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Article

Identification and Characterization of Antiyeast Organic Acids Produced by Lactiplantibacillus plantarum 3121M0s Derived from Mongolian Traditional Fermented Milk, Airag

by
Md. Bakhtiar Lijon
1,
Yuko Matsu-ura
1,
Takumi Ukita
1,
Kensuke Arakawa
1,* and
Taku Miyamoto
1,2,3,4
1
Graduate School of Environmental and Life Science, Okayama University, Okayama 7008530, Japan
2
Faculty of Food Culture, Kurashiki Sakuyo University, Okayama 7100292, Japan
3
Microbial Fermentation Research Center, Minori Co., Ltd., Okayama 7011221, Japan
4
Functional Food Creation Research Institute Co., Ltd., Okayama 7161241, Japan
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(9), 2017; https://doi.org/10.3390/microorganisms13092017
Submission received: 4 July 2025 / Revised: 12 August 2025 / Accepted: 23 August 2025 / Published: 29 August 2025
(This article belongs to the Special Issue Microbial Fermentation in Food Processing)
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Abstract

Lactic acid bacteria are beneficial for food biopreservation by inhibiting not only bacteria but also fungi. However, reports on the control of fungi, especially yeasts, by lactic acid bacteria are limited. In this study, strain 3121M0s derived from Mongolian traditional fermented milk, airag, was selected with relatively high antiyeast activity among 236 strains, and identified as Lactiplantibacillus plantarum. The activity was exhibited under acidic conditions and remained stable after heating. It was also highly resistant to catalase and proteases, indicating that the primary antiyeast substances of 3121M0s were neither H2O2 nor peptides. Then, organic acids (lactic acid, acetic acid, 4-hydroxyphenyllactic acid, 4-hydroxybenzoic acid, and 3-phenyllactic acid) were detected and quantified in the ethyl acetate extract of the 3121M0s culture supernatant. Among them, only acetic acid showed antiyeast activity on its own, and the activity was enhanced by lactic acid or 3-phenyllactic acid. Compared to the type strain of L. plantarum, the production of lactic acid from 3121M0s was almost equal, but acetic acid and 3-phenyllactic acid were about 1.5 times higher. These results suggest that strain 3121M0s would be useful as a biopreservative starter for fermented foods susceptible to yeast contamination due to being produced in open environments without final sterilization.

1. Introduction

Microbial contamination of food poses health risks as well as leads to economic losses and social issues such as food waste. Biological food contaminants are primarily bacteria, but fungi should also be given careful attention because they also cause food poisoning and spoilage [1,2]. From the perspective of preventing food poisoning, it is necessary to control fungi, particularly molds such as some Aspergillus spp. and Fusobacterium spp., which have the potential to produce mycotoxins such as aflatoxins and fumonisins [3,4,5].
Another major fungus, yeast, has long been used in the production of various fermented foods since ancient times. The most common species is Saccharomyces cerevisiae, and other known species include Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, Wickerhamomyces anomalus, and Zygosaccharomyces rouxii; most of which are considered safe [6,7,8]. However, these yeasts are often reported to be involved in food spoilage, and therefore should be controlled when undesirable [6,8,9,10,11,12,13,14,15,16,17,18,19]. In addition, although there are very few cases of food poisoning caused by yeasts, they can cause foodborne illness such as candidiasis by Candida albicans and its relatives [8,20]. Other species, including Rhodotorula mucilaginosa and even S. cerevisiae, have also been reported to rarely cause infection through food, particularly in immunocompromised individuals [21,22].
Yeasts associated with food are generally sensitive to heat, but relatively resistant to moderately low pH, low water activity, and osmotic stress such as high salt and sugar concentrations [15,16,23]. Therefore, yeasts are prone to deteriorating foods, even if they have been fermented, concentrated, and salted. This is particularly noticeable in foods produced under open environments without final heat treatment, because undesirable yeasts can easily contaminate and survive in such products. Dairy products such as cheese and traditional fermented milk are prime examples [6,9,10,12,13,14,17,18,19,24,25,26,27].
To effectively inhibit yeasts, chemical food additives such as calcium sorbate, sodium benzoate, calcium propionate, and sulfur dioxide are commonly used [28,29]. However, consumers have increasingly avoided synthetic preservatives, and a food preservation method using naturally derived preservatives with a long history of safe consumption, known as biopreservation and biopreservatives, has been gaining attention [29,30,31,32]. In particular, the use of lactic acid bacteria (LAB), which are safe, familiar to humans, and possess high antimicrobial effects, is anticipated for food biopreservation. LAB biopreservatives have been studied for their effectiveness mainly against bacteria, and there are also various reports against molds in dairy products, bread, grains, fruits, and vegetables. However, reports on yeasts are comparatively limited. Furthermore, most of these reports focus on LAB screening and food application tests, and a few have examined the details of antiyeast substances. To date, acetic acid (AA), 3-phenyllactic acid (PLA), hydroxy fatty acids, 3,5-dicaffeoylquinic acid, reuterin, cyclic dipeptides, bacteriocin, and proteinaceous compounds, including bacteriocin-like inhibitory substances, have been reported as reliable antiyeast substances [33,34,35,36,37,38,39,40,41,42].
By the way, LAB inhabit various environments, and traditional fermented dairy products are one of the most representative sources for isolating useful LAB strains. Mongolian traditional fermented milk, airag, which is produced by natural fermentation or by backslopping from previous batches without adding any commercial starter LAB strains, has been found to have health benefits. In recent years, its fermentation microbiota has been increasingly characterized using culture-dependent [43,44,45,46,47] and culture-independent methods [48,49]. In the references, it is shown that the main fermentation microorganisms in airag were LAB, and the most abundant species was consistently Lactobacillus helveticus. Other LAB species, such as Lentilactobacillus kefiranofaciens, Lentilactobacillus parakefiri, Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Leuconostoc mesenteroides, and Streptococcus thermophilus, and yeast species such as K. marxianus, Kazachstania unispora, S. cerevisiae, Issatchenkia orientalis, and Pichia manshurica, were frequently detected as well. In addition, some LAB strains isolated from airag have been characterized as potential contributors to its beneficial effects in fermentation and health promotion [50,51,52,53]. Furthermore, LAB strains producing bacteriocins have also been isolated and characterized [39,54,55,56,57], and their potential applications extend beyond fermentation starters and probiotics to biopreservation in food.
This study aimed to select a strain with relatively high antiyeast activity from our LAB library containing strains derived from airag, and to identify and characterize antiyeast substances produced by the strain. LAB with antiyeast activity are expected to be used effectively as starter cultures for fermented foods, especially produced under open environments without final heat treatment, such as cheese and traditional fermented milk [17]. Stated differently, antiyeast LAB strains could serve as biopreservative starters.

