ELSEVIER FEMS Microbiology Letters 128 (1995) 59-62

Effect of pH and hydrogen peroxide produced by LactobaciZZus hilgardii on Pediococcus pentosaceus growth

Ana V. Rodriguez a, Maria C. Manta de Nadra a9b3 *

’ Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145,4#0 San Miguel de Tucumh, Argentina b Facultad de Bioquhica, Quimica y Farmacia, Universidad National de Tucumbn, San Miguel de Tucumbn, Argentina

Received 29 November 1994; revised 31 January 1995; accepted 13 February 1995

Abstract

The growth of Lactobncillus hiZgurdii X,B and Pediococcus pentosuceus 12p, isolated from Argentinian wines, were studied in pure and mixed cultures. In the mixed culture, an amensalistic growth response was observed: Pediococcus pentosuceus growth was inhibited until 24 h; after this time, no viable cells were detected. In pure and mixed cultures, Lactobucillus hilgurdii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. Hydrogen peroxide and increased acidity were responsible for Pediococcus pentosucew inhibition in the mixed culture.

Keywords: Hydrogen peroxide; Luctobucillus hilgurdii; Pediococcus pentosuceus

1. Introduction

Lactic acid bacteria (LAB) play an important role as food preservatives. They can produce a variety of inhibitory compounds which prevent the develop- ment of unwanted or pathogenic bacteria as well as L4B themselves [l]. The acidity produced by LAB is a fundamental factor in food safety. In mixed cultures different acidifying activity and acid toler- ance of the strains may lead to the dominance of the more acid resitant strains. However, the inhibitory capacity of LAB not only results from their ability to lower the pH and the nature of the organic acids that they produce [2,3], but also depends on the produc- tion of other antimicrobials such as hydrogen perox- ide [4,5] and bacteriocins [6,7].

* Corresponding author. Tel: +54 (81) 310465; Fax: +54 (81) 310465.

Federation of European Microbiological Societies. SSDIO378-1097(95)00083-6

In Agentine wines LAB grow in multispecies populations which develop successively or simulta- neously[8]. Different types of interactions between LAB isolated from wine have been reported 19,101, but their antagonistic abilities are not known. The aim of the present work was to study the effect of L. hilgardii X,B on P. pentosaceus 12p growth. Hy- drogen peroxide production was investigated allow- ing a better understanding of the bacterial interac- tion.

2. Materials and methods

2.1. Strains and culture conditions

Lactobacillus hilgardii X, B and Pediococcus pentosaceus 12p were isolated from Argentinian red wines as previously described by Strasser de Saad and Manca de Nadra Ill].

60 A. K Rodriguez, M.C. Manca de Nadra / FEMS Microbiology Letters 128 (1995) 59-62

They were grown at 30” C for 60 h in basal medium containing (g 1 -I or ml l- ’ in distilled

water): yeast extract, 10.0; Tween 80, 1.0 and com- mercial grape juice (5” Brix), 170. The pH of the medium, 5.50, was adjusted with 1.0 M NaOH prior sterilization. In mixed cultures the inoculation rate

was 1: 1 (L. hilgardii-P. pentosaceus).

2.2. Bacterial growth

Growth was measured by changes in optical den- sity at 560 nm with a Bausch and Lomb 20 spec- trophotometer. Viable cells were enumerated by sur- face spreading 0.1 ml samples on basal medium

supplemented with 20.0 g 1-l agar. The enumeration was performed on the base of colony morphology and rate of growth By 48 h L. hilgardii X,B pro- duced colonies with entire margins, convex, smooth and glistening and rods in chains. By 72 h P. pentosaceus 12p produced smooth, round and white colonies and spherical cells in pairs or tetrads.

2.3. Measurement of the inhibitory activity

For the production of antimicrobial substances in liquid cultures L. hifgardii X,B was grown in basal medium, incubated at 30” C for 24 h. The cells were

then removed by centrifugation at 6000 rev min-’ for 20 min.

