Evaluating the Probiotic Potential of Lactic Acid Bacteria Isolated from Nipponia nippon Feces

doi:10.21203/rs.3.rs-4984548/v1

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Evaluating the Probiotic Potential of Lactic Acid Bacteria Isolated from Nipponia nippon Feces

https://doi.org/10.21203/rs.3.rs-4984548/v1

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This study aims to identify an optimal lactic acid bacterial strain from the feces of healthy Nipponia nippon. From the fecal samples, twenty isolates were obtained. The isolates were subjected to biochemical identification, acid and bile tolerance tests, in vitro inhibition of pathogenic bacteria assays, cell surface hydrophobicity assessment, antibiotic susceptibility test, and hemolytic activity evaluation to determine their probiotic potential. The results indicated that six isolates (D1, D2, D6, E7, D8, D9) could survive in low acid and high bile salt conditions. Except for D8, all six isolates exhibited inhibitory activity against tested pathogens. Isolates D6 and E7 showed the least resistance to antibiotics, and only E7 demonstrated moderate hydrophobicity. The E7 strain was further studied in depth and identified as L. plantarum through 16srRNA sequencing. To assess its safety, mice were fed with the E7 strain, and the results showed no deaths or adverse effects on blood cellular components.

Whole genome sequencing of Lactobacillus plantarum E7 using Nanopore PromrthION48 and the Illumina Novaseq revealed a ring chromosome and two ring plasmids. The chromosome encodes 3024 genes, some associated with cell adhesion, acid and bile salt tolerance, antioxidant enzymes, and secondary metabolites. Plasmids contained fewer coding genes. The VFDB database detected only a few virulence genes related to adherence, stress survival, exoenzyme production, immune modulation, and regulation factors. KEGG database analysis indicated that the genes of this bacterium are primarily involved in carbohydrate metabolism, amino acid metabolism, vitamin and cofactor metabolism, environmental information processing and genetic processing. This study lays a theoretical foundation for the clinical application and development of probiotics.

Lactic acid bacteria

Nipponia nippon

Probiotic

antibacterial activity

the complete genome

Lactic acid bacteria (LAB) are among the most significant and extensively researched probiotics. LAB can ferment carbohydrates to produce lactic acid, which enhances their suitability for processing, storage, and survival through the gastrointestinal tract^{[1, 2]}. Studies have demonstrated that LAB can strengthen the intestinal epithelial barrier^[3], secrete antibacterial substances^[4] (such as bacteriocin, organic acid, hydrogen peroxide), regulate intestinal flora^[5], and participate in immunity responses^[6], thereby exerting their probiotic properties. Wang et al.^[7] reported that Tibetan-derived lactic acid bacteria, when used as a probiotic additive for poultry, could improve the growth performance and intestinal barrier health of broilers. Further, Peng et al.^[8] found that Lactobacillus rhamnosus could combat salmonella infection in chickens, serving as a new antibiotic alternative product for farm animals.

Nipponia nippon also known as the Oriental gem. is a vulnerable, endemic species that is extremely popular worldwide. As an omnivorous bird affected by environmental and human factors, it is prone to various intestinal diseases^[9]. While the prevention and treatment of these diseases typically rely on antibiotics, the development of resistance necessitates alternative solutions. LAB, as beneficial bacteria in the gut of the crested ibis (Nipponia nippon), have not yet been fully explored.

Therefore, this study aims to isolate lactic acid bacteria from the feces of Nipponia nippon, conduct in vitro tests to evaluate their probiotic property (biochemical identification, tolerance tests, antibacterial tests, drug sensitivity tests, hydrophobic tests and in vivo safety evaluations) and analyze complete genome sequencing information. The goal is to identify a strain with strong probiotic properties. This study represents the first step toward developing potential probiotics from the crested ibis, paving the way for extensive laboratory and clinical evaluations in the future.

1.1 Strains and Feces Samples

Feces samples were collected from four healthy Nipponia nippon (Emei Mountain Nipponia nippon breeding base in Sichuan province). Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923 and Salmonella typhimurium ATCC14418 were obtained from the pharmaceutical laboratory (Sichuan Agricultural University).

1.2 Isolation of LAB

One gram of each fecal sample was homogenized in 9 mL of sterile saline, serially diluted, and plated onto MRS agar. The plates were incubated at 37°C for 24–48 h in an anaerobic environment. Colonies with different morphologies were selected and purified through multiple restreaking on MRS agar^[10]. Gram-positive pure isolates were subcultured in MRS broth for further study. Initial biochemical identification was conducted by Hangzhou Microbial Reagent co., Ltd. referencing Bergey’s Manual of Systematic Bacteriology and Isolation and Identification and Test Methods of Lactic Acid Bacteria.

1.3 Acid and Bile Tolerance Test

For the tolerance tests, isolates were subcultured in MRS broth for 24 h. Equal volumes of suspension were added to MRS broth adjusted to pH 1.0, 2.0, and 3.0 with 1 M HCl and containing bile salts at 0.3, 1.0, and 2.0% (w/v). The broth was incubated at 37°C and viable counts were conducted at 0 and 3 h for acid tolerance tests and at 0 and 4 h for bile tolerance tests.

