Biological Characteristics of a Probiotic Wickerhamomyces anomalus Isolated from Pickled Vegetables and Its Function in Preventing Hyperuricemia in Mice

Abstract

Hyperuricemia (HUA) is a metabolic disorder that can easily lead to gout or kidney disease, and it is believed that it can be treated effectively using probiotics. This study evaluated the safety, probiotic, and functional properties of Wickerhamomyces anomalus YFJ252, isolated from pickled vegetables, including its in vitro inhibitory activity against xanthine oxidase (XO) and in vivo uric acid-lowering activity in mice, using virulence factor screening, plate counting, and colorimetric assays. Meanwhile, the potential anti-HUA mechanism was also investigated using untargeted metabolomics and whole-genome analysis. The results show that YFJ252 is non-hemolytic and does not produce DNase, gelatinase, or biogenic amine. It has potential probiotic properties: 85.83% DPPH radical scavenging, 39.94% α-amylase inhibition, 35.32% α-glucosidase inhibition, 20.73% anti-inflammatory ability, and 84.15% XO inhibition capacity. Animal experiments indicated that early intake of YFJ252 could maintain serum uric acid levels at 165.08 μmol/L (p < 0.05), lower than the HUA group (212.19 μmol/L), and significantly decrease creatinine and urea nitrogen levels (p < 0.05). The hypothetical anti-HUA potential of YFJ252 might be due to the production of antioxidant, hypoglycemic, and XO-inhibitory metabolites during growth, as well as a purine-degrading pathway that the strain inherited. This study provides a theoretical basis for using W. anomalus YFJ252 as a food ingredient with preventive effects against HUA.

1. Introduction

Hyperuricemia (HUA), a result of an excessive level of serum uric acid (UA), is caused by abnormal purine metabolism, UA production, and excretion [1]. With the development of society and changes in people’s lifestyles, especially the intake of high-purine foods such as seafood, roasted meat, and beer, the number of HUA patients worldwide has significantly increased in recent years [2], making its alleviation and treatment methods a current research hotspot [3]. At present, the main therapeutic drugs for HUA include allopurinol, febuxostat, and benzbromarone, which mainly achieve the treatment goal by reducing the synthesis and increasing the excretion of UA, but long-term use of these drugs can cause serious side effects, including allergic reactions and liver and kidney damage [4]. Therefore, finding safe and efficient methods for treating HUA is key.

Current research has shown that many natural products, such as herbal extracts [5], food-derived peptides [6], and probiotics and their metabolites [7], can serve as potential methods for treating HUA. In recent years, many studies have reported that probiotics from various sources can alleviate HUA via different mechanisms. For instance, Limosilactobacillus fermentum NCUH003018, Limosilactobacillus reuteri NCUH064056, and Lactobacillus gasseri NCUH066006 isolated from infant feces and fermented vegetables can effectively degrade adenosine and guanosine, and inhibit xanthine oxidase (XO) activity [8]. Lactiplantibacillus plantarum 1155 and Lactobacillus mucilaginosus 2644 obtained from traditional fermented dairy products reduce the production of uric acid not only by inhibiting xanthine oxidase (XO) activity but also by upregulating the expression of uric acid transport proteins, accelerating uric acid excretion, and achieving an anti-HUA effect [9]. Another study found that Lacticaseibacillus paracasei CPU202306 inhibited the growth of harmful species, such as Desulfovibrionaceae and Clostridium innocuum, while promoting the growth of the beneficial species Muribaculaceae [7].

Although many studies have examined the alleviation of HUA using LAB, research on the probiotic function of yeast and its applications in alleviating HUA is rare. Yeast is commonly found in fermented foods (bread, soy sauce, beer, etc.) and is an essential part of the fermentation industry [10]. Yeast also plays an important role in the human gut microbiome, despite its richness being relatively low [11]. With the expansion of research, more and more scholars have found that yeast species such as Saccharomyces cerevisiae [12], Hanseniaspora uvarum [13], and Pichia silvicola [13] have diverse probiotic potential, partially due to the variety of bioactive metabolites, such as phenols, alkaloids, isoprenoids, and γ-aminobutyric acid, and some are unique to yeast; meanwhile, a small amount of yeast can produce extracellular hydrolytic enzymes such as β-glucosidase, amylase, and protease [14]. These secondary metabolites and extracellular hydrolytic enzymes often function as antibiotics, antioxidants, and regulators of carbohydrate metabolism [15]. For instance, one study confirmed that live Saccharomyces cerevisiae could be used as an antibiotic substitute when added to feed as a growth promoter [16]. Broilers were fed diets containing live Saccharomyces cerevisiae, and not only was their growth performance enhanced but also the activity of SOD and CAT in serum was significantly increased compared to that in the antibiotic group; other research has shown that cell-free extracts of Pichia silvicola UL-6 and Sporobolomyces carnicolor 402-JB-1 isolated from traditional fermented dairy products have a hypoglycemic function, exhibiting 72.3% and 69.9% inhibition rates toward α-glucosidase, respectively. Moreover, due to the resistance of yeasts to bacteriophages and antibiotics, they have broader applications than LAB [17,18]. Yeast was also proven to have anti-inflammatory [19] properties and the ability to regulate UA metabolism [19]; in particular, “Jiangshui” Pichia kudriavzevii YS711 showed good UA degradation ability, and in vitro studies showed that it could achieve a degradation rate of 31.2% for UA to ammonium within 24 h under the catalytic reaction of uric acid metabolism-related key enzymes. However, our understanding of the uric acid-lowering role of probiotic yeast is limited; more yeast strains need to be explored.

