Identification of Antifungal Compounds Producedby Lactobacillus casei AST18

Hongjuan Li • Lu Liu • Shuwen Zhang •

Wenming Cui • Jiaping Lv

Received: 14 January 2012 / Accepted: 19 April 2012 / Published online: 13 May 2012

� Springer Science+Business Media, LLC 2012

Abstract Lactobacillus casei AST18 was screened as an

antifungal lactic acid bacteria which we have reported

before. In this research, the antifungal properties of cell-

free culture filtrate (CCF) from L. casei AST18 were

detected, and the antifungal compounds of CCF were

prepared by ultrafiltration, and semi-preparative HPLC,

and then determined by GC–MS. CCF was sensitive to pH

and heat treatment but it was not affected by the treatment

of trypsin and pepsin. Through the treatment of ultrafil-

tration and semi-preparative HPLC there were two parts of

CCF which showed antifungal activities: part 1 and part 4.

Lactic acid was identified as the main antifungal compound

in part 1. In part 4, three small molecular substances were

detected with GC–MS. The three potential antifungal

substances were cyclo-(Leu-Pro), 2,6-diphenyl-piperidine,

and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-

a;10,20-d]pyrazine. The antifungal activity of L. casei AST18

was a synergistic effect of lactic acid and cyclopeptides.

Introduction

Molds and yeast are able to grow on almost all kinds of food

including cereals, milk, fruits, vegetables, nuts, fats, and

products of these. Filtenborg et al. [4] concluded that a very

limited number of fungal species (2–12) have been associ-

ated with the spoilage of food and they are mainly Penicil-

lium, Aspergillus, and Fusarium. For example, the most

important spoilage species of cheese without preservatives

added are Penicillium commune and P. nalgiovense. Cur-

rently, biopreservation is widely used to extend the shelf-life

and enhance safety of food obtained by the natural or added

microflora or their antimicrobial products [15].

Lactic acid bacteria (LAB) have a long history of use in

food products and feeds. LAB can decrease the pH value and

produce antibacterial compounds. In the past few years, many

researchers have found that LAB strains have the ability to

inhibit the growth of molds and yeasts. Ryan et al. [14] used

antifungal Lactobacillus plantarum strains to reduce the

amount of calcium propionate in bread. A strong synergistic

effect occurred when calcium propionate and antifungal

sourdoughs were combined into the bread formulation.

Several antifungal compounds have been isolated from

bacterial cultures. Magnusson and Schnurer [9] firstly

reported that Lactobacillus coryniformis subsp. corynifor-

mis strain produced a broad-spectrum proteinaceous anti-

fungal compound. Gerez et al. [5], Magnusson et al. [10],

Strome et al. [16], and Lavermicocca et al. [6] have all

reported a potential antifungal compound known as phen-

yllactic acid. Rouse et al. [13] have separated a possible

cyclic dipeptide from Pc. pentosaceous. Yang and Chang

[18] firstly reported antifungal activities of cyclo(Leu–Leu)

produced by LAB, a class of 2,5-diketopiperazines.

Recently, 2,5-diketopiperazines have attracted attention

due to their biological properties [11]. Dalbello et al. [1]

reported the identification of lactic acid, phenyllactic acid,

and two cyclic dipeptides (cyclo (L-Leu-L-Pro) and cyclo

(L-Phe-L-Pro)) as the major components responsible for the

antifungal activity of L. plantarum FST 1.7. But as anti-

fungal activities in other fractions have been observed, they

believed that more antifungal compounds were present in

addition to the ones that had been detected.

H. Li � L. Liu � S. Zhang � W. Cui � J. Lv (&)

Key Laboratory of Agricultural Product Processing and Quality

Control, Institute of Agro-food Science and Technology,

Chinese Academy of Agricultural Sciences, No. 2 Yuan Ming

Yuan West Road, Haidian District, P. O. Box 5109,

Beijing 100094, People’s Republic of China

e-mail: lvjp586@gmail.com

123

Curr Microbiol (2012) 65:156–161

DOI 10.1007/s00284-012-0135-2

Lactobacillus casei AST18 was screened as an anti-

fungal lactic acid bacteria which we have reported before

[7]. The aims of this study were to detect the antifungal

properties of CCF and to determine the antifungal com-

pounds produced by L. casei AST18.

