ANA MARIA CHAUX GUTIÉRREZ 

 

 

 

 

 

 

CRIOGÉIS DE PROTEÍNA-PECTINA COMO CARREADORES DE ÓLEO 

ESSENCIAL DE PIMENTA ROSA: APLICAÇÃO EM EMBALAGENS ATIVAS  

 

 

 

 

 

Relatório de Pós-doutorado realizado na 
Universidade Estadual Paulista (UNESP), 
Faculdade de Engenharia (FEIS), Ilha 
Solteira. 
 
 
Supervisor(a): Profa. Dra.  Marcia Regina 
de Moura Aouada 
 
Cossupervisor(a): Profa. Dra. Natália 
Soares Janzantti 
 

 

 

 

CAPES-PrInt - Nº do Processo 00.889.834/0001-08 

 

 

 

Ilha Solteira 

2024 



1 
 

 

 

 

 

 

Relatório de atividades referente ao período de Outubro/2023 a Setembro/2024 

 

Jovem Talento com experiência no exterior (JTEE): Projeto CAPES-PrInt-

Unesp 

Edital PROPG/PROPE/AREX nº 40/2023 

 

 

Criogéis de proteína-pectina como carreadores de óleo essencial de pimenta 

rosa: aplicação em embalagens ativas 

 

  

 

 

 

 

Dra. Ana María Chaux Gutiérrez                    Dra. Marcia Regina de Moura Aouada 

               Bolsista                                                                Supervisora 

 

Instituição Sede: Universidade Estadual Paulista “Júlio de Mesquita Filho” – 

Facultade de Engenharia (FEIS) - Departamento de Química e Física PPG Ciência 

dos Materiais (PPGCM) 

 

 

 

2024 



2 
 

Resumo do plano da proposta original  

 

O projeto busca desenvolver uma nova tecnologia de embalagem ativa para 

aumentar a vida útil de morangos, utilizando criogéis de proteína-pectina como 

carreadores do óleo essencial de pimenta rosa. Os criogéis, que são hidrogéis 

secos por liofilização, serão produzidos a partir de duas proteínas diferentes: 

albumina e proteína de grão de bico, combinadas com pectina. A escolha dessas 

proteínas se baseia em suas propriedades funcionais e capacidade de formar géis 

com características específicas. O óleo essencial de pimenta rosa, rico em 

compostos bioativos como monoterpenos e sesquiterpenos, será incorporado aos 

criogéis durante o processo de produção. A encapsulação do óleo essencial visa 

protegê-lo da degradação e permitir sua liberação gradual na embalagem, 

garantindo a atividade antimicrobiana por um período prolongado. A pesquisa 

investigará a influência da composição dos criogéis (tipo de proteína e concentração 

de pectina) nas propriedades físico-químicas, térmicas, atividade antimicrobiana e 

antioxidante. Além disso, será avaliada a eficiência de encapsulação do óleo 

essencial e sua liberação em diferentes condições de armazenamento. A aplicação 

dos criogéis em embalagens de morangos será avaliada em estudos de vida útil, 

monitorando as características físico-químicas (cor, textura, pH) e microbiológicas 

dos frutos durante o armazenamento. Espera-se que os resultados da pesquisa 

contribuam para o desenvolvimento de uma alternativa inovadora e sustentável para 

a conservação de alimentos, com potencial aplicação em diferentes frutas e 

hortaliças, além de gerar conhecimento científico sobre a interação entre 

biopolímeros, óleos essenciais e microrganismos em sistemas de embalagens 

ativas. 

. 

 

 

 



3 
 

Etapas realizadas durante o período 01/10/2023 a 30/09/2024 

 

Atividade Descrição 

1 Compra das matérias primas e insumos para a pesquisa. 

2 Avaliação da albumina, a proteína de grão de bico e a pectina de baixo 
metoxilo amidada para a obtenção de géis. 

3 Submissão do curso tópicos especiais “Aplicação de biopolímeros em 
embalagens ativas para alimentos” ao conselho do PPGCM. 

4 Produção dos criogéis a partir de albumina e pectina carregados com óleo 
essencial de pimenta rosa.  

5 Avaliação das propriedades físico-químicas, térmicas e microbiológicas 
dos criogéis carregados com óleo de pimenta rosa. 

6 Ministrar o curso tópicos especiais “Aplicação de biopolímeros em 
embalagens ativas para alimentos” 

7 Análises de resultados, redação e submissão do artigo científico a 
periódico qualis A.  

8 Treinamento em cromatografia gasosa. 

9 Início etapa de aplicação dos criogéis carregados com óleo de pimenta 
rosa em embalagens ativas. 

 

Descrição das atividades desenvolvidas durante o período 01/10/2023 a 

30/09/2024 

 

Durante o período foram desenvolvidas todas as etapas descritas anteriormente, a 

fim de dar cumprimento aos objetivos planejados para o projeto de pesquisa.  

A atividade 1 consistiu na aquisição das matérias-primas e insumos necessários 

para o desenvolvimento da pesquisa. A seleção dos fornecedores priorizou 

empresas com prazos de entrega mais curtos, os quais variaram, em média, entre 

duas e quatro semanas. 

A atividade 2 consistiu em testar diferentes concentrações de albumina (ALB) e 

proteína de grão de bico (BIC), bem como a temperatura de desnaturação da 

proteína. Diante da indisponibilidade comercial da proteína de grão de bico, 



4 
 

procedeu-se à sua extração a partir da farinha, visando à obtenção da proteína 

isolada. Adicionalmente, foram avaliadas diferentes proporções de pectina de baixa 

metoxilação amidada (PEC) para a formação dos géis. 

Os testes preliminares indicaram que a concentração de 5% de ALB e a temperatura 

de desnaturação de 85°C foram as mais adequadas para a formação de géis. Com 

base nesses resultados, foram preparados géis de ALB pura e com as seguintes 

proporções de ALB:PEC: 70:30, 50:50 e 30:70.  

Cabe ressaltar que a proteína de grão de bico, após extraída, não apresentou 

propriedades adequadas para a formação de géis, sendo, portanto, descartada para 

as etapas subsequentes da pesquisa. 

Em relação à atividade 3, foi elaborado o plano de aula da disciplina "Tópicos 

Especiais: Aplicação de biopolímeros em embalagens ativas para alimentos", 

com carga horária de 8 créditos e 120 horas. O plano foi submetido à aprovação do 

Conselho do Programa de Pós-Graduação em Ciência dos Materiais, obtendo 

parecer favorável, conforme registrado na Deliberação nº 283/2023. 

A atividade 4 dedicou-se à obtenção dos criogéis carregados com óleo essencial de 

pimenta rosa. Os testes realizados determinaram que a concentração ideal de óleo 

essencial para incorporação nos criogéis seria de 3% (m/m). 

A atividade 5 consistiu na caracterização dos criogéis obtidos, utilizando as 

condições definidas nas atividades 2 e 4. Foram avaliadas a eficiência de 

encapsulação, propriedades térmicas, atividade antioxidante e antimicrobiana, 

microestrutura e estruturais.  Os dados completos da caracterização dos criogéis 

encontram-se disponíveis no repositório institucional, acessível através do seguinte 

link: 

https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ

?usp=drive_link 

A atividade 6, realizada entre 15 de abril e 03 de maio de 2024, consistiu em 

ministrar a disciplina "Tópicos Especiais: Aplicação de biopolímeros em embalagens 

ativas para alimentos". Com carga horária de 26 horas, a disciplina foi oferecida aos 

https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ?usp=drive_link
https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ?usp=drive_link


5 
 

alunos de mestrado e doutorado do Programa de Ciência dos Materiais, contando 

com a participação de 6 discentes. 

A atividade 7 compreendeu a análise dos resultados obtidos na atividade 5, 

culminando na redação e submissão de um artigo científico ao periódico 

“Processes”, o que resultou na publicação. Chaux-Gutiérrez, A.M.; Pérez-

Monterroza, E.J.; Cattelan, M.G.; Nicoletti, V.R.; Moura, M.R.d. Encapsulation of 

Pink Pepper Essential Oil (Schinus terebinthifolius Raddi) in Albumin and Low-

Methoxyl Amidated Pectin Cryogels. Processes 2024, 12, 1681. 

https://doi.org/10.3390/pr12081681. A publicação em questão possui Qualis A4, 

atestando sua relevância e contribuição científica.  

A atividade 8, que visa dar continuidade à fase de aplicação dos criogéis em 

embalagens ativas, depende da análise dos compostos voláteis do óleo essencial 

por cromatografia gasosa. Essa técnica permite identificar e quantificar os 

compostos com potencial antimicrobiano para a conservação do morango. No 

entanto, o cronograma da atividade sofreu atrasos devido a alguns imprevistos. 

Inicialmente, houve atraso na entrega do ar sintético, essencial para o 

funcionamento do cromatógrafo. Posteriormente, o equipamento apresentou 

problemas com a bateria e vazamentos, demandando a contratação de um técnico 

especializado. A liberação da verba para o serviço e o agendamento da visita do 

técnico também contribuíram para o atraso na execução da atividade.  

Superadas essas dificuldades, o treinamento para utilização do equipamento foi 

realizado e a fase de aplicação dos criogéis conseguiu ser iniciada. 

