Bozzetti Marianna, Cerri Carolina, Morandi Sara, Rocchetti Gabriele, Mussio Chiara, Barbieri Federica, Tabanelli Giulia, Bassi Daniela
Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, 26100 Cremona, Italy.
Department of Animal Science, Food and Nutrition, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy.
Foods. 2025 Mar 24;14(7):1112. doi: 10.3390/foods14071112.
Folate deficiency is a widespread nutritional issue, and biofortifying dairy products through lactic acid bacteria (LAB) is a promising strategy to enhance natural folate levels. This study aimed to develop a reliable method for selecting and subsp. strains with enhanced folate production for use as functional starter cultures. Initially, a traditional microbiological assay (MA) was used to measure folate production in 36 LAB strains isolated from fermented milks. Due to MA's limitations, an untargeted and semi-quantitative method combining ultra-high-performance liquid chromatography (UHPLC) with high-resolution mass spectrometry (HRMS) was developed for a more comprehensive folate screening. The MA showed higher folate production in strains (309-639 µg/L) compared to subsp. (up to 48 µg/L). Subsequently, nine selected LAB strains were further analyzed using the UHPLC-HRMS approach, which enabled the identification and semi-quantification of six folate metabolites, namely dihydrofolate, tetrahydrofolate (THF), 10-formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF. Lab-scale yogurt production using the top-performing strains, as identified through the HRMS method, demonstrated an increase in folate content over a 14-day shelf life. These findings revealed the potential of UHPLC-HRMS as a high-throughput alternative method for folates detection, offering a promising tool for screening folate-enhanced LAB strains for biofortification.
叶酸缺乏是一个普遍存在的营养问题，通过乳酸菌对乳制品进行生物强化是提高天然叶酸水平的一种有前景的策略。本研究旨在开发一种可靠的方法，用于筛选和挑选叶酸产量提高的亚种菌株，以用作功能性发酵剂培养物。最初，使用传统微生物测定法（MA）来测量从发酵乳中分离出的36株乳酸菌菌株的叶酸产量。由于MA的局限性，开发了一种将超高效液相色谱（UHPLC）与高分辨率质谱（HRMS）相结合的非靶向半定量方法，用于更全面的叶酸筛选。MA显示，亚种（高达48μg/L）相比，菌株（309 - 639μg/L）的叶酸产量更高。随后，使用UHPLC - HRMS方法对九株选定的乳酸菌菌株进行了进一步分析，该方法能够鉴定和半定量六种叶酸代谢物，即二氢叶酸、四氢叶酸（THF）、10 - 甲酰基 - THF、5,10 - 亚甲基 - THF、5,10 - 亚甲基 - THF和5 - 甲基 - THF。使用通过HRMS方法鉴定出的表现最佳的菌株进行实验室规模的酸奶生产，结果表明在14天的保质期内叶酸含量有所增加。这些发现揭示了UHPLC - HRMS作为一种高通量叶酸检测替代方法的潜力，为筛选用于生物强化的叶酸增强型乳酸菌菌株提供了一种有前景的工具。
