Microencapsulation of Microbial Transglutaminase by Ultrasonic Spray-Freeze Drying

  • Hilal IslerogluEmail author
  • Izzet Turker
  • Banu Koc
  • Mehmet Tokatli
Original Paper


Microencapsulation of partially purified microbial transglutaminase (mTG) was investigated using ultrasonic spray-freeze drying (USFD), and the optimum coating materials (gum arabic, maltodextrin, inulin) ratio and the process parameters (flow rate and nozzle frequency) were determined using a D-optimal combined design. Also, the microencapsulated samples by USFD were compared with microencapsulated samples by conventional freeze drying (CFD) and conventional spray drying (CSD) in terms of microencapsulation efficiency, enzyme stability at extreme pH and high temperature conditions, and the presence of metal ions, physical (moisture content, particle morphology, particle and pore size, surface area, pore volume distribution, density and flow properties, caking degree, color), and reconstitution (wettability and solubility) properties. As a result, the optimum coating materials composition was determined as 60% gum arabic and 40% inulin, and process conditions were found to be flow rate of 6.83 ml/min and nozzle frequency of 48 kHz applying desirability function method. Microcapsules with smaller particle size, pore volume, and porosity, with lower moisture content and good reconstitution characteristics, were obtained by USFD with a maximum microencapsulation efficiency of ~ 97%.


Ultrasonic spray-freeze drying Transglutaminase Inulin Particle morphology Pore volume distribution 


Funding Information

This study was financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project Number: 115O216) and Tokat Gaziosmanpasa University Scientific Research Projects (Project Number: 2016/62).