1.4 In vitro inhibition of pathogenic bacteria test

The antibacterial activity of the isolates against Escherichia coli, Staphylococcus aureus and Salmonella was assessed using the punch method. Isolates were grown in MRS broth at 37°C for 24 h, and the cell-free supernatant (CFS) was collected by centrifugation (12,000×g, 10 min). Freshly grown pathogen cultures (100 µ L, 10⁶ CFU/mL) were spread on NA agar plates, and 8 mm diameter holes were punched into the agar. 100 µL CFS was added to each hole, and the plates were incubated at 37°C for 12 hours. The inhibition zones were measured and recorded^[11].

1.5 Antibiotic Susceptibility Test

Antibiotic susceptibility was evaluated using the disk diffusion method. Isolates and an antibiotic-impregnated paper disk (Hangzhou Microbial Reagent Co., Ltd.) were placed on MRS agar plates. After 12 hours of incubation at 37°C, the inhibition zones were measured and compared to Performance Standards for Antimicrobial Disk Susceptibility Tests^[12]. Antibiotics tested included Penicillin (10U), Cefoperazone (75ug), Cefradine (30ug), Carbenicillin (100ug), chloramphenicol (30ug), tetracycline (30ug), doxycycline (30ug), erythromycin (15ug), ciprofloxacin (5ug), gentamicin (10ug), kanamycin (30ug), vancomycin (30ug).

1.6 LAB cell surface hydrophobicity assay

The affinity of LAB to hydrocarbons could reflect the hydrophobicity of the strain surface^[13]. The isolates were inoculated in MRS broth for 24 h. The culture was centrifuged (12,000 × g, 10 min) to harvest the cells and washed 2–3 times by PBS (pH7.4) buffer, and then resuspended in PBS to adjust the absorbance value A600nm to 0.3 − 0.26. The 3mL bacterial suspension and 1 mL xylene were mixed with a vortex for 5min, and the absorbance value at 600nm was measured after 30 min. The surface hydrophobicity of LAB was calculated by formula 1, and three independent tests were repeated.

Hydrophobicity%=[(A₀-A)/A₀]×100%

A0: absorbance value of LAB before xylene treatment; A: Absorbance value of LAB after xylene treatment;

1.7 Hemolytic Activity

Hemolytic activity was tested by streaking fresh bacterial cultures onto blood agar media (containing 5–10% sheep blood) and incubated for 24 h at 37℃. ETEC CVCC 196 was used as a positive control. The presence of clear zones indicated beta hemolysis, greenish zones indicated alpha hemolysis, and the absence of zones indicated gamma hemolysis. Only isolates showing gamma hemolysis were selected^[14].

1.8 Molecular Identification

The isolates were inoculated at 4 ml MRS broth at 37°C overnight and the culture was centrifuged (12,000 × g, 10 min) to harvest the cells and washed 2–3 times by sterile saline. Genomic DNA was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech, China). The primers 27F and 1492R were used for PCR amplification, and the DNA fragments were sequenced (Tsingke biotechnology). Sequences were compared to the GenBank database using the BLAST program, with species-level identification requiring > 99% identity in the 16S rRNA gene sequence.

1.9 In vivo safety assessment of mice

4-weekold male Kunming mice (20.0 ± 0.5 g) and an antibiotic-free basal diet were obtained from Dashuo Biotechnology Co. Ltd. (Chengdu, China). The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (approval number: DY2021203027).

The mice were pre-fed for 7 days in a well-ventilated environment with a normal light-dark cycle and good illumination (temperature 22 ± 1.5°C; free access to water). Subsequently, 30 mice were randomly and equally divided into three groups: NC Group (Negative control group, fed with 0.2 mL of saline alone with the standard diet), BL Group (low-dose group, fed with 0.2 mL of 4.0×10⁸ CFU/mL of L. plantarum E7 solution), and BH Group (high-dose group, fed with 0.2 mL of 4.0×10¹⁰ CFU/mL of L. plantarum E7 solution). Each group was housed in two cages with five mice per cage. Each mouse was fed 3–5 g of diet per day. The health status, feed intake, and fecal condition were recorded daily.

The formal experimental period lasted for 14 days. The number of deaths, average daily weight gain (ADG) of each group of mice were recorded and measured. On day 15, the whole blood and serum of mice were collected, and then the mice were euthanized. The hearts, livers, spleens, kidneys, and lung were taken to measure organ indices. Whole blood samples were used to detect hematological parameters by an Automatic Blood Cell Analyzer (Shenzhen Jingxintai Electronic Equipment CO., Ltd, Shenzhen, China).

1.10 Genome sequencing, assembly and annotation

Genomic DNA was extracted using the CTAB method, and genome sequencing was performed using the Nanopore PromethION48 and Illumina Novaseq platforms. The genome sequencing was then performed by Personal Biotechnology Company (Shanghai, China) by using the Nanopore PromrthION48 and the Illumina Novaseq. Flye and Unicycler software^[15] was used to assemble the data obtained by Nanopore platform sequencing. Subsequently, all assembled results were integrated to generate a complete sequence. Finally, the genome sequence was acquired after rectification by using pilon software

CGview^[16] was used to give an overview of the genome information, while the genome sequences were uploaded to the NCBI database with an accession number of CP158675. The Carbohydrate-Active enzymes (CAZy) database^[17] was used to predict Carbohydrate-Active enzymes. Function annotation was completed by blast search against Kyoto Encycolpedia of Gene and Genomes(KEGG)^[18] databases. AntiSMASH is used to search for secondary metabolites. The Virulence Factors of Pathogenic Bacteria (VFDB) database and the Comprehensive Antibiotic Resistance (CARD) database^[19] were used to retrieve the pathogenicity and antibiotic resistance genes.

