RESSALVA Atendendo solicitação da autora, o texto completo desta tese será disponibilizado somente a partir de 01/03/2025 Thais Cristina Benatti Gallo Interfacial and antioxidant activities of Sorghum protein extracts: protein-polyphenol complexes to stabilize lipid compounds São José do Rio Preto 2023 Câmpus de São José do Rio Preto Thais Cristina Benatti Gallo Interfacial and antioxidant activities of Sorghum protein extracts: protein-polyphenol complexes to stabilize lipid compounds Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Engenharia e Ciência de Alimentos, junto ao Programa de Pós- Graduação em Alimentos, Nutrição e Engenharia de Alimentos, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CAPES Orientadora: Profª. Drª. Vânia Regina Nicoletti Coorientadora: Profª. Drª. Claire Berton- Carabin São José do Rio Preto 2023 G172i Gallo, Thais Cristina Benatti Interfacial and antioxidant activities of Sorghum protein extracts: protein- polyphenol complexes to stabilize lipid compounds / Thais Cristina Benatti Gallo. -- São José do Rio Preto, 2023 164 f. : il., tabs., fotos Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto Orientadora: Vânia Regina Nicoletti Coorientadora: Claire Berton-Carabin 1. Tecnologia de alimentos. 2. Plantas proteínas. 3. Polifenóis. 4. Emulsões. 5. Estabilidade. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. Thais Cristina Benatti Gallo Interfacial and antioxidant activities of Sorghum protein extracts: protein-polyphenol complexes to stabilize lipid compounds Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Engenharia e Ciência de Alimentos, junto ao Programa de Pós- Graduação em Alimentos, Nutrição e Engenharia de Alimentos, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CAPES Comissão Examinadora/Examining Board Profª. Drª. Vânia Regina Nicoletti UNESP – Câmpus de São José do Rio Preto Orientadora Prof. Dr. Eduardo Basílio de Oliveira Universidade Federal de Viçosa Profª. Drª. Louise Emy Kurozawa Universidade Estadual de Campinas Profª. Drª. Mirian Tiaki Kaneiwa Kubo Université de Technologie de Compiègne Prof. Dr. Paulo José do Amaral Sobral Universidade de São Paulo São José do Rio Preto 01 de março de 2023 4 To my mother and grandmother, Josirene and Maria Irene, who taught me to be the best version of myself, to be patient and respect everyone, to work hard, and who provided me the opportunity for a quality education, even in difficult times. ACKNOWLEDGEMENTS I thank God, for the gift of life, for the blessings and opportunities granted during my journey, and for guiding me through this challenging journey. I thank my husband, Artur, for his patience and good humor on my difficult days, for always supporting me and believing in me, and for the countless video calls and conversations during my exchange year. I thank my family, especially my mother Josirene, my sisters Tamires and Maria Eduarda, my grandmother Maria Irene, my stepfather Abraão, and my in-laws for all the support and encouragement during my graduate studies. I thank my supervisor, Vânia, for working with me and guiding me for eleven years, always helping me with seriousness, competence, and dedication, and being a friend in the right moments. I thank my co-advisor, Claire, for welcoming me so well and being so patient while teaching me new techniques, guiding me through and explaining them with endless colorful post-its. I really appreciated all our meetings and discussions. I thank professors Drs. Eduardo Basílio and Mirian Kubo for suggestions and corrections after the qualification exam, and professors Drs. Eduardo Basílio, Louise Kurozawa, Miriam Kubo, and Paulo Sobral for accepting my invitation to participate in the examination board and to contribute in this thesis. I thank the Interfaces et Systèmes Dispersés (ISD) team from INRAE for hosting me during my journey in France, for welcoming me, and teaching me some French, especially the technicians Valérie Beaumal, who worked with me during my entire stay in Nantes, and Lucie Birault, who shared the office and the benches during lipid analyses with me. I thank the Polyphénols, Réactivité, Procédés (PRP) team from INRAE for welcoming me for two months to learn about polyphenols, especially the 6 research director Sylvain Guyout, who taught everything I know about tannins, and the technician Hélène Sotin, who taught me the liquid chromatography technique. I thank the researcher Hamza Mameri from the Ingénierie des Agropolymères et Technologies Émergentes (IATE) team from INRAE for enriching my work with the analyses performed on my powder samples. I thank all and every colleague from the Physical Measurement and ISD laboratories, with whom I shared the benches, good conversations and moments, and rich scientific discussions, especially Poliana and Sungil, who helped me in the beginning of my academic and scientific life and shared good times with me during long days of analysis, and Eléna, who shared the office in Nantes with me, made me laugh during some stressful moments and taught me how to make a good croissant. I thank all faculty and technicians from the Department of Food Engineering and Technology for all knowledge shared. I thank Valéria Aparecida Vieira Queiroz from Embrapa Milho e Sorgo (Sete Lagoas/Minas) for the partnership with the sorghum flours. And, finally, I thank everyone who contributed, directly or indirectly, to the realization of this work. This research was possible thanks to the scholarship granted from Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), in the scope of Program CAPES-PrInt, process number 88887.194785/2018-00, mobility number 88887.570753/2020-00. “Far better it is to dare mighty things, to win glorious triumphs, even though checkered by failure, than to take rank with those poor spirits who neither enjoy much nor suffer much, because they live in the gray twilight that knows not victory nor defeat.” Theodore Roosevelt (1924, p.4) RESUMO A formulação de emulsões baseadas em materiais naturais visando a estabilização de compostos lipídicos tem sido amplamente investigada, porém tais sistemas são termodinamicamente instáveis, o que causa uma dependência do uso de agentes que possam ajudar tanto em sua formação, quanto em sua estabilização. Há uma crescente demanda por moléculas derivadas de fontes naturais que apresentem atividade superficial e possam ser utilizadas para esse fim. Proteínas e polifenóis podem interagir entre si para compor sistemas supramoleculares com atividade interfacial e a presença de polifenóis pode, também, conferir atividade antioxidante complementar a esses complexos. Nesse contexto, este trabalho propõe avaliar o potencial do uso dos complexos naturais proteína-polifenol encontrados em cultivares de sorgo ricas em taninos condensados como agentes tensoativos/antioxidantes para a estabilização de compostos lipídicos. A fim de evidenciar o impacto dos taninos condensados na funcionalidade dos extratos proteicos de sorgo, cultivares sem taninos também foram avaliadas. Os extratos proteicos obtidos a partir das diferentes cultivares de sorgo apresentaram não somente altos teores de proteína (50-67%, fator N calculado), como, também, quantidades substanciais de lipídios (18.7-26%), em sua maioria ácidos graxos livres. Como os extratos proteicos não são solúveis em água, um pré-tratamento por homogeneização à alta pressão foi aplicado em suas suspensões aquosas, melhorando