P. pentosaceus 12p culture of 20 h at a concentra- tion of about 8 X lo8 cfu ml- ’ (O.D. at 560 nm approx 0.70) was overlaid on basal medium agar (15.0 g 1-l agar). The supematant fluid of L. hilgar- dii X,B was tested on these plates into which 10 mm holes has been punched, and filled with 100 ~1 of the solution under test. The supematant fluid was divided into three fractions: A) untreated (control); B) neutralized and treated with 1000 U ml-’ cata- lase (Sigma Chemical Co., St. Louis, MO) and incu-

bated at 25” C for 1 h; C) neutralized with 1.0 mol 1-l NaOH. Th e p a es 1 t were incubated at 30” C for 48 h and examined for inhibition zones.

2.4. Hydrogen peroxide quantification

Samples were taken from the L. hilgurdii, P. pentosuceus and mixed cultures flasks, at different times and were centrifuged at 6000 rev min-’ for 20

min. Hydrogen peroxide was determined as de- scribed previously [ 101.

2.5. Determination of minimum inhibitory concentra- tion and minimum bactericidal concentration of hy- drogen peroxide on Pediococcus pentosaceus culture

P. pentosaceus 12p culture of 20 h (O.D. at 560 nm approx 0.70) was incubated in a series of tubes

containing decreasing concentrations of hydrogen peroxide. Growth was measured by changes in opti-

cal density at 560 nm. The highest dilution of hydro- gen peroxide showing no visible growth is defined as the minimum inhibitory concentration (MIC). To determine if the inhibition is reversible or permanent,

cells from tubes showing no growth after 24 h of incubation at 30” C were subcultured onto solid me- dia lacking hydrogen peroxide. The growth was de-

tected by enumerating colony forming units in basal medium plates incubated at 30” C. The lowest hydro- gen peroxide concentration that kills the test organ- ism is defined as the minimum bactericidal concen-

tration (MBC).

3. Results and discussion

Growth of Lactobacillus hilgardii X,B and Pe- diococcus. pentosaceus 12p were determined (Fig.

0 12 24 36 48 40

Tlmd (h)

Fig. 1. Growth of L. hilgardii X,B and P. pentosaceus 12p in

pure and mixed cultures. L. hilgardii growth ( 0 ); P. pentosaceus growth (0) in pure (solid line) and mixed (dotted line) cultures.

Each value is the mean of three replicates that are reproducible

within f 5%.

A. V. Rodrigueq M.C. Mama de Nadra / FEMS Microbiology Letters 128 (1995) 59-62 61

1). In pure culture, L. hilgurdii grew more actively than P. pentosaceus: the specific growth rates were 0.21 h-land 0.17 h-‘, respectively.

In mixed culture L. hilgurdii had the same growth as that of pure cultureand P. pentosuceus inhibition was observed: cells numbers decreased from 2.6 X

lo8 cells ml-’ (in pure culture) to 7.0 X lo7 cells ml-’ (in mixed culture) in 12 h. At 24 h cells numbers diminished to 1.2 X lo7 cells ml-‘, and after this time no viable cells were detected. This is an amensalistic growth response by which the growth of one strain is inhibited by the presence of the other (Fig. 1)

The inhibitory activity of L. hitgurdii X,B against P. pentosuceus 12p is shown in Fig. 2. When cata- lase was added to neutralized supematant (pH = 7.01, the zones of inhibition did not occur, indicating the inhibitory effect of hydrogen peroxide in mixed cultures (Fig. 2).

Generation of hydrogen peroxide during the growth of P. pentosaceus 12pand L. hilgurdii X,B in pure and mixed cultures, was examined (Fig. 3). In pure cultures P. pentoscueas did not produce hydrogen peroxide and L. hilgurdii X,B produced 7.79 pg ml-’ in 12 h, reaching the maximum at 24 h: 47.43 pg ml-‘. In mixed culture the production of hydrogen peroxide did not change with respect to pure cultum: 1.72 pmol of hydrogen peroxide/l X

lo9 cells and 1.68 pmol/l X lo9 cells were de- tected at 24 h in pure and mixed culture, respec- tively. Autoinhibition by peroxide hydrogen was not observed in L. hilgurdii; these results confirm those obtained for other LAB which were capable of growth in the presence of a high concentration of hydrogen peroxide [ 121.

