Analytical and Bioanalytical Chemistry

, Volume 411, Issue 6, pp 1181–1192 | Cite as

In vitro assessment of pediococci- and lactobacilli-induced cholesterol-lowering effect using digitally enhanced high-performance thin-layer chromatography and confocal microscopy

  • Rohawi Nur Syakila
  • Siong Meng Lim
  • Snezana Agatonovic-Kustrin
  • Fei Tieng Lim
  • Kalavathy RamasamyEmail author
Research Paper


The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19–59% in the absence and 14–69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study.

Graphical abstract

(A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB


Probiotics Cholesterol reduction Cholesterol assimilation Bile salts High-performance thin-layer chromatography Confocal microscopy 



This work was supported by the Ministry of Higher Education Malaysia [600-RMI/LRGS 5/3 (2/2012)].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Veatch SL, Keller SL. Organization in lipid membranes containing cholesterol. Phys Rev Lett. 2002;89:68101. Scholar
  2. 2.
    Brown DA, London E. Structure and function of sphingolipid-and cholesterol-rich membrane rafts. J Biol Chem. 2000;275:17221–4.Google Scholar
  3. 3.
    Hu J, Zhang Z, Shen W-J, Azhar S. Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutr Metab. 2010;7:47. Scholar
  4. 4.
    Cohen DE. Balancing cholesterol synthesis and absorption in the gastrointestinal tract. J Clin Lipidol. 2008;2:S1–3.Google Scholar
  5. 5.
    Leonarduzzi G, Sottero B, Poli G. Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects. J Nutr Biochem. 2002;13:700–10.Google Scholar
  6. 6.
    Nordestgaard BG, Chapman MJ, Ray K, Borén J, Andreotti F, Watts GF, et al. Lipoprotein (a) as a cardiovascular risk factor: current status. Eur Heart J. 2010;31:2844–53.Google Scholar
  7. 7.
    World Health Organization. Raised cholesterol: situation and trends. (2017). Accessed 22 Nov 2018.
  8. 8.
    Ford ES, Li C, Pearson WS, Zhao G, Mokdad AH. Trends in hypercholesterolemia, treatment and control among United States adults. Int J Cardiol. 2010;140:226–35.Google Scholar
  9. 9.
    Woodward M, Martiniuk A, Lee CMY, Lam TH, Vanderhoorn S, Ueshima H, et al. Elevated total cholesterol: its prevalence and population attributable fraction for mortality from coronary heart disease and ischaemic stroke in the Asia-Pacific region. Eur J Cardiovasc Prev Rehabil. 2008;15:397–401.Google Scholar
  10. 10.
    Yen ST, Tan AK, Feisul MI. Awareness and prevalence of diabetes, hypertension, and hypercholesterolemia in Malaysia. J Diabetes. 2016;9:874–83.Google Scholar
  11. 11.
    Mannu S, Zaman JM, Gupta A, Rehman HU, Myint PK. Evidence of lifestyle modification in the management of hypercholesterolemia. Curr Cardiol Rev. 2013;9:2–14.Google Scholar
  12. 12.
    Steinberg D. Earlier intervention in the management of hypercholesterolemia. J Am Coll Cardiol. 2010;56:627–9.Google Scholar
  13. 13.
    Cohen JD, Brinton EA, Ito MK, Jacobson TA. Understanding Statin Use in America and Gaps in Patient Education (USAGE): an internet-based survey of 10,138 current and former statin users. J Clin Lipidol. 2012;6:208–15.Google Scholar
  14. 14.
    Kumar M, Nagpal R, Kumar R, Hemalatha R, Verma V, Kumar A, et al. Cholesterol-lowering probiotics as potential biotherapeutics for metabolic diseases. Exp Diabetes Res. 2012;2012:1–14.Google Scholar
  15. 15.
