Probiotic and safety characterization of Lactiplantibacillus plantarum ZZM01 isolated from mink

Abstract

In recent years, probiotics have attracted increasing attention due to their roles in gut health regulation, antimicrobial activity, and metabolic benefits. Given the high-fat dietary characteristics of mink, their intestinal microbiota represents a potential source of functionally valuable lactic acid bacteria. In this study, we evaluated the probiotic properties, safety profile, and functional potential of Lactiplantibacillus plantarum ZZM01 isolated from mink intestine. ZZM01 exhibited good tolerance to simulated gastrointestinal conditions, with survival rates of 71.92% at pH 2.0 and 84.71% in 0.3% bile salts. The strain showed inhibitory activity against several pathogenic bacteria, no hemolytic activity, and susceptibility to ceftriaxone and chloramphenicol. Its auto-aggregation ability increased over time (up to 93.85% at 24 h), and the surface hydrophobicity reached 33.19%. Additionally, ZZM01 displayed strong antioxidant activity, with a DPPH radical scavenging rate of 84.62%. In vitro cholesterol removal reached 44.94%, although no bile salt hydrolase activity was detected. Whole-genome analysis revealed multiple genes associated with stress tolerance, including acid, bile salt, oxidative, osmotic, heat, and cold stress responses, which were consistent with the observed gastrointestinal tolerance phenotype. Genome annotation also identified genes involved in carbohydrate metabolism, amino acid biosynthesis, and putative bacteriocin production. Safety-related genome mining identified multiple antibiotic resistance-associated genes and putative virulence factor-related genes, while no toxin- or invasion-associated genes were detected. Overall, ZZM01 exhibits strong gastrointestinal tolerance, antimicrobial activity, antioxidant capacity, adhesion-related properties, and favorable genomic characteristics, supporting its potential application as a probiotic strain.

Introduction

In recent years, probiotics have attracted considerable attention due to their multiple beneficial effects on host health, including modulation of gut microbiota, antimicrobial activity, antioxidant capacity, and immune regulation, thereby contributing to the prevention of various chronic diseases (Aminlari et al. 2019; Kavyani et al. 2024; Hashish et al. 2025). These multifunctional properties have positioned probiotics as promising candidates for functional food development and health promotion. Among them, lactic acid bacteria (LAB) are among the most extensively studied and widely utilized probiotic microorganisms and are commonly found in a variety of fermented foods (Aljohani et al. 2025). Among LAB, Lactiplantibacillus plantarum is one of the most widely distributed and well-studied species owing to its remarkable environmental adaptability and ability to colonize diverse ecological niches (Behera et al. 2018). Previous studies have shown that L. plantarum can modulate intestinal microbiota, inhibit potential pathogens, and contribute to host health and disease management (Fidanza et al. 2021). In addition, it exhibits strong tolerance to gastrointestinal conditions, which supports its survival and persistence in the gastrointestinal tract (Garcia-Gonzalez et al. 2021). These characteristics have made L. plantarum an attractive candidate for probiotic research and application.

Probiotics derived from different ecological niches often exhibit strain-specific functional characteristics. As typical carnivores, mink diets contain a high proportion of animal-derived ingredients such as fish and meat (Broom and Fraser 2015). Unlike the characteristic of most livestock and poultry where over 70% of energy is supplied by carbohydrates, Tauson et al. (Tauson et al. 1994) found that the proportion of energy supplied by fat in mink is higher than that of carbohydrates. This unique nutritional environment may shape a distinctive intestinal microbiota with potential functional and adaptive traits. Previous studies have reported that mink-derived lactic acid bacteria may exhibit cholesterol-related activities. However, available data remain limited, and systematic evaluations integrating functional properties, safety assessment, and genomic analysis are still insufficient (Liu et al. 2013).

With the rapid advancement of whole-genome sequencing (WGS) technologies, genome-based approaches have become powerful tools for elucidating the functional potential and safety characteristics of probiotic strains. Through functional annotation using databases such as GO, KEGG, and NR, WGS enables systematic identification of genes associated with stress adaptation, antimicrobial activity, and other probiotic-related traits, as well as safety-related features such as antibiotic resistance and virulence-associated genes.

Therefore, the present study aimed to comprehensively evaluate the probiotic properties, safety characteristics, and genomic features of Lactiplantibacillus plantarum ZZM01 isolated from mink intestinal contents. This study provides a systematic characterization of strain ZZM01 and offers a scientific basis for its potential application in functional food development and probiotic-related applications.

Materials and methods

Isolation and identification of Lactiplantibacillus plantarum ZZM01

Intestinal contents were collected from ten healthy adult minks obtained from Haiyang Furui Mountain Mink Breeding Co., Ltd. (Qingdao, China). The animals were humanely euthanized prior to sampling, and all procedures were approved by the Animal Ethics Committee of Qingdao Agricultural University (Approval No. DKY20231009). Samples were collected under aseptic conditions, transferred to sterile tubes, and transported to the laboratory on ice. Approximately 1 g of intestinal content was suspended in sterile saline and homogenized. The suspension was inoculated into MRS broth (Solarbio, Beijing, China) and incubated at 37 °C with shaking at 200 r/min for 24 h. The cultures were serially diluted and spread onto MRS agar plates, followed by incubation at 37 °C for 24–48 h. Distinct colonies were selected and purified by repeated streaking on MRS agar plates. A total of nine lactic acid bacterial isolates were obtained, among which strain ZZM01 was selected for further study based on its probiotic potential. Colony morphology was observed, and preliminary identification was performed by Gram staining and microscopic examination.

