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. 



22 
 

 
 

2 OBJECTIVES 

 

2.1 General objective 

 

The overall objective of this work was to explore the use of sorghum protein 

extracts as a surfactant agent in emulsions stabilization, investigating the effects of 

protein-polyphenol complexes in both interfacial properties and oxidative stability of 

the lipid phase. 

 

2.2 Specific objectives 

 

The specific objectives include: 

i. production and characterization of protein extracts obtained from different 

sorghum cultivars free or rich in endogenous tannins; 

ii. investigation of the interfacial properties of sorghum fractions rich in proteins 

and lipids as a function of the presence or absence of complexed tannins; 

iii. production and characterization of O/W emulsions prepared with sorghum 

protein extracts free or rich in endogenous tannins, studying their stability and 

microstructure; 

iv. study of the oxidative stability of canola oil in emulsions formulated with 

sorghum protein extracts free or rich in endogenous tannins.



23 
 

 
 

3 THESIS ORGANIZATION 

 

This thesis is divided into five chapters. Chapter 1 is composed by a literature 

review describing the concepts used and discussed throughout the thesis. 

In Chapter 2, sorghum protein extracts were obtained from four different sorghum 

cultivars, in which two of them presented tannin and the other two were tannin-free 

samples. Both flours and their respective protein extracts were deeply characterized 

regarding their composition, assessing also their use for production of protein 

suspensions. As sorghum proteins present a low solubility, a pre-treatment with high-

pressure homogenization (HPH) was applied to analyze its effects on the protein 

dispersibility in aqueous media. 

Chapter 3 presents the interfacial properties and rheological behavior of sorghum 

proteins at oil-water interfaces, as proteins can be used as emulsifiers and produce 

stable emulsions if they are able to form a strong network at interfaces. This chapter 

shows how the pre-treatment by HPH and protein concentration can affect interfacial 

and rheological behavior.  

In Chapter 4, the application of the characterized sorghum proteins was studied 

through the production of O/W emulsions, in which canola oil was used as the lipid 

compound. The emulsions were evaluated by their droplet size, morphology, and 

interfacial composition. The physical stability of these emulsions was investigated for 

28 days, and the pH stability was analyzed for pHs between 3 and 7. 

Chapter 5 presents the oxidative stability of O/W emulsions stabilized by sorghum 

proteins or endogenous sorghum protein-polyphenol complexes, as well as the 

characterization of such polyphenols. The antioxidant activity of the protein extracts 

was also evaluated in order to correlate the results obtained for the tannin-rich 

samples. 



24 
 

 
 

CHAPTER 1 – LITERATURE REVIEW 

 

1.1 Emulsions 

 

Emulsions are dispersed systems composed of two immiscible liquids, typically 

water and oil, in which one of the liquids is dispersed in the other, using mechanical 

energy, resulting in small droplets (0.1 – 100 m). The phase distributed in the droplets 

is called the dispersed phase, whereas the phase in which the droplets are dispersed 

is known as the continuous phase. Emulsions can be classified, basically, as oil-in-

water (O/W) or water-in-oil (W/O). In the food industry, examples of O/W emulsions 

are milk and various dairy products, creams, mayonnaise and soups, while butter and 

margarine, for example, are W/O emulsions (BERTON-CARABIN; SCHROEN, 2015; 

CHEN et al., 2020; LAM; NICKERSON, 2013). 

During the production of an emulsion, the interfacial area between the immiscible 

compounds increases, which increases the interfacial energy of the system, and 

requires the use of energy to compensate this increase. The energy is first transferred 

to the interfacial region to help with the droplet formation, and then it is transferred from 

the continuous phase to the particles to help reduce the size of the already formed 

droplets (WALSTRA, 2003). 

Considered as thermodynamically unstable systems, in which the phases seek 

to arrange themselves towards a more stable state that minimizes free energy – i.e., 

the separation into more dense aqueous layer and a less dense upper layer –, 

emulsions present several possible destabilizing mechanisms, such as coalescence, 

creaming, sedimentation and/or flocculation, among others (CAPEK, 2004; MWANGI 

et al., 2020; TADROS, 2009). To reduce the interfacial tension and maintain the 

stability of the emulsions, the use of emulsifiers is essential (MWANGI et al., 2020; SHI 

et al., 2020). Such compounds have surfactant capacity, adsorbing at the interface, 

which prevents the coalescence of newly formed droplets and thus maintains the 

kinetic stability of the emulsion. In addition, emulsifiers are also able to decrease the 

interfacial tension between immiscible liquids, which facilitates the breakdown of the 

droplets into smaller ones  (MCCLEMENTS, 2005; WALSTRA, 2003). 

Following the trend and preference for consumption and/or use of natural 

products, the use of natural or non-synthetic surfactants is in great evidence. Thus, 



25 
 

 
 

proteins have gained prominence in this area, since they have amphiphilic nature and 

emulsifying ability and meet the growing demand for natural products (LAM; 

NICKERSON, 2013). Proteins tend to diffuse slowly to the interface (Figure 1.1A), 

unlike emulsifiers with low molecular weight, rearranging themselves so as to position 

their hydrophobic groups in contact with the oil phase and their hydrophilic sites in 

contact with the aqueous phase (Figure 1.1B) (MCCLEMENTS, 2005; SURH; 

DECKER; MCCLEMENTS, 2006; WALSTRA, 2003). However, when they reach the 

interface, it is common to develop strong viscoelastic films (Figure 1.1C), which can 

resist to mechanical stress and provide stabilization by electrostatic effects (Figure 

1.2A) and by steric hindrance (Figure 1.2B) (TCHOLAKOVA et al., 2006).  

 

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. 

 

Source: Author’s compilation. 

 



26 
 

 

Figure 1.2. Mechanisms of emulsion stabilization: (A) electrostatic effects and (B) 

steric hindrance. 

 

Source: Author’s compilation. 

 

The functionality as an emulsifier is directly linked to the surface hydrophobicity 

of the protein, which influences the ability to adsorb at the oil interface (KIM; DECKER; 

MCCLEMENTS, 2005). The surface charge of the protein, on the other hand, 

influences its solubility in the aqueous phase. After the formation of the viscoelastic 

film, the pH of the emulsion can cause the droplets to take on positive charges (below 

the isoelectric point) or negative ones (above the isoelectric point). The greater the 

electrostatic repulsion between the oil droplets, the greater the stability of the emulsion. 