This study investigates the probiotic potential of Wickerhamomyces anomalus YFJ252, isolated from pickled vegetables, particularly its in vitro and in vivo uric acid-degrading ability, and explores its potential HUA-alleviating mechanism based on volatile metabolite profiles and whole-genome sequencing data of the strain. This study will provide a theoretical basis for using W. anomalus YFJ252 as a food ingredient with preventive effects against HUA.

2. Materials and Methods

2.1. Chemicals and Reagents

Xanthine oxidase, allopurinol (APL), and potassium oxonate (PO) were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). α-amylase, α-glucosidase, and xanthine were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Pepsin, trypsin, and phenanthroline were purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). The uric acid (UA), creatinine (Cr), and blood urea nitrogen (BUN) detection kits were provided by the Nanjing Jiancheng Biotechnology Research Institute (Nanjing, China). Other materials included yeast peptone glucose broth (YPD) medium; LB broth purchased from Aobo Xing Biotechnology Co., Ltd. (Beijing, China); and Columbia blood agar purchased from Haibo Biotechnology Co., Ltd. (Qingdao, China).

2.2. YFJ252 Identification

YFJ252 (CGMCC NO. 28267) was cultivated on YPD agar plates at 28 °C for 72 h, and its cell morphology was observed. The DNA of YFJ252 was extracted using the yeast genome DNA extraction kit. After that, PCR amplification was performed using the DNA of YFJ252 as the template and NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and (NL-4:5′-GGTCCGTGTTTCAAGACGG-3′) as primers. The amplification conditions were as follows: 95 °C, 30 s; 58 °C, 30 s; 72 °C, 90 s, 35 cycles; 72 °C for 5 min [20]. PCR products were purified and sequenced. The sequencing results were compared and analyzed using the BLASTN tool (2.16.0) in the NCBI database, and a phylogenetic tree was constructed in MEGA 11.0 using the Maximum Likelihood method.

2.3. Safety Assessment of YFJ252

2.3.1. Hemolytic

The hemolytic assay was performed according to the reported method [21]. YFJ252 was first cultured in YPD liquid medium at 28 °C for 72 h. Then, the cultured YFJ252 was inoculated onto a Columbia agar plate containing 5% sheep blood using the streak plate method and incubated at 28 °C for 48 h. Staphylococcus aureus was used as a positive control. The presence of green areas around the colony indicates α-hemolysis; transparent areas indicate β-hemolysis; and no significant change indicates γ-hemolysis or non-hemolysis.

2.3.2. DNase-Producing Ability

YFJ252 was first cultured in YPD liquid medium at 28 °C for 72 h. Subsequently, 1 mL of YFJ252 suspension was inoculated onto DNase agar medium using the streak plate method and incubated at 28 °C for 48 h. After incubation, 1 mol/L HCl was added dropwise to the medium. The formation of a clear, transparent zone around the bacterial growth was interpreted as indicative of DNase production, whereas the absence of such a zone indicated the absence of DNase activity [22].

2.3.3. Gelatinase Producing Ability

YFJ252 was activated as described above, and the culture broth was inoculated into gelatin medium and incubated at 28 °C for 5–7 days. Staphylococcus aureus was used as the positive control. After incubation, the medium was incubated at 4 °C for 1 h. If liquefaction occurred in the puncture area, it was concluded that the strain had gelatinase activity [23].

2.3.4. Biogenic Amine Producing Ability

The Oxford Cup method was used to determine the ability of YFJ252 to produce biogenic amines [24]; 100 μL of cell-free supernatant of YFJ252 was injected into the preformed holes of the medium with biogenic amine (peptone, 0.5%; yeast extract, 0.5%; NaCl, 0.5%; CaCl₃, 0.1%; bromocresol purple, 0.006%; His, 0.2%; Tyr, 0.2%; Lys, 0.2%; Trp, 0.2%; Agar, 1.5%; pH = 5.3 ± 0.2). If the color around the hole turned purple after cultivation at 28 °C for 48 h, it indicated the presence of biogenic amines. One of the spoilage Hafnia strains, which produced biogenic amine, was used as the positive control.

2.3.5. Antibiotic Sensitivity

The paper diffusion method was used to evaluate the antibiotic sensitivity of the strains [25]; antibiotics including penicillin (10 μg), tetracycline (30 μg), ampicillin (10 μg), norfloxacin (10 μg), kanamycin (30 μg), clarithromycin (15 μg), gentamicin (10 μg), streptomycin (10 μg), erythromycin (15 μg), chloramphenicol (30 μg), clindamycin (2 μg), and vancomycin (30 μg) and the diameter of the inhibition zone were measured after incubation at 28 °C for 24–48 h. The standards are expressed as insensitive (inhibition zone diameter ≤ 15 mm), moderately sensitive (16–20 mm), and sensitive (inhibition zone diameter ≥ 21 mm).

2.4. Probiotic Properties

2.4.1. Tolerance to Acid and Bile Salts Analysis

YFJ252 was first cultured in YPD liquid medium at 28 °C for 72 h and was then inoculated (2%, v/v) into YPD liquid media with different pH values (pH 2, pH 3, pH 4, pH 5) and concentrations of bile salts (0.1%, 0.2%, and 0.3%) and cultivated at 28 °C for 24 h, and OD_600nm was measured every 2 h to acquire the growth curve plot [26].

