透過口服藥物及益生菌來改善腸胃疾病，相較於單獨藥物的使用較為有效，然而腸胃消化道是一個複雜且多變的環境，除了蠕動、排空的現象會影響藥物作用的時間，pH值的劇烈變化以及膽鹽的濃度，也會影響益生菌的存活率。為使藥物或益生菌能夠在腸胃消化道中有更好的發揮，本研究選用海藻酸凝膠來作為包覆的材料，達到藥物傳輸及保護的用途。
海藻酸凝膠顆粒具有良好的生物相容性，且在酸性的環境下表現穩定，適合用來作為藥物載體或提供細胞及微生物保護的生醫材料。利用3D列印微流體來製備微米顆粒，相較於傳統擠壓法，此法所製備的海藻酸鈉凝膠顆粒能精準控制大小且形狀更均一，因此微流體乳化法成為製備海藻凝膠顆粒的最佳方法之一。
本研究使用3D列印技術來製作微流體，列印出具有流體聚焦結構的流道，經拋光處理後透過酒精及紫外光作用與壓克力板結合形成微流體。顆粒製備上採用微流體乳化法，先使海藻酸鈉溶液在蓖麻油中形成液滴，再利用”外部凝膠法”使液滴固化成凝膠顆粒。藉由在海藻酸鈉溶液中加入四環黴素或羅伊氏乳桿菌(Lactobacillus reuteri )，對藥物及益生菌進行包覆。並分析此方法所製備之海藻酸凝膠顆粒的大小、外觀、包埋率、藥物釋放率與益生菌存活率，藉此研究最適合在模擬腸胃道環境中釋放藥物及維持益生菌活性的海藻凝膠微粒製備條件。
我們預期此微流道平台可以製備適合藥物和益生菌遞送的海藻膠微粒，並將擴大其在藥物傳輸與食品工業的應用範圍。

Improve gastrointestinal diseases through oral drugs and probiotics, which is more effective than the use of single drugs. However, the gastrointestinal environment is complicated and varied. While Gastric motility and emptying may affect the duration of drugs, varied pH value and concentration of bile salts may decrease the viability of probiotics. To improve the efficiency of drugs and probiotics in gastrointestinal environment, alginate hydrogels were used as encapsulating material which can improve the drug delivery and provide protection.
Due to their good biocompatibility and stability under acidic environment, alginate hydrogel microbeads have become a promising biomaterial for drug delivery and cell delivery applications. Alginate hydrogel microbeads were prepared by 3D printed microfluidic device in this study. Alginate microbeads prepared via microfluidic devices exhibit more uniform in size and shapes when comparing to the traditional extrusion methods.
In this study, an inkjet 3D printer which featured rapid formation and high resolution was used to fabricate the “flow-focusing” microfluidic channels. After polishing, these channel models were bonded with PMMA sheet through ethanol and UV curing to form the microfluidic platform. A microfluidic emulsification method was used to prepare the alginate microbeads. First the alginate droplets were form in castor oil, and then were gelled to microbeads by external gelation process.
Tetracycline and Lactobacillus reuteri were encapsulated in the alginate microbeads respectively by mixing these agents in the alginate solution. The drug-loaded and probiotic-loaded microbeads were analyzed and characterized by their appearance and encapsulation efficiency. The release kinetics of the antibiotics and the viability of probiotics in the microbeads were evaluated in a simulated gastrointestinal environment.
We anticipated this novel microfluidic platform can prepare the appropriate sizes and shapes of alginate microbeads and expand their future applications in drug delivery and food industry.

參考資料
[1] G. M. Whitesides. The origins and the future of microfluidics. Nature 442, (2006) 368–373.
[2] C. N. Jones, A. N. Hoang, L. Dimisko, B. Hamza, J. Martel, D. Irimia. Microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood. J. Vis. Exp. (2014). (88):51215.
[3] I. Bernacka-Wojcik, P. Lopes, A.C. Vaz, B. Veigas, P.J. Wojcik, P. Simões, D. Barata, E. Fortunato, P.V. Baptista, H. Águas. Bio-microfluidic platform for gold nanoprobebased DNAdetection-application to mycobacterium tuberculosis. Biosens. Bioelectron. (2013), 48, 87–93.