However, the closer to the isoelectric point, the greater the emulsion instability due to 

flocculation and aggregation of the droplets, which may cause partial or complete 

coalescence (MCCLEMENTS, 2005; TCHOLAKOVA et al., 2006). Because they have 

lower density, the largest droplets, formed by coalescence, separate from the 

continuous phase and migrate upward, causing creaming (ROBINS, 2000). The 

viscosity of the emulsion increases due to the presence of proteins in the continuous 

phase, which reduces the mobility and diffusion of the oil droplets (JAFARI; 

BEHESHTI; ASSADPOOR, 2012). Nevertheless, the stability of protein-stabilized 

emulsions tend to decrease over time, since during aging, proteins undergo 

conformational changes and then form non-covalent bonds with neighboring proteins 

(KIM; DECKER; MCCLEMENTS, 2005; TCHOLAKOVA et al., 2006). 

Plant-based foods are highlighted nowadays driven by the perceived health and 

environmental benefits. Thus, proteins extracted from plants are being studied and 

applied in different food products to replace animal-based proteins and to follow the 



27 
 

 
 

clean-label trend. Many proteins from plants are being evaluated regarding their 

surface activity to produce stable emulsions, such as soybean, pea, lupin, lentil and 

faba bean proteins (BENJAMIN et al., 2014; BURGER; ZHANG, 2019; CHANG et al., 

2015; CHAPLEAU; DE LAMBALLERIE-ANTON, 2003; CHEN et al., 2019; GUMUS; 

DECKER; MCCLEMENTS, 2017; LADJAL ETTOUMI et al., 2017; LAM et al., 2018; 

LIU; PEI; HEINONEN, 2022; MA et al., 2022; NISHINARI et al., 2014; PRIMOZIC et 

al., 2017; SHEN; HONG; LI, 2022), among others less studied, such as sorghum and 

maize proteins (XIAO et al., 2016; XIAO; LU; HUANG, 2017; ZHAO et al., 2020), which 

are prolamins. 

Despite the increasing tendency to study and apply these proteins as emulsifiers, 

they are often sparingly soluble in water, contributing to rise the challenge in their use 

and demanding treatments that can modify their structure and functionally. High 

pressure homogenization can be a promising technology, once it can modify the 

particle size and the structure of the protein, improving their techno-functionalities 

(BADER; BEZ; EISNER, 2011), as it was observed in studies using pea, faba bean, 

potato, and quinoa proteins (BURGER et al., 2022; LEVY; OKUN; SHPIGELMAN, 

2022; LUO et al., 2022; MELCHIOR et al., 2022; MOLL et al., 2021; YANG et al., 2018).  

 

1.2 Sorghum 

 

Sorghum (Sorghum bicolor L.) (Figure 1.3) is a versatile grain, commonly grown 

in arid regions of Africa, Asia, Australia, and North and South America (GIRARD; 

AWIKA, 2018), and its cultivation is profitable due to its ability to adapt to different 

environmental conditions. According to data available by the Food and Agriculture 

Organization (FAO), the cereal was the sixth most produced in the world in 2020, with 

a total production of 58.705.915 tons, with the largest producers being the United 

States, Nigeria and Ethiopia, and Brazil occupying the seventh position (FAOSTAT, 

2022). According to data from the Brazilian Institute of Geography and Statistics 

(IBGE), sorghum was the seventh most produced grain in Brazil in the years 2021 and 

2022, and its production in 2022 had an increase of 18.3 % (2.850.368 tons) compared 

to 2021 (2.409.724 tons), with emphasis on the Midwest and Southeast regions, 

responsible for approximately 85 % of the total grain production (IBGE, 2022). 

 



28 
 

 

Figure 1.3. Sorghum grain (Sorghum bicolor L. Moench). 

 

Source: Robert Soreng, hosted by the USDA-NRCS PLANTS Database, 2021. 

 

In the semi-arid zones of Africa, South America and Asia, large populations 

consume sorghum as a staple food (PROIETTI; FRAZZOLI; MANTOVANI, 2015; 

QUEIROZ et al., 2011), but its production is still mostly destined for industrial 

conversion into alcohol and for animal feed (LIU et al., 2018). In countries such as 

Japan, the United States and Brazil, sorghum has been studied and has gained 

popularity for use in human food, since it has a low production cost and adapts easily 

to dry and/or hot climates (QUEIROZ et al., 2014). Due to its high concentration of 

phenolic compounds (anthocyanins and tannins) and the content of dietary fiber, this 

cereal presents itself as a good alternative to the consumption of conventional cereals.  

Sorghum grains can be classified into three types: type I relates to grains without 

significant levels of condensed tannins; type II relates to grains in which tannins are 

extracted only in acidified methanol; and type III relates to grains in which tannins are 

extracted both in methanol and acidified methanol (PRICE; SCOYOC; BUTLER, 

1978). Sorghum has the highest levels of high molar mass tannins among cereals 



29 
 

 
 

(DYKES et al., 2005; SERNA-SALDIVAR; ROONEY, 1995), and such substances 

have powerful antioxidant activity, either in vitro or in vivo, and the ability to interact 

with proteins (BARROS; AWIKA; ROONEY, 2014). 

Sorghum is mainly composed of carbohydrates (75 %), and its dietary fiber 

content is about 6 %. Lipids account for approximately 3 %, with the highest amount 

of polyunsaturated fatty acids: oleic acid (31.1 - 48.9 %), linoleic acid (27.6 - 50.7 %), 

palmitic acid (11.7 - 20.2 %) and linolenic acid (1.7 - 3.9 %) (MEHMOOD et al., 2008). 

Regarding the protein content, the grain represents an important vegetable protein 

source among cereals, with contents ranging from 6 to 18 % (DE MESA-

STONESTREET; ALAVI; BEAN, 2010). According to a study conducted at Empraba 

Milho e Sorgo in Sete Lagoas/MG/Brazil, protein contents of eight Brazilian sorghum 

genotypes ranged between 8.57 and 11.59 % (QUEIROZ et al., 2014). 

 

1.2.1 Sorghum protein 

 

Kafirin, the most abundant protein in sorghum, belongs to the prolamin family, 

which resembles zein in solubility, molar mass, amino acid composition and 

polypeptide structure, but is more hydrophobic, allowing the production of films with  

superior gas and water vapor barrier characteristics (XIAO et al., 2015). Kafirins are 

soluble in aqueous alcohol and acetic acid solutions, have low digestibility and the 

ability to self-organize into biomaterials with controlled morphology (ESPINOSA-

RAMÍREZ; SERNA-SALDÍVAR, 2016; TAYLOR et al., 2009; XIAO et al., 2016). 

