Sulfur Compounds in Foods

Chapter 11

Kinetics of the Release of Hydrogen Sulfide from Cysteine and Glutathione During Thermal Treatment

Downloaded by GEORGE MASON UNIV on December 19, 2014 | http://pubs.acs.org Publication Date: July 29, 1994 | doi: 10.1021/bk-1994-0564.ch011

Yan Zheng and Chi-Tang Ho Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903

0.1M of cysteine or glutathione solutions with a pH of 3.0, 5.0, 7.0 or 9.0 were incubated at 80°C, 90°C, 100°C or 110°C for a certain period of time. The amount of hydrogen sulfide released during the thermal treatment was determined using a sulfide/silver electrode. The results showed that both reactions followed first order kinetics. The rate constants of the reactions increased with the pH value. The release of hydrogen sulfide from these two compounds was catalyzed by the hydroxide ion, while the βelimination was favored at higher pH. The activation energies for both reactions were calculated using the Arrhenius equation; 31.3, 31.8, 32.2 and 29.4 kcal/mol for cysteine and 18.8, 30.8, 22.8 and 19.9 kcal/mol for glutathione at pH 3.0, 5.0, 7.0 and 9.0,and respectively. The lower activation energies for glutathione implies that the molecular structure and micro-environmental condition of the amino compound plays a significant role in the release of hydrogen sulfide.

Sulfur-containing compounds are well-recognized as major contributors to meat flavor. Among the sulfur-containing compounds, hydrogen sulfide was one of the first identified in early meat flavor studies (1-4). Apart from its direct contribution to meat flavor, hydrogen sulfide participates in the formation of other sulfurcontaining volatiles, thus contributing indirectly to the meat flavor (5-7). Mabrouk et al. (8) used extraction, dialysis, and gel-permeation chromatography to fractionate an aqueous extract prepared from fat-free lyophilized meat. Out of the twelve resulting fractions, seven had a broiled beef aroma. Those fractions with an intense aroma contained cysteic acids (cysteine + cystine). Two fractions which contained methionine but no cysteic acid did not have any broiled beef flavor. Other reports (9-10) demonstrated that both cysteine 0097-6156/94/0564-0138$08.00/0 © 1994 American Chemical Society

In Sulfur Compounds in Foods; Mussinan, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

11. ZHENG AND HO

Hydrogen Sulfide Release During Thermal Treatment


and glutathione are the major precursors of hydrogen sulfide and other sulfurcontaining flavor compounds in meat. Previous studies have focused on the formation of flavor from cysteine and glutathione under various processing conditions (12-13). The kinetics of the formation of hydrogen sulfide and other sulfur-containing compounds have not been systematically studied. The objective of this research was to study the rate of release of hydrogen sulfide from cysteine and glutathione during thermal treatment. We hoped that the information obtained would enable us to manipulate the quality and quantity of the desired meat flavor formed during thermal processing of meat. Experimental Chemicals. All chemicals were purchased from Sigma Chemical Co. except as indicated. The water used in this study was prepared from distilled water with a Milli-Q deionized water system. Water was degassed by sonication for 15 minutes prior to use. Reactions. 0.1M cysteine or glutathione solutions were prepared in 0.1M citratephosphate or 0.1M phosphate-sodium hydroxide buffer with pH values of 3.0, 5.0, 7.0, and 9.0. Ten mL of the solution were then transferred into a reaction vessel (Kimax brand glass test tube with a PTFE-coated liner screw). The headspace of the tubes wasflushedwith nitrogen to expel the oxygen. The tubes were capped and tightened. The tubes were then incubated in a glycerine bath, set at temperatures of 80°C, 90°C, 100°C or 110°C. Two tubes were withdrawn from the bath at 10 minute intervals for the first 30 minutes and at 30 minutes intervals thereafter. The test tubes withdrawn from the bath were immersed immediately in an ice-water bath to quench the reaction. The hydrogen sulfide generated was determined by the sulfide/silver electrode method described below. Determination of hydrogen sulfide. The determination of hydrogen sulfide produced in the reactions was performed with an Orion Sulfide/Silver ion selective electrode connected to an Orion digital pH/mV meter. The electrode includes a silver/sulfide sensing element which develops an electrode potential in contact with a solution containing either silver or sulfide ions. The measured potential corresponding to the level of silver or sulfide ions in solution is described by the Nemst equation: Ε = E + S χ log (A) 0

