Preliminary investigation on the presence of peptides inhibiting the growth of Listeria innocua and Listeria monocytogenes in Asiago d’Allevo cheese

Abstract

Bacteriocins produced by some strains of lactic acid bacteria or antimicrobial casein fragments can be released into the water-soluble fraction of cheeses during ripening. However, there is a lack of experimental evidence which supports the theory that these peptides can exert an effective antimicrobial effect. The aim of this study was to investigate the anti-listerial activity of such peptides in Asiago d’Allevo cheese. The water-soluble fraction of Asiago was ultrafiltrated through 10 kDa cut-off membranes before being dialyzed (100–500 Da cut-off) to remove proteins, salts, and organic acids, prior to freeze-drying. The growth of Listeria innocua LRGIA 01 and of Listeria monocytogenes strain 162 in Brain Heart Infusion (BHI) at 30 °C in the presence or absence of 5 to 40 mg⋅mL⁻¹ of cheese water-soluble extracts (WSEs) lyophilisate was monitored spectrophotometrically. WSEs lyophilisates from cheeses of different production systems (total mixed-ration-fed cows and cheese-making in a dairy plant in May; alpine-grazing cows and cheese-making in situ in July and September) and ripened for 6, 12, and 18 months were tested. A limited dose-dependent inhibition of growth was observed at all the assayed concentrations. The addition of cheese WSEs lyophilisates to BHI broth inhibited the growth of <50% of L. innocua LRGIA 01 and <10% of L. monocytogenes strain 162. Anti-listerial activity was only significantly affected by ripening time (it was maximal at 6 months). Quantification of peptides with a sequence similar to the antimicrobial casein fragments α_S1-CN f(1–23) (isracidin) and α_S2-CN f(183–207) in cheese WSEs suggests that their anti-listerial activity is likely also to be due to other peptides.

肽对 Asiago d’Allevo 干酪中李斯特英诺克菌和李斯特单胞菌的抑制作用

摘要 某些乳酸菌能产生抗菌素,此外,在干酪成熟过程中会产生一些水溶性的、具有抗菌性能的酪蛋白片段。尽管理论上认为这些肽具有杀菌的作用,但还是缺少实验数据证明这种可能性。本文研究了肽对 Asiago d’Allevo 干酪中李斯特菌的抑制作用。Asiago d’Allevo 干酪的水溶性部分经过 10kDa 的膜超滤,超滤液又经过 100-500 Da 的渗透膜渗析后除去蛋白、盐和有机酸,超滤液冻干。将李斯特英诺克 LRGIA 01 和李斯特单胞菌 162 分别接种于含有5-40 mg⋅mL^-1 冻干的干酪水溶性提取物 (WSEs) 的BHI培养基中,在 30 °C 下培养,并检测李斯特菌的生长情况。WSEs 冻干物分别来源于不同的原料和成熟期的 Asiago d’Allevo 干酪,原料奶的来源分别为 5月(种植牧草)、7月和9月(高山自然放牧),成熟期为 6、12、18 个月。在所有的浓度范围内,部分数据显示出剂量与抑制作用的关系。WSEs冻干物对李斯特英诺克 LRGIA 01 生长的抑制率 <50%, 对李斯特单胞菌 162 的抑制率 <10%。干酪的成熟时间(至少 6 个月)显著地影响 WSEs 冻干物对李斯特菌的抑制作用。采用与 WSEs 序列类似的并具有抗菌作用的酪蛋白片段 α_S1-CN f(1–23) (isracidin) 和 α_S2-CN f(183–207) 进行定量试验,结果证明了,WSEs 冻干物具有抑制李斯特菌的特性可能是来源于其他的肽。