RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 06/04/2023. UNIVERSIDADE ESTADUAL PAULISTA - UNESP FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL DEVELOPMENT OF ALGINATE MICROPARTICLES COATED WITH HPMCAS CONTAINING THE ANTIMICROBIAL PEPTIDE Ctx(Ile21)-Ha FOR APPLICATION AS A NATURAL ORAL ADDITIVE IN LAYING HENS AGAINST SYSTEMIC Salmonella Enteritidis INFECTION Cesar Augusto Roque Borda Biotechnology Engineer 2021 UNIVERSIDADE ESTADUAL PAULISTA - UNESP FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL DESENVOLVIMENTO DE MICROPARTÍCULAS DE ALGINATO REVESTIDAS COM HPMCAS CONTENDO O PEPTÍDEO ANTIMICROBIANO Ctx(Ile²¹)-Ha PARA APLICAÇÃO COMO ADITIVO ORAL NATURAL EM GALINHAS DE POSTURA CONTRA INFECÇÃO SISTÊMICA POR Salmonella Enteritidis Discente: Cesar Augusto Roque Borda Orientador: Prof. Dr. Eduardo Festozo Vicente 2021 Dissertação apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Mestre em Zootecnia. Roque-Borda, Cesar Augusto B727d Development of alginate microparticles coated with HPMCAS containing the antimicrobial peptide Ctx(Ile21)-Ha for application as a natural oral additive in laying hens against systemic Salmonella Enteritidis infection / Cesar Augusto Roque Borda. – – Jaboticabal, 2021 78 p. il. tabs. Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, 2021 Orientador: Eduardo Festozo Vicente Banca examinadora: Marlus Chorilli, Simara Márcia Marcato Bibliografia 1. Poultry industry By-products. 2. Biotechnology. 3. Poultry Diseases. 4. Microencapsulation. 5. Antimicrobial Cationic Peptide. I. Título. II. Jaboticabal- Zootecnia. Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Jaboticabal/SP - Karina Gimenes Fernandes - CRB 8/7418 UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Jaboticabal DEVELOPMENT OF ALGINATE MICROPARTICLES COATED WITH HPMCAS CONTAINING THE ANTIMICROBIAL PEPTIDE Ctx(Ile21)-Ha FOR APPLICATION AS A NATURAL ORAL ADDITIVE IN LAYING HENS AGAINST SYSTEMIC Salmonella Enteritidis INFECTION TÍTULO DA DISSERTAÇÃO: CERTIFICADO DE APROVAÇÃO AUTOR: CESAR AUGUSTO ROQUE BORDA ORIENTADOR: EDUARDO FESTOZO VICENTE Aprovado como parte das exigências para obtenção do Título de Mestre em ZOOTECNIA, pela Comissão Examinadora: Prof. Dr. EDUARDO FESTOZO VICENTE (Participaçao Virtual) Departamento Engenharia de Biossistemas-FCE/UNESP / Tupã/SP Prof. Dr. MARLUS CHORILLI (Participaçao Virtual) Departamento de Fármacos e Medicamentos-FCFAr/UNESP / Araraquara/SP Profa. Dra. SIMARA MÁRCIA MARCATO (Participaçao Virtual) Centro de Ciências Biológicas / UEM - Maringá, PR Jaboticabal, 06 de abril de 2021 Faculdade de Ciências Agrárias e Veterinárias - Câmpus de Jaboticabal - Via de Acesso Professor Paulo Donato Castellane, s/n, 14884900 https://www.fcav.unesp.br/#!/pos-graduacao/programas-pg/zootecnia/CNPJ: 48.031.918/0012-87. AUTHOR CURRICULUM DATA Cesar Augusto Roque Borda, nascido no dia 15 de maio de 1992 no Peru, filho de Augusto Rodolfo Roque Rivera e Rosalia Borda Campos. Bacharel em Engenharia Biotecnologica com período sanduiche pela Universidad Católica de Santa María (UCSM), Arequipa - Perú, título obtido em maio de 2018. Durante a graduação foi líder de grupo de pesquisa ABBC conformada por estudantes sob direção do E- Quipu Santa María, em 2015 ganhou financiamento do Projeto de pesquisa “Evaluación del efecto hepatoprotector anticancerígeno y anticitotóxico de la Avena sativa” pela UCSM, RESOLUÇÃO N° 201381-R2015, em 2016 foi convidado para estagiar no “Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), Argentina”, temática baseada em caracterização de compostos fenólicos e antioxidantes, no mesmo ano foram ministradas 03 disciplinas de curta duração certificadas pelo E-Quipu Santa María, em 2017, foi convidado novamente ao CIDCA como estagiário de pesquisa em criopreservação em colaboração do Banco de Germoplasma de Balcarce, do Instituto de Tecnologia Agraria (INTA) – Argentina, sendo validado como o período sanduíche. Em 2019 ingressou no PPG em Zootecnia pela Universidade Estadual Paulista (UNESP) e foi bolsista pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) até o dia de defesa da presente dissertação. Em 2019 foi estagiário docente da Faculdade de Ciências e Engenharias - FCE/UNESP da disciplina de Bioquímica e Química Orgânica, e no mesmo ano recebeu a Premiação de Menção Honrosa pela apresentação do trabalho no Evento Renorbio 2019 - II Encontro de Biotecnologia do Nordeste da Rede Nordeste de Biotecnologia - RENORBIO / FIOCRUZ-CE. Atualmente faz parte do grupo de pesquisa “Peptídeos: Síntese, Otimização e Estudos Aplicados – PeSEAp” liderado pelo Prof. Dr. Eduardo Festozo Vicente, orientador desta dissertação. “Entre ser o no ser, Yo soy” Teresa “Só existem dois dias do ano em que você não pode fazer nada: um se chama ontem