    Food and Agriculture Organization of the United Nations, World Health Organization. Probiotics in food: health and nutritional properties and guidelines for evaluation. Rome: Food and Agriculture Organization of the United Nations; 2006.Google Scholar
  16. 16.
    Ooi L-G, Liong M-T. Cholesterol-lowering effects of probiotics and prebiotics: a review of in vivo and in vitro findings. Int J Mol Sci. 2010;11:2499–522.Google Scholar
  17. 17.
    Ramasamy K, Majeed ABA, Wan HY, Mani V, Shafawi ZM. Hypocholesterolaemic effects of probiotics. In: Saad M, editor. Complementary therapies for the contemporary healthcare. London: IntechOpen; 2012. Scholar
  18. 18.
    Ebel B, Lemetais G, Beney L, Cachon R, Sokol H, Langella P, et al. Impact of probiotics on risk factors for cardiovascular diseases. A review. Crit Rev Food Sci Nutr. 2014;54:175–89.Google Scholar
  19. 19.
    Jones ML, Tomaro-Duchesneau C, Martoni CJ, Prakash S. Cholesterol lowering with bile salt hydrolase-active probiotic bacteria, mechanism of action, clinical evidence, and future direction for heart health applications. Expert Opin Biol Ther. 2013;13:631–42.Google Scholar
  20. 20.
    Huang Y, Wang X, Wang J, Wu F, Sui Y, Yang L, et al. Lactobacillus plantarum strains as potential probiotic cultures with cholesterol-lowering activity. J Dairy Sci. 2013;96:2746–53.Google Scholar
  21. 21.
    Ding W, Shi C, Chen M, Zhou J, Long R, Guo X. Screening for lactic acid bacteria in traditional fermented Tibetan yak milk and evaluating their probiotic and cholesterol-lowering potentials in rats fed a high-cholesterol diet. J Funct Foods. 2017;32:324–32.Google Scholar
  22. 22.
    Yadav R, Puniya AK, Shukla P. Probiotic properties of Lactobacillus plantarum RYPR1 from an indigenous fermented beverage Raabadi. Front Microbiol. 2016;7:1683. Scholar
  23. 23.
    Iranmanesh M, Ezzatpanah H, Mojgani N. Antibacterial activity and cholesterol assimilation of lactic acid bacteria isolated from traditional Iranian dairy products. LWT Food Sci Technol. 2014;58:355–9.Google Scholar
  24. 24.
    Rudel LL, Morris M. Determination of cholesterol using o-phthalaldehyde. J Lipid Res. 1973;14:364–6.Google Scholar
  25. 25.
    Khatun R, Hunter H, Magcalas W, Sheng Y, Carpick B, Kirkitadze M. Nuclear magnetic resonance (NMR) study for the detection and quantitation of cholesterol in HSV529 therapeutic caccine candidate. Comput Struct Biotechnol J. 2017;15:14–20.Google Scholar
  26. 26.
    Coreta-Gomes FM, Vaz WL, Wasielewski E, Geraldes CF, Moreno MJ. Quantification of cholesterol solubilized in dietary micelles: dependence on human bile salt variability and the presence of dietary food ingredients. Langmuir. 2016;32:4564–74.Google Scholar
  27. 27.
    Zhou G, Guan Y. An on-column enzyme mediated fluorescence-amplification method for plasma total cholesterol measurement by capillary electrophoresis with LIF detection. Chromatographia. 2016;79:319–25.Google Scholar
  28. 28.
    Chitra J, Ghosh M, Mishra H. Rapid quantification of cholesterol in dairy powders using Fourier transform near infrared spectroscopy and chemometrics. Food Control. 2017;78:342–9.Google Scholar
  29. 29.
    Bauer LC. de A Santana D, dos S Macedo M, Torres AG, de Souza NE, Simionato JI. Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD. J Braz Chem Soc. 2014;25:161–8.Google Scholar
  30. 30.
    Al-Balaa D, Rajchl A, Grégrová A, Ševčík R. Čížková H. DART mass spectrometry for rapid screening and quantitative determination of cholesterol in egg pasta. J Mass Spectrom. 2014;49:911–7.Google Scholar