Where: E: E: 0

measured electrode potential. reference potential (a constant)


139

140

SULFUR COMPOUNDS IN FOODS

A: S:

level of silver or sulfide ion in solution electrode slope

A standardized Na^ solution in sulfur antioxidation buffer (SAOB, consisting of 2N NaOH, IN EDTA and 4% ascorbic acid) was used to established a standard curve. Samples were diluted with the SAOB to an electrode potential within the range of the standard curve. The H S concentration in the samples could then be calculated from the electrode potential and the dilution factor.


2

Results and Discussion Experimental data on the release of H S from cysteine solution at different temperatures and pHs were fitted with zero-order,first-orderand second-order methods. As shown in Figures 1 to 4, the reaction followedfirst-orderkinetics. The R s of thefirst-orderregressions as shown in Table I were in the range of 0.955 to 0.999. The results show that the rates of H S release from cysteine were influenced by the pH of the solution. With the increase of pH from acidic condition to basic condition, the rate constant increased (Table I). The results indicate that the release of H S from cysteine is easier under a basic environment. A similar observation has been reported for the release of H S from chicken muscles (10). It is believed that the sulfhydryl group in cysteine undergoes a βelimination reaction at basic pH conditions (14). The initial step in β-elimination is the abstraction of the proton by the hydroxide ion from the α-carbon atom (the β position to the sulfhydryl group). Dehydroalanine is then formed with the subsequent loss of the sulfhydryl group which could combine with the proton to form hydrogen sulfide. A linear relationship between the rate constants and the corresponding pH or hydroxide ion concentration was observed except for those reactions at pH 3.0. The equations for the relationship are listed in Table II. The nonconformity of reactions at pH 3.0 may mean that a different reaction mechanism is involved in the release of hydrogen sulfide from cysteine at pH 3. The release of hydrogen sulfide from glutathione during the thermal treatment was similar to that of cysteine. The rate constants derived from firstorder kinetics, however, were higher than those for cysteine under the same reaction conditions. The rate constants for glutathione and the corresponding R values are listed in Table III. This result confirmed earlier reports that glutathione evolves H S more rapidly than cysteine (77). The molecular environment could be the main reason for the difference. It has been reported that β-elimination would be preferred if the α-amino group of cysteine is acetylated or the carboxyl group is esterified (75). In the case of glutathione, the negative charge of carboxyl group is further away from the α-carbon than it is in cysteine. Therefore, the hydrogen atom at the α-carbon is more readily abstracted. This mechanism is further supported by the activation energies obtained in this study. 2

2

2

2

2

2

2


ZHENG AND HO

Hydrogen Sulfide Release During Thermal Treatment 141


0.001

-0.006

30

60

90 120 Time (minutes)

150

180

210

Figure 1. First-order plot of the release of hydrogen sulfide from cysteine at 80°C.

0.005

-0.030

30

60

90 120 Time (minutes)

150

180

210

Figure 2. First-order plot of the release of hydrogen sulfide from cysteine at 90°C.




142


11.