2.4.2. Hydrophobicity Analysis

YFJ252 was first cultured in YPD liquid medium at 28 °C for 72 h and then centrifuged at 8944× g, 4 °C for 10 min to obtain the cells. The cells were washed twice with 50 mM K₂HPO₄ (pH 6.5) and resuspended in a 0.8~1.0 to OD₅₆₀ nm solution; the absorbance was denoted as OD₁. Then, 3 mL of the above solution was mixed with 1 mL of xylene, n-hexane, or ethyl acetate, vortexed for 2 min, and incubated at 28 °C for 3 h to allow the two phases to separate [27]. OD _560nm of the water phase was measured and recorded as OD₂. Hydrophobicity was calculated as equation (Equation (1)):

$$\mathrm{Hydrophobic} \left(\%\right) = (1-{\mathrm{OD}}_2/{\mathrm{OD}}_1) \times 100$$ (1)

2.4.3. Self-Aggregation and Co-Aggregation Ability

To test the self-aggregation ability of YFJ252, the strain was collected as in Section 2.4.2 and then resuspended to form a 0.5 OD_600nm solution. Then, the solution was cultivated at 28 °C, and the OD_600nm of the supernatant was measured every 2 h and denoted as OD₂. Self-aggregation was calculated using Equation (2) [28].

$$\text{Self-aggregation} \mathrm{rate}\left(\%\right)={(\mathrm{OD}}_1-{\mathrm{OD}}_2)/{\mathrm{OD}}_1 \times 100$$ (2)

To test co-aggregation, the suspensions of E. coli (isolated from a spoiled fish) and YFJ252 were prepared according to Section 2.4.3. Then, 2 mL of each strain was mixed and vortexed for 10 s, and the OD_600nm of the supernatant was measured every 2 h. Co-aggregation was calculated using Equation (3) [28].

$$\text{Co-aggregation} \mathrm{rate}\left(\%\right)=(1-{\mathrm{OD}}_2/{\mathrm{OD}}_1) \times 100$$ (3)

where OD₁ is the absorbance value of the supernatant at 0 h, and OD₂ is the absorbance value of the supernatant at different time intervals.

2.4.4. Antioxidant Capacity Analysis

The antioxidant capacity of the strains was preliminarily evaluated by analyzing their ability to scavenge DPPH, ABTS⁺, and hydroxyl radicals.

For DPPH scavenging, 100 μL of YFJ252 supernatant was mixed with 100 μL of 0.2 mmol/L DPPH in ethanol, and 1 mL of PBS buffer (0.01 mol/L, pH 7.2) was used as a control; 1 mL of ascorbic acid (Vc) was used as a positive control instead of the YFJ252 supernatant. The mixture was incubated at 25 °C in the dark for 30 min; then, the OD_{517 nm} was measured. The DPPH radical scavenging rate was calculated using Equation (4).

$$\mathrm{DPPH} \mathrm{radical} \mathrm{scavenging} \mathrm{rate}\left(\%\right)=(1-{\mathrm{OD}}_1/{\mathrm{OD}}_2) \times 100$$ (4)

OD₁ represents the absorbance value of the sample, and OD₂ represents the absorbance value of the control group.

For the hydroxyl radical scavenging ability, 2 mL of PBS (pH 7.4), 1 mL of 0.25 mmol/mL phenanthroline, 1 mL of 0.75 mmol/L FeSO₄ solution, and 1 mL of YFJ252 supernatant were mixed, then 1 mL of H₂O₂ (12%) was added, and the reaction was allowed to proceed at 37 °C for 60 min; finally, the OD_{536 nm} was measured. The same volume of PBS, instead of the YFJ252 supernatant, was used as the control. At the same time, in the blank group, we used distilled water in place of the YFJ252 supernatant and the other components in the reaction mixture, and the hydroxyl radical scavenging rate was calculated using Equation (5) [29].

$$\mathrm{Hydroxyl} \mathrm{radical} \mathrm{scavenging} \mathrm{rate} (\%)=[1-({\mathrm{OD}}_1-{\mathrm{OD}}_0)/({\mathrm{OD}}_2{-\mathrm{OD}}_0\left)\right] \times 100$$ (5)

OD₀ is the absorbance value of the control group, OD₁ is the absorbance value of the sample, and OD₂ is the absorbance value of the blank group.

The ABTS⁺ solution was prepared by mixing 7 mM ABTS with 2.5 mM K₂S₂O₈ (v:v = 1:1) and incubating the mixture in the dark for 24 h. This solution was subsequently diluted with ethanol or buffer until the absorbance reached 0.700 ± 0.02 at 734 nm. For the assay, 1 mL of the ABTS⁺ solution was combined with 500 μL of the supernatant from strain YFJ252. After a 10 min incubation at 25 °C, the absorbance at 734 nm was recorded. Equation (6) was used to calculate the ABTS⁺ free radical scavenging rate [30].

$${\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}}^{+} \mathrm{radical} \mathrm{scavenging} \mathrm{rate}\left(\%\right)=[1-({\mathrm{OD}}_1{-\mathrm{OD}}_2)/{\mathrm{OD}}_0] \times 100$$ (6)

where OD₀, OD₁, and OD₂ represent the same as for the hydroxyl radical scavenging assay.

2.5. Functional Analysis

2.5.1. α-Amylase Inhibition Ability

Briefly, equal volumes (500 μL) of the sample and α-amylase (0.1 U/mL) were mixed completely, and then the mixture was incubated at 37 °C for 10 min. After that, 500 μL of 1% soluble starch was added to the mixture, which was then incubated at the same temperature for 10 min. After the reaction was completed, DNS (1 mL) was added, and the mixture was boiled for 5 min to terminate the reaction. Finally, 10 mL of distilled water was added, and the OD_{540 nm} was measured. The α-amylase inhibition rate is calculated using Equation (7) [31].

$$\text{α-amylase} \mathrm{inhibition} \mathrm{rate} \left(\%\right)=[1-{(\mathrm{OD}}_1-{\mathrm{OD}}_2)/{\mathrm{OD}}_0] \times 100$$ (7)

where OD₁ refers to the absorbance of the experimental sample (samples and α-amylase), OD₂ indicates the absorbance of the blank (samples without α-amylase), and OD₀ represents the absorbance of the control (with α-amylase and without samples).

