# Antifungal Activity ofLactobacillus sp. Bacteria in the Presence of Xylitol and Galactosyl-Xylitol

Authors: Lidia Lipińska; Robert Klewicki; Elżbieta Klewicka; Krzysztof Kołodziejczyk; Michał Sójka; Adriana Nowak
Journal: BioMed Research International
 (2016)

---

## Abstract

Lactic acid fermentation is a natural method of antimicrobial food protection. Antagonistic activity ofLactobacillus sp. bacteria, taking part in this process, is directed mainly against the same or other microorganisms. In this work we determine the impact of the presence of xylitol and galactosyl-xylitol on the antagonistic activity of 60Lactobacillus sp. strains against indicator molds (Alternaria alternata,Alternaria brassicicola,Aspergillus niger,Fusarium latenicum,Geotrichum candidum, andMucor hiemalis) and yeasts (Candida vini). We used double-layer method to select antifungal strains ofLactobacillus bacteria and poisoned medium method to confirm their fungistatic properties. Additionally, we examined the inhibition ofAlternaria brassicicola byLactobacillus paracasei ŁOCK 0921 cultivated with xylitol or galactosyl-xylitol directly on wild cherries. The presence of xylitol and its galactosyl derivative led to increase of spectrum of antifungal activity in most of the studied plant-associated lactobacilli strains. However, no single strain exhibited activity against all the indicator microorganisms. The antifungal activity ofLactobacillus bacteria against molds varied considerably and depended on both the indicator strain and the composition of the medium. The presence of xylitol and galactosyl-xylitol in the growth medium is correlated with the antifungal activity of the studiedLactobacillus sp. bacteria against selected indicator molds.


---

## Body

## 1. Introduction

Lactic acid fermentation constitutes one of the oldest methods of protecting food from undesirable microflora. However, lactic acid bacteria (LAB) generate antimicrobial compounds inhibiting the growth of related species and show low antagonistic activity towards fungi, the most widespread food spoilage factors. To improve the antifungal effect ofLactobacillus sp., genus belonging to LAB, researchers modify their growth medium to stimulate metabolism towards production of antifungal compounds [1–3].Our preliminary studies confirmed that polyols (glycerol, lactitol, erythritol, sorbitol, and mannitol) may enhance the antifungal activity of LAB [2]. Furthermore, some polyols such as sorbitol, xylitol, erythritol, and lactitol are applied in the food industry and benefit human health [4]. An example of such compound is xylitol, a five-carbon polyol [5], which has a GRAS status (Generally Recognized as Safe) and is applied in pharmaceutical and food industry as an antidiabetic substitute for sucrose [6]. As a low-calorie compound, xylitol is added to sweets [7–9], chewing gum, toothpaste, and dental sealants due to its anticaries properties [10–13]. However, the effect of xylitol and its galactosyl derivative on antifungal properties of LAB has not been studied in detail. Besides, galactosyl-xylitol represents a modern prebiotic and can improve condition of gastrointestinal track.The objective of our study was to determine the antagonistic activity of 60Lactobacillussp. strains in the presence of xylitol and its galactosyl derivative (galactosyl-xylitol) against selected food-contaminating fungi.


## 2. Materials and Methods

### 2.1. Bacteria of the GenusLactobacillusand Indicator Fungal Strains

The study material consisted of 60 bacterial (Lactobacillussp.) and 8 fungal strains deposited with the Pure Cultures Collection of Industrial Microorganisms of the Institute of Fermentation Technology and Microbiology, Lodz University of Technology (ŁOCK 105).ExaminedLactobacillussp. strains were divided into four groups depending on the species (L. acidophilusstrains andL. casei/paracaseistrains) and origin (plant strains and strains isolated from humans). First group consisted of 20 strains ofL. acidophilus designated as 7, 0840, 0842, 0926–0939, and 0941–0943; second one consisted of 23 strains ofL. casei/paracasei:L. casei 0848, 0901–0907, 0909–0911, and 0919, Paris, NCDO206, andL. paracasei 0912, 0913, 0917, 0918, 0920–0922, 0924, and 0985; third group consisted of 13 plant-associated strains:L. plantarum0981, 0982, 0989, and 0990,L. pentosus0979 and 0991,L. brevis0944, 0980, 0983, and 0984,L. helveticus,L. delbrueckii0854, andL. casei1020; and the last one consisted of 4 strains isolated from humans:L. casei0919,L. delbrueckii0987,L. mucosae 0988, andL. rhamnosus0908.Bacteria.L. acidophilus,L. casei,L. helveticus,L. paracasei, andL. rhamnosus were incubated at 37°C under aerobic conditions, the strains isolated from humans at 37°C in the presence of CO2, and the plant-associated strains at 30°C under aerobic conditions. All bacteria were stored at −20°C in 20% (v/v) glycerol.The Yeasts Candida vini 0008 and 0009 and the Molds.Mucor hiemalis 0519,Geotrichum candidum 0511,Alternaria alternata 0409,Alternaria brassicicola 0412,Aspergillus niger 0433, andFusarium latenicum 0508 constituted the indicator microorganisms. They were kept at 4°C on Sabouraud 4%-dextrose agar slants (Merck).


### 2.2. Synthesis of the Galactosyl Derivative of Xylitol

The galactosyl derivative of xylitol was obtained by enzymatic transglycosylation usingβ-galactosidase EC 3.2.1.23 fromKluyveromyces lactis (Novozymes A/S, Bagsvaerd, Denmark). The procedure for the synthesis of galactosyl-xylitol was described by Klewicki [14].


### 2.3. Selection of AntifungalLactobacillusStrains in the Presence of Xylitol and Galactosyl-Xylitol

The antagonistic activity ofLactobacillusbacteria against indicator fungi was tested by the double-layer method. 24-hour cultures of a givenLactobacillus sp. strain were drop plated (with droplets of 10 μL) on MRS agar medium (Merck or BTL) with or without 10 g of xylitol, galactosyl-xylitol, or galactose per liter. The control group consisted of MRS agar plates (Merck) without LAB cultures. After 18–24 hours the plates were overlaid with Sabouraud 4%-dextrose agar (Merck) inoculated with an indicator fungal strain (105–106 spores mL−1). We measured inhibition zones of the indicator strain around colonies ofLactobacillussp. after 24–72 hours (30°C, aerobic conditions). The results were given as fungal inhibition diameters minus the diameter ofLactobacillus sp. colonies.