e outro amanhã”. Dalai Lama ACKNOWLEDGEMENTS A minha avó Eugenia Rivera pelo suporte constante dia a dia desde Peru, desde meu início como graduando e até hoje sempre me alentando por ser sempre melhor. Sou grato aos meus amigos feitos durante a graduação da faculdade no Peru, que até hoje dividem suas alegrias e angústias comigo. Em especial aos meus amigos Marcelo Valdivia, Fernando Dias, Sebastian Vega, Gary Villanueva, Piero Silva, Sofia Mansilla, Yuli Briceño pelos momentos de desabafo, filosofia e de diversão. Amigos feitos no Brasil, Família Giangrecco (Jaboticabal), Família Barradas (Pompeia), Família Baute (Tupã), aos pinkys friends do laboratório de patologia veterinária a Jonathan dos Santos da FCAV, pelo suporte e força constante. As amizades obtidas na FCFar/UNESP e UNIARA, Bruna Fernandes, Andreia Meneguin, Leonardo Di Filippo, Jonatas Lobato e em geral ao pessoal que facilitou minha estância e compartilhou experiencias comigo. Principalmente meus agradecimentos à FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo pela bolsa de mestrado concedida pelo Processo n° 2018/25707-3 vinculado ao Projeto Jovem Pesquisador Processo n° 2016/00446- 7, sendo esses recursos essenciais para a obtenção destes resultados. Ao meu querido orientador, Eduardo Festozo Vicente, quem tem demonstrado ser um excelente profissional e amigo, sempre confiando na minha expertise para fazer mais. Ao grupo de pesquisa PeSEAp e as técnicas de laboratório da FCE/UNESP Tupã pelo apoio constante no desenvolvimento dos experimentos. Minha gratidão para os que colaboraram com o desenvolvimento deste projeto, Priscilla Ayleen Bustos Mac-Lean (FCE), Hernane Barud (UNIARA), Marlus Chorilli (FCFar) e Angelo Berchieri Junior (FCAV). Dedico À minha avó, Eugenia Rivera. Guerreira, rainha, e inspiração, que sempre me incentivou incondicionalmente em tudo e que, durante o período que este trabalho estava sendo desenvolvido, desde Peru ligando todos os dias, venceu o COVID-19 com muita força e fé inabalável na vitória, me inspirando ainda mais a seguir na luta, e a todas as pessoas que sempre acreditaram em mim! SUMMARY RESUMO.......................................................................................................... 1 ABSTRACT ...................................................................................................... 2 CHAPTER 1 – General considerations ............................................................. 3 1. INTRODUCTION ........................................................................................... 3 2. OBJETIVOS ................................................................................................... 4 3. LITERATURE REVIEW .................................................................................. 5 REFERENCES ................................................................................................... 16 CHAPTER 2 – Alginate-based microparticles coated with HPMCAS cellulose- derivative enable the Ctx(Ile21)-Ha antimicrobial peptide application as a feed additive................................................................................................................ 25 GRAPHICAL ABSTRACT.................................................................................... 25 ABSTRACT.......................................................................................................... 26 1. INTRODUCTON............................................................................................... 27 2. MATERIAL AND METHODS ........................................................................... 28 3. RESULTS AND DISCUSSION ........................................................................ 34 4. CONCLUSIONS ............................................................................................... 45 REFERENCES ..................................................................................................... 47 CHAPTER 3 - Application of HPMCAS-coated Ctx(Ile21)-Ha peptide microparticles as a potential use to prevent systemic infection caused by Salmonella Enteritidis in poultry .................................................................................................................. 53 GRAPHICAL ABSTRACT.................................................................................... 53 ABSTRACT.......................................................................................................... 54 1. INTRODUCTON............................................................................................... 55 2. MATERIAL AND METHODS ........................................................................... 56 3. RESULTS......................................................................................................... 