1.11 Statistical Analysis

All data were expressed as means and standard deviations and analyzed using SPSS version 19.0. The difference was evaluated by one-way ANOVA and statistical significance was set at P < 0.05.

2.1 Morphological and Phenotypic Characteristics

A total of 20 isolates were obtained from Nipponia nippon feces (Table 1), characterized by their typical small pinpointed, creamy white colonies, Gram-positive nature, catalase, hippuric acid, and gelatin liquefaction negativity, and both coccus and rod-shaped morphology.

Table 1

Morphological, physiological and biochemical characteristics of lactic acid bacterial strains isolated from the fecal samples of *Nipponia nippon*
Characteristic	1	2
isolates	D1、D2、D3、D4、D5、D6、D7、D8、D9、D11、D12、D13、D14、D15、D16、D17、D18、D19、D20	E7
Morphology	coccus	rod
Gram reaction	་	་
catalase	-	-
gelatin liquefaction	-	-
Sucrose	6/19	་
Xylose	17/19	་
salicin	་	་
glucose	་	་
lactose	9/19	་
Inulin	16/19	་
raffinose	-	་
cellobiose	་	་
esculin	་	་
maltose	10/19	་
sorbitol	5/19	་
mannitol	7/19	་
hippuric acid	-	-
+: positive or weakly positive reaction
−: negative reaction
Number: the number of positive reactions
2.2 Acid tolerance

The tolerance test results for twenty LAB isolates at different pH levels were shown in Table 2. At pH 3.0, most isolates demonstrated high tolerance. At pH 2.0, all isolates except strain D8 maintained viable bacterial counts above 10⁴ CFU/mL. Under pH 1.0 conditions, viable bacterial counts were detected only in strains D1, D6, E7 and D20, exceeding 10³ CFU/mL while no viable counts were detected in the remaining strains.

Table 2

Acid resistance capacity of LAB isolates under different acid conditions (log CFU/mL)
strains	Initial concentration	pH3	pH 2	pH 1
D1	8.67 ± 0.04	6.61 ± 0.04	4.96 ± 0.24	3.31 ± 0.54
D2	8.74 ± 0.14	7.15 ± 0.01	4.66 ± 0.10	-
D3	8.57 ± 0.07	6.22 ± 0.11	4.49 ± 0.15	-
D4	8.38 ± 0.46	6.31 ± 0.26	4.08 ± 0.11	-
D5	8.56 ± 0.15	5.66 ± 0.04	4.55 ± 0.32	-
D6	8.63 ± 0.08	6.72 ± 0.04	5.92 ± 0.13	3.26 ± 0.36
E7	8.44 ± 0.10	6.32 ± 0.02	5.16 ± 0.40	3.07 ± 0.39
D8	8.73 ± 0.06	6.85 ± 0.03	-	-
D9	8.63 ± 0.10	6.86 ± 0.06	4.96 ± 0.25	-
D10	8.78 ± 0.13	5.67 ± 0.27	4.42 ± 0.12	-
D11	8.69 ± 0.04	6.21 ± 0.20	5.42 ± 0.23	-
D12	8.58 ± 0.25	5.70 ± 0.18	4.88 ± 0.09	-
D13	8.73 ± 0.16	6.76 ± 0.05	5.20 ± 0.44	-
D14	8.48 ± 0.06	6.76 ± 0.06	5.04 ± 0.35	-
D15	8.66 ± 0.08	7.29 ± 0.15	5.32 ± 0.37	-
D16	8.39 ± 0.23	6.26 ± 0.22	4.31 ± 0.06	-
D17	8.73 ± 0.18	6.58 ± 0.18	4.32 ± 0.26	-
D18	8.62 ± 0.34	6.56 ± 0.06	4.55 ± 0.24	-
D19	8.66 ± 0.31	5.52 ± 0.29	4.43 ± 0.11	-
D20	8.58 ± 0.03	6.91 ± 0.03	5.32 ± 0.37	3.02 ± 0.31
1-, No viable bacteria were detected
2.3 Bile tolerance

The tolerance results of twenty LAB isolates under different concentrations of bile salt were shown in Table 3. All strains showed high tolerance at 0.3% bile salt, with viable bacterial counts above 10⁶ CFU/m L. At 1% bile salt, all strains except D3, D4, D12, D17, and D18 maintained counts above 10⁴ CFU/mL. At 2% bile salt, only isolates D1, D2, D6, E7, D8, and D9 had viable counts above 10² CFU/mL, with no viable bacteria detected in other strains. Strain E7 had a particularly high viable count exceeding 10⁵ CFU/mL. Among the twenty strains, only six demonstrated good tolerance to low pH and high bile salts, warranting further analysis.

