RagusanoおよびPecorino Sicilianoチーズから分離したLactobacillus属株のプロバイオティクス特性

Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses.

抄録

本研究では，RagusanoおよびPecorino Sicilianoチーズから分離した177株のLactobacillus属について，in vitroでプロバイオティクス特性をスクリーニングし，その特性を市販株のLactobacillus rhamnosus GGの特性と比較検討した．酸性および胆汁酸塩耐性に基づき、13株のLactobacillusが選択された。Multiplex-PCR法により、9株がL. rhamnosus属、4株がLactobacillus paracasei属に属することが明らかとなった。さらに、選択したすべての菌株について、模擬上部消化管での通過性、ヒト腸管細胞株への接着能、疎水性、共凝集・自己凝集性、抗菌活性について検討した。さらに、安全性評価のために、抗生物質耐性、溶血性、胆汁酸塩ヒドロラーゼ活性を調査した。模擬胃・十二指腸通過後の生菌数測定から、選択した乳酸菌は全体的に酸性ジュースよりも膵液や胆汁酸塩に耐性があることが判明した。特に、L.rhamnosus 3株（FS10、FS2、PS11）およびL.paracasei 1株（PM8）は、模擬胃ろう通過後に細胞密度が増加した。また、L.paracasei1株（PM8）は、大腸菌との共凝集や自己凝集を高率に示した。Staphylococcus aureusとE.coliの両方に有効であり、病原性指標株として用いたListeria monocytogenesとEnterococcus faecalisに対しては、すべての菌株が変動性を示した。また，接着力や疎水性については菌株によって異なる挙動を示したが，これらは必ずしも連動しておらず，強い菌株依存性が認められた。安全性の面では，溶血活性や胆汁酸ヒドロラーゼ活性を示す菌株は1株を除いてなく，ほとんどの菌株が広範な臨床用抗生物質に感受性を示した．L.rhamnosus FS10とL.paracasei PM8はプロバイオティクスとして有望であり、今後のin vivo研究の対象となることが示唆された。

In the present study 177 Lactobacillus spp. strains, isolated from Ragusano and Pecorino Siciliano cheeses, were in vitro screened for probiotic traits, and their characteristics were compared to those of Lactobacillus rhamnosus GG, commercial strain. Based on acidic and bile salt resistance, thirteen Lactobacillus strains were selected. The multiplex-PCR application revealed that nine strains belonged to L. rhamnosus species and four to Lactobacillus paracasei species. All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal tract (GI), for adhesion capacity to human intestinal cell lines, for hydrophobicity, for co-aggregation and auto-aggregation and for antimicrobial activities. Moreover, antibiotic resistance, hemolytic and bile salt hydrolase activities were investigated for safety assessment. Viable counts after simulated gastric and duodenal transit revealed that overall the selected lactobacilli tolerated better pancreatic juice and bile salts than acidic juice. In particular, three L. rhamnosus strains (FS10, FS2, and PS11) and one L. paracasei strain (PM8) increased their cell density after the simulated GI transit. The same strains showed also high percentage of auto-aggregation and co-aggregation with Escherichia coli. All strains were effective against both Staphylococcus aureus and E. coli and variability was achieved versus Listeria monocytogenes and Enterococcus faecalis used as pathogenic indicator strains. Different behavior was revealed by strains for adhesion ability and hydrophobicity, which are not always linked each other and are strongly strain-dependent. From the safety point of view, no isolate showed hemolytic and bile salt hydrolase activities, except one, and most of the strains were sensitive to a broad range of clinical antibiotics. This work showed that the L. rhamnosus FS10 and the L. paracasei PM8 are good promising probiotic candidates for further in vivo investigations.