  31. 31.
    Chen Y-Z, Kao S-Y, Jian H-C, Yu Y-M, Li J-Y, Wang W-H, et al. Determination of cholesterol and four phytosterols in foods without derivatization by gas chromatography-tandem mass spectrometry. J Food Drug Anal. 2015;23:636–44.Google Scholar
  32. 32.
    Sandhoff R, Brügger B, Jeckel D, Lehmann WD, Wieland FT. Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J Lipid Res. 1999;40:126–32.Google Scholar
  33. 33.
    Honda A, Yamashita K, Miyazaki H, Shirai M, Ikegami T, Xu G, et al. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS. J Lipid Res. 2008;49:2063–73.Google Scholar
  34. 34.
    Šabović I, De Toni L, Tescari S, De Filippis V, Menegazzo M. Detection of cholesterol and its oxidized derivatives in human sperm membranes through a fast and reliable LC-MS method. J Clin Lab Med. 2017;2:1–7.Google Scholar
  35. 35.
    Li L, Han J, Wang Z, Liu JA, Wei J, Xiong S, et al. Mass spectrometry methodology in lipid analysis. Int J Mol Sci. 2014;15:10492–507.Google Scholar
  36. 36.
    Gachumi G, El-Aneed A. Mass spectrometric approaches for the analysis of phytosterols in biological samples. J Agric Food Chem. 2017;65:10141–56.Google Scholar
  37. 37.
    Kinter M, Herold D, Hundley J, Wills M, Savory J. Measurement of cholesterol in serum by gas chromatography/mass spectrometry at moderate mass resolution, with a nonendogenous cholesterol isomer as internal standard. Clin Chem. 1988;34:531–4.Google Scholar
  38. 38.
    Taranto MP, Fernandez Murga ML, Lorca G, de Valdez GF. Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri. J Appl Microbiol. 2003;95:86–91.Google Scholar
  39. 39.
    Rohawi N, Ramasamy K, Agatonovic-Kustrin S, Lim S. A new high-performance thin-layer chromatographic method for determining bile salt hydrolase activity. J Chromatogr B. 2018;1092:145–51. Scholar
  40. 40.
    Patravale VB, D'Souza S. Narkar Y. HPTLC determination of nimesulide from pharmaceutical dosage forms. J Pharm Biomed Anal. 2001;25:685–8.Google Scholar
  41. 41.
    Thoppil SO, Cardoza RM, Amin P. Stability indicating HPTLC determination of trimetazidine as bulk drug and in pharmaceutical formulations. J Pharm Biomed Anal. 2001;25:15–20.Google Scholar
  42. 42.
    Rohawi N, Ramasamy K, Agatonovic-Kustrin S. Lim SM. A new high-performance thin-layer chromatographic method for determining bile salt hydrolase activity. J Chromatogr A. 2018;1092:145–51.Google Scholar
  43. 43.
    Agatonovic-Kustrin S, Morton DW. High-performance thin-layer chromatography HPTLC-direct bioautography as a method of choice for alpha-amylase and antioxidant activity evaluation in marine algae. J Chromatogr A. 2017;1530:197–203. Scholar
  44. 44.
    Agatonovic-Kustrin S, Morton DW. Determination of free phenolic acids in plant-derived foods by high-performance thin-layer chromatography with direct 2,2′-diphenyl-1-picrylhydrazyl assay. J Planar Chromatogr Mod TLC. 2016;29:121–6.Google Scholar
  45. 45.
    Agatonovic-Kustrin S, Ortakand DB, Morton DW, Yusof AP. Rapid evaluation and comparison of natural products and antioxidant activity in calendula, feverfew, and German chamomile extracts. J Chromatogr A. 2015;1385:103–10. Scholar
  46. 46.
    Gallo FR, Multari G, Federici E, Palazzino G, Giambenedetti M, Petitto V, et al. Chemical fingerprinting of Equisetum arvense L. using HPTLC densitometry and HPLC. Nat Prod Res. 2011;25:1261–70.Google Scholar
  47. 47.