A atividade 9 marcou o início da fase de aplicação dos criogéis como embalagens 

ativas. Com base nos resultados da atividade 5, foram selecionados dois tipos de 

criogéis, com proporções 50:50 e 30:70 de ALB:PEC, para serem aplicados nas 

embalagens. Os testes foram conduzidos a 4°C durante 7 dias, e as seguintes 

propriedades físico-químicas foram avaliadas cor, pH, acidez titulável, textura e 

conteúdo de antocianinas. Além disso, foram realizadas análises microbiológicas 

para avaliar a eficácia dos criogéis na inibição do crescimento microbiano. 

https://doi.org/10.3390/pr12081681


6 
 

Apesar de todos os dados terem sido coletados, o período de pós-doutorado se 

encerrou antes que fosse possível analisar os resultados e redigir um segundo 

artigo científico. Os dados encontram-se no link   

https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ

?usp=drive_link 

Além das atividades descritas anteriormente para o desenvolvimento da pesquisa, 

outras atividades foram realizadas durante este período, incluindo a coautoria do 

seguinte artigo científico:  

Cavalcante Neto, A.A., Chaux-Gutiérrez, A.M., Pérez-Monterroza, E.J. et 

al. Powdered cuxá sauce from Hibiscus sabdariffa L. leaves obtained by foam-mat 

drying. J Food Sci Technol (2024). https://doi.org/10.1007/s13197-024-06067-0.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ?usp=drive_link
https://drive.google.com/drive/folders/1WtASxq41gSNy9Dm9xc54anGqzo3GvYEZ?usp=drive_link
https://doi.org/10.1007/s13197-024-06067-0


7 
 

1. Introdução  

Atualmente, tem crescido o interesse pelas substâncias extraídas de fontes naturais 

como ervas e especiarias, devido a que apresentam propriedades antimicrobiana, 

antioxidante, antiviral e inseticida (Asbahani et al., 2015; Calo et al., 2015; Cook & 

Lanaras, 2016; Gonçalves et al., 2017). Especificamente, a pimenta rosa (Schinus 

terebinthifolia Raddi), um fruto nativo do Brasil, é usada na cozinha como 

condimento devido a seu sabor adocicado e picante, destaca-se pelas suas 

propriedades antioxidantes, antidiabéticas e anti-inflamatórias (Barreira et al., 2023; 

Oliveira et al., 2020).  O óleo essencial extraído da pimenta rosa, possui atividade 

antimicrobiana, devido aos monoterpenos β-mirceno, β-pineno, α-pineno e 

limoneno e ao sesquiterpeno β-cubebeno, os quais têm a capacidade de reduzir a 

quantidade de células viáveis de L. monocytogenes, Escherichia coli, S. 

Typhimurium (Dannenberg et al., 2019) (Dannenberg e de bactérias mesófilas 

aeróbias (Locali-Pereira et al., 2021). Porém, a estabilidade destes compostos se 

vê afetada por fatores tais como temperatura, luz e umidade o que acelera a 

degradação e diminui a suas propriedades antioxidante e antimicrobiana (Ali 

Shabkhiz et al., 2021; Bao et al., 2019; Benavides et al., 2016; Deng et al., 2020; 

Locali-Pereira et al., 2020). Por esta razão, tem-se usados os criogels (materiais 

obtidos por liofilização de hidrogéis) (Betz et al., 2012; Horvat et al., 2017), 

produzidos a partir de biopolímeros como os polissacarídeos e as proteínas, os 

quais fornecem grande proteção como material de parede, ajudando a reter 

compostos voláteis (Ali Shabkhiz et al., 2021; Alsakhawy et al., 2022; Atencio et al., 2020; 

Caló & Khutoryanskiy, 2014; Cui et al., 2021; Farjami et al., 2015). Em esta proposta 

pretende-se desenvolver criogéis   a partir de proteína de albumina (ALB) e de grão 

de bico (BIC) junto à pectina de baixa metoxilação amidada (PEC), com boa 

capacidade para manter a atividade antioxidante e antimicrobiana do óleo essencial 

de pimenta rosa. Além disso, deseja-se estudar a aplicabilidade dessa matriz 

(proteína-óleo-polissacarídeo) em forma de sachê para ser incorporado em um 

sistema de embalagem do tipo ativa, para seu uso na proteção do morango, com o 

objetivo de inibir ou reduzir o crescimento microbiano e aumentar o tempo de vida 

de prateleira (Batista et al., 2019; Locali-Pereira et al., 2021; Manzoor et al., 2022).  



8 
 

 

2. Objetivos 

• Desenvolver criogéis de albumina - pectina de baixa metoxilação amidada 

(ALB-PEC), e de proteína de grão de bico - pectina de baixa metoxilação 

amidada (BIC-PEC), com características estruturais adequadas para a 

encapsulação e liberação controlada dos compostos bioativos de óleo de 

pimenta rosa.  

• Desenvolver embalagens ativas a partir dos criogéis e do óleo essencial de 

pimenta rosa para aumentar o tempo de vida de prateleira do morango.   

 

3. Materiais 

Óleo essencial de pimenta rosa foi fornecido por Ferquima (Vargem Grande 

Paulista, SP, Brasil). A farinha de grão de bico (BIC) e a pectina de baixa metoxilação 

amidada foram obtidas de Ingredientes Online (São Paulo, SP, Brasil). A albumina 

comercial de clara de ovo foi obtida de Natuovos Ltda.  

3.1 Obtenção de proteína de grão de bico  

O processo de obtenção da proteína isolada de grão de bico foi realizado segundo 

Papalamprou et al. (2010) com modificações. A farinha de grão de bico foi submetida 

a um processo de eliminação da gordura. Posteriormente, foi preparada uma 

suspensão ao 10% m/v, e o pH foi ajustado a 8 com uma solução de NaOH 2M. A 

suspensão foi submetida a agitação constante por 1 hora, e posteriormente 

centrifugada a 4500 x g durante 30 minutos a 25°C, o pH do sobrenadante foi 

ajustado a 4,3 com HCL 2N e centrifugado a 4500 x g por 20 minutos a 4°C. O 

sedimento obtido foi dissolvido em água e neutralizado com NaOH 0.1 M, depois foi 

liofilizado e armazenado a 8°C para as posteriores análises. O conteúdo de proteína 

do extrato concentrado foi determinado pelo método Kjeldahl. 

 

 



9 
 

3.2 Preparação do criogel 

A encapsulação do óleo essencial de pimenta rosa foi realizada acordo com a 

metodologia proposta por Volic et al. (2018) e Chaux-Gutiérrez et al. (2019) com 

algumas modificações. 

Inicialmente foram preparadas soluções de albumina (ALB) (5 % m/m), proteína de 

grão de bico (BIC) (5 % m/m) e de pectina de baixa metoxilação amidada (PEC) (5 

% m/m) separadamente, as quais foram mantidas sob agitação até dissolução 

completa. O pH das soluções foi ajustado em 8 usando HCl (0,1 M) ou NaOH (0,1 

M). As soluções de ALB e BIC foram aquecidas a 85 °C por 15 minutos e resfriadas 

até 40°C e, posteriormente, foram misturadas com a de PEC segundo o 

planejamento experimental (Chaux-Gutiérrez 2019). Finalmente, foi adicionado o 

óleo de pimenta rosa (3 % m/m), e emulsionado usando um homogeneizador (T-25 

ULTRATURRAX®, IKA, Alemanha) a 18.000 rpm por 4 minutos (Locali Pereira et 

al., 2019). À dispersão preparada foi adicionado cloreto de cálcio (2 % m/v). Os géis 

foram colocados em caixas de Petri e armazenados a 4 °C até a formação do gel. 

Finalmente os géis foram congelados a -18 °C e liofilizados. 

3.2 Caracterização dos criogéis carregados com óleo de pimenta rosa 

3.2.1 Espectrometria de infravermelho FTIR 

Os espectros de infravermelho foram obtidos em um espectrômetro Spectrum One 

Spectrum (Perkin-Elmer Corp, Shelton, CT, USA). A varredura será conduzida de 

4000 a 400 cm-1. 

3.2.2 Conteúdo de óleo de pimenta rosa e eficiência de encapsulação (EE) 

A eficiência de encapsulamento dos criogéis foi determinada de acordo com a 

metodologia de Abreu et al. (2012) com algumas modificações. Cem miligramas de 

criogéis foram dispersos em 5 mL de etanol. A mistura foi centrifugada a 9000 rpm 

por 5 min a 25 °C usando uma separação centrífuga (Z 326 K, HERMLE 

Labortechnik GmbH, WehinGen, Baden-Würtemberg, Alemanha). O teor de óleo 

essencial de pimenta rosa foi medido usando um espectrofotômetro UV-vis (SP-200, 

Biospectro, Curitiba, Paraná, Brasil) em um comprimento de onda de 291 nm, 



10 
 

usando uma curva de calibração (y = 0,7269x + 0,223, R2 = 0,9949) de óleo 

essencial em solução de etanol (0,2–1,2 mg/mL). A eficiência de encapsulamento 

(EE%) foi calculada de acordo com a Equação 1.  

 

𝐸𝐸(%) =
𝑀

𝑀0
× 100                                                                                                    (1) 

onde M é a quantidade (mg) de óleo nos criogéis carregados e Mo é a quantidade 

inicial de óleo (mg) adicionada à preparação do criogéis. 

3.2.3 Atividade antioxidante  

Método de DPPH 

A capacidade antioxidante do óleo essencial encapsulado e não encapsulado foi 

determinada pelo método de DPPH. Os extratos serão misturados com 1 mL de 

uma solução de DPHH (90 μM), depois será completado até volume final de 4 mL 

com metanol ao 95 %. A absorbância foi determinada a 515 nm em 

espectrofotômetro UV/vis (Hussain et al., 2008). 

Método ABTS*+ 

A capacidade antioxidante do óleo essencial encapsulado e não encapsulado foi 

também determinada pelo método ABTS*+ segundo a metodologia proposta por Re 

et al. (1999). Os extratos foram preparados usando uma solução de metanol: água. 

A leitura da absorbância será realizada a 730 nm em espectrofotômetro. A 

quantificação foi realizada por meio de uma curva padrão usando uma solução 

aquosa de Trolox (0-200 μM/L). 

Compostos fenólicos totais  

O conteúdo de compostos fenólicos presentes no emulgel foi determinado usando 

o método de Folin-Ciocalteu (Singleton et al., 1999). Os extratos foram preparados 

usando metanol. As medidas de absorbância foram realizadas a 725 nm em 

espectrofotómetro UV/vis. A quantificação foi realizada usando una curva de 

calibração de soluciones aquosas de ácido gálico (0-500 ppm). Os resultados foram 



11 
 

expressos em mg de ácido gálico equivalente (EAG) por gramo. As análises foram 

realizadas em triplicata. 

3.2.4 Análises térmicas 

O comportamento térmico do óleo essencial de pimenta rosa e dos criogéis foi 

avaliado em um calorímetro diferencial de varredura (Pyris Series, Perkin Elmer), 

previamente calibrado com índio. A varredura foi realizada de 50 a 200 °C a uma 

velocidade de 10 °C/ min (Locali-Pereira et al., 2020) 

3.2.5 Atividade Antimicrobiana 

O efeito antimicrobiano in vitro dos criogéis contendo óleo de pimenta rosa foi 

determinado segundo a metodologia descrita por Locali-Pereira et al. (2020) para 

bactérias Gram-positivas (Staphylococcus aureus e Bacillus cereus) e Gram-

negativas (Escherichia coli). 

