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Article

Inhibitory Mechanism of Lactic Acid Bacteria Cell-Free Supernatant Against Stored Grain Molds

by
Can Cui
,
Xiaopeng Fu
,
Tianjie Qi
,
Tianci Zou
,
Yijun Liu
,
Yanfei Li
,
Yan Zhao
and
Haoxin Lv
*
School of Food and Strategic Reserves, Henan University of Technology, Zhengzhou 450001, China
*
Author to whom correspondence should be addressed.
Foods 2026, 15(4), 655; https://doi.org/10.3390/foods15040655
Submission received: 22 January 2026 / Revised: 3 February 2026 / Accepted: 7 February 2026 / Published: 11 February 2026
(This article belongs to the Section Grain)
Browse Figures
Versions Notes

Abstract

Grain is highly vulnerable to contamination by fungi during storage, leading to reduced product quality and substantial economic losses. Lactic acid bacteria (LAB) have the potential to be used as antifungal agents; however, strains aimed at inhibiting stored grain molds remain limited, and the inhibitory mechanisms require further investigation. To solve this problem, 71 LAB strains were isolated from various samples. Among these, 17 strains exhibiting inhibitory activity against A. flavus, A. niger and P. citrinum were selected using a dual-layer plate assay. Based on morphological characterization and 16S rRNA gene sequence analysis, these strains were classified as Weissella cibaria, Pediococcus pentosaceus, and Lactiplantibacillus paraplantarum. Further investigations involving pH adjustment, catalase, and proteinase K treatments confirmed that organic acids were the primary antifungal substances in LAB cell-free supernatant (CFS). HPLC quantification identified acetic acid, malic acid, lactic acid, phenyllactic acid and citric acid contained in the CFS. Antifungal assays verified that acetic acid and lactic acid exhibited the strongest inhibitory effects against P. citrinum and A. flavus, whereas phenyllactic acid and acetic acid demonstrated the most potent suppression against A. niger. These findings established a theoretical basis for the application of LAB CFS in grain storage.

1. Introduction

Grain constitutes a fundamental cornerstone for maintaining social stability. It has been estimated that 25–35% of grains are lost after harvest annually, among which fungi infection accounts for 9–13% [1]. Stored grains are particularly susceptible to contamination by species of Aspergillus, Fusarium and Penicillium [2]. These molds not only degrade the quality of grains, but also synthesize mycotoxins, posing a significant threat to human and animal health [3]. According to FAO, around 25% of global grain production is contaminated with mycotoxin each year, reaching hundreds of billions of dollars worldwide [4]. Consequently, there is an urgent need to develop effective strategies to mitigate and prevent fungal contamination during grain storage.
To mitigate the impact of fungal contamination, diverse strategies have been developed. These encompass physical approaches such as mechanical ventilation, cold plasma technology, and electron beam irradiation [5,6,7]. However, physical techniques are limited by high equipment costs, substantial energy consumption, and insufficient technical validation. Chemical inhibition methods, including hydrogen sulfide fumigation and solid fungicides for example benzoic acid, sorbic acid, and sodium diacetate, have also been employed [8,9]. However, these chemical agents may adversely affect the sensory characteristics and nutritional quality of grains, pose negative environmental impacts, and increase the risk of cardiovascular diseases with excessive intake [10,11].
In recent years, the application of lactic acid bacteria (LAB) and their bioactive metabolites for preventing fungi contamination has gradually attracted attention due to their potent antifungal activity, favorable safety profile, cost-effectiveness, and broad-spectrum applicability. Krishnan et al. demonstrated that the cell-free supernatant (CFS) from Weissella confusa strains ML2 and BF2 effectively inhibited the germination of Fusarium verticillioides NCIM 1100 conidia, thereby preventing early-stage infection in wheat [12]. CFS from Lactiplantibacillus pentosus CW5 has been shown to serve a dual purpose, it effectively delays the softening and decay of kiwifruit in storage and concurrently reduces the population of postharvest pathogenic fungi [13]. The peptide mixture within the CFS of Lactobacillus plantarum TE10 exhibits significant antifungal activity, effectively inhibiting the growth of Aspergillus flavus on fresh corn and extending its shelf life [14]. Similarly, Møller et al. reported that Levilactobacillus spp. 3QB398 and Levilactobacillus brevis 2QB422 can inhibit the infection by Aspergillus parasiticus [15]. In another study, Cao et al. identified a strain of Lactobacillus plantarum AR524 that showed strong antagonistic activity against Fusarium graminearum [16].
The antifungal activity of LAB has been attributed to the production of bioactive compounds such as organic acids, hydrogen peroxide and proteinaceous antimicrobials. Organic acids could lower the environmental pH, disrupt fungal cell membrane integrity, and inhibit spore germination and mycelial growth [17,18]. Additionally, hydrogen peroxide exerts antifungal effects by attacking cell membranes and inducing oxidative stress [19]. While proteinaceous antimicrobials were able to inhibit fungal cell wall synthesis and interfere with key metabolic enzymes [20,21]. Although these mechanisms are well-documented in high-moisture substrates such as fruits and dairy products [22,23], their efficacy and dominance in the low-water-activity, complex environment of stored grains remain unclear.
In this study, LAB strains with antifungal activity were isolated from various environmental samples. Then the selected LAB strains were characterized morphologically and identified molecularly based on 16S rDNA sequencing. The antifungal activity of the CFS was assessed, and the primary bioactive substances were identified. Furthermore, the underlying antifungal mechanism of the LAB CFS was investigated, thereby establishing a theoretical foundation for developing biological preservatives for grain storage.