In terms of molar mass and solubility, kafirin can be classified into -kafirin (66 - 

80 %), with masses of 23 and 25 kDa, -kafirin (5 - 8 %), with masses of 16, 18 and 

20 kDa and -kafirin (9 - 12 %), with a mass of 28 kDa. Regarding the secondary 

structure, kafirin contains 40 to 60 % -helix with some  sheets and disordered 

structures. The secondary structure of -kafirin is mainly -helix based, whereas for -

kafirin, such structure is a mixture of random coil and  sheets. Factors that can affect 

the intermolecular  sheet ratio are, mainly, the extraction method and its drying 

conditions to recover the protein and tannin complexation (BELTON et al., 2006; 

ESPINOSA-RAMÍREZ; SERNA-SALDÍVAR, 2016; TAYLOR; TAYLOR, 2018; XIAO; 

CHEN; HUANG, 2017). Kafirins are located in spherical protein bodies, embedded in 

glutelin and surrounded by starch granules (Figure 1.4), which range in diameter from 



30 
 

 

0.4 to 2 µm, with an outer layer composed mainly of interconnected - and -kafirins, 

and an inner layer composed predominantly of -kafirin (Figure 1.5) (DE MESA-

STONESTREET; ALAVI; BEAN, 2010). 

 

Figure 1.4. Scheme for localization of kafirin in gluten matrices. 

 

Source: Adapted from de Mesa-Stonestreet; Alavi; Bean, 2010. 

 

Figure 1.5. Scheme of kafirin protein body. 

 

Source: Adapted from de Mesa-Stonestreet; Alavi; Bean, 2010. 

 

 



31 
 

 
 

1.2.2 Sorghum polyphenols 

 

Polyphenols originate from plant secondary metabolites and generally have an 

important role in natural plant defense against pathogens and pests (GIRARD; AWIKA, 

2018; NACZK; SHAHIDI, 2004). Some sorghum grain cultivars contain polyphenols 

and have higher levels of these than most other cereal grains. The dominant 

polyphenols present in sorghum are structurally classified as phenolic acid derivatives 

or flavonoids (GIRARD; AWIKA, 2018). Other phenolic compounds are also present in 

sorghum, which include hydroxybenzoic acids, such as gallic, p-hydroxy, salicylic, 

vanillic, and xarinic acids, and hydroxycinnamic acids, such as ferulic, caffeic, p-

coumaric, cinnamic, and sinapic acids. The concentration and distribution of such 

compounds varies with the type of sorghum, but they are mainly present in bound form. 

Gallic, cinnamic, vanillic and p-hydroxybenzoic acids are present in low amounts 

(between 0.83 - 5.07 mg/100g) in different sorghum varieties, while ferulic acid is more 

abundant (10.46 - 34.26 mg/100g) (RAO et al., 2018). 

In sorghum grains containing a pigmented testa, the most abundant polyphenols 

are condensed tannins (proanthocyanidins), which have the ability to bind to proteins 

to form insoluble complexes  (BARROS; AWIKA; ROONEY, 2014). According to 

Barros, Awika e Rooney (2012), such substances, specifically those of high molar 

mass, have greater antioxidant activity in vitro and in vivo than simple phenols and 

other natural antioxidants. Even when complexed with proteins, the tannins present in 

sorghum still showed at least 50 % of their antioxidant activity (AWIKA; ROONEY, 

2004). Despite the presence of condensed tannins in other cereal grains, as red wheat, 

barley, millets, and pigmented rice, the type III sorghums can present tannin levels that 

are more than 10-fold higher than reported for other cereal grains (GIRARD; AWIKA, 

2018). 

Condensed tannins are structured as irregular oligomers and polymers of 

polyhydroxyflavan-3-ol monomer units linked by acid-labile 4 → 6 or 4 → 8 bonds. The 

common monomers units for procyanidins are catechin and epicatechin (PORTER, 

1992). Tannins can be divided by their chemical structure (SERRANO et al., 2009), as 

shown in Figure 1.6. Those present in sorghum are mainly polymerized products of 

flavan-3-ols and/or flavan-3,4-diols. Catechin is commonly the most present monomer 

and procyanidin B1 is the most reported dimer in sorghum (AWIKA et al., 2003). 



32 
 

 

 

Figure 1.6. Division of tannins concerning to their chemical structure. 

 

Source: Adapted from Serrano et al., 2009. 

 

Considering the ability of polyphenols to bind to proteins and their antioxidant 

activity, the use of protein-polyphenols complexes to stabilize emulsions and, at the 

same time, inhibit or delay the oxidation of lipid compounds are being studied (GU et 

al., 2017; LI et al., 2023; LIU et al., 2016b; WAN et al., 2014; WANG et al., 2015; YI et 

al., 2016) and have showed promising results. However, in those studies, the 

polyphenol is added to the system, which is not the case in sorghum cultivars that 

present endogenous tannins in their structure. Despite the irreversible binding between 

tannins and proteins is precisely the reason for considering them as antinutritional 

factors, the use of sorghum proteins extracted from cultivars with the presence of 

polyphenols, presenting natural protein-polyphenol complexes, becomes interesting 

due to their low cost and possible beneficial effects, such as their antioxidant capacity. 

 



33 
 

 
 

1.3 Lipid Oxidation 

 

Food emulsions must be formulated taking into account the consumer 

preferences, which are largely linked to healthier and more natural choices. 

Considering this fact, the choice of the oil to produce such emulsions must be made 

according to its nutritional value. The consumption indication is directly linked to their 

complex composition – triacylglycerols, saturated and unsaturated fatty acids, 

phospholipids, pigments, phytosterols, and tocopherols. It is recommended that oils 

with a high content of unsaturated fatty acids be consumed, since high levels of 

saturated fatty acids are related to cardiovascular disease (GANESAN; 

SUKALINGAM; XU, 2018). 

Canola oil (Brassica napus L.), obtained from the seeds of the Canadian 

rapeseed variety, with low erucic acid (< 2 %) and glucosinalate (< 30 mol/g) contents, 

is the third most consumed oil in the world (FAOSTAT, 2022). Due to its high nutritional 

value and a good ratio (2-3) of linoleic (-6) and linolenic (-3) acids, which are long-

chain polyunsaturated fatty acids (PUFAs) considered essential, as they are not 

synthesized by our body (KAUR et al., 2019; KONUSKAN; ARSLAN; OKSUZ, 2019), 

it is considered one of the healthiest oils for consumption (RAMOS et al., 2017; 

SÁNCHEZ; FERNÁNDEZ; NOLASCO, 2018). Canola oil composition is mainly based 

on oleic acid – C18:1 (59.7 - 68.2 %), linoleic acid – C18:2 (17.2 - 23.2 %) and linolenic 

acid – C18:3 (6.2 - 9.1 %) and contains, on average, 64 % monounsaturated fatty 

acids, 28 % polyunsaturated fatty acids and only 7 % saturated fatty acids (CHEW, 

2020; JALILI et al., 2018; KAUR et al., 2019; KIRALAN; RAMADAN, 2016; RAMOS et 

al., 2017; ROSZKOWSKA et al., 2015; SÁNCHEZ; FERNÁNDEZ; NOLASCO, 2018, 

2019; SIGER; KACZMAREK; RUDZIŃSKA, 2015; SYMONIUK; RATUSZ; KRYGIER, 

2019; WINKLER-MOSER; LOGAN; BAKOTA, 2014). Due to its high content of 

unsaturated fatty acids, canola oil is widely susceptible to lipid oxidation, even when 

used in low amounts in different food systems (FRANKEL, 2005). 