sua dispersibilidade. Interfaces óleo-água formadas pelos extratos proteicos de sorgo apresentaram comportamentos elásticos ou viscosos, com grande efeito da concentração proteica e da presença dos ácidos graxos. As emulsões óleo-em-água preparadas com óleo de canola e estabilizadas pelos diferentes extratos proteicos apresentaram estabilidade física durante 28 dias, mostrando pequenos problemas de floculação. A estabilidade oxidativa das emulsões também foi analisada, apresentando menores níveis de oxidação nos extratos proteicos ricos em taninos, evidenciando, assim, o seu potencial uso como emulsificante natural e, ao mesmo tempo, apresentando uma notável capacidade antioxidante. Palavras-chave: Kafirina. Taninos. Emulsão. Estabilidade. Reologia interfacial. Oxidação lipídica. ABSTRACT Formulation of emulsions based on natural materials for stabilization of lipid compounds has been widely investigated in recent years; however, such systems are thermodynamically unstable, which causes a dependence on the use of agents that can help both in their formation and stabilization. There is an increasing demand for surface-active molecules derived from natural sources that present superficial activity and that can be used for this purpose. Proteins and polyphenols can interact with each other to compose supramolecular systems with interfacial activity. The presence of polyphenols may also impart a complementary antioxidant activity to these complexes. In this context, this work proposes to evaluate the potential use of the natural protein- polyphenol complexes found in sorghum cultivars rich in condensed tannins as emulsifier/antioxidant agents for stabilization of lipid compounds. In order to evidence the impact of condensed tannins on the functionality of sorghum protein extracts, non- tannin cultivars are assessed as well. The protein extracts obtained from the different sorghum cultivars showed not only high protein contents (50-67 wt.%, calculated N factor), but also substantial amounts of lipids (18.7-26 wt.%), mostly free fatty acids. As the protein extracts are not soluble in water, a pre-treatment by high pressure homogenization was applied to their aqueous suspensions, improving their dispersibility. Oil-water interfaces formed by sorghum protein extracts showed elastic or viscous behaviour, with great effect of protein concentration and the presence of fatty acids. The oil-in-water emulsions prepared with canola oil and stabilized by the different protein extracts showed physical stability for 28 days, presenting only minor flocculation. The oxidative stability of the emulsions was also assessed, showing lower levels of oxidation in the protein extracts rich in tannins, thus evidencing its potential use as a natural emulsifier and, at the same time, presenting a remarkable antioxidant capacity. Keywords: Kafirin. Tannins. Emulsion. Stability. Interfacial rheology. Lipid oxidation. LIST OF FIGURES Figure 1.1. Representation of protein behavior in emulsion stabilization: (A) migration to the oil-water interface, (B) reorientation of the protein hydrophobic and hydrophilic groups, and (C) formation of the viscoelastic film. ..................................................... 25 Figure 1.2. Mechanisms of emulsion stabilization: (A) electrostatic effects and (B) steric hindrance................................................................................................................... 26 Figure 1.3. Sorghum grain (Sorghum bicolor L. Moench). ......................................... 28 Figure 1.4. Scheme for localization of kafirin in gluten matrices. ............................... 30 Figure 1.5. Scheme of kafirin protein body. ............................................................... 30 Figure 1.6. Division of tannins concerning to their chemical structure. ...................... 32 Figure 2.1. Content in the different lipid classes found in sorghum flours (suffix F) and their protein extracts (suffix PE). BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ...................................................................................................................... 49 Figure 2.2. Tocopherol contents in the lipid phases extracted from sorghum flours (suffix F) and their protein extracts (suffix PE). BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ...................................................................................................................... 50 Figure 2.3. FTIR spectra of two sorghum flours (BR501(T-) F and SC782(T+) F) and their respective protein extracts (BR501(T-) PE and SC782(T+) PE): (A) full spectrum, and (B) amide I region, which was used for deconvolution to observe differences in the protein structure. ....................................................................................................... 52 Figure 2.4. Electrophoresis (SDS-PAGE) of sorghum proteins present in the raw flours (suffix F) and in their protein extracts (suffix PE) under reducing conditions. ............ 53 Figure 2.5. Protein size distribution in sorghum flours and in their respective protein extracts for SDS-soluble (SSP) (A and B) and SDS-insoluble (SIP) (C and D) fractions; and protein quantification in the flours (E) and protein extracts (F) by size exclusion – high performance liquid chromatography (SE-HPLC). BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. .............................................................................................................. 55 Figure 2.6. Particle size distribution (distribution in volume frequency determined by static light scattering) in the suspensions before (square) and after (triangles) high pressure homogenization (HPH): (A) BR501(T-), (B) BRS310(T-), (C) BRS305(T+), (D) SC782(T+); and (E) macroscopic images of the suspensions before (NP) and after (HPH) homogenization. Pictures were taken 24 hours after suspension preparation (and homogenization, when applicable). ................................................................... 57 Figure 2.7. Optical microscopy and CLSM images of two sorghum protein suspensions: (A, B) BR501(T-) before HPH, (C, D) BR501(T-) after HPH, (E, F) SC782(T+) before HPH, and (G, H) SC782(T+) after HPH. Proteins were stained with fast green and are visualized in green, whereas lipids were stained with Nile red and are visualized in red. White arrows indicate starch granules. .................................... 58 Figure 2.8. Polarized light microscopy images for the SC782(T+)-based suspensions: (A) flour, (B) protein suspension before HPH, and (C) protein suspension after HPH. .................................................................................................................................. 59 Figure 2.9. FTIR spectra of two sorghum protein suspensions (BR501(T-) and SC782(T+)) and of their respective supernatants after centrifugation (10000×g for 30 min at 10 °C) before (NP) and after homogenization (HPH): (A) full spectrum, and (B) amide I region (for the flours, F; protein extracts, PE; suspensions, and supernatants), which was used to for deconvolution to observe differences in the protein structure. .................................................................................................................................. 