2. Materials and Methods

2.1. Materials

The LAB strains were isolated from various samples including soil, corn kernel, pickled cabbage and wheat bran samples. Reference fungal strains A. flavus MA (accession number of 18S rRNA gene sequence: MZ557834), A. niger BQM (accession number of 18S rRNA gene sequence: MT903388) and P. citrinum MB (accession number of 18S rRNA gene sequence: MZ557835), previously isolated and preserved in the laboratory, were employed to screen LAB strains exhibiting antifungal activity. DeMan, Rogosa, and Sharpe (MRS) and Potato dextrose agar (PDA) media were sourced from Beijing Aobo Star Biotechnology Co., Ltd. (Beijing, China). PCR Mix (with dye) was purchased from Beijing Liuhe Biotechnology Co., Ltd. (Beijing, China). Tris-acetic acid electrophoresis buffer was obtained from Leagene Biotechnology Co., Ltd. (Beijing, China). Agarose was bought from Biowest (Loire valley, France). Catalase and proteinase K were procured from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Lactic acid, acetic acid, propionic acid, L-3-phenyl-lactic acid, citric acid and malic acid (all HPLC grade) were acquired from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Screening of LAB Strains for Antifungal Activity

2.2.1. Preliminary Screening of LAB Strains for Antifungal Activity

A. flavus MA, A. niger BQM and P. citrinum MB were plated onto PDA plates and grown at a constant temperature of 28 °C for 3 days to allow abundant spore production. Spores were collected by rinsing the PDA plates with sterile distilled water, and the resulting suspension was homogenized using a vortex mixer. The spore concentration was adjusted to 1 × 105–5 × 105 CFU/mL and stored at 4 °C prior use [24].
Around 5 g of each sample (soil, corn kernels, fermented cabbage and wheat bran) was added into 45 mL of sterile saline. The mixture was vortexed at 180 rpm for one hour. The suspension was subjected to serial diluted into five gradients: 10−1, 10−2, 10−3, 10−4 and 10−5 using sterile saline [25]. A 100 μL aliquot of the mold spore suspension was transferred into a sterile Petri dish, and then 20 mL of MRS agar medium cooled to 50 °C was added. After the agar being solidified, 100 μL of each sample dilution was spread onto the surface. A control plate was prepared using an equivalent volume of sterile saline instead of the sample dilution. Cultivation was carried out on all plates at 37 °C with a duration of 48 h [26]. Colonies exhibiting inhibition of mold growth were selected from each plate based on morphological characteristics such as color, glossiness, size and the capacity of growth under co-culture conditions [27].

2.2.2. Evaluation of the Antifungal Activity of LAB Strains

The modified dual-culture assay was applied to assess the antifungal activity of LAB strains against A. flavus MA, A. niger BQM and P. citrinum MB [28]. Briefly, 10 mL of MRS agar medium was dispensed into sterile Petri dishes and allowed to solidify. Following solidification, two parallel lines spaced 3 cm apart were inoculated with LAB strains on MRS plates, which were then incubated at 37 °C for 24 h. Then, 10 mL of PDA semi-solid medium supplemented with a mold spore suspension at a concentration of 1 × 105 to 5 × 105 CFU/mL was overlaid onto the plates. After solidification of the overlay medium, the plates were incubated at 28 °C for 48 h. Plates without LAB inoculation were used as the control group. All experiments were performed in triplicate. The diameter of inhibition zone perpendicular to the LAB inoculation lines was measured using a vernier caliper.

2.3. Identification of LAB Strains with Antifungal Activity

2.3.1. Morphological Identification of LAB Isolates

Physiological and biochemical identification of LAB strains with antifungal activity was conducted in accordance with the procedures outlined in Bergey’s Manual of Systematic Bacteriology. The LAB strains were streaked onto MRS solid medium and incubated at 37 °C for 48 h. Colony morphology was observed and recorded, and single colonies were selected for Gram staining [29].

2.3.2. Molecular Identification of LAB Isolates

Genomic DNA of selected LAB strains was extracted using the DNeasy Blood and Tissue kit (QIAGEN). PCR amplification of the 16S rRNA gene was performed with the universal primers set (Eu8f, 5′-AGAGTTTGATCCTGGCTCAG-3′; Eu1492r, 5′-GGCTACCTTGTTACGACTT-3′), each at a final concentration of 0.2 µM [30]. Following amplification, the PCR products were subjected to electrophoretic separation on a 2% (w/w) agarose gel at a constant voltage of 120 V. Upon completion of electrophoresis, purified PCR products were submitted to the Beijing Genomics Institute for Sanger sequencing of the 16S rRNA gene. The resulting sequences were submitted to the EzBioCloud database (https://www.ezbiocloud.net/ accessed on 5 June 2023) for similarity comparison with closely related type strains [31]. The MEGA 12.1 program was utilized for conducting phylogenetic and molecular evolutionary analysis [32].

