Professional Documents
Culture Documents
Evaluation of Free D-Glutamate in Processed Foods: Department Chemisty, University of Missouri-Rolla, Rolla, Missouri
Evaluation of Free D-Glutamate in Processed Foods: Department Chemisty, University of Missouri-Rolla, Rolla, Missouri
Most a-amino acids contain a stereogenic center and exist (fermentation, aging, etc.),a'o and (3) food sources which
as the L- and D-enantiomers. Although it has been generally naturally contain amino acids (e.g., certain seafoods). ''
accepted that the free amino acids and proteins found in higher This present study examines the enantiomeric composition
organisms are composed exclusively of the L-enantiomers of as well as the total percent of free glutamic acid in a variety of
amino acids, the mirror image D-forms are known to be human food products using precolumn derivatization and
present in some naturally occurring peptide antibiotics and in HPLC. Glutamic acid is a naturally occurring, nonessential
the cell walls of bacteria.',' Recent studies have shown that amino acid that is commonly used as a food additive in the form
free a amino acids are ubiquitous (although not necessarily in of its monosodium salt (monosodium glutamate, MSG). MSG
high concentrations) in the physiological fluids of humans and is added to many processed foods at levels of 0 . 2 4 9 %by
rodents.s7 A variety of factors were found to affect the rela- weight to increase palatability and only the L-enantiomer pro-
tive levels of amino acids present in mammals including cesses flavor enhancement characteristics.l3 Free glutamate
disease, pregnancy, sex, young age, and diet.4 Of these fac- is also ubiquitous in nature; it is naturally present at relatively
tors, the diet was thought to be the most important factor high levels in foods such as tomatoes, peas, mushrooms, and
governing the relative ratios of amino acid enantiomers exam- potatoes. The goal of this study is to evaluate and comparethe
ined in the blood and excreted in the urine of laboratory enantiomeric composition of glutamate (MSG, glutamic acid)
rodents. These studies were made possible by recent ad- in fermented foods, foods containing added MSG, and nonfer-
vances in the analytical methodologies of enantiomericsepara- mented foods to which MSG has not been added.
tions and quantitation of trace levels of amino acid enanti-
omers (see the Experimental Section.) MATERIALS AND METHODS
Concurrently there has been expanded interest in the oc-
currence of D-&O acids in food and beverage products. Apparatus
There have been several recent reports of the presence of Enantiomeric analyses were carried out using a coupled
amino acids in milk products8p9and fermented foods and HPLC system described previo~sly.~ It consisted of 2 pumps
beverages." In these studies, the fermented foods were (LC-6A, Shimadzu, Kyoto, Japan), a fluorescence detector
found to contain unusually high relative levels of free D-&O (RF-535, Shimadzu), UV detector (SPD-2AM, Shimadzu),
acids which were attributed to the fermentation process. Sig- switching valve (Rheodyne, Cotati, USA), and 20 p1 injection
nificant levels of o-alanine, ~-glutamicacid, and D-glutamine valve (Rheodyne.) The columns used were 250 x 4.6 mm C18
(analyzed together as glutamic acid in these studies) and D-as- and y-cyclodextrin and were supplied by Advanced Separation
partic acid and D-asparagine (analyzed together as aspartic Technologies, Inc. (Whippany, NJ).
acid) were found in these products. Interestingly, these are
the predominant D-&O acids present in bacterial cell
walls.'~~ D - ~ acidO enantiomers seem to be most prevalent
in at least three types of foods: (1) highly processed foods Received for publication December 29,1993;accepted February 8,1994.
Address reprint requests to Dr. Daniel W.Armstrong, Department of Chem-
(that is, those which undergo extremes in pH, temperature, istry, College of Arts and Sciences. University of Missouri-Rob, 341 Schrenk
etc.), l1 (2) food products exposed to microbial degradation Hall,Rolla, MO 65401.
0 1994 Wiley-Liss, Inc.
