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. 2023 Feb 14;32(4):589–598. doi: 10.1007/s10068-023-01268-3

Gut microbiota modulation via short-term administration of potential probiotic kefir yeast Kluyveromyces marxianus A4 and A5 in BALB/c mice

Hye-Young Youn 1, Hyeon-Jin Kim 1, Dong-Hyeon Kim 1, Yong-Seok Jang 1, Hyunsook Kim 2, Kun-Ho Seo 1,✉
PMCID: PMC9992467  PMID: 36911334

Abstract

Kefir yeast, Kluyveromyces marxianus, has been evaluated for its potential probiotic properties—survivability, non-pathogenicity, and antioxidant and anti-microbial activities. However, host gut microbiota modulation of kefir yeasts remains unclear. Here, we compared kefir yeast strains K. marxianus A4 (Km A4) and K. marxianus A5 (Km A5) with Saccharomyces boulardii ATCC MYA-796 (Sb MYA-796) by investigating their adherence to colorectal adenocarcinoma (Caco-2) cells and gut microbiota modulation in BALB/c mice. The kefir yeast strains exhibited higher intestinal cell adhesion than Sb MYA-796 (p < 0.05). Bacteroidetes, Bacteroidales, and Bacteroides were more abundant in the 1 × 108 CFU/mL of Km A4 treatment group than in the control group (p < 0.05). Moreover, 1 × 108 CFU/mL of Km A5 increased Corynebacteriales and Corynebacterium compared to the 1 × 108 CFU/mL of Km A4 treatment group (p < 0.01). The results showed that Km A4 and Km A5 had good Caco-2 cell adhesion ability and modulated gut microbiota upon short-term administration in healthy mice.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-023-01268-3.

Keywords: Kefir, Kluyveromyces marxianus, Cell adhesion, Gut microbiota, Probiotics

Introduction

Kefir, a fermented milk product prepared using kefir grains, represents a complex microbial symbiosis of lactic acid bacteria (LAB; Lactobacillus, Lactococcus, Leuconostoc, and Streptococcus spp.), yeasts (Kluyveromyces and Saccharomyces spp.), and acetic acid bacteria (Acetobacter spp.) (Kim et al., 2020). Owing to positive interactions among these microorganisms, various benefits such as anti-microbial and anti-obesity effects and improvements in lactose tolerance and the host’s immune system have been reported (Guzel-Seydim et al., 2011).

Kluyveromyces marxianus, which constitutes over 95% of the yeast population of Korean kefir milk, has been investigated for its antioxidant and anti-microbial effects (Bae et al., 2020; Cho et al., 2018; Kim et al., 2020). In particular, K. marxianus has been extensively investigated for its physiological properties, including its survivability under aerobic and anaerobic conditions and high growth rate compared with Saccharomyces spp. (Wang et al., 2017). Microorganisms with high survivability can inhabit extreme gastrointestinal (GI) environments for a long time and attach to the intestinal surface and successfully colonize it (Van der Aa Kühle et al., 2005). Youn et al. (2022a) reported that K. marxianus A4 (Km A4) and K. marxianus A5 (Km A5) showed survivability greater than 2.1-fold in various GI conditions simulated in vitro, suggesting their potential to survive in the GI tract.

The human GI tract harbors over 1014 microorganisms, which are collectively referred to as the “gut microbiota.” (Sekirov et al., 2010). Probiotics positively modulate the host gut microbiota by suppressing harmful pathogens and promoting the growth of beneficial bacteria (Kumar et al., 2011). To date, Saccharomyces boulardii is the only probiotic yeast recognized through double-blind studies and reported to be effective as a preventive and therapeutic agent for antibiotic-associated diarrhea and recurrent Clostridioides difficile infections (Kotowska et al., 2005; Surawicz et al., 2000). Previous studies reported that administration of kefir milk substantially increased the LAB population of gut microbiota in the host (Kim et al., 2015; Kim et al., 2019). To understand the gut microbiota modulation role of individual microorganisms, kefir LAB such as Lactobacillus kefiranofaciens DN1, Lentilactobacillus kefiri DH5, and Leuconostoc mesenteroides LCM4 have been identified in several studies (Jeong et al., 2017; Kim et al., 2017; Kwon et al., 2019). However, to the best of our knowledge, no studies have investigated gut microbiota modulation after kefir yeast administration in a healthy mouse model.