3.3 Aplicação dos criogéis carregados com óleo de pimenta rosa na 

embalagem do morango 

A avaliação dos efeitos de embalagens ativas contendo óleo essencial de pimenta 

rosa encapsulado em criogéis no armazenamento de morangos foi feita ao longo de 

7 dias, a 4 ºC. Amostras foram coletadas para avaliação de suas características 

físico-químicas em intervalos de 0, 3, 5 e 7 dias a partir do armazenamento. 

Amostras de cerca de 100 g de morango serão armazenadas em pequenas caixas 

de poli(tereftalato de etileno) termo formadas. Partículas de criogéis liofilizados 

contendo óleo de pimenta rosa serão colocadas em pequenos envoltórios de tecido 

(sachês) no fundo das embalagens (Locali-Pereira et al., 2021)  As caixas (duas 

para cada período de amostragem) foram armazenadas em câmaras de 

temperatura controlada. Também foram avaliadas amostras controle, isto é, 

armazenadas sem a presença de criogel. 

Durante o armazenamento foram avaliadas as seguintes propriedades físico-

químicas dos morangos: perda de massa, sólidos solúveis, acidez triturável, pH, cor, 

firmeza, e análises microbiológicas. 



12 
 

3.3.1 Perda de massa 

A perda de massa foi expressa como uma porcentagem da massa inicial. A mudança 

será calculada seguindo a equação 2 (Wigati et al., 2023).  

𝑃𝑒𝑟𝑑𝑎 𝑑𝑒 𝑚𝑎𝑠𝑠𝑎 (%) =
𝑀𝑎𝑠𝑠𝑎 𝑖𝑛𝑖𝑐𝑖𝑎𝑙−𝑀𝑎𝑠𝑠𝑎 𝑑𝑢𝑟𝑎𝑛𝑡𝑒 𝑜 𝑎𝑟𝑚𝑎𝑧𝑒𝑛𝑎𝑚𝑒𝑛𝑡𝑜

𝑀𝑎𝑠𝑠𝑎 𝑖𝑛𝑖𝑐𝑖𝑎𝑙 
× 100                   (2) 

3.3.2 Sólidos solúveis totais, pH e acidez titulável 

Os sólidos solúveis foram determinados usando um refratômetro (Atago, Japão) e 

o pH usando pHmetro Pro-03-0820 (Akso, Brasil) a 25 °C. A acidez titulável foi 

determinada pesando-se 2 g de morangos previamente homogeneizados e diluídos 

em 20 mL de água destilada, a mistura foi titulada usando uma solução de NaOH 

(0.1 M) e fenolftaleína (0.1 %) como indicador (AOAC, 1995) 

3.3.3 Análises de antocianinas 

A extração das antocianinas foi realizada utilizando solução aquosa de metanol e 

acetona (2:2:1 v/v) acidificada com ácido fórmico (0,1%). O teor de antocianina 

monomérica foi determinado pelo método espectrofotométrico diferencial de pH. O 

pH foi ajustado para 1,0 e 4,5 utilizando tampão cloreto de potássio (0,025 M) e 

tampão acetato de sódio (0,04 M). A absorbância foi medida a 520 e 700 nm usando 

espectrômetro UV/Vis (UV-3000, Shanghai Mapada Instruments Co., Ltd., 

Shanghai, China) (Martynenko & Chen, 2016). 

3.3.4 Cor 

A cor da superfície dos morangos será medida com um colorímetro ((Konica Minolta, 

Japão), previamente calibrado. Os parâmetros de cor obtidos serão L*, a* e b* 

((Wigati et al., 2023)a partir dos quais serão calculados o chroma e o ângulo hue. 

3.3.5 Firmeza 

A firmeza das frutas foi medida usando analisador de textura (TA.XT/Plus/50), com 

probe do tipo êmbolo cilíndrico. Os valores serão apresentados em Newton  (Locali-

Pereira et al., 2021). 

 



13 
 

3.3.6 Análises microbiológicas 

As análises microbiológicas dos morangos foram realizadas para a quantificação de 

bolores e leveduras e mesófilos anaeróbios.  

3.3.7 Cromatografia gasosa 

A quantificação dos compostos presentes no óleo essencial de pimenta rosa e dos 

respectivos teores conteúdos nos criogéis será realizada usando um cromatógrafo 

equipado com detector de ionização de chama (GC-FID) (CG-2014, Shimadzu, 

Japan), usando uma coluna capilar de sílica Rtx-5 (30 m X 0.25 mm X 0.25 µm); o 

nitrogênio será usado como gás de arrastre a uma velocidade de 1 mL/min. A 

identificação dos compostos voláteis foi realizada por cromatografia gasosa 

acoplada à espectrofotometria de massa (GC – MS QP2010 SE, Shimadzu, Japão). 

Uma coluna capilar de sílica fundida Rtx-5MS (30 m × 0,25 mm × 0,25 μm) foi usado 

com hélio como gás de arraste a uma vazão constante de 1 mL/min. Os mesmos 

parâmetros cromatográficos descritos para GC-FID foram utilizados em GC-MS. As 

temperaturas da interface e da fonte de ionização foram 240 °C e 230 °C, 

respectivamente, com impacto de elétrons a 70 eV e massa entre 35 e 350 m/z. A 

identificação do pico foi realizada por comparação do índice de retenção linear 

usando o NIST Chemistry WebBook e bibliotecas espectrais de massa (NIST MS 

Search versão 2.0 e banco de dados Wiley). Uma mistura padrão de alcanos (C7-

C30) (Sigma-Aldrich®), diluída 1:2 em hexano, foi injetada em GC-FID e GC-MS 

nas mesmas condições para cálculo do índice de retenção linear (Locali-Pereira et 

al., 2020) 

 

 4. Resultados  

 

Os resultados e discussão do objetivo: Desenvolver criogéis de albumina - pectina 

de baixa metoxilação amidada (ALB-PEC), e de proteína de grão de bico - pectina 

de baixa metoxilação amidada (BIC-PEC), com características estruturais 

adequadas para a encapsulação e liberação controlada dos compostos bioativos de 



14 
 

óleo de pimenta rosa, são apresentados no artigo Encapsulation of Pink Pepper 

Essential Oil (Schinus terebinthifolius Raddi) in Albumin and Low-Methoxyl 

Amidated Pectin Cryogels https://doi.org/10.3390/pr12081681.  

Em relação ao objetivo: Desenvolver embalagens ativas a partir dos criogéis e do 

óleo essencial de pimenta rosa para aumentar o tempo de vida de prateleira do 

morango são apresentados a seguir:  

Para atingir o objetivo proposto, inicialmente foram avaliadas diferentes condições 

de operação do cromatógrafo gasoso, incluindo parâmetros como fluxo de gás de 

arraste, split (30, 50 e 100), temperatura do injetor e do detector, e taxa de 

aquecimento. A Figura 1 apresenta o cromatograma do óleo essencial de pimenta 

rosa, obtido com as seguintes condições cromatográficas, selecionadas por sua 

capacidade de separação dos compostos: temperatura do injetor: 240°C, volume de 

injeção: 1 µL, taxa de injeção (modo split): 1:30, temperatura inicial da coluna: 60°C 

(mantida por 2 minutos), rampa de aquecimento: 2°C/min até 180°C (permanecendo 

por 4 minutos), seguida de 10°C/min até 240°C. Após a definição das condições 

cromatográficas, foram identificados os compostos majoritários presentes no óleo 

essencial de pimenta rosa: α-pineno, β-pineno, α-felandreno e D-limoneno. 

 

 

Figura 1. Cromatograma do óleo essencial de pimenta rosa. 

https://doi.org/10.3390/pr12081681


15 
 

Posteriormente, foi aplicado os criogéis nas embalagens ativas para a conservação 

de morango. Na Tabela 1 são apresentados os parâmetros de cor do morango 

durante o armazenamento usando os criogéis como embalagem ativas.  

Tabela 1. Parâmetros de cor do morango armazenado em embalagens ativas 

usando criogéis carregados com óleo de pimenta rosa.  

 

Parâmetros de 

cor 

Tempo 

armazenamento 

(dia) 

Tratamentos 

Controle 50:50 30:70 

L* 

0 33,32 ± 3,30 33,32 ± 3,30 33,32 ± 3,30 

3 34,50 ± 3,11 33,04 ± 3,42 33,93 ± 4,68 

5 36,66 ± 4,77 32,28 ± 3,03 31,69 ± 3,78 

7 33,15 ± 2,89 32,91 ± 5,14 32,69 ± 2,93 

a* 

0 36,77 ± 2,23 36,77 ± 2,23 36,77 ± 2,23 

3 39,92 ± 2,12 39,61 ± 2,30 38,75 ± 2,56 

5 38,38 ± 4,76 37,19 ± 2,32 37,83 ± 2,61 

7 38,43 ± 2,65 37,27 ± 2,71 37,14 ± 2,56 

b* 

0 27,14 ± 3,96 27,14 ± 3,96 27,14 ± 3,96 

3 31,03 ± 3,31 29,16 ± 3,81 28,51 ± 5,19 

5 32,93 ± 2,88 26,76 ± 4.09 29,99 ± 4,50 

7 29,34 ± 3,36 28,51 ± 5,76 28,31 ± 4,11 

Croma 

0 45,79 ± 3.53 45,79 ± 3.53 45,79 ± 3.53 

3 50,62 ± 3,22 49,24 ± 3,85 48,24 ± 4,53 

5 50,57 ± 5,56 45,88 ± 4,00 48,34 ± 4,62 

7 48,13 ± 3,52 47,04 ± 5,46 46,75 ± 4,32 

Hue 

0 36,27 ± 3,60 36,27 ± 3,60 36,27 ± 3,60 

3 37,79 ± 2,57 36,21 ± 2,63 36,75 ± 4.37 

5 40,82 ± 4,16 35,54 ± 3,01 38,20 ± 2,81 

7 37,15 ± 2,53 37,00 ± 3,96 37,14 ± 2,64 

 

Os resultados demonstraram que a cor dos morangos foi preservada durante todo 

o período de armazenamento (7 dias), mantendo a coloração avermelhada 

característica da fruta (Figura 2). Isso indica que a presença do criogel na 



16 
 

embalagem não interfere na cor, um parâmetro de grande importância na pós-

colheita do morango, pois influencia diretamente a sua qualidade e a percepção do 

consumidor. Esse resultado é promissor, uma vez que sugere a viabilidade da 

aplicação dos criogéis como embalagens ativas para a conservação da fruta. 

