Identification and Flavor Properties of Some 3-Oxazolines and 3

9 Identification a n d F l a v o r Properties of Some 3-Oxazolines a n d 3-Thiazolines Isolated from C o o k e d Beef CYNTHIA J. MUSSINAN, RICHARD A. WILSON, IRA KATZ, ANNE HRUZA, and MANFRED H. VOCK Downloaded by UNIV OF PITTSBURGH on October 1, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0026.ch009

International Flavors and Fragrances, Inc., 1515 Highway 36, Union Beach, N. J. 07735

The f l a v o r of meat r e s u l t s from the chemical r e a c t i o n s o c c u r r i n g when a complex mixture of f l a v o r precursors i s heated i n an aqueous environment. A t tempts to e l u c i d a t e the s p e c i f i c pathways involved have shown that while M a i l l a r d type r e a c t i o n s are important for the development of meat f l a v o r , they are only part of a complex process. Recent work, therefore, has been i n c r e a s i n g l y concerned w i t h the isolation and identification of the volatile f l a v o r chemicals from cooked meat. As these studies continue, more and more heteroc y c l e s c o n t a i n i n g s u l f u r and/or n i t r o g e n are being identified. T y p i c a l of these are the compounds shown i n Table I which have been reported as c o n s t i t u e n t s of beef volatiles. Trithioacetaldehyde, trithioacetone, and t h i a l d i n e , according to the patent literature (1), are useful for chicken and beef f l a v o r s . According to Brinkman et al (2), the t h i a l d i n e "can be considered as a c o n t r i b u t o r to beef broth f l a v o r " . The 3, 5-dimethyl-1, 2, 4-trithiolan was reported by Chang et al (3) i n 1968 i n b o i l e d beef. 1-Methylthioethanethiol was r e ported i n the headspace volatiles of beef broth by Brinkman et al (2) and was described as having the odor of fresh onions. The other two mercaptans shown i n Table I were first reported by Wilson et a l (4) and were subsequently patented for use i n meat and roasted meat f l a v o r s ( 5 ) . We b e l i e v e that one of the reasons why more of these compounds have not yet been reported i s that most of them are present i n only trace q u a n t i t i e s . Since these are new compounds, t h e i r identification is even more difficult due to the lack of a v a i l a b l e r e f erence s p e c t r a . The instability evidenced by many of them may a l s o be a f a c t o r . It i s the purpose of t h i s paper to present the identification and f l a v o r p r o p e r t i e s of some new s u l f u r

133 In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS IN FOOD FLAVORS

134

TABLE I CHEMICALS ISOLATED FROM COOKED BEEF CH

J Trithioacetaldehyde

3

s ^ s

Downloaded by UNIV OF PITTSBURGH on October 1, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0026.ch009

CH^ SA

CH

3

Trithioacetone

CH CH CH_ 3

3

3

Thialdine s

CH

s

3

CH

r

0

Η 3,5-Dimethyl-l,2,4-Tr i t h i o l a n e S

S

CH / ~ 3 \ S S\ C H 3 r

1-Methylthioethanethiol

Q

ÇH^ CH -S-CH-SH 3

Η

3-Methyl-2-Butanethiol

^Η ^ 3 CH -CH-CH-CH 3

3

93

2-Methyl-l-Butanethiol

H

CH -CH -CH-CH -SH 3

2

2

In Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors; Charalambous, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

9.

M u s s i N A N ET AL.

3-Oxazolines and 3-Thiazolines in Cooked Beef


and n i t r o g e n c o n t a i n i n g h e t e r o c y c l e s , s p e c i f i c a l l y 3 o x a z o l i n e s and 3 - t h i a z o l i n e s , from cooked b e e f . The f i r s t r e p o r t o f a 3 - o x a z o l i n e i n meat was made by Chang et a l (3) i n 1968. These workers i s o l a t e d and i d e n t i f i e d 2 , 4 , 5 - t r i m e t h y l - 3 - o x a z o l i n e from b o i l e d beef. The v o l a t i l e s were s e p a r a t e d by a c o m b i n a t i o n o f f l a s h v a p o r i z a t i o n and e v a p o r a t i o n from a t h i n heated f i l m and f r a c t i o n a t e d by p r e p a r a t i v e g c . The compound was then i s o l a t e d from one o f two f r a c t i o n s t h a t , a c c o r d i n g to t h e s e a u t h o r s , p o s s e s s e d a " c h a r a c t e r i s t i c b o i l e d beef aroma".