2. Materials and Methods

2.1. Microbial Strains and Culture Conditions

A total of 236 LAB strains, which were formerly isolated from various animals, plants, and traditional fermented foods and stored in our laboratory, were used for screening based on their antiyeast activity. The LAB strains were cultivated in de Man, Rogosa, and Sharp (MRS) broth (Oxoid; Thermo Fisher Scientific Inc.; Waltham, MA, USA) at their respective optimal temperatures (30 or 37 °C) for 24–48 h before use. Eleven strains of food spoilage yeasts (Table 1 and Table 2) used as indicators in the antiyeast activity assay were cultivated in Yeast and Mold (YM) broth (Difco; Becton, Dickinson and Company; Franklin Lakes, NJ, USA) at 25 °C for 48 h. All culture media except reconstituted skim milk (RSM) were sterilized at 121 °C for 15 min. RSM was prepared using 10% (w/v) skim milk powder (Megmilk Snow Brand Co., Ltd.; Sapporo, Hokkaido, Japan) and autoclaved at 110 °C for 20 min.

2.2. Antiyeast Activity Assay

The overlay method described by Magnusson and Schnürer (2001) [42], with a few modifications, was used primarily to screen for the 236 LAB strains with their antiyeast activity. Each LAB strain culture was centrifuged at 1600× g for 20 min to collect the cell suspension by removing the supernatant, and then 5 µL of the suspension was inoculated onto a plate of MRS agar (Thermo Fisher Scientific Inc.), followed by incubation at 30 °C for 48 h under anaerobic conditions using AnaeroPack (Sugiyama-Gen; Tokyo, Japan). The MRS plate was then overlaid with 10 mL of YM agar (Becton, Dickinson and Company) containing 1.0 × 104 CFU/mL of an indicator yeast strain. After solidification of the agar, the plate was incubated at 25 °C for 48 h. The antiyeast activity was evaluated by the size of the inhibitory zone formed around the inoculated LAB colony.
The strains selected by the overlay assay were further screened for their antiyeast activity using the agar–well diffusion method [42]. Before the assay, samples were prepared as follows. Each LAB strain culture was centrifuged (1600× g, 20 min), and filtrated with a 0.45 µm filter (Membrane Solutions, LLC; Auburn, WA, USA) to make cell-free culture supernatant (CFS). Next, CFS was lyophilized and then dissolved in citrate-phosphate buffer (pH 4.0) to make 10× tenfold concentrated CFS (10× CFS). In the assay, an indicator strain culture was inoculated into 20 mL of YM agar at a density of 1.0 × 104 CFU/mL, and then the agar was poured into a plate. After solidification, 6 mm diameter wells were made in the agar plate, and then 50 µL of 10× CFS was added into each well. After that, the plate was incubated at 25 °C for 48 h. The antiyeast activity was evaluated by the size of the inhibitory zone formed around the well.
Following the same procedure as the agar–well diffusion method described above, the antiyeast activity of 10× CFS (pH 4.0) of a control strain and organic acids (OAs) contained in 10× CFS was also assayed against R. mucilaginosa JCM 8115T. The OA solution was adjusted to pH 4.0 using 1–6 M NaOH and HCl before the assay.

2.3. Cocultivation in Skim Milk

The antiyeast effect of the selected LAB strain, 3121M0s, was verified by cocultivation with a food spoilage yeast strain, R. mucilaginosa JCM 8115T, in RSM. The LAB and yeast strains were inoculated into RSM together at 5–6 × 107 and 1 × 102 CFU/mL, respectively. As a control, RSM inoculated with either 3121M0s or JCM 8115T was used. After inoculation, RSM was incubated at 25 °C for 72 h, and culture pH and viable cell counts were measured at 0, 12, 24, 48, and 72 h incubation. The viable cell counts of the LAB and yeast strains were measured using cultivation in MRS agar supplemented with 10 ppm of cycloheximide and YM agar with 100 ppm of chloramphenicol at 30 and 25 °C for 48 h, respectively.

2.4. Species Identification

To identify the species of the selected strain, 3121M0s, its 16S rRNA gene was amplified using PCR with 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1525R (5′-AAGGAGGTGATCCAGCC-3′) primers [58], and sequenced. Total DNA of the strain was extracted and purified as described by Klaenhammer (1993) [59]. For the PCR, KAPA Taq EXtra HotStart ReadyMix (Kapa Biosystems, Inc.; Wilmington, MA, USA) and SimpliAmp Thermal Cycler (Applied Biosystems; Waltham, MA, USA) were used. The PCR conditions were followed according to the reference. The resulting PCR amplicon was extracted using MagExtractor DNA Fragment Purification Kit (Toyobo Co., Ltd.; Osaka, Japan) after agarose gel electrophoresis, and then submitted to the DNA sequencing service at Eurofins Genomics K.K. (Tokyo, Japan). The obtained 16S rRNA gene sequence was analyzed using the Basic Local Alignment Search Tool (BLAST; Ver. 2.16.0) of the National Center for Biotechnology Information (NCBI). Then, it was used to construct a phylogenetic tree with 16S rRNA gene sequences of the type strains of all species of the genus Lactiplantibacillus and the outgroup Lactobacillus delbrueckii subsp. bulgaricus and Escherichia coli, after trimming to align the lengths using the Gblocks program (Ver. 0.91b) [60]. The tree was constructed by the maximum-likelihood method with the Jukes–Cantor model using the CLC Main Workbench software (Ver. 6.9.2; Qiagen N.V.; Venlo, The Netherlands).
Since it is impossible to identify the species of L. plantarum-group LAB based on only 16S rRNA gene sequences, an additional multiplex PCR [61] concerning the species-specific recA gene was performed. Four reference strains, L. plantarum JCM 1149T, Lactiplantibacillus argentoratensis JCM 16169T, Lactiplantibacillus paraplantarum JCM 12533T, and Lactiplantibacillus pentosus JCM 1558T, and four PCR primers, paraF (5′-GTCACAGGCATTACGAAAAC-3′), pentF (5′-CAGTGGCGCGGTTGATATC-3′), planF (5′-CCGTTTATGCGGAACACCTA-3′), and pREV (5′-TCGGGATTACCAAACATCAC-3′), were used. The PCR conditions were followed according to the reference, and the resulting amplicons were profiled by electrophoresis to identify the species.
After the species identification, the obtained 16S rRNA sequence was submitted to the DNA Data Bank Japan (DDBJ) to obtain an accession number (LC884927).

2.5. Determination of Optimal Culture Conditions

To determine the optimal culture conditions of the selected LAB strain, 3121M0s, the bacterial growth was evaluated by sequentially measuring the culture pH at 0, 4, 8, 12, 16, 18, 24, 36, 48, and 72 h of incubation in MRS broth at 25, 30, and 37 °C. The culture turbidity (at OD 620 nm) and antiyeast activity were also measured at 0, 4, 8, 12, 18, 24, 36, 48, and 72 h of incubation at 30 °C. The antiyeast activity was assayed using the agar–well diffusion method with 10× CFS samples against R. mucilaginosa JCM 8115T.

