Publicação
Development of an alginate and shellac microencapsulation system for phages: targeting intestinal foodborne bacterial pathogens on ruminant livestock
| Resumo: | This work consisted in the development of a microencapsulation system for the oral administration of bacteriophages (phages) to ruminants, to protect them during their passage through the rumen and the abomasum, releasing them later in the intestine. Ruminants are a major reservoir for some of the most relevant bacterial foodborne pathogens, such as human pathogenic Escherichia coli (e.g., Shiga Toxin-producing E. coli - STEC). Infections with these types of bacteria are often related to foodborne outbreaks, causing complications that can be fatal. Oral administration of phages, which are natural and highly selective antibacterial agents, is one of the most promising strategies to prevent these contaminations. The encapsulation method used was based on the extrusion of the polymers and allowed to form spherical microparticles, through the prilling by vibration technique followed by ionic gelation. Two natural, biodegradable, and biocompatible polymers were used, alginate and shellac (considered safe food additives by the European Food Safety Authority), under conditions of temperature, pH, and mechanical stress not harmful for phages. The microparticles optimized were composed of a mixture of 2 % (w/v) alginate and 3 % (w/v) shellac. By Wide-Field Microscopy, it was possible to determine that their average diameter was 488 ± 16 μm and that they had a spherical morphology. In vitro stability tests showed that the polymeric matrix remained intact at the temperature (38.5 °C) and pH values corresponding to the rumen (pH 5.8, 6.5 and 7) and abomasum (pH 3) and disintegrated at the pH corresponding to the intestine (pH 7.5). Scanning Electron Microscopy was used to verify that the microparticles had a porous surface and a compact interior. Confocal microscopy analysis showed that the 200 nm diameter fluorescent nanospheres, previously encapsulated to simulate phages remained encapsulated for three days of storage and were released when placed in TRIS buffer pH 7.5. The two phages selected as proof of concept were the E. coli phage T4 as a model and the phage CBA120, which is specific for STEC O157:H7, the most common strain of STEC. Phages were encapsulated within the polymeric matrix, then stability tests with encapsulated and free phages were performed, proving that encapsulated phages resisted more at pH 3, compared to free phages, which were totally inactivated at the same pH. These results allow us to conclude that the alginate and shellac microparticles developed in this work, present great potential as an encapsulation system that could help phages to reach the intestine in viable and effective quantities, thus eliminating the target bacteria present there. Subsequently, the method developed here may be applied to different phages and pathogenic bacteria to reduce the high number of foodborne diseases that occur worldwide and are considered a public health issue by the World Health Organization, representing a high socio-economic cost. |
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| Autores principais: | Leopoldo,Yolanda Patrícia Manuel |
| Assunto: | Alginate Foodborne zoonoses Phage encapsulation Ruminants Shellac Alginato Encapsulamento de fagos Ruminantes Zoonoses alimentares |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | This work consisted in the development of a microencapsulation system for the oral administration of bacteriophages (phages) to ruminants, to protect them during their passage through the rumen and the abomasum, releasing them later in the intestine. Ruminants are a major reservoir for some of the most relevant bacterial foodborne pathogens, such as human pathogenic Escherichia coli (e.g., Shiga Toxin-producing E. coli - STEC). Infections with these types of bacteria are often related to foodborne outbreaks, causing complications that can be fatal. Oral administration of phages, which are natural and highly selective antibacterial agents, is one of the most promising strategies to prevent these contaminations. The encapsulation method used was based on the extrusion of the polymers and allowed to form spherical microparticles, through the prilling by vibration technique followed by ionic gelation. Two natural, biodegradable, and biocompatible polymers were used, alginate and shellac (considered safe food additives by the European Food Safety Authority), under conditions of temperature, pH, and mechanical stress not harmful for phages. The microparticles optimized were composed of a mixture of 2 % (w/v) alginate and 3 % (w/v) shellac. By Wide-Field Microscopy, it was possible to determine that their average diameter was 488 ± 16 μm and that they had a spherical morphology. In vitro stability tests showed that the polymeric matrix remained intact at the temperature (38.5 °C) and pH values corresponding to the rumen (pH 5.8, 6.5 and 7) and abomasum (pH 3) and disintegrated at the pH corresponding to the intestine (pH 7.5). Scanning Electron Microscopy was used to verify that the microparticles had a porous surface and a compact interior. Confocal microscopy analysis showed that the 200 nm diameter fluorescent nanospheres, previously encapsulated to simulate phages remained encapsulated for three days of storage and were released when placed in TRIS buffer pH 7.5. The two phages selected as proof of concept were the E. coli phage T4 as a model and the phage CBA120, which is specific for STEC O157:H7, the most common strain of STEC. Phages were encapsulated within the polymeric matrix, then stability tests with encapsulated and free phages were performed, proving that encapsulated phages resisted more at pH 3, compared to free phages, which were totally inactivated at the same pH. These results allow us to conclude that the alginate and shellac microparticles developed in this work, present great potential as an encapsulation system that could help phages to reach the intestine in viable and effective quantities, thus eliminating the target bacteria present there. Subsequently, the method developed here may be applied to different phages and pathogenic bacteria to reduce the high number of foodborne diseases that occur worldwide and are considered a public health issue by the World Health Organization, representing a high socio-economic cost. |
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