Lipid oxidation is one of the most important deterioration processes in oils and 

fats and can lead to large economic losses in the food industry, as it can impair the 

nutritional value and the quality of the product, and lead to formation of potentially toxic 

products. This deterioration process is a reaction between unsaturated fatty acids and 

oxygen, under certain physical-chemical conditions, which is divided into 3 stages: (i) 



34 
 

 

initiation, in which unsaturated fatty acids lose a hydrogen atom and form a lipoyl or 

alkyl free radical; (ii) propagation, in which the radical formed at the first stage react 

with triplet oxygen, generating peroxyl radicals; and (iii) termination, in which the 

radicals react together and form stable nonradical compounds (BERTON-CARABIN; 

ROPERS; GENOT, 2014). Some oxidation products are formed during the oxidation 

process, such as hydroperoxides and conjugated dienes, as primary oxidation 

products, and thiobarbituric acid reactive substances (TBARS), as secondary oxidation 

products. 

In emulsions, lipid oxidation can occur generally earlier and faster, due to possible 

overheating during the emulsification process, the possible contact between the 

unsaturated lipids and prooxidant compounds at the oil-water interface, and an 

accessibility to the oxygen dissolved in the aqueous phase (BERTON-CARABIN; 

ROPERS; GENOT, 2014). Thus, the use of antioxidants, especially synthetic ones, 

has become commonplace, once they act to block the chain reaction sequence in the 

lipid oxidation process (SCHAICH, 2020). However, considering the high demand for 

healthy foods based on natural products in the actual days, the use of antioxidants 

obtained from natural sources, such as sorghum polyphenols, is a promising 

alternative.  

 

In view of the thesis' division into Chapters, it is important to point out that the 

topics addressed in the present literature review were not exhaustively discussed, 

since they will be inserted and commented on in the following Chapters according to 

the analyses involved.



35 
 

 
 

CHAPTER 2 – HIGH PRESSURE HOMOGENIZATION AS AN EFFICIENT 

MEANS TO IMPROVE THE AQUEOUS DISPERSIBILITY OF 

TANNIN- AND LIPID-RICH SORGHUM PROTEIN EXTRACTS 

 

Thais Cristina Benatti Galloa,b,*, Valérie Beaumalb, Bérénice Houinsou-Houssoub, 

Michèle Viaub, Lucie Ribourg-Biraultb, Joëlle Bonicelc, Adeline Boireb, Valéria 

Aparecida Vieira Queirozd, Hamza Mameric, Alain Riaublancb, Vânia Regina 

Nicolettia, Claire Berton-Carabinb,e 

 

a São Paulo State University (Unesp), Institute of Biosciences, Humanities and Exact 

Sciences, São José do Rio Preto, Department of Food Engineering and Technology, 

15054-000 São José do Rio Preto, São Paulo, Brazil 

b INRAE, UR1268 Biopolymères Interactions Assemblages (BIA), Interfaces et 

Systèmes Dispersés (ISD), 44316 Nantes cedex 3, France 

c INRAE, UMR 1208 Ingénierie des Agropolymères et Technologies Emergentes 

(IATE), F-34060 Montpellier, France 

d Embrapa Milho e Sorgo, 35701-970 Sete Lagoas, Minas Gerais, Brazil 

e Wageningen University & Research, Laboratory of Food Process Engineering, 

6700 AA Wageningen, The Netherlands



36 
 

 
 

ABSTRACT 

Sorghum protein ingredients can have promising applications, as sorghum is a resilient 

crop. Its grains not only have a good protein content, but also other components of 

potential functional value, such as phenolic compounds - mainly tannins - in some 

genotypes. The present work aimed to investigate the behavior of aqueous 

suspensions produced with sorghum proteins extracted from tannin-rich cultivars in 

comparison to tannin-free ones. The use of high-pressure homogenization to improve 

the dispersibility and technological properties of such suspensions was also assessed. 

Protein extracts presented protein contents ranging from 50 to 67 wt.% (total basis), 

with kafirin as a dominant component for all samples. SE-HPLC analyses showed that 

flours contained soluble and insoluble protein fractions, whereas for the protein 

extracts there were only soluble fractions. The total lipid content in the protein extracts 

varied between 18 and 26 wt.%, with a high contribution of free fatty acids (68 to 76 

g/100 g lipids). These values are substantially higher than the total lipid content in 

sorghum grains, suggesting an enrichment of lipid fractions over the protein extraction 

process, which can have important consequences on the functional properties of the 

ingredients. The tocopherol content ranged from around 1370 to more than 2000 µg/g 

total lipids, with higher values for the protein extracts compared to the flours, showing, 

as was seen for lipids, a substantial accumulation upon the extraction process. When 

applied in aqueous media, the dispersibility of the protein extracts was quite limited, 

yet high-pressure homogenization was efficient to decrease the average size of the 

particles in suspension, from 9 - 66 µm to 1.8 - 2.5 µm, and changed their colloidal 

morphology, as observed by microscopy. Such a processing step was also effective to 

enhance protein dispersibility (i.e., the protein fraction that does not sediment under 

given centrifugation conditions), by up to 288 %. 

 

KEYWORDS 

Kafirin • Tannins • Composition • Sorghum lipids • Particle size • Solubility



37 
 

 
 

2.1 Introduction 

 

Sorghum (Sorghum bicolor L.) is a versatile grain, commonly cultivated in arid 

regions of Africa, Asia, Australia, and North and South America  (Girard & Awika, 

2018), with a rentable growing due to its easy adaptation to different environmental 

conditions. Sorghum was the fifth most produced cereal in the world in 2019, with a 

total production of almost 58 million tons (FAOSTAT, 2022). In semi-arid zones of 

Africa, South America, and Asia, sorghum is consumed as a staple food (Proietti, 

Frazzoli, & Mantovani, 2015; Queiroz et al., 2011), but its production is mainly intended 

for industrial conversion into alcohol and for animal feed (Liu et al., 2018). In some 

countries, such as Japan, the United States and Brazil, this grain has been studied and 

has been recommended as a good alternative for use in human food. Sorghum grains 

are composed mainly of carbohydrates (75 wt.%) and their dietary fiber content is 

about 6 wt.%. Lipids correspond to approximately 3 wt.%, with a typical main fatty acid 

composition as follows: oleic acid (31.1 - 48.9 %), linoleic acid (27.6 - 50.7 %), palmitic 

acid (11.7 - 20.2 %) and linolenic acid (1.7 - 3.9 %) (Mehmood et al., 2008). 