Materials and Methods

Lactobacillus casei AST18 and Indicator Fungal

Lactobacillus casei AST18 (Accession no.: HM773423)

was cultured on deMan Rogosa and Sharpe (MRS) agar or

in MRS broth (Land Bridge Technology Co., China) at

37 �C for 24 h and maintained for a longer storage at

-80 �C in MRS supplemented with 25 % (v/v) glycerol.

Penicillium sp. isolated from spoilage cheese was used as

an indicator fungal. It was maintained on potato dextrose

agar (PDA) (Land Bridge Technology Co., China) at 30 �C

for 4 days and stored at 4 �C.

Preparation of the Spore Solution

Molds grew on PDA slants at 30 �C for 7 days until

sporulation formed. Then the spores were collected after

vigorously shaking the slants with sterile water which

contained 0.05 %(v\v) Tween-80. The concentration of

molds was measured by a hemocytometer (Qiujing Com-

pany, Shanghai, China) and adjusted to 106 mL/L.

Preparation of CCF and Concentrated CCF

Lactobacillus casei AST18 was inculcated in MRS broth at

37 �C for 72 h. The culture was centrifuged at 4,0009g for

10 min. The supernatant was filtered through a 0.22 lm

sterile filter (Mili Company, Shanghai, China), and then

freeze-dried. After freeze-drying, the supernatant was

concentrated 15-folds with respect to their initial volume

and used for the further experiments.

Antifungal Activity Assays

The modified agar well-diffusion method [9] was used to

detect antifungal activities. The penicillium sp. was used as

directed molds. For the agar well-diffusion method, 15 mL

PDA was added to a plate. Then 100 lL spore solution was

sprayed on the coagulative PDA. The 7.5 mm (outside

diameter) wells were settled down for 5 min. Then 200 lL

culture supernatant was added into the wells. The plates

were incubated at 30 �C for 48 h and examined for clear

zones of inhibition around the wells with vernier caliper

(Shanghai Shengliang Suring tools Co., China). The sterile

MRS broth adjusted to the lowest pH of the culture

supernatant was used as a control.

Effects of Temperature, pH, and Proteolytic Enzymes

on Antifungal Activities

The antifungal activities of CCF after the treatment of

temperature, different pH values, or proteolytic enzymes

were determined with the agar well diffusion assay.

CCF was divided into four samples and they were

heated to 50, 70, 100, and 121 �C for 10 min, respectively.

After cooling, the antifungal activity of each sample was

detected.

The pH value of CCF was adjusted to 2.5, 3.0, 4.0, 4.5,

5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with concentrated HCl and

NaOH. The MRS broth adjusted to the same pH values

served as a control.

CCF was treated with trypsin or pepsin (BioDee Bio-

Tech Co., China). Samples were adjusted to the optimum

pH values for each enzyme, i.e., 7.6 and 2.0 for trypsin and

pepsin, respectively [9]. After adjustment of pH, CCF was

treated with 100 lg of the respective enzyme per mL and

incubated at 37 �C for 1 h. Before evaluating the anti-

fungal activity the pH value of CCF was readjusted to the

initial pH (3.8). The MRS broth without the addition of

proteolytic enzymes and with the same pH values was used

as the control group.

Ultrafiltration of Concentrated CCF

The concentrated CCF was ultrafiltrated by 10, 3, and

1 KDa membrane gradually with an ultrafiltration system

(PALL Minimate, USA). Each part of the filtration was

collected and then the antifungal activity was determined.

Semi-Preparative HPLC

The ultrafiltrated CCF was further separated using semi-

preparative HPLC (VARIAN Prostar 218, USA) equipped

with a Pursuit Rs-5u-C18 column (150 9 21.2 mm2).

