Isolation and identification of Pediococcus acidilactici BPA03 derived from human milk and evaluation of its antioxidant and anti-aging probiotic properties

doi:10.12122/j.issn.1673-4254.2026.07.14

Abstract

Abstract:

Objective To identify Pediococcus acidilactici BPA03 strain isolated from healthy women's milk and evaluate its safety and probiotic properties. Methods BPA03 strain was isolated from breast milk using dilution plating and streaking on MRS agar. Identification was performed using Gram staining, biochemical assays (BD card), 16S rRNA sequencing, and phylogenetic analysis. Safety assessment of the strain was conducted by evaluation of its hemolytic activity on blood agar, antibiotic susceptibility (Kirby-Bauer), and acute toxicity in zebrafish embryos. The probiotic properties of the bacterium were evaluated by measuring lactic acid production and assessing its protective effects against UV-induced photoaging and D-galactose-induced aging in zebrafish models. Results The BPA03 strain was Gram-positive with a spherical or ovoid shape, and identified as Pediococcus acidilactici. This strain showed no hemolytic activity and was sensitive to all the tested antibiotics (penicillin, ampicillin, and vancomycin). Growth curve analysis showed a lag phase of 2 h, a logarithmic phase starting from 4 to 12 h, and a stationary phase after 20 h in aerobic culture. Lactic acid concentration reached 23.6 mmol/L after culture for 8 h in MRS medium. In zebrafish embryos, BPA03 exhibited no significant acute toxicity, and the embryos showed a hatching rate above 95% even in the presence of 10⁸ CFU/mL BPA03 (LD₅₀>10⁸ CFU/mL). In UV-exposed zebrafish models, BPA03 obviously alleviated UV-induced caudal fin damage, significantly reduced reactive oxygen species and malondialdehyde levels, enhanced antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), and downregulated mRNA expression of pro-inflammatory cytokines (IL‑1β, IL‑6, IL‑8, and TNF‑α). Conclusion Pediococcus acidilactici BPA03 strain isolated from breast milk of healthy women exhibits good probiotic properties and safety.

Key words: Pediococcus acidilactici, isolation and identification, safety evaluation, probiotic properties

Longbin CAO, Peixiang HUANG, Zikang ZHANG, Weimin SUN, Feng QIU. Isolation and identification of Pediococcus acidilactici BPA03 derived from human milk and evaluation of its antioxidant and anti-aging probiotic properties[J]. Journal of Southern Medical University, 2026, 46(7): 1601-1610.

Figures/Tables 11

Tab.1 Sequences of the primers for qRT-PCR

Gene	Forward (5'—3')	Reverse (5'—3')
IL-1β	GTCACACTGAGAGCCGGAAG	GCAGGCCAGGTACAGGTTAC
IL-6	TGAAGGGGTCAGGATCAGCA	CACGTCAGGACGCTGTAGATT
IL-8	GTCGCTGCATTGAAACAGAA	CTTAACCCATGGAGCAGAGG
TNF-α	GGCTGGAAAACAACGAGATCA	AAGATCAAAGACGGCTCCAA
β-actin	AGGGAAATCGTGCGTGACATCA	ACTCATCGTACTCCTGCTTGCTGA

Fig.1 Colonial morphology of human milk-derived P. acidilactici BPA03. A, B: Colony morphology of BPA03 strain. C: Micrograph of Gram-stained BPA03 (Original magnification: ×200). D: Field emission scanning electron microscopy (FESEM) of BPA03 strain.