2.6. Evaluation of Effects of pH, Heating, and Enzymes on Antiyeast Activity

To evaluate the effect of pH on the antiyeast activity of the selected strain, 3121M0s, the lyophilized CFS was dissolved in hydrochloric acid-potassium chloride buffer (pH 2.0), in citrate-phosphate buffer (pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0), and carbonate-bicarbonate buffer (pH 9.0 and 10.0) to prepare each 10× CFS, followed by filtration through a 0.45 µm filter. The 10× CFS (pH 2.0–10.0) were used for the agar–well diffusion antiyeast activity assay against R. mucilaginosa JCM 8115T.
Next, to evaluate the effect of heating, the 10× CFS (pH 4.0) was treated at 95 °C for 10 min and 121 °C for 15 min. Then, it was used for the activity assay in the same manner as above.
Moreover, to evaluate the effects of enzymatic treatments, 1 mL of CFS of the selected strain, 3121M0s, was reacted with 5 mg of 47,031 U/mg catalase (pH 7.0, 25 °C; Sigma Aldrich; St. Louis, MO, USA), 1650 U/mg pepsin (pH 2.0, 37 °C; Sigma-Aldrich), 1300 U/mg trypsin (pH 8.0, 37 °C; Fujifilm Wako Pure Chemical Corporation; Osaka, Japan), 66 U/mg α-chymotrypsin (pH 7.8, 25 °C; Nacalai Tesque, Kyoto, Japan) and 38.6 U/mg proteinase K (pH 7.5, 37 °C; Nacalai Tesque) for 1 h. After the reaction, the CFS was inactivated by heating at 95 °C for 10 min. Then, each 10× CFS (pH 4.0) was prepared and assayed for the antiyeast activity in the same manner as above.

2.7. Ethyl Acetate Extraction

CFS of the selected strain, 3121M0, and the control strain, JCM 1149T, were adjusted to pH 2.0 using 1–6 M HCl, and then mixed well with ethyl acetate (Fujifilm Wako Pure Chemical) at a ratio of 1:2 in a separating funnel. After leaving them for 15 min, the upper layer (organic fraction) was collected and further mixed well with ethyl acetate again. This step was repeated three times. After that, ethyl acetate solvent was removed using a rotary evaporator (Tokyo Rikakikai Co., Ltd.; Tokyo, Japan), and the concentrated extracts were diluted to one-tenth the volume of the original CFS using citrate-phosphate buffer (pH 4.0). The extracts obtained here were also assayed using the agar–well diffusion method against R. mucilaginosa JCM 8115T.

2.8. HPLC and MS

To separate OA, the ethyl acetate extracts obtained above were subjected to reverse-phase HPLC (Shimadzu Corporation, Kyoto, Japan) with a YMC Pack ODS-AM column (4.6 mm × 250 mm; YMC Co., Ltd.; Kyoto, Japan) at 30 °C. Elution was performed with aqueous acetonitrile containing 0.05% (v/v) trifluoroacetic acid at a flow rate of 1 mL/min. The gradient program for the elution was as follows: 0–5 min, 5% (v/v) acetonitrile; 5–40 min, 5 to 90% (v/v) acetonitrile; and 40–45 min, 90% (v/v) acetonitrile. The eluate was monitored at a wavelength of 220 nm. As analytical standards for qualification and quantification, special grade reagents of AA (Nacalai Tesque), benzoic acid (Fujifilm Wako Pure Chemical), caffeic acid (Tokyo Chemical Industry Co., Ltd.; Tokyo, Japan), 4-hydroxybenzoic acid (OHBA; Tokyo Chemical Industry), 4-hydroxyphenyllactic acid (OHPLA; Tokyo Chemical Industry), 3-(4-hydroxyphenyl)propionic acid (Tokyo Chemical Industry), DL-lactic acid (LA; Nacalai Tesque), L-malic acid (Nacalai Tesque), PLA (Tokyo Chemical Industry), and succinic acid (Nacalai Tesque) were used.
LA was further distinguished between D- and L-forms using chiral reverse-phase HPLC with a Sumichiral OA-5000 column (4.6 mm × 150 mm; Sumika Chemical Analysis Service, Ltd.; Osaka, Japan) at 25 °C. Elution was isocatically performed with 5% (v/v) 2-propanol containing 2 mM copper sulfate at a flow rate of 1 mL/min. The eluate was monitored at a wavelength of 254 nm. As analytical standards for qualification and quantification, special grade reagents of D- and L-LA (Tokyo Chemical Industry) were used.
The OAs separated by HPLC and tentatively identified based on their retention times were further reliably identified based on their molecular weight information obtained using a mass spectrometer, JMS-700N (JEOL Ltd.; Tokyo, Japan), in the Common Facilities Division of the Office for Research Initiative and Development at Nagasaki University, Japan. Among the OA, only LA was analyzed by the positive-ion mode of fast atom bombardment mass spectrometry (FAB-MS) with glycerol as a matrix. The other OAs were analyzed by electron ionization mass spectrometry (EI-MS).

2.9. Evaluation of Effects of Adding Tyrosine and Phenylalanine into Culture Medium

It is known that the addition of L-tyrosine (Tyr) and L-phenylalanine (Phe) to the medium increases the production of OHPLA and PLA by LAB, respectively [62]. To evaluate the effects of the two amino acids on OHPLA and PLA production and the antiyeast activity of the 10× CFS, modified MRS broth supplemented with 0.4% Tyr and/or Phe was used to cultivate the selected strain, 3121M0s. After cultivation at 30 °C for 48 h, the CFS and 10× CFS (pH 4.0) were prepared. Then, the CFS was analyzed to quantify the content of OHPLA and PLA, and the 10× CFS was used for the antiyeast activity assay against R. mucilaginosa JCM 8115T in the same manner as above.

2.10. Statistical Analysis

Data are presented as the mean ± standard deviation. The data were statistically evaluated using a two-way analysis of variance (ANOVA) with Tukey’s test in IBM SPSS Statistics 20.0. Differences were considered significant at p < 0.05.

3. Results

3.1. Selection of Strain 3121M0s with High Antiyeast Activity

As the first screening, 236 LAB strains were assayed for their antiyeast activity using the overlay method against the three indicator yeast strains. Among them, four strains (3121M0s, 3272B0, 3273B0, and 3461M0), which were formerly isolated from Mongolian traditional fermented milk, airag [56], showed relatively high activity against the two food spoilage yeast strains, Rhodotorula mucilaginosa JCM 8115T and Candida parapsilosis JCM 1612T (Table 1). The four strains were next subjected to the antiyeast activity assay using the agar well diffusion method. As a result, only strain 3121M0s showed antiyeast activity against R. mucilaginosa JCM 8115T (Table 1), and, therefore, this strain was selected for further experiments.
The selected strain, 3121M0s, was assayed for the antiyeast activity against the other eight strains of food spoilage yeasts. The strain exhibited activity against the five and four yeast strains on the overlay and agar–well diffusion methods, respectively (Table 2). The activity was particularly high against the type strain of Debaryomyces hansenii, generally with high tolerance to osmotic stress, similar to that against R. mucilaginosa.