[4] Z. Peng, B. Young, A.E. Baird, S.A. Soper. Single-pair fluorescence resonance energy transfer analysis ofmRNA transcripts for highly sensitive gene expression profiling in near real time. Anal. Chem. (2013), 85, 7851–785
[5] K.S. Elvira, X. Casadevall, I. Solvas. The past, present and potential for microfluidic react or technology in chemical synthesis, Nat.Chem.5(2013) 905–915.
[6] A.J. Hughes, R.K. Lin, D.M. Peehl. Microfluidic integration for automated targeted proteomic assays, Proc. Natl. Acad. Sci. USA109 (2012) 5972–5977.
[7] J. Yang, H. Giessen, P. Lalanne, Simple analytical expression for the peak- frequency shifts of plasmonic resonances for sensing, Nano Lett.15 (2015) 3439–3444.
[8] P. Cui, S. Wang. Application of microfluidic chip technology in pharmaceutic alanalysis: A review. Journal of Pharmaceutical Analysis 9 (2019)238–247
[9] W. G Patrick., A. A. K. Nielsen, S. J. Keating, T. J. Levy, C.-W. Wang, J. J. Rivera. DNA assembly in 3D printed fluidics. PLoS ONE(2015), 10(12)
[10] J. Liu, T. Pan, A. T. Woolley, and M. L. Lee. Surface-modified poly(methylmethacrylate) capillary electrophoresis microchips for protein and peptide analysis, Analytical Chemistry (2004) 76 (23), 6948-6955
[11] S. Waheed, J.M. Cabot, N.P. Macdonald, T. Lewis, R.M. Guijt, B. Paull, M.C.Breadmore, 3D printed microfluidic devices: enablers and barriers, Lab Chip(2016)16 (11) 1993–2013
[12] I. S. L. A. Hamid, B. K. Khim, S. S. Hamid, M. F. A. Rahman, A. A. Manaf. Implementation of a single emulsion mask for three-dimensional (3D) microstructure fabrication of micromixers using the grayscale photolithography technique. Micromachines(2020), 11, 548
 
[13] J.-H. Lee, S.-K. Kim, I. A. Khawar, S.-Y. Jeong, S. Chung, H.-J. Kuh. Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of stroma-mediated cell motility and drug resistance. Experimental & Clinical Cancer Research (2018) 37:4
[14] J. Lauri, C. Liedert, A. Kokkonen, T. Fabritius. Effect of solvent lamination on roll-to-roll hot-embossed PMMA microchannels evaluated by optical coherence tomography. Mater. Res. Express 6 (2019) 075333
[15] N. Zhang, J. Liu, H. Zhang, N. J. Kent, D. Diamond, M. D. Gilchrist. 3D Printing of metallic microstructured mould using selective laser melting for injection moulding of plastic microfluidic devices. Micromachines(2019) ,10, 595
[16] I. G. Reichenbach, M. Bohley, F. J. P. Sousa, J. C. Aurich. Micromachining of PMMA—manufacturing of burr-free structures with single-edge ultra-small micro end mills. Advanced Manufacturing Technology (2018) 96:3665–3677
[17] M. Singh, H. M. Haverinen, P. Dhagat and G. E. Jabbour. Inkjet printing—process and its applications. Adv. Mater (2010), 22, 673.
[18] http://www.custompartnet.com/wu/jetted-photopolymer
[19] K. K. B. Hon, L. Li, I. M. Hutchings. Droplet analysis in an inkjet-integrated manufacturing process for nylon 6. CIRP Ann (2008), 57, 601–620.
[20] A. Pfister, R. Landers, A. Laib, U. Hübner, R. Schmelzeisen, R. Mülhaupt, J. Polym. Biofunctional rapid prototyping for tissue‐engineering applications: 3D bioplotting versus 3D printing. Sci., Part A: Polym. Chem. (2004), 42, 624–638.
