n o f alkylpyrazines formed. For i n v e s t i g a t i n g the e f f e c t o f type o f amino acid, t h i s work focused on two amino acids, l y s i n e and asparagine. l y s i n e was chosen because i t contains two amino groups a v a i l a b l e f o r reaction. Asparagine was chosen because Koehler and Odell (22) reported t h a t y i e l d s o f pyrazines were greatest when t h i s amino a c i d was reacted with glucose, verses alanine, g l y c i n e o r l y s i n e . For i n v e s t i g a t i n g the e f f e c t o f sugar, glucose, fructose and ribose were chosen f o r t h i s study. Glucose and fructose were chosen t o provide two common hexoses i n foods, one an aldose and one a ketose. The pentose r i b o s e was chosen because o f i t s reportedly high degree o f r e a c t i v i t y i n meat reaction f l a v o r systems (Dwidedi, 23). The present i n v e s t i g a t i o n used a headspace concentration c a p i l l a r y gas chromatographic technique with nitTOgen-phosphorus detection. Advantages o f t h i s technique are t h a t the procedure was r a p i d (about 30 minutes), p o t e n t i a l f o r a r t i f a c t formation i s minimized and sample requirements are small (15 ml). Experiinental Section The amino acids, sugars, borate s a l t s and solvents were a l l reagent grade, obtained from commercial sources. The amino acid-sugar combinations investigated i n the present study included asparagine-fructose, asparagine-glucose, lysine-fructose, lysine-glucose and lysine-ribose a t concentrations o f 0.1M f o r both amino a c i d and sugar, i n a pH 9.0 0.1M borate b u f f e r (24). Ten ml o f each s o l u t i o n were heated i n Teflon-capped 25 mm (o.d.) χ 150 mm Pyrex t e s t tubes i n a water bath a t 75, 85, and 95°C f o r up t o 24 h. Samples were taken a t 7 t o 8 time i n t e r v a l s . Eighteen t o 20 t o t a l samples per temperature were analyzed. Although the amino acid/sugar solutions were buffered i n an e f f o r t t o maintain pH, a drop i n pH was encountered with increasing r e a c t i o n times. Therefore, a f t e r heat treatment, each sample was adjusted t o pH 9.0 with 0.1N NaOH. One ml o f a s o l u t i o n containing 2-methoxypyrazine i n d i s t i l l e d water (2 ppm) was added as an i n t e r n a l standard. F i n a l sample volume was 15 ml. Pyrazines were then i s o l a t e d , separated and q u a n t i f i e d using an



7.

L E A H Y AND REINECCIUS

Kinetics of Formation ofAlkylpyrazines

automated headspace concentration sampler (Hewlett Packard 7675A Purge and Trap) coupled t o a Hewlett Packard 5880A gas cixromatograph with nitrogen-phosphorus detection. The 15 ml sample was attached t o the purge and t r a p sampler and purged f o r 10 min with hydrogen a t a flow r a t e o f 90 ml/min. V o l a t i l e s were adsorbed on a 4" by 1/4" o.d. s t a i n l e s s s t e e l precolumn packed with Tenax (Hewlett Packard Co., Avondale, PA). A t the end o f the purge period, the sample was removed from the sampler before manually switching t o the desorb c y c l e . E l u t i o n o f the concentrated organic v o l a t i l e s from the Tenax precolumn onto the GC column was accomplished by heating the precolumn t o 180°C with a hydrogen flow o f 90 ml/min. The combined flow o f hydrogen and v o l a t i l e s was s p l i t 50:1 and passed onto the GC column, a 25 m by 0.32 mm (i.d.) DB 225 fused s i l i c a c a p i l l a r y column ( J & W S c i e n t i f i c Inc., Rancho Cordova, CA). The column head pressure was maintained a t 15 p s i g which provided a l i n e a r v e l o c i t y o f 45 cm/sec and a flow r a t e o f 4 ml/min. The v o l a t i l e s were desorbed f o r 3 min from the Tenax column onto the chromatographic column. During t h i s period, the v o l a t i l e s were cold-trapped a t the head o f the chromatographic column by immersing a 20 cm loop o f the column i n a 2 3/8" i . d . χ 4 1/2" Dewar f l a s k containing l i q u i d nitrogen. The oven temperature was isothermal a t 50°C f o r the run, with a post value o f 200°C f o r 1.5 min. The i n j e c t i o n port and NPD temperatures were 225°C and 280°C, respectively. Figure 1 gives a t y p i c a l chrcmatogram obtained by t h i s procedure. Q u a n t i f i c a t i o n o f the pyrazines was accomplished u s i n g an i n t e r n a l standard method. 2-methcxypyrazine was chosen as i n t e r n a l standard due t o i t s s i m i l a r p h y s i c a l properties (MW, s o l u b i l i t y ) t o the pyrazines o f i n t e r e s t and because methoxypyrazines have never been reported t o form as a r e s u l t o f the M a i l l a r d reaction. The amounts o f each pyrazine present i n the sample were determined by the r e l a t i o n s h i p :