### 2.4. Antifungal Activity Assay on Wild Cherries (Prunus avium)

Antifungal effect of sterile supernatants after 30 hours of lactic acid fermentation ofLactobacillus paracaseiŁOCK 0921 in the presence of xylitol or galactosyl-xylitol againstAlternaria brassicicolawas estimated. Examined bacterial strain was chosen based on the results of screening of antifungal lactobacilli. Indicator microorganisms of the genusAlternariawere chosen because they are frequently isolated from wild cherries [15] and they can produce mycotoxins [16]. Wild cherries (average weight 4.8–5.3 g) were washed and soaked in sterile distilled water and left to dry for 30 minutes under sterile conditions (laminar flow chamber BioHazard type II). The fruits (wild cherries) were treated withA. brassicicola (suspension of conidia in 0.85% NaCl, 1
∗
10
5 cells·mL−1) by dissecting needle (0.5 mm in diameter, one puncture per fruit). Then, cell-free supernatant (10 μL, 20 μL, and 100 μL) ofL. paracaseiŁOCK 0921, cultivated with 1% (w/v) xylitol or galactosyl-xylitol (48 hours, 37°C), was dropped in infected fruit. Corresponding experiments were conducted with cell-free supernatant ofL. paracaseiŁOCK 0921 in the growing medium with glucose only, followed by the medium with galactose alone. The mold-inoculated wild cherries were incubated for 10 days at 23.4
±
0.2
°
C. Incubation time has been prolonged on the grounds of using temperature lower than in other studies, typical for storing wild cherries, and to check the appearance of the possible secondary growth of indicator molds.


### 2.5. Antifungal Effect of Postcultivation Supernatant ofLactobacillussp. Poisoned Media Method

We modified poisoned media method described by Manici et al. [17]. MoldAlternaria brassicicola was cultivated on Petri plates with Sabouraud 4%-dextrose agar (Merck) supplemented with 10, 20, 30, 50, and 70% (v/v) cell-free supernatant obtained after 30 hours of lactic acid fermentation ofLactobacillus pentosusŁOCK 0979, strain with antifungal activity towards most indicator fungi, orLactobacillus paracaseiŁOCK 0921, used in the study on wild cherries and shown in Figure 2, with 1% (w/v) xylitol or galactosyl-xylitol. Molds were incubated for 8 days (30°C, aerobic conditions). We measured the diameter of their colonies every day and estimated linear growth index using the following formula:(1)
T
=
A
D
+
b
1
d
1
+
⋯
+
b
x
d
x
,where T is linear growth index; A is diameter of fungal colony [mm]; D is time [days]; b
1
,
…
,
b
x are increase of fungal colony [mm]; d
1
,
…
,
d
x are time between measurements.The fungistatic activity of cell-free supernatant was estimated according to Abbot’s formula [18, 19]:(2)
I
=
K
-
A
K
∗
100
%
,where I is inhibition/stimulation rate according to Abbot’s formula; K is diameter of fungal colony on control plate; A is diameter of fungal colony on experimental plate.


### 2.6. Statistical Analysis

The tests were done in triplicate. The experimental data is expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and the Bonferronipost hoc test (p
≤
0.05) were applied to find differences between groups.


## 2.1. Bacteria of the GenusLactobacillusand Indicator Fungal Strains

The study material consisted of 60 bacterial (Lactobacillussp.) and 8 fungal strains deposited with the Pure Cultures Collection of Industrial Microorganisms of the Institute of Fermentation Technology and Microbiology, Lodz University of Technology (ŁOCK 105).ExaminedLactobacillussp. strains were divided into four groups depending on the species (L. acidophilusstrains andL. casei/paracaseistrains) and origin (plant strains and strains isolated from humans). First group consisted of 20 strains ofL. acidophilus designated as 7, 0840, 0842, 0926–0939, and 0941–0943; second one consisted of 23 strains ofL. casei/paracasei:L. casei 0848, 0901–0907, 0909–0911, and 0919, Paris, NCDO206, andL. paracasei 0912, 0913, 0917, 0918, 0920–0922, 0924, and 0985; third group consisted of 13 plant-associated strains:L. plantarum0981, 0982, 0989, and 0990,L. pentosus0979 and 0991,L. brevis0944, 0980, 0983, and 0984,L. helveticus,L. delbrueckii0854, andL. casei1020; and the last one consisted of 4 strains isolated from humans:L. casei0919,L. delbrueckii0987,L. mucosae 0988, andL. rhamnosus0908.Bacteria.L. acidophilus,L. casei,L. helveticus,L. paracasei, andL. rhamnosus were incubated at 37°C under aerobic conditions, the strains isolated from humans at 37°C in the presence of CO2, and the plant-associated strains at 30°C under aerobic conditions. All bacteria were stored at −20°C in 20% (v/v) glycerol.The Yeasts Candida vini 0008 and 0009 and the Molds.Mucor hiemalis 0519,Geotrichum candidum 0511,Alternaria alternata 0409,Alternaria brassicicola 0412,Aspergillus niger 0433, andFusarium latenicum 0508 constituted the indicator microorganisms. They were kept at 4°C on Sabouraud 4%-dextrose agar slants (Merck).


## 2.2. Synthesis of the Galactosyl Derivative of Xylitol

The galactosyl derivative of xylitol was obtained by enzymatic transglycosylation usingβ-galactosidase EC 3.2.1.23 fromKluyveromyces lactis (Novozymes A/S, Bagsvaerd, Denmark). The procedure for the synthesis of galactosyl-xylitol was described by Klewicki [14].


## 2.3. Selection of AntifungalLactobacillusStrains in the Presence of Xylitol and Galactosyl-Xylitol

The antagonistic activity ofLactobacillusbacteria against indicator fungi was tested by the double-layer method. 24-hour cultures of a givenLactobacillus sp. strain were drop plated (with droplets of 10 μL) on MRS agar medium (Merck or BTL) with or without 10 g of xylitol, galactosyl-xylitol, or galactose per liter. The control group consisted of MRS agar plates (Merck) without LAB cultures. After 18–24 hours the plates were overlaid with Sabouraud 4%-dextrose agar (Merck) inoculated with an indicator fungal strain (105–106 spores mL−1). We measured inhibition zones of the indicator strain around colonies ofLactobacillussp. after 24–72 hours (30°C, aerobic conditions). The results were given as fungal inhibition diameters minus the diameter ofLactobacillus sp. colonies.