 

 Controle 50:50 30:70 

0d 

 

  

3d    

5d    

7d    

 

 



17 
 

No que diz respeito à textura, observou-se que a presença do criogel na embalagem 

ativa influenciou essa característica (Tabela 2). Embora a firmeza dos morangos 

tenha diminuído com o aumento do tempo de armazenamento, a presença do óleo 

essencial provocou uma redução ainda mais acentuada nesse parâmetro, em 

comparação com a amostra controle. Essa diferença sugere que o óleo essencial 

pode ter causado danos fitotóxicos aos morangos, resultando em amolecimento dos 

frutos durante o armazenamento (Wigati et al., 2023).  

Tabela 2. Firmeza do morango armazenado em embalagens ativas usando criogéis 

carregados com óleo de pimenta rosa.  

Tempo 

armazenamento 

(dia) 

Firmeza 

Controle 50:50 30:70 

0 1,613 ± 0,357 1,613 ± 0,357 1,613 ± 0,357 

3 1,535 ± 0,387 1,168 ± 0,491 0,634 ± 0,153 

5 1,690 ± 0,742 0,694 ± 0,172 0,840 ± 0,290 

7 0,869 ± 0,349 0,593 ± 0,344 0,565 ± 0,198 

 

Com relação ao conteúdo de antocianinas presentes nos morangos (Tabela 3), 

observou-se que todas as amostras mantiveram altos níveis desses compostos 

bioativos durante o armazenamento. É importante destacar que a presença dos 

criogéis carregados com óleo essencial de pimenta rosa não interferiu na 

concentração de antocianinas nos frutos, indicando que a tecnologia de embalagem 

ativa não prejudicou os compostos bioativos presentes no morango.  

 

 

 

 

 



18 
 

Tabela 3. Conteúdo de antocianinas no morango armazenado em embalagens 

ativas usando criogéis carregados com óleo de pimenta rosa.  

Tempo 

armazenamento (dia) 

Antocianinas (mg cianidina-3-O-glicosídeo) /g amostra) 

Controle 50:50 30:70 

0 80,462 ± 10,838 80,462 ± 10,838 80,462 ± 10,838 

3 86,275 ± 6,941 88,201 ± 4,294 96,230 ± 4,558 

5 79,912 ± 5,182 80,902 ± 3,830 94,738 ± 5,270 

7 82,454 ± 12,606 95,666 ± 6,771 80,350 ± 10,338 

 

 

 

5. Conclusão 

 

Os criogéis demonstram ser uma alternativa promissora para a encapsulação do 

óleo essencial de pimenta rosa. A composição da matriz do criogel influencia 

diretamente a capacidade de carregamento do óleo, a eficiência de encapsulação 

e as atividades antimicrobiana e antioxidante. Embora os criogéis possam ser 

utilizados como embalagens ativas para morangos, estudos mais aprofundados 

sobre o amolecimento dos frutos durante o armazenamento são necessários. 

 

 

 

 

 

 

 

 

 

 

 



19 
 

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Citation: Chaux-Gutiérrez, A.M.;

Pérez-Monterroza, E.J.; Cattelan, M.G.;

Nicoletti, V.R.; Moura, M.R.d.

Encapsulation of Pink Pepper

Essential Oil (Schinus terebinthifolius

Raddi) in Albumin and

Low-Methoxyl Amidated Pectin

Cryogels. Processes 2024, 12, 1681.

https://doi.org/10.3390/pr12081681

Academic Editor: Renata Różyło

Received: 21 July 2024

Revised: 6 August 2024

Accepted: 6 August 2024

Published: 12 August 2024

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

processes

Article

Encapsulation of Pink Pepper Essential Oil
(Schinus terebinthifolius Raddi) in Albumin
and Low-Methoxyl Amidated Pectin Cryogels
Ana María Chaux-Gutiérrez 1,*, Ezequiel José Pérez-Monterroza 2, Marília Gonçalves Cattelan 2,
Vânia Regina Nicoletti 2 and Márcia Regina de Moura 1

1 Faculdade de Engenharia, Universidade Estadual Paulista (UNESP), Ilha Solteira 15385-007, Brazil;
marcia.aouada@unesp.br

2 Instituto de Biociências Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP),
São José do Rio Preto 15054-000, Brazil; eperez494@gmail.com (E.J.P.-M.); marilia.cattelan@unesp.br (M.G.C.);
vania.nicoletti@unesp.br (V.R.N.)

* Correspondence: ana.chaux@unesp.br or anachauxg@gmail.com

Abstract: This study evaluated cryogels from albumin (ALB) and albumin–pectin (ALB:PEC) as
carriers for pink pepper (Schinus terebinthifolius Raddi) essential oil. Cryogels were evaluated through
infrared spectrophotometry, X-ray diffraction, scanning electron microscopy, thermogravimetric
analysis, and differential scanning calorimetry. The bioactivity of the cryogels was analyzed by mea-
suring their encapsulation efficiency (EE%), the antimicrobial activity of the encapsulated oil against
S. aureus, E. coli, and B. cereus using the agar diffusion method; total phenolic content and antioxidant
activity were analyzed by UV-vis spectrophotometry. The EE% varied between 59.61% and 77.41%.
The cryogel with only ALB had the highest total phenolic content with 2.802 mg GAE/g, while the
cryogel with the 30:70 ratio (ALB:PEC) presented a value of 0.822 mg GAE/g. A higher proportion of
PEC resulted in a more significant inhibitory activity against S. aureus, reaching an inhibition zone
of 18.67 mm. The cryogels with ALB and 70:30 ratio (ALB:PEC) presented fusion endotherms at
137.16 ◦C and 134.15 ◦C, respectively, and semicrystalline structures. The interaction between ALB
and PEC increased with their concentration, as evidenced by the decreased intensity of the O-H
stretching peak, leading to lower encapsulation efficiency. The cryogels obtained can be considered a
suitable matrix for encapsulating pink pepper oil.

Keywords: antimicrobial activity; polysaccharides; biopolymers; phenolic compounds; S. aureus

1. Introduction

The interest in technological applications of essential oils (EOs) has been increasing due
to their antioxidant properties and antimicrobial action against foodborne pathogens [1].
Among the EOs, pink pepper essential oil (Schinus terebinthifolius Raddi) stands out for its
antioxidant, antimicrobial, and anti-inflammatory properties, and anticancer capacity [2],
which are attributed to its major constituents that include α-pinene, β-pinene, β-myrcene,
β-cubebene, and limonene, in addition to monoterpenes and sesquiterpenes [3,4]. Pink
pepper essential oil can be affected by external factors such as oxygen, light, humidity, and
pH, which reduce biological activity [5], so encapsulation processes have become a tool to
increase its stability and even improve its release profile [6]. Different technologies, includ-
ing spray drying [7–14], complex coacervation [15–21], emulsion extrusion technique [15],
and emulsification-ionic gelation [22–24] may be used for encapsulating pink pepper essen-
tial oil. However, recently, researchers have become interested in protecting essential oils
using hydrogels and cryogels because they are produced from biopolymers without using
crosslinker agents, while at the same time allowing the design of a controlled release system.
Cryogels are obtained from hydrogels prepared from proteins or polysaccharides dried

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Processes 2024, 12, 1681 2 of 14

by lyophilization [25]. Cinnamon essential oil encapsulated in gelatin hydrogels offers a
constant release over a long period while it maintains a high antimicrobial capacity against
S. aureus and E. coli [26]. Also, the microgels obtained from chitosan maintain the biological
activity of the essential oil of Gaultheria procumbens against the secretion of aflatoxins from
A. flavus [27]. The cryogels offer advantages due to high load capacity and encapsulation
efficiency. Volić et al. [28] reported that calcium alginate used as wall material reached
an encapsulation efficiency of about 85% for thyme essential oil, and the combination of
calcium alginate and soy protein resulted in an efficiency of 80%.

Egg albumin, a globular protein, possesses excellent gel-forming properties. Heat
treatment above 82 ◦C induces protein denaturation, disrupting its structural integrity and
facilitating the formation of high molecular weight aggregates. These aggregates, stabilized
by disulfide, hydrogen, hydrophobic, and electrostatic interactions, contribute to increased
solution viscosity and subsequent gelation [29–31]. Low-methoxyl amidated pectin, a
polysaccharide containing amino groups, exhibits superior gelling characteristics compared
to standard low-methoxyl pectin. It forms strong gels at low calcium concentrations and
can gel under acidic conditions (pH < 3) [32]. There are few studies on the encapsulation
of essential oils in cryogels of egg albumin and low-methoxyl amidated pectin; these
biopolymers stand out for their ability to form gels and protect bioactive compounds [33].
From these considerations, this study aimed to encapsulate pink pepper essential oil in
cryogels prepared from egg albumin and low-methoxyl amidated pectin, as well as to
evaluate the inhibitory properties of this encapsulation system on S. aureus, E. coli, and B.
cereus. In addition, to better understand the performance of the supramolecular assembly,
we analyzed the microstructure and bioactivity of the produced cryogels.

2. Materials and Methods

Pink pepper (Schinus terebinthifolius Raddi) essential oil extract from fruits was ob-
tained from Ferquima (Vargem Grande Paulista, São Paulo, Brazil). Powdered egg al-
bumin (ALB) was purchased from Neovita Foods Eirelli (São Paulo, São Paulo, Brazil;
83.3% protein, 5.0% carbohydrates, and 0.0% fat). Amidated low-methoxyl pectin (PEC)
was purchased from Danisco (GRINDSTED LA 210; 34% esterification degree and 17%
amidation, Barueri, São Paulo, Brazil). Sodium hydroxide, calcium chloride, and hydrochlo-
ric acid were obtained from Panreac (Química, S.A, Castellar de Vallès, Barcelona, Spain).
Mueller–Hinton agar (HiMedia, Sumaré, São Paulo, Brazil). Ethanol (95%, vol/vol) was
supplied by Synth (Diadema, São Paulo, Brazil). All reagents were analytical grade.