During screening of potential probiotic agents, the safety of microbial strains should be considered. Thus far, we have found that Km A4 and Km A5 metabolize a wide range of substrates and show no remarkable safety concerns based on hematological and serological data obtained from BALB/c mice (Youn et al., 2022a; Youn et al., 2022b). Although one of the basic functions of probiotics is modulation of gut microbiota, our previous study mainly focused on the safety attributes of Km A4 and Km A5, given that the safety of probiotic candidates should be a prerequisite to evaluating their health functions (Hotel and Cordoba, 2001; Jang et al., 2023). Herein, to expand our knowledge on the functionality of kefir yeasts, their cell adhesion and gut microbiota modulation abilities were explored in vitro and in vivo, respectively. Modulation of gut microbiota was assessed using frozen-stored fecal samples obtained from a previous study (Youn et al., 2022b) wherein Km A4 and Km A5 were administered to healthy BALB/c mice for 2 weeks to generate a short-term consumption model.

Materials and methods

Yeast strains

We used kefir yeast strains (Km A4 and Km A5) isolated in our previous study (Youn et al., 2022a; Youn et al., 2022b). S. boulardii ATCC MYA-796 (Sb MYA-796) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Cells and cell culture

Human colorectal adenocarcinoma (Caco-2) cells were purchased from ATCC. Caco-2 cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene Inc., Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; GW Vitek Inc., Seoul, Republic of Korea) and 1% penicillin–streptomycin (PS; Welgene Inc., Gyeongsan, Republic of Korea) at 37 °C in a humidified 5% CO2 atmosphere.

Cell adhesion assay

As the adhesion ability of probiotics to gut epithelial cells is an important factor that affects their modulation of the gut microbiota (Monteagudo-Mera et al., 2019), a cell adhesion assay was performed to select potential probiotic yeast strains as described by Maccaferri et al. (2012) and Cho et al. (2018), with some modifications. Caco-2 cells (passage number 12) were seeded at a concentration of 1 × 105 colony forming units (CFU)/mL in 24-well plates (SPL Life Sciences, Gyeonggi, Republic of Korea) and cultured to 95–99% confluency. The Caco-2 monolayers were then washed twice with sterile phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA). The yeast strains Km A4, Km A5, and Sb MYA-796 were cultured in potato dextrose broth (PDB; Oxoid, Hampshire, United Kingdom) at 30 °C for 24 h. The overnight yeast cultures were suspended in 500 μL of free DMEM (DMEM without FBS and PS). Yeast cells (1 × 105 CFU/mL) were added to the Caco-2 cell cultures and incubated in a 5% CO2 atmosphere at 37 °C for 1 h. After incubation, the cells were washed three times with PBS (Sigma-Aldrich) to remove non-adherent yeast cells and lysed with 100 μL of Triton X-100 (1% v/v; Sigma-Aldrich). The lysates were spread on potato dextrose agar (Oxoid) and cultured at 30 °C for 24 h. The number of colonies was expressed as the total number of yeast cells that adhered to Caco-2 cells. Each adhesion assay was conducted in triplicate.

Animals and experimental design

A schematic flow chart of the animal experimental procedure is shown in Supplementary Data 1. The animal experiment, performed in our previous study (Youn et al., 2022b), was approved by the Konkuk University Animal Experiment Ethics Committee (KU20075). All institutional and national guidelines for the care and use of laboratory animals were followed. Briefly, specific pathogen-free 4-week-old female BALB/c mice were purchased from Orient Bio Inc. (Seongnam, Republic of Korea), acclimatized for 1 week, and then randomly divided into seven groups (n = 6 per group). Mice were fed a chow diet (Safe®, Augy, France; nutritional composition: nitrogen-free extract 60.7%, proteins 15.2%, moisture 12.1%, mineral ash 5.0%, cellulose 4.1%, and lipids 2.9%) with autoclaved tap water provided ad libitum. The mice were maintained at 20–25 °C under controlled lighting conditions (12 h light/dark cycle). To avoid cage effects, up to three mice were placed in a cage, aspen bedding was replaced once a week, and sanitized towels were provided for environmental enrichment.

