Development of Functional Foods Based on Chickpea Protein Hydrolysate for Application to Sport Nutrition

Development of Functional Foods Based on Chickpea Protein Hydrolysate for Application to Sport Nutrition Noelia María Rodríguez Martín CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS Instituto de la Grasa TESIS DOCTORAL 1 2 3 Memoria presentada por Dña. Noelia María Rodríguez Martín para optar al grado de Doctor por la Universidad de Sevilla Departamento de Alimentación y Salud Departamento de Biología Molecular e Ingeniería Bioquímica Departamento Bioquímica Médica y Biología Molecular e Inmunología Development of Functional Foods Based on Chickpea Protein Hydrolysate for Application to Sport Nutrition 4 5 Dr. JUSTO JAVIER PEDROCHE JIMÉNEZ, Científico Titular del Departamento de Alimentación y Salud del Instituto de la Grasa del Consejo Superior de Investigaciones Científicas. Dra. MARÍA SOLEDAD FERNÁNDEZ PACHÓN, Profesora Titular del Departamento de Biología Molecular e Ingeniería Bioquímica de la Universidad Pablo de Olavide. CERTIFICAN: Que la Tesis Doctoral titulada “Development of Functional Foods Based on Chickpea Protein Hydrolysate for Application to Sport Nutrition” realizada por Dña. Noelia María Rodríguez Martín para optar al grado de Doctor, ha sido llevada a cabo bajo nuestra dirección. V.º B.º Los Directores Dr. Justo Javier Pedroche Jiménez Dra. Mª Soledad Fernández Pachón Dr. SERGIO MONTSERRAT DE LA PAZ, Profesor del Departamento de Bioquímica Médica y Biología Molecular e Inmunología de la Universidad de Sevilla, CERTIFICA que Dña. Noelia María Rodríguez Martín cumple los requisitos académicos (R.D. 99/2011) para optar al grado de Doctor. V.º B.º El Tutor Dr. Sergio Montserrat de la Paz 6 7 A mi familia 14 2.3.5. Bioactivity of CHPs Using Biochemicals In Vitro Assays .................. 91 2.3.6. Biological Impact of CPHs ................................................................. 95 2.4. Conclusion ............................................................................ 101 2.5. References ............................................................................ 113 Chapter 3 – Hypothesis and Objectives ......................................... 123 3.1. Hipótesis y Objetivos ............................................................. 125 3.2. Hypothesis and Objectives ..................................................... 129 Chapter 4 – Protein Hydrolysate Optimization (Published work).... 133 4.1. Resumen ............................................................................... 135 4.2. Abstract ................................................................................ 136 4.3. Introduction .......................................................................... 137 4.4. Materials and Methods ......................................................... 140 4.4.1. Chemicals and Sampling .................................................................140 4.4.4. Chemical Characterization of Chickpea Products ..........................141 4.4.5. Evaluation of the Degree of Hydrolysis (DH) .................................142 4.4.6. Molecular Profile Elucidation by Fast Protein Liquid Chromatography (FPLC) ............................................................................142 4.4.7. Antioxidant Activities Evaluation ...................................................143 4.4.8. Amino Acid Quantification by Ultra High-Performance Liquid Chromatography (UPLC) ...........................................................................144 4.4.9. Determination of Protein Solubility vs pH .....................................145 4.4.10. In Vitro Protein Digestibility (IPD) ................................................145 4.4.11. Essential Amino Acids (EAAs) and Protein Digestibility-Corrected Amino Acid Score (PDCAAS) Estimation ...................................................146 4.4.12. Statistical and Principal Component Analysis (PCA) ....................146 4.5. Results and Discussion ........................................................... 147 4.5.1. Optimization of Protein Hydrolysis Conditions for Obtaining CPH 147 4.5.2. The Antioxidant Activities of CPH...................................................149 4.5.3. Principal Component Analysis (PCA) ..............................................151 4.5.4. The Production of H30BIO at Pilot Scale: Chemical and Nutritional Characterization ........................................................................................156 15 4.5.5. Protein Solubility of Chickpea Protein Products and Digestibility Evaluation of H30BIO ................................................................................162 Chapter 5 – The Immunomodulatory Potential (Under revision) .... 171 5.1. Resumen ............................................................................... 173 5.2. Abstract ................................................................................ 174 5.3. Introduction .......................................................................... 175 5.4. Material and Methods ........................................................... 178 5.4.1. Material and Reagent .....................................................................178 5.4.2. Cell Culture: Maintenance and Treatment .....................................179 5.4.3. Total Superoxide Reactive Species Measure .................................179 5.4.4. Measurement of Nitric Oxide in Cell Supernatants .......................180 5.4.5. Superoxide Dismutase (SOD) Activity Assay ..................................181 5.4.6. Catalase (CAT) Activity Assay .........................................................181 5.4.7. Glutathione Peroxidase (GPx) Activity Assay .................................182 5.4.8. Glutathione Reductase (GSR) Activity Assay..................................182 5.4.9. GSH/GSSG Ratio Measurement ......................................................182 5.4.10. Cytokines Evaluation ....................................................................183 5.4.11. Gene Expression ............................................................................183 5.4.12. Statistical Analysis ........................................................................184 5.5. Results .................................................................................. 185 5.5.1. The Hydrolysed Chickpea Protein Increases Cellular ROS Production 185 5.5.2. The Chickpea Protein Hydrolysate Enhances Nitric Oxide Release 188 5.5.3. Activity of Antioxidant Enzymes .....................................................190 5.5.4. Mitochondrial Peroxiredoxins and Thioredoxins...........................194 5.5.5. The Effect of H30BIO on Pro-Inflammatory Cytokines ..................196 5.5.6. Key Inflammatory and Regulatory Markers in Cellular Inflammation 198 5.6. Discussion ............................................................................. 200 5.6.1. The Action of H30BIO on Oxidative Milieu: ROS and NO Levels and Related Enzymes .......................................................................................200 16 5.6.2. The effect of H30BIO on Endogenous Antioxidant Defense ..........202 5.6.3. The Effect of H30BIO on The Resolution of Inflammation ............209 5.7. Prospects for Future Research ............................................... 213 5.8. Conclusions ........................................................................... 213 5.9. References ............................................................................ 214 Chapter 6 – Supplementation on Volunteers (Unpublished work).. 219 6.1. Resumen ............................................................................... 221 6.2. Abstract ................................................................................ 222 6.3. Introduction .......................................................................... 223 6.4. Method and Design ............................................................... 227 6.4.1. Sample Size .....................................................................................227 6.4.2. Subjects ...........................................................................................229 6.4.3. Ethical Concerns ..............................................................................230 6.4.4. Inclusion Criteria .............................................................................230 6.4.5. Exclusion Criteria ............................................................................230 6.4.6. Supplement: Production and Composition ....................................230 6.4.7. Experimental Design .......................................................................231 6.4.8. Training Schedule ............................................................................234 6.4.9. Data Collection ................................................................................234 6.4.10. Anthropometric Measurements ...................................................234 6.4.11. Dietary Assessment ......................................................................234 6.4.12. Blood Samples ...............................................................................235 6.4.13. Participants Privacy and Codification ...........................................238 6.4.14. The Access to the Data ..................................................................239 6.4.15. Statistical Analysis ........................................................................240 6.5. Results .................................................................................. 243 6.5.1. Study Population: Participant Eligibility .........................................243 6.5.2. Performance During Supplementation ..........................................245 6.5.3. Descriptive Analysis of Anthropometric and Metabolic Characteristics ...........................................................................................245 17 6.5.4. Descriptive Analysis of Hematological and Biochemical Parameters 247 6.5.5. Descriptive Analysis of Hematological and Biochemical Parameters 250 6.5.6. Descriptive Analysis of Inflammation, Antioxidant and Muscle Damage Markers .......................................................................................254 6.5.7. Principal Component Analysis (PCA) For All Variables ..................256 6.5.8. PCA For Muscle Damage, Proinflammatory and Antioxidant Variables ....................................................................................................259 6.5.9. General Decision Tree Analysis (DTA) ............................................261 6.5.10. Female Subset of DTA ...................................................................264 6.5.11. Male Subset of DTA ......................................................................266 6.5.12. The Importance of DTA Independent Variables ..........................269 6.6. Discussion ............................................................................. 270 6.6.1. Protocol Evaluation .........................................................................270 6.6.2. Anthropometric Characteristics .....................................................272 6.6.3. Dietary Evaluation ..........................................................................272 6.6.4. Hematological, Biochemical Parameters, and Food Safety ...........274 6.6.5. Inflammation, Antioxidant and Muscle Damage Markers ............275 6.6.6. The Signaling Regulation Through IL-6 ...........................................276 6. 7. Limitations and Future Directions ......................................... 278 6.8. Conclusion ............................................................................ 279 6.9. References ............................................................................ 280 Chapter 7 – General Conclusions .................................................. 287 Agradecimientos .......................................................................... 295 APPENDIX (Ethics committee) ...................................................... 299 18 19 Resumen El garbanzo es un alimento versátil y nutritivo, esencial en la dieta mediterránea desde civilizaciones antiguas. La semilla de garbanzo es rica en vitaminas (B1, B2, B3, B6, A, E, K y C), minerales (Ca, Cu, Fe, K, Mg, Na, P, Se, Zn), es fuente de carbohidratos (49.2 %), proteínas (19.3 %) y fibra dietética (14.9 %), con un contenido bajo en grasas (6.3 %). Además, al ser uno de los cultivos más importantes de leguminosas en el mundo genera numerosos subproductos. En este contexto, la hidrólisis proteica enzimática se presenta como una solución al reaprovechamiento de semillas rotas y descascarilladas o bajo calibre que ya no son óptimas para su comercialización mejorando no solo las propiedades tecnológicas sino también nutricionales. El análisis de la literatura existente ha demostrado que los hidrolizados proteicos de semillas vegetales, entre ellos, los hidrolizados proteicos de garbanzo, tienen potencial para la creación de ingredientes funcionales antioxidantes e inmunomoduladores. En este sentido, destaca también la importancia de la obtención de suplementos con dichos efectos que puedan utilizarse durante la práctica deportiva de alto nivel. Dado a que los deportistas de élite están sujetos a un estado proinflamatorio por el sobreentrenamiento que les genera además estrés oxidativo y daño muscular, este suplemento tendría alta aceptación en el mercado de la nutrición deportiva. Por todo ello, este estudio tuvo como objetivo la creación de un hidrolizado de proteína de garbanzo que mantuviera efectos antiinflamatorios y antioxidantes para poder demostrar su efecto en deportistas durante la actividad física de alta intensidad. La elaboración y evaluación de hidrolizados de proteína de garbanzo en laboratorio utilizando enzimas de grado alimentario (Alcalasa y Bioproteasa) permitió identificar las mejores condiciones para su replicación a mayor escala. El análisis de diferentes parámetros (rendimientos, contenido de proteínas, grado de hidrólisis y actividades antioxidantes) permitió elegir el mejor hidrolizado mediante un análisis de componentes principales. El hidrolizado seleccionado (H30BIO) demostró características destacables en cuanto al contenido de proteínas (67.71%), rendimientos de producción, solubilidad (> 80%), alta digestibilidad (89.50%) y actividad antioxidante. Todas estas características se mantuvieron cuando el hidrolizado se escaló a planta piloto. Dada la alta capacidad antioxidante del H30BIO, se decidió estudiar dicha capacidad en cultivos celulares en monocitos humanos. Se evaluaron los niveles 20 de especies reactivas de oxígeno y óxido nítrico, la actividad de enzimas intracelulares, y la expresión y liberación de genes y citoquinas. Notablemente, la exposición al hidrolizado incrementó la liberación de ROS y NO de forma concentración-dependiente a la vez que mantuvo un efecto inhibitorio sobre la superóxido dismutasa mitocondrial, y disminución de la expresión de genes proinflamatorios, a excepción de TNF-α. Los principales hallazgos destacan un aumento en el metabolismo de ROS y NO unido a significativos efectos antiinflamatorios a través de los componentes del inflamasoma NLRP3 y NF-κB en las células THP-1. Finalmente, se estudió el efecto del hidrolizado en futbolistas amateur durante el período de entrenamiento con un ensayo en voluntarios aleatorizado, ciego y cruzado durante 12 semanas. Se administraron placebo (bebida a base de maltodextrina) y bebida proteica (basada en hidrolizado). Se recolectaron muestras de sangre para evaluar varios biomarcadores relacionados con el daño muscular (LDH, CK), marcadores inflamatorios (CRP, IL-8, IL-6), y estado antioxidante plasmático (ORAC, FRAP, TEAC, GPx, GR), junto a otros. El análisis estadístico de los biomarcadores se realizó utilizando varios enfoques multivariantes: análisis de componentes principales y análisis de árbol de decisión, para observar tendencias debidas al consumo del suplemento. La importancia del marcador IL-6 tanto en hombres como en mujeres, así como su correlación con la ingesta de suplemento/placebo, mostraron tendencias interesantes. Considerando el efecto pleiotrópico de IL-6 tanto en la modulación de la inflamación como en el control de la localización de la energía, los datos sugieren que la bebida desarrollada en este trabajo mantiene un efecto antiinflamatorio y ergogénico durante la práctica deportiva. 