## 2.4. Antifungal Activity Assay on Wild Cherries (Prunus avium)

Antifungal effect of sterile supernatants after 30 hours of lactic acid fermentation ofLactobacillus paracaseiŁOCK 0921 in the presence of xylitol or galactosyl-xylitol againstAlternaria brassicicolawas estimated. Examined bacterial strain was chosen based on the results of screening of antifungal lactobacilli. Indicator microorganisms of the genusAlternariawere chosen because they are frequently isolated from wild cherries [15] and they can produce mycotoxins [16]. Wild cherries (average weight 4.8–5.3 g) were washed and soaked in sterile distilled water and left to dry for 30 minutes under sterile conditions (laminar flow chamber BioHazard type II). The fruits (wild cherries) were treated withA. brassicicola (suspension of conidia in 0.85% NaCl, 1
∗
10
5 cells·mL−1) by dissecting needle (0.5 mm in diameter, one puncture per fruit). Then, cell-free supernatant (10 μL, 20 μL, and 100 μL) ofL. paracaseiŁOCK 0921, cultivated with 1% (w/v) xylitol or galactosyl-xylitol (48 hours, 37°C), was dropped in infected fruit. Corresponding experiments were conducted with cell-free supernatant ofL. paracaseiŁOCK 0921 in the growing medium with glucose only, followed by the medium with galactose alone. The mold-inoculated wild cherries were incubated for 10 days at 23.4
±
0.2
°
C. Incubation time has been prolonged on the grounds of using temperature lower than in other studies, typical for storing wild cherries, and to check the appearance of the possible secondary growth of indicator molds.


## 2.5. Antifungal Effect of Postcultivation Supernatant ofLactobacillussp. Poisoned Media Method

We modified poisoned media method described by Manici et al. [17]. MoldAlternaria brassicicola was cultivated on Petri plates with Sabouraud 4%-dextrose agar (Merck) supplemented with 10, 20, 30, 50, and 70% (v/v) cell-free supernatant obtained after 30 hours of lactic acid fermentation ofLactobacillus pentosusŁOCK 0979, strain with antifungal activity towards most indicator fungi, orLactobacillus paracaseiŁOCK 0921, used in the study on wild cherries and shown in Figure 2, with 1% (w/v) xylitol or galactosyl-xylitol. Molds were incubated for 8 days (30°C, aerobic conditions). We measured the diameter of their colonies every day and estimated linear growth index using the following formula:(1)
T
=
A
D
+
b
1
d
1
+
⋯
+
b
x
d
x
,where T is linear growth index; A is diameter of fungal colony [mm]; D is time [days]; b
1
,
…
,
b
x are increase of fungal colony [mm]; d
1
,
…
,
d
x are time between measurements.The fungistatic activity of cell-free supernatant was estimated according to Abbot’s formula [18, 19]:(2)
I
=
K
-
A
K
∗
100
%
,where I is inhibition/stimulation rate according to Abbot’s formula; K is diameter of fungal colony on control plate; A is diameter of fungal colony on experimental plate.


## 2.6. Statistical Analysis

The tests were done in triplicate. The experimental data is expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and the Bonferronipost hoc test (p
≤
0.05) were applied to find differences between groups.


