J. Gen. App!. Microbiol. Vol. 9, No. 2, 1963

PROTECTIVE EFFECT OF GLUTAMIC ACID AND RELATED

COMPOUNDS ON BACTERIAL CELLS SUBJECTED

TO FREEZE-DRYING

TOSHIKI MORICHI, RYOZABURO IRIE, NOBUHIRO YANO and HIROSHI KEMBO

National Institute of Animal Industry Aoba-cho, Chiba, Japan

Received February 18, 1963

In order to increase the survival ratio immediately after the freeze-drying of bacterial suspension and to minimize the death rate of dried organisms during storage, the choice of an appropriate suspending medium is a matter of importance. Most of the suspending media commonly used have been the solutions of complicated substances such as proteins and polysaccharides. Use has also been made of complex natural materials such as immunologically inactivated serum, skim milk or nutrient broth.

MILLER et al. (1) demonstrated that some compounds of low molecular weight, such as sodium glutamate and aspartate protected Bacille Calmette-Guerin (BCG) during freeze-drying. OBAYASHI et al. (2, 8, 4) showed that a dried BCG vaccine prepared with sodium glutamate solution as suspending medium remained biologically and allergenically stable during storage at a temperature as high as 37°. In a field trial undertaken jointly by Tuberculosis Research Office of World Health Organization and Research Institute of the Japan Anti-tuberculosis Association (5), these vaccines could be maintained for as long as 30 days at a tropical temperature without showing any marked loss of allergenic potency. OrA (6) and OBAYASHI et al. (7) reported that the use of sodium glutamate solution as a medium for freeze-drying of Lactobacillus bifidus significantly enhanced the stability of the dried organisms. KOZIMA et al. (8) demonstrated that freeze-dried organisms as inocula for microbiological assays (Latobacillus arabinosus, Leuconostoc mesenteroides and Streptococcus

f aecalis) could be greatly stabilized by sodium glutamate. GREAVES (9) has also drawn attention to this drying medium for gonococcus and paracolon bacillus and found its effectiveness in preserving the viability of these organisms.

Despite these extensive studies on the application of glutamate in the

process of freeze-drying, the mechanism of its protective effect has remained obscure. The present studies were undertaken to clarify this point with special reference to the relationship between the protective activity and the chemical structures of protectants, using a variety of substances chemically related with glutamic acid.

149

150 MORICHI, IRIE, YANO and KEMBO VoL. 9

MATERIALS AND METHODS

Organisms. The following 26 strains were used as test organisms. Streptococcus cremoris H 61, Streptococcus lactis 527, Streptococcus diaceti-

lactis N-7, Streptococcus f aecalis 706 and 904, Lactobacillus lactis L-51, Lactobacillus acidophilus L-54 and Lactobacillus bulgaricus B-1 were obtained from the stock collection of National Institute of Animal Industry. All these strains were originally isolated from milk and milk products (10, 11, 12). Streptococcus cremoris ATCC 9596 and Streptococcus lactis ATCC 7 963 were received from the American Type Culture Collection. Lactobacillus delbri ckii JAM 1085, Lactobacillus arabinosus 17-5 (ATCC 8014), Leuconostoc citrovorum ATCC 8081, Leuconostoc mesenteroides P-60 (ATCC 8042), Micrococcus varians ATCC 399, Micrococcus lysodeikticus ATCC 4698, Staphylococcus aureus FAD 209 P, Sarcina lutea PCI 1001, Corynebacterium equi JAM 1038, Microbacterium flavum JAM 1642, Arthrobacter ureafaciens ATCC 7562, Brevibacterium ammoniagenes ATCC 6871, Escherichia coli ATCC 7009 and 206, Aerobacter aerogenes ATCC 7256 and 8308 were supplied from the Institute of Applied Microbiology, University of Tokyo. Cultivation of organisms. Streptococci and leuconostocs were cultured at 30° in a liquid medium of the following composition : polypeptone, i% ;

