Skip to main content

Microencapsulation of phages to analyze their demeanor in physiological conditions

Folia Microbiologica volume 64, pages 751–763 (2019)Cite this article

Abstract

Nowadays, phage therapy emerges as one of the alternative solutions to the problems arising from antibiotic resistance in pathogenic bacteria. Although phage therapy has been successfully applied both in vitro and in vivo, one of the biggest concerns in this regard is the stability of phages in body environment. Within the scope of this study, microencapsulation technology was used to increase the resistance of phages to physiological conditions, and the resulting microcapsules were tested in environments simulating body conditions. For this purpose, Bacillus subtilis, Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), and Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) phages were isolated from different sources and then microencapsulated with 1.33% (w/v) sodium alginate using a spray dryer to minimize the damage of physiological environment. Stability of microcapsules in simulated gastric fluid and bile salt presence was tested. As a consequence, the maximum titer decrease of microencapsulated phages after 2-h incubation was found to be 2.29 log unit for B. subtilis phages, 1.71 log unit for S. Enteritidis phages, and 0.60 log unit for S. Typhimurium phages, while free phages lost their viability even after a 15-min incubation. Similarly, microencapsulation was found to increase the stability of phages in the bile salt medium and it was seen that after 3 h of incubation, the difference between the titers of microencapsulated phages and free phages could reach up to 3 log unit.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  • Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM (2014) Phage treatment of human infections. Bacteriophage 1 (2):66–85

    Article  Google Scholar 

  • Acar-Soykut E (2007) Identification and classification of virulent bacteriophages of Streptococcus thermophilus and Lactobacillus bulgaricus based on their replication parameters, capsid protein profiles and restriction endonuclease analysis. Dissertation, Ankara University

  • Allouche R, Dupont S, Charriau A, Gervais P, Beney L, Chambin O (2018) Optimized tableting for extremely oxygen-sensitive probiotics using direct compression. Int J Pharm 538:14–20

    CAS  Article  Google Scholar 

  • Ameri M, Maa Y-F (2006) Spray drying of biopharmaceuticals: stability and process considerations. Dry Technol 24:763–768

    CAS  Article  Google Scholar 

  • Bigot B, Lee W-J, McIntyre L, Wilson T, Hudson JA, Billington C, Heinemann JA (2011) Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage. Food Microbiol 28:1448–1452. https://doi.org/10.1016/j.fm.2011.07.001

    CAS  Article  PubMed  Google Scholar 

  • Bowey KE (2009) Alginate microparticles produced by spray drying for oral insulin delivery.Dissertation, Queen’s University

  • Chan ES, Zhang Z (2002) Encapsulation of probiotic bacteria Lactobacillus acidophilus by direct compression. Food Bioprod Process 80:78–82

    CAS  Article  Google Scholar 

  • Chibani-Chennoufi S, Sidoti J, Bruttin A, Kutter E, Sarker S, Brussow H (2004) In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy. Antimicrob Agents Chemother 48:2558–2569. https://doi.org/10.1128/AAC.48.7.2558-2569.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Choińska-Pulit A, Mituła P, Śliwka P, Łaba W, Skaradzińska A (2015) Bacteriophage encapsulation: trends and potential applications. Trends Food Sci Technol 45:212–221. https://doi.org/10.1016/j.tifs.2015.07.001

    CAS  Article  Google Scholar 

  • Colom J, Cano-Sarabia M, Otero J, et al (2015) Liposome-encapsulated bacteriophages for enhanced oral phage therapy against Salmonella. Appl Environ Microbiol AEM-00812

  • Colom J, Cano-Sarabia M, Otero J, Aríñez-Soriano J, Cortés P, Maspoch D, Llagostera M (2017) Microencapsulation with alginate/CaCO3: a strategy for improved phage therapy. Sci Rep 7:41441. https://doi.org/10.1038/srep41441