Table 3

Survivability of the LAB isolates under different concentrations of bile salt (log CFU/mL)
strains	Initial concentration	0.3%	1%	2%
D1	8.51 ± 0.06	6.18 ± 0.44	5.22 ± 0.09	3.56 ± 0.53
D2	8.76 ± 0.17	6.05 ± 0.32	5.08 ± 0.32	2.29 ± 0.78
D3	8.56 ± 0.13	6.70 ± 0.13	-	-
D4	8.32 ± 0.16	6.31 ± 0.09	-	-
D5	8.60 ± 0.10	6.64 ± 0.24	5.61 ± 0.26	-
D6	8.64 ± 0.11	6.92 ± 0.17	5.08 ± 0.43	2.47 ± 0.27
E7	8.79 ± 0.25	6.93 ± 0.14	5.31 ± 0.16	5.24 ± 0.22
D8	8.66 ± 0.11	6.43 ± 0.38	5.70 ± 0.06	4.04 ± 0.19
D9	8.86 ± 0.11	7.08 ± 0.17	5.78 ± 0.15	3.86 ± 0.05
D10	8.61 ± 0.26	6.57 ± 0.29	4.69 ± 0.01	-
D11	8.31 ± 0.24	6.31 ± 0.10	5.63 ± 0.28	-
D12	8.65 ± 0.08	6.41 ± 0.19	-	-
D13	8.64 ± 0.08	6.42 ± 0.38	4.36 ± 0.39	-
D14	8.23 ± 0.26	6.20 ± 0.46	4.79 ± 0.18	-
D15	8.36 ± 0.22	6.42 ± 0.16	4.59 ± 0.11	-
D16	8.37 ± 0.14	6.34 ± 0.32	5.66 ± 0.09	-
D17	8.78 ± 0.16	6.10 ± 0.14	-	-
D18	8.65 ± 0.11	6.51 ± 0.17	-	-
D19	8.57 ± 0.12	6.70 ± 0.29	5.42 ± 0.12	-
D20	8.86 ± 0.11	6.57 ± 0.29	4.69 ± 0.01	-

The mean of 3 values of each sample are presented with ± SD

2.4 Antimicrobial Activity

Six LAB isolates exhibited varying degrees of antibacterial activity against three pathogens (Escherichia coli, Staphylococcus aureus and Salmonella) as shown in Table 4. All isolates, except D8 (Table 4), showed significant inhibition zones (> 14mm) against the pathogens, with D1 exhibiting the strongest inhibitory effect on Salmonella (> 19mm).

Table 4

Antibacterial activities of LAB isolates
strains	Escherichia coli		Salmonella		Staphylococcus aureus
D1	+	14.31 ± 0.21	++	19.83 ± 3.10	++	16.59 ± 0.76
D2	+	14.43 ± 2.08	+	15.43 ± 3.68	++	18.13 ± 2.13
D6	++	16.17 ± 0.60	++	17.36 ± 3.92	++	16.58 ± 3.07
E7	++	16.53 ± 0.86	++	16.59 ± 1.57	++	16.65 ± 1.28
D8	++	16.35 ± 2.17	+	15.60 ± 2.01	+	12.04 ± 1.19
D9	++	16.56 ± 2.39	++	16.87 ± 2.72	++	16.51 ± 0.47

The different letters represent significant diversity and the same letters represent no significant diversity in one queue. The mean of 3 values of each sample are presented with ± SD

¹±: 8 mm < < zone diameters ≤ 12 mm;+༚12 mm < < zone diameters ≤ 16 mm༛++༚16 mm < < zone diameters ≤ 20 mm༛+++20 mm < < zone diameters

2.5 Antibiotic Susceptibility

Selected isolates exhibited multi-drug resistance. All strains were sensitive to penicillin and chloramphenicol and resistant to vancomycin. Isolates D1 and D9 showed resistance to 8 and 6 different antibiotics, respectively, while strains D2 and D8 showed resistance to 4 different antibiotics. Strain E7 was resistant to cefradine, ciprofloxacin and vancomycin. The D6 strain was only resistant to Carbenicillin, Tetracycline, vancomycin. Although LAB are generally considered safe, the possibility of gene transfer in the gut of the crested ibis suggests selecting strains with less resistance. Strains D6 and E7 were safer than others.

Table 5

Susceptibility to antimicrobial agent of LAB isolates
	antibiotics
strains	PNC	CFP	CE	CB	CHL	TE	DC	ERY	CIP	GM	KAN	VAN
ATCC25923	S	S	S	S	S	S	S	S	S	S	S	S
D1	S	M	R	R	S	R	R	M	R	R	R	R
D2	S	M	R	S	S	M	S	M	R	S	R	R
D6	S	S	M	R	S	R	S	M	S	M	S	R
E7	S	S	R	S	S	M	S	M	R	S	M	R
D8	S	S	R	S	S	S	S	S	R	S	R	R
D9	S	S	R	S	S	S	R	S	R	R	R	R

PNC Penicillin, CFP Cefoperazone, CE Cephradine, CB Carbenicillin, CHL Chloramphenicol, TE Tetracycline, DC Doxycycline, ERY Erythromycin, CIP Ciprofloxacin, GM Gentamicin, KAN Kanamycin, VAN vancomycin