278 RUNDLETT AND ARMSTRONG
various food products after column switching. Fig. 1. A chromatogram showing the achiral separation of FMOC-glycyl deriva-
2-Aminoadipic acid was used as an internal standard for tized worcestershire sauce on C,, column. Mobile phase: 700:3002 (v/v/v) water/
acetonitde/acetic acid. UV detection was at 266 nm and the flow rate was 1.5
quantitation of MSG.The amino acids were derivatized with drnin. The arrow designates the peak containing FMOC-GIY-D,L-GIU.
9-fluorenylmethyl chloroformate (FMOC) using the same de-
rivatization procedure as that for FMOC-Gly-CI. The chro-
matographic system used for quantitation was composed of a
column and a fluorescence detector and the mobile phase HPLC-grade water was added to all samples and the sam-
was acetonitnle/water/aceticacidhiethylamine, 300 7003: 1 ples were sonicated for 10 min. For quantitative measure-
(v/v/v/v). Figure 4 shows a typical separation used for quanti- ments, the 2-aminoadipic acid internal standard was added
tation of MSG in a cracker sample. prior to sonication. Aqueous soup and cracker emulsions,
It should be noted that quantitation of extremely low levels most of which contain high total amounts of MSG, were ex-
of D-glutamic acid (<1%) in the presence of large amounts of tracted with hexane [1:1 x 2 (v/v)l to remove fats and oils,
the L-enantiomer, or when simply determining the enantio- derivatized, and passed through a c18 solid phase extraction
meric ratio, it is often inaccurate to simply use the data from cartridge. The amino acid derivatives were eluted with diethyl
the automated peak integrator. This is because the linear ether, the ether was evaporated, and the samples were dis-
dynamic range of the detector is sometimes exceeded for the solved in an acetonitrile/water/aceticacid mobile phase.
larger of the two peaks (see the L-enantiomers in Fig. 2). Acetone and acetic acid were added to all other samples
Hence, the larger peak often must be quantatively measured prior to sonication in order to precipitate proteins. The sam-
in a second determination after accurate serial dilutions. ples were centrifuged at 3000 rpm for 10 min. The superna-
tant was extracted with diethyl ether [l:l X 2 (v/v)l, deriva-
tized, and the derivatives were extracted into diethyl ether
Preparation of Food Samples El: 1 x 2 (v/v)]. The ether was evaporated and the derivatized
All food samples were purchased at local markets. Care amino acids were dissolved in the desired acetonitrile/water/
was taken to prepare and derivatize the samples immediately acetic acid mobile phase for injection. (Solid phase extraction
upon exposure to air in order to avoid erroneous results due was not used for these samples due to fouling and capacity
to bacterial contamination. problems, which gave nonreproducible results. )
EVALUATION OF FREE D-GLUTAMATE 279
%D = 0.22% %D = 0.25%
4 minutes
I
18 v
0
minutes
18
% D = 35.3%
L kI
i %D = 7.7%
h
Ill
- I I
0 13 0 minutes 18
minutes
Fig. 3. A chromatogram showing the enantioseparation of FMOC-glycyl derivatives of MSG in Kikoman Soy Sauce and
Meeter's Kraut Juice (fermented products) on a y-cyclodextrin column. Mobile phase: 850: 150:12:3 (v/v/v/v) acetonitrile/
methanoUtriethylamine/acetic acid. Fluorescence detection as in Figure 3. Flow rates: 3.0 d m i n for the soy sauce and 2.0
d m i n for the kraut juice.
1 2
some amino acids on lab animals have been reported:
D-serine was related to nephrotoxicity in rats15; renal and
hepatic damage in rats was associated with D-proline,16 and
D-glutamate was reported to exhibit immunosuppressive ac-
tivity in mice when administered in large doses." In addition,
for some D-enantiomers of protein amino acids, consumption
of large amounts of a amino acids was reported to interfere
with the normal metabolism of a amino acids. 17q1'
-
dase. l9 Therefore, compared to other D-&O acids, D-gluta-
mate probably contributes very little to the protein nutritional
value of food. It also appears that amino acids are eliminated
from the bodv via the k i d n e ~However.