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Dini C, Islan GA, de Urraza PJ, Castro GR (2012) Novel biopolymer matrices for microencapsulation of phages: enhanced protection against acidity and protease activity. Macromol Biosci 12:1200–1208. https://doi.org/10.1002/mabi.201200109

    CAS  Article  PubMed  Google Scholar 

  • Dubey R, Shami TC, Bhasker Rao KU (2009) 2009 Microencapsulation technology and applications. Def Sci J 59:82–95. https://doi.org/10.14429/dsj.59.1489

    CAS  Article  Google Scholar 

  • Endo EH, Ueda-Nakamura T, Nakamura CV, Filho BPD (2012) Activity of spray-dried microparticles containing pomegranate peel extract against Candida albicans. Molecules 17:10094–10107. https://doi.org/10.3390/molecules170910094

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Fischetti VA (2008) Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol 11:393–400. https://doi.org/10.1016/j.mib.2008.09.012

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Joerger RD (2003) Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poult Sci 82:640–647

    CAS  Article  Google Scholar 

  • Jończyk E, Kłak M, Międzybrodzki R, Górski A (2011) The influence of external factors on bacteriophages. Folia Microbiol (Praha) 56:191–200

    Article  Google Scholar 

  • Kim S, Jo A, Ahn J (2015) Application of chitosan–alginate microspheres for the sustained release of bacteriophage in simulated gastrointestinal conditions. Int J Food Sci Technol 50:913–918

    CAS  Article  Google Scholar 

  • Koo J, DePaola A, Marshall DL (2000) Effect of simulated gastric fluid and bile on survival of Vibrio vulnificus and Vibrio vulnificus phage. J Food Prot 63:1665–1669

    CAS  Article  Google Scholar 

  • Kutateladze M, Adamia R (2010) Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol 28:591–595. https://doi.org/10.1016/j.tibtech.2010.08.001

    CAS  Article  PubMed  Google Scholar 

  • Leung SSY, Parumasivam T, Gao FG, Carrigy NB, Vehring R, Finlay WH, Morales S, Britton WJ, Kutter E, Chan HK (2016) Production of inhalation phage powders using spray freeze drying and spray drying techniques for treatment of respiratory infections. Pharm Res 33:1486–1496. https://doi.org/10.1007/s11095-016-1892-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Leung SSY, Parumasivam T, Gao FG, Carter EA, Carrigy NB, Vehring R, Finlay WH, Morales S, Britton WJ, Kutter E, Chan HK (2017) Effects of storage conditions on the stability of spray dried, inhalable bacteriophage powders. Int J Pharm 521:141–149

    CAS  Article  Google Scholar 

  • Leung SSY, Parumasivam T, Nguyen A, Gengenbach T, Carter EA, Carrigy NB, Wang H, Vehring R, Finlay WH, Morales S, Britton WJ, Kutter E, Chan HK (2018) Effect of storage temperature on the stability of spray dried bacteriophage powders. Eur J Pharm Biopharm 127:213–222

    CAS  Article  Google Scholar 

  • Ma Y, Pacan JC, Wang Q, Xu Y, Huang X, Korenevsky A, Sabour PM (2008) Microencapsulation of bacteriophage Felix O1 into chitosan-alginate microspheres for oral delivery. Appl Environ Microbiol 74:4799–4805

    CAS  Article  Google Scholar 

  • Ma Y, Pacan JC, Wang Q, Sabour PM, Huang X, Xu Y (2012) Enhanced alginate microspheres as means of oral delivery of bacteriophage for reducing Staphylococcus aureus intestinal carriage. Food Hydrocoll 26:434–440

    CAS  Article  Google Scholar 

  • Matinkhoo S, Lynch KH, Dennis JJ, Finlay WH, Vehring R (2011) Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections. J Pharm Sci 100:5197–5205. https://doi.org/10.1002/jps.22715

    CAS  Article  PubMed  Google Scholar 

  • Mirzaei MK, Nilsson AS (2015) Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. PLoS One 10:e0118557

    Article  Google Scholar 

  • Mohan S (2012) Compression physics of pharmaceutical powders: a review. Int J Pharm Sci Res 3:1580