59 3. DISCUSSION .................................................................................................. 60 4. CONCLUSIONS ............................................................................................ 63 REFERENCES ................................................................................................... 65 APLICAÇÃO DO PEPTÍDEO ANTIMICROBIANO Ctx(Ile21)-Ha E SEUS CONJUGADOS EM DIFERENTES FORMULAÇÕES COMO ADITIVO NUTRICIONAL EM PINTAINHAS DE POSTURA RESUMO - A pesquisa por novos compostos que possam aumentar e melhorar o desempenho ou diminuir resíduos químicos em produtos de origem animal torna-se relevante e demandada. Paralelamente, o uso constante de antibióticos sintéticos como promotores de crescimento causa uma grande resistência bacteriana em aves poedeiras, além de rastros e resíduos em seus produtos, os quais podem afetar a sanidade animal e, consequentemente, a saúde humana. Neste sentido, o peptídeo antimicrobiano (PAM) Ctx(Ile21)-Ha, extraído e isolado da rã do cerrado brasileiro Hypsiboas albopunctatus, é uma molécula muito promissora no controle de bactérias patogênicas. No entanto, os PAMs aplicados em sistemas in vivo, podem sofrer degradação ou desnaturação pela presença de proteases ou hidrolise pelo entorno ácido. Assim, este estudo objetivou a síntese e purificação peptídeo Ctx(Ile21)-Ha, e o revestimento de micropartículas de alginato com acetato/succinato de hipromelose (HPMCAS), além de sua caracterização fisioquímica. Foi também avaliada a atividade hemolítica e liberação in vitro gastrointestinal simulada das micropartículas revestidas. O estudo in vivo foi realizado com galinhas de postura recém-nascidas, desafiadas com Salmonella Enteritidis, e tratadas com as micropartículas na ração. Foi realizado a contagem bacteriana em fígado, baço e ceco para verificação dos efeitos do peptídeo. Os resultados obtidos mostraram micropartículas termoestáveis, com eficiência de encapsulação acima do 70%, leve liberação gástrica, ótima liberação intestinal, atividade hemolítica do peptídeo diminuída em 95%, além no experimento in vivo obteve-se diferença significativa na contagem principalmente no fígado, seguida do baço. Portanto, este estudo demonstrou que as micropartículas revestidas contendo o PAM Ctx(Ile21)-Ha possuem alta atividade antimicrobiana e serve de ferramenta para prevenção de infecção sistêmica, sem resíduos contaminantes em comparação com os fármacos convencionais. Palavras-chave: Aditivos. Aves Poedeiras. Micropartículas. Revestimento entérico. Salmonella. Sanidade. APPLICATION OF THE ANTIMICROBIAL PEPTIDE CTX(Ile21)-Ha AND ITS CONJUGATES IN DIFFERENT FORMULATIONS AS A NUTRITIONAL ADDITIVE IN LAYING HENS ABSTRACT - The search for new compounds that can increase and improve performance or reduce chemical residues in products of animal origin is becoming relevant and in demand. At the same time, the constant use of synthetic antibiotics as growth promoters causes great bacterial resistance in laying birds, as well as traces and residues in their products, which can affect animal health and, consequently, human health. In this sense, the antimicrobial peptide (AMP) Ctx(Ile21)- Ha, extracted and isolated from the Brazilian Cerrado frog Hypsiboas albopunctatus, is a very promising molecule in the control of pathogenic bacteria. However, AMPs applied in an in vivo system can undergo degradation or denaturation due to the presence of proteases or hydrolysis by the acidic environment. Thus, this study aimed at the synthesis and purification of the Ctx(Ile21)-Ha peptide, and the coating of alginate microparticles with hypromellose acetate/succinate (HPMCAS), in addition to its physicochemical characterization. Hemolytic activity and simulated in vitro gastrointestinal release of the coated microparticles were also evaluated. In vivo study was carried out with newly hatched laying hens, challenged with Salmonella Enteritidis and treated with microparticles in the feed. Bacterial counts were performed in liver, spleen, and cecum to verify the effects of peptide. The results obtained showed thermostable microparticles, with encapsulation efficiency greater than 70%, slight gastric release, excellent intestinal release, the hemolytic activity of peptide decreased by 95%, and in the in vivo experiment a significant difference was obtained in bacterial count, mainly in liver, followed by spleen. Therefore, this study demonstrated that the coated microparticles containing AMP Ctx(Ile21)-Ha have a high antimicrobial activity and serve as a tool for the prevention of systemic infections, without contaminating residues compared to conventional drugs. Keywords: Additives. Enteric coating. Laying hens. Microparticles. Salmonella. Poultry health. 