21 Abstract Chickpeas are a versatile and nutritious food, essential in the Mediterranean diet since ancient civilizations. The chickpea seed is rich in vitamins (B1, B2, B3, B6, A, E, K, and C), minerals (Ca, Cu, Fe, K, Mg, Na, P, Se, Zn), and is a source of carbohydrates (49.2%), proteins (19.3%), and dietary fiber (14.9%), with a lowfat content (6.3%). Additionally, as one of the world's most important legume crops, it generates numerous by-products. In this context, enzymatic protein hydrolysis presents a solution for the reutilization of broken, dehulled, or undersized seeds that are not for sale, improving not only their technological properties but also their nutritional value. A review of existing literature has demonstrated that the development of chickpea protein hydrolysates hold potential as antioxidant and immunomodulatory functional ingredient. In this sense, the creation of supplements that retain these effects during high-level athletic activities might have acceptation in sport nutrition market, as elite athletes are subjected to a pro-inflammatory state due to overtraining, which causes oxidative stress and muscle damage. Therefore, this study aimed to create a chickpea protein hydrolysate with anti-inflammatory and antioxidant effects to demonstrate its impact on athletes during training periods. The preparation and evaluation of chickpea protein hydrolysates in the laboratory using food-grade enzymes (Alcalase and Bioprotease) allowed for identifying the best conditions for scaling up. The analysis of various parameters (yields, protein content, degree of hydrolysis, and antioxidant activities) enabled the selection of the best hydrolysate through principal component analysis. The selected hydrolysate (H30BIO) exhibited remarkable characteristics in terms of protein content (67.71%), production yields, solubility (> 80%), high digestibility (89.50%), and antioxidant activity. All these characteristics were maintained when the hydrolysate was scaled up to the pilot plant level. Given the high antioxidant capacity of H30BIO, its capacity was studied in human monocyte cell cultures. Reactive oxygen species and nitric oxide levels, intracellular enzyme activity, and gene and cytokine expression and release were evaluated. Notably, exposure to the hydrolysate increased the release of ROS and NO in a concentration-dependent manner while maintaining an inhibitory effect on mitochondrial superoxide dismutase and decreasing the expression of pro-inflammatory genes, except for TNF-α. The main findings highlighted an increase in ROS and NO metabolism coupled with 22 significant anti-inflammatory effects through the NLRP3 inflammasome and NF- κB components in THP-1 cells. Finally, the effect of the hydrolysate was studied in amateur football players during the training period through a randomized, blind, and crossover trial over 12 weeks. A placebo (maltodextrin-based drink) and a protein drink (based on hydrolysate) were administered. Blood samples were collected to evaluate various biomarkers related to muscle damage (LDH, CK), inflammatory markers (CRP, IL-8, IL-6), and plasma antioxidant status (ORAC, FRAP, TEAC, GPx, GR), among others. The statistical analysis of the biomarkers was performed using several multivariate approaches: principal component analysis and decision tree analysis to observe trends due to supplement consumption. The importance of the IL-6 marker in both men and women, as well as its correlation with supplement/placebo intake, showed interesting trends. Considering the pleiotropic effect of IL-6 in modulating inflammation and controlling energy localization, the data suggest that the drink developed in this work maintains an anti-inflammatory and ergogenic effect during athletic practice. 23 Figure List Chapter 1 Figure 1.1. Axial and longitudinal plane of the chickpea seed. Figure 1.2. Chickpea production, dry in the World (total). Figure 1.3. Production quantities of chickpeas, dry by country. Chapter 2 Figure 2.1. Schematic representation of physiological activities of bioactive peptides. Figure 2.2. Author Keyword Network Visualization in the Literature review on Protein Hydrolysates. Figure 2.3. Author Keyword Network Visualization in the Literature review on Protein Hydrolysates. Figure 2.4. Title and Abstract Keyword Network Visualization in the Literature review on Protein Hydrolysates. Figure 2.5. Title and Abstract Keyword Network Visualization in the Literature review on Chickpea Protein Hydrolysates. Figure 2.6. Flowchart of the Review. Chapter 4 Figure 4.1. Principal Component Analysis (PCA) of Hydrolysis Conditions. Figure 4.2. Diagram Flow Production at Pilot-Scale. Figure 4.3. Molecular Protein Profiles by HPLC. Figure 4.4. Solubility Curves of Hydrolysate and Raw Materials. Chapter 5 Figure 5.1. THP-1 cell ROS generation measurement with CellROX. Figure 5.2. Evaluation of Cellular Nitric Oxide (NO) Production and Expression of Associated Genes. Figure 5.3. Cellular Antioxidative Stress Enzyme Profile: Super Oxide Dismutases (SODs) Figure 5.4. Cellular Antioxidative Stress Enzyme Profile. Figure 5.5. Cellular Antioxidative Stress Enzyme Profile: Peroxiredoxins and Tioxiredoxins. Figure 5.6. Anti-inflammatory Effects of Hydrolysate in the THP-1 Cell Line Model. 30 31 Funding This research work has been conducted with the financial support of the following institutions and research projects: Predoctoral Grant/Contract Predoctoral Research Projects Contract. Reference: 202070E273. Funding Entity: Spanish National Research Council (CSIC). Duration: January 1, 2021 – December 31, 2024. Intramural Contract Director: Justo Javier Pedroche Jiménez. Predoctoral Stay International Mobility Call iMOVE 2023. Reference: IMOVE23066. Funding Entity: Spanish National Research Council (CSIC). Host Center: Department of Neuroanatomy and Neuroscience, APC Microbiome Ireland, University College Cork. Location: Cork, Ireland. Duration: May 15, 2023 – August 15, 2023 (3 months). Research Projects Title: Design and Evaluation of New Ingredients Applied to Sports Nutrition Based on Plant Protein Hydrolysates (PLANTPROSPORT). Reference: IDI-20200562. Funding Entity: Centre for the Development of Industrial Technology (CDTI). Participating Entities: Moreno Ruiz Hermanos S.L.-Aurora Intelligent Nutrition, Instituto de la Grasa (CSIC), Pablo de Olavide University. Granted Budget: €48,400. Duration: December 1, 2020 – May 31, 2022. Principal Investigators: CSIC: Justo Javier Pedroche Jiménez; UPO: José Antonio González Jurado and María Soledad Fernández Pachón. Title: Evaluation of New Disruptive Technologies (Steam Explosion) in the Design of Tailor-Made Plant Protein Hydrolysates Applied to Sport Nutrition (PROVERDE). Reference: PID2019-111368RB-I00. 32 Funding Entity: Ministry of Science, Innovation and Universities, State Research Agency, National Plan. Participating Entities: Instituto de la Grasa (CSIC), Pablo de Olavide University, University of Seville, Institute of Biomedicine of Seville, Virgen del Rocío Hospital. Granted Budget: €145,200. Duration: June 1, 2020 – May 31, 2023. Principal Investigators: Justo Javier Pedroche Jiménez and María Soledad Fernández Pachón. 33 34 35 Chapter 1 – General Introduction This chapter includes a brief historical introduction of the chickpea, emphasizing its importance in the Mediterranean diet, a detailed description of the seed in terms of its botanical and nutritional composition, as well as global data on its production, distribution, and consumption patterns, and future perspectives on the use of plant protein hydrolysates. Development of Functional Foods for Application to Sport Nutrition 36 Chapter 1 — General Introduction 37 1.1. History of Chickpea: Its role in the Mediterranean Diet Archaeological records reveal that chickpea was first cultivated in the Fertile Crescent (Turkey, Syria, Lebanon, Afghanistan, India, Ethiopia, Pakistan, Israel and Jordan) around 10,000 years ago (Chandora et al., 2020; Igolkina et al., 2023). Its subsequent evolution in regions such as the Middle East, South Asia, Ethiopia, and the Western Mediterranean remains unclear and cannot be fully understood through historical and archaeological evidence alone (Igolkina et al., 2023). In the Mediterranean area, civilizations such as the Greeks, Romans and Egyptians of the B.C. centuries already planted and consumed chickpeas, making this legume a vital source of nutrients including proteins and carbohydrates, fundamental in diets that was exclusively plant-based. Pliny the Elder (23-79 B.C.), in his work “Naturalis Historia”, referred to nutritional properties and the chickpeas crops in ancient Rome (Bostock & Riley, 1836). During the Middle Ages, chickpeas remained a significant source of nutrients in Europe and the Mediterranean. In fact, the diet of European monks in the Middle Ages often included chickpeas, leading to the creation of recipes that incorporated them into stews and soups. Through centuries, the elements of the Mediterranean Diet (MD) pattern were being shaped, but it was not until the formulation of "The Seven Countries Study” by Dr. Ancel Keys in the 1950s that the traditional MD was studied and developing (Keys et al., 1986). This study ascertained the health benefits of the MD in relation to cardiovascular disease prevention. The MD encompasses a wide array of fresh foods, such as olive oil, vegetables, fruits, nuts, fish, and legumes being chickpeas a fundamental component for balanced nutrition within the mentioned dietary pattern (Trichopoulou & Lagiou, 2009). Contemporary, in the 21st century, MD continues to be lauded for its impact on health and the prevention of chronic diseases, as evidenced by the PREDIMED study, which underscored the role of MD in Development of Functional Foods for Application to Sport Nutrition 38 cardiovascular disease prevention and its beneficial impact on health (Estruch et al., 2013). The MD is a dietary pattern recognized worldwide for being beneficial for human health and for the environment (Kiani et al., 2022). Over the course of centuries, the MD has undergone multiple modifications, incorporating various local ingredients, being influenced by the food habits of different cultures, and adapting to many geographical areas (Guasch‐Ferré & Willett, 2021; Kiani et al., 2022). Today, the MD has adapted to modern times and habits (Kiani et al., 2022). Despite changes in dietary patterns, chickpeas continue to serve as a key nutritional element of this diet and have gained international recognition as a healthy food (Gupta et al., 2017). 1.2. The Chickpea Seed: A Botanic and Nutritional Approach The chickpea seed, scientifically known as Cicer arietinum L., is a member of the Fabaceae family within the genus Cicer. This genus includes both annual and perennial species, categorized into four sections: Acanthocicer, Chamaecicer, Polycicer, and Monocicer—the latter of which includes C. arietinum (Sajja et al., 2017). 1.2.1. Chickpea Seed Morphological Composition Botanically, the seed's shape is often similar to that of a ram's (Aries) head, giving it the name 'arietinum'. However, other shapes also exist, such as globular or quasi-spheric with a distinctive beak. There are two important types of chickpeas within the C. arietinum species: desi and kabuli. The desi chickpea is characterized by angularshaped seeds with a generally dark color that can vary between brown, black, or reddish, while Kabuli chickpeas are a variety characterized by having lighter skin (between various beige shades) and a rounder, larger shape than desi chickpeas (Sajja et al., 2017). In terms of size, desi Chapter 1 — General Introduction 39 chickpea seeds are usually smaller (weigh: 0.2 g per seed) than kabulitype seeds (weigh: 0.3-0.5 g per seed). Chickpea size might be scaled into five groups: very large (>9 mm), large (8-10 mm), medium (7-9 mm), small (6-8 mm), and very small (<6 mm) (Serrano et al., 2017). Chickpea seed exhibits a unique multi-layered structure setting it apart from other legumes (Fig. 1.1). The surface of the seed coat may be smooth or tuberculate (Sajja et al., 2017). The outermost layer, known as the Testa, size and color are a varietal character highly influenced by moisture and heat (Sajja et al., 2017). It serves as a protective barrier for the seed and contains phenolic compounds that may confer antioxidant properties. Figure 1.1. Axial and longitudinal plane of the kabuki chickpea seed. Figure created by the author. Beneath the Testa lies the tegument, which envelops the inner core of the seed. This layer contains fibers and certain anti-nutritional components, such as tannins and phytates (Sajja et al., 2017). The endosperm is absent in these seeds. Lastly, the embryo, situated at the core of the seed, houses the future plant shoot. Composed of cotyledons, this section gives rise to the initial embryonic leaves (the germination is hypogeal with no hypocotyl). The cotyledon pair are mainly in three colors: cream, green or orange (Sajja et al., 2017). Development of Functional Foods for Application to Sport Nutrition 46 Finally, prolamins representing a small fraction are alcohol-soluble proteins. This fraction is characterized by poor foaming and emulsifying properties, and high content of proline and glutamine amino acids (Grasso et al., 2022). 1.2.2.4. Antinutritional Factors and Other Phytochemicals Chickpea seeds, along with other legumes, contain compounds that serve as a defensive mechanism for the seed, such as protease inhibitors, lectins, phytic acid, oligosaccharides, and some phenolic compounds (0.72 - 1.81 mg/g of seed) (Rachwa-Rosiak et al., 2015). These compounds also are recognized as antinutritional factors (Asif et al., 2013; Gupta et al., 2017). Antinutritional factors are substances that reduce the bioavailability of one or multiple nutrients acting in an antagonistic manner. Usually, this phenomenon occurs through the complex formation between the antinutrient and the nutrient (Yegrem, 2021). In this sense, tannins are compounds prevalent in legumes that can interact with proteins forming tannin-protein complexes. Thus, decreasing protein digestibility and protein solubility. Phytate (inositol hexakisphosphate) is a phosphorus-containing compound that binds with minerals (such as zinc, calcium, magnesium, and iron) inhibiting mineral absorption in the gastrointestinal tract (Yegrem, 2021). Concerning protease inhibitors, such as trypsin and chymotrypsin, they can reduce nutrient digestibility by binding to enzymatic complexes, potentially leading to long-term pancreas hypertrophy as it compensates by overproducing enzymes. Chickpeas exhibit lower protease inhibitor activity than other legumes, but they are still present (Yust Escobar, 2006). On the other hand, lectins are proteins characterized by specific carbohydrate-binding properties and notable resistance to enzymatic degradation, attributable to their cysteine Chapter 1 — General Introduction 47 content and the pronounced ability to form disulfide bridges (Harvard University, n.d.). In general, antinutritional factors can be effectively inactivated through heat treatment, and most of them are either eliminated through processing or rendered insignificant by the cooking method. On the other hand, researchers have recently focused on the health benefits of consuming chickpeas owing to certain bioactive functions that have been assigned to different compounds. These compounds also include some identified antinutritional factors like phytic acid, lectins, sterols, saponins, dietary fiber (mainly insoluble dietary) (de Almeida Costa et al., 2006), resistant starch, oligosaccharides, unsaturated fatty acids, amylase inhibitors, carotenoids and isoflavones (Gupta et al., 2017). For instance, the starch-type contained in chickpea flour (mentioned before) plays an important role in glucose release improving glycemic index due to its slow digestion (Chung et al., 2008). The chickpea has the lowest carbohydrate digestibility of the commonly consumed pulses. 