2A5-TRIMETHYL-3-0XAZ0LINE

2-ACETYL-2-THIAZOLI NE

Tonsbeek et a l (6) i s o l a t e d a compound w i t h the aroma o f " f r e s h l y baked bread c r u s t " from beef b r o t h . In t h i s work, beef b r o t h was simmered f o r 2 . 5 h r s . i n a v e s s e l covered with a s t a i n l e s s s t e e l l i d . After f i l t e r i n g the s l u r r y , v a r i o u s t e c h n i q u e s were used to o b t a i n a f a t and f a t t y a c i d - f r e e e x t r a c t from which the d e s i r e d compound was i s o l a t e d . T h i s compound was l a t e r i d e n t i f i e d as 2 - a c e t y l - 2 - t h i a z o l i n e . The i d e n t i f i c a t i o n o f 2 , 4 , 5 - t r i m e t h y l - 3 - o x a z o l i n e and 2 - a c e t y l 2 - t h i a z o l i n e r e p r e s e n t the f i r s t r e p o r t e d i s o l a t i o n o f compounds o f t h i s type from f o o d s . The o c c u r r e n c e o f the t r i m e t h y l o x a z o l i n e i n beef was l a t e r c o n f i r m e d by Pokorny ( 7 ) . The i s o l a t i o n scheme used i n t h i s i n v e s t i g a t i o n i s shown i n F i g u r e 1. F o r t y pounds o f l e a n ground round beef were s l u r r i e d w i t h water and cooked i n a s t a i n l e s s s t e e l v e s s e l at 1 6 2 . 7 ° f o r 15 m i n . A second 40 l b . cook was made at 182 ° . The lower temperature samp l e was f i l t e r e d through c h e e s e c l o t h . The f i l t r a t e was s i m u l t a n e o u s l y a t m o s p h e r i c a l l y steam d i s t i l l e d and continuously extracted with d i s t i l l e d d i e t h y l ether i n a W i l l i a m s apparatus (8). The 182 ° sample was atmos p h e r i c a l l y d i s t i l l e d , and the d i s t i l l a t e was s a t u r a t e d w i t h sodium c h l o r i d e and c o n t i n u o u s l y e x t r a c t e d w i t h d i s t i l l e d diethyl ether. The e x t r a c t i n each c a s e was f