[21] T. Femmer, I. Flack, M. Wessling. Additive manufacturing in fluid process engineering, Chemie Ingenieur Technik 88 (2016) 535–552..
[22] http://www.stratasys.com/materials/polyjet/digital-materials
[23] http://www.custompartnet.com/wu/3d-printing
[24] S. Waheed, J. M. Cabot, N. P. Macdonald, T. Lewis, R. M. Guijt, B. Paullab, M. C. Breadmore. 3D printed microfluidic devices: enablers and barriers. Lab Chip, (2016), 16, 1993
[25] B. R. Ringeisen, R. K. Pirlo, P. K. Wu, T. Boland, Y. Huang, W. Sun, Q. Hamid, D. B. Chrisey. Cell and organ printing turns 15: Diverse research to commercial transitions. MRS Bull. (2013) 38, 834–843.
[26] L. Novakova-Marcincinova, I. Kuric. Basic and advanced materials for fused deposition modeling rapid prototyping technology. Manuf. and Ind. Eng. (2012) 11, 24–27.
[27] H. N. Chia, B. M. Wu. Recent advances in 3D printing of biomaterials. J. Biol. Eng. (2015) 9, 4.
[28] P. J. Bártolo. Stereolithography: Materials, Processes and Applications, Springer, (2011)
[29] Y. M. Huang, S. Kuriyama, C. P. Jiang. Fundamental study and theoretical analysis in a constrained-surface stereolithography system. Int. J. Adv. Des. Manuf. Technol. (2004) 24, 361–369.
[30] F. P. Melchels, J. Feijen, D. W. Grijpma. A review on stereolithography and its applications in biomedical engineering. Biomaterials (2010) 31, 6121–6130.
[31] X. Zheng, J. Deotte, M. P. Alonso, G. R. Farquar, T. H. Weisgraber, S. Gemberling, H. Lee, N. Fang, C. M. Spadaccini. Design and optimization of a light-emitting diode projection micro-stereolithography three-dimensional manufacturing system. Rev. Sci. Instrum. (2012) 83, 125001.
[32] L. Edermaniger. The facts on taking probiotics with antibiotics, and antibiotics with food. Atlas blog (2020) March 19. https://atlasbiomed.com/blog/probiotics-for-antibiotics/
[33] N.T. Annan, A.D. Borza, L. T. Hansen, Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Research International 41 (2008) 184–193.
[34] L. Edermaniger. Why is lactobacillus so important for human health? Atlas blog (2020) Oct 02.
[35] E. C. Verna, Use of probiotics in gastrointestinal disorders: what to recommend? Therap Adv Gastroenterol. (2010) Sep; 3(5): 307–319.
[36] M. Galloway, W. Stryjewski, A. Henry, S.M. Ford, S. Llopis, R.L. McCarley, S.A. Soper. Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules, Analytical Chemistry 74 (2002) 2407–2415.
[37] H.E. Jeong, K.Y. Suh. On the role of oxygen in fabricating microfluidic channels with ultraviolet curable materials. Lab Chip 8 (2008) 1787–1792.
[38] A. Griebel, S. Rund, F. Schönfeld, W. Dörner, R. Konrad, S. Hardt. Integrated polymer chip for two-dimensional capillary gel electrophoresis. Lab Chip 4 (2004) 18–23.
[39] K.F. Lei, S. Ahsan, N. Budraa, W.J. Li, J.D. Mai, Microwave bonding of polymer-based substrates for potential encapsulated micro/nanofluidic device fabrication, Sensors and Actuators A 114 (2004) 340–346.
[40] C.-W. Tsao, D.L. DeVoe, Bonding of thermoplastic polymer microfluidics, Microfluidics and Nanofluidics 6 (2009) 1–16.
[41] D.A. Mair, M. Rolandi, M. Snauko, R. Noroski, F. Svec, J.M.J. Fréchet, Roomtemperature bonding for plastic high-pressure microfluidic chips, Analytical Chemistry 79 (2007) 5097–5102.