  1. Aghbashlo, M., Mobli, H., Madadlou, A., & Rafiee, S. (2013). Influence of wall material and inlet drying air temperature on the microencapsulation of fish oil by spray drying. Food and Bioprocess Technology, 6(6), 1561–1569.CrossRefGoogle Scholar
  2. Allison, S. D., Chang, B., Randolph, T. W., & Carpenter, J. F. (1999). Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Archives of Biochemistry and Biophysics, 365(2), 289–298.PubMedCrossRefGoogle Scholar
  3. Amid, M., Manap, Y., & Zohdi, K. N. (2014). Microencapsulation of purified amylase enzyme from pitaya (hylocereus polyrhizus) peel in arabic gum-chitosan using freeze drying. Molecules, 19, 3731–3743.PubMedPubMedCentralCrossRefGoogle Scholar
  4. Amine, K. M., Champagne, C. P., Salmeri, S., Britten, M., St-Gelais, D., Fustier, P., & Lacroix, M. (2014). Effect of palmitoylated alginate microencapsulation on viability of Bifidobacterium longum during freeze-drying. LWT - Food Science and Technology, 56, 111–117.CrossRefGoogle Scholar
  5. Anchordoquy, T. J., Izutsu, K. I., Randolph, T. W., & Carpenter, J. F. (2001). Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze–drying. Archives of Biochemistry and Biophysics, 390(1), 35–41.PubMedCrossRefGoogle Scholar
  6. AOAC. (1990). Official methods for analysis (15th ed.). Arlington: Association of Official Analytical Chemists.Google Scholar
  7. Bourneow, C., Benjakul, S., & H-Kittikun, A. (2012). Impact of some additives on the stability of microbial transglutaminase from Providencia sp. C1112. Asian Journal of Food and Agro-Industry, 5(3), 226–233.Google Scholar
  8. Breda, M., Vitolo, M., Duranti, M. A., & Pitombo, R. N. (1992). Effect of freezing-thawing on invertase activity. Cryobiology, 29(2), 281–290.CrossRefGoogle Scholar
  9. Cano-Chauca, M., Stringheta, P. C., Ramos, A. M., & Cal-Vidal, J. (2005). Effect of the carriers on the microstructure of mango powder obtained by spray drying and its functional characterization. Innovative Food Science and Emerging Technologies, 6(4), 420–428.CrossRefGoogle Scholar
  10. Carpenter, J. F., & Crowe, J. H. (1989). An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry, 28(9), 3916–3922.PubMedCrossRefGoogle Scholar
  11. Carr, R. L. (1965). Evaluating flow properties of solids. Chemical Engineering Journal, 72, 163–168.Google Scholar
  12. Constantino, H. R., Firouzabadian, L., Wu, C., Carrasquillo, K. G., Griebenow, K., Zale, S. E., & Tracy, M. A. (2000). Protein spray freeze drying: effect of formulation variables on particle size and stability. Journal of Pharmaceutical Science, 17(11), 1374–1383.Google Scholar
  13. D’Addio, S. M., Chan, J. G. Y., Kwok, P. C. L., Prud’homme, R. K., & Chan, H. K. (2012). Constant size, variable density aerosol particles by ultrasonic spray freeze drying. International Journal of Pharmaceutics, 427(2), 185–191.PubMedCrossRefGoogle Scholar
  14. De Boer, J. H. (1958). The structure and properties of porous materials. In Proceedings of the Tenth Symposium of the Colston Research Society Held in the University of Bristol (pp. 68–94). London: Butterworths.Google Scholar
  15. Desai, K. G. H., & Park, H. J. (2005). Recent developments in microencapsulation of food ingredients. Drying Technology, 23, 1361–1394.CrossRefGoogle Scholar
  16. Dima, C., Pătraşcu, L., Cantaragiu, A., Alexe, P., & Dima, Ş. (2016). The kinetics of the swelling process and the release mechanisms of Coriandrum sativum L. essential oil from chitosan/alginate/inulin microcapsules. Food Chemistry, 195, 39–48.PubMedCrossRefGoogle Scholar
  17. Dolly, P., Anishaparvin, A., Joseph, G. S., & Anandharamakrishnan, C. (2011). Microencapsulation of Lactobacillus plantarum (MTCC 5422) by spray-freeze-drying method and evaluation of survival in simulated gastrointestinal conditions. Journal of Microencapsulation, 28(6), 568–574.PubMedCrossRefGoogle Scholar
  18. Eriksson, H. J. C., Hinrichs, W. L. J., van Veen, B., Somsen, G. W., de Jong, G. J., & Frijlink, H. W. (2002). Investigations into the stabilisation of drugs by sugar glasses: I. Tablets prepared from stabilised alkaline phosphatase. International Journal of Pharmaceutics, 249(1), 59–70.PubMedCrossRefGoogle Scholar
  19. Ezhilarasi, P. N., Karthik, P., Chhanwal, N., & Anandharamakrishnan, C. (2013). Nanoencapsulation techniques for food bioactive components: a review. Food and Bioprocess Technology, 6(3), 628–647.CrossRefGoogle Scholar
  20. Feng, H., Barbosa-Cánovas, G. V., & Weiss, J. (2011). Ultrasound technologies for food and bioprocessing. New York: Springer Science and Business Media, 599p.CrossRefGoogle Scholar