2.4. Evaluation of the Antifungal Activity of LAB CFS

The LAB strains were grown in MRS broth at 37 °C with shaking 200 rpm for 24 h. When the OD600 reached approximately 1, a 5% inoculum was transferred into fresh MRS broth and incubated under identical conditions for an additional 48 h. The CFS was collected by centrifugation at 6791× g for 10 min at 4 °C and subsequently filtered through a 0.22 μm sterile membrane. Following pH measurement, the supernatant was stored at 4 °C for further analysis [33].
The antifungal activity of LAB CFS against A. flavus MA, A. niger BQM and P. citrinum MB was evaluated using a 96-well plate assay. A mixture of 190 μL LAB CFS and 10 μL fungal spore suspension (final concentration: 1 × 105 to 5 × 105 CFU/mL) was added to each well, while the control group received 190 μL sterile MRS broth and 10 μL mold spore suspension [34]. The 96-well plates were incubated at 28 °C for 72 h with three replicates per group. The optical density at 600 nm (OD600) was measured using a microplate reader (Powerscan HT, DS Pharma Biomedical Co., Ltd., Osaka, Japan), and the inhibitory rate was calculated to assess the antifungal effect of LAB CFS.
I n h i b i t o r y   r a t e % = 1 − 72   h   O D L A B − 0   h   O D L A B 72   h   O D c o n t r o l − 0   h   O D c o n t r o l × 100 %
where ODLAB represents the OD600 value of the mold spore suspension cultured in LAB CFS at 0 h or 72 h, respectively; and ODcontrol indicates the OD600 value of the mold spore suspension cultured in sterile MRS broth at 0 h or 72 h.

2.5. Effect of pH, Catalase and Proteinase K Treatments on the Antifungal Activity of LAB CFS

2.5.1. Effect of pH Treatments on the Antifungal Activity of LAB CFS

Following the protocol described by Zhang et al. [35,36], the LAB CFS was adjusted to pH 4.0, 5.0 and 6.0, respectively. Untreated LAB CFS and sterile MRS broth were used as controls. The inhibitory effect of pH-treated LAB CFS against A. flavus MA, A. niger BQM and P. citrinum MB was evaluated using the 96-well plate assay [37].

2.5.2. Effect of Catalase Treatment on the Antifungal Activity of LAB CFS

First, 3% (w/v) catalase was dissolved in 0.05 mol·L−1 phosphate buffer (pH 7.0). The enzyme solution was then added to LAB CFS to yield a final concentration of 5 mg/mL, the mixture was then incubated in a water bath at 37 °C for 2 h. Catalase activity in LAB CFS was subsequently inactivated by heating at 100 °C for 5 min. The pH of the LAB CFS was adjusted to pH 6.0, and the antifungal activity was assessed using the 96-well plate assay [38]. Sterile MRS broth and LAB CFS (pH 6.0) without catalase treatment were used as controls, with three replicates performed for each condition [21].

2.5.3. Effect of Protease K Treatment on the Antifungal Activity of LAB CFS

Protease K was dissolved in 0.05 mol·L−1 phosphate buffer (pH 7.0). The enzyme preparation was added to the LAB CFS to achieve a final concentration of 1 mg/mL and then incubated at 37 °C in a water bath for 2 h. Protease K activity in the LAB CFS was subsequently inactivated by heating at 100 °C for 5 min. After treatment, the pH was adjusted to 6.0, and the antifungal activity was evaluated using the 96-well plate assay [39]. Sterile MRS broth and LAB CFS (pH 6.0) without protease K treatment were used as controls, with three replicates performed for each condition [40].

2.6. Analysis of Organic Acids in LAB CFS

The organic acids in LAB CFS were analyzed using HPLC (Waters, e2695, Milford, CT, USA) [41]. The detection wavelength was set at 210 nm. Chromatographic separation was achieved using an Agilent ZORBAX SB-C18 column (25 cm × 4.6 mm, 5 µm). The mobile phase consisted of 0.01 mol/L KH2PO4 (pH 2.7) and methanol at a ratio of 95:5 (v/v). The flow rate was 0.6 mL/min, and the column temperature was kept at 30 °C. The injection volume was 20 µL, and the total elution time was 20 min.
Standard solutions of each organic acid—phenyllactic acid, lactic acid, acetic acid, citric acid, malic acid and propionic acid—were prepared individually at 20 mg/mL by solubilizing the respective pure compounds in ultrapure water. Each standard solution was injected separately into the system to determine its retention time. A mixed organic acid standard solution was then prepared by combining the six individual standard solutions with 0.1% (w/w) phosphoric acid solution. In the mixed solution, standard solutions of phenyllactic acid, lactic acid, malic acid and citric acid were prepared at concentrations of 0.1, 0.5, 1, 2, 5 mg/mL, respectively, while those of acetic acid and propionic acid were 0.5, 1, 2, 5, 10 mg/mL, respectively. The mixed standard solution was filtered through a 0.22 μm sterile filter membrane into an injection vial for sample introduction and analysis. Calibration curves were constructed by plotting the peak area (y-axis) against the corresponding organic acid concentration (x-axis). The instrument-generated data were processed to construct calibration curves and determine the correlation coefficients.
LAB CFS was prepared based on the method described in [42]. In order to ensure that the organic acid concentrations fell within the detection range of the calibration curve established from the standard solutions, the LAB CFS was diluted 5 times with 0.1% (w/w) phosphoric acid solution and filtered through a 0.22 μm sterile membrane prior to instrumental analysis, then transferred into an injection vial.

2.7. Evaluation of the Antifungal Activity of Organic Acids

According to the results of organic acid analysis in LAB CFS, the antifungal activity of five organic acids—lactic acid, acetic acid, citric acid, malic acid and phenyllactic acid—was evaluated. The concentrations of the individual organic acid solutions were adjusted to 0.5, 1, 5, 10 and 20 mg/mL using sterile MRS liquid medium. After filtration through a 0.22 μm sterile membrane filter, the antifungal activity of each solution against A. flavus MA, A. niger BQM and P. citrinum MB was assessed using the 96-well plate method [43]. The experiment was performed in triplicate with sterile MRS liquid medium serving as the control.