135


136

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

d r i e d over sodium s u l f a t e and c o n c e n t r a t e d by c a r e f u l d i s t i l l a t i o n i n a Kuderna-Danish c o n c e n t r a t o r . The c o n c e n t r a t e s were f r a c t i o n a t e d by p r e p a r a t i v e gas chromatography and a n a l y z e d on a H i t a c h i Model RMU6E mass s p e c t r o m e t e r c o u p l e d w i t h a H e w l e t t - P a c k a r d Model 5750 gas chromatograph. The chromatographic columns used were 500 and 1000* χ .03" s t a i n l e s s s t e e l open t u b u l a r columns c o a t e d w i t h CBW 20M and S F - 9 6 . F u r t h e r d e t a i l s o f the i s o l a t i o n p r o c e d u r e s were r e p o r t e d i n two p r e v i o u s p u b l i c a t i o n s ( 4 ) , ( 9 ) . The compounds were i d e n t i f i e d by comparing t h e i r mass s p e c t r a and r e t e n t i o n times w i t h those of a u t h e n t i c compounds s y n t h e s i z e d i n our l a b o r a t o r y . As an a i d i n t h e d e t e c t i o n o f t h e s e h e t e r o c y c l e s , the t o t a l samples and each t r a p were a n a l y z e d on a T r a c o r MT 220 gas chromatograph m o d i f i e d t o s i m u l t a n e o u s l y d e t e c t s u l f u r a n d / o r n i t r o g e n c o n t a i n i n g com pounds . The compounds i d e n t i f i e d and t h e i r IE v a l u e s , o r r e t e n t i o n i n d i c e s r e l a t i v e to a s e r i e s of e t h y l e s t e r s o f normal a l k a n o i c a c i d s (10), a r e g i v e n i n T a b l e I I . The 2 , 4 - d i m e t h y l - 3 - o x a z o l i n e was o n l y t e n t a t i v e l y i d e n t i f i e d due t o t h e p r e s e n c e o f a n o t h e r component i n t h e spectrum o f the unknown. A l s o found were the t r i methyl, 2,4-dimethyl-5-ethyl-and 2 , 5 - d i m e t h y l - 4 - e t h y l 3 - o x a z o l i n e s and the 2 , 4 - d i m e t h y 1 - a n d t r i m e t h y l - 3 thiazolines. The o x a z o l i n e s were a l l found i n b o t h p r e p a r a t i o n s w h i l e t h e t h i a z o l i n e s were o n l y i d e n t i f i e d i n the lower temperature s a m p l e . It would appear from t h e s e d a t a t h a t the t h i a z o l i n e s a r e l e s s s t a b l e t o heat than the oxazolines. In 1960, A s i n g e r e t a l (11) observed t h a t 3 - t h i a z o l i n e c a n be t h e r m a l l y dehydrogenated i n the p r e s e n c e o f e l e m e n t a l s u l f u r t o the c o r r e s p o n d i n g t h i a zole. These t h i a z o l e s were, i n d e e d , found i n b o t h cooks. Of c o u r s e , i t ' s a l s o p o s s i b l e t h a t the t h i a z o l i n e s were p r e s e n t i n t h e h i g h temperature sample, but not d e t e c t e d . None o f these compounds except 2 , 4 , 5 - t r i m e t h y l - 3 - o x a z o l i n e have ever been r e p o r t e d as c o n s t i t u e n t s o f food v o l a t i l e s . The 3 - t h i a z o l i n e s were p r e p a r e d a c c o r d i n g t o t h e method o f A s i n g e r e t a l (12) as shown below f o r 2 , 4 , 5 trimethyl-3-thiazoline. Ammonia was r e a c t e d w i t h t h e a p p r o p r i a t e a l d e h y d e and mercapto k e t o n e . After ref l u x i n g and steam d i s t i l l i n g , the p r o d u c t s were e x -

CH

3


9.


M u s s i N A N ET AL.

137

BEEF + WATER

COOK 182

COOK 162.7 FILTER THROUGH CHEESECLOTH

DISTILLATE SALT SATURATE & EXTRACT

STEAM DISTILL & EXTRACT

DISCARD


ATMOSPHERIC DISTILLATION

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0

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AQUEOUS

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I DISCARD

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9-FRACTIONS

ANALYSIS BY GC & GC-MS

ANALYSIS BY GC & GC-MS

3-QXAZOLINES AND

COMPOUND 2,1-DIMETHYL-3-0XAZ0LINE

Figure 1. Isolation procedures

THIA70LTHFS IN

I

E

KNOWN

RFFF

I UNKNOWN

A

E

COOK

3.66

162.7°, 182°

1.90

1.81

162.7% 182°

2,1-DIMETHYL-5-ETHYL-3-0XAZ0LINE

5.62

5.61

162.7°, 182°

2,5-DIMETHYL-1-ETHYL-3-0XAZ0LINE

5.10

5.11

162.7°, 182°

2,1-DIMETHYL-3-THIAZ0LINE

7.50

7.60

162.7°

2A5-TRIMETHYL-3-THIAZ0LINE

7.35

7.11

162.7°

B

2A5-TRIMETHYL-3-0XAZ0LINE

I Β

E

3.61

S F

"

9 6

- VALUES WERE DETERMINED ON A 500 FT. X 0.03 IN. L B . STAINLESS STEEL OPEN TUBULAR COLUMN COATED WITH CARBOWAX 20M UNLESS OTHERWISE INDICATED. - THIS COMPOUND WAS ONLY TENTATIVELY IDENTIFIED DUE TO THE PRESENCE OF ANOTHER COMPONENT IN THE SPECTRUM OF THE UNKNOWN.