Mice were gavaged with 0.2 mL of 0.9% sterile saline, Km A4, Km A5, and Sb MYA-796 at 16:00 h every day for 2 weeks. As the potential probiotic yeast used in this study does not have a standard amount for consumption, we used two concentrations of the yeast strains (1 × 108 and 1 × 106 CFU/mL) that were considered safe in our previous study (Youn et al., 2022b). Yeast strains were activated by subculturing twice at 30 °C for 24 h in PDB (Oxoid) and two concentrations (1 × 108 and 1 × 106 CFU/mL) were prepared daily. Group I (negative control containing 0.9% sterilized saline; NC_Saline) mice were gavaged with 0.2 mL of sterilized saline, whereas group II (high concentration of Km A4; Km A4_H) and group III (low concentration of Km A4; Km A4_L) mice were gavaged with Km A4 at 1 × 108 and 1 × 106 CFU/mL, respectively, along with 0.2 mL of sterilized saline. Group IV (high concentration of Km A5; Km A5_H) and group V (low concentration of Km A5; Km A5_L) mice were gavaged with Km A5 at 1 × 108 and 1 × 106 CFU/mL, respectively, along with 0.2 mL of sterilized saline. Group VI (high concentration of Sb MYA-796; Sb MYA-796_H) and group VII (low concentration of Sb MYA-796; Sb MYA-796_L) were gavaged with 0.2 mL of sterilized saline containing 1 × 108 and 1 × 106 CFU/mL Sb MYA-796, respectively.

At the end of the experimental period, four mice with an average body weight of 17–18 g were selected from each group, and their fecal samples were collected (Youn et al., 2022b). Briefly, each mouse was placed in a plastic box and 50 mg of feces was collected after natural defecation. The fecal samples were stored at − 80 °C until use. DNA was extracted from the feces using a NucliSENS easyMAG instrument (bioMérieux, Marcy-l'Étoile, France).

Gut microbiota analysis

Modulation of the gut microbiota in mice fecal samples after oral administration of yeast strains was investigated by Macrogen Inc. (Seoul, Republic of Korea). DNA was extracted from the fecal samples and used for metagenomic analysis. The V3-V4 region of the 16S rRNA gene was amplified for sequencing using the following primers: 341F, 5′-CCTACGGGNGGCWGCAG-3′ and 805R, 5′-GACTACHVGGGTATCTAATCC-3′. PCR was performed using Herculase II Fusion DNA polymerase (Agilent Technologies, Santa Clara, CA, USA) and the Nextera XT index kit v2 (Illumina Inc., San Diego, CA, USA). The PCR amplification was performed in the following steps: 1 cycle at 95 °C for 3 min, 25 cycles of denaturation (95 °C for 30 s), annealing (55 °C for 30 s), and extension (72 °C for 30 s), followed by 1 cycle at 72 °C for 5 min. The paired-end reads (2 × 300 bp) were then sequenced using the MiSeq platform (Illumina, San Diego, CA, USA) and FASTQ files were converted using bcl2fastq (version 2.20). After filtering, the obtained sequences were defined as having over 97% sequence homology using the CD-HIT-operational taxonomic unit (OTU) program. The taxonomic information was obtained through OTU picking using the NCBI (16S rRNA database). The alpha and beta diversity of gut microbiota were analyzed through Quantitative Insights into Microbial Ecology (version 1.9.1).

Statistical analysis

SPSS (version 25.0; SPSS Inc., Chicago, IL, USA) was used for data analysis. Data were further analyzed using Shapiro–Wilk’s and Levene’s tests to assess normality and homogeneity, respectively. Parametric data were subjected to one-way analysis of variance followed by Duncan’s multiple range test and non-parametric data were subjected to Kruskal–Wallis test followed by Dunn’s test and Bonferroni correction. Differences were regarded as statistically significant at p < 0.05 and p < 0.01. Data are presented as mean ± standard deviation (SD).