Molecular identification was performed by amplification of the 16 S rRNA gene using the universal primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Beijing, China). The resulting sequences were compared against those in the NCBI database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine taxonomic affiliation. A phylogenetic tree was constructed using MEGA version 12.0 to confirm the genetic relationship of the isolate.

The strain Lactiplantibacillus plantarum ZZM01 has been deposited in the China Center for Type Culture Collection (CCTCC) under the accession number CCTCC NO: M 2,025,695.

Probiotic potential

Growth ability

Activated bacterial cultures were inoculated into MRS broth at a 2% (v/v) inoculation rate and incubated at 37 °C. The optical density at 600 nm (OD₆₀₀) and pH values were measured at regular intervals to monitor growth.

Tolerance to acid and bile salt

Acid and bile salt tolerance assays were performed according to previously described methods (Jiang et al. 2016) with minor modifications. For acid tolerance, an overnight culture of strain ZZM01 was inoculated (1%, v/v) into MRS broth adjusted to pH 2.0, pH 3.0, or pH 4.0 using 1.0 M HCl. Cultures were incubated at 37 °C for 3 h. For bile salt tolerance, ZZM01 was inoculated (1%, v/v) into MRS broth supplemented with 0.3% (w/v) or 0.5% (w/v) bile salts (Solarbio, China) and incubated at 37 °C for 4 h. Acid and bile salt tolerance were assessed by monitoring bacterial survival under stress conditions using OD₆₀₀ measurements. Survival rate was calculated according to the formula:

OD_Initial and OD_final refer to the optical density measured at 0 h and after 3–4 h of incubation, respectively.

Tolerance to simulated gastrointestinal fluids

The tolerance to simulated gastric and intestinal fluids was evaluated according to Argyri et al. (2013) with slight modifications. Bacterial cells were cultured for 18 h, harvested by centrifugation (10,000 × g, 5 min, 4 °C), washed twice with sterile physiological saline, and resuspended in sterile physiological saline. For the simulated gastric condition, the bacterial suspension was incubated in artificial gastric fluid (pH 1.5) containing pepsin (10 mg/mL) at 37 °C for 3 h. Subsequently, the treated cells were collected and transferred into simulated intestinal fluid containing trypsin (10 mg/mL, pH 6.8), followed by incubation at 37 °C for 3 h. Viable cell counts at 0 h and after treatment were determined by plate counting on MRS agar.

Antimicrobial activity

ZZM01 was inoculated into MRS broth and incubated at 37 °C for 18 h, followed by centrifugation at 10,000 r/min for 5 min to obtain the cell-free supernatant (CFS). The supernatant was further filtered through a 0.22 μm membrane filter. The indicator strains (Escherichia coli ATCC 44350, Staphylococcus aureus CMCC 26003, Listeria monocytogenes ATCC 19115, Salmonella enterica ATCC 13311, and Pseudomonas aeruginosa ATCC 10104) were obtained from Guangdong Yuewei Biotechnology Co., Ltd. (Guangzhou, China), cultured in Tryptic Soy Broth (Macklin, Shanghai, China) at 37 °C for 18 h, and adjusted to a final concentration of 1 × 10⁷ CFU/mL. For the antimicrobial assay, 100 µL of each bacterial suspension was evenly spread onto Tryptic Soy Agar (TSA, Macklin, Shanghai, China) plates to obtain a uniform lawn. Wells (6 mm in diameter) were aseptically punched using a sterile cork borer, and 100 µL of CFS was added into each well. The plates were incubated at 37 °C for 24 h, and inhibition zones were measured in millimeters.

In vitro cholesterol assimilation

The cholesterol-lowering ability of Lactiplantibacillus plantarum ZZM01 was assessed based on a modified version of the method described in (Hajime 1972). Cholesterol was added to MRS broth at a final concentration of 0.1 mg/mL. Briefly, 0.2 g of bile salts, 1 mL of Tween-80, and 0.1 g of sucrose octaacetate were dissolved in 5 mL of ice-cold acetic acid and rapidly added to 1,000 mL of MRS broth. ZZM01 (1 × 10⁹ CFU/mL) was inoculated at 1% (v/v) into the MRS broth containing cholesterol, while uninoculated MRS broth served as the control. Cultures were incubated at 37 °C for 18 h, then centrifuged at 12,000 × g for 10 min at 4 °C to collect the supernatant. Cholesterol concentrations in the supernatant and control broth were determined using the o-phthalaldehyde method (Rudel and Morris 1973). Briefly, 1 mL of sample was mixed with 6 mL of 95% ethanol and 4 mL of 50% (w/v) KOH, heated at 60 °C for 10 min, and cooled to room temperature. Subsequently, 5 mL of hexane and 3 mL of distilled water were added, mixed for 1 min, and allowed to stand for 15 min to achieve phase separation. Three milliliters of the hexane layer were collected and evaporated under nitrogen at 60 °C. Residues were dissolved in 4 mL of freshly prepared o-phthalaldehyde solution (0.5 mg/mL in ice-cold acetic acid) and incubated at room temperature for 10 min. Then, 2 mL of concentrated sulfuric acid was carefully added, mixed immediately, and incubated for another 10 min. Absorbance was measured at 550 nm, with blank controls for correction. Cholesterol concentrations were calculated using a standard curve prepared from 0 to 0.2 mg/mL cholesterol solutions. The cholesterol removal rate (%) was calculated as:

R₀ and R₁ represent the cholesterol content in MRS medium at 0 h and 24 h, respectively.