1.3. Chickpea Consumption, Production, and By-products Chickpea is one of the most important harvested pulses in the world to meet the growing demand of plant-based proteins (Samineni et al., 2017). Thus, chickpea cultivation has an annual production of 16 million tons in 2021 (Fig. 1.2.) (FAO, n.d.). The extent of the cultivated area has increased over the years due to a wide range of climate resilient chickpea varieties with sustainable production have been developed to satisfy both growing conditions and the culinary preferences of the population. Development of Functional Foods for Application to Sport Nutrition 48 Figure 1.2. Chickpea production, dry in the World (total). The graph represents the world area harvested (blue color) in million hectares, and world production (red color) in million tons, from 2000 to 2021 (FAOSTAT). This crop is widely cultivated worldwide including regions in the Mediterranean, Central Asia, North America, and Australia (Fig. 1.3.). Chickpeas are also appreciated in various cultures for their versatility in the kitchen. Their consumption is particularly prominent not only in Mediterranean countries such as Spain, Italy, Greece, Turkey, and the Middle East, but also in South Asian countries like India and Pakistan, where they are a fundamental component of the vegetarian diet (Lucas & Fuller, 2014). Chickpeas are an essential part of dishes like hummus and falafel (in the Middle East), and dhal (in India) (Muehlbauer & Sarker, 2017). Chapter 1 — General Introduction 49 Figure 1.3. Production quantities of chickpeas, dry by country. Values are referred to the average from 2020 to 2021 (FAOSTAT). Globally, there are two main chickpea varieties for cultivation: kabuli and desi, which are descendants from Cicer reticulatum (Toker, 2009). The desi chickpea variety is seen as an earlier cultivated form of chickpeas derived from C. reticulatum mutations, and is commonly accepted as the kabuli variety evolving from desy type. This distinction arises from the fact that seeds of the desi type bear a closer resemblance to those of wild chickpeas (C. reticulatum) compared to the kabuli variety. However, genetic studies suggest that kabuli type could have originated from spontaneous mutants of C. reticulatum (Toker, 2009). The desi type prevails in chickpea production, constituting approximately 85% of global chickpea cultivation, while the kabuli type accounts for around 15% (Toker, 2009). Other notable chickpea varieties include bengal gram chickpea (C. arietinum var. bengal gram), which is another variety of desi chickpea, mainly cultivated in the Indian subcontinent; and green chickpea (C. arietinum var. viridifolium), Development of Functional Foods for Application to Sport Nutrition 50 primarily grown in Africa, stands out for its disease and drought resistance (Javaloyes et al., 2016). On the other hand, chickpeas are considered a safe and environmentally friendly food source. They are well-suited to efficient resource management in agriculture, as they can thrive even in moderately dry conditions in general (Berger et al., 2005). As legumes, chickpeas contribute to soil health by forming beneficial partnerships with mycorrhizal fungi, enabling crop rotation, and reducing the need for added fertilizers. Certain chickpea varieties, developed through human selection, are resistant to pests, also eliminating the necessity for chemical pesticides (Yust Escobar, 2006). Furthermore, by combining various farming practices, adopting new technologies and embracing sustainable approaches, this crop can enhance water and nutrient use minimizing environmental impact, and boosting agricultural productivity. As one of the most significant legumes crops globally, trailing behind soybean, peanut, and common bean production (FAO, n.d.), chickpea cultivation generates substantial byproducts during processing and treatment. These byproducts include husks, leaves, stems, and grains that are damaged or unfit for direct commercialization. Thus, their proper use can contribute to reducing waste and circular economy within the chickpea production chain. Generally, to contribute to the circular economy, byproducts from this industry are utilized for animal feed (Bampidis & Christodoulou, 2011). However, in many cases, the product is suitable for human consumption and could be reevaluated by finding new applications. Thus, in recent years, a series of investigations have been conducted to explore different applications of these byproducts. Studies have shown that chickpea flour can be used in the creation of various enhanced products such as baked goods, snacks, extruded products, weaning foods, roasted chickpeas, gluten-free foods, roasted chickpea flour, and plant-based Chapter 1 — General Introduction 51 beverages (Ammar et al., 2022; Boukid, 2021; Gupta et al., 2019; Lopes et al., 2020; Mefleh et al., 2022; Mygdalia et al., 2022). But also, studies have shown that chickpea protein hydrolysates can be used in the formulation of functional foods, enhancing their nutritional profile by providing proteins and bioactive compounds (Yust et al., 2012; Faridy et al., 2020; Grasso et al., 2022; Schettino et al., 2019; Torres-Fuentes et al., 2015). 1.4. Protein Hydrolysates Hydrolysis is a chemical reaction that occurs between water molecules and others, resulting in the breaking of one or more chemical bonds. Proteolytic hydrolysis, therefore, involves the cleavage of the peptide bond in the presence of water breaking the peptide bond. This process leads to the generation of short proteins, peptides, or free amino acids (Vioque & Millán, 2005). Hydrolysis reactions require a certain amount of energy input to proceed, in the case of proteolytic hydrolysis, it can occur also spontaneously, albeit slowly in nature (Toldrá et al., 2018). During the hydrolysis process, some small peptides with physiological activity can be released; these are also known as bioactive peptides (Chalamaiah et al., 2018). The extraction of bioactive peptides can be expedited through various conventional techniques, such as chemical proteolysis and biological proteolysis methods. Additionally, novel approaches have been developed to release bioactive peptides based on physical processes, including ohmic heating, ultrasound, pulse electric fields, subcritical water hydrolysis, and microwave applications (Zaky et al., 2022). However, this work is only focused on conventional approaches, as they are currently used in the food industry and easy to scale. Development of Functional Foods for Application to Sport Nutrition 52 1.4.1. Chemical Hydrolysis Chemical hydrolysis methods encompass acid (HCl) or alkali (NaOH)- mediated hydrolysis procedures. Chemical hydrolysis is recognized as a highly efficient and cost-effective process. Nevertheless, a key challenge associated with chemical hydrolysis lies in the degradation of peptide chains under these extreme pH conditions. Consequently, tryptophan undergoes a full degradation in acid hydrolysis processes and alkaline hydrolysis leads to the degradation of other amino acids such as aspartic acid, glutamic acid, tryptophan, and isoleucine (Szopa et al., 2023). Furthermore, L-amino acids are destroyed, resulting in the formation of D-amino acids and toxic compounds like lysin-alanine. Consequently, the hydrolysates obtained exhibit suboptimal nutritional quality (Vioque & Millán, 2005). Despite these challenges, the application of chemical hydrolysis for the recovery of valuable constituents from waste materials offers the potential to streamline unit operations and selectively separate specific components. Acidic or alkaline hydrolysis yields hydrolysates containing peptides and amino acids for the revalorization of waste generated by diverse industries such as leather processing, hairdressing, meat processing, or animal production ((Szopa et al., 2023)). 1.4.2. Biological Hydrolysis Biological hydrolysis refers to the use of enzymes (proteases) or microorganisms to break down proteins. These methods are preferred over chemical hydrolysis due to their milder reaction conditions and the ability to produce bioactive peptides with enhanced functional and nutritional properties (Tavano, 2013). In this context, the fermentation of bread dough serves as a prime example. This process involves yeast proteases breaking down wheat flour proteins, resulting in improved dough texture and bread quality. Additionally, the curing of meats, fish, and cheeses also relies on Chapter 1 — General Introduction 53 proteases. These enzymes, sourced from microorganisms, are instrumental in developing the desired sensory and chemical properties of cured foods (Vioque & Millán, 2005). Likewise, the role of proteases extends to the processing of table olives, where they reduce bitterness to enhance sensory appeal (Vioque & Millán, 2005). Although these processes are controlled, their success is contingent upon the choice of microorganisms and the enzymes they produce for food modification. Thus, enzymatic protease action has been appreciated because it takes part in known food processing (Akbarian et al., 2022). In this sense, the manufacture of protein hydrolysates entails the controlled decomposition of proteins into shorter proteins, peptides, and amino acids by directly employing enzymes or blends of enzymes. These proteases are derived from plant or animal by-products, and developed to generate products for specific applications within the food industry and affording them novel uses, as previously mentioned (Vioque & Millán, 2005). A significant benefit of generating hydrolysates via proteases lies in the removal of epitopes that could trigger responses in the autoimmune system, yielding a product conducive to the formulation of hypoallergenic dietary options (Tavano, 2013). Furthermore, the digestibility of such hydrolysates surpasses that of the original protein source. Given that these are partially digested products, the digestive system expends reduced effort towards their complete breakdown (Tavano, 2013). Additionally, these hydrolysates present numerous advantages relative to their native proteins, such as ease of incorporation into food matrices attributed to their low viscosity, along with major agitation, dispersion, and solubility capabilities (Grasso et al., 2022; Shevkani et al., 2019). Owing to these properties, protein hydrolysates find extensive application across a broad spectrum of food products, including infant formulas, nutritional supplements, enteral diets, sports nutrition, and Development of Functional Foods for Application to Sport Nutrition 54 specific dietary formulations. Additionally, enzymatic hydrolysis is recognized as a potent method for the recovery of intact amino acids, facilitating the production of bio-fertilizers, a process documented by Sun et al. (2019). 1.4.2.1. Food-Grade Proteases Proteolytic enzymes used in the food industry can be classified into different categories based on their origin (bacterial, fungal, animal, or plant), their mode of catalytic action (endoprotease or exoprotease activity), or depending on the catalytic site (serine protease, cysteine protease, aspartate protease, metalloprotease) (Benitez et al., 2008; Vioque & Millán, 2005). Table 1.5. summarizes some commercial foodgrade enzymes. Endoproteases, act on the internal peptide bonds of proteins. This results in the formation of smaller peptide fragments along the original polypeptide chain, generating a mixture of peptides of different sizes (Benitez et al., 2008; Vioque & Millán, 2005). On the other hand, exoproteases or exopeptidase proteases act on the ends of polypeptide chains. This action releases amino acids and/or dipeptides from the ends of proteins (Benitez et al., 2008; Vioque & Millán, 2005). Regarding the classification based on the nature of the catalytic center, the action differs according to the amino acids and other ligands involved in the formation of the enzyme-substrate complex (Benitez et al., 2008). Thus, they are named as serine proteases, cysteine proteases, and aspartate proteases, based on the amino acid present in the active center (serine, cysteine, or aspartic acid, respectively) (Benitez et al., 2008). Metalloproteases are those whose activity is promoted by a metallic cation in the active center of the enzyme, where Zinc is commonly found. Generally, all serine proteases have endoactivity, while metalloproteases are mainly exoactivity (Benitez et al., 2008). 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Frontiers Media S.A. https://doi.org/10.3389/fnut.2021.815640 67 Chapter 2 – Theoretical Framework The theoretical framework described in this chapter develops the role of protein hydrolysates in health, presenting them as immunonutrients. To achieve this, a detailed literature review is conducted in databases (Scopus, PubMed) with the additional support of computer tools (VOSviewer) for a global assessment. Development of Functional Foods for Application to Sport Nutrition 68 Chapter 2 — Theoretical Framework 69 2.1. Protein Hydrolysates and Health Protein hydrolysates are highly valued due to the presence of peptides that may exhibit biological activity. In general, bioactive peptides are protein fragments that benefit the body systems and overall human health. Most bioactive peptides range between two and 20 amino acid residues resulting in a molecular mass of 0.4–4 kDa (Chalamaiah et al., 2018), longer bioactive peptides are also possible (Zaky et al., 2022). Peptide size is related to the degree of hydrolysis of the protein, and the more the degree of hydrolysis the more the biological activity they might have, as mentioned in chapter 1. However, extensive hydrolysis can break off proteins and peptides into free amino acids with little activity (Akbarian et al., 2022). Therefore, it is crucial to control all conditions maintaining a balance between the release of small peptides and free amino acids. The bioactivity of peptides has been increasingly studied in the last 30 years because peptides possess several activities to human health, such as anti-hypertensive, anti-inflammatory and immunomodulatory, lipid lowering, anti-diabetic, anti-thrombotic, anti-amnesic and neuroprotective, opioid, anti-cancer, analgesic, and antioxidant effects, as summarized in Fig. 2.1 (Akbarian et al., 2022; Zaky et al., 2022). Additionally, they might improve mineral absorption or maintain antimicrobial activity. Development of Functional Foods for Application to Sport Nutrition 70 Figure 2.1. Schematic representation of physiological activities of bioactive peptides. 