    Morlock GE, Ristivojevic P, Chernetsova ES. Combined multivariate data analysis of high-performance thin-layer chromatography fingerprints and direct analysis in real time mass spectra for profiling of natural products like propolis. J Chromatogr A. 2014;1328:104–12. Scholar
  48. 48.
    Paci A, Mercier L, Bourget P. Identification and quantitation of antineoplastic compounds in chemotherapeutic infusion bags by use of HPTLC: application to the vinca-alkaloids. J Pharm Biomed Anal. 2003;30:1603–10.Google Scholar
  49. 49.
    Puri A, Ahmad A, Panda BP. Development of an HPTLC-based diagnostic method for invasive aspergillosis. Biomed Chromatogr. 2010;24:887–92.Google Scholar
  50. 50.
    John J, Reghuwanshi A, Aravind UK, Aravindakumar C. Development and validation of a high-performance thin layer chromatography method for the determination of cholesterol concentration. J Food Drug Anal. 2015;23:219–24.Google Scholar
  51. 51.
    Broszat M, Ernst H. Spangenberg B. A simple method for quantifying triazine herbicides using thin-layer chromatography and a CCD camera. J Liq Chromatogr Relat Technol. 2010;33:948–56.Google Scholar
  52. 52.
    Angmo K, Kumari A, Bhalla TC. Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LWT Food Sci Technol. 2016;66:428–35.Google Scholar
  53. 53.
    Tomaro-Duchesneau C, Jones ML, Shah D, Jain P, Saha S, Prakash S. Cholesterol assimilation by Lactobacillus probiotic bacteria: an in vitro investigation. BioMed Res Int. 2014;2014:380316. Scholar
  54. 54.
    Dambekodi P, Gilliland S. Incorporation of cholesterol into the cellular membrane of Bifidobacterium longum. J Dairy Sci. 1998;81:1818–24.Google Scholar
  55. 55.
    Anandharaj M, Sivasankari B, Santhanakaruppu R, Manimaran M, Rani RP, Sivakumar S. Determining the probiotic potential of cholesterol-reducing Lactobacillus and Weissella strains isolated from gherkins (fermented cucumber) and South Indian fermented koozh. Res Microbiol. 2015;166:428–39.Google Scholar
  56. 56.
    Choi EA, Chang HC. Cholesterol-lowering effects of a putative probiotic strain Lactobacillus plantarum EM isolated from kimchi. LWT Food Sci Technol. 2015;62:210–7.Google Scholar
  57. 57.
    Lye H-S, Rahmat-Ali GR, Liong M-T. Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. Int Dairy J. 2010;20:169–75.Google Scholar
  58. 58.
    Craig IF, Via DP, Mantulin WW, Pownall HJ, Gotto A, Smith LC. Low density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole fluorophore. J Lipid Res. 1981;22:687–96.Google Scholar
  59. 59.
    Sigma-Aldrich. Anisaldehyde solution. 2017. Accessed 22 Nov 2018.
  60. 60.
    Milz B, Spangenberg B. 2D-thin layer chromatography (2D-TLC) flash test of 17α-ethinylestradiol and related steroids detected by fluorescence densitometry. J Liq Chromatogr Relat Technol. 2013;36:2378–86.Google Scholar
  61. 61.
    Simon RE, Walton LK, Liang Y, Denton MB. Fluorescence quenching high-performance thin-layer chromatographic analysis utilizing a scientifically operated charge-coupled device detector. Analyst. 2001;126:446–50.Google Scholar
  62. 62.
    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Validation of analytical procedures: text and methodology Q2(R1). Geneva: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2005. p. 11–2.Google Scholar
  63. 63.
    Ramasamy K, Abdul Rahman NZ, Sieo CC, Alitheen NJ, Abdullah N, Ho YW. Probiotic potential of lactic acid bacteria from fermented Malaysian food or milk products. Int J Food Sci Technol. 2012;47:2175–83.Google Scholar