¹R-Resistance;M-medium sensitivity༛S-Sensitive

2.6 Cell surface hydrophobicity

The cell surface hydrophobicity of LAB, a measure of adhesion to intestinal epithelial cells, varied among the six isolates (Fig. 1). The E7 isolate exhibited significant moderate hydrophobicity (54.62%), while D2, D6, D8 isolates also showed moderate hydrophobicity (31.04%, 39.66%, 30.74%, respectively). The D1, D9 isolates exhibited low hydrophobicity (27.80%, 34.70%, respectively). The cell hydrophobicity of the strain E7 was the most significant among the six isolates (P < 0.05). Based on the above tests, strain E7 was selected for subsequent studies.

Values expressed as mean ± SD. Different letters represent significant difference, P < 0.05.

2.7 Hemolytic activity

On the blood agar plates, ETEC CVCC196 was γ hemolyzed (Fig. 2A). However, the strain E7 showed no hemolytic activity (Fig. 2B).

2.8 Molecular identification of LAB strains

Strain E7 was further identified by 16S rRNA sequencing and phylogenetic analysis, showing 99% sequence similarity to L. plantarum based on BLASTn. The sequence of the strain E7 was uploaded to the China Center for Type Culture Collection (CCTCC M 2024523), and the phylogenetic tree was constructed using MEGA7.0 (Fig. 3).

2.9 Growth performance of mice

During experimental period, all mice were healthy with no organ abnormalities. Analysis of body weight, final body weight, and ADG showed that L. plantarum E7 supplementation had no negative effect on growth performance (Fig. 4). No significant differences were found in organ indices (heart, liver, spleen, lungs, and kidneys) between the NC, BL, and BH groups (P > 0.05) (Table 6).

Table 6

The organ indices of each group mice (mg/g).
	Heart	Liver	Spleen	lungs	kidneys
NC group	7.5 ± 0.16	45.2 ± 0.29	3.7 ± 0.13	7.2 ± 0.13	15.5 ± 0.16
BL group	8.8 ± 0.20	44.1 ± 0.25	3.1 ± 0.12	7.2 ± 0.09	14.9 ± 0.2
BH group	7.7 ± 0.16	46 ± 0.77	3.1 ± 0.14	7.4 ± 0.14	15.5 ± 0.13

2.10 Analysis of hematological parameters

Hematological parameters are shown in Table 7. Except for PLT, there were no significant differences between BL and BH groups compared to the NC group (P > 0.05). The PLT in BL group and BH group were significantly higher than the NC group (P<0.05).

Table 7

The hematological parameters of each group of mice.
Parameters	NC group	BL group	BH group	Parameters	NC group	BL group	BH group
WBC, 10⁹/L	4.18 ± 1.29	4.93 ± 1.00	5.05 ± 0.85	HGB, g/L	149.33 ± 15.16	150.83 ± 6.4	147.60 ± 8.14
Lymph, 10⁹/L	3.33 ± 1.26	4.08 ± 0.64	4.32 ± 0.96	HCT, %	49.21 ± 4.88	48.81 ± 2.15	48.18 ± 3.13
Mon, 10⁹/L	0.12 ± 0.04	0.15 ± 0.05	0.14 ± 0.1	MCV, fL	50.18 ± 1.27	48.75 ± 1.56	49.62 ± 0.59
Gran, 10⁹/L	0.73 ± 0.27	0.80 ± 0.20	1.08 ± 0.45	MCH, pg	15.15 ± 0.36	15.05 ± 0.50	15.14 ± 0.32
Lymph,%	78.9 ± 4.62	81.65 ± 2.99	77.36 ± 3.37	MCHC,g/L	303.83 ± 4.70	305.17 ± 2.14	306 ± 4.36
Mon, %	3.05 ± 0.80	3.02 ± 0.34	3.23 ± 0.77	RDW, %	14.42 ± 0.30	14.5 ± 0.81	14.9 ± 0.45
PDW, %	16.25 ± 0.3	16.43 ± 0.2	16.58 ± 0.16	PLT, 10⁹/L	1613 ± 126.18^a	1819.66 ± 149.37^b	1825.4 ± 144.63^b
RBC,10¹²/L	9.81 ± 0.94	10.10 ± 0.27	9.72 ± 0.74	MPV, fL	5.6 ± 0.32	5.63 ± 0.34	5.88 ± 0.34

Values were shown as mean ± SD and ^{a, b} indicated a significant difference compared to NC group (^{a, b}p < 0.05).