## 3. Results and Discussion

### 3.1. Screening of AntifungalLactobacillussp. Strains

Antifungal activity of examined bacterial strains depends on growth medium and fungal indicator strain. None of the testedLactobacillussp. strains appreciably inhibited growth of yeastsCandida vini. We observed slight inhibition (1.0
±
0.0
m
m) of indicator yeasts by three strains of lactobacilli:L. mucosae0988,L. delbrueckii0987, both isolated from human, and plant-associatedL. pentosus0979.Lactobacillussp. bacteria poorly suppressed three out of six testing mold strains. Growth of the most resistant mold,Geotrichum candidum 0511, was inhibited only in the presence of xylitol in the range from 1.3 to 3.1 mm (Figure 1(c)). Origin of lactobacilli strains had no significant impact on their antagonistic activity againstG. candidum. Growth inhibition ofA. niger(Figure 1(a)) andM. hiemalis(Figure 1(e)) was moderate. Plant-associated bacterial strains showed the strongest antagonistic effect againstAspergillus niger(0.9–6.3 mm of the inhibition zone diameter). Growth of this mold was not suppressed in the sample containing galactose in MRS agar (BTL, without glucose). MoldM. hiemalis was the most inhibited in the control sample and in the presence of xylitol (especially by the plant-associated strains) and galactosyl-xylitol (by both plant-associated and human strains) and the least inhibited in the presence of galactose (Figure 1(e)). MoldsA. alternata(Figure 1(b)),A. brassicicola(Figure 1(d)), andF. latenicum (Figure 1(f)) exhibited high sensitivity toLactobacillus sp. bacteria in the control samples. However, the presence of xylitol and galactosyl-xylitol increased their susceptibility to products of lactic acid fermentation. The growth ofA. brassicicolawas completely inhibited in the presence of galactosyl-xylitol.Figure 1
Inhibition zones of indicator fungal strains caused byLactobacillus sp. in the presence of xylitol, galactosyl-xylitol, and galactose.
(a)
Aspergillus niger 0433
(b)
Alternaria alternata 0409
(c)
Geotrichum candidum 0511
(d)
Alternaria brassicicola 0412
(e)
Mucor hiemalis 0519
(f)
Fusarium latenicum 0508Figure 2
Antagonistic activity of selectedLactobacillus bacteria strains againstA. brassicicola in (a) the control sample without xylitol; (b) the control sample with only xylitol; (c)Lactobacillussp. without xylitol; (d)Lactobacillussp. with xylitol; (e)Lactobacillussp. and galactosyl-xylitol; (f)Lactobacillussp. and galactose with glucose; (g)Lactobacillussp. and galactose without glucose. LAB strains: “172”L. acidophilus0926, “62”L. paracasei0921, “–”L. paracasei0917, and “291”L. casei0904.
(a)
(b)
(c)
(d)
(e)
(f)
(g)Among the studied fungal indicators, theA. alternata was the most susceptible to LAB metabolites. In turn, in the presence of galactose that strain was only slightly inhibited, mostly by the plant-associatedLactobacillusstrains. In the case of the antagonism ofL. acidophilus andL. casei/paracaseiagainst the moldA.brassicicola, the inhibition zones increased in the presence of galactose, but not as much as in the presence of galactosyl-xylitol (Figure 1(d)). In this study,Lactobacillusbacteria isolated from plant and human sources have shown the strongest average antifungal activity against indicator molds exceptG. candidum. Crowley et al. [20] described similar correlations between lactobacilli origin and their antifungal effect. They screened 70 samples isolated from various sources, isolated LAB, and observed the strongest antifungal effect ofLactobacillus sp. obtained from plants and humans. Strong correlation between origin of lactobacilli and their antagonistic activity against fungi was not found in other studies. In our review article [21] current knowledge about antifungal activity of lactobacilli is presented. ManyLactobacillusspecies have antifungal activity against proper mold or even yeast; however this activity is usually poor or moderate. Lactobacilli species, which are best described as producers of antifungal compounds, areL. acidophilus,L. brevis,L. casei,L. fermentum,L. plantarum,L. reuteri,L. rhamnosus, andL. sakei.The primary goal of our study was to collate the antifungal