    CAS  Google Scholar 

  • Nobrega FL, Costa AR, Santos JF, Siliakus MF, van Lent JWM, Kengen SWM, Azeredo J, Kluskens LD (2016) Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine. Sci Rep 6:39235. https://doi.org/10.1038/srep39235

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Poshadri a KA (2010) Microencapsulation technology: a review. J Res ANGRAU 38:86–102

    Google Scholar 

  • Ramirez K, Cazarez-Montoya C, Lopez-Moreno HS, Castro-del Campo N (2018) Bacteriophage cocktail for biocontrol of Escherichia coli O157: H7: stability and potential allergenicity study. PLoS One 13:e0195023

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold spring harbor laboratory press

  • Shoyele SA, Cawthorne S (2006) Particle engineering techniques for inhaled biopharmaceuticals. Adv Drug Deliv Rev 58:1009–1029

    CAS  Article  Google Scholar 

  • Soto MJ, Retamales J, Palza H, Bastías R (2018) Encapsulation of specific Salmonella Enteritidis phage f3αSE on alginate-spheres as a method for protection and dosification. Electron J Biotechnol 31:57–60

    CAS  Article  Google Scholar 

  • Szekalska M, Amelian A, Winnicka K (2015) Alginate microspheres obtained by the spray drying technique as mucoadhesive carriers of ranitidine. Acta Pharma 65:15–27

    CAS  Article  Google Scholar 

  • Tayyarcan EK (2017) Biocontrol of foodborne pathogenic bacteria by using the combination of phage therapy and phytotherapy. Dissertation, Hacettepe University

  • Vandenheuvel D, Singh A, Vandersteegen K, Klumpp J, Lavigne R, van den Mooter G (2013) Feasibility of spray drying bacteriophages into respirable powders to combat pulmonary bacterial infections. Eur J Pharm Biopharm 84:578–582. https://doi.org/10.1016/j.ejpb.2012.12.022

    CAS  Article  PubMed  Google Scholar 

  • Vandenheuvel D, Meeus J, Lavigne R, Van Den Mooter G (2014) Instability of bacteriophages in spray-dried trehalose powders is caused by crystallization of the matrix. Int J Pharm 472:202–205. https://doi.org/10.1016/j.ijpharm.2014.06.026

    CAS  Article  PubMed  Google Scholar 

  • Vinner GK, Vladisavljević GT, Clokie MRJ, Malik DJ (2017) Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release. PLoS One 12:e0186239

    Article  Google Scholar 

  • Walbeck AK (2013) Methods for drying bacteriophage and bacteriophage-containing compositions, the resulting dry compositions, and methods of use

Download references

Funding

This study was financially supported by The Scientific and Technological Research Council of Turkey (Project No. 114Z680).

Author information

Affiliations

  1. Yeniçağa Yaşar Çelik Vocational School, Bolu Abant İzzet Baysal University, Bolu, Turkey

    Esra Acar Soykut

  2. Food Engineering Department, Hacettepe University, Beytepe, Ankara, Turkey

    Emine Kübra Tayyarcan, Şefika Evran & İsmail Hakkı Boyacı

  3. Food Engineering Department, Bolu Abant İzzet Baysal University, Bolu, Turkey

    İbrahim Çakır

  4. National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Zone Urbaine Nord, 1080, Tunis, Tunisia

    Maha Khaaladi & Sami Fattouch

Authors
  1. Esra Acar Soykut
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emine Kübra Tayyarcan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Şefika Evran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. İsmail Hakkı Boyacı
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. İbrahim Çakır
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maha Khaaladi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sami Fattouch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to İsmail Hakkı Boyacı.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Acar Soykut, E., Tayyarcan, E.K., Evran, Ş. et al. Microencapsulation of phages to analyze their demeanor in physiological conditions. Folia Microbiol 64, 751–763 (2019). https://doi.org/10.1007/s12223-019-00688-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12223-019-00688-1