61 Figure 2.10. Solubility of sorghum proteins in aqueous suspensions (ultrapure water) before and after treatment by HPH (suspensions were homogenized for 6 minutes at 400 bars). NP: non-treated by high pressure homogenization; HPH: treated by high pressure homogenization. ......................................................................................... 62 Figure 2.11. Protein composition determined by optical density analysis of SDS-PAGE gels of: (A) total suspensions, and (B) respective supernatants (suffix SN), before (NP) and after homogenization (HPH). Samples were denatured and reduced by heating at 95 °C and using β-mercaptoethanol. ......................................................................... 64 Supplementary Figure 2.1. Particle size distributions determined during preliminary tests to optimize conditions for high-pressure homogenization (HPH) treatment of sorghum protein aqueous suspensions: (A) tests using different pressures; (B) tests using P = 300 bars with different times; (C) tests using P = 400 bars with different times. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ...................................... 70 12 Figure 3.1. Adsorption kinetics of sorghum protein extracts at oil-water interface (25 °C) for free-tannin samples BR501(T-) (A) and BRS310(T-) (B), and rich-tannin samples BRS305(T+) (C) and SC782(T+) (D). HPH and NP represent samples treated and non-treated by high-pressure homogenization, respectively, and 0.1 and 1g/L indicate the protein concentration in the aqueous suspension. ................................. 80 Figure 3.2. Dilatational elastic modulus (E’d) and loss tangent at oil-water interfaces (25 °C) stabilized with sorghum protein extracts BR501(T-) (sample with tannins) and SC782(T+) (sample with tannins), for applied deformation at fixed frequency (0.02 Hz) (A, B), and applied frequency at fixed amplitude (5 %) (C, D). HPH and NP represent samples treated and non-treated by high-pressure homogenization, respectively, and 0.1 and 1g/L indicate the protein concentration in the suspension. ........................... 83 Figure 3.3. Lissajous plots for interfacial rheology analysis at oil-water interfaces (25 °C) stabilized with sorghum protein extracts BR501(T-) (A, B, C), BRS310(T-) (D, E, F), BRS305(T+) (G, H, I), and SC782(T+), for applied deformation at fixed frequency (0.02 Hz). BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. HPH and NP represent samples treated and non-treated by high-pressure homogenization, respectively, and 0.1 and 1g/L indicate the protein concentration in the suspension. .................................................................................................................................. 87 Supplementary Figure 3.1. Dilatational elastic (E’d) and viscous (E”d) moduli at oil- water interfaces stabilized with sorghum protein extracts, at room temperature (25 °C), for applied deformation at fixed frequency of 0.02 Hz for BR501(T-) (A) and SC782(T+) (B) samples and applied frequency at fixed amplitude of 5 % for BR501(T-) (C) and SC782(T+) (D) samples. BR501(T-) is one of the cultivars without tannins, whereas SC782(T+) is one of the cultivars with tannins. HPH and NP represent samples treated and non-treated by high pressure homogenization, respectively, and 0.1 and 1g/L indicate the protein concentration in the suspension. ................................................ 94 Figure 4.1. Droplet size distribution (volume frequency) in the undiluted (square and dotted lines) and SDS-diluted (solid lines) emulsions produced with sorghum protein suspensions non-treated by HPH (A, C, E, and G) and HPH pre-treated (B, D, F, and H) for different sorghum cultivars: BR501(T-) (A, B) , BRS310(T-) (C, D), BRS305(T+) (E, F), and SC782(T+) (G, H). BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with endogenous tannins. .................................................................................................................... 103 Figure 4.2. Optical and CLSM micrographics of emulsions stabilized by untreated (A, B, E, F) and HPH-treated (C, D, G, H) protein suspensions from sorghum cultivars BR501(T-) (A, B, C, D) and SC782(T+) (E, F, G, H). Lipids were stained with Nile red and are visualized in red, whereas proteins were stained with fast green and are visualized in green. BR501(T-) is a tannin-free cultivar and SC782(T+) is a tannin-rich one. White arrows indicate starch granules. ............................................................ 105 Figure 4.3. Protein surface load in washed cream phase for sorghum protein-based emulsions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. The suffix NP represents emulsions produced with suspensions non-treated by HPH. .................................. 107 Figure 4.4. Protein composition of the washed cream phase for sorghum protein-based emulsions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. The suffix NP represents emulsions produced with suspensions non-treated by HPH. .................................. 108 Figure 4.5. Droplet size distribution (in volume frequency, no dilute in SDS), macroscopic aspect and optical micrographics of emulsions stabilized with sorghum protein, at pH 7, stored at 20 °C for 28 days for samples BR501(T-) (A, C, and E), and SC782(T+) (B, D, and F), at days 0 (I), 7 (II), and 28 (III). BR501(T-) is one of the cultivars without tannins, whereas SC782(T+) is one of the cultivars with tannins. . 110 Figure 4.6. Droplet size distribution (in volume frequency, no dilute in SDS), macroscopic aspect and optical micrographics of emulsions stabilized with sorghum protein, stored at 20 °C, for samples BR501(T-) (A, C, and E), and SC782(T+) (B, D, and F), at pHs 7 (I), 6 (II), 5 (III), 4 (IV), and 3 (V). BR501(T-) is one of the cultivars without tannins, whereas SC782(T+) is one of the cultivars with tannins. ............... 113 Figure 4.7. Contour plots of creaming index (CI) (in percentage) considering pH × storage time for emulsions stored at 20 °C, produced with sorghum protein suspensions of four different cultivars: untreated (A) and HPH-treated (B) BR501(T-); HPH-treated BRS310(T-) (C), HPH-treated BRS305(T+) (D); and untreated (E) and HPH-treated (F) SC782(T+). BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ........ 115 Figure 4.8. Budding-like behavior on emulsions stabilized by sorghum protein, at pH 3, produced with tannin-rich samples BRS305(T+) (A) and SC782(T+) prepared with untreated (B) and HPH-treated (C) suspensions. .................................................... 116 14 Supplementary Figure 4.1. Preliminary tests to optimize conditions for high-pressure homogenization (HPH) treatment in: emulsions produced with sorghum protein suspensions without pre-treatment by HPH for samples BR501(T-) (A), BRS310(T-) (B), BRS305(T+) (C), and SC782(T+) (D); emulsions produced with sorghum protein suspensions pre-treated by HPH for samples BR501(T-) (E), BRS310(T-) (F), BRS305(T+) (G), and SC782(T+) (H); and final tests for emulsions produced with sorghum protein suspensions pre-treated by HPH for P = 100 bars (I) and P = 200 bars (J). BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ......................................................... 121 Supplementary Figure 4.2. Surface load measured in non-washed cream phase for sorghum protein-based emulsions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. The suffix NP represents emulsions produced with suspensions non-treated by HPH. ................................................................................................................................ 