Bile salt hydrolase activity

Bile salt hydrolase (BSH) activity of the selected strains was determined according to the method described by Nguyen et al. (2007). Briefly, overnight cultures were streaked onto MRS agar supplemented with 0.37 g/L CaCl₂ and 0.5% (w/v) sodium taurodeoxycholate (Macklin, Shanghai, China). The plates were incubated at 37 °C for 48 h. BSH activity was evaluated based on the formation of precipitation zones surrounding the colonies, indicating hydrolysis of bile salts.

Cell surface hydrophobicity

The cell surface hydrophobicity was determined by following the established protocol (Polak-Berecka et al. 2014). Strains were cultured in MRS broth at 37 °C for 18 h and harvested by centrifugation at 10,000 r/min for 10 min at 4 °C. The collected cells were washed twice with phosphate-buffered saline (PBS, pH 7.0) and resuspended in the same buffer. The initial absorbance (A₀) of the suspension was measured at 600 nm. Subsequently, 5 mL of the bacterial suspension was mixed with 1 mL of xylene and incubated at 37 °C for 1 h. After phase separation, the absorbance of the aqueous phase (A₁) was measured at 600 nm. Cell surface hydrophobicity was calculated based on the initial and final absorbance values using the following formula:

A₀ and A₁ are the absorbances at 0 h and 1 h, respectively.

Auto-aggregation assay

The auto-aggregation ability of ZZM01 was evaluated according to Krausova et al. (2019). Briefly, cultures grown in MRS broth at 37 °C for 18 h were harvested, washed twice with PBS, and centrifuged at 6000 r/min for 8 min. The cell pellets were resuspended in PBS, and the absorbance at 600 nm (OD₆₀₀) was adjusted to 0.8 ± 0.05 (A₀). The suspensions were incubated at room temperature without disturbance, and the absorbance at 600 nm (Aₜ) was measured after 2, 6, 10, 20 and 24 h. The auto-aggregation rate was calculated using the following formula:

A₀ and A_t are the absorbances at 0 h and 2, 6, 10, 20, 24 h, respectively.

DPPH radical scavenging activity assay

The DPPH free radical scavenging capacity of ZZM01 was measured using the DPPH free radical scavenging capacity assay kit (Grace, Suzhou, China). Briefly, 10 µL of bacterial extract was mixed with 190 µL of DPPH methanol working solution, mixed for 60 s, and incubated in the dark at room temperature for 30 min before measuring the absorbance at 517 nm. The calculation formula is as follows: The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of strain ZZM01 was determined using a commercial assay kit (Grace, Suzhou, China) according to the manufacturer’s instructions with slight modifications. Briefly, 150 µL of bacterial suspension was mixed with 150 µL of DPPH working solution (prepared by dissolving DPPH powder in anhydrous ethanol). For the control, 150 µL of bacterial suspension was mixed with 150 µL of 80% methanol. The blank consisted of 150 µL of 80% methanol and 150 µL of DPPH working solution. All mixtures were vortexed and incubated at room temperature in the dark for 30 min, followed by centrifugation at 12,000 × g for 5 min. Subsequently, 200 µL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 517 nm. The DPPH radical scavenging activity (%) was calculated as follows:

A_sample: Absorbance of the sample mixed with working solution.

A_control: Absorbance of the sample mixed with 80% methanol solution.

A_blank: Absorbance of the working solution mixed with 80% methanol solution.

Safety assessment

Hemolytic activity

Hemolytic activity was detected by streak inoculation of isolated strains cultured overnight on blood agar plates containing 7% (v/v) sheep blood (Macklin, Shanghai, China). The plates were then incubated at 37 °C for 48 h, and the hemolytic zones around the colonies were observed (Pieniz et al. 2014).

Antibiotic susceptibility

Antibiotic susceptibility was evaluated using the Kirby–Bauer (K–B) disk diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines, as previously described (Anisimova and Yarullina 2019). An aliquot of 200 µL of an overnight culture of ZZM01 (18 h) was evenly spread onto the surface of agar plates. Antibiotic disks (Solarbio, BeiJing, China) were then placed in close contact with the agar surface using sterile forceps. After standing at room temperature for 5 min to allow initial diffusion, three disks were positioned on each plate. The plates were left at room temperature for 1.5 h and subsequently incubated at 37 °C for 24 h. Following incubation, the diameters of the inhibition zones surrounding the disks were measured using a vernier caliper and recorded. Each strain was tested in triplicate for each antibiotic, and the mean values were calculated.

In vivo safety evaluation

A total of 20 five-week-old male C57BL/6J mice were obtained from Xingkang Biotechnology Co., Ltd. (Jinan, China). The animals were housed in standard plastic cages under controlled environmental conditions with a 12-h light/dark cycle. Mice had free access to standard rodent chow and water throughout the study. Following a one-week acclimation period, the mice were randomly divided into two groups (n = 10). The control group received sterile saline, and the treatment group was administered Lactiplantibacillus plantarum ZZM01 at a concentration of 1 × 10⁹ CFU/mL. Oral gavage was performed once daily with a volume of 0.2 mL per mouse. On day 27, all mice were fasted for 12 h and then anesthetized and humanely euthanized by an intraperitoneal overdose of sodium pentobarbital (Sigma-Aldrich, USA) at 150 mg/kg body weight, a dose commonly used to ensure deep anesthesia and humane euthanasia in rodents. Whole blood was collected from the retro-orbital venous plexus. Serum was obtained by centrifugation at 3,000 × g for 15 min at 4 °C, aliquoted, and stored at − 80 °C for subsequent biochemical analyses. All animal procedures were approved by the Animal Ethics Committee of Qingdao Agricultural University (Approval No. DKY20231009) and conducted in accordance with institutional guidelines for the care and use of laboratory animals.