2.1.1. Protein Hydrolysates and the Antioxidant Activity In the context of healthy effects, we focus on antioxidant activity. The production of free radicals serves as a signaling molecule by cells, considering that oxidative stress occurs when there is an imbalance between pro-oxidant and antioxidant molecules. Thus, the Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) challenge the antioxidant defense systems (Tauffenberger & Magistretti, 2021). Antioxidant enzymes such as catalase, superoxide dismutase and glutathione reductase are considered the primary defense barrier of cells. When this primary defense is insufficient, small molecules with antioxidant activity can react with free radicals to produce less dangerous molecules, as a second defense barrier (Grauers Wiktorin et al., 2020; Nocella et al., 2023), such as vitamins or peptides. Oxidative stress led to biological damage and inflammation, which are closely related to non-communicable diseases (Forrester et al., 2018; Tauffenberger & Magistretti, 2021) including cancer, diabetes, cardiovascular diseases, neurological disorders, inflammatory bowel disease, and pulmonary diseases (Chalamaiah et al., 2018; He et al., 2015). In this regard, plant-based protein hydrolysates and peptides can Chapter 2 — Theoretical Framework 71 exert antioxidant activity, but their biological mechanisms are not fully elucidated (Akbarian et al., 2022). Several works have studied the in vitro antioxidant potential of protein hydrolysates and peptides, demonstrating that they can inhibit oxidation by eliminating free radicals and chelating metal ions. In general, all protein amino acids per se possess the potential to interact with free radicals (Matemu et al., 2021), besides the antioxidant potential increase in peptides in comparison with the amino acids themselves (Akbarian et al., 2022). Notable examples include peptides derived from casein, which have been shown to reduce lipid oxidation and modulate the activity of antioxidant enzymes (Bamdad et al., 2017). Not only the amino acid content but also the peptide sequence and size play important roles in the antioxidant properties of peptides since aromatic and hydrophobic amino acids confer recognized bioactivity at peptides. For instance, hydrophobic amino acids can neutralize fatsoluble free radicals. The presence of hydrophilic amino acids in the N- terminal, along with specific amino acids (Tyr, Val, Met, Leu, Ileu, Glu, and Trp) in the C-terminal position, is associated with antioxidant effects. The amino acids His, Met, Cys, Tyr, Phe, Trp, can act as protons or electron donors, thus converting free radicals into stable molecules (Akbarian et al., 2022; Matemu et al., 2021). On the other hand, few studies have shown that an increase of oxidative stress have a protective role, as ROS, Nitric Oxide (NO), and RNS play complex roles in molecular mobilization. The physiological role of ROS and RNS molecules as adaptive response are needed to differentiate between oxidative “eustress” (good oxidative stress) versus “distress” (bad oxidative stress), and the relation to “hormesis” effect (possessing antioxidant effect at low doses but becomes toxic at higher doses), as further explained in Chapter 4 (Calabrese & Kozumbo, 2021; Moghaddam et al., 2019; Sies et al., 2022). Thus, an oxidant effect may be essential to induce the eustress response Development of Functional Foods for Application to Sport Nutrition 78 Figure 2.3. Author Keyword Network Visualization in the Literature review on Protein Hydrolysates. This figure displays the author keyword co-occurrence network generated by VOSviewer, based on bibliographic data from the analysis of 4,592 scientific articles published between 1942 and 2024. The selected term “chickpea” shows the relationship between other terms. Chapter 2 — Theoretical Framework 79 To deep into more detailed data, a similar analysis based on text was conducted using binary counting of title and abstract fields of most relevant works (1,963) excluding those works that include terms like biomass/bioethanol generation, microbiology or are focused on carbohydrates terms. The analysis presented in Fig. 2.4. shows that out of 535 terms, 69 keywords were selected and categorized into four clusters. Each cluster was represented by a different color: cluster 1 red (28 items), cluster 2 green (18 items), cluster 3 blue (15 items), and cluster 4 yellow (8 items). The terms selection was conducted avoiding general words, methodology terms, duplicates terms (using a thesaurus file), and terms concerning infant nutrition, milk, fish, meat, or collagen hydrolysates. Cluster terms are presented below: Cluster 1 (28 items): allergenicity, anti-inflammatory, antidiabetic, bioactive compound, blood glucose, cancer, cardiovascular disease, cat, cereal, chia, cholesterol, clinical trial, elisa, gene expression, hemp, hyperglycemia, immunomodulatory, in vivo, inflammation, insulin, insulin resistance, legume, lipid metabolism, lypopolysacharide, metabolic syndrome, obesity, prevention, soy. Cluster 2 (18 items): alcalase, amylase, bean, bitternes, bromelain, chickpea, chymotrypsin, diabetes, dppiv, flavourzyme, in silico analysis, lentil, molecular docking, neutrase, papain, peanut, pepsin pancreatin, protamex. Cluster 3 (15 items): abts, antioxidant, cytotoxicity, dpph, frap, gsh, hydroxyl radical scavenging, lipid oxidation, mda, metal chelating activity, mung bean, nitric oxide, orac, radical scavenging activity, sod. Cluster 4 (8 items): ace, antihypertensive, gastrointestinal digestion, nutraceutical, pancreatin, pepsin, seed protein, synthetic peptide. The nodes, whose size varies proportionally to the frequency of occurrence of each keyword, highlight the terms “antioxidant”, “alcalase”, “pepsin”, “ace”, and “dpph” as some of the most prominent Development of Functional Foods for Application to Sport Nutrition 80 within the literature (Fig. 2.4). The interconnecting lines between these nodes indicate a strong co-occurrence, suggesting a close relationship among these themes in studies on protein hydrolysates and peptides. In terms of raw materials, soy is predominantly used for producing hydrolysates, but other seeds such as beans, chickpea, lentil, wheat, chia, hemp, quinoa, peanut, and rice are also utilized. Furthermore, the health-related topics most frequently investigated in the context of "protein hydrolysates" encompass metabolic and non-communicable diseases, including diabetes, hypertension, cardiovascular diseases, and cancer, which are closely associated with lipid metabolism disorders, inflammation linked to oxidative stress, and chronic inflammation. Studies also explore antimicrobial activity, metal chelating capabilities, and the activity of metabolites derived from simulated gastrointestinal digestion. Furthermore, a focused analysis on the term 'chickpea' within the network analysis of co-occurring words in titles and abstracts was conducted to identify related keywords (Fig. 2.5). There is an association with terms such as “antioxidant”, “dpph”, “abts”, “lipid oxidantion”, “antihypertensive”, “ace”, “diabetes”, “dppiv”. This finding may indicate that the investigation using chickpea hydrolysates and peptides are focused on the measure of: i) antioxidant activity though DPPH, ABTS and lipid oxidation assays; ii) the antihypertensive activity using ACE inhibitory activity; and iii) antidiabetic potential with the measure of DPP-IV. Moreover, from the terms “alcalase”, “flavourzyme”, and “pepsin” it can be deduced that Alcalase is the enzyme most used in the production of chickpea hydrolysates and peptides. In contrast to author keyword analysis, it could not be deduced (Fig. 2.2. and 2.3.). This analysis underscores the importance of assays aimed at the antihypertensive and metabolic effect potential, as well as the antioxidant character of bioactive peptides, providing a solid foundation for future research in these areas. Chapter 2 — Theoretical Framework 81 Figure 2.4. Title and Abstract Keyword Network Visualization in the Literature review on Protein Hydrolysates. This figure displays a keyword co-occurrence network generated by VOSviewer, based on text data from the analysis of 1,963 scientific articles published between 1942 and 2024. Lines between nodes signify the co-occurrence of terms within the same articles, illustrating the interconnection between different research areas and each term. Development of Functional Foods for Application to Sport Nutrition 82 Figure 2.5. Title and Abstract Keyword Network Visualization in the Literature review on Chickpea Protein Hydrolysates. This figure displays a keyword co-occurrence network generated by VOSviewer, based on text data from the analysis of 1,963 scientific articles published between 1942 and 2024. The term “chickpea” is highlighted to observe related keywords. Lines between nodes signify the co-occurrence of terms within the same articles, illustrating the interconnection between different research areas and “chickpea” term. Chapter 2 — Theoretical Framework 83 2.3. Review of Chickpea Protein Hydrolysates Focusing on chickpea protein hydrolysates (CPHs), the PubMed literature review reveals that the term 'Chickpea protein hydrolysate' yields 63 published works, while Scopus search revealed 107 works (PubMed, n.d.; Scopus, n.d.). However, only 33 of these PubMed plus 1 Scopus works were selected for our investigation based on the inclusion criteria: they either measure antioxidant activity (biochemistry assays), involve bioactivity assays measured in vitro (cellular models) or in vivo (animal models), and/or involve peptide purification or identification. Exclusion criteria were studies that only showed chemical characterization or technological properties, and review articles were not considered. The comprehensive review of the full texts of selected works indicates the need to exclude one work and to include previous work that were not listed in these reviews. Ultimately, 33 studies from both databases were included. A work on the in vitro properties of CPHs from the SciELO database was used to complete the background (Scielo, n.d.), but not included in tables. The complete methodology for both analyses, the VOSviewer analysis (based on the author keyword and title and abstract words) and the complete revision of CPHs, was summarized in the diagram represented in Fig.2.6. The 34 studies included about CPHs are summarized in Tables 2.1. and 2.2. located at the end of this chapter. Development of Functional Foods for Application to Sport Nutrition 84 Figure 2.6. Flowchart of the Review. Terms selection (1) for analysis based on VOSviewer using terms from author keywords, or key words derived from the title and abstract for plant-based hydrolysates. Terms selection (2) for the selection of works included in the review of chickpea protein hydrolysates. 2.3.1. Raw Material and Pretreatment Conditions Among the selected studies, not only kabuli-type but also desi-type chickpea flours were used for protein concentration and isolation. Only two studies have reported the specific chickpea genotypes which are desi-type as raw material (Milán-Noris et al., 2018; Navarro-Leyva et al., 2023) . With respect to pre-treatment conditions, most studies (14 works) have reported employing a pre-treatment step prior to protein isolation. These pre-treatments include germination and sprouting (Chandrasekaran & Gonzalez de Mejia, 2022, 2023; Milán-Noris et al., Chapter 2 — Theoretical Framework 85 2018; Serrano-Sandoval et al., 2023), precooking (Acevedo Martínez & Gonzalez de Mejia, 2021a; Acevedo-Martinez & Gonzalez de Mejia, 2021b; Arnal et al., 2023), cooking (Acevedo Martínez & Gonzalez de Mejia, 2021a; Arnal et al., 2023; Medina-Godoy et al., 2012), high hydrostatic pressure treatment or autoclaving (Arnal et al., 2023; Moreno et al., 2020; Zhang et al., 2012), and extrusion (Chávez- Ontiveros et al., 2022). Among these, 14 studies also indicate a defatting treatment of the flour before protein isolation, as fat reduce the efficiency of extraction. However, this chemical process involves petroleum-derived solvents that produce highly polluting waste, difficult to manage and, on an industrial scale, incur significant costs. In some cases (5 works), a preheating to 80 ºC is reported to inactivate native enzymes of the raw material for a duration ranging between 5 and 30 minutes (Felix et al., 2020; Navarro-Leyva et al., 2023; Quintero- Soto et al., 2021; Zan et al., 2023a; Zan, et al., 2023a). 2.3.2. Protein Isolation and Hydrolysis Conditions Typically, the two-step method utilized for protein isolation comprises an initial solubilization at alkaline pH, followed by subsequent precipitation at the isoelectric point (4-4.5). Additionally, some authors have reported isolating protein fractions (6 works) using the Osborne method (Mukhamedov et al., 2023) or albumin/globulin isolation (Medina-Godoy et al., 2012; Mukhamedov et al., 2021; Navarro-Leyva et al., 2023; Quintero-Soto et al., 2021; Xue et al., 2012) probably due to the isoelectric point of albumins and globulins are 4-4.5. Regarding hydrolysis conditions, authors predominantly utilized enzymes, finalizing the hydrolysis process with enzyme deactivation. Alcalase has been identified as the enzyme most frequently used in CPHs (nearly 50% of the studies reviewed), as evidenced by the networking analysis as well as by Tables 2.1 and 2.2, followed by pancreatin, pepsin, papain, and trypsin. Enzymes less utilized in these studies include Development of Functional Foods for Application to Sport Nutrition 86 Bromelain, Flavourzyme, Ficin, Neutrase, Protamex, and Chymotrypsin. Furthermore, some studies have adopted sequential hydrolysis with combinations such as Alcalase-Flavourzyme (Megías et al., 2007; Xue et al., 2012; Yust et al., 2012), Papain-Trypsin-Alcalase (Mukhamedov et al., 2021), Pepsin-Pancreatin-Bromelain (Acevedo Martínez & Gonzalez de Mejia, 2021a), or Pepsin-Pancreatin. The use of enzyme mixtures may increase the diversity of peptide sequences in chickpea hydrolysates due to the variety of enzymatic activity. Notably, the sequential application of Pepsin-Pancreatin is often associated with simulated gastrointestinal digestion (SGID). 2.3.3. Methods for Simulated Gastrointestinal Digestion (SGID) The SGID methods are used to assess the digestibility of foods, enabling the calculation of theoretical metrics such as the protein digestibility corrected amino acid score (PDCAAS) for evaluating the protein quality of newly developed foods. Indeed, these methods can even be compared to animal models as Nosworthy et al. (2019) have demonstrated. In the reviewed works, the most frequently utilized SGID method is the pepsin-pancreatin protocol (Acevedo Martínez & Gonzalez de Mejia, 2021; Acevedo-Martinez & Gonzalez de Mejia, 2021b; Chandrasekaran et al., 2020; Chandrasekaran & Gonzalez de Mejia, 2022; Moreno et al., 2020; Navarro-Leyva et al., 2023) reported by Akeson & Stahmann (1964). This method involves a straightforward two-step protocol designed to replicate gastrointestinal conditions, with pH values set to 2 in the first step and 6.8 in the second, alongside a constant temperature of 37°C. The Pepsin-Pancreatin digestion method offers a rapid and precise estimation of protein quality, demonstrating high correlation with the biological value for rats (Akeson & Stahmann, 1964). Chapter 2 — Theoretical Framework 87 An alternative, increasingly and recognized method is the INFOGEST protocol developed by Brodkorb et al. (2019). Three studies have reported using this method for SGID (Arnal et al., 2023; Zan, et al., 2023a; Zan, et al., 2023b). The INFOGEST 2.0 static in vitro method streamlines the simulation of digestion, facilitating the analysis of digestion products and micronutrient release (Brodkorb et al., 2019). This approach enhances our comprehension of the impacts of food structure and composition on human health. The method is more complex, involving a three-phase digestion process that encompasses oral, gastric, and intestinal stages, utilizing simulated fluids for each phase and complex enzymatic mixtures to emulate the complete digestion process at specific temperatures and pH levels for each phase. Other SGID methods used for the digestion are the Pepsin-Corolase (which is a bacterial enzyme mixture possessing pancreatic, trypsin and quimotrypsin activities, as explained in Chapter 1 (section 1.4.2.2)). This method is used in one work (Felix et al., 2020). Two articles (Chávez- Ontiveros et al., 2022; Milán-Noris et al., 2018) used methods similar to INFOGEST, the method described by Ortega et al. (2009) which are derived from the method by Gil-Izquierdo et al. (2002). These encompass the mimic of oral, gastric, and intestinal stages using fluids and enzymes but also include static and dynamic dialysis to eliminate water. Remarkably, the Pepsin-Corolase method is focused on the evaluation, digestibility and bioaccessibility of polyphenols (Felix et al., 2020). All digestibility methods mentioned exhibit high reproducibility, with even the simplest methods demonstrating a strong correlation in measurements compared to in vivo studies. Selection of a particular method should be based on the measurement requirements as well as the constituents of the sample to be assessed. A method designed for the digestion of protein-rich samples initiates different reactions compared to those created for fat-rich samples, where lipases are primarily required. In the case of studies with CPHs, many of them aim Development of Functional Foods for Application to Sport Nutrition 94 investigate antidiabetic properties of CPHs. A unique study addressed the measurement of alcohol dehydrogenase activity, which is significant in the context of alcohol metabolism. This study suggests the use of CPH as a natural anti-alcoholic ingredient to prevent alcoholic liver disease (Zan, et al., 2023b). In 10 of the reviewed studies, the focus extends beyond measuring enzyme inhibition to include molecular docking analyses which are used to verify or predict the binding sites between peptides and these enzymes (Acevedo Martínez & Gonzalez de Mejia, 2021a; Acevedo- Martinez & Gonzalez de Mejia, 2021b; Arnal et al., 2023; Chandrasekaran et al., 2020; Chandrasekaran & Gonzalez de Mejia, 2022; Quintero-Soto et al., 2021; Zan, et al., 2023a; Zan, et al., 2023b). Thus, emphasizing the significance of in silico assays and molecular docking as critical tools for predicting the bioactivity of these peptides and hydrolysates. 