Amt. C = Amt. ISTD RF

X

AC C AC ISTD

(1)

where RF = response f a c t o r o f the compound, C, r e l a t i v e t o the i n t e r n a l standard, ISTD AC = area count Amt. = amount i n jug/ml Response f a c t o r s were e m p i r i c a l l y determined by adding known amounts o f each cxnpound t o 15 ml o f pH 9.0 borate b u f f e r , followed by a purge o f the sample under usual conditions o f analysis. Pyrazine peak i d e n t i f i c a t i o n was i n i t i a l l y accomplished by cochromatography with standards, (Pyrazine S p e c i a l t i e s , Atlanta, GA) then f u r t h e r confirmed by gas cdiromatography/mass spectrometry using the following sample, equipment and operating conditions. A 10 ml sample o f 0.1M glucose-lysine heated a t 95°C f o r 6 h was analyzed. Following pH adjustment t o 9.0 with 0.1N NaOH, the volume was brought up t o 15 ml. The sample was analyzed u s i n g the Hewlett Packard 7675A Purge & Trap sampler interfaced with a Carlo


79

80


1

DEVELOPMENTS

2

1

Pyrazine

2

2-Methylpyrazine

3

ISTD, 2-^thoxypyrazine

4 2,5-Diirethylpyrazine Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: February 21, 1989 | doi: 10.1021/bk-1989-0388.ch007

5

2,6-diitethylpyrazine

6 2,3-Dinethylpyrazine

Figure 1.

OirOmatogram o f pyrazines detected under standard conditions o f a n a l y s i s


7.

LEAHY AND REINECCIUS

Kinetics ofFormation ofAlkylpyrazines 81

Erba gas crirxxnatograph connected t o a Kratos MS 25 mass spectrometer. Helium was used as c a r r i e r a t a pressure o f 90 kPa. The same column was used f o r routine pyrazine analysis, a 25 m χ 0.32 mm i . d . DB 225 fused s i l i c a c a p i l l a r y column. The run was isothermal a t 50°C with a post value o f 200°C f o r 2 min. Spectra were recorded a t 70 e l e c t r o n v o l t s . The spectra were compared t o published spectra (25) as w e l l as those obtained from reference standards. The k i n e t i c s o f the formation o f pyrazines were determined using the b a s i c equation f o r the r a t e o f change o f A with time:


dA = kAn

where A * k η

= = = =

(2)

concentration o f pyrazine (ppm) time (h) r a t e constant r e a c t i o n order

Integrating t h i s equation between A Q , the concentration o f A a t time zero, and A, the concentration o f A a t time 0, y i e l d s A = A Q + kO (3) f o r a zero order r e a c t i o n . T h i s implies t h a t the r a t e o f formation o f A i s constant with time and independent o f the concentration o f reactants. For a f i r s t order r e a c t i o n t h i s y i e l d s the r e l a t i o n s h i p : In Α ΝτΛο + *β·

(4)

In t h i s case, the r a t e f o r formation o f A i s dependent on the œncentration o f reactants remaining. Reactions i n foods have been found t o follow pseudo zero o r f i r s t order k i n e t i c s (26). One i s generally s a f e r when d i s c u s s i n g r e a c t i o n orders i n foods i n using the term "pseudo", due t o the complexity o f the system. Pseudo r e a c t i o n orders i n foods are generally assigned because a high c o r r e l a t i o n ( r ) f o r a mathematical r e l a t i o n s h i p between formation o f product and time e x i s t s . The formation o f pyrazines appears t o b e t t e r f i t a pseudo zero order r e a c t i o n rather than f i r s t order r e a c t i o n . P l o t t i n g concentrations o f pyrazines formed versus time o f r e a c t i o n gave the b e t t e r f i t o f the l i n e , u s u a l l y with a c o e f f i c i e n t o f determination ( r ) o f greater than 0.95. For a pseudo f i r s t order reaction, a curve rather than a l i n e was obtained. General l e a s t squares a n a l y s i s o f the data was used t o compute r a t e constants (27). Two zero points were used f o r each regression. Duplicate samples were t e s t e d a t the e a r l y sampling times v s . t r i p l i c a t e samples a t l a t e r times, as v a r i a t i o n s i n concentration among r e p l i c a t e s increased with increased r e a c t i o n time. Each data p o i n t c o l l e c t e d was treated separately i n the regression analyses. A c t i v a t i o n energies f o r the formation o f pyrazines were c a l c u l a t e d using the Arrhenius relationship, which r e l a t e s the r a t e constant, k, t o temperature: 2

