J. Biol. Macromol., 11 (1), 3-13 (2011)

ARTICLEEFFECTS OF CO-CULTURING LAB ON GABA PRODUCTION
Yuko Watanabe,¶ Kiyoshi Hayakawa,§ and Hiroshi Ueno *¶¶Laboratory of Applied Microbiology and Biochemistry, Nara Women’s University, Nara,
630-8506 JAPAN§CHA Research Center, Fukujuen Co., Kyoto, 619-0223 JAPANReceived January 12, 2011; Accepted February 4, 2011

Two strains of lactic acid bacteria, Streptococcus thermophilus IFO13957 and Lactobacillus delbrueckii subsp. bulgaricus IAM1120, were examined for the effects ofco-culturing on acid resistance and production of GABA.

When S. thermophilus

and L. bulgaricus were grown separately, the medium became acidic at around pH 5
and 4, respectively.

For the latter it became highly acid-sensitive.

When bothstrains were co-cultured, they appeared to be acid resistant and grew well.

The

co-culturing raised GABA level in the medium, which apparently coincided with theacidification of the medium.

When each of the strains was cultured in the pH 3.5

and 6 medium, only S. thermophilus produced a large amount of GABA at pH 3.5 andsurvived.

The results suggest that co-culturing of the two strains raises the eachother’s properties of the acidification of the medium and production of GABA or a cid resistance.
Key words: GABA, acid tolerance, mixed cultured, S. thermophilus, L. bulgaricus*

To whom correspondence should besent: E-mail: hueno@cc.nara-wu.ac.jp,
Phone no.: +81-742-20-3493, Fax no.:+81-742-20-3448

Abbreviations: GABA, γ-aminobutyric acid;
GAD, glutamate decarboxylase; LAB, lactic
acid bacteria; MSG, monosodium glutamate;
OPA, O-phthalaldehyde; BCP, bromocresol

purpleGABA-production in co-culturing

4

γ-Aminobutyric acid (GABA) has neuro-

been used for food production; however,transmission, antihypertensive, and relaxa-

their detailed mechanism of GABA pro-tion activities that are beneficial to humanduction has not been fully understood.

Owing to these health benefits,

In the production process of yo-

food production enriched with GABA is ingurt, two strains of LAB, L. delbrueckii

high demand (2, 3).

GABA is synthe-

subsp. bulgaricus and S. thermophilus,

sized by decarboxylation reaction of glu-

were co-cultured in order to facilitate thehealth (1).

tamate with the enzyme glutamate decarboxylase (GAD) which is widely distrib-

growth (14-16) and enhance lactate fermentation (17, 18).

L. bulgaricus digests

uted among mammals, plants and microorganisms, including lactic acid bacteria(LAB) (4-9).

GABA production in LAB

casein, the milk protein, to make aminoacids which feed to S. thermophius (19).

relies upon GAD and is induced when pH

S. thermophilus produces formate whichbecomes acidic.

feeds to L. bulgaricus (20).

Some of LAB are capa-

As a result,ble of surviving under strongly acidic con-an efficient and effective lactate fermenta-

ditions due to the presence of active GADtion was accomplished.

that utilizes intracellular proton to make
LAB acid-resistance.

L. bulgaricus (10, 21) and S.

The level of acidity
thermophilus (10) were isolated fromduring the lactate fermentation depends
upon LAB strains in which the productioncommercial yogurt.

When they were

GAD iso-cultured separately, a minute amount oflated from LAB exhibits an optimum pH at

GABA, less than 0.2 mg/ml, was produced

around pH 4-5. For example, the optimum

When both strains were co-cultured, apH for GAD from i.e., L. brevis and L. lac-quantitative conversion of glutamate toof lactate is highly variable.

tis are pH 4.2-4.5 (9) and pH 4.7 (6), respectively.

The GABA production by L.

GABA was observed (10).

The mecha-

nism of the co-culturing enhanced GABA

brevis (8, 10, 11, 12 )and by L. Buchner(13) was reported and those LAB haveproduction has not been fully characterized.

Watanabe, Y., et al.

In this study, we examined the co-culturing

medium.

and evaluated the relationship between thegrowth and acid-resistance.