 
[42] H. H. Tran, W. Wu, N. Y. Lee. Ethanol and UV-assisted instantaneous bonding of PMMA assemblies and tuning in bonding reversibility. Sensors and Actuators B 181 (2013) 955– 962
[43] S. H. Ching, N. Bansal, B. Bhandari. Alginate gel particles–A review of production techniques and physical properties. Critical Reviews in Food Science and Nutrition, (2017)57:6, 1133-1152
[44] A. Zhang, K. Jung, A. Li, J. Liu, and C. Boyer. Recent advances in stimuli-responsive polymer systems for remotely controlled drug release. Progress in Polymer Science (2019) vol. 99, 101164
[45] A. Sosnik, Alginate particles as platform for drug delivery by the oral route: state-of-the-art. ISRN Pharmaceutics (2014) vol. 2014, 926157
[46] H. F. Helander, L. Fändriks,. Surface area of the digestive tract - revisited. Scandinavian Journal of Gastroenterology. (2014) 49 (6): 681–689
[47] R. L. Drake, W. Vogl, A. W.M. M. Tibbitts, P. Richardson. Gray's anatomy for students (3rd ed.). Philadelphia: Elsevier/Churchill Livingstone. (2015) p. 312.
[48] S.H. Ching, N. Bansal, B. Bhandari, Alginate gel particles-A review of production techniques and physical properties. Crit Rev Food Sci Nutr (2017) 57(6): p. 1133-1152.
[49] M. Cardoso, R. Costa, and J. Mano, Marine origin polysaccharides in drug delivery systems. Marine Drugs (2016) vol. 14, no. 2, p. 34.
[50] S. V. Bhujbal, P. de Vos, and S. P. Niclou, Drug and cell encapsulation: alternative delivery options for the treatment of malignant brain tumors, Advanced Drug Delivery Reviews (2014) vol. 67-68, pp. 142–153
[51] A. M. A. Rokstad, I. Lac´ık, P. de Vos, and B. L. Strand, Advances in biocompatibility and physico-chemical characterization of microspheres for cell encapsulation, Advanced Drug Delivery Reviews (2014) vol. 67-68, pp. 111–130.
[52] L. Mazutis, R. Vasiliauskas, D.A. Weitz, Microfluidic production of alginate hydrogel particles for antibody encapsulation and release, Macromol. Biosci. 15 (2015) 1641–1646
[53] X. Guo, S. Huang, J. Sun, and F. Wang, Comparison of the cytotoxicities and wound healing effects of hyaluronan, carbomer, and alginate on skin cells in vitro, Advances in Skin & Wound Care (2015) vol. 28, no. 9, pp. 410–414.
[54] T. Wang, Y. Zheng, Y. Shi, and L. Zhao, pH-Responsive calcium alginate hydrogel laden with protamine nanoparticles and hyaluronan oligosaccharide promotes diabetic wound healing by enhancing angiogenesis and antibacterial activity, Drug Delivery and Translational Research (2019) vol. 9, no. 1, pp. 227–239.
 
[55] D. M. Hariyadi and N. Islam. Current status of alginate in drug delivery. Advances in Pharmacological and Pharmaceutical Sciences, vol. (2020) Article ID 8886095.
[56] C.-H. Choi, J.-H. Jung, T.-H. Yoon, D.-P. Kim, C.-S. Lee. The effect of microfluidic geometry for in situ generating monodispersed hydrogels. Journal of Chemical Engineering of Japan. (2008) Vol.41, No.7, p.649-654.
[57] A. Nakatsuka, A. Matsuo, T. Kanai. Preparation of monodisperse solid fat microspheres in a microfluidic device. (2016) Vol 49, No 6, p. 541-543.
[58] M. Yamada, M. Seki. Multiphase microfluidic processes to produce alginate-based microparticles and fibers. Journal of Chemical Engineering of Japan. (2016) Vol 51, No 4, p. 318-330
[59] A.F. Hofmann, L.R. Hagey. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol Life Sci (2008) 65:2461–83.
[60] V. Urdaneta, J. Casadesús. Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts. Front. Med.(2017) 4:163.
[61] J.C. van Velkinburgh, J.S. Gunn. PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp. Infect Immun (1999) 67:1614–22.