2.8. Statistical Analysis

Data were primarily organized and descriptive statistics were calculated using Microsoft Excel 2019. Statistical analysis was performed using SPSS 23. Significant differences among groups were evaluated by Duncan’s multiple range test at a threshold of p < 0.05. Figures were plotted and prepared using Origin 2018 and final compositing was done with Adobe Photoshop CS6 for improved presentation clarity. All experiments were conducted in triplicate, and data are expressed as mean ± standard deviation.

3. Results and Discussion

3.1. Screening of LAB Strains with Antifungal Activity

3.1.1. Preliminary Screening of LAB Strains with Antifungal Activity

Figure 1 illustrates that the control group’s MRS agar plate presented a uniform lawn of mold colonies (Figure 1a), whereas several smooth, round and white colonies—presumed to be LAB with antifungal potential—appeared on the MRS agar plate of the experiment group (Figure 1b). According to distinct morphological characteristics such as color, size, shape and morphology, a collection of 71 strains was obtained from diverse materials, including soil, corn kernel, fermented cabbage and wheat bran samples (Table 1). The isolation strategy of this study leverages the broad natural distribution of LAB [44]. This integrated niche-sampling approach serves a dual purpose: LAB isolated directly from grain substrates may exhibit enhanced environmental adaptability for direct application in storage ecosystems, while those from highly competitive environments like soil and fermented pickles are more likely to yield robust strains, naturally selected for strong acid production or novel antifungal mechanisms [45]. The considerable biodiversity of LAB underpins their significant application potential [46]. Therefore, this study aims to systematically screen strains from these distinct sources and elucidate their inhibitory mechanisms against key storage molds, thereby establishing a theoretical and resource foundation for developing targeted, efficient bioprotective agents.

3.1.2. Evaluation of Antifungal Activity of LAB Strains

According to the inhibition zone diameter [47], the antifungal activity of LAB strains was divided into four levels: no inhibitory effect (−), defined as the absence of an inhibition zone; low inhibitory effect (+), with a diameter less than 20 mm; moderate inhibitory effect (++), ranging from greater than 20 mm to less than 30 mm; high inhibitory effect (+++), higher than 30 mm (Figure S1).
A modified dual-culture assay was used to screen the inhibitory potential of the 71 LAB strains against the three mold species: A. flavus MA, A. niger BQM, and P. citrinum MB. The results showed that 17 LAB strains demonstrated inhibitory effects against all three pathogenic molds (Table 2). As shown in Table 2, 11 LAB strains inhibited the growth of A. flavus MA, with strain P3 demonstrating the strongest inhibitory effect and yielding an inhibition zone diameter exceeding 30 mm. Moreover, 14 LAB strains showed inhibitory effect on A. niger BQM, among which, strain P3 had the highest inhibitory effect. In addition, 17 strains inhibited P. citrinum MB, with 12 strains, including DA1, DA2, DA4, DB2, DB5, DB6, DB11, DC5, DC6, P3, P5, P6, had strong inhibitory effect, and strain DC5 produced the largest inhibition zone. Similarly, Zhang et al. reported that Lactococcus lactis PLL15 and Lactobacillus sakei PLL12 could significantly inhibit the germination of A. niger spores and disrupt the integrity of the cell membrane and cell wall [48]. Concurrently, Kim et al. found that Lactiplantibacillus plantarum G2 and Levilactobacillus brevis H8 efficiently suppressed the growth of Aspergillus species in ham [49].

3.2. Analysis of LAB Strains with Antifungal Activity

3.2.1. Morphological Identification

Table 3 summarizes the key morphological traits of the antifungal LAB strains. Colonies were circular, smooth, white or cream-white, and either semi-transparent or non-transparent and all isolates were Gram-stain-positive. These characteristics were consistent with the established morphological profile of typical LAB strains. As a defined group of Gram-positive, non-sporulating bacteria, they are usually in the form of cocci or rods [50].

3.2.2. Molecular Identification

Table 4 presents the results of molecular identification for the LAB strains using 16S rDNA sequencing. It is indicated that 17 LAB strains were divided into 3 genera and 3 species. The 16S rDNA sequences of strains DA1, DA2, DA4, DB3, DC5 and DC6 were most closely related to that of the reference strain Weissella cibaria KACC 11862 T, with sequence identity exceeding 99%. Based on both molecular and morphological characteristics, these six strains were identified as Weissella cibaria. Strains DB1, DB2, DB5, DB6, DB7, DB8, DB11, DB12 and P6 showed the closest phylogenetic relationship to Pediococcus pentosaceus DSM 20336 T, with sequence similarities also higher than 99%. Integrating molecular data with morphological features, these nine strains were identified as Pediococcus pentosaceus. The 16S rDNA sequences of strains P3 and P5 were closest to that of Lactiplantibacillus paraplantarum DSM 10667 T, with sequence similarities of 99.59% and 99.25%, respectively. According to their morphological characteristics, both strains were identified as Lactiplantibacillus paraplantarum. Vasundaradevi et al. demonstrated that organic acids produced by Lactobacillus paraplantarum significantly suppress the growth of uropathogens [51]. Furthermore, Wang et al. demonstrated that applying the CFS of Weissella cibaria effectively delays quality deterioration in sweet corn [52]. Separately, Pediococcus pentosaceus has also been employed in litchi postharvest preservation to mitigate postharvest losses [53]. Collectively, these studies confirm that Weissella cibaria, Pediococcus pentosaceus, and Lactiplantibacillus paraplantarum exhibit antifungal properties. However, their potential in controlling major molds specifically associated with grain spoilage during storage remains underexplored. Therefore, this study aimed to elucidate the antifungal mechanisms of these LAB strains against key storage-borne pathogenic fungi.