Serum biochemical parameters, including total protein (TP), albumin (ALB), glucose (GLU), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), were determined using Jiancheng kits (Nanjing, China) according to the manufacturer’s instructions, and measurements were performed with an automated biochemical analyzer (Beckman Coulter AU5800, USA).

Complete genome sequencing analysis

Genomic DNA of strain ZZM01 was extracted using a modified cetyltrimethylammonium bromide (CTAB) method. DNA concentration and purity were determined using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

For whole-genome sequencing, both short-read and long-read technologies were employed. A paired-end library was constructed using the TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina NovaSeq platform to obtain high-quality short reads. A long-read library was prepared according to the Oxford Nanopore Technologies protocol and sequenced on the PromethION platform (Oxford Nanopore Technologies, Oxford, UK). Raw reads were filtered to remove adapters and low-quality sequences. Hybrid de novo assembly was performed using SPAdes and Flye, and the assembled genome was polished using Pilon to obtain the final complete circular chromosome.

Gene prediction was conducted using GeneMarkS. Transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were identified using tRNAscan-SE and Barrnap, respectively, and CRISPR loci were detected using CRISPR Finder. Functional annotation was performed through BLAST searches against the KEGG, GO, COG, and Swiss-Prot databases(Kanehisa et al. 2017; van Heel et al. 2018). Carbohydrate-active enzyme (CAZyme) genes were identified using hmmscan against the CAZy database (Krogh et al. 1994).

Potential virulence factors and antibiotic resistance genes were predicted by BLASTp searches against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD), respectively, with thresholds set at E-value ≤ 1e − 6, sequence identity ≥ 45%, and alignment coverage ≥ 70%. Prophage regions and genomic islands were predicted using PhiSpy and IslandViewer4. A circular genome map was generated using CGView.

Statistical analysis

Data were tested for normality using the Shapiro–Wilk test. Differences between two groups were analyzed using an unpaired Student’s t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was performed. All data are presented as mean ± standard deviation (SD), and differences were considered statistically significant at P < 0.05. Statistical analyses were conducted using IBM SPSS Statistics for Windows, version 27.0 (IBM Corp., Armonk, NY, USA).

Results

Identification of ZZM01

Strain ZZM01 formed circular, smooth-edged, centrally raised, milky white colonies on MRS agar medium (Fig. 1A). Under microscopic observation, strain ZZM01 appeared as rod-shaped, non-spore-forming, Gram-positive bacteria (Fig. 1B). The 16 S rRNA sequencing results of ZZM01 were compared with the NCBI database (Fig. 1C), showing 100% similarity to Lactiplantibacillus plantarum strains JCM 1231, DSM 21,115, LMG 24,282, and LMG 23,560. Therefore, strain ZZM01 was identified as Lactiplantibacillus plantarum.

Fig. 1.

Morphology, staining and phylogenetic tree of strain ZZM01.A Morphology of strain ZZM01 on MRS medium. B Gram staining results of strain ZZM01.C Neighbor-joining phylogenetic tree of strain ZZM01

Evaluation of the probiotic potential of ZZM01

Growth curves

As shown in Fig. 2A, the strain grew slowly during the first 0–4 h of cultivation, corresponding to the lag phase. From 4 to 14 h, the OD₆₀₀ increased rapidly, indicating an exponential growth phase. After 24 h, the growth rate leveled off and entered the stationary phase. The pH began to decline sharply after 4 h and decreased more gradually after 24 h, corresponding to the onset of the stationary phase. This acidification pattern is consistent with the typical fermentation characteristics of lactic acid bacteria.

Fig. 2.

Probiotic potential evaluation of strain ZZM01 A Growth and acid-producing curve; B Acid resistance; C Bile salt resistance; D Gastrointestinal tract models; E Auto aggregation ability; F Cell surface hydrophobicity rate; G DPPH radical scavenging rate; H Cholesterol-removal rate; I Hemolysis results on the front and back sides of blood agar plates for Staphylococcus aureus and ZZM01; J Bile salt hydrolase (BSH) activity of ZZM01. *P < 0.05 and **P < 0.01

Tolerance to acid and bile salt

As shown in Fig. 2B and C, the survival rates of strain ZZM01 after 3 h of exposure were 71.92%, 84.34%, and 88.81% at pH 2.0, 3.0, and 4.0, respectively. Regarding bile salt tolerance, ZZM01 exhibited survival rates of 84.71% and 72.54% after 4 h in media containing 0.3% and 0.5% bile salts.

Tolerance to simulated gastrointestinal fluids

As shown in Fig. 2D, Lactiplantibacillus plantarum ZZM01 exhibited high survival rates after 3 h of exposure to simulated gastrointestinal conditions. The survival rate in simulated gastric fluid was 90.55 ± 1.01%, while a slightly higher survival rate of 92.68 ± 1.41% was observed in simulated intestinal fluid, indicating strong tolerance to gastrointestinal environments.

Auto aggregation activity and cell surface hydrophobicity

As shown in Fig. 2E, the auto-aggregation activity rates of Lactiplantibacillus plantarum ZZM01 at 2, 6, 10, 20 and 24 h were 23.97%,35.97%, 50.88%, 93.09%, and 93.85%, respectively, with extremely significant differences between groups (P < 0.01), and increased significantly with incubation time. The CSH of strain ZZM01 after xylene treatment was 33.19% (Fig. 2F).

