波兰青霉CK2023-1食品安全性的系统评价

Systematic assessment of Penicillium polonicum CK2023-1 for food safety

摘要:

目的 评估城口老腊肉风味形成关键菌株波兰青霉(Penicillium polonicum) CK2023-1的食用安全性，为开发腊肉的微生物发酵剂提供科学依据。方法 采用三代联合二代测序技术测定P. polonicum CK2023-1的基因组DNA序列，并利用GO、KEGG和antiSMASH等数据库进行注释。在腌肉表面涂抹CK2023-1孢子液(1×10⁹ spores/kg)，于城口当地熏制26 d，采用LC-MS/MS分析非挥发性代谢物。用小鼠开展急性经口毒性(7 d)、亚急性经口毒性(28 d)试验以评价菌株的致病性。结果 CK2023-1基因组大小为33.27 Mb，N₅₀为5 236 196 bp，G+C含量为52.91%，预测到10 901个编码基因，占基因组全长的49.02%。基因组中预测到83种次级代谢产物合成基因簇(biosynthesis gene clusters, BGCs)，含已知的真菌毒素verrucosidin合成基因簇。与产毒株P. polonicum X6相比，CK2023-1株的此BGC发生倒置。实地发酵腊肉中未检测到真菌毒素，急性、亚急性致病性试验也未导致小鼠发病、死亡。结论 多组学分析与致病性试验结果表明，P. polonicum CK2023-1基因组结构清晰，不产生真菌毒素，无致病性，满足食品级微生物菌种的安全要求。

Abstract:

Objective To evaluate the edible safety of Penicillium polonicum CK2023-1, a key strain responsible for flavor formation of traditional bacon in Chengkou, thus providing a scientific basis for developing microbial starter for bacon fermentation.Methods The genomic DNA sequence of P. polonicum CK2023-1 was determined via third-generation sequencing combined with second-generation sequencing and annotated via GO, KEGG, and antiSMASH databases. The spore suspension (1×10⁹ spores/kg) of CK2023-1was applied to cured meat surfaces, followed by 26 d local smoking in Chengkou. Non-volatile metabolites were analyzed via LC-MS/MS. Acute oral toxicity (7 d) and subacute oral toxicity (28 d) tests in mice were conducted to assess pathogenicity.Results The genome of CK2023-1 was 33.27 Mb, with the N₅₀ of 5 236 196 bp and the G+C content of 52.91%, containing 10 901 predicted coding genes (49.02% of the genome). Eighty-three biosynthetic gene clusters (BGCs) for secondary metabolites were identified, including known mycotoxin (verrucosidin) BGC. Compared with that in the toxigenic strain P. polonicum X6, the BGC in CK2023-1 exhibited an inversion. No mycotoxins were detected in fermented bacon, and neither acute nor subacute toxicity tests caused morbidity or mortality in mice.Conclusion P. polonicum CK2023-1 has a well-defined genomic structure, produces no mycotoxins, and is non-pathogenic, meeting the safety criteria for food-grade microbial strains.