122 Supplementary Figure 4.3. Protein composition measured in non-washed cream phase for sorghum protein-based emulsions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. The suffix NP represents emulsions produced with suspensions non-treated by HPH. ................................................................................................................................ 123 Supplementary Figure 4.4. Droplet size distribution (distribution in volume frequency determined by static light scattering) of emulsions stabilized with sorghum protein, at pH 7, stored at 20 °C for 28 days for BR501(T-) produced with non-treated (A) and pre- treated (B) suspensions, BRS310(T-) produced with pre-treated suspension (C), BRS305(T+) produced with pre-treated suspension (D), and SC782(T+) produced with non-treated (E) and pre-treated (F) suspensions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. .................................................................................................................... 124 Supplementary Figure 4.5. Droplet size distribution (distribution in volume frequency determined by static light scattering) of emulsions stabilized with sorghum protein, stored at 20 °C, for study of pH stability for BR501(T-) produced with non-treated (A) and pre-treated (B) suspensions, BRS310(T-) produced with pre-treated suspension (C), BRS305(T+) produced with pre-treated suspension (D), and SC782(T+) produced with non-treated (E) and pre-treated (F) suspensions. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ............................................................................................................ 125 Supplementary Figure 4.6. Zeta potential of sorghum protein suspensions. BR501(T-) and BRS310(T-) are free-tannin samples and BRS305(T+) and SC782(T+) are rich- tannin samples. ....................................................................................................... 126 Figure 5.1. Formation of conjugated dienes – CD (A) and hydroperoxides – HPX (B) during incubation of the emulsions stabilized with sorghum proteins, at 40 °C, in the presence of FeSO4/EDTA (1/1; M/M; 200 µM). BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. *Error bars represent standard deviations. ........................... 137 Figure 5.2. Formation of malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS) during incubation of the emulsions stabilized by tannin-free and tannin-rich sorghum proteins (A) and by tannin-rich sorghum proteins on an enlarged scale (B), at 40 °C, in the presence of FeSO4/EDTA (1/1; M/M; 200 µM). BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ......................................................... 139 Figure 5.3. Effect of lipid oxidation on the normalized tryptophan (Trp) fluorescence (A) (excitation = 290 nm, emission = 320 nm) and 3D fluorescence maps of BR501(T-) (B) and SC782(T+) (C) samples during incubation of the emulsions stabilized by sorghum proteins, at 40 °C, in the presence of FeSO4/EDTA. BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ........................................................................................ 141 Figure 5.4. Reversed-phase chromatograms, at 280 nm, of the acidified methanol extract of sorghum protein samples BR501(T-) (A), BRS310(T-) (B), BRS305(T+) (C) and SC782(T+) (D). BR501(T-) and BRS310(T-) are the cultivars without endogenous tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ........ 143 Supplementary Figure 5.1. Level of oxidation measured in the lipid phase extracted from the sorghum protein extracts used to stabilize oil-in-water emulsions (O/W) regarding the formation of primary oxidation products. Secondary products (TBARS and MDA) were also measured but were found to be below the quantification threshold (10 nmol/g lipid), for all samples. ............................................................................. 152 LIST OF TABLES Table 2.1. Composition (g/100 g total matter) of sorghum flours and their protein extracts. BR501(T-) and BRS310(T-) are the cultivars without tannins, whereas BRS305(T+) and SC782(T+) are the cultivars with tannins. ...................................... 45 Supplementary Table 2.1. Amino acid composition (g/kg of powder) of sorghum protein extracts. Free-tannin and rich-tannin cultivars are represented with (T-) and (T+), respectively. .............................................................................................................. 71 Supplementary Table 2.2. Fatty acid composition (% of total fatty acids) of the lipids extracted from sorghum flours and their respective protein extracts. Free-tannin and rich-tannin cultivars are represented with (T-) and (T+), respectively. The three major fatty acids in these samples are indicated in bold font. ............................................. 72 Table 5.1. LC-UV-Visible/MS identification of the flavanols in sorghum protein extracts rich in endogenous tanninsa. ................................................................................... 145 Table 5.2. Concentrations of flavanols and percentage of terminal and extension flavanol subunits in sorghum protein extracts rich in endogenous tanninsa............. 145 SUMMARY 1 INTRODUCTION ................................................................................................ 20 2 OBJECTIVES .................................................................................................... 22 2.1 General objective .............................................................................................. 22 2.2 Specific objectives ............................................................................................ 22 3 THESIS ORGANIZATION .................................................................................. 23 CHAPTER 1 – LITERATURE REVIEW .................................................................. 24 1.1 Emulsions .......................................................................................................... 24 1.2 Sorghum ............................................................................................................ 27 1.2.1 Sorghum protein ........................................................................................... 29 1.2.2 Sorghum polyphenols ................................................................................... 31 1.3 Lipid Oxidation .................................................................................................. 33 CHAPTER 2 – HIGH PRESSURE HOMOGENIZATION AS AN EFFICIENT MEANS TO IMPROVE THE AQUEOUS DISPERSIBILITY OF TANNIN- AND LIPID- RICH SORGHUM PROTEIN EXTRACTS ................................................................. 35 2.1 Introduction ....................................................................................................... 37 2.2 Materials and Methods ..................................................................................... 38 2.2.1 Characterization of sorghum flours and freeze-dried protein extracts .......... 39 2.2.2 Lipid extraction and composition analysis .................................................... 39 2.2.3 Fourier transform infrared (FTIR) spectrophotometry ................................... 41 2.2.4 Protein composition analysis ........................................................................ 