2.5.2. α-Glucosidase Inhibition Ability

We mixed the sample solution (40 μL) thoroughly with 40 μL of α-glucosidase solution (1 U/mL). After culturing at 37 °C for 10 min, 20 μL of p-nitrophenyl-α-D-glucopyranoside solution (16 mM) was added and incubated at 37 °C for 15 min; then, 40 μL of Na₂CO₃ was added to the solution (0.2 M) to terminate the reaction. The OD_405nm was recorded, and Equation (8) was used to calculate the α-glucosidase inhibition rate [32].

$$\text{α-glucosidase} \mathrm{inhibition} \mathrm{rate}\left(\%\right)=[1-({\mathrm{OD}}_1-{\mathrm{OD}}_2)/{\mathrm{OD}}_3] \times 100$$ (8)

OD₁ is the absorbance of the sample group; OD₂ is the background control group without α-glucosidase; and OD₃ is the blank control group without the sample.

2.5.3. Inhibition of Bovine Serum Albumin Denaturation

We mixed 100 μL of the sample with 400 μL of 5% bovine serum albumin, adjusted the pH to 6.5 using 1 M HCl, and incubated the mixture at 37 °C for 20 min and then at 51 °C for 20 min; after cooling, 2.5 mL of PBS (0.01 M, pH 7.2) was added, and the OD_{416 nm} of the mixture was measured. The calculation formula for the protein denaturation inhibition rate is given by Equation (9) [33].

$$\mathrm{Protein} \mathrm{denaturation} \mathrm{inhibition} \mathrm{rate}\left(\%\right)=({\mathrm{OD}}_1-{\mathrm{OD}}_2)/{\mathrm{OD}}_1 \times 100$$ (9)

OD₁ is the absorbance value of the control group; OD₂ is the absorbance value of the sample.

2.6. Assessment of Uric Acid-Lowering Ability

2.6.1. In Vitro XO Inhibition Ability

YFJ252 was first cultured in YPD liquid medium at 28 °C for 72 h and centrifuged at 7104× g for 10 min to obtain the yeast cells; then, the precipitate yeast cells were resuspended in PBS (0.1 mol/L, pH = 7.0) and incubated at 37 °C for 12 h. After centrifugation under the same conditions as above, the supernatant was collected and used to perform the XO-inhibitory assay. The reaction mixture for the blank group consisted of 20 μL of xanthine oxidase (0.1 U/mL), 20 μL of 0.15 mM xanthine, and 160 μL of PBS. The sample group was prepared by adding 20 μL of cell-free supernatant to 140 μL of reduced PBS, while the positive control contained 20 μL of allopurinol and 140 μL of reduced PBS. The absorbance of each mixture was subsequently measured at 293 nm. The XO inhibition rate was calculated using Equation (10) [8].

$$\mathrm{XO} \mathrm{inhibition} \mathrm{rate}\left(\%\right)=[1-\left({\mathrm{A}}_{\mathrm{s}}-{\mathrm{A}}_{\mathrm{s}0}\right)/{(\mathrm{A}}_{\mathrm{b}}-{\mathrm{A}}_{\mathrm{b}0})] \times 100$$ (10)

A_s0 and A_s are the absorbance values of the sample group and positive control group at 0 min and 10 min of reaction, respectively. A_b0 and A_b are the absorbance values at 0 min and 10 min of the blank control reaction, respectively.

2.6.2. In Vivo Uric Acid-Lowering Ability Based on the HUA Mouse Model

We selected 6-week-old Kunming mice weighing 20–25 g. Before the experiment, the mice were acclimated to a standard environment (24 ± 2 °C, 50 ± 5% humidity) for 1 week. At the same time, they were subjected to a 12/12 h light/dark cycle, during which they were free to consume food and water. The study was approved by the ethical review committee of Jinzhou Medical University, and the license was SYXK [Liao] 2024-0012.

We evenly divided 60 mice into 5 groups (n = 12 per group). The control group was fed a standard diet and received a daily gavage of physiological saline. In contrast, the model group was fed a high-sugar diet (10% fructose) and received a daily gavage of potassium oxonate (PO) (250 mg/kg) to induce HUA. The YFJ252 and APL groups, which were designed to evaluate the UA-lowering ability, underwent an induction phase (days 1–7) identical to that of the model group, followed by an intervention phase (days 8–21) during which potassium oxonate was co-administered with 1 × 10⁹ CFU/mL YFJ252 [8] or 20 mg/kg APL, respectively. The HUA+YFJ252 group, which was designed to show the protective effects of YFJ252 under a uric acid-elevating diet, received the high-sugar diet and simultaneous administration of potassium oxonate and YFJ252 for all 21 days.