3.2. Evaluation of Antiyeast Effect of Strain 3121M0s in Skim Milk

To verify the antiyeast effect of strain 3121M0s in actual food, cocultivation with R. mucilaginosa JCM 8115T was performed in RSM. As a result, strain 3121M0s inhibited the growth of R. mucilaginosa while maintaining its own acid production or growth (Figure 1).

3.3. Species Identification of Strain 3121M0s

To identify the species of strain 3121M0s, its 16S rRNA gene was sequenced. The resulting sequence (LC884927) had almost 100% identity to strains of the L. plantarum-group LAB. A similar result was also observed in a phylogenetic tree constructed based on the 16S rRNA gene sequences of strain 3121M0s and the type strains of all species of the genus Lactiplantibacillus (Figure S1). Next, for further identification, the species-specific multiplex PCR was performed. The resulting electrophoretic profile shown in Figure S2 concluded that strain 3121M0s was identified as L. plantarum.

3.4. Determination of Optimal Culture Conditions of Strain 3121M0s

To determine the optimal incubation temperature for strain 3121M0s, the culture turbidity was measured sequentially during cultivation at 25, 30, and 37 °C for 72 h. The growth at 30 °C was faster and higher than at 25 and 37 °C, respectively (Figure S3). From the result, 30 °C was selected as the incubation temperature for subsequent experiments.
Culture pH and cell viability at 30 °C were also measured with the antiyeast activity of 10× CFS of strain 3121M0s against R. mucilaginosa JCM 8115T to determine the optimal incubation time. The growth peaked at 12–36 h, but the activity was relatively higher at 24–72 h (Figure 2). Therefore, the subsequent incubation time was set to 48 h.

3.5. Evaluation of pH, Heating, and Enzymes on Antiyeast Activity of Strain 3121M0s

To evaluate the effect of pH on the antiyeast activity of strain 3121M0s, the activity assay of the 10× CFS was performed against R. mucilaginosa JCM 8115T under pH 2.0–10.0. The activity was observed at pH 2.0–4.0, not pH 5.0–10.0 (Figure 3). No activity was also observed in 10× MRS broth at pH 2.0–10.0 used as a control.
Next, the effect of heating (95 °C for 10 min and 121 °C for 15 min) was evaluated. As a result, 121 °C for 15 min slightly reduced the activity, but 95 °C for 10 min was unaffected (Figure 4).
Then, the effects of catalase and proteases were also evaluated. Catalase had no observable effect, while trypsin, α-chymotrypsin, and proteinase K, among the proteases, slightly reduced the activity (Figure 4). In addition, pepsin markedly reduced the activity, but sufficient intensity remained after the reaction. These results meant that the primary antiyeast substances of strain 3121M0s were neither H2O2 nor peptides.

3.6. Identification and Quantification of Organic Acids Produced by Strain 3121M0s

Antiyeast substances produced by strain 3121M0s were separated using ethyl acetate extraction. The antiyeast activity was strongly detected in the organic fraction (upper layer), while weakly detected in the aqueous fraction (lower layer). The organic fraction was subjected to HPLC after evaporation, followed by redissolving for further analysis.
In the HPLC analysis, several peaks were detected, and five OAs (LA, AA, OHPLA, OHBA, and PLA) were roughly identified based on their retention times compared to those of the standard chemicals (Figure 5). More reliable identification was achieved using EI- and FAB-MS (Figure S4). In addition, the chirality (D- and L-forms) of LA was identified using another HPLC (Figure 6). Then, the concentrations of the five OAs and the two isomers were calculated from the HPLC peak areas obtained. In the CFS of strain 3121M0s, LA (16,053.0 mg/L = 178 mM) was the most abundant OA, and the ratio of the D- and L-forms was 2:1 (Table 3). The second most abundant OA was AA (1437.9 mg/L = 23.9 mM), followed by PLA (99.1 mg/L = 0.60 mM), OHBA (26.9 mg/L = 0.195 mM), and OHPLA (28.7 mg/L = 0.158 mM).

3.7. Antiyeast Activity of Organic Acids Produced by Strain 3121M0s

Based on the results in Section 3.6 and Table 3, the concentrations of each OA in the 10× CFS were calculated, and the antiyeast activity of the individual and mixed OA solutions was assayed against R. mucilaginosa JCM 8115T. The total OA mixture in the 10× CFS exhibited slightly lower activity than that of the whole 10× CFS, but the difference was not so large (Table 4).
In the assay with the individual OA, only AA showed low activity, while no activity was observed with the other OA (Figure 7A). Next, the antiyeast activity of the mixtures excluding one OA was assayed. The mixtures, excluding AA or LA, completely or partially lost the activity, respectively (Figure 7B). Exclusion of OHPLA, OHBA, or PLA had no observable effect on the activity. Moreover, a combination of AA and another OA was tested. The combination of AA and LA exhibited complete activity equal to the total OA mixture (Figure 7C). The combination of AA and PLA showed moderate activity, and OHPLA and PHBA had no observable effect on the activity of AA. These results indicated that the main antiyeast substance produced by 3121M0s was AA, and that LA and PLA enhanced the activity of AA. In addition, there was no difference in effect between D- and L-forms of LA (Figure 7B,C).

3.8. Comparison of Organic Acid Productivity and Antiyeast Activity Between Strains 3121M0s and JCM 1149T

To verify the superiority of strain 3121M0s in terms of OA productivity and antiyeast activity, it was compared with the type strain (JCM 1149T) of L. plantarum. Strain JCM 1149T showed nearly identical levels (16,244.4 mg/L = 180 mM) of LA production to 3121M0s, and their D/L-form ratio was also similar, at approximately 2/1 (Table 3). However, the production levels of AA (902.2 mg/L = 15.0 mM), OHPLA (3.0 mg/L = 0.016 mM), and PLA (69.2 mg/L = 0.42 mM) in JCM 1149T were clearly lower than those in 3121M0s. Conversely, the production of OHBA was higher (34.4 mg/L = 0.249 mM) in JCM 1149T. Regarding antiyeast activity, the 10× CFS and the total OA mixture of JCM 1149T were significantly inferior to those of 3121M0s (Table 4). Thus, strain 3121M0s had superior antiyeast activity, as well as AA, OHPLA, and PLA productivity, compared to strain JCM 1149T.