yeast extract (Difco), 0.5°° ; glucose, 1% ; sodium succinate (hexa-hydrated) 2°° (13). Streptococcus faecalis was grown at 37°. Lactobacilli were grown at 37° in a liquid medium containing: polypeptone, i 0 ; yeast extract (Difco), 0.5% ; glucose, 2% ; Tween 80, 0.1% ; L-cysteme, 0.01% ; sodium acetate (tri-hydrated) i°0 (14). Escherichia and Aerobacter were grown without aeration at 37° in a liquid medium consisting of: polypeptone, i% ; glucose, 0.2° ; NaCI, 0.25%. The other organisms were cultured at 30° on a solid agar slant containing: meat extract 1% ; polypeptone, 1% ; NaCI, 0.5°° ; powdered agar, i%. Staphylococcus aureus was cultured at 37°. All the media were adjusted to pH 7.0. One per cent of powdered agar was added to the said broth medium to make plating medium.

Compounds examined. Forty-five compounds examined are listed in Tables 3, 4, 5 and 7. DL-a-Aminopimelic acid, a-methyl-L-glutamic acid, p-amino-benzoyl-L-glutamic acid and L-glutamic acid-r-methyl ester were purchased from Nutritional Biochemicals Corporation. D-Glutamic acid and DL-5-pyrro-lidone-2-carboxylic acid were obtained from Aji-no-moto Co., Ltd. N-Dimethyl--L-glutamic acid and N-dimethyl-L-aspartic acid were the generous gifts of

Dr. Y. Izumi, Institute for Protein Research, Osaka University. Aminomalonic acid was kindly supplied by Dr. H. Nagayama, Research Institute for Tuberculosis and Leprosy, Tohoku University. N-Acetyl-L-glutamic acid and

L-glutamic acid-r-benzyl ester were synthesized according to the ordinary

procedure. All the other reagents were commercial products of guaranteed grade.

Preparation of cell suspension. In preliminary experiments, it was

1963 Freeze-drying of bacteria in presence of glutamate etc. 151

confirmed that the survival ratio after freeze-drying of bacterial suspension was the highest at 0.06 M of glutamate adjusted to pH 7. Therefore, all the suspending media were made up to contain 0.06 M of the test compounds, and adjusted to pH 7 with NaOH or HC1. The suspending media were never subjected to heat treatment to avoid the chemical modification, if any, of the substances. The bacterial cells were harvested at the stationary phase and washed twice with sterile distilled water, or sometimes with physiological saline. The washed cells were then resuspended in the suspending media to make the final cell concentration to 1-2 x 10`' viable cells per ml. One ml samples of each bacterial suspension were transferred into a series of sterile vials and were subjected to freeze-drying. Procedure of freeze-drying. Freeze-drying was carried out in a chamber-type freeze-drying equipment, Stokes model 2003 F-2, with initial freezing at -30° for l hr , and desiccation under vacuum of 50 to 100,aHg for 6 to 8 hr

followed by the last heating up to 30° for 2 hr. Estimation of protective effect. The number of viable cells was determined by the agar plate method. Just before plating, each sample of freeze-dried bacteria was rehydrated to the original fluid volume with sterile distilled water. The protective effect of suspending media during freeze-drying were compared with each other on the basis of percentage of survivors. In most experiments, the mean values of 5 trials are presented with standard deviations.

RESULTS

Protective effect of glutamic acid The survival ratios of freeze-dried bacteria prepared with glutamic acid and 4 related compounds as suspending media are shown in Table 1. With many kinds of bacteria tested, glutamic acid was found to exert a remarkable

protective action against the death caused by the process of freeze-drying. By contrast, some other compounds, such as glutamine, norvaline, r-amino-butyric acid and glutaric acid, did not serve as effective protectants. Although the survival ratios after freeze-drying varied greatly according to the species of bacteria tested, glutamic acid exhibited a relatively strong protective action in most cases, including those of Gram-negative bacteria which, in general, were highly sensitive to freeze-drying under various conditions. Similar results were obtained with 14 strains of lactic acid bacteria (Table 2), among which an exception was L. bulgaricus, whose death could not be protected satisfactorily by glutamic acid. As may be seen from Table 2, no appreciable protective action could be observed with glutaric acid, norvaline and r-aminobutyric acid. This seems to indicate that the favorable effect of glutamic acid is due to its amino- and two carboxyl-groups.