Tab.2 Biochemical reaction results of isolated P. acidilactici BPA03 strain

Biochemical test	BPA03	Biochemical Test	BPA03	Biochemical Test	BPA03
A_ARARR	-	A_GLPRB	+	A_LALT	+
A_LARGH	+	A_LHIST	-	A_LISO	+
A_LLEUH	+	A_LPHET	+	A_LPROB	-
A_LPYR	-	A_LTRY	+	A_META	+
C_3MGA	-	C_CLST	-	C_DFRU	+
C_DGUA	-	C_DMNT	-	C_IMN	-
C_KGA	-	C_MAA	-	C_PXB	-
C_THY	-	M_ADGLU	-	M_BDCEL	+
M_BDGAL	-	M_BDGLC	-	M_BDGLU	+
M_NAG	+	M_PHOS	-	M_PHOT	-
N_ALALH	-	N_LPROT	-	N_VAALA	-
P_ADGLU	-	P_PHOL	-	R_BGEN	-
R_DEX	+	R_DSUC	-	R_DTAG	-
R_DTRE	-	R_MAL	-	R_MTT	-
R_NGU	+	S_URE	-	T_ESC	-
R_MGP	-

Fig.2 Phylogenetic tree of P. acidilactici BPA03.

Fig.3 Test results of hemolytic activity of P. acidilactici BPA03. 1: Negative control bacterium: non-pathogenic Escherichia coli (ATCC 25922). 2: Positive control bacterium: Staphylococcus aureus (ATCC 29213). 3: P. acidilactici BPA03.

Tab.3 Drug sensitivity tests of P. acidilactici BPA03 (Mean±SD)

Antibiotics	Drug mass (μg)	Disk diffusion test results (mm)	Drug sensitivity
Penicillin	10	28.10±0.53	Sensitive
Ampicillin	10	25.17±0.93	Sensitive
Imipenem	10	26.63±0.42	Sensitive
Vancomycin	30	30.73±0.38	Sensitive
Erythromycin	15	24.63±0.84	Sensitive
Clindamycin	2	30.00±0.85	Sensitive
Linezolid	30	22.33±0.74	Sensitive

Fig.4 Growth curve and lactic acid production of P. acidilactici BPA03. A: Growth curve of BPA03 [R: relative growth (A600 nm)]. B: Lactic acid production by P. acidilactici BPA03 in fermentation test.

Fig.5 　Toxicity test of P. acidilactici BPA03 in zebrafish embryos.A: Morphological changes of zebrafish embryos exposed todifferent concentrations of BPA03 (Scale bar=1000 μm). B:Effects of BPA03 exposure on hatchability of zebrafish embryos (n=5). No significant changes were observed in hatchability at96 h (one-way ANOVA).

Fig.6 Effect of P. acidilactici BPA03 on ultraviolet-induced aging in zebrafish. A: Morphological changes of the zebrafish with UV exposure with and without P. acidilactici BPA03 treatment. B: Effect of P. acidilactici BPA03 on caudal fin damage in zebrafish larvae exposed to UV. C: Comparison of caudal fin area (pixels) among the 4 groups. ***P<0.001.

Fig.7 Effect of P. acidilactici BPA03 on D-galactose-induced aging in zebrafish larvae. A: Intracellular ROS production in zebrafish larvae detected by DCFH‑DA staining. B: Quantitative analysis of ROS levels (n=10). C: Superoxide dismutase (SOD) activity in zebrafish larvae. D: Catalase (CAT) activity in zebrafish larvae. E: Glutathione peroxidase (GSH-Px) activity in zebrafish larvae. F: Malondialdehyde (MDA) content in zebrafish larvae. Data are expressed as Mean±SD of 5 samples, with each sample containing 10 zebrafish. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001.

Fig.8 Effects of P. acidilactici BPA03 on inflammatory factors in a zebrafish larvae model of D‑galactose‑ induced aging. A: Expression level of IL‑1β mRNA in zebrafish larvae. B: Expression level of IL‑6 mRNA in zebrafish larvae. C: Expression level of IL‑8 mRNA in zebrafish larvae. D: Expression level of TNF‑α mRNA in zebrafish larvae. Data are presented as Mean±SD of 5 samples, each containing 10 zebrafish larvae. Statistical analysis was performed using one‑way ANOVA followed by Tukey's multiple comparison test for group‑wise validation; *P<0.05, **P<0.01, ***P<0.001.

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