WBC, white blood cell counts; Lymph, lymphocytes; Mon, monocytes; PDW, platelet; RBC, red blood cell counts; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width; PLT, platelet counts; MPV, mean platelet volume;

2.11 Complete genome information of L. plantarum E7

The L. plantarum E7 genome is a circular chromosome with a total length of 3197533 bp and an average GC content of 46.50%. There were two circular plasmids, one with a total length of 56484 bp and an average GC content of 39.74%, and the other with a total length of 2503 bp and an average GC content of 55.97% (Table S1). The chromosome contains 3024 protein-coding sequences, accounting for 83.54% of the total length, as well as six 5srRNAs, five 16srRNAs, five 23 sRNAs, 72 tRNAs, 54 ncRNAs, and one CRISPRs. Plasmid1 contained 58 protein-coding sequences, accounting for 83.37% of the total length, six ncRNAs, and no 5S sRNAs, 16srRNAs, 23SrRNA, tRNA, ncRNA, or CRISPR. Plasmid 2 contained three protein-coding sequences, accounting for 55.97% of the total length, one ncRNA, and no 5SrRNA, 16SrRNA, 23SrRNA, tRNA, ncRNA, or CRISPR (Table S1), and its genome lacks CRISPRs. From inside to outside, the diagrammatic of the genome circle shows that the first circle represents scale, the second circle represents GC Skew, the third circle represents GC-content, the fourth and seventh circles represent COG to which each CDS belongs, the fifth and sixth circles represent the positions of CDS, tRNA, and rRNA on the genome (Fig. 5). The complete genome sequence was uploaded to NCBI (accession number CP158575.1).

The genome of L. plantarum E7 was annotated with 113 genes in the CAZy (Fig. 6), which showed that 32 genes, accounting for 0.0106 percent, were associated with glycosyltransferase (GTS). There was one gene related to polysaccharide lyase (PLs), accounting for 0.0003%. Genes associated with sugar esterase (CEs) were 13, accounting for 0.0043%, secondary activity (AAs) related genes were eight, accounting for 0.0026%, and carbohydrates related genes (CBMs) were 11, accounting for 0.0036%. There were 48 genes related to glycoside hydrolases (GHs), accounting for 0.0159%; however, the plasmid had no carbohydrate active enzymes. These findings suggest that the metabolic activity of L. plantarum E7 is mainly focused on the breakdown of carbohydrates to provide energy for the body.

In total, 2688 KEGG annotation genes were identified in the genome of L. plantarum E7 (Fig. 7A). These genes were assigned to 8 different signaling pathways. The distributing of these genes among various categories was as follows: 877 genes related to metabolism, 217 genes related to environmental information processing, 196 genes related to genetic information processing, 78 genes related to cellular processes, 78 genes related to human diseases, 991 genes related to Brite Hierarchies, 218 genes were related to Not Included in Pathway or Brite, and 33 genes were related to Organismal System. Among the metabolism-related genes, the main annotations were carbohydrate metabolism (270), amino acid metabolism (164), metabolism of cofactors and vitamins (87), energy metabolism (87), lipid metabolism (50), nucleotide metabolism (70), metabolism of other amino acids (38), biosynthesis of other secondary metabolites (34), xenobiotics biodegradation and metabolism (29), metabolism of terpenoids and polyketides (23) and glycine biosynthesis and metabolism (28).

In total, 29 KEGG annotation genes were identified in the Plasmid 1 genome of L. plantarum E7 (Fig. 7B). These genes were assigned to 4 different signaling pathways. The distribution of these genes among various categories was as follows: 12 genes related to metabolism, 1 gene related to environmental information processing, 11 genes related to Brite Hierarchies, 5 genes related to Not Included in Pathway or Brite. Among the metabolism-related genes, the main annotations included carbohydrate metabolism (4), amino acid metabolism (1), energy metabolism (3) and nucleotide metabolism (4). The plasmid 2 genomes had no functional annotation genes These findings suggest that L. plantarum E7 has strong abilities in carbohydrates and amino acid metabolism, which could be valuable for applications in various industries such as Food Engineering and biologics.

Based on gene annotation results, the genome of L. plantarum E7 contains numerous probiotic marker genes related to acid and bile tolerance, temperature tolerance, oxidative stress, riboflavin synthesis, exopolysaccharide secretion, and cell adhesion (Table S2). AntiSMASH analysis detected four secondary metabolite synthesis genes clusters namely RiPP-like, T3PKS, terpene and cyclic-lactone-autoinducer in the chromosome, with no similar gene clusters found in the plasmids (Table 8, Fig. 8). There were no potential probiotic genes in the plasmids.

Similar gene clusters were analyzed using MiBIG database and ClusterBlast algorithm (Fig. 8). The RiPP-like gene cluster of strain E7 and Lactiplantibacillus plantarum strain XJ25, Lactiplantibacillus plantarum strain SRCM10347 and Lactiplantibacillus plantarum strain LpYC41 have 100% homology. The T3PKS gene cluster has only 97% homology with Lactiplantibacillus plantarum strain RI-113. There are no gene clusters in the plasmids. These results indicate that L. plantarum E7 has the ability to produce new antibacterial substances.

Table 8

Secondary metabolite synthesis gene cluster of *L. plantarum* E7
Region	Type	From	to
Region1	RiPP-like	361955	374105
Region2	T3PKS	1814322	1855491
Region3	terpene	2874410	2895090
Region4	cyclic-lactone-autoinducer	3132211	3152813

Compared to the CARD database, 40 genes related to antibiotic resistance accounting for 0.628% were identified in the genome, with no resistance genes in the plasmids. There were 11 genes related to antibiotic targets, accounting for 0.364%. There was 1 gene related to antibiotic biosynthesis, accounting for 0.033%. There were 25 genes related to the total genes, accounting for 0.827%. There were no resistance genes in the plasmids (Table 9).