2.3.5.4. Non-Enzymatic Assays A non-enzymatic-based assay that evaluates the in vitro micellar solubility of cholesterol in the presence of CPHs was observed, highlighting the higher hypocholesterolemic and antioxidant activities of CPHs than intact proteins (Yust et al., 2012). 2.3.5.5. Antifungal and Antibacterial Activities Antibacterial and antifungal activities are other areas of investigation, typically assessed through assays on bacterial and fungal strains. Heymich et al. (2021a) identified two peptides, named Leg1 (RIKTVTSFDLPALRFLKL) and Leg2 (RIKTVTSFDLPALRWLKL), with Leg2 exhibiting notable antioxidative activity through mechanisms of radical scavenging and reduction. These peptides have the potential to be applied as preservatives in food and other products against bacterial and fungal spoilage, as well as oxidative degradation (Chang et al., 2011; Heymich et al., 2021a; Heymich et al., 2021b). Chapter 2 — Theoretical Framework 95 Furthermore, two smaller peptides derived from Leg2, named KTA and KTR, have demonstrated effectiveness in inactivating gram-positive bacteria, such as Staphylococcus aureus. Their action mechanisms include disrupting the bacterial membrane and interfering with peptidoglycan synthesis, highlighting their potential as antimicrobial agents (Zhang et al., 2024). 2.3.6. Biological Impact of CPHs The evaluation of CPHs through studies in cellular cultures and animal models constitutes a fundamental pillar for understanding their therapeutic potential which are complementary to biochemical assays explained before. 2.3.6.1. In-Vitro Studies: Cellular Culture In the context of cell cultures, CPHs and chickpea peptides (CPs) have been shown to possess various biological activities across multiple cell lines and primary cultures. According to the reviewed articles, the evaluation of cellular viability was conducted with three main objectives: to assess the safety of exposure to the hydrolysates, to evaluate tumor inhibition, and to gauge growth inhibition during or after UV exposure. Other studies evaluated different biological activities such as antioxidant, antidiabetic, antihypertensive, or anti-inflammatory. Cellular Viability Evaluation by MTT and SRB assays. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay is one of the most widely used methods for assessing cell viability, proliferation, and cytotoxicity, and exhibit interesting redox behavior (Stockert et al., 2018). Assessment with MTT has found that non-cytotoxic effects from different CPH fractions or CPs were observed using the murine macrophage RAW 264.7 cell line (Milán-Noris et al., 2018), human epithelial human colon Caco-2 cells at concentrations below 2000 µM for Leg1 peptide and 1000 µM for Leg2 peptide Development of Functional Foods for Application to Sport Nutrition 96 (Heymich et al., 2021), and primary mouse spleens (Xue et al., 2012). On the other hand, cell inhibitory effects on human lung cancer A549 cells by the MTT assay have been interpreted as tumor reduction following exposure to oxovanadium-CPs (Mukhamedov et al., 2021). Moreover, Giron-Calle et al. (2010) observed that exposure to pepsin-pancreatin CPHs can inhibit the growth of Caco-2 cells by up to 45% and THP-1 cells by up to 78%, as determined through MTT assays, suggesting cancer inhibitory effects. Conversely, the co-culture of Caco-2 and THP-1 actually enhanced the proliferation of THP-1 cells by up to 66%, indicating immunomodulatory activity (Girón-Calle et al., 2010). Another method, less commonly used to investigate cell viability in the reviewed articles, is the Sulforhodamine B (SRB) assay, which demonstrated a low cytotoxic effect of ultrafiltrate CPH in murine peritoneal macrophages (Meza Márquez et al., 2022). The SRB method is chosen for its cost-effectiveness in screenings and is substantially simplified compared to the MTT method, as it does not rely on measuring metabolic activity (Orellana & Kasinski, 2016). Antioxidant Potential Evaluation by MTT and CAA assays. The MTT assay may also be used to investigate redox processes and study redox biology since the reduction of MTT in cells should primarily be interpreted as an indicator of glycolytic NAD(P)H production within the endoplasmic reticulum, cytosol, and plasma membranes (Stockert et al., 2018). For instance, exposure of primary dermal fibroblast HDFa cell line to CPHs smaller than 10 kDa inhibited collagen degradation after UVA radiation, suggesting that these CPHs possess significant cellular antioxidant, and consequently, anti-photoaging activity which were confirmed using other assays (Serrano-Sandoval et al., 2023). The Cellular Antioxidant Activity (CAA) assay quantifies the capacity of compounds to inhibit the conversion of Dichlorohydrofluorescin (DCFH), which is non fluorescent, to fluorescent Dichlorofluorescein (DCF) by Chapter 2 — Theoretical Framework 97 peroxyl radicals generated by 2,2′-Azobis(2-amidinopropane) dihydrochloride (ABAP) (Wolfe & Liu, 2007). A study using HDFa cell line confirmed the high antioxidant potential of chickpea protein-derived compounds (Serrano-Sandoval et al., 2023). The CAA assay was also utilized to assess the antioxidant activity of CP in Caco-2 cells, demonstrating that CPs with the strongest FRAP and DPPH activities also maintain the highest antioxidant activities in cell culture (Torres-Fuentes et al., 2015). The Evaluation of Anti-Inflammatory Activity (Cytokines and NO). NO levels measured in cellular supernatants, using Griess reaction (Moreno et al., 2020), from certain studies indicate that its inhibition reflects antioxidant activity and anti-inflammatory properties. This was evidenced by data obtained from murine peritoneal macrophages (Meza Márquez et al., 2022) and RAW 264.7 cell line (Milán-Noris et al., 2018). In the work of Meza Márquez et al. (2022), the assessment of extracellular concentrations of TNF-α and IL-1β via ELISA assays revealed that peptide fractions smaller than 10 kDa significantly reduce the release of these cytokines at a higher dosage (5 mg/mL). Furthermore, the inhibition of IL-1β by fractions smaller than 5 kDa was observed at all tested dosages demonstrating anti-inflammatory potential (Meza Márquez et al., 2022). The in-vitro Evaluation of Anti-diabetic and Anti-hypertensive activities Additionally, efforts to evaluate the antidiabetic and antihypertensive potential of CPHs and CPs were highlighted. Glucose uptake, along with sucrase-maltase-isomaltase activity, and DPP-IV inhibition rate were measured in the cellular supernatant, revealing a dose-dependent inhibition of glucose uptake by three peptides (VVFW, GEAGR, and FDLPAL) (Chandrasekaran & Gonzalez de Mejia, 2023). Another study highlighted those peptides preselected through in silico analyses Development of Functional Foods for Application to Sport Nutrition 98 (AAWPGHPEF, IAIPPGIPYW, PPGIPYW) exhibit significant DPP-IV inhibitory activity (Zan, et al., 2023a). Study of Organoleptic Properties Using Cell Culture (Bitter Receptors) Given the significance of organoleptic changes associated with peptide release, one study has been found that evaluates the potential stimulation of bitterness receptors. This finding is particularly important considering that small peptides and amino acids may exhibit a bitter flavor, as previously mentioned. In the cited study, an analysis of protein expression via western blot for T2R4 and T2R14 receptors was conducted, which indicated concurrent inhibition of the bitter receptor signaling pathway (Chandrasekaran & Gonzalez de Mejia, 2023). Advantages and Limitations of Cellular Models The use of cellular models to study the potential of protein hydrolysates as functional foods presents a blend of distinct advantages and limitations that are crucial for researchers to consider. On one hand, these models offer a controlled environment that allows for highthroughput screening, enabling the rapid assessment of numerous compounds. This, coupled with their ease of cultivation and manipulation, provides a relatively cost-effective and reproducible method for investigating the bioactive effects of protein hydrolysates (Onuh & Aluko, 2019; Tenenbaum et al., 2023). Such models are particularly useful for evaluate molecular mechanisms, as they allow for the application of specific inhibitors or gene modifications to explore biochemical pathways (Onuh & Aluko, 2019; Tenenbaum et al., 2023). On the other hand, the limitations of cellular models stem from their inability to fully mimic the complexity of living organisms (Li-Chan, 2015). The use of immortalized cell lines may not accurately reflect genetic uniformity, natural cellular behavior, and the lack of systemic interactions, as well as the influence of the microbiome, which are not considered. Despite these limitations, in vitro cellular models remain Chapter 2 — Theoretical Framework 99 vital for initial bioactivity assessments and mechanism of action studies serving as an approach to guides subsequent research in more complex systems. 2.3.6.2. In-Vivo Studies: Animal Models Two studies conducted on animal models have provided evidence that the consumption of CPHs may possess anticancer activity. Sánchez- Chino et al. (2019) proposed that the consumption of CPH might confer a protective effect against colon carcinogenesis using azoxymethane (AOM)-induced carcinogenesis in mice. Similarly, one study involving H- 22 tumor-bearing mice (KM) indicate protective roles against hepatic carcinoma (Xue et al., 2012). Additionally, both studies also concluded that animals treated with a high-fat diet and hydrolysates showed reduced concentrations of LDL-cholesterol and triglycerides in blood serum, whereas HDL-cholesterol did not undergo significant changes showing hypolipidemic effect of CPHs (Sánchez-Chino et al., 2019; Xue et al., 2012). Considering the antidiabetic potential of CPHs, extensively analyzed in ex vivo assays by the reviewed works (Acevedo Martínez & Gonzalez de Mejia, 2021a; Acevedo-Martinez & Gonzalez de Mejia, 2021b; Arnal et al., 2023; Chandrasekaran et al., 2020; Chandrasekaran & Gonzalez de Mejia, 2022; Felix et al., 2020; Quintero-Soto et al., 2021), as well as through cellular cultures (Chandrasekaran & Gonzalez de Mejia, 2023; Zan et al., 2023a), the hypo- and anti-hyperglycemic activities were investigated in BALB-c mice. The findings of this study suggest that the consumption of pepsin-pancreatin hydrolyzed proteins retains antihyperglycemic activity (Navarro-Leyva et al., 2023). Furthermore, the research explores anti-inflammatory potential of Alcalase CPHs. Additionally, the antihypertensive potential was evaluated in male spontaneously hypertensive rats, revealing that CPHs from extruded flour effectively lower systolic blood pressure (Chávez-Ontiveros et al., 2022). This finding is consistent with several ex vivo assays measuring Development of Functional Foods for Application to Sport Nutrition 100 ACE inhibitory activity (Arnal et al., 2023; Boschin et al., 2014; Chandrasekaran & Gonzalez de Mejia, 2022; Felix et al., 2020; Medina- Godoy et al., 2012; Mukhamedov et al., 2023). Advantages and Limitations of Animal Models In the context of animal models to study the bioactive effects in functional foods, it is important to consider both their advantages and disadvantages. The physiological similarity and the capability for experimental control that animal models offer facilitate the understanding of the mechanisms by which bioactive compounds exert their effects in complex organisms (Robinson et al., 2019). However, the specific differences between species, ethical concerns, and logistical and economic challenges present significant limitations for the direct extrapolation of results to humans (Andersen & Winter, 2019; Hartung, 2008; Robinson et al., 2019). Moreover, advancements in cellular and 3D models, alongside reduction and refinement techniques, and the development of computational models, emerge as promising and complementary alternatives (Cencič & Langerholc, 2010). These approaches not only address the limitations of animal models but also reflect a growing commitment to ethics and animal welfare in scientific research. Therefore, the selection of an appropriate model should be guided by careful consideration of these factors, always seeking a balance between investigative efficacy and ethical responsibility. 2.3.6.3. In-Vivo Studies: Human Supplementation To date, at least 70 studies registered on ClinicalTrials (Clinical Trials, n.d.) have been identified that aim to investigate various beneficial aspects of consuming chickpeas, chickpea flour, or flours derived from chickpea by-products, alongside the study of the consumption of other legumes and cereals or those related to the MD. Human interventional studies on legume-based diet are associated with metabolic and inflammatory changes, such as a reduction of C-reactive protein and complement C3, and are accompanied by significant weight loss in Chapter 2 — Theoretical Framework 101 obese subjects (Hermsdorff et al., 2011). Moreover, the inclusion of chickpeas in an intervention diet have been shown to result lower serum total and low-density lipoprotein cholesterol levels (Pittaway et al., 2006, 2007), lower glycemic index eliciting protracted digestion, and enhanced satiety responses (Chen et al., 2022; Hafiz et al., 2022; Johnston et al., 2021). In studies involving human dietary interventions using plant-based hydrolysates, a considerable reduction in blood pressure was observed in human subjects after consuming pea hydrolysates (Li et al., 2011). Studies on protein hydrolysates from pea, gluten, rice, and soy, have demonstrated enhanced insulin secretion and a correspondingly low plasma glucose response (Claessens et al., 2009). Supplementation with Vicia faba hydrolysates significantly improves strength recovery and restoration, reduces fatigue, and positively modulates alterations in markers related to muscle homeostasis in healthy male subjects (Kerr et al., 2023). However, no studies have been found in any population where chickpea protein hydrolysates were administered. 