Sorghum grain represents an important vegetable protein source, with a content 

ranging from 6 to 18 wt.% (total basis) (de Mesa-Stonestreet, Alavi, & Bean, 2010). 

This fact highlights the potential of using sorghum proteins as a sustainable substitute 

of animal proteins, or as a natural functional ingredient in foods, in the context of the 

global clean label trend. Certain sorghum cultivars present a significant content of 

polyphenols, of which some are condensed tannins (proanthocyanidins) (Barros, 

Awika, & Rooney, 2014), which have capacity to bind to proteins and form insoluble 

complexes. Even though this fact is precisely the reason for considering tannins as 

antinutritional factors, this potentially adverse effect could be counterbalanced by other 

positive functionalities of the tannins, such as antioxidant potential. 

Kafirin, the most abundant protein in sorghum, belongs to the family of prolamins, 

which resembles zein in solubility, molar mass, amino acid composition and 

polypeptide structure, although being more hydrophobic (Xiao et al., 2015). In terms of 

molar mass, kafirin can be classified into -kafirin (66 - 80 wt.%), with molecular 

weights of 23 and 25 kDa, -kafirin (5 - 8 wt.%), with 16, 18 and 20 kDa, and -kafirin 

(9 - 12 %), with 28 kDa.  



38 
 

 

The low solubility of sorghum proteins in water can be a problem for their 

application in food products; therefore, dedicated strategies should be implemented to 

facilitate their dispersibility in aqueous media, which implies some modifications of the 

structure and functionality of the proteins. For instance, high pressure homogenization 

can be used to modify both the size and structural organization of protein-based 

supramolecular assemblies (aggregates, particles) in suspensions. This mechanical 

treatment induces an intense shear on the protein suspension by forcing it to pass 

through a small gap (Zamora & Guamis, 2015), which can disrupt protein aggregates 

(Balny & Masson, 1993). Thus, the use of high pressure homogenization can improve 

the techno-functionalities of proteins (Bader, Bez, & Eisner, 2011), for example their 

emulsifying properties, which would make this processing step a relevant pre-

treatment for proteins from plants (Burger & Zhang, 2019; Levy, Okun, & Shpigelman, 

2022; Melchior, Moretton, Calligaris, Manzocco, & Nicoli, 2022). 

This research therefore aimed to characterize the suspensions produced with 

proteins extracted from the flours of four sorghum cultivars, with and without tannins in 

their intrinsic composition, and to investigate the impact of high pressure 

homogenization on particle size and protein dispersibility in aqueous suspensions. 

Therefore, this work is a step towards facilitating the future utilization of this relevant 

crop in food products. 

 

2.2 Materials and Methods 

 

Flours of four different sorghum cultivars (two cultivars without tannins, BR501 

and BRS310, and two cultivars rich in tannins, BRS305 and SC782) were provided by 

Embrapa Milho e Sorgo (Sete Lagoas, MG, Brazil). For clarity, the suffixes (T-) and 

(T+) are added to the sample codes to recall the absence or presence of endogenous 

tannins, respectively. 

Protein-rich fractions were extracted from the sorghum flours following the 

methodology described by Taylor, Taylor, Dutton & De Kock (2005). Each flour was 

dispersed in an aqueous solution containing 70 wt.% ethanol, 0.5 wt.% sodium 

metabisulfite and 0.35 wt.% NaOH, at 70 °C for 1 hour, under constant stirring, at a 

ratio of 1:5 (w/w, flour:aqueous phase), followed by centrifugation at 1000×g for 5 

minutes at 25 °C. The supernatant was collected and placed overnight in a fume 

cupboard, at room temperature, for solvent evaporation, until a viscous liquid sediment 



39 
 

 
 

was formed. The protein slurry was added to cold distilled water (< 10 °C) and the pH 

was adjusted to 5.0 with HCl 1 N to precipitate the protein. Protein was recovered by 

filtration under vacuum, collecting the material on the top of the filter and dispersing it 

in distilled water to neutralize (7.0) the pH with NaOH 1 N before subsequent freeze-

drying. Differently from the reference procedure described by Taylor et al. (2005), the 

starting flours were not defatted with hexane in order to evaluate the functional 

properties of protein-rich extracts in the presence of lipids, which is relevant in a context 

of minimal fractionation routes. 

 

2.2.1 Characterization of sorghum flours and freeze-dried protein extracts 

 

Flours and protein extracts were characterized according to AOAC (2006) official 

methods, for protein, water, and ash contents. The protein content was evaluated by 

Kjeldahl, and the Nitrogen to Protein conversion factor (N:P factor) was computed as 

the ratio of total anhydrous mass of amino acids to the total mass of nitrogen (Sosulski 

& Imafidon, 1990). The Aspagarine (Asn) and Glutamine (Gln) were assayed in their 

acidic form, as Asparagic acid (Asp) and Glutamic acid (Glu). The relative content in 

Asp, Asn, Gln and Glu was estimated using kafirin sequences (uniprot reference: 

P14690, P14691 and P14692). Water content was analyzed by indirect method of 

water removal by drying using a vacuum oven at 60 °C for 48 h. The ash content was 

evaluated by incineration in a muffle furnace at 550 °C until the residue was white or 

light gray. The carbohydrate content, including fibers, was calculated by difference. 

The total lipid content was determined by cold extraction using 

dichloromethane/methanol as solvents. The next sections describe the detailed 

procedure for lipid and protein composition analyses. 

 

2.2.2 Lipid extraction and composition analysis 

 

For lipid extraction, 1 g of powder sample was weighed (in triplicate) in a tube 

and 8 mL of ultrapure water were added. The mixture was stirred on a vortex overnight 

at 4 °C. The dichloromethane/methanol mixture (2:1) was then added (10 mL) and the 

system was stirred on a vortex for 1 h at room temperature. After centrifugation at 

1700×g for 10 min at 20 °C (Eppendorf 5810 R, Hamburg, Germany), the lower organic 



40 
 

 

phase was recovered in a volumetric flask. This procedure was repeated two more 

times, and the lower phases were pooled in the same volumetric flask. The solvent 

was evaporated under vacuum in a rotary evaporator at 40 °C and the lipid extract was 

recovered washing the flask with 10 mL of dichloromethane/methanol solution (2:1) 

twice. The mixture was put in a separation funnel with the addition of 0.73 wt.% sodium 

chloride solution (5 mL) and stored at 4 °C overnight. The lower phase was recovered 

in another volumetric flask, previously weighed, using a hopper with glass wool and 

anhydrous sodium sulfate. The solvent was evaporated in a rotary evaporator at 40 °C 

and the sample was dried with nitrogen flow. The lipid content was calculated by weight 

difference and expressed in g lipids/g sample. Chloroform was added to the volumetric 

flask to obtain a concentration of approximately 10 mg lipid extract/mL and the solution 

was stored at -80 °C until subsequent analysis of fatty acid methyl esters (FAMEs), 

tocopherols, and quantification of lipid classes. 