Linear gradient elution was used with methanol/0.05 %

trifluoroacetic acid-solvent A (Sigma-Aldrich) and water/

0.05 % trifluoroacetic acid-solvent B at 1 mL min-1 and

A/B ratios of 10: 90, 100: 0, and 100: 0, with run time of 0,

30, and 33 min, respectively. The eluate was monitored

using UV detector at 210 nm [8].

Analysis of Organic Acids

Organic acids were analyzed by D20NEX ICS-3000 ion

chromatography system (Dionex, Sunnyvale, USA)

equipped with an electroconductivity detector. Ion chro-

matography was carried out using the AG11-HC column

H. Li et al.: Identification of Antifungal Compounds 157

123

(4 9 250 mm). The mobile phase, 0.8 mM KOH, was at a

flow rate of 1.0 mL/min and at room temperature. The

organic acids were detected by chemical suppressed con-

ductivity using an anion-ICE micromembrane suppressor.

Identification of the Antifungal Compounds by GC/MS

For gas chromatography/mass spectrometry (GC/MS),

samples of fractions obtained from semi-preparative HPLC

were evaporated with an N2 stream and dissolved in

CH3OH. A QP2010-plus GC/MS instrument (Shimadzu,

Kyoto, Japan), equipped with a nonpolar capillary column

RTX-5MS (30 m by 0.25 mm inner diameter; film thick-

ness, 0.25 mm), was used for the analysis. The GC oven

was held at 50 �C for 1 min and then increased to 275 at

5 �C per min for 3 min. Helium was used as the carrier gas

with a flow rate of 30 mL/min. The identification of the

compounds was based on 90 % similarity between the MS

spectra of unknown and reference compounds in an MS

spectra library.

Results

Antifungal Properties of CCF

The antifungal activity was partly lost during heat treat-

ment and there was no inhibition activity observed after the

treatment of 121 �C, 10 min (Table 1). Maximum effi-

ciency of antifungal activity was observed when pH values

were between 2.5 and 4.0, but it decreased rapidly when pH

values were between 4.0 and 7.0, and then it was totally

lost at higher pH values (Fig. 1). This indicated that either

organic acids or other pH-dependent antifungal compounds

were responsible for the antifungal effect. The antifungal

activity was not sensitive toward trypsin and pepsin

(Table 2).

Purification and Identification

The concentrated CCF of L. casei AST18 was ultrafiltrated

by 10, 3, and 1 KDa membrane. The antifungal activity

was detected in the low-molecular weight substances

which was \1 KDa. There were no activities detected in

the retentate of each part. Further fractionations of the

liquid filtration were carried out using recycling of semi-

preparative HPLC.

The fraction 2 and 4 of the injection showed the anti-

fungal activity. The antifungal activity of fraction 4 in

comparison with fraction 2 of the same amount is shown in

Fig. 2. There was no antifungal activity detected in the

fraction 1, 3, and 5.

To determine if the antifungal activity was due to

organic acid production, IC analysis of organic acids in the

fractions was performed. There were no organic acids

detected in fraction 1, 3, 4, and 5. Four kinds of organic

acids were detected in fraction 2. Lactic acid showed the

highest quantity (93.70 mg/mL), followed by tartaric acid

Table 1 Effects of various temperatures on antifungal activity

Temperature (�C) 50 70 100 121

Inhibition zone (mm) 12.68 ± 0.85 11.78 ± 0.59 9.20 ± 0.51 ND

ND not detected

Fig. 1 Effects of various pH values on antifungal activity

Table 2 Effects of various proteinase on antifungal activity

Samples Culture supernatants Pepsase Trypsase

Treatment samples Control Treatment samples Control

Inhibition zone (mm) 19.05 ± 0.58a 15.65 ± 2.23a 16.29 ± 1.74a 18.52 ± 1.82a 17.53 ± 0.73a

Number of samples n = 3. Values in the same column with different small letters are significantly different (P \ 0.05)

158 H. Li et al.: Identification of Antifungal Compounds

123

(9.59 mg/mL), citric acid (1.29 mg/mL), and acetic acid

(2.42 mg/mL).