138

PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

t r a c t e d w i t h e t h e r . The s o l v e n t was removed and the p r o d u c t was p u r i f i e d by p r e p a r a t i v e gas chromatography. The o x a z o l i n e s were s y n t h e s i z e d i n a s i m i l a r manner as d e s c r i b e d by Jassman and S c h u l z (13) s u b s t i t u t i n g the a p p r o p r i a t e a c y l o i n f o r the mercapto k e t o n e . This i s i l l u s t r a t e d below f o r 2 , 4 , 5 - t r i m e t h y l - 3 - o x a z o l i n e . The r e a c t i o n m i x t u r e was s t i r r e d a t room temperature, and the p r o d u c t s were e x t r a c t e d w i t h e t h e r . A f t e r r e moving the s o l v e n t , the p r o d u c t was p u r i f i e d by f r a c t i o n a l atmospheric d i s t i l l a t i o n . Downloaded by UNIV OF PITTSBURGH on October 1, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0026.ch009

CH NH

il

+

3

CHCH

1

+

3

II

CHCH CCH 3

3

Γ M

3 F i g u r e s 2, 3, and 4 show the mass s p e c t r a o f the o x a z o l i n e s and t h i a z o l i n e s found by us i n cooked b e e f . The s p e c t r a o f a l l compounds except 2 , 4 - d i m e t h y l o x a z o l i n e were o b t a i n e d on a CEC-103C mass s p e c t r o m e t e r w i t h an i o n i z a t i o n v o l t a g e of 70eV and s o u r c e and i n l e t temperatures o f 150°. The spectrum o f 2 , 4 - d i m e t h y l o x a z o l i n e was o b t a i n e d on a H i t a c h i RMU-6E mass s p e c t r o m e t e r o p e r a t e d under the same co n d i t i o n s . We b e l i e v e t h a t i t ' s p o s s i b l e t h a t t h e s e com pounds form i n beef i n a manner s i m i l a r t o t h a t by which they a r e s y n t h e s i z e d . In the c a s e o f 2 , 4 , 5 - t r i m e t h y l - 3 - o x a z o l i n e , the s t a r t i n g m a t e r i a l s , ammonia, a c e t a l d e h y d e , and a c e t o i n have a l l been r e p o r t e d as c o n s t i t u e n t s o f cooked meat. The same i s t r u e f o r the corresponding t h i a z o l i n e . In t h i s c a s e , however, the a l p h a mercapto ketones have not been r e p o r t e d i n beef o r any o t h e r n a t u r a l p r o d u c t . This i s not too s u r p r i s i n g s i n c e these compounds a r e p r o b a b l y h i g h l y r e a c t i v e . N e v e r t h e l e s s , the mercapto ketones c o u l d e x i s t as t r a n s i e n t s p e c i e s formed from a v a i l a b l e p r e c u r s o r s . I t has been s u g g e s t e d and c o n f i r m e d i n our l a b o r a t o r y t h a t hydrogen s u l f i d e can r e a c t w i t h and r e p l a c e a c a r b o n y l function. Thus, as shown below, w i t h a d i c a r b o n y l such as d i a c e t y l which does o c c u r i n beef, one c o u l d expect a m i x t u r e o f p r o d u c t s i n c l u d i n g the t h i a z o l i n e p r e c u r s o r 2 - k e t o - 3 - b u t a n e t h i o l . T h i s compound, t o g e t h e r w i t h SH 0

οο

CHC CCH 3

3

+

HS 2

*

;s

CHCH ι CCH 3

3

II


9. MussiNAN ET AL.

139 3-Oxazolines and 3-Thiazolines in Cooked Beef


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Identification and Flavor Properties of Some 3-Oxazolines and 3

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