For FAMEs preparation and analysis, 100 L of lipid extract (around 1 mg) were 

mixed with 100 L of internal standard solution (1 mg/mL in chloroform) in a glass tube 

and the solvent was evaporated under nitrogen flow. Then, 1 mL of toluene and 1 mL 

of BF3 reagent at 14 % in methanol (B-1252, Sigma) were added and the mixture was 

incubated at 100 °C for 45 min. After cooling, 1 mL of cyclohexane and 0.5 mL of 

ultrapure water were added, and the mixture was vortexed for 20 s. After centrifugation 

at 900×g for 5 min at 20 °C (Eppendorf 5810 R, Hamburg, Germany), the upper phase, 

containing cyclohexane and FAMEs, was recovered and subjected to gas 

chromatography according to Meynier et al. (2014). The results were expressed in mg 

fatty acids/g lipids. 

The tocopherol content was measured, in triplicate, by high-performance liquid 

chromatography (HPLC) as described by Meynier et al. (2014) with slight 

modifications, in which 40 µL were injected onto a normal phase column (Acclaim Polar 

Advantage II, Dionex, Si 3 µm, 250 mm x 3 mm), and the separation was achieved in 

isocratic mode. Hexane/tert-butyl-ether (90/10 v/v) was used for mobile phase, a flow 

rate of 0.5 mL/min was set, and 295 nm and 330 nm were used as wavelengths of 

excitation and emission, respectively. The identification and concentration of the 

tocopherol isomers in the samples was determined comparing their retention time with 

those of standard compounds and using external calibration curves of tocopherol 

isomers and -tocotrienol from commercial standards, respectively. Although the 



41 
 

 
 

detection limit is low (about 1-2 ng injected, depending on the isomers), the 

quantification was only possible from 5 ng injected for α-, β-, and -tocopherol, and 10 

ng for -tocopherol and for -tocotrienol. The results were expressed in g 

tocopherols/g lipids. 

Lipid classes were analyzed by high performance liquid chromatography (HPLC). 

Approximately 5 μg of lipid in 10 μL chloroform were analyzed by HPLC with a modular 

UltiMate 3000 RS system (Dionex, Voisins Le Bretonneux, France) equipped with an 

Uptisphere Strategy column (150mm×4.6 mm, 2.2 μm, 100 Å; Interchim, Montluçon, 

France) and coupled to an evaporative light scattering detector (ELSD) Sedex 85 

(Sedere S.A., Alfortville, France). Chromatographic conditions, identification and 

quantification of lipids were described by Fogang Mba et al. (2017). 

 

2.2.3 Fourier transform infrared (FTIR) spectrophotometry 

 

A FTIR spectrophotometer Thermo Nicolet IS50 (Thermo Scientific, USA) 

connected to an attenuator of total reflectance (FTIR-ATR) with deuterated triglycine 

sulfate (DTGS) was used to obtain FTIR spectrum for the sorghum flours and the 

respective freeze-dried protein-rich extracts. Samples were placed in a single-bounce 

diamond crystal, and fifty scans were collected in the range of 4000 to 650 cm-1, with 

4 cm-1 resolution. 

 

2.2.4 Protein composition analysis 

 

Protein composition in flours and protein extracts was determined by sodium 

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion - 

high performance liquid chromatography (SE-HPLC). 

For SDS-PAGE for powders, total kafirins were extracted from raw flour according 

to D’Almeida et al. (2021) and the protein solutions were freeze-dried before being 

dispersed at 1.5 g/mL during 15 minutes at 70 °C in 10 mM borate pH 10.0 buffer 

containing 2 % SDS and 20 mM dithioerythritol (DTE). Equivalents of 12 µg of 

protein/sample were loaded on a pre-casted gel and run for 2 h using a Bio-Rad Mini 

Protean 3 Cell (USA) system. Pre-stained protein markers, with molecular weights 

ranging from 10 to 250 kDa (Biorad), were loaded in a 4-20% gradient gel. Then, the 



42 
 

 

gel was stained and decolored following the method described by Neuhoff, Arold, 

Taube, & Ehrhardt (1988). 

For SE-HPLC, the procedure was carried out as described by Morel et al. (2002), 

with some modifications. In brief, a protein extraction step was performed to recover 

soluble proteins, by dispersing 50 mg of powder (flour or protein extract) in 1 mL 10 

mM Na-borate buffer pH 10.0. The mixture was stirred for 20 min at 20 °C, and 

centrifuged at 20000×g for 10 min at 20 °C. The supernatant was then recovered and 

added into a new tube, repeating this procedure two more times. The pellet resulted 

from the centrifugation was dispersed in 1 mL of buffer complemented with 20 mM of 

DTE, following the same parameters as described above for recovery of the insoluble 

proteins. The protein analysis was performed at 25 °C, on the Alliance Waters chain 

equipped with a TSK G4000 (Toso bioscience) (7.8 X 300 mm) column and a TSK gel 

SWXL (6 mm X 40 mm) pre-column, eluted with the phosphate buffer, 0.1 M, pH 6.8, 

SDS 0.1%. A volume of 15 μL was injected and eluted to 0.7 ml/min, the detection of 

different proteins was done at 214 nm. The data were collected with the help of 

Empower software. The mass calibration of the device was carried out by injecting a 

standard protein of known molecular weight and the area under the curve was 

integrated, using a response coefficient determined experimentally, to determine the 

protein content. 

 

2.2.5 Production and characterization of protein suspensions 

 

Suspensions containing 2 wt.% protein extracts were prepared in ultrapure water. 

The suspensions were stirred overnight at 4 °C to ensure maximum hydration. The 

suspensions were then treated by high pressure homogenization (HPH) at 400 bars 

for 6 min, corresponding to 24 passes through the homogenizer (Panda plus 1000, 

GEA) in one continuous cycle. These conditions were preliminary optimized in order to 

have a particle size and distribution fairly comparable among the different samples 

(Supplementary Figure 2.1). Both non-treated and HPH-treated suspensions were 

assessed regarding particle size distribution, morphology, FTIR spectra and 

composition of soluble and insoluble protein fractions. 

The particle size distribution and mean volume diameter (d43) were determined 

by static light scattering (Horiba LA-950). The refractive indices were set to 1.450 for 

the dispersed (protein) phase and 1.333 for the continuous (aqueous) phase. Particle 



43 
 

 
 

morphology was evaluated by optical microscopy (Zeiss Axioskop, equipped with 

Prosilica Digital Camera 5DCAM 1.31, and image software), and confocal laser 

scanning microscopy (CLSM) (Inverted Nikon A1 laser scanning confocal microscope) 

using Nile red and fast green to stain the lipids and proteins, respectively. FTIR 

analysis was performed as described in Section 2.2.3, with only one modification, using 

ZnSe crystal instead of diamond crystal.  

