Folate production in lactic acid bacteria and determination of folate derivatives by HPLC
Yıl 2025,
Cilt: 31 Sayı: 4, 694 - 700, 25.08.2025
Meryem Nur Zeydanlı
,
Zehranur Yüksekdağ
,
Berat Çınar Acar
Öz
Folate is a B-group vitamin that humans cannot synthesize and must be taken externally. Although some types of Lactic Acid Bacteria can produce folate, there is insufficient information about the production of folate by yoghurt starter cultures. In this study, four bacteria belonging to two Lactobacillus, and two Streptococcus genera were isolated from yoghurt samples, and the isolates were biochemically identified by 16S rDNA with an API 50 CHL kit. Intracellular and extracellular folate production of the bacteria was designated in various media and buffer applications. The highest intracellular folate production was observed in Lactobacillus delbrueckii subsp. bulgaricus ZN951 (126.3 μg/L) and Streptococcus. thermophilus Z151 (177.1 μg/L) strains in MRS/Elliker medium with potassium phosphate buffer. L. delbrueckii subsp. bulgaricus ZN541 strain showed the highest extracellular folate production (105.1μg/L) in the same medium and buffer, but S. thermophilus Z151 strain exhibited 86.4 μg/L folate productions. Whether the strains contained folate derivatives and pABA was investigated by HPLC analysis and pABA was not detected in any of the strains. Among cultures, the highest total folate content was detected as 110.5μg/L by L. delbrueckii subsp. bulgaricus ZN951 strain. This strain was found to have concentrations of 5-MeTHF and 5-FmTHF. Therefore, the use of these bacteria can be an alternative to naturally increase folate content in foods.
Kaynakça
-
[1] Liu M, Chen Q, Sun Y, Zeng L, Wu H, Gu Q, Li P. “Probiotic potential of a folate-producing strain Latilactobacillus sakei LZ217 and ıts modulation effects on human gut microbiota”. Foods, 11(2), 234, 2022.
-
[2] Zahed O, Khosravi-Darani K, Mortazavian AM, Mohammadi A. “Effects of cultivation conditions on biofortification of yoghurt with natural folate by Propionibacterium freudenreichii”. Biocatalysis and Agricultural Biotechnology, 39, 102267, 2022.
-
[3] Laiño JE, LeBlanc JG, de Gior GS. “Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yoghurts”. Canadian Journal of Microbiology, 58(5), 581-588, 2012.
-
[4] Mahara FA, Nuraida L, Lioe HN. “Fermentation of milk using folate-producing lactic acid bacteria to ıncrease natural folate content: a review”. Journal of Applied Biotechnology Reports, 6(4), 129-136, 2019.
-
[5] Pophaly SD, Tomar SK, De S, Singh R. “Multifaceted attributes of dairy propionibacteria: a review”. World Journal of Microbiology and Biotechnology,
28(11), 3081-3095, 2012.
-
[6] Yuksekdag ZN, Zeydanlı M. “Folat eksikliği ve probiyotikler”. Nevşehir Bilim ve Teknoloji Dergisi,
2(2), 21-36, 2013.
-
[7] Zhang J, Cai D, Yang M, Hao Y, Zhu Y, Chen Z, Aziz T, Sarwar A, Yang Z. “Screening of folate-producing lactic acid bacteria and modulatory effects of folate-biofortified yoghurt on gut dysbacteriosis of folate-deficient rats”. Food Function, 11, 6308-6318, 2020.
-
[8] Donnelly JG. “Folic acid”. Critical Reviews in Clinical Laboratory Sciences, 38(3), 183-223, 2001.
-
[9] Pannia E, Hammoud R, Simonian R, Kubant R, Harvey Anderson G. “Folate dose and form during pregnancy may program maternal and fetal health and disease risk”. Nutrition Reviews, 80(11), 2178-2197, 2022.
-
[10] Mattarelli P, Biavati B, Holzapfel WH, Wood BJB. The Bifidobacteria and Related Organisms: Biology, Taxonomy, Applications. Elsevier, London, United Kingdom Academic Press, 2018.
-
[11] Sybesma W, Starrenburg M, Tijsseling L, Hoefnagel MHN, Hugenholtz J. “Effect of cultivation conditions on folate production by lactic acid bacteria”. Applied and Environmental Microbiology, 69(8), 4542-4548, 2003a.
-
[12] Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, Hugenholtz J. “Increased production of folate by metabolic engineering of Lactococcus lactis”. Applied and Environmental Microbiology, 69(6), 3069-3076, 2003(b).