3.3. Analysis of the Antifungal Activity of LAB CFS

Table 5 presents a comparative summary of the antifungal activity exhibited by the CFS of 17 LAB strains against A. flavus MA, A. niger BQM and P. citrinum MB. Among these strains, the CFS of strains DA2, DA4, DB2, DB11, DB12, DC5, P3 and P5 exhibited inhibitory rates exceeding 99%. These results align with precious report that Lactobacillus plantarum strains were used as biocontrol agents against A. niger, A. flavus, P. chrysogenum in wheat and fresh maize seeds; Pediococcus pentosaceous strain was utilized to control the growth of P. expansum in pear, grape and plum [54]; and W. cibaria strains have previously been employed to prevent the growth of foodborne pathogens [55]. Moreover, molecular identification analysis revealed that these eight high-performing strains belong to three distinct genera and three species, highlighting their taxonomic diversity and providing a sound rationale for selecting them for mechanistic investigation.

3.4. Analysis of the Effect of pH, Catalase and Proteinase K Treatments on the Antifungal Activity of LAB CFS

3.4.1. Analysis of the Effect of pH Treatments on the Antifungal Activity of LAB CFS

The inhibitory activity of the CFS from 8 LAB strains at different pH values against A. flavus MA, A. niger BQM and P. citrinum MB is displayed in Figure 2. A marked dependence on pH was observed for the inhibitory activity of the LAB CFS. At pH 4, the inhibitory rates against all three molds remained comparable to those of the control, with no significant difference (p > 0.05), maintaining at approximately 99%. However, when pH value was increased to 5 and 6, the inhibitory rate of LAB CFS was markedly reduced (p < 0.05). At pH 6, the inhibitory rate ranged from 12.42% to 39.28%, indicating a substantial decrease (p < 0.05). Similar results were reported by Szczerbiec et al., who demonstrated that Lactobacillus spp. strains exhibited significant inhibitory activity against Proteus mirabilis; however, the antifungal activity of the CFS was entirely eliminated at pH 6.0 [56]. In an acidic environment, organic acids could disrupt transmembrane proton gradients and inhibit essential metabolic enzymes, causing cell death or growth arrest; when the environmental pH rises, organic acids cannot easily cross the lipid bilayer, thereby losing their antifungal efficacy [57]. Additionally, LAB CFS may contain other metabolites which possess antifungal activity or exhibit synergistic effects with organic acids.

3.4.2. Analysis of the Effect of Catalase Treatment on the Antifungal Activity of LAB CFS

Figure 3 illustrates the inhibitory activity against A. flavus MA, A. niger BQM, and P. citrinum MB exhibited by LAB CFS following catalase treatment. Compared to untreated LAB CFS, no significant change in inhibitory activity against the three molds was found following catalase treatment (p > 0.05), indicating that hydrogen peroxide was not secreted by the eight LAB strains. The study by Ma et al. also indicated that the CFS from Pediococcus acidilactici JY03, Weissella paramesenteroides JT13 and Lactobacillus sucicola JT03 remained antifungal activity following catalase treatment, thus indicating that hydrogen peroxide was not included among its antifungal substances [58].

3.4.3. Analysis of the Effect of Protease K Treatment on the Antifungal Activity of LAB CFS

The inhibitory activity of LAB CFS treated with protease K against A. flavus MA, A. niger BQM and P. citrinum MB is demonstrated in Figure 4. Protease K treatment of the LAB CFS showed no significant difference in inhibitory rate relative to the untreated control against all three molds (p > 0.05). Therefore, it was presumed that the antifungal substances in LAB CFS are not proteinaceous. Similarly, it has been demonstrated that the antifungal activity of the CFS from Lactiplantibacillus plantarum G2 showed no significant change after protease K treatment, confirming that the antifungal substances in this CFS are not proteins [49].

3.5. Analysis of Organic Acid Composition of LAB CFS

3.5.1. HPLC Analysis of Organic Acids

Previous studies have demonstrated that organic acids including phenyllactic acid, lactic acid, acetic acid, citric acid, malic acid and propionic acid were produced by LAB [26,59]. Therefore, HPLC calibration curves (Figure 5) for these six kinds of organic acid standards were constructed to investigate the content of each organic acid in LAB CFS. As can be seen from Figure 5, the retention times of the organic acid standards were as follows: phenyllactic acid at 5.075 min, lactic acid at 6.158 min, acetic acid at 6.766 min, citric acid at 7.133 min, malic acid at 8.081 min and propionic acid at 15.183 min. The calibration curves and correlation coefficient (R2) of the organic acid standards are summarized in Table 6. The results demonstrate that the R2 values ranged from 0.9981 to 0.9999, confirming an excellent linear correlation between analyte concentration and chromatographic response. Under the selected chromatographic conditions, all six organic acids exhibited well-defined peaks and were effectively separated. Therefore, the developed calibration curves are suitable for the quantitative determination of organic acids in LAB CFS.