Antioxidant activity

The DPPH free radical scavenging rate of strain ZZM01 was 84.62 ± 1.66% (Fig. 2G).

Cholesterol-removal rate

As shown in Fig. 2H, the in vitro cholesterol removal rate of ZZM01 was 44.94 ± 15.45%.

Bile salt hydrolase (BSH) activity

The strain ZZM01 showed no bile salt hydrolase activity on MRS agar medium supplemented with 0.37 g/L CaCl₂ and 0.5% (w/v) sodium taurodeoxycholate (Fig. 2J).

Antimicrobial activity

The ability of strain ZZM01 to inhibit harmful indicator bacteria is shown in Table 1. Lactiplantibacillus plantarum ZZM01 effectively inhibited the growth and proliferation of five pathogenic bacteria. The cell-free supernatant showed significantly greater inhibition against Salmonella enterica than against Escherichia coli and Listeria monocytogenes (P < 0.05), and significantly greater inhibition against Staphylococcus aureus than against Listeria monocytogenes (P < 0.05).

Table 1.

Antimicrobial activity of Lactiplantibacillus plantarum ZZM01

Items	Inhibition circle diameter of cell free supernatant(mm)
Escherichia coli	11.60 ± 1.30bc
Staphylococcus aureus	13.81 ± 2.10ab
Salmonella enterica	14.90 ± 1.63a
Listeria monocytogenes	10.77 ± 0.85c
Pseudomonas aeruginosa	12.77 ± 2.01abc

Different superscript lowercase letters indicate significant differences among means at P < 0.05. Values sharing at least one common letter are not significantly different. Values are expressed as mean ± SD (n = 3)

Safety assessment

Hemolytic activity

As shown in Fig.2I, Lactiplantibacillus plantarum ZZM01 exhibited negative hemolytic activity, with Staphylococcus aureus serving as the positive control.Antibiotic Susceptibility

Antibiotic susceptibility

According to the CLSI standards for antimicrobial susceptibility, the antibiotic sensitivity of the tested strain was evaluated. As shown in Table 2, Lactiplantibacillus plantarum ZZM01 was resistant to vancomycin, streptomycin, ciprofloxacin, amikacin, and ampicillin, intermediately resistant to penicillin, but highly sensitive to ceftriaxone and chloramphenicol.

Table 2.

Antibiotic resistance of ZZM01

Antimicrobial classes	Antibiotics name	antibacterial ring diameter (mm)	Judgment standard: antibacterial ring diameter (mm)			Susceptibility
			Resistance (R)	Intermediate (I)	Sensitive (S)
β-lactams antibiotics	Ceftriaxone	23.15 ± 0.71	≤ 19	20 ~ 22	≥ 23	S
	Penicillin	18.20 ± 1.71	≤ 15	16 ~ 20	≥ 21	I
	Ampicillin	9.65 ± 0.65	≤ 12	13 ~ 15	≥ 16	R
Broad-spectrum antibiotics	Chloramphenicol	19.73 ± 1.63	≤ 12	13 ~ 17	≥ 18	S
Fluoroquinolone antibiotics	Ciprofloxacin	0	≤ 13	14 ~ 18	≥ 19	R
Aminoglycosides antibiotics	Streptomycin	0	≤ 11	13 ~ 17	≥ 15	R
	Amikacin	0	≤ 15	16 ~ 17	≥ 18	R
Glycopeptides	Vancomycin	8.87 ± 0.24	—	—	≥ 15	R

R Resistant, I Intermediate, S Susceptible

Growth performance and serum biochemical parameters

As shown in Fig. 3, no significant differences were observed in body weight between the two groups. TP, ALB, and GLB levels also did not differ significantly between groups (P > 0.05). The ZZM01 group exhibited significantly higher GLU compared with the control group (P < 0.05). Regarding lipid metabolism, ZZM01 group markedly reduced TC (P < 0.01) and LDL-C (P < 0.01). HDL-C was also significantly decreased in the ZZM01 group (P < 0.05), whereas TG showed no significant difference between groups (P > 0.05). Serum AST and ALT levels were comparable between groups (P > 0.05).

Fig. 3.

Lactiplantibacillus plantarum ZZM01 safe evaluation in vivo. A Body weight; B Total protein (TP); C Albumin (ALB); D Globulin (GLB); E Glucose (GLU); F Total cholesterol (TC); G Triglycerides (TG); H High-density lipoprotein cholesterol (HDL-C); I Low-density lipoprotein cholesterol (LDL-C); J Aspartate aminotransferase (AST); K Alanine aminotransferase (ALT). *P < 0.05, **P < 0.01

Complete genome sequencing analysis

Genomic characterization of Lactiplantibacillus plantarum ZZM01

The complete genome of Lactiplantibacillus plantarum ZZM01 was sequenced using second and third generation sequencing technologies, and the genome map was drawn using cgview (Fig. 4). The total base length of ZZM01’s complete genome was 3,118,339 bp, with a GC content of 44.72%, and 6 plasmids were assembled, with plasmid lengths ranging from 2,411 bp to 60,512 bp (Table 3). The genome contained 3,005 protein-coding sequences (CDS) and 67 non-coding RNAs. Prediction of special structures included 77 secreted proteins and 15 prophages. To obtain comprehensive gene function information, we performed functional annotation analysis using multiple databases.

Fig. 4.

