Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt Microbial cell encapsulation as a strategy for the maintenance of stock cultures Joseane C. Bassani a , Vidiany A. Queiroz Santos a , Aneli M. Barbosa-Dekker b , Robert F.H. Dekker c , Mário Antônio A. da Cunha a,* , Edimir A. Pereira a,* a Departamento de Química, Universidade Tecnológica Federal do Paraná, Via do Conhecimento, km 01, CEP, 85503-390, Pato Branco, PR, Brazil b Departamento de Química, Universidade Estadual de Londrina, CP 10.011, CEP, 86057-970, Londrina, PR, Brazil c Programa de Pós-Graduação em Engenharia Ambiental, Universidade Tecnológica Federal do Paraná, Câmpus Londrina, CEP, 86036-370, Londrina, PR, Brazil ARTICLE INFO Keywords: Calcium alginate Cell entrapment Culture preservation E. coli E. aerogenes ABSTRACT A cellular immobilization technique was developed as a new proposal for the maintenance and preservation of microbial cultures. Cells of enterobacteria, Escherichia coli (ATCC 25922) and Enterobacter aerogenes (ATCC 13048) were entrapped in calcium alginate beads, which were subjected to different storage conditions. Non- dehydrated beads crude devoid of bacterial cells were kept under refrigeration at 5 °C, or frozen at -60 °C, and freeze-dried beads were stored at -18 °C. The viability and biochemical stability of the entrapped cells during storage time was assessed by the Petrifilm™ procedure and VITEK 2 system. Morphological aspects of the beads were evaluated by optical and scanning electron microscopy. Cell entrapment within alginate beads was a promising technique for maintaining E. coli and E. aerogenes stock cultures. E. aerogenes cells entrapped within calcium alginate beads and maintained under refrigeration or frozen at -18 °C were viable for 240 days, and maintained their biochemical characteristics. Encapsulated E. aerogenes cells maintained at -60 °C remained viable for up to 150 days. The viability of the E. coli cells was maintained throughout the storage period under all storage conditions studied, but this was accompanied by a loss of synthesis capacity of the enzymes, β-ga- lactosidase and β-glucuronidase. 1. Introduction Advances in microbiology have contributed to the bioprospecting of different microorganisms of industrial interest, and for the proper ex- ploitation of the microbial potentialities it is necessary that the cultures be available, be easy to transport and maintain their biochemical characteristics and cellular viability. Microbial preservation aims to maintain the viability of microbial cultures without incurring any morphological, physiological or genetic changes, thus providing cell stability. In this context, studies are needed to avoid genetic mutation, loss of biochemical characteristics, or cell viability during storage. Several methods have been developed to pre- serve microbial cultures, but no method is adequate to preserve all microorganisms. The most appropriate method for preserving a mi- crobial culture should be determined experimentally, taking into ac- count the characteristics and specificities of each microorganism. The most widely used techniques for microbial preservation are cryopre- servation and lyophilization, but they are not applicable to all microorganisms and can have some limitations (Alonso, 2016). Cell immobilization techniques have been extensively studied in bioprocesses to increase cell effectiveness by protecting cells against adverse conditions of the medium or cultivation conditions. In addition, probiotic microorganisms are immobilized on different supports aimed at the prolongation of cell viability (Gul & Dervisoglu, 2016; Mitropoulou, Nedovic, Goyal, & Kourkoutas, 2013). Among the different methods of cell immobilization, entrapment in porous matrices such as calcium alginate gel has been proposed to obtain cells with high activity and stability for use in bioprocesses. Cell entrapment in alginate gel is a widespread technique because alginate gelation takes place immediately with no changes in temperature, pH, and osmotic pressure, thus allowing the preservation and viability of microorganisms (Chalfoun, 2010). The use of alginate as a matrix for the encapsulation of microbial strains has been shown to be promising because this material, besides being non-toxic, protects the microbial cell against stressful conditions (sudden fluctuations in temperature, humidity and pressure) common https://doi.org/10.1016/j.lwt.2018.12.058 Received 25 July 2018; Received in revised form 13 December 2018; Accepted 18 December 2018 * Corresponding authors. Permanent Address: Departamento de Química, Universidade Tecnológica Federal do Paraná, Via do Conhecimento, km 01, Bairro Fraron, CEP, 85503-390, Pato Branco, PR, Brazil. E-mail addresses: mcunha@utfpr.edu.br, mario.utfpr@gmail.com (M.A.A. da Cunha), edimir@utfpr.edu.br (E.A. Pereira). LWT - Food Science and Technology 102 (2019) 411–417 Available online 21 December 2018 0023-6438/ © 2018 Elsevier Ltd. All rights reserved. T