Fig. 2. Inhibition of P. pentosaceus 12p by L. hilgardii X,B

cell-free supernatant. Holes contained supematam fluids, which were untreated (A); neutralized and treated with catalase (B), and

neutralized (0.

action of hydrogen peroxide on P. pentosuceus 12~: MIC = 6.12 pg ml-’ and MBC = 34.00 pg ml-‘. In mixed culture 4.22 pg ml-’ of hydrogen peroxide was produced in 12 h and 46% of inhibition of P. pentosaceas growth was observed in this time (Figs. 3 and Table 1). 4.08 pg ml-’ of hydrogen peroxide produced only an inhibition of 11% (Table 1); thus, the inhbitory effect observed in mixed culture must be resulted from the combination of two or more factors. These results are different from those obtained with another mixed culture where hydrogen peroxide production by L. hilgurdii totally inhibited Leuconostoc oenos growth [lo].

Table 1 shows the MIC and MBC values for the When the pH of the supematant was adjusted to

Table 1 Effect of hydrogen peroxide on Pediococcus penrosaceur 12p growth

Hydrogen peroxide Increase in absorbance

(cLg ml-‘) at56Onm’

P. pentosaceus

(o/o) b

2.&I ct.48 100

4.08 0.26 89.0

6.12 0.02 62.0 8.50 0.01 25.8 17.00 0.01 5.8 34.00 0.00 0.0

a The increase in absorbance at 560 mn was estimated after incubation for 24 h at 30” C. b Percent of bacteria remaining viable after 24 h

of incubation in relation to control c&me without hydrogen peroxide. Each value is the mean of three repiicates that are reproducible within

f 5%.

62 A. V. Rodriguez, M. C. Mama de Nadra / FEMS Microbiology Letters 128 (1995) 59-62

OH Hydr~en oemxlde (WI ml - ’ I

- 60

5

- 60

- 40

4

- 30

t /,, ;/

0 __._. --+.._.__- -e- --4 ,/-

3 I

20

. Y.l IO

0 12 24 36 48 bo”

Time (h)

Fig. 3. Hydrogen peroxide production and pH of L. hilgardii XrB

and P. pentosaceus 12p in pure and mixed cultures. Hydrogen

peroxide production by P. pentosaceus (* ), by L. hilgardii (X ) and by mixed culture (0). pH of P. pentosaceus (Cl), pH of L.

hilgardii ( - ) and pH of mixed culture (0). Each value is the

mean of three replicates that are reproducible within f 2%.

7.0 a significant reduction in the inhibition zone with

respect to the control was observed (Fig. 21, indicat- ing that the inhibitory effect could be partially due to acid. In pure culture of P. pentosaceus pH changed from 5.40 to 4.20 in 24 h; in mixed culture an increased acidity was observed: from 5.40 to 3.80 in the same time (Fig. 3). L. hilgardii, heterofermenta- tive, produced lactic and acetic acids from its sugars metabolism (data not shown). It is known that in- hibitory effect of organic acids is due to the undisso- ciated acid [13,14]. The cell membrane is imperme- able to ionized hydrophilic acid, while non-ionized hydrophobic acids diffuse passively [ 151. Thus, weak

organic acids, such as acetic acid (pK, = 4.75) will be present at the same concentration on both side of the membrane. The intracellular medium is basic in comparison to the external medium, and will favor the ionization of these molecules producing an in- crease in their intracellular concentration. This causes an acidification of the cytoplasm and an inhibition of some enzymes [151.

Under the conditions described in this study, L hilgardii produces antimicrobial compounds which inhibit P. pentosaceus growth. However, future stud- ies would provide some direct evidence about the production of hydrogen peroxide in vinification con- ditions and if it is responsible for some antagonistic effect observed between LAB.

Acknowledgements

This work was supported by CONICET and CIUNT, Argentina.

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