5

Cell counts - Cell population was
estimated by counting the cell numbers
after diluting the culture medium including
0.25% yeast extract, 0.5% polypeptone,

MATERIALS AND METHODS
Culture medium - Skim milk me-

0.1% glucose, 0.1% polysorbate 80, 0.83

dium contained 10% skim milk and 1%

mM cysteine, 0.1 mM bromocresol purple

monosodium glutamate (MSG).

and 1.5% agarose and incubating for 48h at

GYP

medium, a rich nutritional medium, con-37°.

tained 1% yeast extract, 0.5% polypeptone,

GABA analysis in the medium -1% glucose, 15 µM sodium acetate 3H2O,

GABA was detected as a single peak on

0.41 µM MgSO4 4H2O, 0.36 µM FeSO4

amino acids analysis. Amino acid analy-

7H2O, 1.7 µM NaCl, and 1% MSG.

For

sis was carried out on a Shimadzu HPLC

experiments examining the effects of acid-(Kyoto, Japan) that employed pre-column

ity on the growth and GABA production,

derivatization (22).

the initial pH of the medium was adjustedof the culture medium, 1 ml aliquot of thewith HCl.

supernatant was mixed with 1 ml acetoni-

After centrifugation

Bacteria species - LAB strains, L.

trile and the mixture was centrifuged again.

bulgaricus IAM1120 and S. thermophilus

An aliquot of 30 μl supernatant was mixed

IFO 13957, were from the stock in our la-with 300 μl each of O-phthalaldehyde

boratory.

(OPA) solution and mercaptoethanol solu-

Culture condition - LAB strainstion and allowed for 120 sec for derivatiza-

were pre-cultured in GYP medium for 24 htion. OPA solution was prepared by dis-

at 37 °C, then 1/1000th of the aliquot ofsolving 20 mg OPA in 3 ml acetonitrile andthe culture medium was cultured in the

7 ml of 0.5 M potassium borate buffer, pH

GABA-production in co-culturing6

10.0. Mercaptoethanol solution was pre-TSK-gel 80TM column that had beenpared by mixing 50 μl mercaptoethanol inequilibrated with 4 mM sodium citrate

10 ml of 0.5 M potassium borate buffer,buffer at pH 6.0 in 7% acetonitrile solution.

An aliquot of 30 μl of the

OPA labeled amino acids were eluted byreaction mixture was applied onto araising acetonitrile concentration from 7 to

pH 10.0.

Fig. 1. The effect of the mixed culture on the growth and GABA production.
L. bulgaricus IAM1120 and S. thermophilus IFO13957 were inoculated in 10%
skim milk medium with 1% MSG at 37 ˚C.

Watanabe, Y., et al.

7

40% in a linear manner at a flow rate of 0.8

IAM1120 showed the typical growth curve

ml/min.

Peaks were monitored by Shi-

with the initial log-phase growth followedmadzu RF-4000 fluorescence detector thatby the stationary-phase up to 100 h cultur-

was equipped with 350 nm excitation filtering.

and 455 nm emission filter.

The amounticus IAM1120 alone showed the initialof GABA was estimated by comparing the

log-phase growth followed by the shortpeak area with the corresponding standard

stationary-phase, and then declined. The

GABA.

declining of the growth appeared to coin-

In contrast, the growth of L. bulgar-

cide with the acidification of the medium,
RESULTS AND DISCUSSION

which

suggeststhatL.

bulgaricus

The effect of co-culturing on

IAM1120 strain seems to be acid-sensitive.

GABA production – Both L. bulgaricus

As the acidity of the L. bulgaricus

IAM1120 and S. thermophilus IFO13957

IAM1120 growth medium reached down tostrains were originally isolated from yogurt

pH 4, lactate synthesized by this strain

by one of the authors (K.Y.) and distin-

could be accumulated enough to kill theguished from previously described strains,

cells.

L. delbrueckii subsp. bulgaricus (10, 21)

is a part of LAB family that should haveand S. thermophilus (10).

Two strains

acid tolerance built-in, this strain may not

were cultured in skim milk medium indi-

possess full capability of acid-resistancevidually or together.

system.

The cell growth, pH

Although L. bulgaricus IAM11120

change in the medium, and GABA con-

S. thermophilus IFO13957 by itself

centration in the medium were analyzed

grew well, but the pH of its medium was(Fig. 1).

shifted down only to 5, the pH being sig-

andThe growth of S. thermophilus alone

nificantly higher than that observed for L.

co-culturing

bulgaricus IAM1120.

with

L.