Structural features of protective compounds

To clarify more in detail the chemical structure essential for the protective

152 MORICHI, IRIS, YANO and KEMBO VOL. 9

Table 1. Protective effect of glutamic aci

for various bacteria during

d and 4 related freeze-drying

compounds

Survival (%)

Table 2. Protective effect

for lactic acid

of glutamic acid

bacteria during

and 4 related freeze-drying

compounds

Survival (%)

1963 Freeze-drying of bacteria in presence

action, 45 compounds related with glutamic acid for their effects (Tables 3-7). a-Amino-dicarboxylic acids. From Table 3,

of glutamate etc.

were examined

it can be seen

153

systematically

that glutamic

General structural formula for test compounds is HOOC-C-(CH2)n-COON. n : 0-4,

NH2 X : -H or -CH3. For experimental conditions see Table 1. Each value in this and the following tables represents mean value of 5 trials with standard deviation.

acid is the most effective and aspartic acid the next among the series of a-amino-dicarboxylic acids tested. These two compounds were very effective on all the species of bacteria tested independent of their Gram-staining characteristics. a-Aminopimelic acid was effective for Gram-positive bacteria but not for Gram-negatives. Aminomalonic acid was slightly effective only for Gram-positive organisms. Increment or decrement of one or two methylenes in glutamic acid molecule caused only a minor modification of its protective action, whereas five-carbon compound (glutamic acid) was the most effective. Substitution of the methyl group for hydrogen at the a-carbon of glutamic acid did not influence its high protective activity (see the result obtained with a-methyl-L-glutamic acid). Significance of -NH2 group of glutamic acid. The results obtained with several non-substituted and substituted dicarboxylic acids are presented in Table 4. Non-substituted dicarboxylic acids showed generally no protective effect, with an exception of malonic acid which was effective for both Gram-positive and -negative bacteria. The protective effect of aspartic acid decreased only slightly when its -NH2 group was substituted by -OH, as exemplified by malic acid which was effective for both Gram-positive and -negative bacteria . Tartronic acid, the oxygen analog of aminomalonic acid, was fairly effective for Gram-positive bacteria but not for Gram-negatives. Although we did not examine the effectiveness of a-hydroxyglutaric acid,

Table 3. Protective effect of a-amino-dicarboxylic acids

Survival (%)

154 MORICHI, IRIE, YANG and KEMBO VOL. 9

General structural formula for test compounds

X : -H, -OH, -SH, -NH-CO-CH3i -NH-CO-C6H5-NH

H

is HOOC-C-(CH2),1-000H. n:

X

2 or N\CH3.

0-2,

these results seem to suggest that in the case of the dicarboxylic acids the substi-tution of the -NH2 group with the -OH group does not result in a significant change in the favorable effects of the acids. Worth noticing in this connection are the facts that a-ketoglutaric acid has an effect favorable for Gram-positive bacteria, and that the conversion of -OH of malic acid to an -SH group

(thiomalic acid) reduced strikingly the protective activity of the acid. The protective activity of glutamic acid seems to be hardly modified by the acetylation of its -NH2 group. The results obtained with p-aminobenzoyl

glutamic acid were rather complicated ; whereas it was as effective as glutamic acid in the cases of two spherical organisms, it was less effective in the cases of Gram-negative bacteria, and showed little or no protective effect in the case of L, arabinosus. We have no experimental evidence at present as to whether or not the acyl-derivatives of the acid are decomposed by acylase. The effect of the -NH-CO- group which is contained in the acyl-compounds in question will be discussed later. It may be added that the N-dimethyl derivatives of

glutamic and aspartic acids were found to be fairly effective except in the cases of two or three bacterial species tested. Significance of r- and a-COOH groups of glutamic acid. As may be seen

