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Development of sensitive and specific multiplex PCR method for the detection of microcystin producing cyanobacteria in spirulina food supplements

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Abstract

Spirulina has emerged as the next-generation dietary supplement owing to its health benefits. Despite the advantages, there have been reports of contamination by cyanotoxins such as microcystins that can adversely affect human health. Hence, there is a need to develop a robust, efficient, and cost-effective method to detect microcystin-producing cyanobacteria in these food supplements. In this study, we have demonstrated a multiplex polymerase chain reaction (PCR) method for identification of microcystin-contamination in spirulina dietary supplements. This method involves simultaneous amplification of phycocyanin and microcystin B encoding genes (pcb, mcyB). The sensitivity of the multiplex PCR was assessed, and the limit of detecting mcyB along with pcb was found to be 250 fg/µL. The presence of microcystin was detected in five out of seven fish food supplements indicating poor culture conditions. Hence, rigorous quality control is required for monitoring the spirulina food supplements.

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References

  • Akao Y, Ebihara T, Masuda H, Saeki Y, Akazawa T, Hazeki K, Hazeki O, Matsumoto M, Seya T. Enhancement of antitumor natural killer cell activation by orally administered spirulina extract in mice. Cancer Sci. 100: 1494–1501 (2009)

    Article  CAS  PubMed  Google Scholar 

  • Barón-Sola A, Ouahid Y, del Campo FF. Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions. Ecotoxicol. Environ. Saf. 75(1): 102–108 (2012)

    Article  CAS  Google Scholar 

  • Belay A, Ota Y, Miyakawa K, Shimamatsu H. Current knowledge on potential health benefits of spirulina. J. Appl. Phycol. 5: 235–241 (1993)

    Article  Google Scholar 

  • Bittencourt-Oliveira M. Detection of potential microcystin-producing cyanobacteria in brazilian reservoirs with a mcyb molecular marker. Harmful Algae 2(1): 51–60 (2003)

    Article  Google Scholar 

  • Bláha L, Babica P, Maršálek B. Toxins produced in cyanobacterial water blooms—toxicity and risks. Interdiscip. Toxicol. 2: 36–41 (2009)

    Article  Google Scholar 

  • Corbel S, Mougin C, Bouaïcha N. Chemosphere cyanobacterial toxins: modes of actions, fate in aquatic and soil ecosystems, phytotoxicity and bioaccumulation in agricultural crops. Chemosphere 96: 1–15 (2014)

    Article  CAS  PubMed  Google Scholar 

  • Douma M, Ouahid Y, Loudiki M, del Campo FF, Oudra B. The first detection of potentially toxic microcystis strains in two middle Atlas mountains natural lakes (Morocco). Environ. Monit. Assess. 189(1): 39 (2017)

    Article  CAS  PubMed  Google Scholar 

  • Gilroy DJ, Kauffman KW, Hall RA, Huang X, Chu FS. Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements. Environ. Health Perspect. 108: 435–439 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grobbelaar JU. Quality control and assurance: crucial for the sustainability of the applied phycology industry. J. Appl. Phycol. 15: 209–215 (2003)

    Article  CAS  Google Scholar 

  • Heussner AH, Mazija L, Fastner J, Dietrich DR. Toxin content and cytotoxicity of algal dietary supplements. Toxicol. Appl. Pharmacol. 265: 263–271 (2012)

    Article  CAS  PubMed  Google Scholar 

  • Jiang Y, Xie P, Chen J, Liang G. Detection of the hepatotoxic microcystins in 36 kinds of cyanobacteria spirulina food products in china. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 25: 885–894 (2008)

    Article  CAS  PubMed  Google Scholar 

  • Joventino IP, Alves HG, Neves LC, Pinheiro-Joventino F, Leal LK, Neves SA, Ferreira FV, Brito GA, Viana GB. The microalga Spirulina platensis presents anti-inflammatory action as well as hypoglycemic and hypolipidemic properties in diabetic rats. J. Complement. Integr. Med. 9: 1 (2012)

    Article  Google Scholar 

  • Karkos PD, Leong SC, Karkos CD, Sivaji N, Assimakopoulos DA. Spirulina in clinical practice: evidence-based human applications. Evid. Based Complement. Alternat. Med. 2011: 4 (2011)

    Article  Google Scholar 

  • Kulshreshtha A, Zacharia AJ, Jarouliya U, Bhadauriya P, Prasad GB, Bisen PS. Spirulina in health care management. Curr. Pharm. Biotechnol. 9: 400–405 (2010)

    Article  Google Scholar 

  • Manali KM, Arunraj R, Kumar T, Ramya M. Detection of microcystin producing cyanobacteria in spirulina dietary supplements using multiplex HRM quantitative PCR. J. Appl. Phycol. 29: 1279–1286 (2017)

    Article  CAS  Google Scholar 

  • Mathew B, Sankaranarayanan R, Nair PP, Varghese C, Somanathan T, Amma BP, Amma NS, Nair MK. Evaluation of chemoprevention of oral cancer with Spirulina fusiformis. Nutr. Cancer. 24: 197–202 (1995)

    Article  CAS  PubMed  Google Scholar 

  • Mazokopakis EE, Papadomanolaki MG, Fousteris AA, Kotsiris DA, Lampadakis IM, Ganotakis ES. The hepatoprotective and hypolipidemic effects of spirulina (Arthrospira platensis) supplementation in a Cretan population with non-alcoholic fatty liver disease: a prospective pilot study. Ann. Gastroenterol. 27: 387–394 (2014)