3.9. Effects of L-Tyrosine and L-Phenylalanine on Organic Acid Productivity and Antiyeast Activity of Strain 3121M0s

The effects of adding Tyr and Phe into the culture broth on the OHPLA and PLA production from strain 3121M0s were evaluated. The addition of Tyr increased the OHPLA production (28.7 to 125.4 mg/L = 0.688 mM) but significantly decreased the PLA production (99.1 mg/L to trace) (Table 5). Conversely, the addition of Phe increased the PLA production (99.1 to 126.5 mg/L = 0.76 mM), but inhibited the OHPLA production (28.7 mg/L to trace). Furthermore, the co-addition of both amino acids suppressed the increased OHPLA production with Tyr alone (125.4 to 84.9 mg/L), and reduced the PLA production even compared to the control without adding them (99.1 to 88.9 mg/L). Although such changes in the OHPLA and PLA production were observed with the addition of Tyr and Phe, no significant changes were observed in the antiyeast activity against R. mucilaginosa JCM 8115T of their 10× CFS.

4. Discussion

With the aim of developing a useful antiyeast biopreservative starter for fermented foods, strain 3121M0s with comparatively high activity was selected from 236 strains in our LAB library (Table 1). Although the strain exhibited no inhibitory effects against I. orientalis, P. kudriavzevii, and W. anomalus, it showed low antiyeast activity against each type strain of C. parapsilosis and S. cerevisiae, and pronounced activity against R. mucilaginosa, D. hansenii, Hanseniaspora uvarum, K. marxianus, and Z. rouxii (Table 1 and Table 2). The relatively substantial inhibition observed particularly against R. mucilaginosa and D. hansenii aligns with previous reports highlighting their high sensitivity to antiyeast LAB [63,64,65,66,67]. Many studies have consistently reported that the main yeast contaminants in raw milk and non-fermented dairy products, such as cream and butter, were overwhelmingly Candida spp. [9,12,13,14]. However, in traditional fermented milk and cheese, Candida spp. were not the only prevalent yeasts; rather, D. hansenii, K. marxianus, and Kluyveromyces lactis were commonly detected at high abundance and considered to be the primary spoilage yeasts [6,9,10,14,17,18,19,24,25]. Also, in airag, K. marxianus was reported to be the most dominant yeast [45]. Although Rhodotorula spp. are generally non-dominant yeasts, they have been widely detected in raw milk and non-fermented and fermented dairy products [6,9,10,12,13,14,17,18,19,24,25]. In addition, Rhodotorula spp. are known to deteriorate the appearance and flavor of products due to their reddish-pink color and high lipolytic activity [9,13]. Figure 1 shows that strain 3121M0s completely inhibited 102 CFU/mL of R. mucilaginosa cocultivated in RSM. The effect was notably stronger than previously reported results of the other antiyeast LAB [63,66,67]. It is known that the number of contaminating yeast in traditional fermented milk and cheese can often reach 104 CFU/mL or more, but the yeast counts in raw milk and dairy products in the early fermentation stages are less than 102–3 CFU/mL [6,9,18,19,24,25,66,67]. Therefore, the significant inhibitory effects not only on R. mucilaginosa but also on D. hansenii and K. marxianus in the agar–well diffusion assay (Table 1 and Table 2), and the inhibition on R. mucilaginosa in the cocultivation test (Figure 1) demonstrate a high practical potential of strain 3121M0 for biopreservation in fermented dairy products.
The species of strain 3121M0s was identified as L. plantarum (Supplemental Figures S1 and S2). L. plantarum is one of the most versatile LAB species with ecological and metabolic adaptability based on its larger genome and higher genetic diversity compared to other LAB species, and inhabits a wide range of ecological niches, including plants, meats, the mammalian gastrointestinal tract, and fermented foods, including traditional products such as airag [68,69,70,71]. Due to such versatility, L. plantarum strains are used as safe and highly functional starter and probiotic cultures in dairy, meat, and many plant-based fermentations such as Feta cheese, dry sausage, sauerkraut, pickles, sourdough, and kimchi. In fact, various fermentation starter cultures, including L. plantarum, are commercially available.
L. plantarum is also known as the most frequently reported LAB species with antifungal activity [16,30,31]. In this study, the optimal culture conditions for strain 3121M0s were determined at 30 °C for 48 h based on its antiyeast activity in order to streamline the experiment (Supplemental Figure S3 and Figure 2). The antiyeast activity of 3121M0s-CFS was detected on and after 12 h of incubation at 30 °C, and was maintained at high levels for 24–72 h. Traditional fermented milk products are generally produced by incubation at ambient temperature, depending on climatic conditions, 15–45 °C, from one night to three days [25,26,27,72]. In the case of airag, incubation at 20–30 °C for 12–24 h is general [46,49,72]. These findings indicate that some antiyeast substances would be produced by L. plantarum in traditional fermented milk, including airag. This indication is supported by our previous study that another L. plantarum strain derived from airag significantly decreased the ethanol production from S. cerevisiae 4C cocultivated in RSM [50]. In addition, the primary antiyeast substance from strain 3121M0s was determined to be AA (Figure 7). AA production has actually been confirmed in airag in the other reports [46,73].
The antifungal effect of AA involves lowering the environmental pH beyond the growth limits of target organisms, similar to other OAs [30,31]. In addition, AA is unique in that its higher pKa allows a greater proportion of undissociated molecules at comparable pH levels, facilitating membrane permeation. Once inside the cell, AA dissociates, causing intracellular acidification and accumulation of anions, leading to metabolic inhibition and stronger growth suppression than other OA. In addition, AA is known to have a synergistic antimicrobial effect with LA [74,75], and this phenomenon was clearly observed also in this antiyeast study (Figure 7B,C). The antifungal effect of LA itself has not been clearly investigated, and to our knowledge, its effect alone against yeast has not been confirmed. In fact, no effects were observed in this study for LA alone or LA combined with any OA used here except AA (Figure 7A,B). The synergistic effect of LA and AA is thought to be exerted when a large amount of LA lowers the environmental pH, thereby reaching a pH range that increases the amount of undissociated AA [74,75]. In addition, it is thought that LA, together with AA, permeates the membrane, further lowering the intracellular pH and inhibiting metabolism of the target cells. Similar to LA, PLA did not show any antiyeast activity on its own, but a synergistic effect was observed with AA (Figure 7A,C). The reason that the removal of PLA had no observed effect on the activity of the mixture in Figure 7B is thought because the