2


82


DEVELOPMENTS

k = ko-Ea/KT where ko Ea R Τ

= = = =

(5)

a pre-exponential (absolute) r a t e constant a c t i v a t i o n energy i n kcal/mole gas constant, 1.986 c a l / n o l e °K temperature i n °K


Results and Discussion Table I l i s t s the regression data f o r the formation o f pyrazines with time f o r the d i f f e r e n t sugar-amino a c i d combinations. An increase i n r a t e s o f formation occurred with an increase i n temperature. The e f f e c t o f temperature on the formation o f pyrazine i n the lysine-glucose system i s g r a p h i c a l l y depicted i n Figure 2. Formation o f pyrazines best f i t a pseudo zero order reaction, with c o e f f i c i e n t s o f determination u s u a l l y greater than 0.90. T h i s suggests t h a t the r a t e o f formation o f pyrazines i s independent o f reactant concentration. Using 0.1M s o l u t i o n s o f both reactants (sugar and amino a c i d ) , œncentrations o f reactants are q u i t e high, e s p e c i a l l y r e l a t i v e t o pyrazines formed and reactants consumed. Mthough the r a t e o f formation must be a function o f reactant concentration, i t may not be apparent due t o the r e l a t i v e l y high reactant/product r a t i o , m u l t i p l i c i t y o f steps i n pyrazine formation and competing s i d e reactions. Pigment formation i n the M a i l l a r d reaction, which i s a l s o a multi-step process, has a l s o been shown t o e x h i b i t pseudo zero order k i n e t i c s by many researchers, including Labuza e t a l . (28) and Warmbier e t a l . (29) when reactant concentrations were not l i m i t i n g f o r the r a t e o f formation o f brown pigment. Loss o f reactants has generally been shown t o e x h i b i t f i r s t order k i n e t i c s (29) as w e l l as formation o f Amadori compounds, which i s e s s e n t i a l l y a s i n g l e step process as f a r as reactants are œroerned (30). Activation energies for alkylpyrazine formation were c a l c u l a t e d from the slope o f Arrhenius p l o t s , ranging from 27 t o 45 kcal/mole (see Table I I ) . A c t i v a t i o n energies have been reported f o r other aspects o f the M a i l l a r d r e a c t i o n . The a c t i v a t i o n energy f o r browning as measured f o r pigment production ranges from 15.5 kcal/mole f o r a glycine-glucose system (31) t o 33 kcal/mole f o r a s o l i d intermediate moisture model food system (29). In the same system, Warmbier e t a l . (29) reported the a c t i v a t i o n energies f o r both l y s i n e and glucose l o s s t o be 25 kcal/mole. The current research i n d i c a t e s t h a t the a c t i v a t i o n energies f o r pyrazine formation are higher, suggesting a d i f f e r e n t rate-œntrolling step. The mean a c t i v a t i o n energies f o r the dimethylpyrazines are slightly higher than those of the unsubstituted and 2-methylpyrazines. An a n a l y s i s o f variance was performed t r e a t i n g the f i v e sugar-amino a c i d substrate examinations as a block and pyrazine, 2-methylpyrazine and 2,5-cUitethylpyrazine as treatments. No d i f f e r e n c e i n response was found among the substrates a t a 95% level of significance. A d i f f e r e n c e was found t o e x i s t among treatments, a t an cX o f 0.05. Duncan's m u l t i p l e range t e s t was performed t o determine which a c t i v a t i o n energies d i f f e r e d a t the same l e v e l o f s i g n i f i c a n c e (0.05). I t was found t h a t the


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LEAHY AND REINECCIUS

Kinetics of Formation ofAlkylpyrazines 83

Table I . Regressions f o r the e f f e c t o f type o f sugar and amino a c i d on the formation o f pyrazines Number o f Model System Temperature k frxm/hr) kn (intercept) samples


LySINE-GIUQOSE pyrazine

r

2

95 C 85 C 75 C

3.596 0.490 0.214

0.0596 0.458 0.279

22 22 20

0.994 0.960 0.965

2-methylpyrazine

95 C 85 C 75 C

2.837 0.422 0.159

-0.104 0.142 0.0910

22 22 20

0.995 0.967 0.941

2,5-dimethylpyrazine

95 C 85 C 75 C

0.186 0.0247 0.00668

-0.0457 -0.00604 -0.00536

20 22 16

0.995 0.985 0.942

2,3-dimethylpyrazine

95 C 85 C 75 C

0.0229 0.00309 0.000677

-0.00569 -0.00173 -0.00860

16 18 12

0.948 0.978 0.958

pyrazine

95 C 85 C 75 C

1.359 0.395 0.134

-0.226 -0.0991 -0.103

20 22 21

0.995 0.995 0.963

2-methylpyrazine

95 C 85 C 75 C

1.105 0.388 0.116

0.301 0.119 -0.0343

20 22 21

0.995 0.945 0.968

2,5-

Kinetics of Formation of Alkylpyrazines - American Chemical Society

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