[62] M. Begley, R.D. Sleator, C.G. Gahan, C. Hill. Contribution of the three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes. Infect Immun (2005) 73:894–904
[63] A. Vander, J. Sherman, D. Luciano. Human Physiology: The mechanisms of body function. 8th ed. Boston: McGraw-Hill (2001)
[64] H. Zhang, E. Tumarkin, R. M. A. Sullan, G. C. Walker, E. Kumacheva. Exploring microfluidic routes to microgels of biological polymers, Macromol. Rapid Commun (2007)., 28, 527–538
[65] D. H. Lee, W. Lee, E. Um, J. K. Park. Microbridge structures for uniform interval control of flowing droplets in microfluidic networks. Biomicrofluidics (2011) 5, 34117–341179
[66] C. Kim, J. Park, J. Y. Kang. A microfluidic manifold with a single pump system to generate highly mono-disperse alginate beads for cell encapsulation. Biomicrofluidics (2014) 8, 066504
[67] P. Garstecki, M.J. Fuerstman, H.A. Stone, G.M. Whitesides, Formation of droplets and bubbles in a microfluidic T-junction-scaling and mechanism of break-up, Lab Chip 6 (2006) 437–446.
[68] M.K. Mulligan, J.P. Rothstein. The effect of confinement-induced shear on drop deformation and breakup in microfluidic extensional flows, Phys. Fluids 23 (2011) 022004.
 
[69] K. Churski, M. Nowacki, P.M. Korczyk, P. Garstecki, Simple modular systems for generation of droplets on demand, Lab Chip 13 (2013) 3689–3697.
[70] Y. Zeng, M. Shin, T.Wang, Programmable active droplet generation enabled by integrated pneumatic micropumps, Lab Chip 13 (2013) 267–273.
[71] S. Mashaghi, A. Abbaspourrad, D. A.Weitz, A. M. van Oijen. Droplet microfluidics: A tool for biology, chemistry and nanotechnology. Trends in Analytical Chemistry 82 (2016) 118–125.
[72] Z.Wu, H. Chen, X. Liu, Y. Zhang, D. Li, H. Huang, Protein adsorption on poly(Nvinylpyrrolidone)- modified silicon surfaces prepared by surface-initiated atom transfer radical polymerization, Langmuir 25 (2009) 2900–2906.
[73] L. Mazutis, A.D. Griffiths, Selective droplet coalescence using microfluidic systems, Lab Chip 12 (2012) 1800–1806.
[74] J.-C. Baret, Surfactants in droplet-based microfluidics, Lab Chip 12 (2012) 422–433.
[75] S.B. Singh, K. Young, L.L. Silver. What is an "ideal" antibiotic? Discovery challenges and path forward. Biochem Pharmacol (2017) 133: p. 63-73.
[76] D. Brown. Antibiotic resistance breakers: can repurposed drugs fill the antibiotic discovery void? Nat Rev Drug Discov (2015) 14(12): p. 821-32.
[77] W. Yang. TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics. J Biol Chem (2004) 279(50): p. 52346-52.
[78] R. A. Buckler, M.M.P, J. C.Gallagher. Beta-lactams and tetracyclines side effects of drugs. Annual (2017) 39: p. 217-227.
[79] I. Chopra. Tetracycline analogs whose primary target is not the bacterial ribosome. Antimicrob Agents Chemother (1994) 38(4): p. 637-40.
[80] D.M. Lilly, R.H. Stillwell. Probiotics: Growth-promoting factors produced by microorganisms. Science. (1965) 147:747–748.
[81] T.C. Wallace, F. Guarner, K. Madsen, M.D. Cabana, G. Gibson, E. Hentges, M.E. Sanders. Human gut microbiota and its relationship to health and disease. Nutr Rev. (2011) Jul; 69(7):392-403.
[82] E. Isolauri, Y. Sütas, P. Kankaanpää, H. Arvilommi, S. Salminen. Probiotics: Effects on immunity. Am J Clin Nutr. (2001) 73(Suppl 2):S444–S450.