After the experiment, the mice were euthanized, and their blood was collected for subsequent experiments. Serum UA, Cr, and BUN were tested according to the manufacturer’s protocols (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.7. Untargeted Metabolomics Analysis of YFJ252 Metabolites

A 100 μL aliquot of the fermentation supernatant was transferred to a microcentrifuge tube, followed by the addition of 400 μL of extraction solvent (methanol–acetonitrile = 1:1, v/v) containing isotopically labeled internal standards. The mixture was vortexed for 30 s and sonicated for 10 min in an ice-water bath. After incubation at −40 °C for 1 h, samples were centrifuged at 13,800× g for 15 min at 4 °C. The supernatant was then transferred to an injection vial for analysis. Chromatographic separation was performed on a Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 50 mm, 1.7 μm). The mobile phases consisted of (A) aqueous 25 mmol/L ammonium acetate and 25 mmol/L ammonium hydroxide and (B) acetonitrile. The autosampler temperature was maintained at 4 °C, and the injection volume was 2 μL. Pooled quality control (QC) samples were prepared by mixing equal aliquots of each experimental sample to monitor instrument stability and analytical reproducibility. Throughout the analytical sequence, a QC sample was injected after every 10 experimental samples, and blank solvent and matrix blank analyses were inserted at the beginning, at the end, and at intervals within the sequence to ensure data reliability. Metabolite identification followed the internationally accepted four-level confidence scheme: Level 1 (requiring matching of MS1, MS2, and retention time) is considered the gold standard; Level 2 (matched by high-resolution MS1 and MS2 spectra) also provides high confidence; and Level 3 and Level 4 identifications are suitable for exploratory research and offer clues for uncovering novel functions within the metabolomics “dark matter”. Raw data were acquired using an Orbitrap Exploris 120 mass spectrometer (Thermo Scientific) and preprocessed with SIMACA software (13.0). Pathway enrichment analysis was performed using MetaboAnalyst 4.0 (https://www.metaboanalyst.ca/, accessed on 24 January 2024). The metabolic pathways associated with the compounds were clarified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.genome.jp/kegg, accessed on 24 January 2024).

2.8. Whole-Genome Analysis of YFJ252

YFJ252 was cultured to the logarithmic growth phase. Cells were harvested via centrifugation at 8000× g for 10 min at 4 °C, washed twice with PBS, and flash-frozen in dry ice. Whole-genome sequencing was performed using a Nanopore sequencer. The assembled genome was annotated by aligning it against the GO and KEGG databases using DIAMOND to predict gene functions and metabolic pathways.

2.9. Statistical Analysis

All experiments were performed in triplicate, and results are presented as mean ± SD. Statistical analysis was performed using SPSS 18.0. A Student t-test was used for comparisons between two groups; a one-way ANOVA and Duncan multiple-comparison test were used to identify significant differences between groups. Statistical significance was set at p < 0.05. Data visualization was performed using GraphPad Prism 10.1.2. In the animal study, mice were randomly assigned to four groups (n = 12 per group). The experimenters were blinded to group allocation during administration, sample collection, and behavioral assessment to minimize bias.

3. Results and Discussions

3.1. Identification of YFJ252

YFJ252 was screened from pickled vegetables; after 72 h of cultivation on YPD agar, it appeared as a spherical and thick colony, with a milky white color, moist and rough surface, and creamy and viscous texture (Figure 1a). Agarose nucleic acid gel electrophoresis showed that the DNA target bands of YFJ252 appeared around 600 bp (Figure 1b), which was the target band of fungi. The phylogenetic tree results showed that YFJ252 had the highest homology of 98% with Wickerhammyces anomalus HN1 (Figure 1c); hence, it was designated as W. anomalus.

Figure 1.

Identification of YFJ252. (a) Morphology; (b) nucleic acid electrophoresis; (c) phylogenetic tree.

3.2. Safety Evaluation

As shown in Table 1, YFJ252 was γ-hemolytic and did not produce DNase, gelatinase, or biogenic amines. In this study, YFJ252 showed antibiotic resistance, consistent with other research [12], indicating that the simultaneous use of such yeast during antibiotic treatment does not affect its survival or related functions. Moreover, antibiotic resistance genes do not transfer between yeast and bacteria [34].

Table 1.

Safety analysis of strain.

Safety Indicators	Results
Hemolysis	γ
DNase enzyme	-
Bioamine	-
Gelatinase	-
Penicillin	-
Tetracycline	-
Ampicillin	-
Norfloxacin	-
Kanamycin	-
Clarithromycin	-
Gentamicin	-
Streptomycin	-
Erythromycin	-
Chloramphenicol	-
Clindamycin	-
Vancomycin	-

“γ” represents γ-hemolysis; for DNase enzyme, bioamine, and gelatinase, “-” indicates negative; for the antibiotics, “-” indicates insensitivity.

3.3. Probiotic Characteristics of YFJ252

3.3.1. Tolerance to Acid and Bile Salts

Currently, most probiotic formulations remain dependent on oral administration, which requires the strains to withstand the harsh gastrointestinal environment—particularly the strongly acidic conditions of the stomach and the high bile salt concentrations in the intestine. The efficacy of probiotics thus hinges on their ability to survive under such extreme acidity and bile salt stress [35]. Hence, the survival rate of YFJ252 under various pH conditions and at different bile salt concentrations was assessed. As shown in Figure 2a, YFJ252 grew well at pH 3, 4, and 5; however, its survival was lower at pH 2 than at other pH conditions. Other research also showed that yeast could grow well under acidic conditions [36]. The bile salt tolerance of the strain is shown in Figure 2b; it can be seen that different concentrations (0.1%, 0.2%, and 0.3%) prolong the duration of the logarithmic phase of YFJ252, consistent with the characteristics of Torulaspora delbrueckii [37]. The results indicated that the strain adapted to the high-bile-salt environment.

Figure 2.

The probiotic properties of YFJ252. (a) Acid tolerance; (b) bile salt tolerance; (c) hydrophobicity; (d) self-aggregation and co-aggregation ability. CK is the group without any treatment. Different lowercase letters (a–c) indicate statistically significant differences (p < 0.05).