-
[13] Mo H, Kariluoto S, Piironen V, Zhu Y, SandersMG, Vincken JP, Wolkers-Rooijackers J, Nout MJ. “Effect of soybean processing on content and bioaccessibility of folate, vitamin B12 and isoflavones in tofu and tempeh”.
Food Chemistry, 141(3), 2418-2425, 2013.
-
[14] Scaglione F, Panzavolta G. “Folate, folic acid and 5-methyltetrahydrofolate are not the same thing”. Xenobiotica, 44(5), 480-488, 2014.
-
[15] Pandey SM, Mishra H. “Optimization of the prebiotic & probiotic concentration and incubation temperature for the preparation of synbiotic soy yoghurt using response surface methodology”. LWT-Food Science and Technology, 62(1), 458-467, 2015.
-
[16] Zeydanli MN, Yuksekdag Z, Cinar Acar B. “Production of intra-/extracellular folate in Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria isolated from traditional turkish yoghurts”. KSU Journal of Agriculture and Nature, 27(3), 704-717, 2024.
-
[17] dos Santos HRM, Argolo CS, Argolo-Filho RC, Loguercio LLA. “16S rDNA PCR based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information”. BMC Microbiology, 19, 74, 2019.
-
[18] Aswathy R, Ismail B, John RP, Nampoothiri KM. “Evaluation of the probiotic characteristics of newly ısolated lactic acid bacteria”. Applied Biochemistry and Biotechnology, 151, 244-255, 2008.
-
[19] Horne DW, Patterson D. “Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates”. Clinical Chemistry, 34(11), 2357–2359, 1988.
-
[20] Wilson SD, Horne DW. “Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid”. Clinical Chemistry, 28(5), 1198-1200, 1982.
-
[21] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[22] Laiño JE, Juarez del Valle M, Savoy de Giori G, LeBlanc JGJ. “Development of a high folate concentration yoghurt naturally bio-enriched using selected lactic acid bacteria”. LWT Food Science and Technology, 54(1), 1-5, 2013.
-
[23] Strandler HS. “Determination of Folate for Food Composition Data”. Swedish University of Agricultural Sciences, Uppsala, Licentiate Thesis, 15-30, 2012. ISBN 978-91-576-9061-6.
-
[24] de Crécy-Lagard V, El Yacoubi B, Diaz de la Garza R, Noiriel A, Hanson AD. “Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations”. BMC Genomics, 8, 245, 2007.
-
[25] Cucick ACC, Gianni K, Todorov SD, de LeBlanc AM, LeBlanc J, Franco BDGM. “Evaluation of the bioavailability and intestinal effects of milk fermented by folate producing lactic acid bacteria in a depletion/repletion mice model”. Journal of Functional Foods, 66, 103785, 2020.
-
[26] Albano C, Silvetti T, Brasca M. “Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment”. FEMS Microbiology Letters, 367(9), 1-9, 2020.
-
[27] Laiño JE, Levit R, LeBlanc AM, Giori GS, LeBlanc JG. “Characterization of folate production and probiotic potential of Streptococcus gallolyticus subsp. macedonicus CRL415”. Food Microbiology, 79, 20-26, 2019.
-
[28] Mahara FA, Nuraida L, Lioe HN. “Folate in milk is fermented by lactic acid bacteria from different food sources”. Preventive Nutrition and Food Science,
26(2), 230-240, 2021.
-
[29] Sarma JD, Duttagupta C. “Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis”. Food Biological Contaminants, 78(5), 1173-1176, 1995.
-
[30] Holt DL, Wehling RL, Zeece MG. “Determination of native folates in milk and other dairy products by high-performance liquid chromatography”. Journal of Chromatography A, 449, 271-279, 1988.
-
[31] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[32] Meucci A, Rossetti L, Zago M, Monti L, Giraffa G, Carminati D, Tidona F. “Folates biosynthesis by Streptococcus thermophilus during growth in milk”. Food Microbiology, 69, 116-122, 2018.
-
[33] KEGG. “Kyoto Encyclopedia of Genes and Genomes”. http://www.genome.jp/kegg (13.03.2024).
-
[34] Rossi M. Amaretti A, Raimondi S. “Folate production by probiotic bacteria”. Nutrients, 3(1), 118-134, 2011.
-
[35] Crittenden RG, Martinez NR, Playne MJ. “Synthesis and utilisation of folate by yoghurt starter cultures and probiotic bacteria”. International Journal of Food Microbiology, 80(3), 217-222, 2003.