3.5.2. Quantitative Determination of Organic Acid Content in LAB CFS

As can be seen from Table 7, five organic acids including phenyllactic acid, lactic acid, acetic acid, citric acid and malic acid were detected in the CFS of DA2, DA4, DB2, DB11, DB12, DC5 and P3, while only four organic acids such as phenyllactic acid, lactic acid, acetic acid and malic acid were found in P5 CFS. Among the CFS of the eight selected LAB strains, acetic acid and lactic acid were the main organic acids, followed by malic acid, citric acid and phenyllactic acid. L. paraplantarum P3 and P5 secreted the highest concentrations of lactic acid, with corresponding levels of 17.84 mg/mL and 17.82 mg/mL, respectively. The highest acetic acid contents were observed in the CFS of W. cibaria DC5 and DA4, at 11.73 mg/mL and 10.33 mg/mL, respectively. According to the study by Garnier et al., the primary organic acids synthesized by Lactobacillus CIRM-BIA1952 were identified as lactic acid and acetic acid [60]. Similarly, a study by Vasundaradevi et al. quantitatively analyzed the organic acid profile in the CFS of Lactiplantibacillus plantarum MYSVCF3, revealing citric acid as the predominant metabolite, which also demonstrated the strongest inhibitory efficacy against Fusarium oxysporum [51]. To identify the key antifungal contributor, a further investigation was conducted to evaluate the individual activity of each identified organic acid against the target molds.

3.6. Analysis of the Antifungal Activity of Organic Acids

The inhibitory activities of five organic acids were evaluated against A. flavus MA, A. niger BQM and P. citrinum MB at concentrations of 0.5, 1, 5, 10 and 20 mg/mL. As shown in Figure 6, acetic acid and lactic acid exerted higher efficacy against A. flavus MA and P. citrinum MB than the other three organic acids (p < 0.05). While acetic acid and phenyllactic acid displayed the highest inhibitory efficacy against A. niger BQM (p < 0.05). At a concentration of 20 mg/mL, the inhibitory rate of all five organic acids against the three molds reached approximately 98%~99%. It was indicated that acetic acid and lactic acid may be the primary antifungal components in CFS against storage molds. Lactic and acetic acids, as small monobasic acids, penetrate the fungal membrane due to their lipid solubility, causing intracellular acidification and metabolic disruption that ultimately inhibits growth. This mechanism is consistent with studies confirming hyphal damage and loss of membrane integrity [61,62]. Concurrently, phenyllactic acid demonstrated notable specificity against A. niger. Beyond acidification, evidence suggests it may interfere with specific fungal enzyme systems and signal transduction pathways [63]. Importantly, at a concentration of 10 mg/mL, the inhibitory rate of any single organic acid remained lower than that observed for the CFS of strains such as Pediococcus pentosaceus DB11, Weissella cibaria DA2, Weissella cibaria DA4. It was inferred that the remarkable antifungal activity of the CFS is attributable not to a single compound, but to a synergy between various organic acids. These compounds presumably act on complementary fungal targets, producing a stronger and broader-spectrum inhibitory effect than any individual acid [34,64]. Based on these findings, a conceptual framework for a multi-target biocontrol strategy can be proposed. This antifungal agent employs acetic and lactic acids as a broad-spectrum bases, supplemented with phenyllactic acid for the specific targeting of A. niger. The efficacy and practicality of this approach warrant further validation through simulated grain models or pilot-scale storage trials.

4. Conclusions

In this study, a total of 71 LAB strains originated from distinct materials such as soil, corn kernels, fermented cabbage and wheat bran. Among these, 17 LAB strains demonstrated antifungal activity against three harmful molds such as A. flavus MA, A. niger BQM and P. citrinum MB. They were classified as three genera and species, W. cibaria, P. pentosaceus and L. paraplantarum, based on morphological and molecular identification. Of the 17 LAB strains, the CFS of 8 LAB strains inhibited the growth of all three molds by over 99%. Their antifungal activity was significantly reduced following pH treatment, while it was not affected by catalase or proteinase K treatment, indicating that the antifungal substances in LAB CFS were mainly organic acids. In addition, HPLC analysis revealed that the LAB CFS consists of five kinds of organic acids: malic acid, acetic acid, lactic acid, phenyllactic acid and citric acid. Among them, acetic acid and lactic acid showed strongest antifungal activity against A. flavus MA and P. citrinum MB, while acetic acid and phenyllactic acid demonstrated the highest inhibitory efficacy against A. niger BQM. This research reveals the antifungal mechanism of LAB strains against dominant fungal strains, providing a theoretical foundation for their application in mold prevention and postharvest grain quality preservation. Further research is planned to assess the application of the LAB CFS in grain storage, thereby laying the groundwork for its development as a practical antifungal agent.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/foods15040655/s1. Figure S1: Evaluation of the antifungal activity of LAB strains. (a) no inhibitory effect; (b) low inhibitory effect; (c) moderate inhibitory effect; (d) high inhibitory effect.