Table 9 Antimicrobial resistance genes detected in the genome of L. plantarum E7

Property	Number of Genes	Percentage (%)
Antibiotic Resistance	19	0.628
Antibiotic Target	11	0.364
Antibiotic Biosynthesis	1	0.033
Total genes	25	0.827

Comparing the chromosome genome sequence of L. plantarum E7 with the VFDB database (Table S3). Only a few virulence genes related to adherence, stress survival, exoenzyme activity, immune modulation, and regulation were detected in the genome data of L. plantarum E7, with none in the plasmids (Table S3).

2.11 Statistical Analysis

All data were expressed as means and standard deviations and analyzed using SPSS version 27.0. The difference was evaluated by one-way ANOVA and statistical significance was set at P < 0.05.

LAB have been extensively utilized due to their ability to maintain the balance of intestinal microbes and promote intestinal health^[5]. Previous studies have demonstrated that LAB, when used as feed supplements, have a significant positive impact on animal husbandry. However, LAB need to be characterized and identified to ensure their safety and efficacy. Studies indicate that the gut of Nipponia nippon is rich in LAB, but it remains underexplored. Hence, in this present study, twenty isolates were obtained from Nipponia nippon feces, and their probiotic properties were investigated.

Probiotic strains must endure acid and bile salt environments to survive in the gastrointestinal tract and remain functional^[20]. Gastric acid pH ranges from 1.5 to 4.5, and food retention time in the stomach is approximately three hours. Bile salt concentrations in the upper intestine range from 0.03–0.3% (wt/vol)^[13], and food takes about four hours to pass through the small intestine. In this study, six out of twenty isolates showed good tolerance to low pH and high bile salts, aligning with previous findings by Akinyemi, who isolated 23 lactic acid bacteria strains from goat milk in Nigeria, with only six tolerating acidic and bile salt conditions ^[1].

Infections with intestinal pathogens can lead to diarrhea and inflammatory bowel disease in both humans and animals^[21]. Common pathogens include E. coli, Salmonella, and Staphylococcus aureus. Antibacterial activity against these pathogens is a key probiotic requirement^[8]. In this study, six LAB isolates exhibited varying degrees of antibacterial activity against three pathogens, excluding D8. Kaewchomphunuch found^[22] that the CFS from LAB strains such as L. acidophilus KMP, L. plantarum KMP and P. pentosaceus KMP inhibited the growth of pathogenic E. coli isolated from pigs in Thailand. Haghshenas^[23] found that silymarin-enriched Lactobacillus strains displayed significant antibacterial and antifungal activity against 12 pathogenic bacteria and A. flavus.

Although LAB are generally recognized as safe (GRAS) microorganisms, their safety properties must be evaluated before administration. In this study, six isolates were tested for antibiotic sensitivity. An ideal probiotic strain should lack resistance genes to avoid the risk of gene transfer in the gut of Nipponia nippon. Strains D6 and E7 showed resistance to only three antibiotics, suggesting they are safe candidates.

Surface hydrophobicity, a measure of probiotic adhesion to intestinal epithelial cells, was another criterion assessed^[23]. Among the six isolates, E7 exhibited significant moderate hydrophobicity. Farid isolated three Lactobacillus acidophilus strains (WFA1, WFA2, WFA3) from indigenous yogurt, which were strongly hydrophobic. Yang found that L. plantarum 4–10 had good hydrophobicity, indicating that this trait is strain-specific^[24]. Strain E7 was further studied for this reason.

Hemolytic activity is an important virulence factor, facilitating pathogen acquisition of iron and other growth factors, which can lead to host anemia or edema. Strain E7 exhibited no hemolytic activity, consistent with previous reports that most LAB strains are non-hemolytic^{[25, 26]}.

In vivo safety assessment is crucial for screening potential probiotics^[27]. Most studies suggest that strains in the range of 10⁸ to 10⁹ CFU/mL are more effective^{[28, 29]}. This study examined low-dose (4.0×10⁸ CFU/mL) and high-dose (4.0×10¹⁰ CFU/mL) supplementation. When the body suffers a bacterial infection, the liver and spleen become enlarged^[30]. High-dose L. plantarum E7 supplementation did not negatively affect growth performance or organ development in mice.

Hematological parameters are commonly used in histopathology. Bacterial infections typically increase white blood cell counts, triggering an inflammatory response^[31]. In this study, leukocyte counts in all groups were within the normal range (0.8–6.8 ×10⁹/L), indicating that L. plantarum E7 does not cause inflammation. Platelet counts increased after L. plantarum E7 supplementation^[32], suggesting it may promote platelet production, consistent with previous research^[33]. Additionally, L. plantarum E7 had no effect on erythrocyte-related indicators, supporting previous hemolysis test results showing no toxic effects on red blood cells.