3.3.2. Hydrophobicity and Self/Co-Aggregation Ability

The hydrophobicity and self-aggregation abilities of probiotics are closely related to their adhesion to intestinal epithelial cells, making it easier for them to colonize the intestine [38]. Their co-aggregation ability is related to their function in blocking colonization by harmful microorganisms in the gut and thus preventing infection [39]. The hydrophobicity of YFJ252 toward xylene, n-hexane, and ethyl acetate reached 74.71%, 83.19%, and 37.21%, respectively (Figure 2c). The self-aggregation rate increased over time: 37.53% at 2 h, 95.71% at 4 h, and 96.40% at 8 h. The co-aggregation rate of YFJ252 and E. coli (Figure 2d) showed no significant difference between 2 and 8 h, ranging from 71.00% to 73.00%. Based on the above results, we can infer that YFJ252 can colonize the intestine relatively easily and has the potential to prevent colonization by pathogens.

3.4. Functional Capacity of YFJ252

3.4.1. Antioxidant Capacity

Figure 3a shows that YFJ252 achieved scavenging rates of 85.53%, 63.73%, and 56.52% for DPPH radicals, hydroxyl radicals, and ABTS radicals, respectively; the clearance rates at a 1 mg/mL Vc concentration reached 96.40%, 98.88%, and 100%, respectively. Oxidation is associated with aging and some forms of body inflammation; thus, the antioxidants in probiotics might offer a new way to alleviate oxidative stress [40]. Meanwhile, elevated UA levels induce oxidative stress, mitochondrial dysfunction, and inflammation, which collectively contribute to renal tubular injury and interstitial fibrosis [41]. Given that mitigating oxidative stress has been shown to alleviate renal damage and enhance UA excretion, targeting this pathway represents a novel approach to lowering UA levels [42].

Figure 3.

Functional capacity of YFJ252. (a) The scavenging rate of DPPH, hydroxyl radicals, and ABTS radicals; (b) α-amylase and α-glucosidase inhibition ability, and anti-inflammatory ability. Different lowercase letters (a, b) indicate statistically significant differences (p < 0.05).

3.4.2. Hypoglycemic and Anti-Inflammatory Ability

The ability to inhibit α-amylase and α-glucosidase could be used to evaluate the hypoglycemic capacity of the probiotics [43]. Figure 3b shows that YFJ252 demonstrated an inhibitory ability of 39.94% against α-amylase and 35.32% against α-glucosidase. Numerous clinical studies have established a significant association between diabetes and HUA. Specifically, individuals with prediabetes exhibit markedly higher serum UA levels compared to their normoglycemic counterparts [44]. Insulin resistance (IR) has been identified as a central pathophysiological mechanism in this comorbidity. The enzymatic hydrolysis of dietary carbohydrates by α-amylase and α-glucosidase produces substantial glucose loads, which, in turn, induce metabolic stress in pancreatic β-cells and exacerbate insulin resistance. This metabolic dysfunction, in turn, impairs renal uric acid excretion via tubular transport mechanisms, thereby creating a pathological cycle that promotes the progression of HUA [45]. The ability of YFJ252 to inhibit α-amylase and α-glucosidase would help prevent IR, thereby maintaining stable UA levels.

Inflammation is a major factor in disease, and the anti-inflammatory potential of W. anomalus YFJ252 was evaluated using an in vitro protein denaturation inhibition assay. The results (Figure 3b) show that the inhibition rate of YFJ252 reached 20.73%. Current research suggests that inhibiting inflammation-related pathways can alleviate HUA. For instance, Lactiplantibacillus plantarum LLY-606 reduces uric acid levels by suppressing the TLR4/MyD88/NF-κB pathway, accompanied by a significant decrease in the ratios of phosphorylated p-IκB and p-NF-κB [46]. Additionally, Lactobacillus acidophilus F02 modulates the gut microbiota by increasing the relative abundance of genera such as Bacteroides, Ruminococcus, and Lactobacillus. This modulation reverses the LPS-induced elevation and reduces the expression levels of IL-10, IFN-γ, IL-1β, and the inflammasome NLRP3, thereby lowering systemic uric acid levels [47]. Furthermore, ameliorating the NLRP3 inflammasome-triggered cascade response can also prevent stem cell apoptosis, among other beneficial effects [48]. Hence, these functions might play an important role in YFJ252’s ability to prevent HUA; however, these findings are based on in vitro experiments. Whether YFJ252 shows these functions in vivo and whether it jointly participates in metabolic regulation require further validation through in vivo studies.

3.5. The Uric Acid-Lowering Ability of YFJ252

3.5.1. In Vitro Inhibition Ability of XO

Abnormal UA metabolism is a cause of HUA, and XO is a key enzyme in UA synthesis. Adenine and guanine are converted into hypoxanthine and xanthine under the action of XO, ultimately producing UA [49]. As shown in Figure 4a, we found that YFJ252 had an inhibitory ability of 84.15% toward XO.

Figure 4.

Uric acid-lowering ability of YFJ252. (a) In vitro XO inhibition ability; (b) serum uric acid level of mice; (c) creatinine content of mice; (d) BUN content of mice. Different lowercase letters (a–c) indicate statistically significant differences (p < 0.05).