Results and discussion

Adhesion ability of yeast strains to Caco-2 cells

Km A4 and Km A5 isolated from kefir milk and Sb MYA-796 adhered to Caco-2 cells in 1 h (Fig. 1). Km A4 and Km A5 reached a cell density of 4.96 and 4.99 log CFU/mL, respectively, displaying higher adhesion ability to Caco-2 cells. Furthermore, the adhesion ability of Sb MYA-796 allowed its growth to a density of 4.35 log CFU/mL. The adhesion ability of the kefir yeast strains to Caco-2 cells was significantly higher than that of Sb MYA-796 (p < 0.05).

Fig. 1.

Fig. 1

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Adhesion of kefir yeast strains (Kluyveromyces marxianus A4 and K. marxianus A5) and Saccharomyces boulardii ATCC MYA-796 to colorectal adenocarcinoma (Caco-2) cells. The yeast cells (1 × 105 CFU/mL) were added to Caco-2 cell cultures and incubated in a 5% CO2 atmosphere at 37 °C for 1 h. After incubation, cells were washed, lysed, and inoculated on potato dextrose agar. Data are presented as the mean ± standard deviation. * indicates significant differences (p < 0.05) compared with the S. boulardii ATCC MYA-796 using the one-way analysis of variance followed by Duncan’s multiple range test. Km A4, K. marxianus A4; Km A5, K. marxianus A5; Sb MYA-796, S. boulardii ATCC MYA-796

Consistent with the results of the present study, a previous study revealed that the adherence ability of all kefir-derived K. marxianus strains (KU140723-01, 02, 04, and 05) to Caco-2 cells is approximately 5 log CFU/well, which is greater than that of Lactobacillus acidophilus and Saccharomyces cerevisiae ATCC 6037 (approximately 4 log CFU/well) (Cho et al., 2018). In another study, K. marxianus B0399 isolated from the whey and curds of cow milk demonstrated a 5.4-fold higher adhesion to Caco-2 cells than L. mesenteroides C5 (Maccaferri et al., 2012).

Adhesion to the host cell is a potential probiotic agent selection criterion for inducing colonization and promoting beneficial functions including immunomodulatory effects, stimulating the intestinal barrier, and modulating the gut microbiota (Monteagudo-Mera et al., 2019). As these functions are triggered through the maintenance of host–microbial interactions via intestinal mucosal adhesion, a high adherence capacity of microorganisms to intestinal epithelial cells could prevent immediate clearance by peristalsis and provide a competitive advantage in the host gut ecosystem (Kos et al., 2003). Consistently, the above study showed that K. marxianus B0399, which survives in a colon model system with high adhesion to intestinal epithelial cells, induces an increase in the population of Bifidobacterium spp. (Maccaferri et al., 2012).

Cell adhesion ability is closely related to the cell surface hydrophobicity, biofilm formation, and auto-aggregation capacity of microorganisms (Krasowska and Sigler, 2014). The hydrophobicity of the probiotic surface, which can change due to differences in the expression of various surface-binding proteins between strains, is essential for non-specific interactions with receptors on intestinal epithelial cells (Dianawati et al., 2016). The hydrophobicity of the cell surface is also linked to microbial auto-aggregation, which is a state between the planktonic and biofilm forms (Al Azzaz et al., 2020). Microorganism aggregation is known to favor biofilm formation by facilitating rapid conversion to biofilm-like phenotypes (Trunk et al., 2018). In our previous study, Km A4 and Km A5 showed significantly higher cell surface hydrophobicity and biofilm-forming and auto-aggregation abilities than Sb MYA-796 (Youn et al., 2022a). Therefore, the superior adhesion capacity of Km A4 and Km A5 could be a result of these factors, which might be attributed to the cell adhesion ability of yeast strains.