ZHENG AND HO


Table L First-order Rate Constants (K), the Corresponding Regression Coefficients (R ) and the Half-Life (T ) of the Release of Hydrogen Sulfide from Cysteine during Thermal Processing at Different pH Value 2


Vi

Temp

pH

Κ (min )

80°C

pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH 3.0 pH 5.0 pH 7.0 pH 9.0

1.87 χ ΙΟ" 2.02 χ 10" 9.71 χ 10" 2.95 χ ΙΟ" 8.30 χ 10" 1.24 χ ΙΟ" 5.99 χ 10" 1.33 χ ΙΟ 4.15 χ ΙΟ 2.65 χ 10" 2.30 χ 10" 4.53 χ 10" 5.23 χ 10" 7.97 χ 10 3.35 χ 10" 7.45 χ ΙΟ

90°C

100°C

110°C

1

6

6 6

5 6

5

5 4

5 5 4

4 5 5 4 4

R

T., (hrs)

2

0.999 0.998 0.998 0.991 0.995 0.997 0.996 0.985 0.998 0.998 0.995 0.996 0.973 0.985 0.955 0.995

6171.2 5713.4 1188.5 391.3 1395.2 930.1 192.9 86.7 278.2 435.6 50.3 25.5 220.8 144.8 34.5 15.5

Table IL Linear Relationship between the Rate Constants of the Release of Hydrogen Sulfide from Cysteine and the pH Temp. 80°C 90°C 100°C 110°C

Relationship Κ = 6.87 χ Κ = 3.01 χ Κ = 1.07 χ Κ = 1.66 χ

R 6

5

10" x log[OH] + 6.19 χ 10 10- x log[OH] + 2.78 χ 10" ΙΟ" χ log[OH] + 9.88 χ 10' ΙΟ" χ log[OH] + 1.55 χ 10' 5

4

4

4

4

4

2

0.939 0.985 0.999 0.982


144


Table ID. First-order Rate Constants (K), the Corresponding Regression Coefficients (R ) and the Half-Life (T*) of the Release of Hydrogen Sulfide from Glutathione during Thermal Treatment at Different pH Values


2

Temp

pH

80°C

pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH3.0 pH 5.0 pH 7.0 pH 9.0 pH 3.0 pH 5.0 pH 7.0 pH 9.0 pH3.0 pH 5.0 pH 7.0 pH 9.0

90°C

100°C

110°C

T., (hrs)

1

Κ (min ) 4.04 4.11 3.25 1.34 1.17 1.57 7.69 2.44 2.28 4.94 2.26 7.74 4.57 7.46 3.89 1.09

6

χ 10' χ 10" χ 10" χ ΙΟ" χ 10" χ ΙΟ" χ 10" χ ΙΟ" χ 10" χ 10' χ 10" χ ΙΟ" χ 10 χ 10" χ 10" χ ΙΟ 6

5

4

5

5

5

4

5

5

4 4

5

5

4 3

2859.5 2810.8 355.5 86.2 987.4 735.8 150.2 47.3 506.7 233.9 51.1 14.9 252.8 154.9 29.7 10.6

2

R

0.996 0.996 0.996 0.989 0.987 0.992 0.997 0.989 0.965 0.991 0.993 0.995 0.979 0.956 0.992 0.990

The linear relationship between rate constants and pH was also observed for the reactions of glutathione in the range from pH 5.0 to pH 9.0 (Table IV). Again, the relationship between the rate constants and the pH at pH 3.0 did not follow those observed in the higher pH conditions. The activation energies for the hydrogen sulfide released from cysteine and glutathione at pH 3.0, 5.0, 7.0 and 9.0 were calculated using the Arrhenius equation: Κ = K χ Exp (-E./RT) Where: K = pre-exponential (absolute) rate constant E = activation energy in kcal/mol R = gas constant, 1.987 cal/mol/°K Τ = temperature in °K 0

0

a

Table V shows the activation energies for the elimination of hydrogen sulfide from cysteine and glutathione. The fact that the activation energy for the release of H S from glutathione is lower than for cysteine again suggested the structural effect on the release of hydrogen sulfide. The result also indicate that pH affected the activation energy for the elimination. As the pH increased from 2



11.