combination of AA and LA in the mixture already reached the maximum activity. PLA is known to disrupt membrane integrity, causing leakage of intracellular components, and to inhibit DNA replication through intercalation [76]. Based on this, the synergistic effect of AA and PLA can be explained as follows: AA lowers the pH, increasing the proportion of undissociated PLA (pKa 3.5–3.8), which improves membrane permeability and, as a result, facilitates the penetration of AA into the target bacterial cells [32]. However, in this research, pH was adjusted to 4.0 in most cases, so the synergistic effect between AA and the other two OAs is unlikely to be due to an increase in their undissociated forms. Conversely, the increase in antiyeast activity observed by lowering solution pH is not due to the pH falling below the growth-permissible range of the target yeast strain (R. mucilaginosa JCM 8115T), because no activity was observed in the pH-adjusted control without OAs (Figure 3). Instead, it is likely due to an increase in the undissociated forms of each OA. Additionally, OHPLA and OHBA were detected in HPLC (Figure 5 and Table 3), but due to their low concentrations, neither showed individual activity nor synergistic effects with the other OAs (Figure 7). Also, other OAs known to have antiyeast activity, such as those used as standards in HPLC, were not detected by this analytical method.
L. plantarum is a facultative heterofermentater known to produce relatively high levels of AA, compared to other LAB that are not obligate heterofermentaters [77]. Although the mechanism of AA production by L. plantarum has not been fully elucidated, it can be generally described as follows [77,78,79,80]. During the exponential phase under static cultivation in milk or MRS broth, both of which are rich in glucose (hexose), L. plantarum metabolizes glucose to pyruvic acid through anaerobic glycolysis and subsequently converts it almost exclusively to LA by lactate dehydrogenase. However, mainly in the early stationary phase, as glucose decreases and NADH accumulates, repression by catabolite control protein A (CcpA) is alleviated, leading to the upregulation of enzymes involved in mixed acid fermentation. Consequently, pyruvic acid is transformed into acetyl phosphate anaerobically by pyruvate formate lyase and phosphotransacetylase, or aerobically by pyruvate oxidase. Finally, AA is generated from acetyl phosphate by acetate kinase. In strain 3121M0s, although LA production was equivalent to that of the type strain, JCM 1149T, AA production was approximately 1.6 times higher (Table 3). This may be due to the differences in the activity of AA biosynthesis-related enzymes and/or their expression levels affected by CcpA. In addition, since ATP is generated by acetate kinase in AA production, it is predicted that the bacterial growth would improve. In fact, between the two strains, although there was no significant difference in culture pH because lactic acid production was equivalent, there was a clear difference in culture turbidity (Supplementary Figure S5). Considering the future use of strain 3121M0s, further optimization of cultivation conditions is required to achieve higher AA production, as it is known that AA production increases by oxygenating as well as restricting glucose [77,78,79,80].
Besides, strain 3121M0s produced approximately 1.4 times more PLA than the type strain (Table 3). This is also thought to contribute to the higher antiyeast activity of 3121M0s (Table 4). The PLA production furthermore increased approximately 1.3-fold (Table 5) when 0.4% Phe was added to the medium based on previous reports [62]. Similarly, the addition of 0.4% Tyr increased OHPLA production by 4.4-fold. However, no increase in activity was observed with the addition. Phe and Tyr are oxidatively deaminated to phenylpyruvic acid and 4-hydroxyphenylpyrubic acid by an aromatic aminotransferase, and subsequently they are converted to PLA and OHPLA by a hydroxyl acid dehydrogenase, respectively [81,82,83]. These reactions, which are NADH-dependent, explain the increased PLA and OHPLA production by the addition of Phe and Tyr, and also contribute to the elimination of the accumulated NADH. Interestingly, the Phe addition led to an increase in PLA alongside a significant decrease in OHPLA production, while the Tyr addition resulted in an increase in OHPLA production alongside a significant decrease in PLA production. Similar reports have been published in the past, and this is thought to be due to the use of the same enzymes in the production of both OAs [62]. Despite the increased production of PLA and OHPLA, the activity remained unchanged, which is thought to be due to their effect being masked by the already high effect of the combination of AA and LA.
In this study, the CFS of strain 3121M0s was extracted with ethyl acetate, and the organic layer was used for qualitative and quantitative analyses of OA. A slight difference in activity was observed between 10× CFS and the corresponding OA mixture (Table 4), and heat (at 121 °C for 15 min) and protease treatments led to a slight decrease in activity (Figure 4). These results suggest the presence of proteinaceous antiyeast substances, similar to those previously reported [34,35,36,37,38,84]. Since these substances might be present in another (aqueous) layer on the ethyl acetate separation, current efforts are focused on identifying antifungal substances in the layer.
In conclusion, this study is the first to comprehensively characterize the antiyeast activity and synergistic effect of OAs produced by L. plantarum 3121M0s isolated from traditional Mongolian fermented milk, airag. The detailed analysis of both individual and combined effects of LA, AA, and PLA provided clear evidence of their synergistic antiyeast activity, which had previously only been vaguely described. Given the large genome and complex metabolism of L. plantarum [85], further studies are needed to fully elucidate its antifungal capabilities. As L. plantarum 3121M0s is capable of milk fermentation, it holds promise as a biopreservative starter for fermented dairy foods produced in open environments without final sterilization, where yeast contamination is a significant concern.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microorganisms13092017/s1, Figure S1: Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strain 3121M0s with the type strains of all species of the genus Lactiplantibacillus; Figure S2: Electrophoresis profile of the recA multiplex PCR amplicons; Figure S3: Changes in culture turbidity of strain 3121M0s cultivated in MRS broth at 25, 30, and 37 °C (light blue, red, and yellow green, respectively) for 72 h; Figure S4: FAB-MS (a,b) and EI-MS (c–j) spectra of standard chemicals (a,c,e,g,i) and fractions of HPLC peaks (b,d,f,h,j) described in Figure 5; Figure S5: Changes in culture pH (broken lines) and turbidity (solid lines) of Lactiplantibacillus plantarum 3121M0s (light blue) and JCM 1149T (red) cultivated in MRS broth at 30 °C for 72 h.