[83] P.T. Shyu, G.G. Oyong, E.C. Cabrera. Cytotoxicity of probiotics from Philippine commercial dairy products on cancer cells and the effect on expression of cfos and cjun early apoptotic-promoting genes and interleukin-1β and tumor necrosis factor-α proinflammatory cytokine genes. Biomed Res Int. (2014):491740.
[84] W.D. Chey, ACG Clinical Guideline: Treatment of helicobacter pylori infection. Am J Gastroenterol (2017) 112(2): p. 212-239.
 
[85] P.K. Singh, I.P. Kaur. Synbiotic (probiotic and ginger extract) loaded floating beads: A novel therapeutic option in an experimental paradigm of gastric ulcer. J Pharm Pharmacol. (2012);64:207–217.
[86] Q. Mu, V. J. Tavella and X. M. Luo, Role of Lactobacillus reuteri in human health and diseases. Front. Microbiol.(2018) 9:757
[87] F. Franceschi, A. Cazzato, E. C. Nista, E. Scarpellini, D. Roccarina, G. Gigante. Role of probiotics in patients with Helicobacter pylori infection. Helicobacter (2007) 12(Suppl. 2), 59–63.
[88] R. Francavilla, L. Polimeno, A. Demichina, G. Maurogiovanni, B. Principi, G. Scaccianoce. Lactobacillus reuteri strain combination in Helicobacter pylori infection: a randomized, double-blind, placebo-controlled study. J. Clin. Gastroenterol. (2014) 48, 407–413.
[89] J. V. D. Abeele, J. Rubbens, J. Brouwers, P. Augustijns, The dynamic gastric environment and its impact on drug and formulation behavior. European Journal of Pharmaceutical Sciences 96 (2017) 207–231
[90] M. Camilleri, A.E. Bharucha, C. Di lorenzo, W.L. Hasler, C.M. Prather, S.S. Rao, A. Wald. American neurogastroenterology and motility society consensus statement on intraluminal measurement of gastrointestinal and colonicmotility in clinical practice.Neurogastroenterol. Motil. 20 (2008) 1269–1282.
[91] D. Cassilly, S. Kantor, L.C. Knight, A.H. Maurer, R.S. Fisher, J. Semler, H.P. Parkman. Gastric emptying of a non-digestible solid: assessment with simultaneous SmartPill pH and pressure capsule, antroduodenal manometry, gastric emptyingscintigraphy. Neurogastroenterol. Motil. 20 (2008) 311–319.
[92] P. Sachdeva, S. Kantor, L.C. Knight, A.H. Maurer, R.S. Fisher, H.P. Parkman, Use of a high caloric liquid meal as an alternative to a solid meal for gastric emptying scintigraphy. Dig. Dis. Sci. 58 (2013) 2001–2006.
[93] B. L. Dekkers , E. Kolodziejczyk , S. Acquistapace , J. Engmann and T. J. Wooster. Impact of gastric pH profiles on the proteolytic digestion of mixed βlg-Xanthan biopolymer gels. Food Funct. (2016) 7, 58-68
[94] J R Malagelada, V L Go, W H Summerskill. Different gastric, pancreatic, and biliary responses to solid-liquid or homogenized meals. Dig. Dis. Sci. (1979) 24, 101–110
[95] Y.T. Hsu, Evaluation of the inhibitory effect of tetracycline-alginate floating beads on the growth of H. pylori in a simulated gastric environment. (2019) Chapter 4: p.55
[96] H. Duong, P. C. Chen. Novel solvent bonding method for creation of a three-dimensional, non-planar, hybrid PLA/PMMA microfluidic chip. Sensors and Actuators A 280 (2018) 350–358
[97] F. Flamminii, C.D. Di Mattia, M. Nardella, M. Chiarini, L. Valbonetti, L. Neri, G. Difonzo, P. Pittia. Structuring alginate beads with different biopolymers for the development of functional ingredients loaded with olive leaves phenolic extract, Food Hydrocolloids (2020)
[98] A. F. Oliveira, R. G. Bastos, L. G. de la Torre. Bacillus subtilis immobilization in alginate microfluidic-based microparticles aiming to improve lipase productivity. Biochemical Engineering Journal 143 (2019) 110–120.