Table 4. Significance of -NH2 group of glutamic acid in the protective activity

Survival (%)

1963 Freeze-drying of bacteria in presence of glutamate etc. 155

from Table 5, the neutral amino acids listed as the first group (from glycine to DL-norvaline) were found to be practically ineffective. The amino acids classified as the second group (from L-cysteine to DL-threonine) might be con-sidered to have some functional group(s) in place of j3-COON group in the aspartic acid molecule. Among these, cysteine and methionine showed no effect, while cysteic acid having a -S03H group instead of -SH in cysteine, showed a remarkable protective activity for both Gram-positive and -negative

Table 5. Significance of Y-000H and a-COON of glutamic acid in the protective activity

156 MORICHI, IRIE, YANG and KEMBO VOL. 9

bacteria. This seems to suggest that the /3-COON group of aspartic acid could effectively be replaced by an -SO3H group. Serine having an -OH

group instead of -SH in cysteine was ineffective, whereas threonine was found to be fairly effective for both Grampositive and -negative bacteria. These observations together with the fact that homoserine and a-aminobu-tyric acid showed no effect, seem to indicate that the protective activity of threonine is due to the presence of J3-CH3 and secondary -OH groups in the molecule.

Comparing the effectiveness of the amino acids classified as the third group in Table 5, (from L-asparagine to L-glutamic acid-r-benzyl ester) glutamine was found to be completely ineffective as already shown in Tables 1 and 2. Succinamide also was ineffective. By contrast, asparagine showed an appreci-

able effect (to an extent that was one-sixth of that of aspartic acid) on E. coli, although it showed only a slight protective effect upon Gram-positive bacteria. As it was the case in the amidation of r-COON group in glutamic acid, negative effects were obtained on esterification of the same group as exemplified by the results obtained with the r-methyl or benzyl esters of the acid.

Among the substances belonging to the fourth group in Table 5 (from

~3-alanine to L-lysine), lysine was the most effective, but only for Gram-positive bacteria. The effectiveness of l3-alanine and r-aminobutyric acid for M. lysodeikticus was comparable to that of lysine, but they were rather ineffective for other bacteria. These results seem to indicate that the a--COOH group of

glutamic acid is essential for its protective activity. Compounds containing -NH-GO-group. Although glutamine was found to be ineffective, the heating of glutamine solution at 100° for 1 hr resulted in the exhibition of remarkable protective activity. The results are presented in Table 6. VICKERY et al. (15) observed that when a solution of glutamine was heated at 100° for 1 hr at pH 6.5, 86/ of glutamine was converted to pyrrolidonecarboxylic acid. Ac-cordingly, the effect of this acid was examined using a chemically pure sample of 5-pyrrolidone-2-carboxylic acid. The result presented in Table 7 shows that this acid has actually a considerable protective effect both for Gram-positive and -negative bac-

teria. Investigations were, therefore, made of the protective activities of

Table 6. Protective effect of heated glutamine

Survival (00)

1963 Freeze-drying of bacteria in presence of glutamate etc. 157

several other compounds having the -NH-CO- group (Table 7). 2-Pyrrolidine-carboxylic acid (proline) was effective for M. lysodeikticus but ineffective for other bacteria tested. Hydroxyproline, i. e., 3-hydroxy-2-pyrrolidonecarboxylic acid, and a-pyrrolidone were found to be ineffective. These results suggested that at least the presence of -NH-CO- together with -COON in pyrrolidone-carboxylic acid would be necessary for its protective activity. However, the tests made of several other compounds having both -NH-CO- and -COOH

groups gave somewhat erratic results. As described already, N-acetylglutamic and p-aminobenzoylglutamic acids were appreciably effective. Also effective was acetylglycine, but no effect was observed with orotic acid, glycylglycine and hippuric acid. Protective effect of D-glutamic acid. Protective effect of L- and D-isomers of gultamic acid was compared (Table 8). The results obtained showed that both isomers have practically the same protective effect.