Complete genome map of L. plantarum ZZM01.Circles from inner to outer represent: the first circle indicates scale; the second circle represents GC Skew; the third circle represents GC content; the fourth and seventh circles represent COG categories of each CDS; the fifth and sixth circles represent the position of CDS, tRNA and rRNA on the genome

Table 3.

Genome assembly of Lactiplantibacillus plantarum ZZM01

Type	Length (bp)
Total genome size (bp)	3,241,598
Chromosome size (bp)	3,118,339
Plasmids	6
Total plasmid size (bp)	2,411–60,512
Total Protein-Coding Genes (CDS)	3,005
rRNA	6 × 5 S, 5 × 16 S, 5 × 23 S
tRNA	62
ncRNA	67
Chromosome GC content (%)	44.72

Functional annotation analysis of the Lactiplantibacillus plantarum ZZM01

The annotation statistics are shown in the Fig. 5. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, gene annotations covered multiple metabolic pathways, with carbohydrate metabolism and amino acid metabolism being the most annotated pathways. Additionally, in the Gene Ontology (GO) functional enrichment analysis, the main enriched functional categories included catalytic activity and binding function in molecular function, cell membrane and intracellular components in cellular components, and metabolic processes and cellular processes in biological processes. The Clusters of Orthologous Groups (COG) functional annotation results showed that the most annotated functions were transport and metabolism, followed by energy production and conversion. In the Carbohydrate-Active enZYmes (CAZy) database, multiple enzyme categories were annotated, with glycoside hydrolases (GHs) being the most numerous, followed by glycosyltransferases (GTs).

Fig. 5.

Functional prediction of L.plantarum ZZM01.A KEGG analysis; B CAZy analysis; C COG of proteins functional; D GO analysis

Lactiplantibacillus plantarum ZMM01 tolerance-related genes

Genome analysis revealed that Lactiplantibacillus plantarum ZZM01 harbors 4 pH stress-related genes, 4 osmotic stress-related genes, 4 metal stress-related genes, 3 heat stress-related genes, 2 cold shock-related genes, 4 bile salt resistance-related genes, and 6 oxidative stress-related genes (Table 4).

Table 4.

Tolerance-related genes of Lactiplantibacillus plantarum ZZM01

Stress type	Gene	Gene Name	Gene Function
pH stress	chr_156	nhaC	Na+/H+ antiporter NhaC
	chr_327	napA	Na+/H+ antiporter
	chr_624	nhaK	sodium: proton antiporter
	chr_873	mleS	NAD-dependent malic enzyme
Osmotic stress	chr_306	opuCB	glycine/betaine ABC transporter permease
	chr_307	yehX	glycine betaine uptake ATP-binding protein
	chr_1238	opuCA	betaine/proline/choline ABC transporter ATP-binding protein
	chr_1240	opuCC	osmoprotectant ABC transporter substrate-binding protein
Metal stress	chr_219	mntH	divalent metal cation transporter
	chr_670	zur	zinc-specific metallo-regulatory protein
	chr_850	mntB	manganese transport ATP-binding protein
	chr_852	psaA	zinc ABC transporter substrate-binding protein
Heat stress	chr_544	groEL	chaperonin GroEL
	chr_1589	dnaJ	molecular chaperone DnaJ
	chr_1590	dnaK	molecular chaperone DnaK
Cold shock	chr_28	cspL	cold shock protein 2
	chr_907	csp	cold shock protein 1
Bile salt resistance	chr_53	yxeI	choloylglycine hydrolase family protein
	chr_1642	cbh	choloylglycine hydrolase family protein
	chr_1672	clpX	ATP-dependent Clp protease ATP-binding subunit
Oxidative stress	chr_181	gpo	glutathione peroxidase
	chr_196	trxA	thioredoxin
	chr_308	garB	glutathione reductase
	chr_578	trxB	thioredoxin-disulfide reductase
	chr_688	ohrR	organic hydroperoxide resistance regulator
	chr_1038	msrA1	peptide-methionine (S)-S-oxide reductase

Lactiplantibacillus plantarum ZMM01 safety-related genes

As shown in Tables 5 and 6, multiple antibiotic resistance-related genes and putative virulence factor-related genes were identified in Lactiplantibacillus plantarum ZZM01. The resistance-related genes were distributed across several drug classes, including 4 genes associated with fluoroquinolone resistance, 2 with rifamycin resistance, 1 with cycloserine resistance, 3 with multidrug resistance, 1 with glycopeptide resistance, 1 with aminoglycoside resistance, and 2 with daptomycin-related resistance. Putative virulence factor-related genes were also detected, including genes annotated to stress response, transcriptional regulation, cell envelope biosynthesis, and central metabolism.No toxin- or invasion-related genes were detected.

Table 5.

Antibiotic resistance-related genes in Lactiplantibacillus plantarum ZZM01

Drug Class	Gene	Identity (%)	Coverage (%)	Functional Annotation
Fluoroquinolones	gyrA	58.91	98.71	DNA gyrase subunit A
	gyrB	65.13	97.67	DNA gyrase subunit B
	parC	60.37	98.65	Topoisomerase IV subunit A
	parE	66.52	98.2	Topoisomerase IV subunit B
Rifamycins	rpoB	70.86	96.02	RNA polymerase beta subunit
	rpoC	69.13	97.94	RNA polymerase beta’ subunit
Cycloserine	alr	53.76	99.73	Alanine racemase
Multidrug	mdtG	46.05	93.14	Drug efflux transporter
	lmrC	47.11	99.83	ABC transporter
	lmrD	55.91	80.51	ABC transporter
Glycopeptides	vanR	50.44	98.7	Response regulator
Aminoglycosides	StrA	68.38	100	Aminoglycoside phosphotransferase
Daptomycin-related	pgsA	48.96	100	Phospholipid biosynthesis
	cls	48.75	97.17	Cardiolipin synthetase

Table 6.

