人乳源乳酸片球菌BPA03的分离鉴定及其抗氧化与抗衰老益生特性评价

doi:10.12122/j.issn.1673-4254.2026.07.14

摘要/Abstract

摘要：

目的对健康妇女乳汁中分离得到的菌株BPA03进行鉴定，并评价其安全性与益生特性。方法通过稀释平板涂布法和划线分离法在血琼脂培养基和MRS固体培养基上对乳汁中的菌株进行分离纯化和菌落形态观察；采用革兰氏染色鉴别菌株的形态与革兰氏阴阳性；采用BD鉴定卡对菌株BPA03进行生理生化性质的鉴定；结合16S rRNA测序与系统发育分析，对菌株BPA03进行了分子生物学鉴定；采用血琼脂平板检测菌株BPA03的溶血性；采用KB法检测菌株BPA03对抗生素的敏感性；使用生化分析仪检测其发酵液中的乳酸浓度；斑马鱼胚胎急性毒性实验评价其安全性；利用紫外线及D-半乳糖诱导的斑马鱼衰老模型，评价菌株BPA03的益生特性。结果菌株BPA03在MRS培养基上生长良好，菌落呈乳白色半透明、圆形、表面光滑、边缘整齐；该菌革兰氏染色为阳性，球形或卵圆形，多呈单、双或短链状排列，直径0.5~1.0 μm。菌株BPA03经形态学、生理生化及16S rRNA系统发育分析，被鉴定为乳酸片球菌。溶血性试验结果显示，该菌在血琼脂平板上无溶血环；药敏试验对青霉素、氨苄西林、万古霉素等测试抗生素均敏感。菌株BPA03成人血培养需氧瓶中35 ℃培养时，延滞期约2 h，对数生长期为4~12 h，20 h后进入稳定期，在MRS培养基中培养约8 h乳酸产量达到饱和终浓度为23.6 mmol/L。斑马鱼胚胎急性毒性实验表明，即使在10⁸ CFU/mL浓度下，胚胎孵化率仍高于95%，LD₅₀>10⁸ CFU/mL，无显著急性毒性。在益生功能方面，BPA03在紫外线诱导的斑马鱼光老化模型中，能有效抑制尾鳍损伤并改善光老化；在D-半乳糖诱导的衰老模型中，BPA03能显著降低机体ROS与丙二醛（MDA）含量，提升超氧化物歧化酶（SOD）、过氧化氢酶（CAT）及谷胱甘肽过氧化物酶（GSH-Px）的抗氧化酶活性，并下调促炎细胞因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）的mRNA表达水平（与模型组比较，P<0.05）。结论从健康妇女乳汁中分离得到的菌株BPA03被鉴定为乳酸片球菌，其具有良好的益生效应和安全性。

关键词: 乳酸片球菌, 分离鉴定, 安全性评价, 益生效应

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

操龙斌, 黄佩香, 张子康, 孙卫民, 邱峰. 人乳源乳酸片球菌BPA03的分离鉴定及其抗氧化与抗衰老益生特性评价[J]. 南方医科大学学报, 2026, 46(7): 1601-1610.

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.

图/表 11

表1 qRT-PCR引物序列

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

图1 人乳来源乳酸片球菌BPA03菌落形态特征

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.

表2 乳酸片球菌BPA03生化反应结果

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	-

图2 人乳来源乳酸片球菌BPA03基因序列系统发育树

Fig.2 Phylogenetic tree of P. acidilactici BPA03.

图3 人乳来源乳酸片球菌BPA03溶血反应检测结果

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.

表3 人乳来源乳酸片球菌BPA03药敏性

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

图4 乳酸片球菌 BPA03生长及产酸曲线

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.

图5 　乳酸片球菌 BPA03对斑马鱼毒性试验

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).

图6 乳酸片球菌BPA03对紫外线诱导斑马鱼衰老的影响

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.

图7 乳酸片球菌BPA03对D-半乳糖诱导斑马鱼幼虫衰老的影响

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.

图8 乳酸片球菌BPA03对D-半乳糖诱导斑马鱼幼虫衰老体内炎症因子表达的影响

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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