Author Contributions

Conceptualization, K.A. and T.M.; methodology, K.A., Y.M.-u. and T.M.; experiments, M.B.L., Y.M.-u. and T.U.; data analysis, M.B.L., Y.M.-u. and K.A.; resources, K.A. and T.M.; writing—original draft preparation, M.B.L. and Y.M.-u.; writing—review and editing, K.A.; supervision, K.A. and T.M.; project administration, K.A. and T.M.; funding acquisition, K.A. and T.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by JSPS KAKENHI, grant numbers 25450408 for T.M. and 22K02091 for K.A., and by a management expense grant to the Laboratory of Animal Applied Microbiology, Faculty of Agriculture, Okayama University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genetic data from this study have been deposited to DDBJ under accession number LC884927. The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We sincerely thank Nobuaki Tsuda in the Common Facilities Division of the Office for Research Initiative and Development at Nagasaki University, Japan, for his proper operation of the mass analysis.

Conflicts of Interest

Author Taku Miyamoto was employed by the companies Minori Co., Ltd., and Functional Food Creation Research Institute Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
LABLactic acid bacteria
AAAcetic acid
PLA3-Phenyllactic acid
MRSDe Man, Rogosa, and Sharp
YMYeast and Mold
RSMReconstituted skim milk
S.D.Standard deviation
CFUColony forming unit
CFSCell-free culture supernatant
10× CFSTenfold concentrated cell-free culture supernatant
OAsOrganic acids
rRNARibosomal ribonucleic acid
PCRPolymerase chain reaction
DNADeoxyribonucleic acid
BLASTBasic Local Alignment Search Tool
NCBINational Center for Biotechnology Information
DDBJDNA Data Bank Japan
ODOptical density
HPLCHigh performance liquid chromatography
OHBA4-Hydroxybenzoic acid
OHPLA4-hydroxyphenyllactic acid
LALactic acid
MSMass spectrometry
FAB-MSFast atom bombardment mass spectrometry
EI-MSElectron ionization mass spectrometry
TyrTyrosine
PhePhenylalanine
ANOVAAnalysis of variance
NADHReduced nicotinamide adenine dinucleotide
CcpAcatabolite control protein A
ATPAdenosine triphosphate

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Microorganisms 13 02017 g001
Figure 1. Changes in culture pH (gray), and cell viability of strain 3121M0s (light blue) and Rhodotorula mucilaginosa JCM 8115T (yellow green) during mono- (A,B) or co-cultivation (C) in reconstituted skim milk at 25 °C for 72 h.
Figure 1. Changes in culture pH (gray), and cell viability of strain 3121M0s (light blue) and Rhodotorula mucilaginosa JCM 8115T (yellow green) during mono- (A,B) or co-cultivation (C) in reconstituted skim milk at 25 °C for 72 h.
Microorganisms 13 02017 g001
Microorganisms 13 02017 g002
Figure 2. Changes in culture pH (light blue), cell viability (red), and antiyeast activity against Rhodotorula mucilaginosa JCM 8115T (yellow green) of strain 3121M0s cultivated in MRS broth at 30 °C for 72 h.
Figure 2. Changes in culture pH (light blue), cell viability (red), and antiyeast activity against Rhodotorula mucilaginosa JCM 8115T (yellow green) of strain 3121M0s cultivated in MRS broth at 30 °C for 72 h.
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Microorganisms 13 02017 g003
Figure 3. Effect of pH (2–10) on antiyeast activity against Rhodotorula mucilaginosa JCM 8115T of strain 3121M0s. Different lowercase letters denote a significant difference (p < 0.05).
Figure 3. Effect of pH (2–10) on antiyeast activity against Rhodotorula mucilaginosa JCM 8115T of strain 3121M0s. Different lowercase letters denote a significant difference (p < 0.05).
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Microorganisms 13 02017 g004
Figure 4. Effects of heating (121 °C, 15 min) and enzymatic treatment (catalase and four proteases) on antiyeast activity against Rhodotorula mucilaginosa JCM 8115T of strain 3121M0s. Different lowercase letters denote a significant difference (p < 0.05).
Figure 4. Effects of heating (121 °C, 15 min) and enzymatic treatment (catalase and four proteases) on antiyeast activity against Rhodotorula mucilaginosa JCM 8115T of strain 3121M0s. Different lowercase letters denote a significant difference (p < 0.05).
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Microorganisms 13 02017 g005
Figure 5. HPLC chromatogram of extract from the cell-free culture supernatant of strain 3121M0s with ethyl acetate. MRS broth extract was used as a control. Standards: (A) DL-lactic acid; (B) acetic acid; (C) 4-hydroxyphenyllactic acid; (D) 4-hydroxybenzoic acid; and (E) 3-phenyllactic acid.
Figure 5. HPLC chromatogram of extract from the cell-free culture supernatant of strain 3121M0s with ethyl acetate. MRS broth extract was used as a control. Standards: (A) DL-lactic acid; (B) acetic acid; (C) 4-hydroxyphenyllactic acid; (D) 4-hydroxybenzoic acid; and (E) 3-phenyllactic acid.
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Microorganisms 13 02017 g006
Figure 6. Chiral HPLC chromatogram of extract from the cell-free culture supernatant of strain 3121M0s with ethyl acetate. Reagents D-and L-lactic acids were used as standards.
Figure 6. Chiral HPLC chromatogram of extract from the cell-free culture supernatant of strain 3121M0s with ethyl acetate. Reagents D-and L-lactic acids were used as standards.
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Microorganisms 13 02017 g007
Figure 7. Antiyeast activity of the tenfold concentrated solutions (pH 4.0) of single (A) and mixed (B,C) organic acids contained in the cell-free culture supernatant of strain 3121M0s. (B) The activity of tenfold mixtures of all organic acids except one. (C) The activity of tenfold mixtures containing acetic acid and another organic acid. The antiyeast activity was measured against Rhodotorula mucilaginosa JCM 8115T. Used organic acids: LA, lactic acid; AA, acetic acid; OHPLA, 4-hydroxyphenyllactic acid; OHBA, 4-hydroxyphenylbenzoic acid; PLA, 3-phenyllactic acid; and Mix, mixture of these organic acids. Different lowercase letters denote a significant difference (p < 0.05).
Figure 7. Antiyeast activity of the tenfold concentrated solutions (pH 4.0) of single (A) and mixed (B,C) organic acids contained in the cell-free culture supernatant of strain 3121M0s. (B) The activity of tenfold mixtures of all organic acids except one. (C) The activity of tenfold mixtures containing acetic acid and another organic acid. The antiyeast activity was measured against Rhodotorula mucilaginosa JCM 8115T. Used organic acids: LA, lactic acid; AA, acetic acid; OHPLA, 4-hydroxyphenyllactic acid; OHBA, 4-hydroxyphenylbenzoic acid; PLA, 3-phenyllactic acid; and Mix, mixture of these organic acids. Different lowercase letters denote a significant difference (p < 0.05).