Modulation of gut microbiota

Information on body weight, food intake, and water intake for all groups from animal experiments conducted in our previous study is presented in Supplementary Data 2 (Youn et al., 2022b). The filtered sequence information, such as total bases, read count, N (%), GC (%), Q20 (%), and Q30 (%), for each sample is shown in Supplementary Data 3. We evaluated the gut microbiota community by comparing the Chao1, Shannon, inverse Simpson, and Good’s coverage diversity indices (Fig. 2). The Chao1, Shannon, and inverse Simpson indices showed no significant differences among all groups (p > 0.05). The Good’s coverage diversity index was > 0.997 for fecal samples obtained after yeast consumption for all groups. Regarding the weighted UniFrac PCoA, the microbiota of all groups were not significantly separated (Fig. 3; p > 0.05). However, the unweighted UniFrac PCoA indicated significant clustering in the groups that consumed high concentrations of Km A4 and Km A5 compared with that in the NC_Saline group (Fig. 3D; p = 0.023 and p = 0.035, respectively).

Fig. 2.

Fig. 2

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Alpha diversity indices of the gut microbiota using BALB/c mice fecal samples. Gut microbiota analysis was performed using BALB/c mice feces collected on day 14. There are no significant differences among the alpha diversity indices using one-way analysis of variance followed by Duncan’s multiple range test (p > 0.05). NC_Saline: Negative control with 0.9% sterilized saline; Km A4_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of Kluyveromyces marxianus A4; Km A4_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A4; Km A5_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A5; Km A5_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A5; Sb MYA-796_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of Saccharomyces boulardii ATCC MYA-796; Sb MYA-796_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of S. boulardii ATCC MYA-796

Fig. 3.

Fig. 3

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Beta diversity of gut microbiota using BALB/c mice fecal samples. Gut microbiota analysis was performed using BALB/c mice feces collected on day 14. Weighted UniFrac principal coordinate analysis (PCoA) of (A) all groups and (B) the compared groups (negative control group and high concentrations of Kluyveromyces marxianus A4 and K. marxianus A5) and unweighted UniFrac PCoA of (C) all groups and (D) the compared groups. NC_Saline: Negative control with 0.9% sterilized saline; Km A4_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of Kluyveromyces marxianus A4; Km A4_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A4; Km A5_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A5; Km A5_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A5; Sb MYA-796_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of Saccharomyces boulardii ATCC MYA-796; Sb MYA-796_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of S. boulardii ATCC MYA-796

The relative taxonomy abundance of gut microbiota composition after Km A4, Km A5, and Sb MYA-796 gavage was analyzed at the phylum, order, and genus level (Fig. 4). At the phylum level, the abundance of Cyanobacteria, Deferribacteres, Firmicutes, Proteobacteria, and Tenericutes was not significantly different among all groups (p > 0.05). Significant alterations at phylum, order, and genus level in the gut microbiota are shown in a heatmap (Fig. 5). The abundance of Bacteroidetes showed significant differences after Km A4_H administration compared with that in the NC_Saline group (p < 0.05). Moreover, significant differences were observed in the abundance of Actinobacteria after the administration of Km A5_H compared to that in the Km_A4_L group (p < 0.05). At the order level, the abundance of Bacteroidales was significantly higher in the Km A4_H group than in the NC_Saline group (p < 0.05), whereas that of Corynebacteriales was significantly higher in the Km A5_H group than in the Km A4_H group (p < 0.01). At the genus level, the abundance of Bacteroides was higher, whereas that of Murimonas was significantly lower in the Km A4_H group than that in the NC_Saline group (p < 0.05). Furthermore, the abundance of Corynebacterium increased significantly in the Km A5_H group than in the Km A4_H group (p < 0.01).

Fig. 4.

Fig. 4

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Effect of Kluyveromyces marxianus A4, K. marxianus A5 and Saccharomyces boulardii ATCC MYA-796 on the BALB/c mice fecal microbiota. Gut microbiota analysis was performed using BALB/c mice feces collected on day 14. Relative taxonomy abundance of the gut microbiota at the (A) phylum, (B) order, and (C) genus level. NC_Saline: Negative control with 0.9% sterilized saline; Km A4_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A4; Km A4_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A4; Km A5_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A5; Km A5_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A5; Sb MYA-796_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of S. boulardii ATCC MYA-796; Sb MYA-796_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of S. boulardii ATCC MYA-796

Fig. 5.