Prediction of virulence factors in Lactiplantibacillus plantarum ZZM01

Virulence Factor ID	Gene	Putative Function
VF0594	groEL	Stress response and protein folding
VF0244	hasC	Polysaccharide biosynthesis
VF0074	clpP	Stress tolerance and protein quality control
VF0073	clpE	Stress response
VF0713	dnaK	Heat shock response
VF1060	eno1	Glycolysis and adhesion-related function
VF0877	tuf	Protein synthesis; potential adhesion role
VF0361	glf	Cell wall biosynthesis
VF0659	rfbP	Cell envelope formation
VF0792	arlR	Environmental adaptation
VF0257	sigA	Transcription regulation
VF0560	gnd	Central metabolism
VF0350	mdh	Energy metabolism

Discussion

For probiotics to survive in the human gastrointestinal tract, they must first be able to tolerate the host’s defense mechanisms, including low pH in gastric juice and high bile salts in the small intestine. In this experiment, Lactiplantibacillus plantarum ZZM01 showed a survival rate of 71.92% after three hours of exposure to acidic conditions (pH 2.0), and 84.71% after four hours of exposure to 0.3% bile salt environment. Compared with the study by Karasu et al. (2010), in which multiple Lactiplantibacillus plantarum strains exhibited 3 h survival rates of 58–79% at pH 3, ZZM01 in the present study showed superior acid tolerance under the same or even lower pH conditions. Considering that bile salt concentrations in the small intestine typically range from 0.03% to 0.3% (Kailasapathy et al. 2008), these results indicate that ZZM01 possesses strong tolerance to gastrointestinal stress. Genome analysis further supports these phenotypic characteristics. Several stress response-related genes were identified, including acid resistance-associated genes (nhaC, napA, and nhaK), molecular chaperones (groEL, dnaK, and dnaJ), and bile tolerance-related genes such as yxeI and cbh. These genes are known to contribute to intracellular pH homeostasis, protein folding, and cellular stress adaptation, thereby enhancing bacterial survival under acidic and bile stress conditions. Collectively, these findings suggest that the gastrointestinal tolerance of ZZM01 is mediated by general stress response systems rather than specific enzymatic pathways.

The antibacterial activity of lactic acid bacteria may be due to organic acids, hydrogen peroxide, bacteriocins, or other inhibitory substances in metabolites (Caplice and Fitzgerald 1999; Saboori et al. 2022). In in vitro inhibition experiments, we demonstrated that Lactiplantibacillus plantarum ZZM01 has ideal antagonistic effects against five common pathogens Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Pseudomonas aeruginosa, which is consistent with previous reports by Zhao et al. (2025). Notably, the inhibitory effect of ZZM01 against Salmonella enterica was the most pronounced among all tested indicator strains, suggesting a potentially stronger and more specific anti-Salmonella activity. This inhibitory performance was higher than the average activity reported for 26 isolated from milk (Zhang et al. 2022), as well as stronger than that observed in multispecies direct-fed microbial (DFM) preparations, where inhibition of Salmonella enterica was relatively limited (Gresse et al. 2024). Moreover, Barbosa et al. (2021) demonstrated that lactic acid bacteria (LAB) are capable of producing bacteriocins—low-molecular-weight, heat-stable, ribosomally synthesized antimicrobial peptides that have been shown to inhibit various foodborne pathogens. Li et al. (2016)reported that most pln genes are involved in the biosynthesis of bacteriocins. Through the BAGEL4 database (van Heel et al. 2018), we found 6 proteins in the ZZM01 genome that are 100% identical to class II bacteriocins (Plantaricins A, Plantaricins E, Plantaricins F, Plantaricins J, Plantaricins K, and Plantaricins N), which may explain its broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, particularly its strong inhibitory effect on Salmonella enterica.

Safety is an important criterion for screening probiotics. Lactic acid bacteria are beneficial to health (Garbacz 2022; Abdul Hakim et al. 2023).however, when these bacteria appear in the food chain and the intestines of animals and humans, they may also become repositories for mobile antibiotic resistance genes, thereby transferring these genes to pathogens (McInnes et al. 2020). Therefore, bacteria used as human or animal probiotics should not carry any transferable antibiotic resistance genes (Authority (EFSA) 2008). In the present study, ZZM01 was sensitive to ceftriaxone and chloramphenicol, moderately sensitive to penicillin, and resistant to ciprofloxacin, amikacin, vancomycin, and streptomycin. Genome analysis using the CARD database (Alcock et al. 2020) identified resistance-related genes such as gyrA, parC, and vanR. CARD-based annotation further indicated that these resistance determinants were primarily located on the chromosome, with no resistance genes detected on plasmids, suggesting a low potential for horizontal gene transfer.Previous studies have shown that many Lactiplantibacillus species exhibit intrinsic resistance to antibiotics such as ciprofloxacin, streptomycin, and vancomycin (Gogry et al. 2021). Virulence factor analysis using BLAST against a curated virulence database showed that the identified genes were mainly associated with basic cellular functions, such as stress response and metabolism, rather than pathogenicity-related functions, indicating a low virulence potential. Intrinsic resistance results from inherent structural or physiological characteristics of bacteria and generally shows a low potential for horizontal gene transfer, thereby posing limited safety concerns in non-pathogenic microorganisms (Campedelli et al. 2018; Bottery et al. 2021). Hemolysis testing is an important indicator of probiotic safety. Lactic acid bacteria are generally considered to be non-hemolytic (Chen et al. 2022). In this study, Lactiplantibacillus plantarum ZZM01 did not exhibit hemolytic activity, consistent with these reports, indicating that Lactiplantibacillus plantarum ZZM01 is non-toxic. In vivo safety assessment further confirmed the non-toxic nature of ZZM01. No significant differences in body weight or key serum biochemical parameters were observed between treated and control groups, indicating no adverse effects on liver function or general metabolism.