2.4. Conclusion Collectively, these studies dilucidated a comprehensive framework of the multifaceted health benefits of CPHs. The CPHs and CPs have potential bioactivities, such as antioxidant, anti-inflammatory, and mineral-chelating properties, antidiabetic, antihypertensive, immunomodulatory, and anticancer, as observed in the scientific networking analysis and the comprehensive review of CPHs works. Therefore, CPHs and CPs hold promise in various therapeutic applications as functional ingredients or supplements. Noteworthy, the comprehensive review of the literature underscores a critical need to further explore the antioxidant and anti-inflammatory capabilities of chickpea protein hydrolysates. This is because little is known about their cellular mechanisms. Thus, more investigations are Development of Functional Foods for Application to Sport Nutrition 102 needed to unravel the real potential of these bioactive compounds in cellular models, but also require standardization, sensory evaluation, and well-designed clinical trials to provide robust evidence for supporting these biological activities (Li-Chan, 2015). Based on the foregoing, a hypothesis is formulated and objectives for our research are established, which are presented in the following chapter. Chapter 2 — Theoretical Framework 103 Table 2.1. Summary of in vitro studies based on chickpea protein hydrolysates (CPHs). The table includes raw material, pre-treatment (before performing hydrolysis), the hydrolysis conditions, the main results about antioxidant or biological activity, and the purification or peptide. Raw Material Pretreatment Hydrolysis conditions Main Results Purification or Peptide Identification Source Chickpea protein isolates (Isoelectric point) Alcalase (2.4 L, 0.3 AU/g), 50 ºC, pH: 8, 60 min; subsequent hydrolysis of Flavourzyme (100 LAPU/g), 50 ºC, pH: 7, 200 min. Inact: 80 °C for 20 min. High copper chelating and β-carotene oxidation inhibition activities. (Megías et al., 2007) Chickpea protein isolates High hydrostatic pressure (100- 600 MPa, 2–40 min, 50ºC) Alcalase 2.4 L FG (1:20 (w/v) E/S ratio. During or after the pretreatment, 50 ºC, pH: 8, 360 min in 0.5 M NaOH. Inact: 95ºC, 10 min. High FRAP and superoxide radical scavenging activities. (Zhang et al., 2012) Globulin and albumin from fresh and hard-to- cook chickpeas Cooked Alcalase: 50 ◦C, pH: 9.0. Or Papain: T:40 ◦C, pH: 6.5. Or Pancreatin: T: 39 ◦C, pH: 8.0. 2 h, pH 4.0, inact: 95 ◦C for 10 min. High ACE inhibitory and antioxidative (DPPH, ABTS) activities using both materials (fresh and hard- to-cook). (Medina- Godoy et al., 2012) Chickpea protein isolate (isoelectric point) Defatted flour Subsequential treatment with Alcalase (4.6 mL /L, temp. 50ºC, pH 8 for 1 h) and Flavourzyme (1.85 mL/L, temp. 50ºC, pH 7 for 2 h). High hypocholesterolaemic and antioxidant (βcarotene, FRAP, and DPPH) activities. (Yust et al., 2012) Table 2.1. continues in the next page Development of Functional Foods for Application to Sport Nutrition 110 Raw Material Pretreatment Hydrolysis conditions Main Results Purification or Peptide Identification Source Desi chickpea (ICC5613) Flour from 5 days germinated or sprouted chickpeas. Defatted and protein extraction (isoelectric point) SGID using method 10.1021/jf900591q The chickpea samples have shown antiinflammatory potential in the LPS activated murine RAW 264.7 cell line (higher cell viability and nitric oxide inhibition rate). The peptide profile and phenolic compounds revealed the importance of glutamine residues. RP-LC-MS for fractionation, Peptide identification by nano-UPLC–ESI-MS/MS. Relevant peptides: LHQNIGSSSSPDIYNPQAGR, LHQNIGSSSSPDIYNPQAGRIK, QQSQETDVIVK, VLLEEQEQKPK, DVGQKTKEVGQDIQAK (Milán- Noris et al., 2018) Chickpea albumin, globulin, and glutelin (precipitation) Defatted and denaturalization (TRIS, urea, and 1.4- dithiothreitol (1h, 30min)) Enzymatic hydrolysis (enzyme/protein 1:50) was performed for 16 h at 25 °C using chymotrypsin from bovine pancreas. The identified peptides showed antibacterial, antifungal, and antioxidative activities (DPPH, FRAP). Additionally, assays on the Caco-2 cell line revealed non cytotoxicity. Peptide identification using micro-LC–ESI–QTOF-MS/MS. Relevant peptides Leg1: RIKTVTSFDLPALRFLKL, Leg2: RIKTVTSFDLPALRWLKL. (Chang et al., 2011; Heymich et al., 2021a; Heymich et al., 2021b) Chickpea protein (Isoelectric point) Flour Extrusion (at 150 C and screw speed of 240 rpm), and defatted. SGID using method 10.1021/jf900591q The extruded hydrolysate showed a more potent antihypertensive effect than the unextruded one in male spontaneously hypertensive rats (SHRs). The samples were fractionated by ultrafiltration with a cut-off of 10 kDa. (Chávez- Ontiveros et al., 2022) Table 2.2. continues in the next page Chapter 2 — Theoretical Framework 111 Raw Material Pretreatment Hydrolysis conditions Main Results Purification or Peptide Identification Source Albumin from desi chickpea (ICC3761) Albumin fraction extraction from chickpea seeds. Preheated at 80◦C for 5 min Alcalase (0.3 AU/g protein): 50 ◦C, pH: 8.0, 180 min Or SGID using pepsinpancreatin method Both have high antioxidant activities (ABTS and DPPH), as well as hepatoprotective and hypoglycemic effects in BALB-c mice. Only the SGID hydrolysate reduced the in-vivo postprandial hyperglycemia. Both demonstrated anti-inflammatory activity. (Navarro- Leyva et al., 2023) Kabuli chickpea seeds Germination with 2 mg Na2SeO3/100 g of seeds for 2 days, and protein precipitation Alcalase: 60 ◦C, pH: 8.0. Or Pepsin: 37 ◦C, pH: 2.2. Or Trypsin: 37 ◦C, pH: 8.0. E/S ratio 4% w/w, 30, 60, 120 min, inact. at 90 C for 5 min. Pepsin and Trypsin showed the highest antioxidant potential (DPPH, ORAC, Superoxide, etc) and stability with Se. Protein hydrolysates < 10 kDa inhibited collagen degradation after UVA radiation in the HDFa cell line. This study highlights their potential use in cosmetics. Membrane < 10 kDa. (Serrano- Sandoval et al., 2023) Albumin from chickpea seeds and oxovanadium complexes Defatted (w/ petroleum) Subsequential hydrolysis of Papain: pH: 6.0, 12 h. Inactivation; Trypsin: pH: 8.0, 12 h; and Alcalase: pH: 10, 12 h. Inact. at 95 ºC for 10 min. In silico analysis of bio-penetrating properties. Cell inhibitory effects on lung cancer A549 cells using metalchelating peptides. Relevant peptides: ESEFDFR, FDDEKEFR, EENPEEAR, SLSDFR, DFSER, YPGSC, AFSD (437–1864 Da) (Mukhame dov et al., 2021) Table 2.2. continues in the next page Development of Functional Foods for Application to Sport Nutrition 112 Raw Material Pretreatment Hydrolysis conditions Main Results Purification or Peptide Identification Source Kabuli variety chickpeas Protein extraction from chickpeas (isoelectric point), dialyzed for salt removal, defatted, preheated 80ºC for 20 min. Hydrolysis using 1 or 2 enzymes after heat inactivation. Neutrase: pH 7.0, 45°C. Protamex: pH 8.0, 55°C. Alcalase: pH 8.5, 55°C. Papain: pH 7.0, 55°C. E/S ratio 8000 U/g. Inact. at 80ºC for 20 min. After hydrolysis and SGID using the INFOGEST method, 4847 peptides were identified. In silico study revealed DPP-IV inhibitory peptides. Only three peptides were selected for in-vitro evaluation in the CACO-2 cell line, exhibiting significant DPP-IV activity. 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K., Cehun, M., Chronopoulos, A., Robertson, I. K., Nestel, P. J., & Ball, M. J. (2006). Dietary Supplementation with Chickpeas for at Least 5 Weeks Results in Small but Significant Reductions in Serum Total and Low-Density Lipoprotein Cholesterols in Adult Women and Men. Annals of Nutrition and Metabolism, 50(6), 512–518. https://doi.org/10.1159/000098143 Pittaway, J. K., Ahuja, K. D. K., Robertson, I. K., & Ball, M. J. (2007). Effects of a Controlled Diet Supplemented with Chickpeas on Serum Lipids, Glucose Tolerance, Satiety and Bowel Function. Journal of the American College of Nutrition, 26(4), 334–340. https://doi.org/10.1080/07315724.2007.10719620 PubMed. (n.d.). Retrieved March 31, 2024, from https://www.ncbi.nlm.nih.gov/ Quintero-Soto, M. F., Chávez-Ontiveros, J., Garzón-Tiznado, J. A., Salazar-Salas, N. Y., Pineda-Hidalgo, K. V., Delgado-Vargas, F., & López-Valenzuela, J. A. (2021). Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates. Journal of Food Science, 86(7), 2962–2977. https://doi.org/10.1111/1750-3841.15778 Robinson, N. B., Krieger, K., Khan, F. M., Huffman, W., Chang, M., Naik, A., Yongle, R., Hameed, I., Krieger, K., Girardi, L. N., & Gaudino, M. (2019). The current state of animal models in research: A review. International Journal of Surgery, 72, 9–13. https://doi.org/10.1016/j.ijsu.2019.10.015 Development of Functional Foods for Application to Sport Nutrition 126 agente antioxidante y antiinflamatorio exógeno evitando el malestar generado por el síndrome de sobreentrenamiento debido al ejercicio de alta intensidad y larga duración. Acumulativamente, se espera que estos efectos promuevan una mejora en la salud general entre los atletas. Considerando lo expuesto anteriormente, a continuación, se presentan los objetivos individuales: Optimizar la producción de hidrolizados proteicos de garbanzo y evaluar sus propiedades antioxidantes. Producir a escala piloto un hidrolizado proteico de garbanzo seleccionado. Evaluar la actividad biológica del hidrolizado proteico de garbanzo en modelos celulares. Evaluar el potencial efecto beneficioso del consumo de un alimento funcional basado en hidrolizado proteico de garbanzo en humanos. Dichos objetivos se desarrollan a continuación. Objetivo 1: Optimizar y Evaluar las Propiedades Antioxidantes de Hidrolizados Proteicos de Garbanzo Uno de los principales objetivos de esta Tesis fue la optimización de los parámetros de hidrólisis en la producción de los hidrolizados proteicos de garbanzo, y caracterización química de los mismos. Para ello se realizó una batería de 36 hidrolizados obtenidos a partir de proteína concentrada comercial. Se evaluaron 2 enzimas de grado alimentario (Alcalase y Bioprotease) a 3 relaciones Enzima/substrato y a 6 tiempos de hidrólisis. Se realizó una caracterización parcial de las muestras (contenido proteico, grado de hidrólisis, perfil proteico), además de calcular el rendimiento del proceso a escala de laboratorio. Se evaluó la actividad antioxidante de los hidrolizados proteicos de garbanzo Chapter 3 —Hypothesis and Objectives 127 obtenidos. Para ello, se realizaron medidas de actividad antioxidante mediante 3 técnicas diferentes (ORAC, FRAP y TEAC). Este objetivo se encuentra desarrollado en el capítulo 4. Objetivo 2: Producir a Escala Piloto un Hidrolizado Proteico de Garbanzo Para la producción en planta piloto se seleccionó previamente el hidrolizado que mejores caracteres presentara en cuanto a rendimientos y actividad antioxidante, utilizando un análisis de los componentes principales, y que fuera posible extrapolar a gran escala para su producción en planta piloto. Una vez seleccionado el hidrolizado de interés se realizó el escalado a planta piloto. Este objetivo se encuentra desarrollado en el capítulo 4. Objetivo 3: Evaluar la Actividad Biológica del Hidrolizado Proteico de Garbanzo en Modelos Celulares Para la consecución de este objetivo se usaron monocitos previamente inflamados y tratados con el hidrolizado proteico producido en planta piloto. Se evaluó tanto la inocuidad del hidrolizado como su actividad antioxidante, a través de la medida de la actividad de enzimas antioxidantes. Además, se evaluó su funcionalidad como inmunomodulador midiendo niveles y expresión de marcadores como TNF-α, IL-1β, e IL-6, y otros genes envueltos en la modulación de rutas proinflamatorias celulares, como el factor de transcripción NF-kB y el inflamasoma NLRP3. Este objetivo se encuentra desarrollado en el capítulo 5. Development of Functional Foods for Application to Sport Nutrition 128 Objetivo 4: Evaluar el Potencial Efecto Beneficioso del Consumo de un Alimento Funcional Basado en Hidrolizado Proteico de Garbanzo en Humanos Para la consecución de este objetivo se diseñó un protocolo de intervención dietética y se reclutó un grupo de futbolistas amateur integrado por hombres y mujeres. Se realizó un estudio aleatorizado, cruzado y controlado por placebo. Se evaluaron en suero sanguíneo parámetros implicados en estatus antioxidante, status antiinflamatorio y de daño muscular además de la seguridad del producto. Se llevó a cabo un protocolo de actuación exhaustivo y un análisis descriptivo de los datos para observar el comportamiento de las variables y tendencias en los datos. Este objetivo se encuentra desarrollado en el capítulo 6. Chapter 3 —Hypothesis and Objectives 129 3.2. Hypothesis and Objectives It has been evidenced that plant protein hydrolysates and their derived peptides possess numerous bioactive properties such as anticancer, antidiabetic, antihypertensive, antioxidant, and anti-inflammatory activities, among others. This thesis arises from the need for technological advancement in the production of bioactive chickpeaprotein hydrolysates in a more efficient and sustainable manner, and from the necessity to delve deeper into the cellular mechanisms. For this purpose, in addition to cell culture studies, it was proposed to evaluate the effect of a functional food on the reduction of inflammatory markers in athletes. The choice of athletes (amateur football players) was made because this group is subjected to high oxidative stress level due to the high-intensity training, which can lead to overtraining syndrome. This functional food is considered safe, as it is a common ingredient in the food industry and low allergenicity has reported. Chickpeas are a good choice as a natural source of plant protein due to their interesting amino acid profile, and that it is healthy and sustainable product. Additionally, the World Health Organization (WHO) recommends that plant protein intake should constitute 75% of the total protein in a healthy and sustainable diet, as stated in its 2023 document. Therefore, the main hypothesis of this thesis is to develop a chickpea protein hydrolysate with bioactive properties that is suitable for inclusion in the formulation of a sports supplement. This supplement, when applied before and after exercise, will have the potential to serve as an exogenous antioxidant and anti-inflammatory agent, preventing overtraining syndrome. Cumulatively, it is expected that these effects will promote an improvement in the overall health of athletes. Development of Functional Foods for Application to Sport Nutrition 130 Considering the above, the following individual objectives are presented: Optimize and evaluate the antioxidant properties of chickpea protein hydrolysates Produce chickpea protein hydrolysate on a pilot scale Evaluate the biological activity of chickpea protein hydrolysate in cell models Evaluate the potential beneficial effects of consuming a functional food based on chickpea protein hydrolysate in humans These objectives are further developed below. Objective 1: Production and Measurement of Antioxidant Properties of Chickpea Protein Hydrolysates One of the main objectives of this thesis was to optimize the hydrolysis parameters in the production of chickpea protein hydrolysates and their chemical characterization. To achieve this, a series of 36 hydrolysates were produced from commercial protein concentrate. Two food-grade enzymes (Alcalase or Bioprotease) were evaluated at three enzyme/substrate ratios and six hydrolysis times. A partial characterization of the samples