Table 7. Protective effect of compounds containing -NH-CO- group

Survival (%)

Table 8. Protective effect of D-glutamic acid

Survival (%)

158

Table

MORICHI,

9. Protective effect

for bacteria during

IRIE, YANO and KEMBO

of glutamic acid and 4 related

freezing and thawing

compounds

Survival

VOL. 9

(°o)

Table 10. Effect of some compounds on rehydration process of freeze-dried bacteria

Survival (/)

1963 Freeze-drying of bacteria in presence of glutamate etc. 159

Proctective effect during freezing and rehydration processes According to SCOTT (16), the death of freeze-dried bacterial cells would take place either during the process of freezing, of drying, of storage or of rehydration. To investigate in which of these processes the protective action of chemical substances comes into play, the following experiments were performed. A part of the test organism was frozen in the presence of the agent and thawed immediately, and, on the other hand, the cells suspended in distilled water were freeze-dried and rehydrated with the solution of the test agent, and the survival ratios in both samples were compared. Under the pre-resent experimental condition, the duration of storage was so short that the influence of this process might be excluded in the evaluation of the results.

In Table 9 are summarized the results obtained with glutamic acid and four related compounds which were given during the freezing-and-thawing

process. As may be seen, glutamic acid was effective for five organisms tested. Considerable protective activity was also observed with glutaric acid and r-aminobutyric acid, which exhibited no activity in the experiments described earlier. Glutamine and norvaline, ineffective agents in freeze-drying, were also ineffective in this treatment.

The experiment, in which the bacteria were feeze-dried in distilled water and rehydrated in the presence of test substances, showed that all the substances (14 in number) had no appreciable protective activity (Table 10). The results indicate that the effective agents, such as glutamic acid and the related compounds, exert their protective action principally in the freezing and drying

processes and not in the rehydration process.

DISCUSSION

The present results and those obtained by several workers (1-3, 6-9) show that glutamic acid has a protective effect on various bacterial species against their death during the process of freeze-drying. Some compounds chemically related with glutamic acid also showed similar protective activities. The protecting effects of these compounds are considered to be of some physico-chemical nature and not of a metabolic one. This reasoning is based on the following facts : (1) Among the effective compounds there are some , such as a-aminopimelic, cysteic and pyrrolidonecarboxylic acids, which can hardly be thought to be metabolized by the test organisms. On the other hand , some substances which are considered to be metabolizable, e. g., glutamine and asparagine, showed no protective activity. L. arabinosus is indeed able to utilize glutamine (non-effective) as well as glutamic acid (effective) but not

pyrrolidonecarboxylic acid (effective) (17). (2) The protective effect of glutamic acid was found to have nothing to do with its optical activity .

WASSERMAN et al. (18) demonstrated the efficiency of succinate and malate solutions (0.05 M, pH 6.0) in increasing the recovery of viable cells from freeze-dried Serratia marcescens. Our experiment showed, however, that glutamic

160 MORICHI, IRIE, YANO and KEMBO VOL. 9

acid and some related compounds applied during the process of rehydration did not give any favorable effect. The effectiveness of the substances seems to have some correlation to their chemical structures. The -NH2 group of glutamic acid molecule could be replaced, without affecting its protective activity, by some other groups having the atom of high electro-negativity (>NH, -OH, =0). These functional

groups have a capacity to form hydrogen bond. DoEBBLER et al. (19) have reported that sugars and glycols inhibited the hemolysis of erythrocytes during freezing and thawing and that the protection was correlated with the concentra-tion of hydrogen-bond generating groups provided by the active solutes. The