41 2.2.5 Production and characterization of protein suspensions .............................. 42 2.2.6 Statistical analysis ........................................................................................ 44 2.3 Results and Discussion .................................................................................... 44 2.3.1 Composition of the sorghum flours and protein extracts .............................. 44 2.3.2 Behavior of the protein extracts in aqueous suspensions ............................ 56 2.4 Conclusions ...................................................................................................... 65 Funding .................................................................................................................... 65 Acknowledgements ................................................................................................. 65 References ............................................................................................................... 66 Appendix – Supplementary Information................................................................ 70 CHAPTER 3 – THE COMPOSITIONAL AND STRUCTURAL COMPLEXITY OF SORGHUM PROTEIN INGREDIENTS DRIVES THEIR INTERFACIAL BEHAVIOR…………………………………………………………………………………. 73 3.1 Introduction ....................................................................................................... 75 3.2 Materials and Methods ..................................................................................... 76 3.2.1 Preparation of materials ............................................................................... 76 18 3.2.2 Interfacial properties ..................................................................................... 77 3.3 Results and Discussion .................................................................................... 78 3.3.1 Adsorption kinetics at the oil-water interface ................................................ 78 3.3.2 Rheological properties of the films formed at the oil-water interface ............ 81 3.4 Conclusions ...................................................................................................... 89 Funding .................................................................................................................... 90 References ............................................................................................................... 90 Appendix – Supplementary Information................................................................ 94 CHAPTER 4 – EMULSIONS PRODUCED WITH TANNIN-FREE AND TANNIN- RICH SORGHUM PROTEIN INGREDIENTS: CHARACTERIZATION AND STABILITY 95 4.1 Introduction ....................................................................................................... 97 4.2 Materials and Methods ..................................................................................... 98 4.2.1 Characterization of emulsions ...................................................................... 99 4.2.2 Physical and pH stabilities of emulsions ..................................................... 101 4.2.3 Statistics ..................................................................................................... 102 4.3 Results and discussion .................................................................................. 102 4.3.1 Production and characterization of emulsions ............................................ 102 4.3.2 Emulsion stability ....................................................................................... 109 4.4 Conclusions .................................................................................................... 116 Funding .................................................................................................................. 117 References ............................................................................................................. 117 Appendix – Supplementary Information.............................................................. 121 CHAPTER 5 – OXIDATIVE STABILITY OF EMULSIONS PRODUCED USING SORGHUM PROTEIN INGREDIENTS WITH NATURAL PROTEIN-POLYPHENOL COMPLEXES…………………. ................................................................................ 127 5.1 Introduction ..................................................................................................... 129 5.2 Materials and Methods ................................................................................... 131 5.2.1 Analysis of lipid oxidation products ............................................................ 132 5.2.2 Polyphenol characterization and antioxidant activity of sorghum protein extracts…. ............................................................................................................... 134 5.2.3 Statistics ..................................................................................................... 135 5.3 Results and discussion .................................................................................. 135 5.3.1 Lipid Oxidation ........................................................................................... 135 5.3.2 Polyphenol characterization and antioxidant activity of sorghum protein extracts…… ............................................................................................................. 142 5.4 Conclusions .................................................................................................... 146 Funding .................................................................................................................. 147 Acknowledgements ............................................................................................... 147 References ............................................................................................................. 147 Appendix – Supplementary Information.............................................................. 152 4 GENERAL DISCUSSION ................................................................................ 153 5 GENERAL CONCLUSIONS ............................................................................ 156 6 SUGESTIONS FOR FUTURE STUDIES ......................................................... 157 REFERENCES ........................................................................................................ 