-
[36] Wu Z, Wu J, Cao P, Jin Y, Pan D, Zeng X, Guo Y. “Characterization of probiotic bacteria involved in fermented milk processing enriched with folic acid”. Journal of Dairy Science, 100(6), 4223-4229, 2017. https://doi.org/10.3168/jds.2017-12640
-
[37] Mahara FA, Nuraida L, Lioe HN, Nurjanah S. “Folate production and its distribution during growth of lactic acid bacteria isolated from fermented food and breast milk”. Food Technology, 47(1), 167-179, 2023.
-
[38] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[39] Sanna MG, Mangia NP, Garau G, Murgia MA, Massa T, Franco A, Deiana P. “Selection of folate-producing lactic acid bacteria for improving fermented goat milk”. Italian Journal of Food Science, 17(2), 143-154, 2005.
-
[40] Khalili M, Rad AH, Khosroushahi AY, Khosravi H, Jafarzadeh S. “Application of probiotics in folate bio-fortification of yoghurt”. Probiotics and Antimicrobial Proteins, 12, 756-763, 2020.
-
[41] Kariluoto S, Edelmann M, Herranen M, Lampi AM, Shmelev A, Salovaara H, Korhola M, Piironen V. “Production of folate by bacteria isolated from oat bran. International Journal of Food Microbiology,143(1-2),41-47, 2010.
-
[42] Delchier N, Herbig AL, Rychlik M, Renard CM. “Folates in fruits and vegetables: contents, processing, and stability”. Comprehensive Reviews in Food Science and Food Safety, 15(3), 506-528, 2016.
-
[43] Castaño E, Pinunuri R, Hirsch S, Ronco AM. “Folate and pregnancy, current concepts: It is required folic acid supplementation?” Revista Chilena de Pediatria,
88(2), 199-206, 2017.
-
[44] Greppi A, Hemery Y, Berrazaga I Almaksour Z, Humblot C. “The ability of lactobacilli isolated from traditional cereal-based fermented food to produce folate in culture media under different growth conditions”. LWT-Food Science and Technology, 86, 277-84, 2017.
Laktik asit bakterilerinde folat üretimi ve HPLC ile folat türevlerinin tayini
Yıl 2025,
Cilt: 31 Sayı: 4, 694 - 700, 25.08.2025
Meryem Nur Zeydanlı
,
Zehranur Yüksekdağ
,
Berat Çınar Acar
Öz
Folat, insanların sentezleyemedikleri ve dışarıdan alınması gereken B grubu bir vitamindir. Bazı Laktik Asit Bakterileri folat üretebilse de, yoğurt starter kültürlerinin folat üretimi hakkında yeterli bilgi yoktur. Bu çalışmada yoğurt örneklerinden iki Lactobacillus ve iki Streptococcus cinsine ait dört bakteri izole edilmiş ve izolatlar API 50 CHL kiti ile 16S rDNA ile biyokimyasal olarak tanımlanmıştır. Bakterilerin hücre içi ve hücre dışı folat üretimi çeşitli ortam ve tampon uygulamalarında belirlenmiştir. En yüksek hücre içi folat üretimi Lactobacillus delbrueckii subsp. bulgaricus ZN951 (126.3 μg/L) ve Streptococcus thermophilus Z151 (177.1 μg/L) suşlarında MRS/Elliker besiortamı ve potasyum fosfat tamponunda belirlenmiştir. L. delbrueckii subsp. bulgaricus ZN541 suşu, aynı ortam ve tamponda en yüksek hücre dışı folat üretimini (105.1 μg/L) gösterirken, S. thermophilus Z151 suşu, 86.4 μg/L folat üretimi sergilemiştir. Suşların folat türevleri ve pABA içerip içermediği HPLC analizi ile araştırılmış ve suşların hiçbirinde pABA tespit edilememiştir. Kültürler arasında en yüksek toplam folat içeriği 110.5 μg/L olarak L. delbrueckii subsp. bulgaricus ZN951 suşunda tespit edilmiştir. Bu suşun 5-MeTHF ve5-FmTHF konsantrasyonlarına sahip olduğu bulunmuştur. Bu nedenle, bu bakterilerin kullanımı gıdalardaki folat içeriğini doğal olarak arttırmaya bir alternatif olabilir.
Kaynakça
-
[1] Liu M, Chen Q, Sun Y, Zeng L, Wu H, Gu Q, Li P. “Probiotic potential of a folate-producing strain Latilactobacillus sakei LZ217 and ıts modulation effects on human gut microbiota”. Foods, 11(2), 234, 2022.
-
[2] Zahed O, Khosravi-Darani K, Mortazavian AM, Mohammadi A. “Effects of cultivation conditions on biofortification of yoghurt with natural folate by Propionibacterium freudenreichii”. Biocatalysis and Agricultural Biotechnology, 39, 102267, 2022.