3 CHAPTER 1 – GENERAL CONSIDERATIONS 1. INTRODUCTION The discovery of new applications brought from Biotechnology area to Animal Production area continues to be an issue that requires a lot of research and dedication. Specifically, poultry sector generates large monetary incomes at the national level, being the United States of America, China and Brazil the largest exporters and producers of this kind of meat. On the other hand, China, USA and India are countries with the highest egg production in the world, and consequently in laying hens’ production (FAO 2020). It is known that the main pathogen considered a public health problem is Salmonella sp., which it must necessarily be controlled to do not generate discomfort in the consumer (Renu et al. 2020). Salmonella sp. transmission is usually due to poor hygiene, by contact with contaminated meat and/or because there is no adequate monitoring of the infected properties (Gaggìa et al. 2010). In recent years, the use of antibiotics has been severely restricted by regulatory institutions, due to the large number of bacteria mutations that occurred over time. In response, they acquired resistance and made conventional antibiotic compounds almost obsolete. Bell (2014), points out that there is a direct relationship between the use of drugs and their resistance, and that this factor is correlated with sectors of population application, such as country, region, state or municipality. Therefore, the use and study of new biological molecules with capacity for spontaneous or kinetic bacterial inhibition superior to conventional drugs has been recommended, since this factor could prevent these bacteria from acquiring or generating resistance (Gong et al. 2020). Many of these biomolecules can be purchased from different natural sources or synthesized using a natural source as a model. Antimicrobial peptides (AMPs) are a great example of biomolecules that are present in virtually all living beings. They are a set of linked amino acids that help in the protection and defense of the organism, proliferate, and increase the useful life and/or neutralize the presence of pathogens. AMP Ctx(Ile21)-Ha, is a peptide from the skin of the Brazilian Cerrado frog Hypsiboas albopunctatus, which has an α-helical amphipathic structure of 21 amino acid residues, is water soluble, and has a positive 4 charge (+3), therefore, it is within the group of cationic antimicrobial peptides (cAMP) and ceratotoxins class (Lorenzón et al., 2012). This peptide has been shown to be relevant due to its antimicrobial activity against public health pathogens with a high affinity for Gram negative bacteria and some fungi, such as Escherichia coli, Staphylococcus aureus and Candida albicans (Vicente et al 2012). It is a peptide described but not studied in depth, so its application and use as an antimicrobial agent could be very promising. AMPs use and application are restricted since (i) these AMPs often have a high rate of antimicrobial activity with such a small amount of sample, which implies excessive waste and lack of dose control, (ii) hemolytic activity of these AMPs frequently can be harmful to animal cells and/or (iii) their active form presents biological activity difficulties which depend on the location and conditions of the action site. To overcome these issues, encapsulation technology was successfully used in food and pharmaceutical technology area to protect bioactive compounds and allow a suitable drug delivery, being an excellent alternative for several research fields, such as Animal Science (Borrmann et al. 2013; Rostami et al. 2019). Microencapsulation, together with biopolymers, can increase the biocompatibility and bioavailability of drugs to treat various diseases. Their molecular structure are easily absorbed. Among techniques at micro or nanoscale, microencapsulation is the most stable (Bratovcic and Suljagic 2019). For these reasons aforementioned, the present study is based on the application of an antimicrobial peptide known as Ctx(Ile21)-Ha, chosen for its potential antimicrobial activity against Salmonella sp. It was microencapsulated and coated with cellulose-based biopolymers and applied to laying chickens, to evaluate its effect on this type of formulation as a food additive with efficient pharmacological activity. 16 REFERENCES Abebe E, Gugsa G (2018) A Review on Poultry Coccidiosis. Abyssinia Journal of Science and Technology 3:1–12. https://doi.org/10.20372/AJST.2018.3.1.76 Akbari MR, Haghighi HR, Chambers JR, et al (2008) Expression of antimicrobial peptides in cecal tonsils of chickens treated with probiotics and infected with Salmonella enterica serovar typhimurium. 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