158 20 1 INTRODUCTION Delivery systems based on oil-in-water (O/W) emulsions consist of dispersing oil as small droplets in a continuous aqueous phase. Such systems are widely used for stabilization and protection of lipophilic food ingredients, but their preparation must be done carefully in order to ensure physical and chemical stability when it is subjected to adverse conditions commonly found in foods, such as high or low temperatures, humidity, and pH variations (LIU et al., 2016a). To ensure such stabilization, it is common to use surfactants, which decrease the interfacial tension, helping to break larger droplets into smaller ones and preventing coalescence, which, in turn, happens when droplets that are not sufficiently stabilized collide (MUIJLWIJK et al., 2017). Among the surfactants that can be used to stabilize emulsions, proteins stand out because they come from natural sources, reflecting the green tendency in food industry. They also present some advantages such as biocompatibility, biodegradability, good amphiphilic and functional properties, besides presenting adsorption sites capable of anchoring at the oil-water interface, inhibiting droplet aggregation and forming viscoelastic films with greater resistance to mechanical stress around the droplets (LAM; NICKERSON, 2013; NICOLETTI TELIS, 2018). Due to their conformation and aggregation state, the general use of proteins as emulsifiers is limited (LIU et al., 2016a) and their use alone may be insufficient to fulfill the requirements needed to confer long term stability to emulsified systems (NICOLETTI TELIS, 2018). In order to overcome possible adverse factors, there is great interest in improving the attributes of proteins as emulsifiers, using, for example, protein- polyphenol complexes, formed through intermolecular interactions, or protein- polyphenol conjugates, formed through covalent interactions, further improving the stability of emulsions (OZDAL et al., 2018; WANG et al., 2015). Polyphenols are secondary metabolites produced by plants for their defense against predators and to reduce pre- and post-harvest stresses (JAKOBEK, 2015; QUEIROZ et al., 2018). The study of these substances have gained emphasis due to their binding characteristics with proteins to form complexes, which can help stabilize colloidal systems and improve oxidative stability in O/W emulsions (KAREFYLLAKIS et al., 2017). The main factors affecting protein-polyphenols interactions are the molecular weight and structural flexibility of the phenolic compound, as well as the 21 number of hydroxyl groups and the type of side chain. The higher the molar weight and the quantity of hydroxyl groups, the higher the affinity of the polyphenol for the protein. Protein-polyphenol interactions can be grouped into covalent, which are mostly irreversible, and non-covalent (hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals interactions), which are reversible (OZDAL et al., 2018). Sorghum is one of the most versatile grains with rentable growing and is an important vegetable source of protein (DE MESA-STONESTREET; ALAVI; BEAN, 2010; GIRARD; AWIKA, 2018), which could be used as natural surfactant to stabilize lipid compounds. Some sorghum cultivars present a native, significant content of polyphenols, known as condensed tannins (proanthocyanidins), that have greater antioxidant activity in vitro and in vivo than simple phenols and other natural antioxidants (BARROS; AWIKA; ROONEY, 2012, 2014). They also have capacity to bond to proteins to form insoluble complexes and may act as natural antioxidants. Thus, the use of sorghum protein-rich fractions extracted from cultivars with the presence of polyphenols is of potential interest to stabilize lipid compounds due to their low cost and beneficial effects. Literature reporting stabilization of emulsions using sorghum protein is scarce, and this area still has high potential for prospection. Only a few works evaluated emulsion stabilization and the antioxidant effects of protein- polyphenol interactions. In addition, to the best of our knowledge, there are no published works investigating the antioxidant activities of native sorghum protein- polyphenol complexes. Considering these facts, the present work aimed to evaluate the effect of protein- polyphenol complexes on the stability and interfacial properties of emulsions, using protein extracts obtained from different sorghum cultivars, with or without the presence of polyphenols. After the characterization of emulsions, the influence of such complexes was also investigated regarding their oxidative stability, seeking to correlate the effect of physical properties of emulsions with the protection and stability of the lipid compound. 156 5 GENERAL CONCLUSIONS In the present thesis, a step-by-step production of sorghum fraction rich in proteins and its use in emulsions has been demonstrated. Such approach enabled to understand possible factors that can influence their behavior and subsequent use, in addition to following the “clean label” tendency. A deeper characterization of the flour and protein extracts enabled a better understanding of the protein extraction process, which reached its primary goal, concentrating the protein, but also leaded to an accumulation of lipids, mostly fatty acids. A pre-treatment by high pressure homogenization (HPH) was applied to increase the dispersibility of sorghum proteins, showing to be a promising alternative for the solubility problem of sorghum proteins in aqueous medium. Then, it was observed that an interconnected network was formed at oil-water interfaces, and this network were able to resist to dilatational deformations without lose its properties, showing the possibility of the sorghum protein ingredient as a good emulsifier. An investigation about the use of sorghum protein extracts as an emulsifier was carried out. It was found that these ingredients can form emulsions and that they are physically stable for 28 days, with minor flocculation and creaming. However, these emulsions are not stable to pH changes, presenting flocculation and creaming for pHs equal to or lower than 6 right after their formation. Finally, the oxidative stability of the emulsions was assessed, showing that natural sorghum protein-tannin complexes can delay or even inhibit lipid oxidation. To conclude, sorghum demonstrated to be a good alternative as plant-based protein, and the presence of tannins in its grain could be a strategy to combine both emulsifier and antioxidant effects in one ingredient. 158 REFERENCES AWIKA, J. M. et al. Processing of sorghum (Sorghum bicolour) and sorghum products alters procyanidin oligomer and polymer distribution and content. Journal of Agricultural and Food Chemistry, v. 51, p. 5516–5521, 2003. AWIKA, J. M.; ROONEY, L. W. Sorghum phytochemicals and their potential impact on human health. Phytochemistry, v. 65, p. 1199–1221, 2004. BADER, S.; BEZ, J.; EISNER, P. Can protein functionalities be enhanced by high- pressure homogenization? – A study on functional properties of lupin proteins. Procedia Food Science, v. 1, p. 1359–1366, 2011. BARROS, F.; AWIKA, J. M.; ROONEY, L. W. Interaction of tannins and other sorghum phenolic compounds with starch and effects on in vitro starch digestibility. Journal of Agricultural and Food Chemistry, v. 60, n. 46, p. 11609–11617, 2012. BARROS, F.; AWIKA, J.; ROONEY, L. W. Effect of molecular weight profile of sorghum proanthocyanidins on resistant starch formation. Journal of the Science of Food and Agriculture, v. 94, n. 6, p. 1212–1217, 2014. BELTON, P. S. et al. Kafirin structure and functionality. Journal of Cereal Science, v. 44, n. 3, p. 272–286, 2006. BENJAMIN, O. et al. Emulsifying Properties of Legume Proteins Compared to β- Lactoglobulin and Tween 20 and the Volatile Release from Oil-in-Water Emulsions. Journal of Food Science, v. 79, p. E2014–E2022, 2014. BERTON-CARABIN, C. C.; ROPERS, M. H.; GENOT, C. Lipid Oxidation in Oil-in- Water Emulsions: Involvement of the Interfacial Layer. Comprehensive Reviews in Food Science and Food Safety, v. 13, n. 5, p. 945–977, 2014. BERTON-CARABIN, C. C.; SCHROEN, K. Pickering emulsions for food applications: Background, trends, and challenges. Annual Review of Food Science and Technology, v. 6, p. 263–297, 2015. BURGER, T. G. et al. Comparison of physicochemical and emulsifying properties of commercial pea protein powders. Journal of the Science of Food and Agriculture, v. 102, n. 6, p. 2506–2514, 2022. BURGER, T. G.; ZHANG, Y. Recent progress in the utilization of pea protein as an emulsifier for food applications. Trends in Food Science & Technology, v. 86, p. 25– 33, 1 abr. 2019. CAPEK, I. Degradation of kinetically-stable o/w emulsions. Advances in Colloid and Interface Science, v. 107, n. 2–3, p. 125–155, mar. 2004. CHANG, C. et al. Effect of pH on the inter-relationships between the physicochemical, interfacial and emulsifying properties for pea, soy, lentil and canola protein isolates. 