2.1. Encapsulation of Pink Pepper Essential Oil in Cryogels

The encapsulation of pink pepper essential oil was carried out according to the method-
ology proposed by Chaux-Gutiérrez et al. [33] and Volić et al. [28] with some modifications.
Individual dispersions of ALB (5% w/w, based on the total mass of the dispersion) and PEC
(5% w/w, based on the total mass of the dispersion) were prepared at room temperature,
maintaining constant stirring for 3 h until complete dispersion using a magnetic stirrer.
For preparing ALB hydrogels, the ALB dispersion was adjusted to pH 8 using HCl (0.1 M)
and NaOH (0.1 M) solutions, heated to 85 ◦C, kept under constant stirring for 15 min, and
cooled to 40 ◦C. Then, 3% of pink pepper essential oil (% w/w, based on the total mass of
the dispersion) was dispersed into the ALB hydrogel at 14000 rpm for 5 min (Ultra-Turrax®

IKA T25, IKA-Werke GmbH, Staufen, Baden-Württemberg, Germany). Finally, the mixture
was stored in a Petri dish at 4 ◦C until gel formation. For preparing ALB-PEC gels, ALB
and PEC dispersions were first prepared as described above, and then mixed at different
ALB:PEC ratios—70:30, 50:50, and 30:70—maintaining constant stirring for 5 min. After
that, 3% of the pink pepper essential oil (% w/w, based on the total mass of the dispersion)
was added, and the mixture was homogenized at 14,000 rpm for 5 min (Ultra-Turrax®

IKA T25, IKA-Werke GmbH, Staufen, Baden-Württemberg Bodense, Germany). Then,
2% (w/w) calcium chloride solution (prepared previously at 2% w/v) was incorporated,
maintaining constant stirring for 10 min. The mixture was stored in a Petri dish at 4 ◦C



Processes 2024, 12, 1681 3 of 14

until gel formation. Finally, the gels were frozen at −18 ◦C for 24 h and then freeze-dried
(model L-101, Liotop, São Carlos, São Paulo, Brazil) at 40 µmHg for 48 h. The lyophilized
samples were stored in metalized bags within desiccators at 25 ◦C. (Figure 1).

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constant stirring for 5 min. After that, 3% of the pink pepper essential oil (% w/w, based 
on the total mass of the dispersion) was added, and the mixture was homogenized at 
14,000 rpm for 5 min (Ultra-Turrax® IKA T25, IKA-Werke GmbH, Staufen, Baden-
Württemberg Bodense, Germany). Then, 2% (w/w) calcium chloride solution (prepared 
previously at 2% w/v) was incorporated, maintaining constant stirring for 10 min. The 
mixture was stored in a Petri dish at 4 °C until gel formation. Finally, the gels were frozen 
at −18 °C for 24 h and then freeze-dried (model L-101, Liotop, São Carlos, São Paulo, Brazil) 
at 40 µmHg for 48 h. The lyophilized samples were stored in metalized bags within 
desiccators at 25 °C. (Figure 1). 

 
Figure 1. Schematic diagram for the preparation of ALB and ALB:PEC cryogels. 

2.2. Encapsulation Efficiency (EE%) 
The encapsulation efficiency of the cryogels was determined according to the 

methodology of Abreu et al. [34] with some modifications. One hundred milligrams of 
cryogels was dispersed in 5 mL of ethanol. The mixture was centrifuged at 9000 rpm for 5 
min at 25 °C using a centrifugal separation (Z 326 K, HERMLE Labortechnik GmbH, 
WehinGen, Baden-Würtemberg, Germany). The pink pepper essential oil content was 
measured using a UV-vis spectrophotometer (SP-200, Biospectro, Curitiba, Paraná, Brazil) 
at a wavelength of 291 nm, using a calibration curve (y = 0.7269x + 0.223, R2 = 0.9949) of 
essential oil in ethanol solution (0.2–1.2 mg/mL). The encapsulation efficiency (EE%) was 
calculated according to Equation (1): 𝐸𝐸 %   100  (1)

where M is the amount (mg) of oil in loaded cryogels and Mo is the initial oil amount (mg) 
added to cryogel preparation. 

2.3. Total Phenolic Content 

Figure 1. Schematic diagram for the preparation of ALB and ALB:PEC cryogels.

2.2. Encapsulation Efficiency (EE%)

The encapsulation efficiency of the cryogels was determined according to the method-
ology of Abreu et al. [34] with some modifications. One hundred milligrams of cryogels
was dispersed in 5 mL of ethanol. The mixture was centrifuged at 9000 rpm for 5 min at
25 ◦C using a centrifugal separation (Z 326 K, HERMLE Labortechnik GmbH, WehinGen,
Baden-Würtemberg, Germany). The pink pepper essential oil content was measured using
a UV-vis spectrophotometer (SP-200, Biospectro, Curitiba, Paraná, Brazil) at a wavelength
of 291 nm, using a calibration curve (y = 0.7269x + 0.223, R2 = 0.9949) of essential oil
in ethanol solution (0.2–1.2 mg/mL). The encapsulation efficiency (EE%) was calculated
according to Equation (1):

EE(%) =
M
M0

× 100 (1)

where M is the amount (mg) of oil in loaded cryogels and Mo is the initial oil amount (mg)
added to cryogel preparation.

2.3. Total Phenolic Content

The phenolic content of the cryogels was determined using the Folin–Ciocalteu
method [35]. Extracts were prepared using methanol, and the absorbance was read at
725 nm in a UV/VIS spectrophotometer (SP-200, Biospectro, Curitiba, Paraná, Brazil).
Quantification was performed using a calibration curve of aqueous solutions of gallic
acid (0–500 ppm). The results were expressed in mg of gallic acid equivalent per gram
(mg GAE/g).



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2.4. Antioxidant Activity

The antioxidant activity was determined by radical ABTS*+ capture methodology,
according to a method proposed by Re et al. [36]. Extracts were prepared using methanol
and the absorbance was measured at 730 nm (SP-200, Biospectro, Brazil). The antioxidant
activity was determined using a calibration curve prepared with an aqueous solution of
Trolox (0–200 µmol/L) and expressed as micromoles of Trolox equivalents (TEs) per gram
(µmol TEs/g).

2.5. In Vitro Antimicrobial Activity

Evaluation of the inhibitory effects of essential oil encapsulated in cryogels on selected
bacteria was performed in vitro using the agar diffusion method according to the Clinical
and Laboratory Standards Institute [37], with some adaptations. Three bacteria were se-
lected: two Gram-positive (Staphylococcus aureus ATCC6538 and Bacillus cereus ATCC11778),
and one Gram-negative (Escherichia coli ATCC8739). Suspensions were prepared for each
of the bacteria at a concentration of 108 CFU/mL. The culture medium used in the Petri
dishes for microbial strains was MMA Growth Agar. In the inoculated agar, wells of 7 mm
diameter were made using a sterile metal cylinder; subsequently, these were filled with
different amounts of the cryogels (1.25, 2.5, 5, 10, 20, and 30 mg). Cryogel without essential
oil was used as blank. The inoculated material was incubated at 37 ◦C for 24 h. After
incubation, inhibition zone diameters were measured in millimeters.

2.6. Fourier Transform Infrared Spectrometry (FT-IR)

FT-IR spectra of cryogels were recorded using a Spectrum One (Perkin-Elmer Corp.,
Shelton, CT, USA) device with an attenuated total reflectance accessory with a ZnSe crystal.
Cryogels were ground into a fine powder using mortar and pestle. Then, samples were
analyzed directly after pressing them on the crystal (80 psi). The scanning was conducted
from 4000 to 400 cm−1 using 20 scans, with a resolution of 4 cm−1 [33].

2.7. X-ray Diffraction

The X-ray diffraction (XRD) patterns of cryogels were determined using a RINT
2000 diffractometer (Rigaku, Tokio, Japan) equipped with a Cu Kα radiation source
(λ = 1.542 Å). Operating conditions were 45 kV and 30 mA. Data were collected from
5◦ and 50◦ (2θ), with step sizes of 0.02◦ and a scan rate of 1 s per step. Before analysis,
cryogel samples were ground into a fine powder using a mortar and pestle.

2.8. Thermal Analysis

Thermogravimetric analysis (TGA) (SDT Q600 Thermogravimetric Analyzers, TA
Instrument, New Castle, DE, USA) was used to estimate the thermal stability of cryogels.
Each 5 mg sample was placed in the platinum pan and heated up by a TGA furnace at
10 ◦C/min under a nitrogen atmosphere from 25 to 200 ◦C. Differential scanning calorime-
try was carried out using a calorimeter (DSC-25, TA Instrument, New Castle, DE, USA)
previously calibrated with indium. An empty aluminum pan was used as a reference. Each
3 mg sample was weighed, cooled from 25 to −50 ◦C at 35 ◦C/min, maintained at this
temperature for 1 min, and subsequently heated at 35 ◦C/min from −50 to 200 ◦C.

2.9. Scanning Electron Microscopy (SEM)

The morphology of the cryogels was examined using scanning electron microscopy
(SEM, EVO LS15, Zeiss, Carl Zeiss, Ostalbkreis, Baden-Württerberg, Germany) at 20 kV.
Samples were prepared by mounting cryogels on double-sided carbon tape and coating
them with a thin layer of gold using a sputter coater (Quorum, model Q150 T, Lewes,
UK) for 1.5 min. Micrographs were acquired from both the surface and the cross-section
of cryogels.



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2.10. Statistical Analysis

All measurements were performed in triplicate and results were expressed as
mean ± standard deviation. Statistical analyses were performed with one-way analy-
sis of variance (ANOVA) using the Minitab 21® program, considering a significance level
of 5%. Tukey’s test was used to compare differences among the mean values of samples.

3. Results and Discussion
3.1. Encapsulation Efficiency (EE%)

Encapsulation efficiency is crucial for assessing the potential of ALB and PEC as
suitable materials for encapsulating pink pepper essential oil. Higher efficiency indicates
a better encapsulation process. It is also considered a measure of the essential oil that
could reach its target site. However, this depends on wall structural characteristics and
its affinity for the essential oil. The encapsulation efficiency of the cryogels with varying
ALB:PEC ratios ranged between 59.6 and 77.4% (Table 1), being almost constant for the
cryogels with higher contents of ALB (pure ALB, 70:30 and 50:50 ALB:PEC). However,
an increase in the PEC concentration, as in the 30:70 (ALB:PEC) ratio, led to a decrease
in efficiency. This suggests a limited concentration for pectin, beyond which the EE%
decreases. These results are attributed to the superior ability of proteins to form films
and trap oil compared to polysaccharides [38]. Studies on oregano essential oil (Origanum
vulgare Linneus) encapsulation exemplify this effect: a 79% encapsulation efficiency was
achieved using zein at 0.2% (w/v) in the wall material [21]. In contrast, chitosan at 1% (w/v)
resulted in a lower efficiency of 24.72% [39]. Similarly, increasing the protein concentration
in systems composed of whey protein isolate (WPI) and carboxymethylcellulose (CMC)
for encapsulation of orange essential oil leads to higher efficiency: the encapsulation
efficiency increased from 3% in the 1:1 (WPI:CMC) ratio to 86% in the 3:1 (WPI:CMC) [40].
Bastos et al. [41] reported a similar trend when encapsulating black pepper essential oil
(Piper nigrum L) using a wall material composed of gelatin and alginate: a ratio of 0.3:0.05
(gelatin: alginate) resulted in an efficiency of 52.26%, while increasing the protein content
to a ratio of 0.9:0.15 (gelatin:alginate) increased efficiency to 82.20%. The encapsulation
efficiency obtained in the present study is within a similar range to those reported by Chen
and Zhong [42] and Rajkumar et al. [43] for encapsulation of mint essential oil in chitosan
nanoparticles, for which they achieved an efficiency of 64%.