-
[3] Laiño JE, LeBlanc JG, de Gior GS. “Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yoghurts”. Canadian Journal of Microbiology, 58(5), 581-588, 2012.
-
[4] Mahara FA, Nuraida L, Lioe HN. “Fermentation of milk using folate-producing lactic acid bacteria to ıncrease natural folate content: a review”. Journal of Applied Biotechnology Reports, 6(4), 129-136, 2019.
-
[5] Pophaly SD, Tomar SK, De S, Singh R. “Multifaceted attributes of dairy propionibacteria: a review”. World Journal of Microbiology and Biotechnology,
28(11), 3081-3095, 2012.
-
[6] Yuksekdag ZN, Zeydanlı M. “Folat eksikliği ve probiyotikler”. Nevşehir Bilim ve Teknoloji Dergisi,
2(2), 21-36, 2013.
-
[7] Zhang J, Cai D, Yang M, Hao Y, Zhu Y, Chen Z, Aziz T, Sarwar A, Yang Z. “Screening of folate-producing lactic acid bacteria and modulatory effects of folate-biofortified yoghurt on gut dysbacteriosis of folate-deficient rats”. Food Function, 11, 6308-6318, 2020.
-
[8] Donnelly JG. “Folic acid”. Critical Reviews in Clinical Laboratory Sciences, 38(3), 183-223, 2001.
-
[9] Pannia E, Hammoud R, Simonian R, Kubant R, Harvey Anderson G. “Folate dose and form during pregnancy may program maternal and fetal health and disease risk”. Nutrition Reviews, 80(11), 2178-2197, 2022.
-
[10] Mattarelli P, Biavati B, Holzapfel WH, Wood BJB. The Bifidobacteria and Related Organisms: Biology, Taxonomy, Applications. Elsevier, London, United Kingdom Academic Press, 2018.
-
[11] Sybesma W, Starrenburg M, Tijsseling L, Hoefnagel MHN, Hugenholtz J. “Effect of cultivation conditions on folate production by lactic acid bacteria”. Applied and Environmental Microbiology, 69(8), 4542-4548, 2003a.
-
[12] Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, Hugenholtz J. “Increased production of folate by metabolic engineering of Lactococcus lactis”. Applied and Environmental Microbiology, 69(6), 3069-3076, 2003(b).
-
[13] Mo H, Kariluoto S, Piironen V, Zhu Y, SandersMG, Vincken JP, Wolkers-Rooijackers J, Nout MJ. “Effect of soybean processing on content and bioaccessibility of folate, vitamin B12 and isoflavones in tofu and tempeh”.
Food Chemistry, 141(3), 2418-2425, 2013.
-
[14] Scaglione F, Panzavolta G. “Folate, folic acid and 5-methyltetrahydrofolate are not the same thing”. Xenobiotica, 44(5), 480-488, 2014.
-
[15] Pandey SM, Mishra H. “Optimization of the prebiotic & probiotic concentration and incubation temperature for the preparation of synbiotic soy yoghurt using response surface methodology”. LWT-Food Science and Technology, 62(1), 458-467, 2015.
-
[16] Zeydanli MN, Yuksekdag Z, Cinar Acar B. “Production of intra-/extracellular folate in Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria isolated from traditional turkish yoghurts”. KSU Journal of Agriculture and Nature, 27(3), 704-717, 2024.
-
[17] dos Santos HRM, Argolo CS, Argolo-Filho RC, Loguercio LLA. “16S rDNA PCR based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information”. BMC Microbiology, 19, 74, 2019.
-
[18] Aswathy R, Ismail B, John RP, Nampoothiri KM. “Evaluation of the probiotic characteristics of newly ısolated lactic acid bacteria”. Applied Biochemistry and Biotechnology, 151, 244-255, 2008.
-
[19] Horne DW, Patterson D. “Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates”. Clinical Chemistry, 34(11), 2357–2359, 1988.
-
[20] Wilson SD, Horne DW. “Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid”. Clinical Chemistry, 28(5), 1198-1200, 1982.
-
[21] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[22] Laiño JE, Juarez del Valle M, Savoy de Giori G, LeBlanc JGJ. “Development of a high folate concentration yoghurt naturally bio-enriched using selected lactic acid bacteria”. LWT Food Science and Technology, 54(1), 1-5, 2013.
-
[23] Strandler HS. “Determination of Folate for Food Composition Data”. Swedish University of Agricultural Sciences, Uppsala, Licentiate Thesis, 15-30, 2012. ISBN 978-91-576-9061-6.