activity of lactobacilli in different medium. We observed strong correlation between the presence of xylitol and galactosyl-xylitol and the antagonistic activity of LAB isolated from different sources. Figure2 presents the significantly disparate results of inhibition zones ofA. brassicicola aroundLactobacillus sp. colonies cultivated in the medium containing xylitol or galactosyl-xylitol and various carbon sources: glucose or galactose.Table1 shows the antagonistic activity of selectedLactobacillus sp. strains against the indicator fungi on all the tested media, MRS agar with glucose, galactose, both glucose and galactose, xylitol, or galactosyl-xylitol. We selectedLactobacillus strains with high inhibitory activity against a given indicator fungal strain. The bacterial strain with the widest spectrum of antifungal activity wasL. acidophilus0927, strongly affectingA. alternatain the presence of xylitol and galactosyl-xylitol and inhibitingA. brassicicolain all of the tests but affectingG. candidumonly in the presence of xylitol (inhibition zone of 11.0 mm). The moldsF. latenicumandM. hiemaliswere most inhibited byL. brevis 0980, while moldA. nigerwas inhibited byL. plantarum0982.Table 1
Inhibition zones of indicator fungal strains caused by selectedLactobacillus sp. strains which have the strongest antagonistic activity against proper mold.
Strains of LAB
LAB medium
Diameter of inhibition zone of indicator molds caused by selected LAB [mm] ± SD1
A. niger
A. alternata
A. brassicicola
F. latenicum
G. candidum
M. hiemalis
L. acidophilus0927
Control
0.5 ± 0.7a
ND
19.0 ± 1.4a
16.0 ± 1.4a
—
3.5 ± 0.7a
Xylitol
0.5 ± 0.7a
>90a
5.0 ± 1.4b
12.5 ± 0.7b
11.0 ± 0.0
6.0 ± 0.0b
Galactosyl-xylitol
2.0 ± 0.0b
>90a
>90c
>90c
—
3.0 ± 0.0a
Galactose + glucose
—
14.0 ± 0.0b
>90c
30.0 ± 0.7d
—
—
Galactose
—
14.0 ± 0.0b
>90c
14.0 ± 0.0a
b
—
—
L. acidophilus 0932
Control
—
—
8.0 ± 2.5a
18.0 ± 2.8a
—
—
Xylitol
—
24.0 ± 0.0a
—
2.0 ± 0.0b
—
—
Galactosyl-xylitol
—
>90b
>90b
>90b
—
3.0 ± 0.0
Galactose + glucose
—
—
—
3.0 ± 0.0b
—
—
Galactose
—
—
—
—
—
—
L. acidophilus 0937
Control
—
10.0 ± 0.0a
19.0 ± 4.2a
1.0 ± 0.0a
—
—
Xylitol
8.0 ± 2.8
30.0 ± 0.0b
NDb
11.0 ± 1.4b
—
2.0 ± 0.0
Galactosyl-xylitol
—
—
>90c
—
—
—
Galactose + glucose
—
2.0 ± 0.0c
—
0.5 ± 0.0a
—
—
Galactose
—
—
—
—
—
—
L. brevis0944
Control
—
25.0 ± 4.2a
1.5 ± 0.7a
5.0 ± 1.4a
—
4.0 ± 1.4a
Xylitol
8.0 ± 0.0a
24.0 ± 0.0a
ND
18.0 ± 0.0b
15.5 ± 0.0a
7.5 ± 2.1a
Galactosyl-xylitol
4.0 ± 0.0b
>90b
>90b
>90c
—
6.0 ± 0.0a
Galactose + glucose
—
13.0 ± 0.0c
>90b
>90c
—
2.0 ± 0.0b
Galactose
—
11.0 ± 0.0d
4.0 ± 0.0c
11.0 ± 0.0d
—
2.0 ± 0.0b
L. brevis0980
Control
—
ND
4.5 ± 0.7a
10.5 ± 0.7a
8.0 ± 0.0
8.0 ± 0.0a
Xylitol
18.5 ± 2.1a
>90a
6.5 ± 0.7a
15.5 ± 0.7b
—
15.0 ± 7.2a
b
Galactosyl-xylitol
3.0 ± 0.0b
>90a
>90b
>90c
—
9.0 ± 0.0b
Galactose + glucose
—
19.0 ± 0.0b
18.0 ± 0.0c
>90c
—
5.0 ± 0.0a
c
Galactose
—
18.0 ± 0.0c
20.0 ± 0.0d
19.0 ± 0.0d
—
4.0 ± 0.0a
c
L. casei1020
Control
—
15.0 ± 0.0a
—
16.0 ± 0.0a
—
—
Xylitol
10.0 ± 0.0a
16.0 ± 0.0b
ND
12.0 ± 1.4b
8.0 ± 0.0
8.5 ± 0.7a
Galactosyl-xylitol
1.0 ± 0.0b
>90c
>90a
>90c
—
7.0 ± 0.0a
Galactose + glucose
—
14.0 ± 0.0d
>90a
>90c
—
—
Galactose
—
12.0 ± 0.0e
10.0 ± 0.0b
16.0 ± 0.0a
—
—
L. paracasei 0921
Control
—
11.0 ± 1.4a
—
10.5 ± 0.7a
—
—
Xylitol
—
20.0 ± 0.0b
>90a
2.0 ± 0.0b
—
—
Galactosyl-xylitol
—
>90c
>90a
7.0 ± 0.0c
—
—
Galactose + glucose
—
5.0 ± 0.0d
2.0 ± 0.0b
8.0 ± 0.0d
—
—
Galactose
—
3.0 ± 0.0e
8.0 ± 0.0c
2.0 ± 0.0b
—
—
L. pentosus0979
Control
—
32.5 ± 3.5a
—
8.0 ± 2.8a
4.0 ± 0.0
9.0 ± 1.4a
Xylitol
15.0 ± 0.0a
>90b
15.0 ± 0.0a
10.0 ± 0.0a
—
—
Galactosyl-xylitol
2.0 ± 0.0b
>90b
>90b
>90b
—
9.0 ± 0.0a
Galactose + glucose
—
21.0 ± 0.0c
30.0 ± 0.7c
>90b
—
4.0 ± 0.0b
Galactose
—
18.0 ± 0.0d
16.0 ± 0.0d
15.0 ± 0.0c
—
3.0 ± 0.0c
L. plantarum0982
Control
6.5 ± 2.1a
28.0 ± 0.0a
ND
5.0 ± 0.0a
—
3.0 ± 0.0a
Xylitol
5.0 ± 1.4a
17.5 ± 0.7b
>90a
9.0 ± 0.0b
—
1.0 ± 0.0b
Galactosyl-xylitol
>90b
>90c
>90a
>90c
—
4.0 ± 0.0c
Galactose + glucose
—
18.0 ± 0.0b
>90a
28.0 ± 0.0d
—
—
Galactose
—
9.0 ± 0.0d
>90a
7.0 ± 0.0e
—
—
SD1: standard deviation.
>90: total inhibition of indicator strain.
a
,
b
,
c
,
d
,
eStatistically significant difference, p
<
0.05.