    PubMed  PubMed Central  Google Scholar 

  • Parikh P, Mani U, Iyer U. Role of spirulina in the control of glycemia and lipidemia in type 2 diabetes mellitus. J. Med. Food. 4: 193–199 (2001)

    Article  PubMed  Google Scholar 

  • Park JH, Lee SI, Kim IH. Effect of dietary Spirulina (Arthrospira) platensis on the growth performance, antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta noxious gas emission, and breast meat quality of broiler chickens. Poult. Sci. 97: 2451–2459 (2018)

    Article  CAS  PubMed  Google Scholar 

  • Parker CH, Stutts WL, Degrasse SL. Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of microcystins in blue-green algal dietary supplements. J. Agric. Food Chem. 63: 10303–10312 (2015)

    Article  CAS  PubMed  Google Scholar 

  • Pekar H, Westerberg E, Bruno O, Lääne A, Persson KM, Sundström FL, Thim A. Fast, rugged and sensitive ultra-high pressure liquid chromatography tandem mass spectrometry method for analysis of cyanotoxins in raw water and drinking water—first findings of anatoxins, cylindrospermopsins and microcystin variants in Swedish source waters and infiltration pond. J. Chromatogr. A. 1429: 265–276 (2016)

    Article  CAS  PubMed  Google Scholar 

  • Preece EP, Moore BC, Swanson ME, Hardy FJ. Identifying best methods for routine ELISA detection of microcystin in seafood. Environ. Monit. Assess. 187: 12 (2015)

    Article  CAS  PubMed  Google Scholar 

  • Ramakrishnan GS, Aliya FA, Ramya M. A rapid and efficient DNA extraction method suitable for marine macroalgae. 3 Biotech. 7(6): 364 (2017)

    Google Scholar 

  • Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111: 1–61 (1979)

    Google Scholar 

  • Saker ML, Welker M, Vasconcelos VM. Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption. Appl. Microbiol. Biotechnol. 73: 1136–1142 (2007)

    Article  CAS  PubMed  Google Scholar 

  • Samuels R, Mani UV, Iyer UM, Nayak US. Hypocholesterolemic effect of spirulina in patients with hyperlipidemic nephrotic syndrome. J. Med. Food. 5: 91–96 (2002)

    Article  CAS  PubMed  Google Scholar 

  • Sangolkar LN, Maske SS, Muthal PL, Kashyap SM, Chakrabarti T. Isolation and characterization of microcystin producing microcystis from a central Indian water bloom. Harmful Algae 8: 674–684 (2009)

    Article  CAS  Google Scholar 

  • Soares RM, Magalhaes VF, Azevedo S. Accumulation and depuration of microcystins (cyanobacteria hepatotoxins) in Tilapia rendalli (Cichlidae) under laboratory conditions. Aquat. Toxicol. 70(1): 1–10 (2004)

    Article  CAS  PubMed  Google Scholar 

  • Sorichetti RJ, McLaughlin JT, Creed IF, Trick CG. Suitability of a cytotoxicity assay for detection of potentially harmful compounds produced by freshwater bloom-forming algae. Harmful Algae 31: 177–187 (2014)

    Article  CAS  PubMed  Google Scholar 

  • Valerio E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R. Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples. Environ. Toxicol. 25(3): 251–260 (2010)

    Article  CAS  PubMed  Google Scholar 

  • Vallone PM, Butler JM. Autodimer: a screening tool for primer-dimer and hairpin structures. BioTechniques. 37: 226–231 (2004)

    Article  CAS  PubMed  Google Scholar 

  • Vichi S, Lavorini P, Funari E, Scardala S, Testai E. Contamination by microcystis and microcystins of blue-green algae food supplements (BGAS) on the Italian market and possible risk for the exposed population. Food Chem. Toxicol. 50: 4493–4499 (2012)

    Article  CAS  PubMed  Google Scholar 

  • Yakes BJ, Handy SM, Kanyuck KM, DeGrasse SL. Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor. Harmful Algae 47: 9–16 (2015)

    Article  CAS  Google Scholar 

  • Yu P, Li JR, Cen PL. Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis. J. Appl. Phycol. 14: 307–310 (2002)

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to acknowledge the support and facility given by SRM University, Tamil Nadu, India, in carrying out this project. Kamath Mukund Manali acknowledges GATE fellowship supported by SRM Institute of Science and Technology for execution of the project.

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Authors and Affiliations

  1. Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankkulathur, Kancheepuram District, Tamil Nadu, 603203, India

    Kamath Mukund Manali, Rex Arunraj, Gautham Subramaniam Ramakrishnan & Mohandass Ramya

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  1. Kamath Mukund Manali
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  2. Rex Arunraj
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  3. Gautham Subramaniam Ramakrishnan
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  4. Mohandass Ramya
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Correspondence to Mohandass Ramya.

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Manali, K.M., Arunraj, R., Ramakrishnan, G.S. et al. Development of sensitive and specific multiplex PCR method for the detection of microcystin producing cyanobacteria in spirulina food supplements. Food Sci Biotechnol 28, 609–614 (2019). https://doi.org/10.1007/s10068-018-0476-0

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  • DOI: https://doi.org/10.1007/s10068-018-0476-0

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