Fig. 5

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Heatmap of the significant differences in gut microbiota at the phylum, order, and genus levels. The heatmap represents the relative taxonomy abundance of gut microbiota and the values of the relative taxonomy abundance ratio at the phylum, order, and genus levels are expressed as a percentage. * indicates significant differences (p < 0.05) compared with the NC_Saline group using the one-way analysis of variance followed by Duncan’s multiple range test. ** indicates significant differences (p < 0.05) compared with the Km A4_L group using Kruskal–Wallis test followed by Dunn’s test and Bonferroni correction. *** indicates significant differences (p < 0.01) compared with the Km A4_H group using Kruskal–Wallis test followed by Dunn’s test and Bonferroni correction. NC_Saline: Negative control with 0.9% sterilized saline; Km A4_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A4; Km A4_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A4; Km A5_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of K. marxianus A5; Km A5_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of K. marxianus A5; Sb MYA-796_H: 0.2 mL of sterilized saline containing 1 × 108 CFU/mL of S. boulardii ATCC MYA-796; Sb MYA-796_L: 0.2 mL of sterilized saline containing 1 × 106 CFU/mL of S. boulardii ATCC MYA-796

The composition of the gut microbiota undergoes rapid changes owing to the diet of the host (Ferrer et al., 2013). Owing to the changeable properties of gut microbiota, various probiotics have been documented as food supplements that target alterations in gut microorganism patterns and populations (Qin et al., 2018). The normal gut microbiota comprises several phyla, including Bacteroidetes and Firmicutes, which constitute more than 90% of the total population of the distal gut microbiota as well as Actinobacteria, Cyanobacteria, Deferribacteres, Proteobacteria, and Tenericutes (Qin et al., 2010).

In the present study, Km A4_H significantly increased the relative taxonomy abundance of the phylum Bacteroidetes, order Bacteroidales, and genus Bacteroides. Bacteroidetes is the largest gut microbiota phylum, and a decrease in its relative taxonomy abundance induces metabolic syndrome, coronary heart disease, and nonalcoholic steatohepatitis (Gibiino et al., 2018). Consistent with the results of the present study, our previous study showed that the number of Bacteroidetes colonies in BALB/c mice was significantly higher in the group treated with kefir milk for 3 weeks than in the control group (Kim et al., 2015). Furthermore, Everard et al. (2014) found that oral administration of S. boulardii CNCM I-745 for 4 weeks to obese and type 2 diabetes-induced mice significantly changed the gut microbial communities by increasing the population of Bacteroidetes. Another recent study reported that the administration of S. boulardii for 72 days to healthy broilers resulted in a higher abundance of Bacteroidetes than that in the control group (Qin et al., 2018). The order Bacteroidales not only contributes to the differentiation of inflammatory T helper 17 cells that regulate inflammation but also produces secretory immunoglobulin A, which plays a critical role in intestinal protection and homeostatic regulation (Butel, 2014). Furthermore, Bacteroidales (including the genus Bacteroides) serves as a source of short-chain fatty acids (SCFAs) including acetate, propionate, and butyrate for beneficial intestinal bacteria (Chakraborti, 2015). Intestinal SCFAs play an important role in ameliorating inflammatory diseases and promoting the health of colonic epithelial cells by inhibiting the production of proinflammatory cytokines, enhancing the expression of interleukin 10, and activating regulatory T cells (Huang et al., 2018). In contrast, the abundance of the genus Murimonas, which belongs to the phylum Firmicutes, decreased significantly in the Km A4_H group compared with that in the NC_Saline group; however, to date, these genera are not well-defined, and their health effects are also unclear (Kläring et al., 2015).