Suspensions treated and non-treated by HPH were also analyzed before and 

after centrifugation at 10000×g for 30 min at 10 °C (Eppendorf 5810 R, Hamburg, 

Germany) regarding the nitrogen content in the supernatant according to the Dumas 

method (Dumas, 1831). The protein solubility (S) was calculated using equation 1. It 

should be pointed out that, by convention, ‘solubility’ is defined as the fraction of 

proteins that remain in the supernatant of a given sample following centrifugation under 

fixed conditions. Yet, such supernatants may contain small particles that do not 

sediment in these conditions, and that are often referred to as ‘soluble aggregates’. 

For simplicity, we will use the terms ‘solubility’, ‘soluble’ in the following, but one should 

bear in mind that this terminology may be a bit stretched compared to the strict 

definition of solubility. 

 

𝑆 =
𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛
× 100%                                  (1) 

 

The protein composition in the total suspensions and in the supernatants after 

centrifugation was determined by SDS-PAGE, as described by Xiao et al. (2015) with 

some modifications. At first, tests were made to adjust the best conditions for not 

saturate the gels, as the gel used for concentration and separation and the amount of 

sample to be loaded on the gel. Then, after adjusting these conditions, samples were 

denatured and reduced by heating at 95 °C for 10 minutes in a buffer solution 

containing 0.01 mol/L Tris-HCl buffer (pH 6.8), 2 % (w/v) SDS, 10 % (v/v) glycerol, and 

5 % (v/v) β-mercaptoethanol (-ME). Pre-stained protein markers, with molecular 

weights ranging from 3 to 198 kDa, and the samples were loaded in gels of 

concentration and separation at 8 % and 16 %, respectively. Migration was made for 

2 h using a Bio-Rad Mini Protean 3 Cell (USA) system. Then, the gel was put in 

contact with a brilliant blue Coomassie G 250 solution and stirred at 35 rpm overnight 

(Rocker 25, Labnet, Labnet International Inc.) for posterior discoloration with solution 



44 
 

 

containing 50 % methanol and 12 % acetic acid. The gel was analyzed by the software 

Multi Gauge V3.0 (Fujifilms). 

2.2.6 Statistical analysis 

 

Results obtained in the analytical determinations in triplicate were submitted to 

variance analysis (ANOVA) and the differences between the means were tested by 

Tukey test at 5 % of probability (XLStat). 

 

2.3 Results and Discussion 

 

2.3.1 Composition of the sorghum flours and protein extracts 

 

2.3.1.1 Proximate composition of the samples 

 

The starting flours had protein contents between 9.7-10.6 wt.% (Table 2.1, 

calculated N factor), and there was no significant difference between one sample 

without tannin – BR501(T-) – and one with tannin – SC782(T+) –, indicating that the 

presence of endogenous tannins does not affect directly the protein content in the 

grains. Martino et al. (2012) studied eight Brazilian sorghum cultivars, developed and 

cultivated by Embrapa Milho e Sorgo (Sete Lagoas/MG) and obtained protein contents 

ranging from 8.6 to 11.9 wt.%, which matches with the contents found in the present 

study. The cultivars BR501(T-), BRS310(T-), and BRS305(T+) were also analyzed by 

Martino et al. (2012), who found slightly different results for the protein content of these 

cultivars. Antunes et al. (2007) also studied the genotype BR501(T-) and obtained 

about 11.2 wt.% protein, which is a bit higher than in the present work and the results 

obtained by Martino et al. (2012). The extraction process presented yield (% of protein 

extracted/protein content in the respective flour) ranging from 46 to 57 %, which agrees 

with the values found by Da Silva & Taylor (2004) and Taylor et al. (2005), whom used 

the same extraction method as in the present work.



45 
 

 
 

 

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. 

Sample 
Proteins¹ (N = 

6.25)  

Proteins² 

(calculated N 

factor) 

Total lipids Water Ash 
Carbohydrates 

(including fibers)† 

Flours 

BR501(T-) 10.98 ± 0.17ef 10.33 ± 0.16ef* 3.51 ± 0.03d 8.27 ± 0.03a 1.40 ± 0.03a 76.49 ± 0.09a 

BRS310(T-) 11.29 ± 0.26e 10.62 ± 0.24e 3.04 ± 0.20de 8.18 ± 0.01a 1.38 ± 0.03ab 76.78 ± 0.15a 

BRS305(T+) 10.21 ± 0,09g 9.67 ± 0.09g 3.12 ± 0.16de 8.19 ± 0.03a 1.29 ± 0.02c 77.73 ± 0.13a 

SC782(T+) 10.77 ± 0.20f 10.12 ± 0.18f 2.58 ± 0.29e 8.04 ± 0.02b 1.20 ± 0.04de 78.06 ± 0.18a 

Protein 

extracts 

BR501(T-) 69.32 ± 0.59b 65.22 ± 0.56b 22.19 ± 0.45b 4.30 ± 0.07c 1.41 ± 0.01a 6.88 ± 0.71d 

BRS310(T-) 71.31 ± 0.38a 67.14 ± 0.35a 21.88 ± 0.12b 3.73 ± 0.06d 1.35 ± 0.01b 5.90 ± 0.64e 

BRS305(T+) 58.06 ± 0.39c 54.99 ± 0.37c 25.84 ± 0.13a 3.39 ± 0.03f 1.27 ± 0.04cd 14.51 ± 0.51c 

SC782(T+) 53.32 ± 1.56d 50.08 ± 1.46d 18.71 ± 0.63c 3.59 ± 0.03e 1.15 ± 0.02e 26.47 ± 0.92b 

¹Protein contents calculated using the traditional N factor value (6.25). ²Protein contents calculated using N factors values determined for each sample in 

the present work: 5.88, 5.88, 5.92, and 5.87 for BR501(T-), BRS310(T-), BRS305(T+) and SC782(T+), respectively. †Calculated by difference. *Values in 

the same column with different letters are significantly different (ANOVA with Tukey test: p < 0.05). All experiments were carried out in triplicate. 