3.5.2. In Vivo Anti-HUA Ability

Based on the mouse model (Figure 4b–d), after 21 days of the experiment, the serum uric acid concentration of the HUA group reached 212.19 μmol/L, significantly higher than that of the (HUA+YFJ252) group (165.08 μmol/L) (p < 0.05). At the same time, the concentrations of Cr and BUN in this group (HUA+YFJ252) were only 20.54 μmol/L and 9.88 mmol/L, compared to those of the HUA model group, which were 27.84 μmol/L and 10.61 mmol/L, respectively. This indicates that administering YFJ252 during the initial modeling stage could help to prevent HUA. Although similar studies have found that some LAB, for instance, Lactobacillus brevis LABC170 and L. fermentum LABC37, isolated from kimchi and other samples during the early stage of inducing HUA, can effectively reduce the levels of UA, Cr, and BUN in the serum of HUA mice, achieving the goal of preventing HUA [50], in another study, administering L. reuteri TSR332 to Wistar rats during the pre-modeling stage also showed the same result [51]. There is no research yet claiming that yeast is capable of preventing HUA.

There is other literature reporting that interactions between probiotics and prebiotics (polysaccharides or dietary fiber) and the fermentation of prebiotics as substrates in the gut alter the gut microbiota and metabolites such as short-chain fatty acids (SCFAs), thereby improving and regulating intestinal diseases and certain metabolic processes. One study confirmed that continuous intake of an α-glucan-type LmEPS secreted by Leuconostoc mesenteroides led to an increased abundance of Bacteroides in the mouse gut and significantly elevated levels of SCFAs (acetate and propionate) in the plasma and cecum; the increase in SCFAs, in turn, promotes the secretion of gut hormones. Subsequently, the incretin hormone GLP-1 binds to G protein-coupled receptors GPR41 and GPR43, thereby maintaining host energy metabolism homeostasis and glucose metabolic stability [52]. Meanwhile, SCFA levels are also associated with the regulation of uric acid metabolism and the alleviation of HUA by inhibiting XOD activity/expression [53], downregulating transporter targets such as URAT1 and GLUT9 to suppress uric acid reabsorption [54], and upregulating ABCG2 or OAT1 to accelerate uric acid excretion [55]. As mentioned above, the interaction of YFJ252 with the gut microbiota may be one way in which it helps lower uric acid levels.

3.6. Metabolomics Analysis of YFJ252

Based on the non-targeted metabolomics analysis, a total of 24 main types of metabolic compounds were detected (Figure 5a), including organic heterocyclic compounds, organic acids and their derivatives, benzene ring compounds, alkaloids, lipids and lipid molecules, amino acids and peptides, fatty acids, shikimic acid and phenylpropane, oxygen-containing organic compounds, phenylpropanoids, and polyketide compounds.

Figure 5.

Metabolomics analysis of YFJ252. (a) The pie chart of the metabolite classification; (b) key metabolic pathway analysis.

In order to further understand the corresponding metabolic pathways, information on the volatile metabolites was imported into the KEGG database for metabolic pathway analysis (Figure 5b). They belong to 15 main metabolic pathways, including phenylalanine metabolism, tyrosine metabolism, arginine and proline metabolism, lysine degradation, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, glycerolic acid and dicarboxylic acid metabolism, nucleotide metabolism, amino acid biosynthesis, 2-oxocarboxylic acid metabolism, carbon metabolism, ABC transporter protein, and pyrimidine metabolism. Among these are five key metabolic pathways, namely, lysine biosynthesis, alanine, aspartate, and glutamate metabolism, glyoxylate and dicarboxylic acid metabolism, arginine and proline metabolism, and pantothenic acid and coenzyme A biosynthesis. The top 20 metabolites can be seen in Supplementary Table S1.

Among these metabolites, arginine levels were notably elevated. Arginine is a conditionally essential amino acid that can be both endogenously synthesized and obtained from dietary sources. It has been shown to partially regulate insulin secretion and to possess antioxidant properties. The presence of arginine may directly or indirectly influence changes in uric acid levels. Studies have shown that L-arginine can inhibit uric acid production by maintaining the activity of antioxidant enzymes, such as SOD, catalase, and GPx, in the liver and kidneys [56].

Furthermore, arginine intake exerts positive effects on gut health by enhancing the resistance of intestinal cells to oxidative stress; promoting the colonization of beneficial bacteria such as Prevotella, Akkermansia, and Faecalibacterium; and increasing the production of SCFAs in the gut—all of which have been confirmed in numerous studies to contribute to lowering uric acid levels and alleviating HUA [57,58]. In addition to arginine, other metabolites such as pyruvate, pyroglutamic acid, and leuctine also accounted for a relatively high proportion in the metabolic profile of YFJ252. Although these compounds have been shown to play important roles in modulating certain diseases and are speculated to influence uric acid levels indirectly [59,60,61], their direct relationship with HUA requires further experimental validation.

Although several metabolites identified in this study have been shown in the existing literature to alleviate oxidative stress damage and modulate host energy metabolism via gut microbiota regulation, thereby providing indirect evidence for the protective role of YFJ252 in maintaining uric acid homeostasis under a uric acid-elevating diet, the current study lacks direct analysis of gut microbiota composition and related gene expression. These aspects should be explored through further investigations in subsequent research. Additionally, the questions of whether other detected metabolites contribute to the functional effects of YFJ252 and whether the lack of therapeutic effect on already elevated uric acid levels is influenced by intervention duration or strain dosage also require further analysis.

3.7. Whole Genome Analysis of YFJ252

The W. anomalus YFJ252 genome is 13.9 Mbp and has a GC content of 34.59% (Figure 6a). The sequence was uploaded to NCBI under the Biosample accession SAMN50025326. The genome sequence length and GC proportion of YFJ252 were then compared with those of other 24 strains of W. anomalus in the NCBI database (https://www.ncbi.nlm.nih.gov/), and the results showed that the genome sequence length (Figure 6b) and GC proportion (Figure 6c) of YFJ252 were consistent.