Table 1. Encapsulation efficiency, total phenolic content, and antioxidant activity of pink pepper
essential oil encapsulated in ALB and ALB: PEC cryogels.

Sample EE (%) Total Phenolic Content (mg GAE/g) ABTS (µmol TEs/g)

ALB 75.0 ± 5.5 a 2.80 ± 0.23 a 6.28 ± 0.49 a

70:30 ALB:PEC 75.2 ± 4.9 a 2.31 ± 0.18 b 1.16 ± 0.16 b

50:50 ALB:PEC 77.4 ± 1.9 a 1.41 ± 0.11 c 0.84 ± 0.05 b

30:70 ALB:PEC 59.6 ± 2.3 b 0.82 ± 0.17 d 0.99 ± 0.24 b

Results expressed as mean (n = 3) ± standard deviation. Different lowercase letters in the column indicate
significant differences between treatments. GAE: gallic acid equivalent. TEs: Trolox equivalents.

3.2. Total Phenolic Compound Content and Antioxidant Activity

Phenolic compounds, abundant secondary metabolites in plants and recognized for
their antioxidant properties, include flavonoids, phenolic acids, and terpenes such as car-
vacrol, eugenol, and p-cinene [44,45]. This study examined the influence of wall material
composition on total phenolic content and their antioxidant activity in cryogels prepared
with either ALB or ALB:PEC. Phenolic content ranged from 2.80 to 0.82 mg GAE/g (Table 1),
with higher values observed in the cryogels with greater ALB content. This result could be
related to protein denaturation during the production of the cryogels. Thermal treatment
enhances protein–polyphenol interactions via hydrogen bonding [46]. Unfolded polypep-
tide chains in the heat-treated protein expose hydrophobic amino acid residues, creating



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a hydrophobic site for entrapped phenolic compounds within the three-dimensional net-
work [47]. Conversely, the negatively charged PEC electrostatically interacts with positively
charged sites on ALB [29], altering protein–phenolic compound interaction and reducing
encapsulation efficiency; this explains the decreasing total phenolic content observed in
cryogels with increasing PEC ratios (70:30, 50:50, 30:70 ALB:PEC). Similarly, Volić et al. [28]
reported greater encapsulation efficiency (80% of the total phenolics) for thyme essential oil
in a 1:1.5 (% w/w) alginate:soy protein blend compared to alginate alone (72% of the total
phenolics). On the other hand, the antioxidant activity did not show significant differences
in cryogels prepared with PEC (p < 0.05). It was observed that the presence of PEC in the
wall material, even at the lower level, decreased the encapsulation of phenolic compounds
contained in the essential oil, which then contributed to a decrease in the antioxidant activ-
ity, ranging from 6.28 to 0.99 µmol TEs/g (Table 1). Authors such as Arslan and Çelik [48]
report that the antioxidant activity of Salvia cidronella Boiss essential oil is associated with
its phenolic compound content.

3.3. Antimicrobial Activity

Pink pepper oil has known antimicrobial properties due to the presence of monoter-
penes such as α-pinene, D-Limonene, α-phellandrene, γ-terpene, and p-cymene, and
sesquiterpenes such as caryophyllene, germacrene-D, and α-Muurolene that can damage
microbial cell walls [49,50]. In the present study, the antimicrobial activity of the pro-
posed encapsulation system was evaluated by measuring inhibition zones formed around
cryogels in contact with gram-positive and gram-negative bacteria cultures [37]. The in-
hibition zone size reflects the amount of essential oil diffusing through the cryogel and
inhibiting microbial growth. The results showed that none of the cryogels were able to
inhibit B. cereus or E. coli growth even at the higher amounts applied. The resistance of
E. coli (gram-negative) cells is due to the envelope; the complex structure, including the
lipopolysaccharide membrane, hinders the penetration of monoterpenes like D-limonene
and γ-terpinene found in pink pepper oil [50,51]. The B. cereus, an endospore-forming
bacterium, offers increased resistance to monoterpenes found in pink pepper essential
oil [52]. However, Dannenberg et al. [53] reported pink pepper essential oil inhibiting B.
cereus, suggesting variations in essential oil composition. Encapsulated pink pepper oil
inhibited the growth of S. aureus, but this effect depended on the mass of cryogels used. The
sensitivity of S. aureus is likely due to its cell wall, which is around 90% peptidoglycan. This
composition makes it less resistant to hydrophobic molecules, allowing them to destabilize
the cell wall and cytoplasm [51]. Phenolic compounds in the essential oil can further alter
the bacteria cell membrane by binding to proteins and disrupting their function [52]. The
study also found a relationship between the concentration of ALB and PEC in the cryogel
wall material and the size of the inhibition zone (Figure 2 and Table 2). Cryogels with a
30:70 (ALB:PEC) ratio required the least mass (only 5 mg) to show inhibition. Conversely,
cryogels with 50:50 (ALB:PEC) ratio, ALB alone, and 70:30 (ALB:PEC) required a minimum
of 10 and 20 mg, respectively. Interestingly, although the cryogels prepared with ALB alone
and with 70:30 and 50:50 (ALB:PEC) ratios had a higher encapsulation efficiency (Table 1),
their required mass for the same inhibitory effect was higher, suggesting that some of the
essential oil was unavailable as an antimicrobial agent, possibly due to interaction with
the unfolded protein in the cryogel wall. Evans et al. [38] suggested that these interactions
correspond to hydrophobic bonds. A higher pectin ratio in the cryogel wall material led to
larger pores, as observed in the SEM analysis (as discussed in Section 3.7), which facilitated
the release and diffusion of the encapsulated oil. The cryogel with a 30:70 (ALB:PEC) ratio
showed a significant increase in the inhibition zone (from 9.33 mm to 18.67 mm) when
the mass increased from 5 mg to 30 mg, compared to cryogels with only ALB or a 70:30
(ALB:PEC) ratio. These results align with previous findings of S. aureus inhibition by pink
pepper oil encapsulated in soy protein isolate/high methoxyl pectin [4]. These results
suggest that amidated low methoxyl pectin is crucial in designing a controlled release
system for pink pepper oil by acting as the release trigger.



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suggested that these interactions correspond to hydrophobic bonds. A higher pectin ratio 
in the cryogel wall material led to larger pores, as observed in the SEM analysis (as 
discussed in Section 3.7), which facilitated the release and diffusion of the encapsulated 
oil. The cryogel with a 30:70 (ALB:PEC) ratio showed a significant increase in the 
inhibition zone (from 9.33 mm to 18.67 mm) when the mass increased from 5 mg to 30 mg, 
compared to cryogels with only ALB or a 70:30 (ALB:PEC) ratio. These results align with 
previous findings of S. aureus inhibition by pink pepper oil encapsulated in soy protein 
isolate/high methoxyl pectin [4]. These results suggest that amidated low methoxyl pectin 
is crucial in designing a controlled release system for pink pepper oil by acting as the 
release trigger. 

 
Figure 2. Inhibitory activity assay agar disk diffusion of pink pepper essential oil encapsulated in 
ALB (a), 70:30 ALB:PEC (b), 50:50 ALB:PEC (c), and 30:70 (d) cryogels against S. aureus (ATCC 6538). 

Table 2. Inhibitory activity (agar disk diffusion) against S. aureus (ATCC 6538) of pink pepper 
essential oil encapsulated in ALB and ALB:PEC cryogels. 

 Inhibition Zone (mm) 
Mass Cryogel (mg) ALB 70:30 ALB:PEC 50:50 ALB:PEC 30:70 ALB:PEC 

1.25 n.d n.d n.d n.d  
2.5 n.d n.d n.d n.d 
5 n.d n.d n.d 9.33 ± 1.12 d 

10 0.50 ± 0.43 ef n.d 9.44 ± 0.88 d 12.44 ± 3.54 c 

20 1.33 ± 0.50 ef 1.89 ± 0.60 ef 15.56 ± 1.94 b 17.22 ± 2.59 ab 

30 1.17 ± 0.41 ef 2.44 ± 0.73 e 18.22 ± 1.39 a 18.67 ± 2.83 a 

Results expressed as mean (n = 6) ± standard deviation. Different lowercase letters in the row 
indicate significant differences between treatments. n.d = not detected. 

3.4. FT-IR Spectroscopy 
The Fourier-transform infrared (FTIR) spectra of the cryogels loaded with essential 

oil revealed characteristic peaks from the stretching of the C–H bond at 2934 and 2872 
cm−1, typically associated with alkenes; peaks between 1440 and 1447 cm−1, indicative of 
C–H and C–O stretching; as well as a peak at 884 cm−1 associated with the C–H out-of-
plane bending vibration due to the presence of monoterpenes present in essential oil 

Figure 2. Inhibitory activity assay agar disk diffusion of pink pepper essential oil encapsulated in
ALB (a), 70:30 ALB:PEC (b), 50:50 ALB:PEC (c), and 30:70 (d) cryogels against S. aureus (ATCC 6538).

Table 2. Inhibitory activity (agar disk diffusion) against S. aureus (ATCC 6538) of pink pepper essential
oil encapsulated in ALB and ALB:PEC cryogels.

Inhibition Zone (mm)
Mass Cryogel (mg) ALB 70:30 ALB:PEC 50:50 ALB:PEC 30:70 ALB:PEC

1.25 n.d n.d n.d n.d
2.5 n.d n.d n.d n.d
5 n.d n.d n.d 9.33 ± 1.12 d

10 0.50 ± 0.43 ef n.d 9.44 ± 0.88 d 12.44 ± 3.54 c

20 1.33 ± 0.50 ef 1.89 ± 0.60 ef 15.56 ± 1.94 b 17.22 ± 2.59 ab

30 1.17 ± 0.41 ef 2.44 ± 0.73 e 18.22 ± 1.39 a 18.67 ± 2.83 a

Results expressed as mean (n = 6) ± standard deviation. Different lowercase letters in the row indicate significant
differences between treatments. n.d = not detected.