bulgaricus

It was probably

8GABA-production in co-culturing

Fig. 2. The effect of the initial pH on the growth and GABA production.
L. bulgaricus IAM1120 or S. thermophilus IFO13957 was inoculated in GYP
medium that contained 1% yeast extract, 0.5% polypeptone, 1% glucose, 15 µM
sodium acetate 3H2O, 0.41 µM MgSO4 4H2O, 0.36 µM FeSO4 7H2O, 1.7 µM
NaCl and 1% MSG at 37 ˚C for 48 h. Indicated pH was the initial pH of themedium.
due to the poor lactate production on thisthe microscope, where S. thermophilus

strain; although the details should wait for

IFO13957 appeared to be predominant,further study.

about 80 to 90% population over L. bul-

When both L. bulgaricus IAM1120 and S.

garicus IAM1120. Thus, it is likely that

thermophilus IFO13957 were co-cultured,

L. bulgaricus IAM1120 was able to sur-the growth pattern looked like that of S.

vive in the acidic environment; hence, it

thermophilus IFO13957 alone whereas the

became acid-resistant by co-culturing.

shift in medium pH was like L. bulgaricus

More drastic difference was observed on

IAM1120 approaching to pH 4.

In thethe production of GABA, where the exten-

co-cultured media, we were able to observe

sively large amount of GABA was pro-the growth of both L. bulgaricus IAM1120

duced in the co-cultured medium.

and S. thermophilus IFO13957 by using

GABA production appeared to correlate

TheWatanabe, Y., et al.

9with the acidification of the medium;

pH adjusted to 3.5 and 6.0. After 48 h

hence, it is probable that two strains col-fermentation, the growth and GABA pro-laborated in the production of GABA as aduction were analyzed (Fig. 2).

result of acid-resistant response.

garicus IAM1120 grew well at pH 6.0, but

L. bulgaricus IAM1120 produced

grew poorly at pH 3.5.

L. bul-On the other hand,little GABA throughout the culturing peri-

S. thermophilus IFO13957 grew well at

od (Fig. 1).

both pH 6.0 and 3.5.

Since GABA production is a

key to bacterial acid-resistance, why the

When S. thermophilus IFO13957

poor growth observed for L. bulgaricus

alone was cultured for nearly 100 h in the

IAM1120, when the medium became acid-medium in which the initial pH was ad-

ic, could be explained because of the lackjusted to 6, pH of the medium approachedof GABA-synthesizing capability.

to near 5 (Fig. 1).

S.

We have initially as-

thermophilus IFO13957 also produced

sumed that S. thermophilus IFO13957 wassmall amount of GABA during the cultur-

not able to lower the pH of the medium

ing condition and was suspected for its in-and might not be able to survive at pH be-capability of making GABA.

In order tolow 5. However, as shown in Fig. 2, S.

answer to this question, we designed thethermophilus IFO13957 was capable ofexperiment described in the next section.

growing at pH 3.5 and it produced signifi-

The effect of acidity on survival ofcant amount of GABA.

This may suggest

LAB and GABA production - To evaluatethat S. thermophilus IFO13957 was likely

the effect of acidity, the growth of the LAB

to be responsible for GABA production

and GABA production were examined onwhen two strains were co-cultured.

L. bulgaricus IAM1120 and S. thermophilus IFO13957.

By co-culturing the two strains, it is

These strains were cul-

apparent that L. bulgaricus IAM1120 and S.

tured separately in the GYP medium withthermophilus IFO13957 compensate each10
GABA-production in co-culturingother’s weakness.

Similar effect ofparticipates in the acid-neutralizing mech-co-culturing was previously reported for S.

anism in the microbes.

thermophilus and L. bulgaricus, in whichcarboxylation of malate decreased thethe production of amino acids and formic

acidity of the medium during the fermenta-acid (14-16), respectively, enhanced thetion of wine (28-30).

production of lactate (17, 18).

For example, de-Co-culturing of microbes is benefi-

The action of GABA production to-cial not only for food production, but also

ward acid-resistance is not unique to S.

for human health since a large number ofthermophilus IFO13957; it is also reported

microbes live together in the animal intes-

in E. coli (23, 24) and Lactobacillus (25),tines, in other word they are co-culturing.

where the mechanism known as glutamate

The microbes may collaborate to carry out

dependent acid resistance (GDAR) plays aimportant biological functions; however,significant role. GAD is the enzyme tovery little is understood.

make GABA from L-glutamate, and utiliz-of the co-culturing of different microbes

es a single proton during the catalysis (4).

needs to be explored and the findings canThis consumption of the intracellular pro-be extended to the industrial application as

ton is responsible for shifting the intracel-

well as the improvement of human health.

The mechanism

lular pH toward neutral; hence, it helps
microbes to survive under the acidic envi-

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Communicated by Ishijima Sumio