159 Food Research International, v. 77, p. 360–367, 1 nov. 2015. CHAPLEAU, N.; DE LAMBALLERIE-ANTON, M. Improvement of emulsifying properties of lupin proteins by high pressure induced aggregation. Food Hydrocolloids, v. 17, n. 3, p. 273–280, 1 maio 2003. CHEN, L. et al. Food-grade pickering emulsions: Preparation, stabilization and applications. Molecules, v. 25, n. 14, 2020. CHEN, M. et al. Study on the emulsifying stability and interfacial adsorption of pea proteins. Food Hydrocolloids, v. 88, p. 247–255, 1 mar. 2019. CHEW, S. C. Cold-pressed rapeseed (Brassica napus) oil: Chemistry and functionality. Food Research International, v. 131, p. 108997, maio 2020. DE MESA-STONESTREET, N. J.; ALAVI, S.; BEAN, S. R. Sorghum proteins: The concentration, isolation, modification, and food applications of kafirins. Journal of Food Science, v. 75, n. 5, 2010. DYKES, L. et al. Phenolic compounds and antioxidant activity of sorghum grains of varying genotypes. Journal of Agricultural and Food Chemistry, v. 53, p. 6813– 6818, 2005. ESPINOSA-RAMÍREZ, J.; SERNA-SALDÍVAR, S. O. Functionality and characterization of kafirin-rich protein extracts from different whole and decorticated sorghum genotypes. Journal of Cereal Science, v. 70, p. 57–65, 2016. FAOSTAT. Food and Agriculture Organization of the United Nations - FAO. Disponível em: . Acesso em: 13 dez. 2022. FRANKEL, E. N. Lipid oxidation. Scotland: The oily press Dundee, 2005. GANESAN, K.; SUKALINGAM, K.; XU, B. Impact of consumption and cooking manners of vegetable oils on cardiovascular diseases – A critical review. Trends in Food Science and Technology, v. 71, p. 132–154, 2018. GIRARD, A. L.; AWIKA, J. M. Sorghum polyphenols and other bioactive components as functional and health promoting food ingredients. Journal of Cereal Science, v. 84, n. October, p. 112–124, 2018. GU, L. et al. Protection of β-carotene from chemical degradation in emulsion-based delivery systems using antioxidant interfacial complexes: Catechin-egg white protein conjugates. Food Research International, v. 96, p. 84–93, 2017. GUMUS, C. E.; DECKER, E. A.; MCCLEMENTS, D. J. Formation and Stability of ω-3 Oil Emulsion-Based Delivery Systems Using Plant Proteins as Emulsifiers: Lentil, Pea, and Faba Bean Proteins. Food Biophysics, v. 12, p. 186–197, 2017. 160 IBGE. Levantamento Sistemático da Produção Agrícola - LSPA. Disponível em: . Acesso em: 13 dez. 2022. JAFARI, S. M.; BEHESHTI, P.; ASSADPOOR, E. Rheological behavior and stability of D-limonene emulsions made by a novel hydrocolloid (Angum gum) compared with Arabic gum. Journal of Food Engineering, v. 109, p. 1–8, 2012. JAKOBEK, L. Interactions of polyphenols with carbohydrates, lipids and proteins. Food chemistry, v. 175, p. 556–67, 2015. JALILI, F. et al. Optimization of Ultrasound-Assisted Extraction of Oil from Canola Seeds with the Use of Response Surface Methodology. Food Analytical Methods, v. 11, n. 2, p. 598–612, 2018. KAREFYLLAKIS, D. et al. Physical bonding between sunflower proteins and phenols: Impact on interfacial properties. Food Hydrocolloids, v. 73, p. 326–334, 2017. KAUR, A. et al. Chemical, thermal, rheological and FTIR studies of vegetable oils and their effect on eggless muffin characteristics. Journal of Food Processing and Preservation, v. 43, n. 7, p. 1–11, 2019. KIM, H. J.; DECKER, E. A.; MCCLEMENTS, D. J. Influence of protein concentration and order of addition on the thermal stability of betalactoglobulin stabilized n- hexadecane oil-in-water emulsions at neutral pH. Langmuir, v. 21, p. 134–139, 2005. KIRALAN, M.; RAMADAN, M. F. Volatile oxidation compounds and stability of safflower, sesame and canola cold-pressed oils as affected by thermal and microwave treatments. Journal of Oleo Science, v. 65, n. 10, p. 825–833, 2016. KONUSKAN, D. B.; ARSLAN, M.; OKSUZ, A. Physicochemical properties of cold‐ pressed sunflower, peanut, rapeseed, mustard and olive oils grown in the Eastern Mediterranean region. Saudi Journal of Biological Sciences, v. 26, n. 2, p. 340–344, 2019. LADJAL ETTOUMI, Y. et al. Legume Protein Isolates for Stable Acidic Emulsions Prepared by Premix Membrane Emulsification. Food Biophysics, v. 12, p. 119–128, 2017. LAM, A. C. Y. et al. Pea protein isolates: Structure, extraction, and functionality. Food Reviews International, v. 34, n. 2, p. 126–147, 2018. LAM, R. S. H.; NICKERSON, M. T. Food proteins A review on their emulsifying properties using a structure-function approach. Food Chemistry, v. 141, n. 2, p. 975– 984, 2013. LEVY, R.; OKUN, Z.; SHPIGELMAN, A. Utilizing high-pressure homogenization for the production of fermented plant-protein yogurt alternatives with low and high oil content using potato protein isolate as a model. Innovative Food Science and Emerging 161 Technologies, v. 75, n. December 2021, p. 102909, 2022. LI, S. et al. Development of Zein/tannic acid nanoparticles as antioxidants for oxidation inhibition of blackberry seed oil emulsions. Food Chemistry, v. 403, n. June 2022, p. 134236, 2023. LIU, C.; PEI, R.; HEINONEN, M. Faba bean protein: A promising plant-based emulsifier for improving physical and oxidative stabilities of oil-in-water emulsions. Food Chemistry, v. 369, n. December 2020, p. 130879, 2022. LIU, F. et al. Development of polyphenol-protein-polysaccharide ternary complexes as emulsifiers for nutraceutical emulsions: Impact on formation, stability, and bioaccessibility of β-carotene emulsions. Food Hydrocolloids, v. 61, p. 578–588, 2016a. LIU, F. et al. Utilization of interfacial engineering to improve physicochemical stability of β-carotene emulsions: Multilayer coatings formed using protein and protein- polyphenol conjugates. Food Chemistry, v. 205, p. 129–139, 2016b. LIU, L. et al. Optimization of extraction of polyphenols from Sorghum Moench using response surface methodology, and determination of their antioxidant activities. Tropical Journal of Pharmaceutical Research, v. 17, n. 4, p. 619–626, 2018. LUO, L. et al. Impact of high-pressure homogenization on physico-chemical, structural, and rheological properties of quinoa protein isolates. Food Structure, v. 32, n. September 2021, p. 100265, 2022. MA, K. K. et al. Functional Performance of Plant Proteins. Foods, v. 11, n. 4, p. 1–23, 2022. MCCLEMENTS, D. J. Food emulsions: principles, practice and techniques. 2nd. ed. USA: CRC Press, 2005. MEHMOOD, S. et al. Fatty acid composition of seed oil of different Sorghum bicolor varieties. Food Chemistry, v. 109, n. 4, p. 855–859, 2008. MELCHIOR, S. et al. High pressure homogenization shapes the techno-functionalities and digestibility of pea proteins. Food and Bioproducts Processing, v. 131, p. 77– 85, 2022. MOLL, P. et al. Impact of microfluidization on colloidal properties of insoluble pea protein fractions. European Food Research and Technology, v. 247, n. 3, p. 545– 554, 2021. MUIJLWIJK, K. et al. Coalescence of protein-stabilised emulsions studied with microfluidics. Food Hydrocolloids, v. 70, p. 96–104, 2017. MWANGI, W. W. et al. Food-grade Pickering emulsions for encapsulation and delivery of bioactives. Trends in Food Science & Technology, v. 100, p. 320–332, 2020. 