  64. 64.
    Hahn-Deinstrop E. Applied thin-layer chromatography: best practice and avoidance of mistakes. 2nd ed. Weinheim: Wiley-VCH; 2007.Google Scholar
  65. 65.
    Shabir GA. Step-by-step analytical methods validation and protocol in the quality system compliance industry. J Valid Technol. 2005;10:314–25.Google Scholar
  66. 66.
    Cayman Chemical. Cholesterol uptake cell-based Aassay kit. 2018. Accessed 29 Aug 2018.
  67. 67.
    Ridlon JM, Harris SC, Bhowmik S, Kang D-J, Hylemon PB. Consequences of bile salt biotransformations by intestinal bacteria. Gut Microbes. 2016;7:22–39.Google Scholar
  68. 68.
    Ridlon JM, Kang D-J, Hylemon PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res. 2006;47:241–59.Google Scholar
  69. 69.
    Zhang R, He L, Zhang L, Li C, Zhu Q. Screening of cholesterol-lowering Bifidobacterium from Guizhou Xiang pigs, and evaluation of its tolerance to oxygen, acid, and bile. Korean J Food Sci Anim Resour. 2016;36:37–43.Google Scholar
  70. 70.
    Anila K, Kunzes A, Bhalla T. In vitro cholesterol assimilation and functional enzymatic activities of putative probiotic Lactobacillus sp. isolated from fermented foods/beverages of north west India. J Nutr Food Sci. 2016;6:467. Scholar
  71. 71.
    Shehata M, El Sohaimy S, El-Sahn MA, Youssef M. Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Ann Agric Sci. 2016;61:65–75.Google Scholar
  72. 72.
    Ilavenil S, Vijayakumar M, Kim DH, Valan Arasu M, Park HS, Ravikumar S, et al. Assessment of probiotic, antifungal and cholesterol lowering properties of Pediococcus pentosaceus KCC-23 isolated from Italian ryegrass. J Sci Food Agric. 2016;96:593–601.Google Scholar
  73. 73.
    Lim FT, Lim SM, Ramasamy K. Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 assimilate cholesterol and modulate ABCA1, CD36, NPC1L1 and SCARB1 in vitro. Benef Microbes. 2017;8:97–109. Scholar
  74. 74.
    Pereira DI, McCartney AL, Gibson GR. An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties. Appl Environ Microbiol. 2003;69:4743–52.Google Scholar
  75. 75.
    Lye H-S, Rusul G, Liong M-T. Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol. J Dairy Sci. 2010;93:1383–92.Google Scholar
  76. 76.
    Begley M, Hill C, Gahan CG. Bile salt hydrolase activity in probiotics. Appl Environ Microbiol. 2006;72:1729–38.Google Scholar
  77. 77.
    Anandharaj M, Sivasankari B. Isolation of potential probiotic Lactobacillus oris HMI68 from mother's milk with cholesterol-reducing property. J Biosci Bioeng. 2014;118:153–9.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Rohawi Nur Syakila
    • 1
    • 2
  • Siong Meng Lim
    • 1
    • 2
  • Snezana Agatonovic-Kustrin
    • 3
    • 4
  • Fei Tieng Lim
    • 2
  • Kalavathy Ramasamy
    • 1
    • 2
    Email author
  1. 1.Faculty of PharmacyUniversity Teknologi MARA (UiTM)Bandar Puncak AlamMalaysia
  2. 2.Collaborative Drug Discovery Research (CDDR) Group, Pharmaceutical and Life Sciences Community of ResearchUniversiti Teknologi MARA (UiTM)Shah AlamMalaysia
  3. 3.Department of Pharmaceutical and Toxicological Chemistry named after Arzamastsev of the Institute of PharmacyI.M. Sechenov First Moscow State Medical University (Sechenov University)MoscowRussia
  4. 4.School of Pharmacy and Applied Science, La Trobe Institute of Molecular SciencesLa Trobe UniversityBendigoAustralia

Personalised recommendations