3.4. FT-IR Spectroscopy

The Fourier-transform infrared (FTIR) spectra of the cryogels loaded with essential oil
revealed characteristic peaks from the stretching of the C–H bond at 2934 and 2872 cm−1,
typically associated with alkenes; peaks between 1440 and 1447 cm−1, indicative of C–
H and C–O stretching; as well as a peak at 884 cm−1 associated with the C–H out-of-
plane bending vibration due to the presence of monoterpenes present in essential oil
(Figure 3) [54,55]. The intensity of the peak at 3275 cm−1, assigned to O-H stretching
corresponding to the stretching of the O–H hydroxyl group [56], decreased with increasing
PEC concentration in the 50:50 and 30:70 (ALB:PEC) cryogels, suggesting a more significant
interaction between the ALB and PEC components in the wall material; this could explain
the lower encapsulation efficiency observed for these formulations. Conversely, in the ALB
and 70:30 (ALB:PEC) cryogels, the absorption bands at 1600 cm−1 corresponding to the
amide I group, which represents the secondary structure of the protein, were shifted to
lower wavenumber. This shift, associated with the stretching vibration of C-N bonds [57]
in the cryogels, suggests an interaction between polyphenols and protein, as evidenced by
the higher phenolic compounds content in these cryogels.



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(Figure 3) [54,55]. The intensity of the peak at 3275 cm−1, assigned to O-H stretching 
corresponding to the stretching of the O–H hydroxyl group [56], decreased with 
increasing PEC concentration in the 50:50 and 30:70 (ALB:PEC) cryogels, suggesting a 
more significant interaction between the ALB and PEC components in the wall material; 
this could explain the lower encapsulation efficiency observed for these formulations. 
Conversely, in the ALB and 70:30 (ALB:PEC) cryogels, the absorption bands at 1600 cm−1 
corresponding to the amide I group, which represents the secondary structure of the 
protein, were shifted to lower wavenumber. This shift, associated with the stretching 
vibration of C-N bonds [57] in the cryogels, suggests an interaction between polyphenols 
and protein, as evidenced by the higher phenolic compounds content in these cryogels. 

 
Figure 3. Infrared spectra of albumin (ALB) and albumin:pectin (ALB:PEC) cryogels with and 
without pink pepper essential oil encapsulated. 

3.5. X-ray Diffraction 
Figure 4 shows the X-ray diffraction (XRD) of the cryogels. All cryogels showed 

characteristic diffraction patterns of semicrystalline structures. Compared to pure 
albumin, the disappearance of the broad peak at 9° in the cryogels indicates alterations to 
the secondary structure of the protein [58]. The intensity of the peaks increased with the 
presence of PEC, due to the more crystalline nature of this polysaccharide (Figure 4f). This 
behavior resembles that observed in studies of edible films produced with egg albumin 
protein (5% w/v) and pectin (5% w/v) [59]. However, the characteristic diffraction pattern 
of pure PEC (Figure 4f) practically disappears in ALB:PEC cryogels due to interactions 
between the unfolded protein chains and the polysaccharide, which alters the crystalline 
regions of the PEC and decreases the crystallinity of the sample, as was also observed by 
Mebarki et al. [60], who found that the casein–pectin interaction leads to a decrease in 
crystallinity. This reduction in crystallinity is generally advantageous for encapsulation 
systems, as it can improve bioactive compound release and bioavailability, according to 
Chen et al. [58] and Ghobadi et al. [61]. 

Wavenumber (cm-1) 
Figure 3. Infrared spectra of albumin (ALB) and albumin:pectin (ALB:PEC) cryogels with and without
pink pepper essential oil encapsulated.

3.5. X-ray Diffraction

Figure 4 shows the X-ray diffraction (XRD) of the cryogels. All cryogels showed
characteristic diffraction patterns of semicrystalline structures. Compared to pure albumin,
the disappearance of the broad peak at 9◦ in the cryogels indicates alterations to the
secondary structure of the protein [58]. The intensity of the peaks increased with the
presence of PEC, due to the more crystalline nature of this polysaccharide (Figure 4f). This
behavior resembles that observed in studies of edible films produced with egg albumin
protein (5% w/v) and pectin (5% w/v) [59]. However, the characteristic diffraction pattern
of pure PEC (Figure 4f) practically disappears in ALB:PEC cryogels due to interactions
between the unfolded protein chains and the polysaccharide, which alters the crystalline
regions of the PEC and decreases the crystallinity of the sample, as was also observed by
Mebarki et al. [60], who found that the casein–pectin interaction leads to a decrease in
crystallinity. This reduction in crystallinity is generally advantageous for encapsulation
systems, as it can improve bioactive compound release and bioavailability, according to
Chen et al. [58] and Ghobadi et al. [61].

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Figure 4. X-ray diffraction patterns of albumin (ALB) (a), 70:30 (ALB:PEC) (b), 50:50 (ALB:PEC) (c), 
and 30:70 (ALB:PEC) (d) cryogels with pink pepper essential oil encapsulated, albumin pure (e), 
and amidated low methoxyl pectin (PEC) pure (f). 

3.6. Thermal Analysis 
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were 

employed to investigate the thermal behavior of the cryogels. These techniques provide 
valuable insights into the material thermal stability and potential first-order or second-
order transitions [62]. TGA analysis showed distinct mass loss patterns between cryogels 
(Figure 5a). The cryogel containing only albumin (ALB) exhibited the lowest overall mass 
loss (7.66%) and the most excellent thermal stability above 80 °C. Conversely, cryogels 
formulated with ALB and PEC displayed higher mass losses of 11.5% (70:30), 12.4% 
(50:50), and 12.5% (30:70). The derivative TGA curves indicated a single significant mass 
loss event for the ALB-only cryogel. In contrast, PEC-containing cryogels showed multiple 
events, suggesting a more complex degradation process (Figure 5b). The analysis of DTG 
curves (the first derivative of TGA) revealed distinct mass loss patterns for the cryogels 
(Figure 5b). The cryogel with only ALB exhibited the highest mass loss rate within the 40–
70 °C temperature range, followed by cryogels with ALB:PEC ratios of 50:50, 70:30, and 
30:70, respectively. Notably, the 70:30 ALB:PEC cryogel displayed two distinct peaks at 
59.13 °C and 154.87 °C, while the others showed three events at 54.59 °C, 101.82 °C, and 
151.48 °C for the ratio 50:50, and 53.84 °C, 122.39 °C, and 148.80 °C for the 30:70 ratio 
(ALB:PEC). The presence of PEC influenced the degradation profile. Cryogels with PEC 
exhibited lower mass loss rates between 20 and 80 °C than the ALB-only cryogel, 
indicating enhanced stability in this range. However, ALB provided superior thermal 
stability at higher temperatures (>80 °C). Finally, all PEC-containing cryogels underwent 
degradation between 170 and 190 °C, with higher PEC content correlating to a lower 
degradation temperature (176.6 °C); this suggests the depolymerization and 
decomposition of PEC units within the cryogel matrix, as previously reported by Liu et 

Figure 4. Cont.



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Figure 4. X-ray diffraction patterns of albumin (ALB) (a), 70:30 (ALB:PEC) (b), 50:50 (ALB:PEC) (c), 
and 30:70 (ALB:PEC) (d) cryogels with pink pepper essential oil encapsulated, albumin pure (e), 
and amidated low methoxyl pectin (PEC) pure (f). 

3.6. Thermal Analysis 
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were 

employed to investigate the thermal behavior of the cryogels. These techniques provide 
valuable insights into the material thermal stability and potential first-order or second-
order transitions [62]. TGA analysis showed distinct mass loss patterns between cryogels 
(Figure 5a). The cryogel containing only albumin (ALB) exhibited the lowest overall mass 
loss (7.66%) and the most excellent thermal stability above 80 °C. Conversely, cryogels 
formulated with ALB and PEC displayed higher mass losses of 11.5% (70:30), 12.4% 
(50:50), and 12.5% (30:70). The derivative TGA curves indicated a single significant mass 
loss event for the ALB-only cryogel. In contrast, PEC-containing cryogels showed multiple 
events, suggesting a more complex degradation process (Figure 5b). The analysis of DTG 
curves (the first derivative of TGA) revealed distinct mass loss patterns for the cryogels 
(Figure 5b). The cryogel with only ALB exhibited the highest mass loss rate within the 40–
70 °C temperature range, followed by cryogels with ALB:PEC ratios of 50:50, 70:30, and 
30:70, respectively. Notably, the 70:30 ALB:PEC cryogel displayed two distinct peaks at 
59.13 °C and 154.87 °C, while the others showed three events at 54.59 °C, 101.82 °C, and 
151.48 °C for the ratio 50:50, and 53.84 °C, 122.39 °C, and 148.80 °C for the 30:70 ratio 
(ALB:PEC). The presence of PEC influenced the degradation profile. Cryogels with PEC 
exhibited lower mass loss rates between 20 and 80 °C than the ALB-only cryogel, 
indicating enhanced stability in this range. However, ALB provided superior thermal 
stability at higher temperatures (>80 °C). Finally, all PEC-containing cryogels underwent 
degradation between 170 and 190 °C, with higher PEC content correlating to a lower 
degradation temperature (176.6 °C); this suggests the depolymerization and 
decomposition of PEC units within the cryogel matrix, as previously reported by Liu et 

Figure 4. X-ray diffraction patterns of albumin (ALB) (a), 70:30 (ALB:PEC) (b), 50:50 (ALB:PEC) (c),
and 30:70 (ALB:PEC) (d) cryogels with pink pepper essential oil encapsulated, albumin pure (e), and
amidated low methoxyl pectin (PEC) pure (f).