  21. Furlán, L. T. R., Lecot, J., Padilla, A. P., Campderrós, M. E., & Zaritzky, N. (2012). Stabilizing effect of saccharides on bovine plasma protein: A calorimetric study. Meat Science, 91(4), 478–485.CrossRefGoogle Scholar
  22. Gaspar, A. L. C., & de Góes-Favoni, S. P. (2015). Action of microbial transglutaminase (MTGase) in the modification of food proteins: A review. Food Chemistry, 171, 315–322.PubMedCrossRefGoogle Scholar
  23. Grasmeijer, N., Stankovic, M., de Waard, H., Frijlink, H. W., & Hinrichs, W. L. (2013). Unraveling protein stabilization mechanisms: vitrification and water replacement in a glass transition temperature controlled system. BBA Proteins and Proteomics, 1834(4), 763–769.PubMedCrossRefGoogle Scholar
  24. Gregg, S. J., & Sing, K. S. W. (1982). Adsorption, surface area and porosity (2nd ed.). London: Academic.Google Scholar
  25. Hausner, H. H. (1967). Friction conditions in a mass of metal powder. Powder Metallurgy, 13, 7–13.Google Scholar
  26. Heidebach, T., Först, P., & Kulozik, U. (2009). Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. Journal of Food Engineering, 98, 309–316.CrossRefGoogle Scholar
  27. Heinrich, Z. (2003). Colour chemistry: syntheses, properties and applications of organic dyes and pigments. Weinheim: Wiley-VCH GmbH & Co. KGaA.Google Scholar
  28. Her, J. Y., Kim, M. S., & Lee, K. G. (2015). Preparation of probiotic powder by the spray freeze-drying method. Journal of Food Engineering, 150, 70–74.CrossRefGoogle Scholar
  29. Hundre, S. Y., Karthik, P., & Anandharamakrishnan, C. (2015). Effect of whey protein isolate and β-cyclodextrin wall systems on stability of microencapsulated vanillin by spray–freeze drying method. Food Chemistry, 174, 16–24.PubMedCrossRefGoogle Scholar
  30. Ishwarya, S. P., & Anandharamakrishnan, C. (2015). Spray-freeze-drying approach for soluble coffee processing and its effect on quality characteristics. Journal of Food Engineering, 149, 171–180.CrossRefGoogle Scholar
  31. Ishwarya, S. P., Anandharamakrishnan, C., & Stapley, A. G. F. (2015). Spray-freeze-drying: a novel process for the drying of foods and bioproducts. Trends in Food Science and Technology, 41(2), 161–181.CrossRefGoogle Scholar
  32. Isleroglu, H., Turker, I., Tokatli, M., & Koc, B. (2018). Ultrasonic spray-freeze drying of partially purified microbial transglutaminase. Food and Bioproducts Processing, 111, 153–164.CrossRefGoogle Scholar
  33. Jinapong, N., Suphantharika, M., & Jamnong, P. (2008). Production of instant soymilk powders by ultrafiltration, spray drying and fluidized bed agglomeration. Journal of Food Engineering, 84(2), 194–205.CrossRefGoogle Scholar
  34. Karthik, P., & Anandharamakrishnan, C. (2013). Microencapsulation of docosahexaenoic acid by spray-freeze drying method and comparison of its stability with spray-drying and freeze-drying methods. Food and Bioprocess Technology, 6, 2780–2790.CrossRefGoogle Scholar
  35. Kumar, R., & Kar, A. (2014). Microencapsulation of nutraceuticals using spray freeze drying method: a brief review. Indo Global Journal of Pharmaceutical Sciences, 4(2), 47–51.Google Scholar
  36. Kurozawa, L. E., Morassi, A. G., Vanzo, A. A., Park, K. J., & Hubinger, M. D. (2009). Influence of spray drying conditions on physicochemical properties of chicken meat powder. Drying Technology, 27(11), 1248–1257.CrossRefGoogle Scholar
  37. Labani, M. M., Rezaee, R., Saeedi, A., & Hinai, A. A. (2013). Evaluation of pore size spectrum of gas shale reservoirs using low pressure nitrogen adsorption, gas expansion and mercury porosimetry: a case study from the Perth and Canning Basins, Western Australia. Journal of Petroleum Science and Engineering, 112, 7–16.CrossRefGoogle Scholar
  38. Maa, Y. F., Nguyen, P. A., Sweeney, T., Shire, S. J., & Hsu, C. C. (1999). Protein inhalation powders: spray drying vs spray freeze drying. Pharmaceutical Research, 16(2), 249–254.PubMedCrossRefGoogle Scholar
  39. MacLeod, C., McKittrick, J., Hindmarsh, J., Johns, M., & Wilson, D. (2006). Fundamentals of spray freezing of instant coffee. Journal of Food Engineering, 74(4), 451–461.CrossRefGoogle Scholar
  40. Motoki, M., & Seguro, K. (1998). Transglutaminase and its use for food processing. Trends in Food Science and Technology, 9(5), 204–210.CrossRefGoogle Scholar
  41. Mujumdar, A. S. (2014). Handbook of Industrial Drying. Boca Raton: CRC Press.CrossRefGoogle Scholar
  42. Nesterenko, A., Alric, I., Violleau, F., Silvestre, F., & Durrieu, V. (2013). A new way of valorizing biomaterials: the use of sunflower protein for α-tocopherol microencapsulation. Food Research International, 53(1), 115–124.CrossRefGoogle Scholar