.~ the human Dhvsio- . <
i/L
logical effects (if any) of frequent consumption of D-glutamate
are unknown, 0 minutes 15
MSG listed
on product
Product label % MSG“ %Db nc
MSG
McCormick Flavor Enhancer Yes 99.9 *
0.26 0.08 7
Accent Flavor Enhancer Yes 99.9 0.21 f 0.06 5
Sigma Monosodium Glutamate - 99.9 *
0.23 0.05 4
Soup products
Wylers Beef Bouillon Yes 3.18 0.52 2 0.05 8
MBT Beef Broth (dried) Yes 7.53 0.82 & 0.38 8
Smack Ramen Beef Broth (dried) Yes 3.86 *
0.23 0.05 6
Wyler’s Chicken Broth (dried) Yes 5.35 0.39 & 0.07 10
Smack Ramen Chicken Broth (dried) Yes 3.36 0.52 2 0.35 6
MBT Onion Broth (dried) Yes 3.59 0.80 5 0.10 4
Campbell’s Chicken Broth (canned) Yes 0.44 0.36 f 0.04 6
Cracker products
Nabisco Chicken in a Biskit Crackers Yes 0.53 0.40 & 0.19 4
Keebler White Cheddar Crackers Yes 0.27 0.54 & 0.11 4
Nabisco Cheese Nips Crackers Yes 0.30 *
2.93 0.70 6
Keebler Munch ’Em Crackers Yes 0.42 0.40 f 0.10 4
Nabisco Sociables Crackers No“ 0.028 *
1.62 0.18 4
Sauces
Heinz 57 Sauce Nd 0.38 0.71 2 0.08 8
A. 1. Steak Sauced No 0.35 6.69 2 0.17 8
Lea and Penin’s Worcestershire Sauced Nd 0.18 5.83 f 0.09 8
French’s Yellow Mustard Nd 0.062 0.95 f 0.07 8
Griffin’s Yellow Mustard Nd 0.065 0.56 * 0.08 8
Tabasco Pepper Sauced No 0.023 5.63 f 0.74 4
Kikoman Soy Sauced No 0.94 7.93 * 0.23 4
La Choy Soy Sauced No 1.04 7.77 f 0.51 4
Vinegars
Reese Red Wine Vinegaf’ No 0.00092 12.6 & 0.80 4
Heinz Red Wine Vinegaf‘ No 0.00018 13.7 ? 1.10 4
Regina Red Wine Viegaf’ No 0.00030 *
18.1 0.70 4
Sauerkraut juice
Meeter’s Kraut Juiced No 0.058 35.7 f 0.6 3
Bush’s Sauerkrautd (drained juice) No 0.026 *
31.1 2.7 8
Del Monte Sauerkrautd (drained juice) No 0.020 21.1 2 1.4 4
Tomato products
V-8 Vegetable Juice No 0.35 0.81 f 0.08 5
Hunt’s Tomato Paste No 0.89 *
0.57 0.03 4
Contadina Tomato Paste No 1.14 *
1.10 0.20 4
Hunt’s Ketchup No 0.47 1.62 f 0.20 9
Heinz Ketchup No 0.91 *
1.52 0.08 8
Milk products
Peevley Buttermilkd No 0.022 6.17 k 0.10 7
Peevley Sour Creamd No 0.010 5.39 f 0.25 8
Dean’s Bacon & Cheddar Dipd Yes 0.15 0.37 f 0.04 5
Dean’s French Onion Dipd Yes 0.14 0.61 f 0.05 4
LITERATURE CITED 11. Friedman, M. Formation, Nutritional value and safety of wamino acids.