[99] C. N. Baroud, F. Gallaire and R. Dangl. Dynamics of microfluidic droplets. Lab Chip 10, (2010) 2032–2045
[100] O. Gul, M. Dervisoglu, Application of multicriteria decision technique to determine optimum sodium alginate concentration for microencapsulation of Lactobacillus casei Shirota by extrusion and emulsification. J. Food Process Eng. (2016) 1-10
[101] M. T Cook, G. Tzortzis, D. Charalampopoulos, & V. V. Khutoryanskiy. Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria. Biomacromolecules 12(7), (2011) 2834-2840.
[102] M. Chávarri, I. Marañón, R. Ares, F.C. Ibáñez, F. Marzo, M. del Carmen Villarán. Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions Int. J. Food Microbiol. 142(1–2), (2010) 185–189
[103] C.-H. Choi, J.-H. Jung, Y. W. Rhee, D.-P. Kim, S.-E. Shim, C.-S. Lee. Generation of monodisperse alginate microbeads and in situ encapsulation of cell in microfluidic device. Biomed Microdevices (2007) 9:855–862
[104] B. Lupo, A. Maestro, J.M. Gutiérrez, C. González, Characterization of alginate beads with encapsulated cocoa extract to prepare functional food: comparison of two gelation mechanisms, Food Hydrocoll. 49 (2015) 25–34.
[105] C. M. Silva, A. Ribeiro, M. Figueiredo, D. Ferreira, F. Veiga. Microencapsulation of hemoglobin in chitosan-coated alginate microspheres prepared by emulsification/internal gelation. AAPS J. (2005). 7:903–913.
[106] R. H. Moghaddam, S. Dadfarnia, A. M. H. Shabani, Z. H. Moghaddam, M. Tavakol. Electron beam irradiation synthesis of porous and non-porous pectin based hydrogels for a tetracycline drug delivery system. Materials Science and Engineering: C, (2019), Volume 102, Pages 391-404
 
[107] Ekhlas A. El-Alfy, Manal K. El-Bisi, Ghada M. Taha, Hassan M. Ibrahim, Preparation of biocompatible chitosan nanoparticles loaded by tetracycline, gentamycin and ciprofloxacin as novel drug delivery system for improvement the antibacterial properties of cellulose based fabrics, International Journal of Biological Macromolecules, (2020) Volume 161, Pages 1247-1260
[108] G. R. Bardajee, A. Pourjavadi, S. Ghavami, R. Soleyman, F. Jafarpour. UV-prepared salep-based nanoporous hydrogel for controlled release of tetracycline hydrochloride in colon, Journal of Photochemistry and Photobiology B: Biology, (2011)Volume 102, Issue 3, Pages 232-240
[109] A. Belščak-Cvitanovic , A. Bušić, L. Barišić, D. Vrsaljko, S. Karlović, I. Špoljarić, A. Vojvodić, G. Mršić, D. Komes. Emulsion templated microencapsulation of dandelion (Taraxacum officinale L.) polyphenols and β-carotene by ionotropic gelation of alginate and pectin. Food Hydrocolloids,(2016) 57, 139–152.
[110] S. E Jones, J. Versalovic. Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiology (2009), 9:35
[111] G.Vantrappen, J. Janssens, J. Hellemans, Y. Ghoos. The interdigestive motor complex of normal subjects and patients with bacterial overgrowth of the small intestine. J. Clin. Invest. 59 (1977) 1158–1166
[112] S.H. Yalkowsky, Yan. He, Handbook of aqueous solubility data: an extensive compilation of aqueous solubility data for organic compounds extracted from the AQUASOL dATAbASE. CRC Press LLC, Boca Raton, FL. 2003., p. 282
[113] https://pubchem.ncbi.nlm.nih.gov/compound/Tetracycline
[114] US EPA; Estimation Program Interface (EPI) Suite. Ver. 4.11. Nov, 2012. Available from, as of Nov 7, 2016: http://www2.epa.gov/tsca-screening-tools