### 3.2. Antifungal Activity Assay ofLactobacillussp. Supernatants on Wild Cherries

We observed inhibition ofAlternaria brassicicolaon wild cherries treated with cell-free supernatant after fermentation ofLactobacillus paracaseiŁOCK 0921 in the presence of xylitol. The presence of galactosyl-xylitol also affected antifungal activity of tested bacterial strain but volume of 10 μL of cell-free supernatant was insufficient to inhibit the growth ofA. brassicicola. However, examined mold was totally inhibited with 20 and 100 μL, while the volume of 10 μL was too low (Figure 3). After the fermentation ofLactobacillus paracasei ŁOCK 0921 in the presence of glucose (Figure 3(c)) or galactose (Figure 3(d)) the cell-free supernatant has not shown antifungal activity againstA. brassicicola at any volume; Figures 3(c) and 3(d) present the highest volume (100 μL) of cell-free supernatant.Figure 3
Inhibition ofAlternata brassicicolaon wild cherries after 10 days of incubation with 10, 20, or 100 μL cell-free supernatant after lactic acid fermentation byLactobacillus paracaseiŁOCK 0921 in the presence of glucose (c), galactose (d), glucose + xylitol (e), or glucose + galactosyl-xylitol (f); (a) negative control (without molds); (b) positive control (infected by molds).
(a)
(b)
(c)
(d)
(e)
(f)


### 3.3. Fungistatic Effect of Cell-Free Supernatant after Lactic Acid Fermentation with Xylitol or Galactosyl-Xylitol

We cultivatedAlternaria brassicicola on Petri dishes with 10, 20, 30, 50, or 70% (v/v) of cell-free supernatant after lactic acid fermentation ofLactobacillus pentosus0979 andLactobacillus paracasei0921 in the presence of xylitol or galactosyl-xylitol. We have measured mold colony diameters for 8 days and estimated linear growth index (T) and inhibition/stimulation rate (I) according to Abbot’s formula (Table 2).Table 2
Linear growth index and inhibition/stimulation rate according to Abbot’s formula of cell-free supernatant after lactic acid fermentation ofLactobacillus pentosus0979 and Lactobacillus paracasei0921 towards Alternaria brassicicola.
LAB
Polyol/gal-polyol
Estimated value∗
Content of cell-free supernatant of LAB (v/v)
0%
10%
20%
30%
50%
70%
L. pentosus 0979
Xylitol
T
14.3
0
0
0
0
0
I [%]
—
100
100
100
100
100
Galactosyl-xylitol
T
26.6
0
0
0
0
0
I [%]
—
100
100
100
100
100
L. paracasei 0921
Xylitol
T
6.4
5.6
1.8
0
0
0
I [%]
—
12.5
71.9
100
100
100
Galactosyl-xylitol
T
6.8
0
0
0
0
0
I [%]
—
100
100
100
100
100
⁢T
∗: linear growth index [—]; I: inhibition/stimulation rate according to Abbot’s formula [%].In the presence of xylitol and galactosyl-xylitol the cell-free supernatant ofL. pentosus0979 which was placed in fungal medium has shown absolute inhibition ofA. brassicicola in all tested cases.L. paracasei0921, mentioned in study on wild cherries and in Figure 2, demonstrated similar effect in the presence of galactosyl-xylitol, but only moderate fungistatic effect cultivated in the presence of xylitol (10% and 20% of supplementation).The antifungal activity ofLactobacillusbacteria depends on the growth medium and is distinctive for all studied fungal species [22]. Klewicka and Klewicki [3] investigated the growth and metabolism of lactic acid bacteria in the presence of some polyols and their galactosyl derivatives. They found that LAB grew poorly in the presence of xylitol; meanwhile in the presence of galactosyl-xylitol their growth was comparable to the controls. This implies that the studied LAB efficiently use galactosyl-polyols to hydrolyze galactosyl bonds enzymatically and to cleave the galactose molecule. In the case of galactosyl-polyol fermentation, galactose is the first saccharide metabolized by the tested bacteria. The amounts of lactic and acetic acids synthesized by lactobacilli in the presence of galactosyl-xylitol were similar to or lower than those observed in the glucose-containing control samples [3]. It may therefore be inferred that the antagonistic effect ofLactobacillus sp. bacteria in the presence of xylitol and galactosyl-xylitol results from xylitol metabolism. It may also be assumed that in the presence of polyols bacterial metabolism is directed towards effective synthesis of some secondary metabolites with antifungal properties.Antifungal activity is linked to the synthesis of specific metabolites including low molecular weight peptides, such as reuterin isolated fromLactobacillus reuteri (MW 148 Da) [23], 3-hydroxy fatty acids [24], phenyllactic acid [25], and cyclic dipeptides [26]. Our preliminary studies suggest that antifungal effect of tested lactobacilli is probably correlated with acids production, especially phenyllactic acid, or with synergistic effect of acids and other antifungal compounds [data not shown]. Almståhl et al. [27] examined the metabolism of polyols, including xylitol, by selected bacterial strains. They isolatedLactobacillussp. strains,L. fermentum,L. casei,L. rhamnosus,L. salivarius,L. acidophilus, andL. gasseri, as well as some unidentified strains, from the human oral cavity. Subsequently, they studied the ability of bacterial strains to ferment sugars (glucose, fructose, and sucrose) and polyols (mannitol, sorbitol, and xylitol). It was found that the bacterial strains grew more vigorously in media containing saccharides than in those containing polyols; also following lactic acid fermentation pH was lower in the former than in the latter [27]. Sugar fermentation led to lower pH as if during that process bacteria generated more lactic acid and other organic acids than during polyol fermentation. A similar correlation between the presence of polyols in the growth media and the metabolism of LAB was observed by Magnusson [26], who reported increased antifungal activity ofLactobacillus coryniformisin the presence of glycerol in the growth medium.In our hypothesis, the use of xylitol or galactosyl-xylitol, instead of chemical preservatives, can be an alternative method to inhibit the growth of fungi in fermented food. Elucidation of the mechanism antagonistic activity ofLactobacillussp. in the presence of xylitol and its galactosyl derivative is a promising avenue of research. Therefore, in the further studies we will focus our attention on the identification of specific lactic acid fermentation products with antifungal properties generated in the presence of polyols and their galactosyl derivatives.