was performed, including protein content, degree of hydrolysis, and protein profile, in addition to calculating the yield of the process at the laboratory scale. The antioxidant activity of the chickpea protein hydrolysates obtained was evaluated. Antioxidant activity was measured using three different techniques: ORAC (Oxygen Radical Absorbance Capacity), FRAP (Ferric Reducing Antioxidant Power), and TEAC (Trolox Equivalent Antioxidant Capacity). This objective is developed in Chapter 4. Objective 2: Pilot Scale Production of the Hydrolysate Chapter 3 —Hypothesis and Objectives 131 For pilot plant production, the hydrolysate with the best characteristics in terms of yield and antioxidant activity was selected using principal component analysis. This selection was based on its feasibility for largescale production. Once the hydrolysate of interest was selected, it was scaled up to the pilot plant. This objective is developed in Chapter 4. Objective 3: Measurement of Biological Activity in Cellular Models To achieve this objective, proinflammatory monocytes were treated with the protein hydrolysate produced at the pilot plant. The cytotoxicity and the antioxidant activity were evaluated by measuring the activity of cellular antioxidant enzymes. Additionally, its functionality as an immunomodulator was assessed by measuring the levels and expression of markers such as TNF-α, IL-1β, and IL-6, as well as other genes involved in the modulation of cellular pro-inflammatory pathways, such as the transcription factor NF-kB and the inflammasome NLRP3. This objective is developed in Chapter 5. Objective 4: Evaluation of the Effect of the Functional Food on Volunteers To achieve this objective, a dietary intervention protocol was designed, and a group of amateur football players was recruiting, including men and women. A randomized, crossover, placebo-controlled study was conducted. Blood serum parameters related to antioxidant status, antiinflammatory status, and muscle damage, as well as the safety of the product, were evaluated. A comprehensive action protocol and a descriptive data analysis were carried out to observe variable behavior and data trends. This objective is developed in Chapter 6. Development of Functional Foods for Application to Sport Nutrition 132 133 Chapter 4 – Protein Hydrolysate Optimization (Published work) Production of chickpea protein hydrolysate at laboratory and pilot plant scales: Optimization using principal component analysis based on antioxidant activities Noelia M. Rodríguez-Martín a, José Carlos Márquez-López a, Isabel Cerrillo b, Francisco Millán a, José Antonio González-Jurado c, María-Soledad Fernández- Pachón b, Justo Pedroche a* (*corresponding autor) a Group of Plant Proteins, Instituto de la Grasa-CSIC, 41013 Seville, Spain b Area of Nutrition and Food Sciences, Department of Molecular Biology and Biochemistry Engineering, Universidad Pablo de Olavide, 41013 Seville, Spain. c Physical and Sport Education, Department of Sport and Computer Science, Universidad Pablo de Olavide, 41013 Sevilla, Spain. Received 2 June 2023, Revised 18 August 2023, Accepted 7 October 2023, Available online 18 October 2023, Version of Record 1 November 2023. Food Chemistry Volume 437, Part 1, 30 March 2024, 137707 https://doi.org/10.1016/j.foodchem.2023.137707 Development of Functional Foods for Application to Sport Nutrition 134 Chapter 4 — Protein Hydrolysate Optimization 135 4.1. Resumen Los garbanzos son una fuente rica en nutrientes con una puntuación óptima y alta de aminoácidos esenciales. Para evaluar su potencial como ingrediente en alimentos funcionales, se produjeron 36 hidrolizados de proteína de garbanzo a escala de laboratorio utilizando enzimas de grado alimentario. Se emplearon parámetros que incluyen rendimientos, contenido de proteínas, grado de hidrólisis y actividades antioxidantes para identificar las condiciones más favorables para la ampliación de la producción a nivel de planta piloto mediante un análisis de componentes principales. El hidrolizado seleccionado demostró características destacables: un contenido sustancial de aminoácidos esenciales y proteínas del 67.71%, notables rendimientos de proteína (73.12%) y peso (72.00%), junto con una solubilidad excepcional que supera el 80% y una digestibilidad destacable del 89.50%. Al pasar a proporciones de planta piloto, el hidrolizado conservó su perfil de proteínas atenuado y al mismo tiempo mostró actividades antioxidantes elevadas: Capacidad Antioxidante Equivalente a Trolox 451.0 ± 16.90 µmol/g, Poder Antioxidante Reductor Férrico 29.09 ± 0.64 µmol/g y Capacidad Antioxidante de Radicales de Oxígeno 141.4 ± 5.95 µmol/g, fortaleciendo así su potencial como ingrediente en alimentos funcionales. Los prometedores resultados sugieren que los hidrolizados de proteína de garbanzo pueden aplicarse en alimentos funcionales como coadyuvantes en el tratamiento y prevención de enfermedades crónicas. Development of Functional Foods for Application to Sport Nutrition 142 moisture analyser Ohaus MB45 (Ohaus Corp, Pine Brook, NJ, USA). Ash content was also obtained from the gravimetric method using ignition at 550 °C during 36 h. Fat was extracted in a Soxhlet with hexane during 16 h. Soluble sugars and polyphenols were measured through an ethanolic extraction where glucose standard curves were used for the quantification of soluble sugars (DuBois et al., 1956) and polyphenols were determined using a standard curve of chlorogenic acid (Moores et al., 1948). Both analyses were performed using a DU 640 Spectrophotometer (Beckman Instruments, Fullerton, CA, USA). Total fiber was determined according to the enzymatic-gravimetric procedure described (Lee et al., 1992). 4.4.5. Evaluation of the Degree of Hydrolysis (DH) The degree of hydrolysis (DH) was determined based on the measurement of released α-amino groups resulting from peptide bond cleavage. The α-amino groups react with TNBS, and the absorbance was measured at 420 nm (Adler-Nissen, 1979). A standard curve of L-Leucine was used for quantification, and the percentage of cleaved bonds was calculated. To establish the total number of amino groups (100% DH), a sample of chickpea meal was hydrolyzed at 110 °C for 24 hours in 6 N HCl. 4.4.6. Molecular Profile Elucidation by Fast Protein Liquid Chromatography (FPLC) The protein molecular weight profile was determined using FPLC Akta Purifier 10 (GE Healthcare Bio-sciences AB, Switzerland). A prepacked chromatography column for high-performance size exclusion, Superose 12 10/300 GL, was utilized. The column was calibrated using proteins with known molecular weights, spanning a range of 1 to 300 kDa. Lyophilized samples were reconstituted to a concentration of 10 mg protein/mL in elution buffer (sodium phosphate 0.05 M, sodium chloride 0.15 M, and sodium azide 0.02% w/v) at pH 7.5. The elution Chapter 4 — Protein Hydrolysate Optimization 143 flow rate was set at 0.8 mL/min, and 500 μL of each sample was injected for analysis. The column effluent was continuously monitored at 280 nm. 4.4.7. Antioxidant Activities Evaluation The antioxidant activities of the chickpea protein hydrolysates were assessed using three different assays. Oxygen Radical Antioxidant Capacity (ORAC) Assay: The ORAC assay was conducted following the method described by Ou et al. (2001) with minor modifications. All reagents were prepared in a phosphate buffer (75 mM, pH 7.4). A sample of 50 μL (0.05 mg hydrolysate/mL) was mixed with 100 μL of sodium fluorescein (2.934 mg/L) and incubated at 37 °C for 15 minutes. Subsequently, 50 μL of AAPH (2,2'-Azobis(2- amidinopropane) dihydrochloride) (221.25 mM) was added, and the fluorescence intensity was measured at excitation wavelength (λex) of 460 nm and emission wavelength (λem) of 515 nm every 5 minutes for 120 minutes. The area under the curve was calculated, and the results were obtained from a standard curve using different concentrations of Trolox (2-38 μM). The ORAC values are expressed as μmol Trolox Equivalent/g sample and recorded using a SynergyTM HT-multimode microplate reader (Biotek Instruments, Winooski, VT). Ferric Reducing Antioxidant Power (FRAP) Assay: The FRAP assay was performed according to Delgado-Andrade et al. (2005). In this assay, 280 μL of prewarmed FRAP reagent was mixed with 20 μL of the sample diluted in distilled water (10 mg hydrolysate/mL). The FRAP reagent contained 2.5 mL of a 10 mM TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine) solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3·6H20, and 25 mL of 0.3 M acetate buffer (pH 3.6). The change in absorbance at the maximum wavelength (595 nm) was monitored for up to 30 minutes at 37 °C. The results were obtained from a calibration curve using different Development of Functional Foods for Application to Sport Nutrition 144 concentrations of Trolox (0.1-1.6 mM) and expressed as μmol Trolox Equivalent/g sample, recorded on a microplate reader. Trolox Equivalent Antioxidant Capacity (TEAC) Assay: The TEAC assay was also performed following the procedure described by Delgado- Andrade et al. (2005). Here, 20 μL of diluted sample (0.35 mg hydrolysate/mL in a water/methanol 1:1 mixture) was mixed with 280 μL of diluted ABTS•+ (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid) solution (7 mM ABTS•+ solution in 5 mM phosphate-buffered saline, pH 7.4). The change in absorbance at the maximum wavelength (730 nm) was measured for up to 30 minutes at 30 °C. The results were obtained from a calibration curve using different concentrations of Trolox (0.06-0.28 mM) and expressed as μmol Trolox Equivalent/g sample, recorded on a microplate reader. 4.4.8. Amino Acid Quantification by Ultra High-Performance Liquid Chromatography (UPLC) Ultra-High-Performance Liquid Chromatography (UPLC) was employed to determine the amino acid content in the chickpea protein hydrolysates. A multi-solvent delivery system (Acquity Arc, Waters, USA) was utilized, and a reversed-phase column (XSelect HSS T3 XP, 2.5 µm, Waters, USA) was employed for the separation and identification of the amino acids. The analysis of acidic amino acids was conducted as previously described by Alaiz et al. (1992). In summary, lyophilized samples containing 4 mg of protein were hydrolyzed with D, L-α- aminobutyric acid as an internal standard in 6 N HCl at 110 °C for 24 hours. Subsequently, the samples were derivatized with diethyl ethoxymethylenemalonate. A binary gradient of 25 mM sodium acetate, 0.02% (w/v) sodium azide at pH 6 buffer, and acetonitrile was used for the separation of the amino acids. Tryptophan content was determined after basic hydrolysis following the method by Yust et al. (2004). An isocratic separation was employed with 25 mM sodium acetate, 0.02% (w/v) sodium azide at pH 6 buffer, and acetonitrile (92:8 v/v). To Chapter 4 — Protein Hydrolysate Optimization 145 quantify the amino acid composition of the chickpea protein samples, individual calibration curves were developed for each amino acid using an amino acid standard (Merk, Madrid, Spain) under the same conditions as described above. Data processing and quantification were performed using EMPOWER software (Waters, USA). 4.4.9. Determination of Protein Solubility vs pH To evaluate the solubility of the chickpea protein hydrolysates at different pH values, a 5% (w/v) suspension of the samples was prepared and maintained under stirring at room temperature. The pH of each suspension was then adjusted to different values (pH 12, 10, 8, 6, 4, and 2) using 1 N NaOH and 1 N HCl solutions. Subsequently, an aliquot of 1.5 mL was taken from each pH-adjusted suspension and centrifuged at 8000 rpm for 10 minutes. The resulting supernatants were then dried in an oven at 110 °C for 24 hours. The protein content of the dried samples was measured using an Elemental Microanalyzer TruSpec Micro CHN. Protein solubility was expressed as the percentage of total protein that remained soluble after centrifugation. 4.4.10. In Vitro Protein Digestibility (IPD) The in vitro protein digestibility of the chickpea protein hydrolysates was assessed using the method described by Sindayikengera & Xia (2006) with minor modifications. Briefly, 100 mg of the sample was incubated under continuous stirring with a pepsin dilution (0.2 mg/mL) at 37 °C and pH 2 for 2 hours. Subsequently, the resulting mixtures were neutralized, and pancreatin dilution (0.5 mg/mL) was added, maintaining stirring at 37 °C and pH 8 for another 2 hours. Finally, trichloroacetic acid was added to the samples, followed by centrifugation (10000 rpm, 20 minutes). The protein content of the supernatant was measured using an Elemental Microanalyzer TruSpec Micro CHN. The data were expressed as a percentage of digestible protein following the formula: Protein Content of the Supernatant x 100/ Total Protein Content. Development of Functional Foods for Application to Sport Nutrition 146 4.4.11. Essential Amino Acids (EAAs) and Protein Digestibility- Corrected Amino Acid Score (PDCAAS) Estimation The content of essential amino acids (EAAs) was calculated based on the experimental values of Thr, Cys, His, Lys, Val, Met, Ile, Leu, and Trp, following the guidelines set by FAO/WHO/UNU (2007). The individual values of each essential amino acid were summed to obtain the total essential amino acid content in mg/g of protein for each sample. To calculate the PDCAAS value, the following equation was used: % 𝑃𝐷𝐶𝐴𝐴𝑆 = 𝐸𝐴𝐴𝑆 (𝑠𝑎𝑚𝑝𝑙𝑒) 𝐸𝐴𝐴𝑆 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 × 𝐼𝑃𝐷 (𝑠𝑎𝑚𝑝𝑙𝑒) Where, EAAS (sample) represents the essential amino acid score of the sample (sum of essential amino acids content in mg/g of protein), EAAS (standard) represents the essential amino acid score of a standard protein with high biological value. In this case, the score of whey protein was used as the reference standard (Banaszek et al., 2019), IPD (sample) represents the in-vitro protein digestibility of the sample. 4.4.12. Statistical and Principal Component Analysis (PCA) The data are expressed as arithmetic means with standard deviations (SD). GraphPad Prism Version 8.0.1 software (San Diego, CA, USA) was used to display the data. Statistical analysis was performed using Twoway ANOVA (not repeated measures) with mean, SD, and N=3. Tukey's test was used for multiple comparisons to observe simple effects within columns. A p-value less than 0.05 was considered statistically significant. A PCA was conducted using R software (R Core Team, 2020). The FactorMinerR package was utilized for generating PCA output, utilizing Pearson correlation to establish the data structure. Data visualization was accomplished by creating graphics with the ggplot2-based framework, using the fviz_pca_biplot() function from the factoextra package. This facilitated the creation of biplots representing individuals Chapter 4 — Protein Hydrolysate Optimization 147 and variables. The chickpea protein hydrolysates (CPHs) data were analyzed both collectively and individually for each protease (Bioprotease or Alcalase) employed in the hydrolysis process. 