Author Contributions

Conceptualization, H.L.; Methodology, C.C.; Software, X.F. and T.Z.; Validation, T.Q. and Y.L. (Yijun Liu); Formal analysis, C.C.; Investigation, X.F. and T.Z.; Resources, Y.Z.; Data curation, T.Q. and Y.L. (Yijun Liu); Writing—original draft preparation, C.C.; Writing—review and editing, C.C. and H.L.; Visualization, C.C. and H.L.; Supervision, Y.L. (Yanfei Li); Project administration, Y.L. (Yanfei Li); Funding acquisition, H.L. and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Department of Science and Technology of Henan Province, grant number 242103810072; China Scholarship Council, grant number 202508410212; Henan University of Technology, grant number 21421239; the Ministry of Science and Technology of the People’s Republic of China, grant number 2021YFD2100604-2.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included within the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Screening of LAB strains with antifungal potential. (a) control group, mold spore suspension with sterile saline; (b) experiment group, mold spore suspension with sample diluent.
Figure 1. Screening of LAB strains with antifungal potential. (a) control group, mold spore suspension with sterile saline; (b) experiment group, mold spore suspension with sample diluent.
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Figure 2. Inhibitory rate of LAB CFS with different pH values against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c). Different small letters denote significant differences (p < 0.05) within the same LAB strain across processing methods.
Figure 2. Inhibitory rate of LAB CFS with different pH values against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c). Different small letters denote significant differences (p < 0.05) within the same LAB strain across processing methods.
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Figure 3. Inhibitory rate of LAB CFS treated with catalase against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
Figure 3. Inhibitory rate of LAB CFS treated with catalase against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
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Figure 4. Inhibitory rate of LAB CFS treated with protease K against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
Figure 4. Inhibitory rate of LAB CFS treated with protease K against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
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Figure 5. HPLC calibration curve of organic acid standards: (1) phenyllactic acid; (2) lactic acid; (3) acetic acid; (4) citric acid; (5) malic acid; (6) propionic acid.
Figure 5. HPLC calibration curve of organic acid standards: (1) phenyllactic acid; (2) lactic acid; (3) acetic acid; (4) citric acid; (5) malic acid; (6) propionic acid.
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Figure 6. Antifungal activity of organic acids against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
Figure 6. Antifungal activity of organic acids against A. flavus MA (a), A. niger BQM (b) and P. citrinum MB (c).
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Table 1. LAB strains isolated from various experimental sources.
Table 1. LAB strains isolated from various experimental sources.
Experimental SourcesNumber of Strains LAB Strains
Rhizosphere soil of wheat7TA1, TA2, TA3, TA4, TA5, TA6, TA7
Rhizosphere soil of vegetables6TB1, TB2, TB3, TB4, TB5, TB6
Rhizosphere soil of garlic11TC1, TC2, TC3, TC4, TC5, TC6, TC7, TC8, TC9, TC10, TC11
Soil from flower beds13TD1, TD2, TD3, TD4, TD5, TD6, TD7, TD8, TD9, TD10, TD11, TD12, TD13
Corn kernel from Northeast China, variety No. 34DA1, DA2, DA3, DA4
Corn kernels from Heihe city, Heilongjiang province, China12DB1, DB2, DB3, DB4, DB5, DB6, DB7, DB8, DB9, DB10, DB11, DB12
Corn kernel from Northeast China, variety No. 68DC1, DC2, DC3, DC4, DC5, DC6, DC7, DC8
Fermented cabbage6P1, P2, P3, P4, P5, P6
Wheat bran4F1, F2, F3, F4
Table 2. The antifungal activity of LAB strains against A. flavus MA, A. niger BQM and P. citrinum MB.
Table 2. The antifungal activity of LAB strains against A. flavus MA, A. niger BQM and P. citrinum MB.
LAB StrainsA. flavus MAA. niger BQM Penicillium citrinum MB
Inhibition Zone Diameter Value (mm)Antifungal ActivityInhibition Zone Diameter Value (mm)Antifungal ActivityInhibition Zone Diameter Value (mm)Antifungal Activity
DA10−0−30.86+++
DA20−30.91+++38.11+++
DA40−28.17++32.23+++
DB115.22+0−29.39++
DB212.23+22.12++30.37+++
DB313.86+21.53++27.31++
DB50−39.43+++31.68+++
DB614.55+26.65++38.04+++
DB713.37+29.87++29.92++
DB80−0−29.81++
DB1113.75+37.87+++30.55+++
DB1219.52+35.81+++29.01++
DB518.75+23.05++46.53+++
DB619.39+25.07++30.53+++
P331.85+++40.63+++42.37+++
P521.41++29.38++38.41+++
P60−26.62++31.69+++
Note: −, no inhibitory effect; +, low inhibitory effect; ++, moderate inhibitory effect; +++, high inhibitory effect.
Table 3. Morphological characteristics of LAB strains with antifungal activity.
Table 3. Morphological characteristics of LAB strains with antifungal activity.
LAB StrainsMorphologyColorTransparencyGram-Stain