Figure 6.

The whole-genome analysis of YFJ252. (a) Genomic information; (b) the length of the gene sequence comparison between YFJ252 and other 24 W. anomalus strains; (c) GC content comparison between YFJ252 and 24 other W. anomalus strains; (d) GO analysis; (e) KEGG analysis; (f) potential purine metabolism pathway of YFJ252. Note: The red dot represents YFJ252, the blue and green dot represent the maximum and minimum value, respectively.

GO analysis (Figure 6d) indicated that, among all genes of W. anomalus YFJ252, the largest number is for biological processes (5941 genes), followed by cellular components (1859 genes) and molecular functions (4986 genes).

The KEGG annotation (Figure 6e) identified 3151 functional genes across the five major pathways. Among them, metabolism includes the most genes, with a total of 1571; followed by genetic information processing (915); cellular processes (489); environmental information processing (144), and biological systems (32). It was found that the metabolic pathways of YFJ252 are mainly distributed in amino acid metabolism, carbohydrate metabolism, lipid metabolism, cofactor and vitamin metabolism, ribose metabolism, polysaccharide biosynthesis and metabolism, other amino acid metabolism, nucleotide metabolism, terpenoid and polyketide compound metabolism, the biosynthesis of other secondary metabolites, and biodegradation and metabolism pathways of exogenous substances.

Research has shown that strains of bacteria have developed mechanisms to cope with oxidative stress, including superoxide dismutase and the glutathione system [62]. The genome of YFJ252 encodes multiple proteins involved in antioxidant activity, including catalase (KatE), peroxidases (PRDX5, PRDX2_4, AHP1), and glutathione peroxidase (GPX). Furthermore, in the whole-genome sequencing results for YFJ252, it was found that the genes of this strain are involved in four energy metabolism pathways, methane metabolism, sulfur metabolism, oxidative phosphorylation, and nitrogen metabolism, as well as seven lipid metabolism pathways: glycerophospholipid metabolism, glyceryl ester metabolism, ether lipid metabolism, sphingolipid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, and arachidonic acid metabolism. This explains why the strain can inhibit glucose metabolism-related indicators such as α-amylase and α-glucosidase activity. In addition, 36 genes in YFJ252 are related to tryptophan metabolism, and 55 genes are related to purine metabolism. Studies have shown that probiotics can improve hyperuricemia by degrading purines and regulating tryptophan metabolism [63], providing a basis for future research on the functional roles of bacterial strains in reducing uric acid.

In addition, whole-genome analysis of YFJ252 revealed a purine-degrading pathway, an essential aspect in evaluating the strain’s ability to reduce uric acid. These results help us further understand the mechanism behind YFJ252’s uric acid reduction (Figure 6f). Guanosine is converted to xanthine by purine nucleoside phosphorylase and guanine deaminase and then converted to urate by XO and xanthine dehydrogenase (XDH). Urate is converted to allantoate by urate oxidase, 5-hydroxyisouricase, and allantoinase, and a portion of allantoate is converted to acetic acid by allantoicase and ureidoglycolate lyase to participate in the glyoxylate cycle; the other part of allantoate is converted into urea through allantoicase, which generates NH₃ and CO₂ under the action of urease. Inosine is converted into hypoxanthine by purine nucleoside phosphorylase, which is then converted into xanthine by XO and XDH. The subsequent process is consistent with that of guanosine. The purine metabolism pathway discovered in the YFJ252 genome provides a new research direction for subsequent analysis of fungal purine metabolism and uric acid-lowering ability.

4. Conclusions

This study demonstrated that W. anomalus YFJ252 is basically safe and has antioxidant, anti-inflammatory, and xanthine oxidase (XO)-inhibitory activities. Animal experiments indicated its potential to prevent a rise in UA in the serum under a diet that could lead to HUA. Moreover, a complete purine metabolic pathway was identified in YFJ252, which possibly converts purines into ammonia, thereby reducing the risk of uric acid accumulation. However, further research is warranted to validate its probiotic effects in long-term and therapeutic (post-HUA) intervention models; to integrate gut microbiome sequencing with host transcriptomics to clarify microbiota–host interactions; to explore its application in food matrices such as fermented foods and functional beverages; and to assess the strain’s stability, safety, and efficacy in human clinical trials. In these studies, we will systematically elucidate the role of YFJ252 in the host metabolic network for the prevention of HUA and apply YFJ252 in anti-HUA functional foods.

Abbreviations

The following abbreviations are used in this manuscript:

APL	Allopurinol
BUN	Urea nitrogen
Cr	Creatinine
GPX	Glutathione peroxidase
LAB	Lactic acid bacteria
HUA	Hyperuricemia
PO	Potassium oxonate
UA	Uric acid
XDH	Xanthine dehydrogenase
XO	Xanthine oxidase

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040744/s1, Table S1: Top 20 metabolites of YFJ252.

Author Contributions

Conceptualization, J.L. and X.L.; methodology, X.T., S.G., and X.C.; software, X.T. and S.G.; validation, L.Y.; formal analysis, L.Y.; investigation, X.C.; data curation, S.G.; writing—original draft preparation, X.T. and S.G.; writing—review and editing, X.T. and S.G.; visualization, X.T., S.G., and L.Y.; funding acquisition, X.T. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was approved by the Ethics Committee (SYXK [Liao] 2024-0012) on 8 April 2025.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Doctoral Start-up Program of Liaoning Province (CN), grant number 2025-BS-0814.