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Table 1. Antiyeast activity of 236 strains and tenfold concentrates (pH 4.0) of the cell-free culture supernatants of the selected four strains of lactic acid bacteria against three strains of food-related yeasts using the overlay and agar–well diffusion methods, respectively.
Table 1. Antiyeast activity of 236 strains and tenfold concentrates (pH 4.0) of the cell-free culture supernatants of the selected four strains of lactic acid bacteria against three strains of food-related yeasts using the overlay and agar–well diffusion methods, respectively.
StrainOverlay Assay * Against Agar–Well Diffusion Assay ** Against
Rhodotorula
mucilaginosa		Candida
parapsilosis		Saccharomyces
cerevisiae		 Rhodotorula
mucilaginosa		Candida
parapsilosis		Saccharomyces
cerevisiae
JCM 8115TJCM 1612T4C JCM 8115TJCM 1612T4C
3121M0s+++++- 10.63 ± 0.45--
3272B0+++++- ---
3273B0+++++- ---
3461M0+++++- ---
123+++±- N.D.N.D.N.D.
IB3781+++±- N.D.N.D.N.D.
NMCB2521+++±- N.D.N.D.N.D.
NMCM2521+++±- N.D.N.D.N.D.
3070+++-- N.D.N.D.N.D.
301102S+++-- N.D.N.D.N.D.
425+++-- N.D.N.D.N.D.
IM376+++- N.D.N.D.N.D.
IB374++- N.D.N.D.N.D.
JCM 1120T-+- N.D.N.D.N.D.
2253RB0-+- N.D.N.D.N.D.
5890--± N.D.N.D.N.D.
50 strains++-- N.D.N.D.N.D.
77 strains+-- N.D.N.D.N.D.
93 strains--- N.D.N.D.N.D.
* -, no activity; ±, growth delay; +, inhibitory diameter (ID) < 11 mm; ++, ID 11–15 mm; and +++, ID > 15 mm. ** The value represents ID length (mean ± S.D.; mm). -, no activity; and N.D., No data.
Table 2. Antiyeast activity of strain 3121M0s against eight strains of food spoilage and fermentation yeasts using the overlay and agar–well diffusion methods.
Table 2. Antiyeast activity of strain 3121M0s against eight strains of food spoilage and fermentation yeasts using the overlay and agar–well diffusion methods.
AssayDebaryomyces
hansenii		Hanseniaspora
uvarum		Issatchenkia
orientalis		Kluyveromyces
marxianus		Pichia
kudriavzevii		Saccharomyces
cerevisiae		Wickerhamomyces
anomalus		Zygosaccharomyces
rouxii
JCM 1990TJCM 2184TJCM 1710TJCM 1617TJCM 3536TJCM 7255TJCM 3585TJCM 2325T
Overlay *+++-+-+-+
Agar–well diffusion **10.83 ± 0.248.67 ± 0.24-7.00 ± 0.00---7.33 ± 0.24
* -, no activity; +, inhibitory diameter (ID) < 11 mm; ++, and ID 11–15 mm. ** Tenfold concentrate (pH 4.0) of the cell-free culture supernatant of strain 3121M0s was used as a sample. The value represents ID length (mean ± S.D.; mm). -, no activity.
Table 3. Contents of organic acids in the cell-free culture supernatants (CFS) of Lactiplantibacillus plantarum 3121M0s and JCM 1149T, calculated based on the area of each HPLC peak.
Table 3. Contents of organic acids in the cell-free culture supernatants (CFS) of Lactiplantibacillus plantarum 3121M0s and JCM 1149T, calculated based on the area of each HPLC peak.
IngredientConcentration (mg/L)
3121M0s-CFSJCM 1149T-CFS
DL-Lactic acid16,053.016,244.4
(D-form:L-form)(10,981.0:5072.0)(11,571.9:4672.5)
Acetic acid1437.9902.2
4-Hydroxyphenyllactic acid28.73.0
4-Hydroxybenzoic acid26.934.4
3-Phenyllactic acid99.169.2
Table 4. Antiyeast activity of the tenfold cell-free culture supernatants (pH 4.0) and organic acids mixtures (pH 4.0) of Lactiplantibacillus plantarum 3121M0s and JCM 1149T against Rhodotorula mucilaginosa JCM 8115T.
Table 4. Antiyeast activity of the tenfold cell-free culture supernatants (pH 4.0) and organic acids mixtures (pH 4.0) of Lactiplantibacillus plantarum 3121M0s and JCM 1149T against Rhodotorula mucilaginosa JCM 8115T.
SolutionAntiyeast Activity (mm; Mean ± S.D.) *
3121M0sJCM 1149TMRS Broth
Tenfold culture supernatant **10.63 ± 0.45 a7.00 ± 0.00 c- d
Tenfold organic acids mixture ***10.00 ± 0.00 b6.83 ± 0.24 cN.D.
* -, no activity; and N.D., no data. Different lowercase letters denote a significant difference (p < 0.05). ** The cell-free culture supernatants were obtained after cultivation of the two strains in MRS broth at 30 °C for 48 h. *** The mixtures of organic acids were prepared on the basis of the values shown in Table 3.
Table 5. Effects of adding L-tyrosine and L-phenylalanine to MRS broth on the production of 4-hydroxyphenyllactic acid and 3-phenyllactic acid, and on the antiyeast activity against Rhodotorula mucilaginosa JCM 8115T in the cell-free supernatant (CFS) of strain 3121M0s.
Table 5. Effects of adding L-tyrosine and L-phenylalanine to MRS broth on the production of 4-hydroxyphenyllactic acid and 3-phenyllactic acid, and on the antiyeast activity against Rhodotorula mucilaginosa JCM 8115T in the cell-free supernatant (CFS) of strain 3121M0s.
Additive to MRS BrothConcentration (mg/L) in 3121M0s-CFSAntiyeast Activity (mm; Mean ± S.D.) of 10-Fold 3121M0s-CFS (pH 4.0) *
4-Hydroxyphenyllactic Acid3-Phenyllactic Acid
No added28.799.110.63 ± 0.45 a
L-Tyrosine125.4Trace11.00 ± 0.00 a
L-PhenylalanineTrace126.511.00 ± 0.00 a
L-Tyrosin and L-phenylalanine84.988.911.00 ± 0.00 a
* Antiyeast activity assay was performed against Rhodotorula mucilaginosa JCM 8115T. Different lowercase letters denote a significant difference (p < 0.05).
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Lijon, M.B.; Matsu-ura, Y.; Ukita, T.; Arakawa, K.; Miyamoto, T. Identification and Characterization of Antiyeast Organic Acids Produced by Lactiplantibacillus plantarum 3121M0s Derived from Mongolian Traditional Fermented Milk, Airag. Microorganisms 2025, 13, 2017. https://doi.org/10.3390/microorganisms13092017

AMA Style

Lijon MB, Matsu-ura Y, Ukita T, Arakawa K, Miyamoto T. Identification and Characterization of Antiyeast Organic Acids Produced by Lactiplantibacillus plantarum 3121M0s Derived from Mongolian Traditional Fermented Milk, Airag. Microorganisms. 2025; 13(9):2017. https://doi.org/10.3390/microorganisms13092017

Chicago/Turabian Style

Lijon, Md. Bakhtiar, Yuko Matsu-ura, Takumi Ukita, Kensuke Arakawa, and Taku Miyamoto. 2025. "Identification and Characterization of Antiyeast Organic Acids Produced by Lactiplantibacillus plantarum 3121M0s Derived from Mongolian Traditional Fermented Milk, Airag" Microorganisms 13, no. 9: 2017. https://doi.org/10.3390/microorganisms13092017

APA Style

Lijon, M. B., Matsu-ura, Y., Ukita, T., Arakawa, K., & Miyamoto, T. (2025). Identification and Characterization of Antiyeast Organic Acids Produced by Lactiplantibacillus plantarum 3121M0s Derived from Mongolian Traditional Fermented Milk, Airag. Microorganisms, 13(9), 2017. https://doi.org/10.3390/microorganisms13092017

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