In: Nutritional and Toxilogical Consequences of Food Processing. Fried-
1. Strominger, J. L., Izaki, K., Matsushsi, M., Tipper, D.J., Peptidoglycan man, M., ed., New York Plenum. 1991:447-481.
transpeptidase and u-alanine carboxypeptidase; penicillin-sensitive en- 12. Preston, R. L. Occurrence of o-amino acids in higher organisms: A survey
zyme reactions. Fed. Proc. 269-22, 1967. of the distribution of 11-aminoacids in marine invertebrates. Comp. Bio-
2. Meister, A. Biochemistry of the Amino Acids. New York Academic chem. Physiol. 87B:55-62, 1987.
Press, 1965114. 13. Yoshida, T. 1.-Monosodium glutamate. In: Encyclopedia of Chemical
3. Armstrong, D. W., Duncan, J. D., Lee, S. H. Evaluation of o-amino acid Technology, Vol. 2. New York John Wiley & Sons, 1978.
levels in human urine and in commercial amino acid samples. Ammo 14. Carpino, L. A., Cohen, B. J., Stephens, K. E., Sadat-Adaee, S., Tien, J.,
Acids 1:97-106, 1991. Langridge, D. C. (9-Fluorenylmethyl)oxy)carbonyl (FMOC) amino acid
4. Armstrong, D. W., Gasper, M., Lee, S., Ercal, N., Zukowski, J. Factors chlorides. J. Org. Chern. 51:3732-3734, 1986.
controlling the level and accurate determination of u-amino acids in the 15. Ganote, C. E., Peterson, D. R., Carone, F. A. The nature of o-serine
urine and serum of several laboratory rodents. J. Amino Acids 4402, induced nephrotoxicity. Am. J. Pathol. 77269-276, 1974.
1993.
16. Kampel, D., Kupferschrmdt, R., Lubec, G. Toxicity of o-proline. In:
5. Armstrong, D. W., Gasper, M., Lee, S. H., Zukowski, J., Ercal, N. Armno Acids: Chemistry, Biology, Medicine. Lubec, G., Rosenthal, G.,
D - ~ acid
O levels in human physiological fluids. Chirality 5375-378,
eds. Leiden: ESCOM, 1990: 1167.
1993.
17. Carefoot, T., Taylor, B., Kalwa, S., Somogyi, G., Hughes, B. Effect of
6. Armstrong, D. W., Zukowski, J., Ercal, N., Gasper, M. Stereochemistry dietary D-amino acids on growth, survival, ammonia excretion and specific
of pipecolic acid found in the urine and plasma of subjects with perixosomal dynamic action in the supralittoral isopod, Ligiapullusii. Comp. Biochem.
deficiencies. J. Pharm. Biomed. Anal. 111:881-886, 1993. Physiol. 103A 559-563, 1992.
7. Zukowski, J., Pawlowska, M., Armstrong, D. W. Efficient Enantiosepa- 18. Bunjapami, S., Mahoney, R., Fagerson, S. Determination of o-amino
ration and determination of trace impurities in secondary amino acids (ie. acids in some processed foods and effect of racemization on in vitro
imino acids.) J. Chromatogr. 333-1, 1992. digestibility of casein. J. Food Sci. 47: 1229-1234, 1982.
8. Gandoffi, I., Palla, G., Deprato, L., DeNisco, F., Marchelli, R., Salva- 19. Bender A. E., Krebs, H. A. The oxidation of various synthetic a-amino
don, C. 0-Amin0acids in nulk as related to heat treatments and bacterial acids by mammalian amino acid oxidase, amino acid oxidase of cobra
activity. J. Food Sci. 57:377-379, 1992. venom and the L- and 0-amino acid oxidases of Neurosporu c~ussu.Bio-
9. Palla, G., Marchelli, R., Dossena, A,, Casnati, G., Occurrence of amino chemJ. 46210-219, 1950.
acids in foods. J. Chromatogr. 475:45-53, 1989. 20. Inoue, Y., Zama, Y., Suzuki, M. u-Amino acids as immunosuppressive
10. Briickner H., Hausch, M. D-Amino acids as ubiquitous constituents in agents. Jpn. J. Expl. Med. 51:363-366, 1981.
fermented foods. In: Amino Acids: Chemistry, Biology and Medicine.
Luec, G., Rosenthal, G. A., eds. ESCOM: Leiden, 1990 1172.