162 NACZK, M.; SHAHIDI, F. Extraction and analysis of phenolics in food. Journal of Chromatography, v. 1054, p. 95–111, 2004. NICOLETTI TELIS, V. R. O/W Emulsions Stabilized by Interactions Between Proteins and Polysaccharides. Encyclopedia of Food Chemistry, p. 494–498, 2018. NISHINARI, K. et al. Soy proteins: A review on composition, aggregation and emulsification. Food Hydrocolloids, v. 39, p. 301–318, 1 ago. 2014. OZDAL, T. et al. Polyphenol-Protein Interactions and Changes in Functional Properties and Digestibility. Encyclopedia of Food Chemistry, p. 566–577, 2018. PORTER, L. J. Structure and Chemical Properties of the Condensed Tannins. In: HEMINGWAY, R. W.; LAKS, P. E. (Eds.). . Plant Polyphenols: Synthesis, Properties, Significance. Boston, MA: Springer, 1992. v. 59p. 104–116. PRICE, M. L.; SCOYOC, S. VAN; BUTLER, L. G. A Critical Evaluation of the Vanillin Reaction as an Assay for Tannin in Sorghum Grain. Journal of Agricultural and Food Chemistry, v. 26, n. 5, p. 1214–1218, 1978. PRIMOZIC, M. et al. Effect of lentil proteins isolate concentration on the formation, stability and rheological behavior of oil-in-water nanoemulsions. Food Chemistry, v. 237, p. 65–74, 15 dez. 2017. PROIETTI, I.; FRAZZOLI, C.; MANTOVANI, A. Exploiting nutritional value of staple foods in the world’s semi-arid areas: risks, benefits, challenges and opportunities of sorghum. Healthcare, v. 3, n. 2, p. 172–193, 2015. QUEIROZ, V. A. V. et al. O sorgo. Revista Brasileira de Milho e Sorgo, v. 10, n. 3, p. 180–195, 2011. QUEIROZ, V. A. V. et al. Potencial do sorgo para uso na alimentação humano. Informe Agropecuário, v. 35, n. 278, p. 7–12, 2014. QUEIROZ, V. A. V. et al. A low calorie and nutritive sorghum powdered drink mix: Influence of tannin on the sensorial and functional properties. Journal of Cereal Science, v. 79, p. 43–49, 2018. RAMOS, L. B. et al. Optimization of Microwave Pretreatment Variables for Canola Oil Extraction. Journal of Food Process Engineering, v. 40, n. 3, 2017. RAO, S. et al. Cereal phenolic contents as affected by variety and environment. Cereal Chemistry, v. 95, n. 5, p. 589–602, 2018. ROSZKOWSKA, B. et al. Variation in the composition and oxidative stability of commercial rapeseed oils during their shelf life. European Journal of Lipid Science and Technology, v. 117, n. 5, p. 673–683, 2015. SÁNCHEZ, R. J.; FERNÁNDEZ, M. B.; NOLASCO, S. M. Hexane-Free Green Solvent Extraction of Canola Oil From Microwave-Pretreated Seeds and of Antioxidant-Rich 163 Byproducts. European Journal of Lipid Science and Technology, v. 120, n. 9, p. 1– 8, 2018. SÁNCHEZ, R. J.; FERNÁNDEZ, M. B.; NOLASCO, S. M. Canola Oil with High Antioxidant Content Obtained by Combining Emerging Technologies: Microwave, Ultrasound, and a Green Solvent. European Journal of Lipid Science and Technology, v. 121, n. 11, p. 1–9, 2019. SCHAICH, K. M. Lipid Oxidation: New Perspectives on an Old Reaction. In: SHAHIDI, F. (Ed.). . Bailey’s Industrial Oil and Fat Products. [s.l: s.n.]. p. 1–72. SERNA-SALDIVAR, S.; ROONEY, L. W. Structure and chemistry of sorghum and millets. In: Sorghum and Millets: Chemistry and Technology. St Paul, MN: American Association of Cereal Chemists, 1995. p. 69–124. SERRANO, J. et al. Tannins: Current knowledge of food sources, intake, bioavailability and biological effects. Molecular Nutrition and Food Research, v. 53, n. SUPPL. 2, p. 310–329, 2009. SHEN, Y.; HONG, S.; LI, Y. Pea protein composition, functionality, modification, and food applications: A review. Advances in Food and Nutrition Research, v. 101, p. 71–127, 1 jan. 2022. SHI, A. et al. Pickering and high internal phase Pickering emulsions stabilized by protein-based particles: A review of synthesis, application and prospective. Food Hydrocolloids, v. 109, n. February, p. 1–15, 2020. SIGER, A.; KACZMAREK, A.; RUDZIŃSKA, M. Antioxidant activity and phytochemical content of cold-pressed rapeseed oil obtained from roasted seeds. European Journal of Lipid Science and Technology, v. 117, n. 8, p. 1225–1237, 2015. SURH, J.; DECKER, E. A.; MCCLEMENTS, J. Properties and stability of oil-in-water emulsions stabilized by fish gelatin. Food Hydrocolloids, v. 20, p. 596–606, 2006. SYMONIUK, E.; RATUSZ, K.; KRYGIER, K. Evaluation of the Oxidative Stability of Cold-Pressed Rapeseed Oil by Rancimat and Pressure Differential Scanning Calorimetry Measurements. European Journal of Lipid Science and Technology, v. 121, n. 2, p. 1–8, 2019. TADROS, T. F. Emulsion Science and Technology. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA, 2009. TAYLOR, J. et al. Kafirin microparticle encapsulation of catechin and sorghum condensed tannins. Journal of Agricultural and Food Chemistry, v. 57, n. 16, p. 7523–7528, 2009. TAYLOR, J.; TAYLOR, J. R. N. Making Kafirin, the Sorghum Prolamin, into a Viable Alternative Protein Source. JAOCS, Journal of the American Oil Chemists’ Society, v. 95, n. 8, p. 969–990, 2018. 164 TCHOLAKOVA, S. et al. Coalescence stability of emulsions containing globular milk proteins. Advances in Colloid and Interface Science, v. 16, p. 123- 126:259–93, 2006. WALSTRA, P. Formation of Emulsions and Foams. In: Physical Chemistry of Foods. New York: Marcel Dekker Inc., 2003. WAN, Z. L. et al. Complexation of resveratrol with soy protein and its improvement on oxidative stability of corn oil/water emulsions. Food Chemistry, v. 161, p. 324–331, 2014. WANG, X. et al. Physicochemical characterisation of β-carotene emulsion stabilised by covalent complexes of α-lactalbumin with (-)-epigallocatechin gallate or chlorogenic acid. Food Chemistry, v. 173, p. 564–568, 2015. WINKLER-MOSER, J. K.; LOGAN, A.; BAKOTA, E. L. Antioxidant activities and interactions of α- and γ-tocopherols within canola and soybean oil emulsions. European Journal of Lipid Science and Technology, v. 116, n. 5, p. 606–617, 2014 . XIAO, J. et al. Structure, morphology, and assembly behavior of kafirin. Journal of Agricultural and Food Chemistry, v. 63, n. 1, p. 216–224, 2015. XIAO, J. et al. Kafirin nanoparticles-stabilized Pickering emulsions: Microstructure and rheological behavior. Food Hydrocolloids, v. 54, p. 30–39, 2016. XIAO, J.; CHEN, Y.; HUANG, Q. Physicochemical properties of kafirin protein and its applications as building blocks of functional delivery systems. Food and Function, v. 8, n. 4, p. 1402–1413, 2017. XIAO, J.; LU, X.; HUANG, Q. Double emulsion derived from kafirin nanoparticles stabilized Pickering emulsion: Fabrication, microstructure, stability and in vitro digestion profile. Food Hydrocolloids, v. 62, p. 230–238, 2017. YANG, J. et al. Effects of high pressure homogenization on faba bean protein aggregation in relation to solubility and interfacial properties. Food Hydrocolloids, v. 83, n. February, p. 275–286, 2018. YI, J. et al. Characterization of catechin-α-lactalbumin conjugates and the improvement in β-carotene retention in an oil-in-water nanoemulsion. Food Chemistry, v. 205, p. 73–80, 2016. ZHAO, Z. et al. Interfacial engineering of pickering emulsion costabilized by zein nanoparticles and tween 20: Effects of the particle size on the interfacial concentration of gallic acid and the oxidative stability. Nanomaterials, v. 10, n. 6, 2020.