The ability to adhere to intestinal mucosa or intestinal epithelial cells and proliferate is a prerequisite for lactic acid bacteria to function in the intestine (Sengupta et al. 2013). Studies have shown that the auto-aggregation ability and hydrophobicity of strains can reflect their adhesion ability to intestinal mucosa or intestinal epithelial cells, and probiotics with strong auto-aggregation ability have better adhesion to cells (Tjandrawinata et al. 2022). In this study, ZZM01 exhibited a high auto-aggregation rate of 93.85% at 24 h and a surface hydrophobicity of 33.19%, which are higher than those reported for lactic acid bacteria isolated from broiler intestines (Li et al. 2024), indicating that ZZM01 has the potential to adhere to the intestinal mucosa and form a gastrointestinal defense barrier. CAZy annotation further demonstrated a high abundance of glycoside hydrolase (GH) genes, suggesting an enhanced capacity for carbohydrate degradation and utilization. This metabolic flexibility may support bacterial growth and persistence under intestinal conditions.

Good antioxidant capacity of strains can not only improve the body’s resistance to oxidative stress responses but also inhibit the formation of pathological conditions associated with oxidative stress. Many previous studies have reported the antioxidant properties of lactic acid bacteria (Yadav et al. 2016; Zhang et al. 2020). In this study, Lactiplantibacillus plantarum ZZM01 exhibited a DPPH radical scavenging rate of 84.62%, which is higher than the values reported for 15 Lactobacillus strains isolated from human feces (ranging from 52.3% to 83.5%) and Streptococcus alactolyticus isolated from chicken cecum. ZZM01 demonstrates superior antioxidant activity when compared to other animal-derived strains, highlighting its potential as a more effective probiotic for oxidative stress alleviation.

Elevated serum cholesterol levels are widely recognized as a major risk factor for cardiovascular diseases. Numerous studies have demonstrated that certain Lactiplantibacillus plantarum strains possess cholesterol-lowering capabilities, which are often associated with bile salt hydrolase (BSH) activity (Ooi and Liong 2010; Wang et al. 2021). BSH catalyzes the deconjugation of bile salts, increasing their hydrophobicity and reducing their intestinal reabsorption, thereby promoting their excretion and indirectly lowering serum cholesterol levels (Begley et al. 2006). In this study, Lactiplantibacillus plantarum ZZM01 exhibited a cholesterol removal rate of 44.94% in vitro. Previous studies have reported that among 66 Bacteroides strains isolated from healthy adult feces, only six strains showed cholesterol assimilation abilities exceeding that of Lacticaseibacillus rhamnosus GG (LGG). Under comparable experimental conditions, ZZM01 demonstrated a relatively higher cholesterol removal capacity. Although putative bile salt hydrolase (BSH)-related genes were identified in the genome, no detectable BSH activity was observed under the experimental conditions. This apparent discrepancy may reflect condition-dependent gene expression, limited catalytic efficiency, or substrate specificity of the encoded enzymes (Xu et al. 2019; Foley et al. 2021).This suggests that ZZM01 may lower cholesterol levels through other pathways. H. Kimoto (2002) reported that Lactococcus lactis subsp. lactis biovar diacetylactis N7 could remove cholesterol from the medium without producing BSH, similar to our findings. Research has found that this strain lowers cholesterol levels by physically adsorbing cholesterol and incorporating it into the cell membrane, a process closely related to cell growth. However, the present study did not employ a high-fat diet model, which limits the ability to directly evaluate the cholesterol-lowering efficacy of ZZM01 under hyperlipidemic conditions. Therefore, future studies using appropriate animal models are required to further validate its in vivo functionality and elucidate the underlying mechanisms.

Conclusions

In conclusion, Lactiplantibacillus plantarum ZZM01 isolated from mink intestine exhibits high tolerance to low pH and bile salts, good auto-aggregation ability, notable surface hydrophobicity, and strong antioxidant capacity. The strain also exerts inhibitory activity against multiple pathogenic bacteria and shows no hemolytic activity, with a low risk of horizontal transfer of antibiotic resistance genes, indicating its good gastrointestinal adaptability and safety. In addition, ZZM01 demonstrates a considerable in vitro cholesterol-removal capacity. Overall, this strain holds promise for applications in food, animal production, and biomedical fields.

Authors’ contributions

Y.W. and C.H. performed data curation, investigation, and methodology, and wrote the original draft. J.W. provided resources and contributed to review and editing of the manuscript. Y.Z. and K.L. assisted with data curation, methodology, and manuscript review. W.S. acquired funding, conceived and supervised the study, and contributed to manuscript review and editing. All authors reviewed and approved the final manuscript.

Funding

This research was supported by the Shandong Provincial Special Economic Animal Industry Technology System (SDARS-21).

Data availability

The raw sequence data have been deposited in the NCBI database under accession number PRJNA1375671.

Declarations

Ethics approval and consent to participate

The animal studies were approved by the Animal Ethics Committee of Qingdao Agricultural University (Approval No.DKY20231009).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.