## 3.1. Screening of AntifungalLactobacillussp. Strains

Antifungal activity of examined bacterial strains depends on growth medium and fungal indicator strain. None of the testedLactobacillussp. strains appreciably inhibited growth of yeastsCandida vini. We observed slight inhibition (1.0
±
0.0
m
m) of indicator yeasts by three strains of lactobacilli:L. mucosae0988,L. delbrueckii0987, both isolated from human, and plant-associatedL. pentosus0979.Lactobacillussp. bacteria poorly suppressed three out of six testing mold strains. Growth of the most resistant mold,Geotrichum candidum 0511, was inhibited only in the presence of xylitol in the range from 1.3 to 3.1 mm (Figure 1(c)). Origin of lactobacilli strains had no significant impact on their antagonistic activity againstG. candidum. Growth inhibition ofA. niger(Figure 1(a)) andM. hiemalis(Figure 1(e)) was moderate. Plant-associated bacterial strains showed the strongest antagonistic effect againstAspergillus niger(0.9–6.3 mm of the inhibition zone diameter). Growth of this mold was not suppressed in the sample containing galactose in MRS agar (BTL, without glucose). MoldM. hiemalis was the most inhibited in the control sample and in the presence of xylitol (especially by the plant-associated strains) and galactosyl-xylitol (by both plant-associated and human strains) and the least inhibited in the presence of galactose (Figure 1(e)). MoldsA. alternata(Figure 1(b)),A. brassicicola(Figure 1(d)), andF. latenicum (Figure 1(f)) exhibited high sensitivity toLactobacillus sp. bacteria in the control samples. However, the presence of xylitol and galactosyl-xylitol increased their susceptibility to products of lactic acid fermentation. The growth ofA. brassicicolawas completely inhibited in the presence of galactosyl-xylitol.Figure 1
Inhibition zones of indicator fungal strains caused byLactobacillus sp. in the presence of xylitol, galactosyl-xylitol, and galactose.
(a)
Aspergillus niger 0433
(b)
Alternaria alternata 0409
(c)
Geotrichum candidum 0511
(d)
Alternaria brassicicola 0412
(e)
Mucor hiemalis 0519
(f)
Fusarium latenicum 0508Figure 2
Antagonistic activity of selectedLactobacillus bacteria strains againstA. brassicicola in (a) the control sample without xylitol; (b) the control sample with only xylitol; (c)Lactobacillussp. without xylitol; (d)Lactobacillussp. with xylitol; (e)Lactobacillussp. and galactosyl-xylitol; (f)Lactobacillussp. and galactose with glucose; (g)Lactobacillussp. and galactose without glucose. LAB strains: “172”L. acidophilus0926, “62”L. paracasei0921, “–”L. paracasei0917, and “291”L. casei0904.
(a)
(b)
(c)
(d)
(e)
(f)
(g)Among the studied fungal indicators, theA. alternata was the most susceptible to LAB metabolites. In turn, in the presence of galactose that strain was only slightly inhibited, mostly by the plant-associatedLactobacillusstrains. In the case of the antagonism ofL. acidophilus andL. casei/paracaseiagainst the moldA.brassicicola, the inhibition zones increased in the presence of galactose, but not as much as in the presence of galactosyl-xylitol (Figure 1(d)). In this study,Lactobacillusbacteria isolated from plant and human sources have shown the strongest average antifungal activity against indicator molds exceptG. candidum. Crowley et al. [20] described similar correlations between lactobacilli origin and their antifungal effect. They screened 70 samples isolated from various sources, isolated LAB, and observed the strongest antifungal effect ofLactobacillus sp. obtained from plants and humans. Strong correlation between origin of lactobacilli and their antagonistic activity against fungi was not found in other studies. In our review article [21] current knowledge about antifungal activity of lactobacilli is presented. ManyLactobacillusspecies have antifungal activity against proper mold or even yeast; however this activity is usually poor or moderate. Lactobacilli species, which are best described as producers of antifungal compounds, areL. acidophilus,L. brevis,L. casei,L. fermentum,L. plantarum,L. reuteri,L. rhamnosus, andL. sakei.The primary goal of our study was to collate the antifungal activity of lactobacilli in different medium. We observed strong correlation between the presence of xylitol and galactosyl-xylitol and the antagonistic activity of LAB isolated from different sources. Figure2 presents the significantly disparate results of inhibition zones ofA. brassicicola aroundLactobacillus sp. colonies cultivated in the medium containing xylitol or galactosyl-xylitol and various carbon sources: glucose or galactose.Table1 shows the antagonistic activity of selectedLactobacillus sp. strains against the indicator fungi on all the tested media, MRS agar with glucose, galactose, both glucose and galactose, xylitol, or galactosyl-xylitol. We selectedLactobacillus strains with high inhibitory activity against a given indicator fungal strain. The bacterial strain with the widest spectrum of antifungal activity wasL. acidophilus0927, strongly affectingA. alternatain the presence of xylitol and galactosyl-xylitol and inhibitingA. brassicicolain all of the tests but affectingG. candidumonly in the presence of xylitol (inhibition zone of 11.0 mm). The moldsF. latenicumandM. hiemaliswere most inhibited byL. brevis 0980, while moldA. nigerwas inhibited byL. plantarum0982.Table 1
Inhibition zones of indicator fungal strains caused by selectedLactobacillus sp. strains which have the strongest antagonistic activity against proper mold.
Strains of LAB
LAB medium
Diameter of inhibition zone of indicator molds caused by selected LAB [mm] ± SD1
A. niger
A. alternata
A. brassicicola
F. latenicum
G. candidum
M. hiemalis
L. acidophilus0927
Control
0.5 ± 0.7a
ND
19.0 ± 1.4a
16.0 ± 1.4a
—
3.5 ± 0.7a
Xylitol
0.5 ± 0.7a
>90a
5.0 ± 1.4b
12.5 ± 0.7b
11.0 ± 0.0
6.0 ± 0.0b
Galactosyl-xylitol
2.0 ± 0.0b
>90a
>90c
>90c
—
3.0 ± 0.0a
Galactose + glucose
—
14.0 ± 0.0b
>90c
30.0 ± 0.7d
—
—
Galactose
—
14.0 ± 0.0b
>90c
14.0 ± 0.0a
b
—
—
L. acidophilus 0932
Control
—
—
8.0 ± 2.5a
18.0 ± 2.8a
—
—
Xylitol
—
24.0 ± 0.0a
—
2.0 ± 0.0b
—
—
Galactosyl-xylitol
—
>90b
>90b
>90b
—
3.0 ± 0.0
Galactose + glucose
—
—
—
3.0 ± 0.0b
—
—
Galactose
—
—
—
—
—
—
L. acidophilus 0937
Control
—
10.0 ± 0.0a
19.0 ± 4.2a
1.0 ± 0.0a
—
—
Xylitol
8.0 ± 2.8
30.0 ± 0.0b
NDb
11.0 ± 1.4b
—
2.0 ± 0.0
Galactosyl-xylitol
—
—
>90c
—
—
—
Galactose + glucose
—
2.0 ± 0.0c
—
0.5 ± 0.0a
—
—
Galactose
—
—
—
—
—
—
L. brevis0944
Control
—
25.0 ± 4.2a
1.5 ± 0.7a
5.0 ± 1.4a
—
4.0 ± 1.4a
Xylitol
8.0 ± 0.0a
24.0 ± 0.0a
ND
18.0 ± 0.0b
15.5 ± 0.0a
7.5 ± 2.1a
Galactosyl-xylitol
4.0 ± 0.0b
>90b
>90b
>90c
—
6.0 ± 0.0a
Galactose + glucose
—
13.0 ± 0.0c
>90b
>90c
—
2.0 ± 0.0b
Galactose
—
11.0 ± 0.0d
4.0 ± 0.0c
11.0 ± 0.0d
—
2.0 ± 0.0b
L. brevis0980
Control
—
ND
4.5 ± 0.7a
10.5 ± 0.7a
8.0 ± 0.0
8.0 ± 0.0a
Xylitol
18.5 ± 2.1a
>90a
6.5 ± 0.7a
15.5 ± 0.7b
—
15.0 ± 7.2a
b
Galactosyl-xylitol
3.0 ± 0.0b
>90a
>90b
>90c
—
9.0 ± 0.0b
Galactose + glucose
—
19.0 ± 0.0b
18.0 ± 0.0c
>90c
—
5.0 ± 0.0a
c
Galactose
—
18.0 ± 0.0c
20.0 ± 0.0d
19.0 ± 0.0d
—
4.0 ± 0.0a
c
L. casei1020
Control
—
15.0 ± 0.0a
—
16.0 ± 0.0a
—
—
Xylitol
10.0 ± 0.0a
16.0 ± 0.0b
ND
12.0 ± 1.4b
8.0 ± 0.0
8.5 ± 0.7a
Galactosyl-xylitol
1.0 ± 0.0b
>90c
>90a
>90c
—
7.0 ± 0.0a
Galactose + glucose
—
14.0 ± 0.0d
>90a
>90c
—
—
Galactose
—
12.0 ± 0.0e
10.0 ± 0.0b
16.0 ± 0.0a
—
—
L. paracasei 0921
Control
—
11.0 ± 1.4a
—
10.5 ± 0.7a
—
—
Xylitol
—
20.0 ± 0.0b
>90a
2.0 ± 0.0b
—
—
Galactosyl-xylitol
—
>90c
>90a
7.0 ± 0.0c
—
—
Galactose + glucose
—
5.0 ± 0.0d
2.0 ± 0.0b
8.0 ± 0.0d
—
—
Galactose
—
3.0 ± 0.0e
8.0 ± 0.0c
2.0 ± 0.0b
—
—
L. pentosus0979
Control
—
32.5 ± 3.5a
—
8.0 ± 2.8a
4.0 ± 0.0
9.0 ± 1.4a
Xylitol
15.0 ± 0.0a
>90b
15.0 ± 0.0a
10.0 ± 0.0a
—
—
Galactosyl-xylitol
2.0 ± 0.0b
>90b
>90b
>90b
—
9.0 ± 0.0a
Galactose + glucose
—
21.0 ± 0.0c
30.0 ± 0.7c
>90b
—
4.0 ± 0.0b
Galactose
—
18.0 ± 0.0d
16.0 ± 0.0d
15.0 ± 0.0c
—
3.0 ± 0.0c
L. plantarum0982
Control
6.5 ± 2.1a
28.0 ± 0.0a
ND
5.0 ± 0.0a
—
3.0 ± 0.0a
Xylitol
5.0 ± 1.4a
17.5 ± 0.7b
>90a
9.0 ± 0.0b
—
1.0 ± 0.0b
Galactosyl-xylitol
>90b
>90c
>90a
>90c
—
4.0 ± 0.0c
Galactose + glucose
—
18.0 ± 0.0b
>90a
28.0 ± 0.0d
—
—
Galactose
—
9.0 ± 0.0d
>90a
7.0 ± 0.0e
—
—
SD1: standard deviation.
>90: total inhibition of indicator strain.
a
,
b
,
c
,
d
,
eStatistically significant difference, p
<
0.05.