Genomic analysis of L. plantarum E7 identified 27 genes encoding probiotic properties. Genes related to acid tolerance (e.g., atpA, atpB, atpC, atpD, atpE, atpF, atpH) ^[34], bile salt hydrolysis (bsh), temperature tolerance (groeL, csp, dnaJ, dnaK), oxidative stress defense (arsC, trxA, trxB, nfrA1), riboflavin synthesis (ribBA, ribE, ribF, ribT), extracellular polysaccharide synthesis (epsF, epsH, rmlC, rmlD), and cell adhesion (luxS, scpA, scpB) were identified, consistent with previous studies. The bsh gene can hydrolyze bile salts and help the strains to survive in the bile salt-rich intestinal environment^[35]. At abnormal temperatures, proteins misfold or aggregate. Molecular chaperone genes (groeL, csp, dnaJ, dnaK) play a role in temperature tolerance by interacting with these abnormal proteins to maintain the correct folding state of the proteins in the cell^[36].

Oxidative stress-related genes (arsC, trxA, trxB, nfrA1) help cells defend against damage caused by reactive oxygen species ROS^[37]. The riboflavin biosynthesis protein gene family (ribBA, ribE, ribF, ribT) is involved in the synthesis pathway of riboflavin, which can promote the absorption and utilization of riboflavin by the host. The extracellular polysaccharide synthesis gene family (epsF, epsH, rmlC, rmlD) is involved in the synthesis, assembly and secretion of extracellular polysaccharide, and plays an important role in the biofilm formation and environmental adaptation of strains^[38].

Good cell adhesion function is an important basis for the biological effects of lactic acid bacteria. Three genes related to cell adhesion (luxS, scpA, scpB) were found in genome of L. plantarum E7. The discovery of these genes related to prebiotic properties is consistent with previous studies^[39].

Annotated results from the CAZy database revealed 113 carbohydrate active enzymes in L. plantarum E7. Carbohydrate active enzymes are a very important group of enzymes, including GHs, GTs, PLs, CEs, AA and CBMs^[40]. GHs are involved in the process of polysaccharide hydrolysis, which releases a lot of energy to supply various activities of bacteria. GTs use activated donors to form glycosidic bonds, transfer sugars to specific receptors such as proteins, lipids, or other glycan, and form new polymers to participate in various physiological processes. GHs and GTs were the most abundant, supporting bacterial growth on various carbon sources and playing a role in host-strain interactions and microecological balance. CEs can catalyze the de-esterification of various carbohydrate substrates.

AAs include a large class of REDOX active enzymes that act on carbohydrates. GHs and GTs were the most abundant in this study. This is similar to the results obtained by Chen et al^[41] in the annotation of multiple carbohydrate hydrolases from B. velezensis TS5. These carbohydrate enzymes contribute to the growth of L. plantarum E7 in a variety of carbon sources and play an important role in the interaction between strains and hosts and the maintenance of microecological balance.

Potential LAB probiotic strains were characterized for the production of inhibitory substances such as bacteriocin, hydrogen peroxide and organic acid. Annotated results of AntiSMASH show four bacteriocin synthesis gene clusters in L. plantarum E7. Jin et al^[42] found that there were bacteriocin gene clusters in the genome of L. plantarum L3.

Studies have shown that the probiotic resistance is inherent and non-metastatic. However, there is a possibility that plasmid-bound probiotics will transfer resistance^[43]. In this study, no resistance genes were found in the L. plantarum E7 plasmid, indicating a low risk of gene transfer. Although some virulence genes were detected, none were virulence-coding, and safety tests confirmed the strain's safety. The discovery of L. plantarum E7 enriches the gene pool of Nipponia nippon intestinal probiotics, laying a theoretical foundation for further research. However, the study has limitations, such as not extracting and preparing antibacterial substances from LAB CFS. Future research should focus on bacteriocin extraction for antibacterial drug development. L. plantarum E7, with its probiotic properties, has great potential as a microecological preparation in livestock production. Conclusion

Taken together, the results obtained from this study showed that twenty isolates were obtained from Nipponia nippon feces and screened in vitro for various probiotic properties, including acid and bile salt tolerance, antibacterial activity, antibiotic sensitivity, hemolytic activity, hydrophobicity, and in vivo safety. Six strains exhibited good probiotic activity, with L. plantarum E7 showing the best activity. L. plantarum E7 had no adverse effects on mouse growth or blood cell composition. Genomic analysis of L. plantarum E7 revealed many genes supporting probiotic roles in host intestinal adhesion, acid and bile salt tolerance, antioxidant production, antimicrobial substances, and hydrolase.

Acknowledgements

We would like to thank Emei Mountain Nipponia nippon breeding base in Sichuan province for providing animals feces samples.

Author contributions

LY, GS, YZ conceived and designed the project. JL, BYZ, YW collected sample.LY, BYZ and YW performed experiments; LY analysis and interpretation the data. LY, YZ wrote the manuscript. YZ, Felix Kwame Amevor and GS revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Key R&D Program Project (2022YFD1600902-4) and Sichuan Province Regional Innovation Cooperation Project (2023YFQ005).

Data Availability

Raw sequencing data have been deposited in the NCBI Sequence Read Archive database (CP158575.1- CP158577.1).

Compliance with Ethical Standards

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (approval number: DY2022203014).

Competing Interests

The authors declare no competing interests.

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No competing interests reported.

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Evaluating the Probiotic Potential of Lactic Acid Bacteria Isolated from Nipponia nippon Feces

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