3.6. Thermal Analysis

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were
employed to investigate the thermal behavior of the cryogels. These techniques provide
valuable insights into the material thermal stability and potential first-order or second-
order transitions [62]. TGA analysis showed distinct mass loss patterns between cryogels
(Figure 5a). The cryogel containing only albumin (ALB) exhibited the lowest overall mass
loss (7.66%) and the most excellent thermal stability above 80 ◦C. Conversely, cryogels
formulated with ALB and PEC displayed higher mass losses of 11.5% (70:30), 12.4% (50:50),
and 12.5% (30:70). The derivative TGA curves indicated a single significant mass loss
event for the ALB-only cryogel. In contrast, PEC-containing cryogels showed multiple
events, suggesting a more complex degradation process (Figure 5b). The analysis of DTG
curves (the first derivative of TGA) revealed distinct mass loss patterns for the cryogels
(Figure 5b). The cryogel with only ALB exhibited the highest mass loss rate within the
40–70 ◦C temperature range, followed by cryogels with ALB:PEC ratios of 50:50, 70:30,
and 30:70, respectively. Notably, the 70:30 ALB:PEC cryogel displayed two distinct peaks
at 59.13 ◦C and 154.87 ◦C, while the others showed three events at 54.59 ◦C, 101.82 ◦C,
and 151.48 ◦C for the ratio 50:50, and 53.84 ◦C, 122.39 ◦C, and 148.80 ◦C for the 30:70
ratio (ALB:PEC). The presence of PEC influenced the degradation profile. Cryogels with
PEC exhibited lower mass loss rates between 20 and 80 ◦C than the ALB-only cryogel,
indicating enhanced stability in this range. However, ALB provided superior thermal
stability at higher temperatures (>80 ◦C). Finally, all PEC-containing cryogels underwent
degradation between 170 and 190 ◦C, with higher PEC content correlating to a lower
degradation temperature (176.6 ◦C); this suggests the depolymerization and decomposition
of PEC units within the cryogel matrix, as previously reported by Liu et al. [63]. Table 3
shows the Tpeak onset, Tpeak endset values, and enthalpy of cryogels. The thermograms
(Figure 5c,d) for cryogels prepared only with ALB and with a 70:30 ALB:PEC ratio revealed
a single endothermic event at 137.16 ◦C and 134.15 ◦C, respectively, attributed to the
formation of agglomerates during cryogel preparation via intermolecular hydrophobic
and hydrogen-bonding interactions between unfolded ALB chains (ALB-ALB and ALB-
PEC). As Jacob et al. [64] suggested, these intermolecular interactions are essential in
stabilizing the denatured protein. These structures exhibit high fusion temperatures and
fusion enthalpies (Table 3) and enhanced thermal stability. In cryogels prepared with
ALB:PEC ratios of 50:50 and 30:70, peaks at 159.54 ◦C and 168.69 ◦C, respectively, were
observed. This peak corresponds to the thermal degradation of the wall material rather
than protein unfolding, suggesting the disruptive effect of PEC on protein assembly within
the cryogel matrix.



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al. [63]. Table 3 shows the Tpeak onset, Tpeak endset values, and enthalpy of cryogels. The thermo-
grams (Figure 5c,d) for cryogels prepared only with ALB and with a 70:30 ALB:PEC ratio 
revealed a single endothermic event at 137.16 °C and 134.15 °C, respectively, attributed to 
the formation of agglomerates during cryogel preparation via intermolecular hydropho-
bic and hydrogen-bonding interactions between unfolded ALB chains (ALB-ALB and 
ALB-PEC). As Jacob et al. [64] suggested, these intermolecular interactions are essential in 
stabilizing the denatured protein. These structures exhibit high fusion temperatures and 
fusion enthalpies (Table 3) and enhanced thermal stability. In cryogels prepared with 
ALB:PEC ratios of 50:50 and 30:70, peaks at 159.54 °C and 168.69 °C, respectively, were 
observed. This peak corresponds to the thermal degradation of the wall material rather 
than protein unfolding, suggesting the disruptive effect of PEC on protein assembly 
within the cryogel matrix. 

  

  

  

Figure 5. TGA curves (a) and DTG (b) of ALB and ALB:PEC cryogels with pink pepper essential oil 
encapsulated and thermogram DSC of ALB cryogel (c), and 70:30 (ALB:PEC) (d), 50:50 (ALB:PEC) 
(e), and 30:70 (ALB:PEC) (f) cryogels with pink pepper essential oil encapsulated. 

Table 3. Thermal behavior of the ALB and ALB: PEC cryogels loaded with pink pepper essential oil. 

Sample Tpeak onset (°C) Tpeak (°C) Tpeak endset (°C) ΔH (W/g) 
ALB 79.58 137.16 193.05 62.83 
70:30 51.18 134.15 169.81 70.96 
50:50 110.7 159.54 193.05 29.52 
30:70 114.89 168.69 193.05 56.43 

  

Figure 5. TGA curves (a) and DTG (b) of ALB and ALB:PEC cryogels with pink pepper essential oil
encapsulated and thermogram DSC of ALB cryogel (c), and 70:30 (ALB:PEC) (d), 50:50 (ALB:PEC) (e),
and 30:70 (ALB:PEC) (f) cryogels with pink pepper essential oil encapsulated.

Table 3. Thermal behavior of the ALB and ALB: PEC cryogels loaded with pink pepper essential oil.

Sample Tpeak onset (◦C) Tpeak (◦C) Tpeak endset (◦C) ∆H (W/g)

ALB 79.58 137.16 193.05 62.83
70:30 51.18 134.15 169.81 70.96
50:50 110.7 159.54 193.05 29.52
30:70 114.89 168.69 193.05 56.43

3.7. Scanning Electron Microscopy

Figure 6 shows the cross-sectional micrographs of the cryogels containing pink pep-
per essential oil. The microparticles exhibit irregular morphology and varied sizes. The
cryogel only with ALB displays a dense, heterogeneous structure (Figure 6(a1,a2)) with
essential oil droplets on its surface (Figure 6(a4)). As the proportion of PEC increases,
the cryogels develop a more porous structure, entrapping essential oil particles within
their cavities (Figure 6(c3,d3)). These structural changes correspond to the essential oil
release profiles, particularly in the 50:50 and 30:70 ALB:PEC cryogels, which exhibited
enhanced antimicrobial activity against S. aureus. The soy protein isolate–pectin encapsula-
tion system demonstrates increased porosity due to pectin presence, enhancing the release
of encapsulated bioactive compounds [65,66].



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3.7. Scanning Electron Microscopy 
Figure 6 shows the cross-sectional micrographs of the cryogels containing pink 

pepper essential oil. The microparticles exhibit irregular morphology and varied sizes. 
The cryogel only with ALB displays a dense, heterogeneous structure (Figure 6(a1,a2)) 
with essential oil droplets on its surface (Figure 6(a4)). As the proportion of PEC increases, 
the cryogels develop a more porous structure, entrapping essential oil particles within 
their cavities (Figure 6(c3,d3)). These structural changes correspond to the essential oil 
release profiles, particularly in the 50:50 and 30:70 ALB:PEC cryogels, which exhibited 
enhanced antimicrobial activity against S. aureus. The soy protein isolate–pectin 
encapsulation system demonstrates increased porosity due to pectin presence, enhancing 
the release of encapsulated bioactive compounds [65,66]. 

 
Figure 6. SEM micrographs of ALB (a), 70:30 ALB:PEC (b), 50:50 ALB:PEC (c), and 30:70 ALB:PEC 
(d) (magnification 150× (1), 400× (2), 1000× (3), and 2500× (4)). 

4. Conclusions 
ALB and ALB:PEC cryogels are suitable matrices for encapsulating the essential oil 

of pink pepper (Schinus terebinthifolius Raddi) and demonstrated antimicrobial activity 
primarily against S. aureus. The inhibition zone size was influenced by both cryogel com-
position and mass, with higher pectin content correlating to larger inhibition zones. The 
pectin plays a crucial role in oil release and diffusion. PEC acts as a trigger in the proposed 
encapsulation system. Its concentration in the wall material significantly influences the 
encapsulation efficiency and total phenolic compound protection. More importantly, it 
can be harnessed to control the release of the essential oil, thereby enhancing its inhibitory 
effects against S. aureus. The interaction between ALB, PEC, and essential oil alters the 
wall material, thermal properties, and morphology. The PEC introduced led to multiple 
degradation steps and a more complex degradation process, reducing the thermal stabil-
ity and increasing its porosity. It is still necessary to establish the optimal concentrations 

Figure 6. SEM micrographs of ALB (a), 70:30 ALB:PEC (b), 50:50 ALB:PEC (c), and 30:70 ALB:PEC (d)
(magnification 150× (1), 400× (2), 1000× (3), and 2500× (4)).

4. Conclusions

ALB and ALB:PEC cryogels are suitable matrices for encapsulating the essential oil
of pink pepper (Schinus terebinthifolius Raddi) and demonstrated antimicrobial activity
primarily against S. aureus. The inhibition zone size was influenced by both cryogel
composition and mass, with higher pectin content correlating to larger inhibition zones. The
pectin plays a crucial role in oil release and diffusion. PEC acts as a trigger in the proposed
encapsulation system. Its concentration in the wall material significantly influences the
encapsulation efficiency and total phenolic compound protection. More importantly, it
can be harnessed to control the release of the essential oil, thereby enhancing its inhibitory
effects against S. aureus. The interaction between ALB, PEC, and essential oil alters the
wall material, thermal properties, and morphology. The PEC introduced led to multiple
degradation steps and a more complex degradation process, reducing the thermal stability
and increasing its porosity. It is still necessary to establish the optimal concentrations of
PEC in the proposed encapsulation system to determine the kinetics and release profile
and to establish its use in food matrices as an alternative for food preservation.

Author Contributions: Conceptualization, A.M.C.-G., E.J.P.-M. and V.R.N.; methodology, A.M.C.-G.
and M.G.C.; formal analysis, A.M.C.-G. and E.J.P.-M.; investigation, A.M.C.-G., E.J.P.-M. and M.G.C.;
resources, V.R.N. and M.R.d.M.; data curation, A.M.C.-G. and E.J.P.-M.; writing—original draft
preparation, A.M.C.-G. and E.J.P.-M.; writing—review and editing, A.M.C.-G., E.J.P.-M., V.R.N.,
M.G.C. and M.R.d.M.; visualization, A.M.C.-G.; supervision, M.R.d.M.; project administration,
M.R.d.M.; funding acquisition, A.M.C.-G. All authors have read and agreed to the published version
of the manuscript.

Funding: This research was funded by the Coordination for the Improvement of Higher Education
Personnel (CAPES) and Capes-Print program for a scholarship (code 88887.890935/2023-00).



Processes 2024, 12, 1681 12 of 14

Data Availability Statement: The original contributions presented in the study are included in the
article; further inquiries can be directed to the corresponding author.

Acknowledgments: The authors are grateful to Mauricio Boscolo for the use of the FT-IR
spectrophotometer.

Conflicts of Interest: The authors declare no conflicts of interest.

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