ZHENG AND HO


Table IV. Linear Relationship between the Rate Constants of the Release of Hydrogen Sulfide from Glutathione and the pH Temp.

Relationship

80°C 90°C 100°C 110°C

Κ = 3.25 χ 10- x Κ = 5.71 χ 10' x K = 1.81 χ 10- x K = 2.54x 10" x

R 5

5

4

4

log[OH] log[OH] log[OH] log[OH]

+ 2.85 χ + 5.12 χ + 1.62 χ + 4.82 χ

4

ioΙΟ IO" io4

3

3

2

0.905 0.933 0.920 0.954

Table V. Activation Energy of Release of Hydrogen Sulfide from Cysteine and Glutathione at Different pH Values Cysteine

pH 3.0 pH 5.0 pH 7.0 pH 9.0

Glutathione

Ea (kcal/mol)

Linearity R

Ea (kcal/mol)

Lmeamy R

31.3 31.8 32.3 29.4

0.940 0.975 0.942 0.967

18.8 30.8 22.8 19.9

0.997 0.996 0.990 0.970

2


2

146


3.0 to 9.0, the activation energy reached a maximum and then declined. For both cysteine and glutathione, the maximum activation energy showed at about 2 pH units above the isoelectric point (pi) of the molecule (i.e. pi of cysteine = 5.07; pi of glutathione = 2.83). Such a phenomenon could be related to the ionization of the molecule. At pH 7.0, cysteine exists mainly as HSCH CH(NH )COO . These molecules could associate with each other so that the attack at the ahydrogen by the hydroxyl ion is slowed down. Subsequently, the activation energy is higher than those at the other pHs. On the other hand, the major structure of glutathione at pH 5.0 is H N(COO)CHCH CH CONHCH(CH SH)CONHCH COO-. In this case, the interaction between the carboxyl group in the glycine residue with the amino group in the glutamate residue will also reduce the attack of hydroxide ion on the α-hydrogen atom. Therefore, the activation energy is higher. +

2

_

3

+


3

2

2

2

2

Acknowledgements New Jersey Agricultural Experiment Station Publication No. D-10205-11-93 supported by State Funds. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Osborne, W.A. Biochem. J. 1928, 22, 1312-1316. Crocker, E.C. Food Res. 1948, 13, 179-183. Bouthilet, R.J. Food Res. 1951, 16, 137-141. Pippen, E.L.; Erying, E.J. Food Technol. 1957, 11, 53-56. Pippen E.L.; Mecchi, E.P. J. Food Sci. 1969, 34, 443-446. Schutte, L.; Koenders, E.B. J. Agric. Food Chem. 1972, 20, 181-184. Boelens, H.; van der Linde, L.M.; de Valois, P.J.; van Dort, J.M.; Takken, H.J. J. Agric. Food Chem., 1974, 22, 1071-1076. Mabrouk, A.F.; Jarboe, J.K.; O'Conner, E.M. J. Agric. Food Chem. 1969, 17, 5-9. Obataka, Y.; Tanaka, H. Agric. Biol. Chem. 1965, 29, 191-195. Mecchi, E.P.; Pippen, E.L.; Lineweaver, H. J. Food Sci. 1964, 29, 393-399. Ohloff, G.; Flament, I.; Pickenhagen, W. Food Rev. Intl. 1985, 1, 99-148. Shu, C.K.; Hagedorn, M.L.; Mookherjee, B.D.; Ho, C.-T. J. Agric. Food Chem. 1985, 33, 442-446. Zhang, Y.; Chien, M.; Ho, C.-T. J. Agric. Food Chem. 1988, 36, 992-996. Tarbell, D.S.; Hamish, D.P. Chem. Rev. 1951, 49, 1-90. Schneider, J.F.; Westley, J. J. Biol Chem. 1969, 244, 5735-5744.

RECEIVED March 23, 1994


Sulfur Compounds in Foods

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