The antifungal activities of the detected organic acids of

fraction 2 were assayed. As shown in Fig. 3, lactic acid with a

concentration of (93.70 mg/mL) showed the same antifungal

activity with fraction 2. The tartaric acid (9.59 mg/mL),

citric acid (1.29 mg/mL), and acetic acid (2.42 mg/mL) did

not show any antifungal activities. Lactic acid was the main

antifungal compound in fraction 2.

The fraction 4 was analyzed by GC–MS. There were

three potential antifungal compounds detected.

The retention times of the three substances were 29.84,

32.34, and 37.88 min. They were designated as I, II, and III

(Fig. 4). As shown in Fig. 4, they were the main substances

existing in part 4 analyzed by GC–MS. The three potential

antifungal substances were: I, ctclo-(Leu-Pro); II, 5,10-

Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;10,20-d]pyrazine; and III, 2,6-diphenyl-piperidine (Fig. 5).

Discussion

The inhibition of the growth of mold and mycotoxin

accumulation by lactic acid bacteria and their metabolites

appear to be a promising biocontrol strategy in perishable

foods or feed which frequently contaminated by toxigenic

fungal strains [2]. However, few reports exist on specific

Fig. 2 The antifungal activities of part 2 and part 4 Fig. 3 The antifungal activities of part 2 and lactic acid

Fig. 4 The GC spectrum of the

three identified compounds in

part 4

H. Li et al.: Identification of Antifungal Compounds 159

123

antifungal compounds produced by LAB. Proteinaceous

compounds and low-molecular mass organic compounds

with antifungal activities are widely distributed among

different genera and species of LAB from different envi-

ronments [18].

In this research, we studied the antifungal properties of

CCF and found the potential antifungal compounds pro-

duced by L. casei AST18. Two parts in CCF which con-

tributed to antifungal activities were found. One part was

lactic acid, which was the main metabolite of lactic acid

bacteria. Large amount of lactic acid can lower the pH and

create an unsuitable growth condition for the fungi.

Organic acids can only penetrate the microbial cell wall in

their undissociated form, The pKa of lactic acid, acetic

acid, 3-phenyl-lactic acid, and caproic acid is 3.8, 4.7, 3.5,

and 4.9, respectively [3]. There was 36 % antifungal

activity loss when the pH ranged from 3.0 to 5.0. This

suggested that organic acid were partly responsible for the

antifungal activities. The other part was an admixture. It

contained three potential antifungal compounds: cyclo-

(Leu-Pro), 2,6-diphenyl-piperidine and 5,10-diethoxy-

2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;10,20-d]pyrazine.

The purification was quite difficult because the con-

centration of the compounds was low and it was hard for

the column to separate them accurately. We need a further

research to confirm the exact antifungal compounds.

Until now, five kinds of cyclic dipeptides with anti-

fungal activities have been isolated from LAB. These

include cyclo(Gly-Leu) [12], cyclo(Phe-Pro) [1], cyclo

(Phe-OH-Pro) [16], cyclo(Leu-Pro) [1], and cyclo(Leu–

Leu) [18]. Yan et al. [17] first reported the isolation of

cyclo (L-Leu-LPro) from the culture medium of Achro-

mobacter xylosoxidans. It was found that low concentra-

tions of this cyclic dipeptide are responsible for inhibition

of aflatoxin production, although higher concentration is

needed to inhibit the growth of Aspergillus parasiticus.

Dalbello et al. [1] first reported the isolation of cyclo

(L-Leu-LPro) from Lactobacillus plantarum. To our

knowledge, this is the first study reporting the isolation of

cyclo (L-Leu-L-Pro) from L. casei and its antifungal activity

of this cyclo dipeptide. In this study, 2,6-diphenyl-piperi-

dine and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyr-

rolo[1,2-a;10,20-d]pyrazine were detected for the first time

in the culture of LAB with antifungal activities.

Acknowledgments This research was supported by the National

Eleventh Five-Year Project of China: Dairy science and technology

support projects (Grant no. 2006BAD04A07).

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