r-COON group of glutamic acid might be replaced by some other acid group (i. e., -SO3H). Modification of r-COOH group to some hydrophobic structures (e, g., ester, and amide) appeared to destroy the protective activity. It is con-sidered that the -NH2 and r-COON groups of glutamic acid might condense in-tramolecularly to form -NH-CO- bond without altering the protective activity. Summarizing the results obtained, it may be inferred that the effectiveness of substances is related to the following chemical structure as found in glutamic acid : (1) presence of a functional group containing the atom of high electro-negativity on the a-carbon, e. g., -NH2, (2) presence of two acid groups, e. g., a-and r-COON, (3) close proximity of -NH2 and r-COON. All the effective com-

pounds except malonic acid were found to have similar structural features to glutamic acid. Malonic acid contains an active methylene but no functional group corresponding to -NH2 of glutamic acid. It may be assumed that the protective solutes have the affinity not only to water but also to hydrogen-bond generating groups of bacterial cells. In this connection, OBAYASHI et al.

(7) pointed out the importance of the high affinity of sodium glutamate to water during the drying process and considered that the salt ensured the retention of adequate moisture in the dried preparation.

SUMMARY

The presence of glutamic acid in a suspending medium prevented remark-ably the death of various bacteria (21 species, 26 strains) subjected to the

process of freeze-drying. Systematic studies were made of the protective effects of 45 compounds related chemically with glutamic acid, and the following compounds were found to be effective for both Gram-positive and -negative organisms tested : aspartic acid, malic acid, cysteic acid, threonine, pyrrolidone-carboxylic acid, acetylglycine, malonic acid, a-methyl-glutamic acid, N-acetyl-

glutamic acid, N-dimethyl-glutamic acid and N-dimethyl-aspartic acid. L- and n-forms of glutamic acid showed equally strong protective activities. Amino-malonic acid, tartoronic acid, a-aminopimelic acid, a-ketoglutaric acid and lysine were effective only for Gram-positive bacteria. The protective effect of glutamic acid and related compounds are considered to be of some physico-chemical nature and not of a metabolic one. Chemical structures of the effec-tive compounds seems to be characterized by the presence of a hydrogen-bond

1963 Freeze-drying of bacteria in presence of glutamate etc. 161

generating group (e, g., a-COON and r-COON).

-NH2 , >NH, -OH or =0) and two acid groups (e. g.,

The authors wish to express their appreciation to Dr. H. Iizuka, Institute of Applied

Microbiology, University of Tokyo, and Dr. K. Komagata, Aji-no-moto Co., Ltd., for

their helpful advices concerning test organisms. We also wish to thank Dr. Y. Izumi,

Institute for Protein Research, Osaka University, and Dr. H. Nagayama, Research Institute

for Tuberculosis & Leprosy, Tohoku University, for their generous gifts of several

reagents and many helpful suggestions. We would like to express our gratitude to

Dr. T. Yanagita, Institute of Applied Microbiology, University of Tokyo, for his helpful

suggestion and criticism during the preparation of this manuscript. We are grateful

to the Rockeff eler Foundation for the donation of the freeze-drying equipment.

(1) (2)

(3) (4)

(5)

(6) (7) (8)

(9)

(10) (11) (12) (13)

(14)

(15)

(16)

(17) (18) (19)

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(1962). H. B. VICKERY, G. W. PUCHER, H. E. CLARK, A. C. CHIBNALL and R. G. WESTALL : Biochem. J., 29, 2710 (1935). W. J. SCOTT: Recent Research in Freezing and Drying, Blackwell Scientific Public-ations Ltd., 1960, p. 188. H. A. HARPER: Arch. Biochem., 15, 433 (1947). A. E. WASSERMAN and W. J. HOPKINS: Appl. Microbiol., 5, 295 (1957). G. F. DOEBBLER and A. P. RINFRET: Biochim. Biophys. Acta., 58, 449 (1962).