## 3.2. Antifungal Activity Assay ofLactobacillussp. Supernatants on Wild Cherries

We observed inhibition ofAlternaria brassicicolaon wild cherries treated with cell-free supernatant after fermentation ofLactobacillus paracaseiŁOCK 0921 in the presence of xylitol. The presence of galactosyl-xylitol also affected antifungal activity of tested bacterial strain but volume of 10 μL of cell-free supernatant was insufficient to inhibit the growth ofA. brassicicola. However, examined mold was totally inhibited with 20 and 100 μL, while the volume of 10 μL was too low (Figure 3). After the fermentation ofLactobacillus paracasei ŁOCK 0921 in the presence of glucose (Figure 3(c)) or galactose (Figure 3(d)) the cell-free supernatant has not shown antifungal activity againstA. brassicicola at any volume; Figures 3(c) and 3(d) present the highest volume (100 μL) of cell-free supernatant.Figure 3
Inhibition ofAlternata brassicicolaon wild cherries after 10 days of incubation with 10, 20, or 100 μL cell-free supernatant after lactic acid fermentation byLactobacillus paracaseiŁOCK 0921 in the presence of glucose (c), galactose (d), glucose + xylitol (e), or glucose + galactosyl-xylitol (f); (a) negative control (without molds); (b) positive control (infected by molds).
(a)
(b)
(c)
(d)
(e)
(f)


## 3.3. Fungistatic Effect of Cell-Free Supernatant after Lactic Acid Fermentation with Xylitol or Galactosyl-Xylitol

We cultivatedAlternaria brassicicola on Petri dishes with 10, 20, 30, 50, or 70% (v/v) of cell-free supernatant after lactic acid fermentation ofLactobacillus pentosus0979 andLactobacillus paracasei0921 in the presence of xylitol or galactosyl-xylitol. We have measured mold colony diameters for 8 days and estimated linear growth index (T) and inhibition/stimulation rate (I) according to Abbot’s formula (Table 2).Table 2
Linear growth index and inhibition/stimulation rate according to Abbot’s formula of cell-free supernatant after lactic acid fermentation ofLactobacillus pentosus0979 and Lactobacillus paracasei0921 towards Alternaria brassicicola.
LAB
Polyol/gal-polyol
Estimated value∗
Content of cell-free supernatant of LAB (v/v)
0%
10%
20%
30%
50%
70%
L. pentosus 0979
Xylitol
T
14.3
0
0
0
0
0
I [%]
—
100
100
100
100
100
Galactosyl-xylitol
T
26.6
0
0
0
0
0
I [%]
—
100
100
100
100
100
L. paracasei 0921
Xylitol
T
6.4
5.6
1.8
0
0
0
I [%]
—
12.5
71.9
100
100
100
Galactosyl-xylitol
T
6.8
0
0
0
0
0
I [%]
—
100
100
100
100
100
⁢T
∗: linear growth index [—]; I: inhibition/stimulation rate according to Abbot’s formula [%].In the presence of xylitol and galactosyl-xylitol the cell-free supernatant ofL. pentosus0979 which was placed in fungal medium has shown absolute inhibition ofA. brassicicola in all tested cases.L. paracasei0921, mentioned in study on wild cherries and in Figure 2, demonstrated similar effect in the presence of galactosyl-xylitol, but only moderate fungistatic effect cultivated in the presence of xylitol (10% and 20% of supplementation).The antifungal activity ofLactobacillusbacteria depends on the growth medium and is distinctive for all studied fungal species [22]. Klewicka and Klewicki [3] investigated the growth and metabolism of lactic acid bacteria in the presence of some polyols and their galactosyl derivatives. They found that LAB grew poorly in the presence of xylitol; meanwhile in the presence of galactosyl-xylitol their growth was comparable to the controls. This implies that the studied LAB efficiently use galactosyl-polyols to hydrolyze galactosyl bonds enzymatically and to cleave the galactose molecule. In the case of galactosyl-polyol fermentation, galactose is the first saccharide metabolized by the tested bacteria. The amounts of lactic and acetic acids synthesized by lactobacilli in the presence of galactosyl-xylitol were similar to or lower than those observed in the glucose-containing control samples [3]. It may therefore be inferred that the antagonistic effect ofLactobacillus sp. bacteria in the presence of xylitol and galactosyl-xylitol results from xylitol metabolism. It may also be assumed that in the presence of polyols bacterial metabolism is directed towards effective synthesis of some secondary metabolites with antifungal properties.Antifungal activity is linked to the synthesis of specific metabolites including low molecular weight peptides, such as reuterin isolated fromLactobacillus reuteri (MW 148 Da) [23], 3-hydroxy fatty acids [24], phenyllactic acid [25], and cyclic dipeptides [26]. Our preliminary studies suggest that antifungal effect of tested lactobacilli is probably correlated with acids production, especially phenyllactic acid, or with synergistic effect of acids and other antifungal compounds [data not shown]. Almståhl et al. [27] examined the metabolism of polyols, including xylitol, by selected bacterial strains. They isolatedLactobacillussp. strains,L. fermentum,L. casei,L. rhamnosus,L. salivarius,L. acidophilus, andL. gasseri, as well as some unidentified strains, from the human oral cavity. Subsequently, they studied the ability of bacterial strains to ferment sugars (glucose, fructose, and sucrose) and polyols (mannitol, sorbitol, and xylitol). It was found that the bacterial strains grew more vigorously in media containing saccharides than in those containing polyols; also following lactic acid fermentation pH was lower in the former than in the latter [27]. Sugar fermentation led to lower pH as if during that process bacteria generated more lactic acid and other organic acids than during polyol fermentation. A similar correlation between the presence of polyols in the growth media and the metabolism of LAB was observed by Magnusson [26], who reported increased antifungal activity ofLactobacillus coryniformisin the presence of glycerol in the growth medium.In our hypothesis, the use of xylitol or galactosyl-xylitol, instead of chemical preservatives, can be an alternative method to inhibit the growth of fungi in fermented food. Elucidation of the mechanism antagonistic activity ofLactobacillussp. in the presence of xylitol and its galactosyl derivative is a promising avenue of research. Therefore, in the further studies we will focus our attention on the identification of specific lactic acid fermentation products with antifungal properties generated in the presence of polyols and their galactosyl derivatives.


## 4. Conclusions

Antagonistic activity of investigatedLactobacillussp. strains depends on both growth media and indicator strains of fungi. None of the bacterial strains demonstrated a wide spectrum of antagonistic activity against the indicator microorganisms, but some of them inhibited the growth of individual fungal strains in the presence of appropriate substrates. Moreover, molds are more susceptible to the presence of lactic acid bacteria and xylitol or galactosyl-xylitol than yeasts. Most of tested lactic acid bacteria have an antifungal effect only in the presence or xylitol or galactosyl-xylitol and have shown lower or no antifungal effect in the presence of glucose and galactose only. Antifungal activity assays of LAB supernatants on wild cherries and using poisoned media method confirmed that xylitol and galactosyl-xylitol can enhance antifungal properties of tested lactobacilli.



---

*Source: 5897486-2016-05-12.xml*