DA1Circular, smoothWhiteSemitransparent+
DA2Circular, smoothWhiteSemitransparent+
DA4Circular, smoothWhiteSemitransparent+
DB1Circular, smoothCream-whiteNon-transparent+
DB2Circular, smoothCream-whiteNon-transparent+
DB3Circular, smoothCream-whiteSemitransparent+
DB5Circular, smoothCream-whiteNon-transparent+
DB6Circular, smoothCream-whiteNon-transparent+
DB7Circular, smoothCream-whiteNon-transparent+
DB8Circular, smoothCream-whiteNon-transparent+
DB11Circular, smoothCream-whiteNon-transparent+
DB12Circular, smoothCream-whiteNon-transparent+
DC5Circular, smoothWhiteSemitransparent+
DC6Circular, smoothWhiteSemitransparent+
P3Circular, smoothCream-whiteNon-transparent+
P5Circular, smoothCream-whiteNon-transparent+
P6Circular, smoothCream-whiteNon-transparent+
Note: +, positive.
Table 4. Molecular identification of LAB strains based on 16S rDNA sequencing.
Table 4. Molecular identification of LAB strains based on 16S rDNA sequencing.
LAB StrainsReference Strains16S rDNA Sequence Identity/%
DA1W. cibaria KACC 11862 T99.19
DA2W. cibaria KACC 11862 T100.00
DA4W. cibaria KACC 11862 T99.85
DB3W. cibaria KACC 11862 T99.26
DC5W. cibaria KACC 11862 T99.92
DC6W. cibaria KACC 11862 T100.00
DB1P. pentosaceus DSM 20336 T99.26
DB2P. pentosaceus DSM 20336 T99.39
DB5P. pentosaceus DSM 20336 T99.12
DB6P. pentosaceus DSM 20336 T99.32
DB7P. pentosaceus DSM 20336 T99.53
DB8P. pentosaceus DSM 20336 T100.00
DB11P. pentosaceus DSM 20336 T99.33
DB12P. pentosaceus DSM 20336 T100.00
P6P. pentosaceus DSM 20336 T99.39
P3L. paraplantarum DSM 10667 T99.59
P5L. paraplantarum DSM 10667 T99.25
Note: W. cibaria, Weissella cibaria; P. pentosaceus, Pediococcus pentosaceus; L. paraplantarum, Lactiplantibacillus paraplantarum. T: Type strain.
Table 5. Antifungal activity of LAB CFS against A. flavus MA, A. niger BQM and P. citrinum MB.
Table 5. Antifungal activity of LAB CFS against A. flavus MA, A. niger BQM and P. citrinum MB.
LAB StrainsInhibitory Rate/%
A. flavus MAA. niger BQMP. citrinum MB
DA195.0899.0595.96
DA299.2999.3599.53
DA499.1399.5099.55
DB198.2297.8891.41
DB299.4999.1199.08
DB397.5299.2391.41
DB597.6995.2393.03
DB697.5998.5492.22
DB798.8998.9098.78
DB898.5898.6398.30
DB1199.2099.1299.88
DB1299.0599.4199.36
DC598.9499.3699.65
DC698.0995.1698.88
P399.4399.8899.81
P599.3199.5099.10
P697.9497.6497.75
Table 6. Regression equation and correlation coefficient of organic acid standards.
Table 6. Regression equation and correlation coefficient of organic acid standards.
Organic Acid StandardsRetention Time (min)Regression EquationCorrelation Coefficient (R2)
Phenyllactic acid5.075y = 1 × 106x + 199,0990.9987
Lactic acid6.158y = 2 × 106x + 22,1190.9999
Acetic acid6.766y = 969,221x + 27,3400.9999
Citric acid7.133y = 2 × 106x + 19,1460.9999
Malic acid8.081y = 948,650x + 3952.90.9981
Propionic acid15.183y = 1 × 106x + 67,5040.9999
Table 7. Organic acid content in LAB CFS.
Table 7. Organic acid content in LAB CFS.
LAB StrainsOrganic Acid Content (mg/mL)
Phenyllactic AcidLactic AcidAcetic AcidCitric AcidMalic AcidPropionic Acid
Weissella cibaria DA21.70 ± 0.23 d13.53 ± 0.13 b8.69 ± 0.45 bc1.00 ± 0.06 bc5.81 ± 0.22 bND
Weissella cibaria DA40.91 ± 0.04 e13.51 ± 0.08 b10.33 ± 0.12 ab4.32 ± 0.05 a5.61 ± 0.27 bND
Pediococcus pentosaceus DB20.31 ± 0.17 f17.65 ± 0.71 a7.52 ± 0.91 c0.97 ± 0.01 c2.32 ± 0.85 cND
Pediococcus pentosaceus DB110.55 ± 0.02 f12.14 ± 0.11 b8.64 ± 0.13 bc1.04 ± 0.02 b6.89 ± 0.35 abND
Pediococcus pentosaceus DB122.44 ± 0.16 c17.36 ± 0.03 a7.76 ± 0.06 c1.04 ± 0.01 b2.61 ± 0.04 cND
Weissella cibaria DC50.86 ± 0.03 e16.26 ± 2.3 a11.73 ± 2.73 a0.98 ± 0.02 c8.10 ± 1.91 aND
Lactiplantibacillus paraplantarum P34.51 ± 0.35 a17.84 ± 0.08 a8.93 ± 0.02 bc0.97 ± 0.01 c2.63 ± 0.05 cND
Lactiplantibacillus paraplantarum P53.92 ± 0.08 b17.82 ± 0.03 a8.48 ± 0.06 bcND2.44 ± 0.06 cND
Note: Data are presented as mean ± standard deviation from three independent experiments. Significant differences (p < 0.05) within the same row are denoted by different lowercase letters (a–f). ND, not detected.
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Cui, C.; Fu, X.; Qi, T.; Zou, T.; Liu, Y.; Li, Y.; Zhao, Y.; Lv, H. Inhibitory Mechanism of Lactic Acid Bacteria Cell-Free Supernatant Against Stored Grain Molds. Foods 2026, 15, 655. https://doi.org/10.3390/foods15040655

AMA Style

Cui C, Fu X, Qi T, Zou T, Liu Y, Li Y, Zhao Y, Lv H. Inhibitory Mechanism of Lactic Acid Bacteria Cell-Free Supernatant Against Stored Grain Molds. Foods. 2026; 15(4):655. https://doi.org/10.3390/foods15040655

Chicago/Turabian Style

Cui, Can, Xiaopeng Fu, Tianjie Qi, Tianci Zou, Yijun Liu, Yanfei Li, Yan Zhao, and Haoxin Lv. 2026. "Inhibitory Mechanism of Lactic Acid Bacteria Cell-Free Supernatant Against Stored Grain Molds" Foods 15, no. 4: 655. https://doi.org/10.3390/foods15040655

APA Style

Cui, C., Fu, X., Qi, T., Zou, T., Liu, Y., Li, Y., Zhao, Y., & Lv, H. (2026). Inhibitory Mechanism of Lactic Acid Bacteria Cell-Free Supernatant Against Stored Grain Molds. Foods, 15(4), 655. https://doi.org/10.3390/foods15040655

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