46 
 

 
 

The extracts had a protein content between 53-71 wt.% (Table 2.1, N factor = 

6.25), and this content is significantly lower for the (T+) extracts compared to the (T-) 

extracts, indicating that the presence of tannins tends to decrease the protein content 

after the extraction process. This can be due to the ability of tannins to bind covalently 

and irreversibly to proteins, thus making it less easy to extract them, and causing a 

lower yield upon the extraction process (Duodu et al., 2003). Taylor et al. (2005) used 

the same extraction method as performed in the present study for sorghum protein 

extraction, and found protein contents in the extracts ranging between 74.6 and 89.3 

wt.%, before and after defatting process, respectively. Da Silva & Taylor (2004) also 

produced protein extracts using the same reagents, with small differences in the 

extraction process, leading to protein contents between 77.9 and 83.1 wt.% without 

the defatting process, and contents between 88.3 and 88.7 wt.% after defatting with 

hexane. Espinosa-Ramírez & Serna-Saldívar (2016), who also used the same 

extraction methodology, found protein contents ranging from 72.7 to 88.6 wt.% for 

extracts from whole sorghum grains. Such results agree with those in the present 

study, even if the protein contents are lower than previously reported, especially for 

tannin-rich cultivars, since the protein content in the extract depends on both the 

cultivar and the extraction methodology. It is important to remember that protein 

contents also depend on the used N conversion factor, which, in turn, depends on the 

protein composition of each sample. A generalized value of 6.25 is commonly used by 

manufacturers/suppliers of plant protein ingredients, including the works cited above, 

but this value is usually higher than the real values for plant proteins, leading to an 

overestimation of the protein content in such plant protein ingredients. For instance, 

Sosulski & Imafidon (1990) studied the amino acid composition of sorghum grain 

proteins and determined a nitrogen-to-protein conversion factor of 5.93. In the present 

work, after analyzing the amino acid composition (Supplementary Table 2.1) and 

correlate the results with the protein sequences, it was possible to determine a specific 

N conversion factor for each sample, which was 5.88 for BR501(T-), 5.88 for 

BRS310(T-), 5.92 for BRS305(T+), and 5.87 for SC782(T+). Comparing the columns 

3 and 4 (Table 2.1), in which the calculation of protein contents was made using the 

traditional N factor of 6.25 and the N factors found in this work for each sample, 

respectively, it is clear that the N factor may mislead the actual protein content in the 

samples. 



47 
 

 
 

The lipid content in the flours varied from 2.5 to 3.5 wt.% (Table 2.1), without a 

particular effect of the presence of tannins. Martino et al. (2012) found lipid contents 

varying from 2.6 to 3.1 wt.% for BR501(T-), BRS310(T-), and BRS305(T+) genotypes, 

which, despite slight differences, overall agrees with the results in the present work. In 

the protein extracts, lipid content was noticeably high (18.7-26 wt.%), with the tannin-

rich samples BRS305(T+) and SC782(T+) having the highest and lowest values, 

respectively. Espinosa-Ramírez & Serna-Saldívar (2016) obtained lipid contents 

ranging between 17.9 and 20.3 wt.%, whereas Da Silva & Taylor (2004) values of 12.8-

16.7 wt.% for protein-rich fractions. However, instead of a cold solvent extraction, the 

authors applied a Soxhlet extraction, which involves heat and may have degraded 

some of the lipid components. The Soxhlet method also does not allow for a 

quantitative extraction of polar lipids, as discussed by Li et al. (2014). It is remarkable 

that the high lipid contents observed in the protein extracts show that the applied 

extraction process not only concentrates the proteins, but also leads to a marked 

accumulation of the lipids initially present in the flours, which can be related to the fact 

that the flours were not defatted. 

The water content was higher for the flours when compared to the respective 

protein extracts, whereas the ash content did not present a significant difference 

between the flours and protein extracts (Table 2.1). This can probably be related to the 

freeze-drying process ultimately used to prepare the protein extracts. Regarding the 

carbohydrate content (i.e., the sum of the starch and fiber contents) we found, in the 

present study, higher results than in the previous work of Martino et al. (2012). Such 

differences in the proximate composition, even for the same cultivars, can be explained 

by variations in the cultivation and storage conditions of the grains. 

 

2.3.1.2 Lipid composition of the samples (dry state) 

 

The accumulation of lipids in the produced sorghum protein extracts can be of 

high importance for the properties and functionality of these ingredients, and therefore 

a detailed analysis of the composition of the lipid extracts was carried out. 

No phospholipids (i.e., polar lipids) were detected in any sample. The most 

abundant lipid class present was free fatty acids (FFAs) (Figure 2.1), ranging from 51.3 

to 75.8% of the total lipids, followed by triacylglycerols (TAGs), ranging from 21.4 to 

44.6%, except for the BR501(T-) flour, which presented more TAGs (54.1%) than FFAs 



48 
 

 

(41.5%). All the flour samples were significantly different from each other regarding 

FFA and TAG contents, not showing a clear effect of the tannins. For protein extracts, 

in which the lipid fraction is mass-wise important (as previously discussed in Section 

2.3.1.1), (T-) samples are significantly different from the (T+) ones for FFA content, 

with the former ones showing the highest values, indicating that the presence of 

tannins could be linked to a lower FFA content. Osagie (1987) and Price & Parsons 

(1975) observed that sorghum grains contained a small quantity of phospholipids and 

glycolipids, with neutral lipids being the main lipid class (> 85%), as reported in this 

study. However, Osagie (1987) observed a large contribution of TAGs in the neutral 

lipids (85%), which was not the case in the present study. The high FFA proportions 

present in our samples come most likely from hydrolysis of TAGs initially present in the 

seeds, which may happen because of endogenous lipases either in planta (i.e., during 

the maturation of the seeds on the sorghum plants) or after harvesting of the 

seeds/upon production or storage of the flours. 

The fatty acid composition (Supplementary Table 2.2) of the sorghum lipids 

presented a high concentration of linoleic acid (45-51% of all fatty acids for the protein 

extracts, and 43-48% for the flours), oleic acid (27-34% for the protein extracts, and 

29-37% for the flours), and palmitic acid (13-15% for the protein extracts and flours), 

thus showing a high content in PUFAs (> 50% of all fatty acids) and MUFAs (> 30%), 

rather than in saturated FAs. There was a slight increase of linoleic acid and a slight 

decrease in oleic and palmitic acids when comparing the fatty acid composition of the 

protein extracts with that of the starting flours, suggesting that there was a slight 

accumulation of PUFAs during the protein extraction process. Hassan, Imran, Ahmad, 

& Khan (2017), Osagie (1987) and Price & Parsons (1975) obtained similar results for 

sorghum grains, with linoleic (43-58 wt.%), oleic (21-37 wt.%), and palmitic (13-19 

wt.%) acids being the main compounds identified in the fatty acid profile. 

 



49 
 

 
 

 

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. 

TAG: triacylglycerol; DAG1: diacylglycerol-1-3; DAG2: diacylglycerol-1-2; MAG: monoacylglycerol; 

FFA: free fatty acid. *Different letters for the same lipid class indicate significant differences (ANOVA 

with Tukey test: p < 0.05). 

 

The total tocopherol content found in the lipid extracts obtained from sorghum 

flours and their respective protein extracts varied between 1371-2039 µg/g total lipids. 

These values are considered very high when compared to PUFA-rich vegetable oils, 

which usually have tocopherol contents be