-
[24] de Crécy-Lagard V, El Yacoubi B, Diaz de la Garza R, Noiriel A, Hanson AD. “Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations”. BMC Genomics, 8, 245, 2007.
-
[25] Cucick ACC, Gianni K, Todorov SD, de LeBlanc AM, LeBlanc J, Franco BDGM. “Evaluation of the bioavailability and intestinal effects of milk fermented by folate producing lactic acid bacteria in a depletion/repletion mice model”. Journal of Functional Foods, 66, 103785, 2020.
-
[26] Albano C, Silvetti T, Brasca M. “Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment”. FEMS Microbiology Letters, 367(9), 1-9, 2020.
-
[27] Laiño JE, Levit R, LeBlanc AM, Giori GS, LeBlanc JG. “Characterization of folate production and probiotic potential of Streptococcus gallolyticus subsp. macedonicus CRL415”. Food Microbiology, 79, 20-26, 2019.
-
[28] Mahara FA, Nuraida L, Lioe HN. “Folate in milk is fermented by lactic acid bacteria from different food sources”. Preventive Nutrition and Food Science,
26(2), 230-240, 2021.
-
[29] Sarma JD, Duttagupta C. “Improved microbiological assay for folic acid based on microtiter plating with Streptococcus faecalis”. Food Biological Contaminants, 78(5), 1173-1176, 1995.
-
[30] Holt DL, Wehling RL, Zeece MG. “Determination of native folates in milk and other dairy products by high-performance liquid chromatography”. Journal of Chromatography A, 449, 271-279, 1988.
-
[31] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[32] Meucci A, Rossetti L, Zago M, Monti L, Giraffa G, Carminati D, Tidona F. “Folates biosynthesis by Streptococcus thermophilus during growth in milk”. Food Microbiology, 69, 116-122, 2018.
-
[33] KEGG. “Kyoto Encyclopedia of Genes and Genomes”. http://www.genome.jp/kegg (13.03.2024).
-
[34] Rossi M. Amaretti A, Raimondi S. “Folate production by probiotic bacteria”. Nutrients, 3(1), 118-134, 2011.
-
[35] Crittenden RG, Martinez NR, Playne MJ. “Synthesis and utilisation of folate by yoghurt starter cultures and probiotic bacteria”. International Journal of Food Microbiology, 80(3), 217-222, 2003.
-
[36] Wu Z, Wu J, Cao P, Jin Y, Pan D, Zeng X, Guo Y. “Characterization of probiotic bacteria involved in fermented milk processing enriched with folic acid”. Journal of Dairy Science, 100(6), 4223-4229, 2017. https://doi.org/10.3168/jds.2017-12640
-
[37] Mahara FA, Nuraida L, Lioe HN, Nurjanah S. “Folate production and its distribution during growth of lactic acid bacteria isolated from fermented food and breast milk”. Food Technology, 47(1), 167-179, 2023.
-
[38] Lin MY, Young CM. “Folate levels in cultures of lactic acid bacteria”. International Dairy Journal, 10(5-6), 409-413, 2000.
-
[39] Sanna MG, Mangia NP, Garau G, Murgia MA, Massa T, Franco A, Deiana P. “Selection of folate-producing lactic acid bacteria for improving fermented goat milk”. Italian Journal of Food Science, 17(2), 143-154, 2005.
-
[40] Khalili M, Rad AH, Khosroushahi AY, Khosravi H, Jafarzadeh S. “Application of probiotics in folate bio-fortification of yoghurt”. Probiotics and Antimicrobial Proteins, 12, 756-763, 2020.
-
[41] Kariluoto S, Edelmann M, Herranen M, Lampi AM, Shmelev A, Salovaara H, Korhola M, Piironen V. “Production of folate by bacteria isolated from oat bran. International Journal of Food Microbiology,143(1-2),41-47, 2010.
-
[42] Delchier N, Herbig AL, Rychlik M, Renard CM. “Folates in fruits and vegetables: contents, processing, and stability”. Comprehensive Reviews in Food Science and Food Safety, 15(3), 506-528, 2016.
-
[43] Castaño E, Pinunuri R, Hirsch S, Ronco AM. “Folate and pregnancy, current concepts: It is required folic acid supplementation?” Revista Chilena de Pediatria,
88(2), 199-206, 2017.
-
[44] Greppi A, Hemery Y, Berrazaga I Almaksour Z, Humblot C. “The ability of lactobacilli isolated from traditional cereal-based fermented food to produce folate in culture media under different growth conditions”. LWT-Food Science and Technology, 86, 277-84, 2017.