  43. Nguyen, C. X., Herberger, J. D., & Burke, A. D. (2004). Protein powders for encapsulation: a comparison of spray-freeze drying and spray drying of darbepoetin alfa. Pharmaceutical Research, 21(3), 507–513.PubMedCrossRefGoogle Scholar
  44. Niwa, T., Shimabara, H., & Danjo, K. (2010). Novel spray freeze-drying technique using four-fluid nozzle-development of organic solvent system to expand its application to poorly water soluble drugs. Chemical and Pharmaceutical Bulletin, 58(2), 195–200.PubMedCrossRefGoogle Scholar
  45. Pang, Y., Duan, X., Ren, G., & Liu, W. (2017). Comparative study on different drying methods of fish oil microcapsules. Journal of Food Quality, 2017, 1–7.CrossRefGoogle Scholar
  46. Parthasarathi, S., & Anandharamakrishnan, C. (2016). Enhancement of oral bioavailability of vitamin E by spray-freeze drying of whey protein microcapsules. Food Bioproducts and Processing, 100, 469–476.CrossRefGoogle Scholar
  47. Popović, L. M., Peričin, D. M., Vaštag, Ž. G., & Popović, S. Z. (2013). Optimization of transglutaminase cross-linking of pumpkin oil cake globulin; improvement of the solubility and gelation properties. Food and Bioprocess Technology, 6(4), 1105–1111.CrossRefGoogle Scholar
  48. Rajam, R., & Anandharamakrishnan, C. (2015). Microencapsulation of Lactobacillus plantarum (MTCC 5422) with fructooligosaccharide as wall material by spray drying. LWT-Food Science and Technology, 60(2), 773–780.CrossRefGoogle Scholar
  49. Ramos, A., Raven, N., Sharp, R. J., Bartolucci, S., Rossi, M., Cannio, R., Lebbing, J., Oost, J. V. D., de Vos, W. M., & Santos, H. (1997). Stabilization of enzymes against thermal stress and freeze-drying by mannosylglycerate. Applied and Environmental Microbiology, 63(10), 4020–4025.PubMedPubMedCentralGoogle Scholar
  50. Ratti, C. (2001). Hot air and freeze-drying of high-value foods: a review. Journal of Food Engineering, 49(4), 311–319.CrossRefGoogle Scholar
  51. Semyonov, D., Ramon, O., Kaplun, Z., Levin-Brener, L., Gurevich, N., & Shimoni, E. (2010). Microencapsulation of Lactobacillus paracasei by spray freeze drying. Food Research International, 43(1), 193–202.CrossRefGoogle Scholar
  52. Sonner, C., Maa, Y. F., & Lee, G. (2002). Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability. Journal of Pharmaceutical Sciences, 91(10), 2122–2139.PubMedCrossRefGoogle Scholar
  53. Stapley, A. G. F. (2008). Freeze drying. In J. A. Blackwell (Ed.), Frozen food science technology. Oxford: Evans.Google Scholar
  54. Sun-Waterhouse, D., Wadhwa, S. S., & Waterhouse, G. I. N. (2013). Spray-drying microencapsulation of polyphenol bioactives: a comparative study using different natural fibre polymers as encapsulants. Food and Bioprocess Technology, 6(9), 2376–2388.CrossRefGoogle Scholar
  55. Tonnis, W. F., Amorij, J. P., Vreeman, M. A., Frijlink, H. W., Kersten, G. F., & Hinrichs, W. L. J. (2014). Improved storage stability and immunogenicity of hepatitis B vaccine after spray-freeze drying in presence of sugars. European Journal of Pharmaceutical Sciences, 55, 36–45.PubMedCrossRefGoogle Scholar
  56. Tonnis, W. F., Mensink, M. A., de Jager, A., van der Voort Maarschalk, K., Frijlink, H. W., & Hinrichs, W. L. J. (2015). Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins. Molecular Pharmaceutics, 12(3), 684–694.PubMedCrossRefGoogle Scholar
  57. Turker, I., Domurcuk, G., Tokatli, M., Isleroglu, H., & Koc, B. (2016). Enhancement of microbial transglutaminase production from Streptomyces sp. Ukrainian Food Journal, 5(2), 306–417.CrossRefGoogle Scholar
  58. van Drooge, D. J., Hinrichs, W. L., Dickhoff, B. H., Elli, M. N., Visser, M. R., Zijlstra, G. S., & Frijlink, H. W. (2005). Spray freeze drying to produce a stable Δ9-tetrahydrocannabinol containing inulin-based solid dispersion powder suitable for inhalation. European Journal of Pharmaceutical Sciences, 26(2), 231–240.Google Scholar
  59. Wanning, S., Süverkrüp, R., & Lamprecht, A. (2015). Pharmaceutical spray freeze drying. International Journal of Pharmaceutics, 488(1), 136–153.PubMedCrossRefGoogle Scholar
  60. Yu, Z., Johnston, K. P., & Williams, R. O. (2006). Spray freezing into liquid versus spray-freeze drying: influence of atomization on protein aggregation and biological activity. European Journal of Pharmaceutical Sciences, 27(1), 9–18.PubMedCrossRefGoogle Scholar
  61. Zhang, D., Zhu, Y., & Chen, J. (2009). Microbial transglutaminase production: understanding the mechanism. Biotechnology and Genetic Engineering Reviews, 26(1), 205–222.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of Engineering and Natural Sciences, Department of Food EngineeringTokat Gaziosmanpasa UniversityTokatTurkey
  2. 2.Fine Arts, Gastronomy and Culinary ArtsGaziantep UniversityGaziantepTurkey

Personalised recommendations