After administration of Km A5_H, the relative taxonomy abundance of the phylum Actinobacteria, order Corynebacteriales, and genera Corynebacterium increased significantly. In a previous study, a pool of LAB probiotics induced an increase in the abundance of Actinobacteria when administered to obese mice for 5 weeks (Bagarolli et al., 2017). Actinobacteria, including Corynebacteriales, maintain intestinal barrier homeostasis and release positive metabolites by degrading hydrocarbons (Binda et al., 2018). Furthermore, although Actinobacteria represents a minority group of commensal bacteria, the production of SCFAs during Actinobacteria fermentation is crucial for providing energy for epithelial cell turnover and inducing their potent anti-microbial activity (Binda et al., 2018; Chakraborti, 2015). Therefore, advantageous bacteria belonging to the phylum Actinobacteria can maintain the gut barrier by increasing SCFAs production (Hardy et al., 2013). Corynebacterium (a genus of the phylum Actinobacteria) provides nutrients to humans and some animals by producing amino acids such as L-glutamic acid and L-lysine (Leuchtenberger et al., 2005). In particular, Corynebacterium glutamicum has been used industrially to fermentatively produce non-proteinogenic amino acids, polyphenols, and medical polymers (Wolf et al., 2021). However, some pathogenic Corynebacterium spp., which prefer warm and moist conditions, can cause skin infections such as pitted keratolysis, erythrasma, and trichomycosis in humans (Blaise et al., 2008). In particular, Corynebacterium diphtheriae, which can potentially produce diphtheria toxin, causes clinical diphtheria with symptoms such as dyspnea, sore throat, and mild fever in humans (Wagner et al., 2012).

The distinct constituents of gut microbiota with regard to host health and diseases are continuously being studied and gut microbiota modulation could depend on the particular microbial strains as well as the doses administered. Dysbiosis exerts harmful effects on the host by changing the qualitative, quantitative, and metabolic activities of the gut microbiota (Holzapfel et al., 1998). Although excessive changes in gut microbiota are not favorable, moderate changes in gut microbiota can be beneficial to the host. In the present study, in addition to the good survivability and safety revealed by our previous studies, Km A4 and Km A5 presented good adhesion to Caco-2 cells and alterations in the gut microbiota were observed after oral administration in healthy mice for 2 weeks compared with mice administered Sb MYA-796. Interestingly, the adhesion ability of Km A4 and Km A5 was higher than that of Sb MYA-796. Furthermore, the consumption of high concentrations of Km A4 resulted in increased Bacteroides, which could be associated with SCFAs and the regulation of inflammation. In addition, Corynebacterium was significantly increased after administration of high concentrations of Km A5. These results suggest that individually administered Km A4 and Km A5 survive safely in the host intestine and modulate the gut microbiota by colonizing the host, showing characteristics of potential yeast probiotic agents.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 109 KB) (107.4KB, docx)

Acknowledgements

This study was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (Grant Number, 2021R1A2C2006817).

Author contributions

Conceptualization: H-YY, K-HS. Data curation: H-YY. Formal analysis: H-YY, H-JK, D-HK, Y-SJ. Methodology: H-YY, D-HK. Software, Validation, Investigation: H-YY, H-JK, Y-SJ. Project administration: HK, K-HS. Supervision: K-HS. Funding acquisition: K-HS, HK. Writing—original draft: H-YY, H-JK. Writing—review and editing: H-YY, H-JK, D-HK, Y-SJ, HK, K-HS.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The ITS sequences of K. marxianus A4 and A5 are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MT793576 and MT793594.

Declarations

Conflicts of interest

The authors declare no conflict of interest.

Ethical approval

All experiments were approved by the Konkuk University Animal Experiment Ethics Committee (KU20075). All institutional and national guidelines for the care and use of laboratory animals were followed.

Footnotes

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Contributor Information

Hye-Young Youn, Email: younv_v123@naver.com.

Hyeon-Jin Kim, Email: khj970923@naver.com.

Dong-Hyeon Kim, Email: i76845@konkuk.ac.kr.

Yong-Seok Jang, Email: ryej9@hanmail.net.

Hyunsook Kim, Email: hyunsk15@hanyang.ac.kr.

Kun-Ho Seo, Email: bracstu3@konkuk.ac.kr.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 109 KB) (107.4KB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. The ITS sequences of K. marxianus A4 and A5 are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MT793576 and MT793594.

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