4.5. Results and Discussion 4.5.1. Optimization of Protein Hydrolysis Conditions for Obtaining CPH In this study, the commercial chickpea protein concentrate (CCPC) was used as the substrate for obtaining chickpea protein hydrolysates (CPH). Two food-grade serine endopeptidases, Alcalase 2.4 L and Bioprotease LA-660, were chosen as the proteolytic enzymes for hydrolysis. These enzymes are cost-effective and have broad catalytic activity, making them suitable for producing protein hydrolysates and bioactive peptides from plant-based proteins, including legumes and (pseudo)cereals (Matemu et al., 2021; Paz et al., 2021; Tacias-Pascacio et al., 2020). Various hydrolysis times and enzyme-to-substrate (E/S) ratios were selected to optimize the production of CPH. Data on protein yield (PY) and weight yield (WY) were calculated for all protein hydrolysates (Table 4.1.). The results indicated that the protein content increased by 1.7% at an E/S ratio of 0.05 UA/g enzyme concentration when using Alcalase, while at a ratio of 0.1 UA/g, it decreased by 1.5%, and further decreased by 5.6% at a ratio of 0.3 UA/g concentration. In the case of using Bioprotease, the protein content decreased by 2.2% at a ratio of 0.05 UA/g enzyme concentration, increased by 2.7% at a ratio of 0.1 UA/g, and then decreased significantly by 10.9% at a ratio of 0.3 UA/g concentration. Overall, the protein content tended to decrease with increasing E/S ratio, with a modest increase in PY and WY observed when Alcalase was used. Development of Functional Foods for Application to Sport Nutrition 148 Table 4.1. Protein yield (PY), and weigh yield (WY) of hydrolysates by Bioprotease or Alcalase at different enzyme/substrate ratios and hydrolysis times. 0.05 UA/g protein Bioprotease 0.10 UA/g protein Bioprotease 0.30 UA/g protein Bioprotease Time (min) PY WY PY WY PY WY 5 53.53 49.53 62.47 53.23 72.06 64.97 10 54.31 47.15 66.07 61.78 74.20 68.05 15 61.51 53.18 71.92 61.53 77.59 72.41 30 56.87 53.80 70.58 62.94 83.57 76.87 45 63.90 61.84 73.64 66.22 82.54 79.83 60 67.31 63.67 76.87 66.85 87.14 80.61 0.05 UA/g protein Alcalase 0.10 UA/g protein Alcalase 0.30 UA/g protein Alcalase Time (min) PY WY PY WY PY WY 5 50.08 45.50 56.93 50.85 72.96 65.17 10 52.56 48.18 63.55 56.77 75.81 70.09 15 58.49 52.47 66.61 61.05 80.77 75.35 30 64.60 57.95 72.07 64.38 83.17 78.63 45 68.81 61.21 72.99 66.04 88.70 83.85 60 71.74 64.09 78.75 71.87 92.38 89.34 Data are expressed as%. To assess the hydrolysis efficiency, the degree of hydrolysis (DH) was determined for each protein hydrolysate, which represents the percentage of peptide bonds cleaved (Adler-Nissen, 1979). The results showed that the highest DH for both substrates and enzymes was achieved at an E/S ratio of 0.3 UA/g after 45-60 minutes of hydrolysis. The DH value variance ranged from around 13-16%, and they were directly proportional to the hydrolysis time and E/S ratio, regardless of the enzyme used. Slightly higher DH values were observed in the protein hydrolysates obtained with Bioprotease (32% at 60 minutes and a ratio of 0.3 UA/g) compared to those obtained with Alcalase (27% at 60 minutes and a ratio of 0.3 UA/g). Similar DH values were reported for another chickpea hydrolysate using Alcalase (Clemente et al., 1999) and kiwicha hydrolysates using Bioprotease (Paz et al., 2021). Chapter 4 — Protein Hydrolysate Optimization 149 The optimization of hydrolysis conditions is crucial in obtaining protein hydrolysates with desirable functional and nutritional properties. The chosen E/S ratio and hydrolysis time significantly influenced the protein content and DH of the CPHs. These results will aid in selecting the most suitable conditions for producing CPHs with specific characteristics for various food applications. 4.5.2. The Antioxidant Activities of CPH The antioxidant activities of the chickpea protein hydrolysates (CPHs) were assessed using the TEAC, FRAP, and ORAC assays to identify hydrolysates with better antioxidant potential. The enzymatic action of both Bioprotease and Alcalase leads to the release of small peptides, which may contain functional amino acid residues and exhibit antioxidant activity (Karami & Akbari-adergani, 2019; Tacias-Pascacio et al., 2020; Waseem et al., 2018). The antioxidant activity values are presented in Tables 4.2. and 4.3. for the hydrolysis by Bioprotease and Alcalase, respectively. Generally, there was an increase in antioxidant activities with increasing E/S ratio and hydrolysis time for both enzymes. In particular, the antioxidant activities measured by the TEAC, FRAP, and ORAC assays were higher in all hydrolyzed samples compared to the non-hydrolyzed samples. This suggests that the hydrolysis process resulted in the generation of antioxidant compounds from the chickpea protein. Notably, there were significant differences (p-value < 0.05) in antioxidant activities between CPHs with low versus high degrees of hydrolysis (DH) or short versus long hydrolysis times. The DH values ranged from 16.54% to 33.12% when Bioprotease was used and from 12.46% to 26.41% when Alcalase was used. Development of Functional Foods for Application to Sport Nutrition 150 Table 4.2. Antioxidant activity of chickpea protein hydrolysates by Bioprotease at different enzyme/substrate ratios and hydrolysis times. 0.05 UA/g Bioprotease Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 e 8.73 ± 0.06 b 93.10 ± 7.28 d 5 457.50 ± 18.40 c 23.83 ± 0.36 a 170.00 ± 8.73 bc 10 459.70 ± 13.40 c 23.13 ± 0.83 a 172.30 ± 4.10 bc 15 426.20 ± 9.20 d 23.45 ± 0.43 a 154.90 ± 11.30 c 30 496.30 ± 7.36 ab 24.67 ± 0.92 a 185.40 ± 13.10 ab 45 511.50 ± 16.40 a 23.52 ± 0.94 a 211.60 ± 0.36 a 60 482.90 ± 2.91 bc 24.25 ± 0.18 a 184.20 ± 10.20 ab 0.10 UA/g Bioprotease Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 d 8.73 ± 0.06 c 93.10 ± 7.28 c 5 541.20 ± 0.00 ab 23.37 ± 0.12 b 221.10 ± 12.40 a 10 521.80 ± 4.33 b 23.90 ± 0.75 b 174.30 ± 4.21 b 15 435.80 ± 7.17 c 24.51 ± 1.00 b 202.30 ± 1.00 ab 30 547.00 ± 7.62 ab 24.98 ± 0.87 b 207.00 ± 3.92 a 45 524.20 ± 17.8 b 28.30 ± 1.87 a 228.20 ± 8.64 a 60 559.70 ± 6.69 a 27.21 ± 0.85 a 219.10 ± 3.29 a 0.30 UA/g Bioprotease Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 d 8.73 ± 0.06 d 93.10 ± 7.28 b 5 518.50 ± 23.1 b 24.90 ± 0.17 c 276.50 ± 21.40 b 10 577.50 ± 0.00 a 24.86 ± 0.55 c 290.90 ± 14.30 ab 15 471.50 ± 20.20 c 27.25 ± 0.61 b 296.90 ± 12.90 ab 30 518.50 ± 8.73 b 29.60 ± 0.51 a 310.20 ± 5.63 a 45 501.30 ± 8.08 b 29.67 ± 0.27 a 272.00 ± 23.60 b 60 521.60 ± 9.59 b 27.31 ± 0.37 b 282.00 ± 18.00 ab Values are presented as the means ± SD (n = 3) and expressed as µmol equivalent of Trolox per gram of protein; those values marked with different letters (from “a” to “e”, considering “a” as the highest value) are significantly different between the same column values (p < 0.05). TEAC: Trolox Equivalent Antioxidant Capacity; FRAP: Ferric Reducing Antioxidant Power Assay; ORAC: Oxygen Radical Absorbance Capacity. Chapter 4 — Protein Hydrolysate Optimization 151 Table 4.3. Antioxidant activity of chickpea protein hydrolysates by Alcalase at different enzyme/substrate ratios and hydrolysis times. 0.05 UA/g Alcalase Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 c 8.73 ± 0.06 f 93.10 ± 7.28 e 5 446.50 ± 6.87 ab 14.32 ± 0.19 e 243.90 ± 0.13 c 10 429.30 ± 2.69 b 15.17 ± 0.10 d 268.20 ± 12.70 b 15 457.20 ± 11.50 a 18.02 ± 0.40 b 262.70 ± 12.80 b 30 450.30 ± 23.20 ab 16.69 ± 0.45 c 197.30 ± 12.00 d 45 471.20 ± 26.70 a 18.53 ± 0.34 ab 312.20 ± 25.90 a 60 470.60 ± 4.79 a 19.20 ± 0.27 a 317.30 ± 18.80 a 0.10 UA/g Alcalase Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 c 8.73 ± 0.06 d 93.10 ± 7.28 b 5 429.90 ± 18.30 b 15.03 ± 0.30 c 280.70 ± 14.40 a 10 463.60 ± 8.30 a 17.69 ± 0.37 b 293.70 ± 15.90 a 15 476.90 ± 3.81 a 18.50 ± 0.19 a 294.20 ± 14.20 a 30 473.80 ± 4.40 a 18.39 ± 0.29 ab 309.40 ± 12.30 a 45 468.70 ± 10.50 a 19.08 ± 0.19 a 305.20 ± 17.30 a 60 487.40 ± 1.35 a 18.67 ± 0.00 a 298.50 ± 16.20 a 0.30 UA/g Alcalase Time (min) TEAC FRAP ORAC 0 299.30 ± 2.91 d 8.73 ± 0.06 d 93.10 ± 7.28 c 5 459.70 ± 11.60 ab 18.97 ± 0.65 c 272.50 ± 6.06 b 10 469.80 ± 3.57 a 18.72± 0.00 c 326.50 ± 12.00 a 15 450.20 ± 7.78 ac 21.99 ± 0.43 b 328.60 ± 21.40 a 30 453.70 ± 12.90 ac 22.27 ± 0.50 b 345.50 ± 32.30 a 45 438.50 ± 1.26 bc 23.15 ± 0.21 a 342.40 ± 14.90 a 60 430.50 ± 5.05 c 23.55 ± 0.43 a 334.10 ± 23.90 a Values are presented as the means ± SD (n = 3) and expressed as µmol equivalent of Trolox per gram of protein; those values marked with different letters (from “a” to “f”, considering “a” as the highest value) are significantly different between the same column values (p < 0.05). TEAC: Trolox Equivalent Antioxidant Capacity; FRAP: Ferric Reducing Antioxidant Power Assay; ORAC: Oxygen Radical Absorbance Capacity. 4.5.3. Principal Component Analysis (PCA) A Principal Component Analysis (PCA) was conducted to identify the optimal conditions for obtaining the best chickpea protein hydrolysate in a cost-efficient and functional manner. The variables used in the PCA were DH, protein content, weight yield (WY), protein yield (PY), and Development of Functional Foods for Application to Sport Nutrition 254 6.5.6. Descriptive Analysis of Inflammation, Antioxidant and Muscle Damage Markers Other parameters related to antioxidant status, inflammation, and muscle damage caught our attention (Table 6.10.). Specifically, IL-6 and IL-8 had a mean concentration of 33.20 ± 29.70 pg/mL and 272.88 ± 488.25 pg/mL with high variations, respectively. In the case of the serum antioxidant status, the FRAP mean was 719.10 ± 91.17 µmol/L, the mean ABTS/TEAC was 8185.44 ± 1080.12 µmol/L, while the ORAC value had a mean of 10208.54 ± 1267.86 µmol/L. GPx and GR enzyme levels had means of 7.83 ± 1.80 U/g Hb and 1.03 ± 0.14 U/L, respectively. LDH averaged 209.72 ± 52.74 U/L, and CK had a mean of 281.51 ± 244.25 U/L. Finally, CRP levels averaged 1.74 ± 4.75 mg/L. Chapter 6 — Supplementation on Volunteers 255 Table 6.10. Values of Other Biochemical Parameters of female and male volunteers. n mean sd median trimmed mad min max range skew kurtosis se IL-6 199 33.20 29.70 22.19 28.46 12.82 9.98 257.27 247.29 3.88 22.44 2.11 IL-8 199 272.88 488.25 73.00 140.71 45.96 0.00 1946.00 1946.0 2.56 5.46 34.61 FRAP 181 719.10 91.17 719.41 719.30 89.98 493.27 940.17 446.90 -0.03 -0.34 6.78 ABTS/TEAC 181 8185.44 1080.12 8225.49 8217.06 1065.44 5555.26 11057.69 5502.4 -0.15 -0.23 80.28 ORAC 181 10208.54 1267.86 10284.35 10208.77 1218.41 6924.80 13720.48 6795.6 -0.04 0.17 94.24 GPx 179 7.83 1.80 7.50 7.77 2.04 4.22 12.14 7.91 0.30 -0.71 0.13 GR 179 1.03 0.14 1.03 1.03 0.10 0.00 1.34 1.34 -2.09 15.84 0.01 LDH 199 209.72 52.74 200.00 204.27 34.10 106.00 586.00 480.00 3.11 17.78 3.74 CK 195 281.51 244.25 199.00 235.80 151.23 21.00 1445.00 1424 2.02 4.83 17.49 RCP 198 1.74 4.75 0.70 0.88 0.59 0.00 58.60 58.60 9.23 102.81 0.34 Interleukin 6 (IL-6), Interleukin 8 (IL-8), Ferric Reducing Ability of Plasma (FRAP), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Trolox Equivalent Antioxidant Capacity (TEAC), Oxygen Radical Absorbance Capacity (ORAC), Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Lactate Dehydrogenase (LDH), Creatine Kinase (CK), C-Reactive Protein (CRP) Development of Functional Foods for Application to Sport Nutrition 256 6.5.7. Principal Component Analysis (PCA) For All Variables A PCA was conducted for 41 normalized variables (Fig. 6.3. and 6.4.). In this case, the PCA performance is not fully appropriate, as despite passing Bartlett’s test of sphericity, it did not pass the KMO test, yielding a value of 0.551. This value suggests potential inadequacies in the model and indicates that its suitability is mediocre. Nevertheless, the analysis was performed to observe the complete dataset and to facilitate the identification of outliers. This analysis showed that the parameter values corresponding to the start of the study are dispersed throughout the graph. Data is more represented on the left side of Dim1, but the high dispersion indicates significant internal variability. The parameter values samples corresponding to the washout period form a diagonal between the upper left and lower right quadrants, passing through the upper right quadrant. While the 95% confidence circle shows a tendency to cluster between the upper and lower left quadrant, the high dispersion indicates significant internal variability similar to the group of variables measured at the beginning of the study. In contrast, the parameter values from individuals who took the supplement and the placebo shakes are concentrated near the origin of the graph, indicating lower internal variability and greater homogeneity in the measured characteristics within these groups. The vectors in the PCA variables plot (Fig. 6.3.) indicate that variables with the longest vectors, such as Hemoglobin, Hematocrit, Red Blood Cells (on the right), and Insulin, Leukocytes or Neutrophils (on the left), contribute significantly to the variance explained by the first principal component (Dim1). On the other hand, variables such as MCHC, MCH, MCV (in the upper quadrants) or RCDW (in the lower quadrant) have vectors mainly pointing towards the upper part, indicating their influence on the second principal component (Dim2). Additionally, variables with vectors in similar directions, such as MCV and MCH, are Chapter 6 — Supplementation on Volunteers 257 positively correlated, while those with vectors in opposite directions, such as MCV and Insulin, show a negative correlation. Figure 6.3. Principal Component Analysis (PCA) Biplot of 41 Variables. The PCA biplot displays the relationship between the variables on the first two principal components, Dim1 and Dim2, explaining the variance of the data. Each arrow represents a variable from the serum measure of all the subjects in this study (including female and male population). The direction and length of the arrows indicate its contribution and correlation with the principal components. The color gradient represents the contribution of each variable, with warmer colors indicating higher contributions. The comparison between the four groups (Start, Supplement/H30BIO, Placebo/Maltodextrin, and Washout), as observed in Fig. 6.4., shows that the variables most influencing the measurements at the start of the study were Hemoglobin, Hematocrit, and Red Blood Cells, while Development of Functional Foods for Application to Sport Nutrition 258 washout samples were influenced by Neutrophils, Leukocytes, and Insulin. Moreover, the supplement and the placebo intakes appear in the middle of the graph, making it difficult to identify the variables they are influenced by. Apparently, the supplement samples are slightly displaced towards the upper right quadrant (with variables like ABTS, FRAP, CREAT, Total Protein), while the placebo samples are directed towards the opposite quadrant (with variables like Glucose, Platelets, Eosinophils) suggesting that these variables are the most influential on the data. Figure 6.4. Principal Component Analysis (PCA) Biplot of Individuals per all variables (41). The PCA biplot displays the projection of individuals on the first two principal components Dim1 and Dim2). Each point represents an individual, colored and shaped according to their treatment group: Start (blue squares), H30BIO or supplement (red circles), Malto (maltodextrin) or placebo (green triangles), and Wash (purple crosses). The ellipses represent the 95% confidence intervals for each group, indicating the spread and central tendency of the individuals within each group. Chapter 6 — Supplementation on Volunteers 259 6.5.8. PCA For Muscle Damage, Proinflammatory and Antioxidant Variables On the other hand, proinflammatory, antioxidant status, and muscle damage markers and related variables were analyzed, including IL-8, IL- 6, CRP, GPx, GR, ABTS, ORAC, FRAP, LDH, CK, creatinine, Alkaline Phosphatase, GOT/ALT, GPT, GGT, and myoglobin. A light different distribution was observed as Fig. 6.5. and 6.6. shows. In this model, both the Bartlett’s test of sphericity (sig. < 0.001) and KMO measure (0.699), indicates the adequacy of conducting PCA (IBM Corp, 20247). Figure 6.5. Principal Component Analysis (PCA) Biplot of 16 Selected Variables. The PCA biplot displays the relationship between the proinflammatory, antioxidative status and muscle damage variables on the first two principal components, Dim1 (23.9%) and Dim2 (11.1%). Each arrow represents a variable from the serum measure of all the subjects in this study (including female and male population). The direction and length of the arrows indicating the principal Development of Functional Foods for Application to Sport Nutrition 260 components contribution and correlation, and the color gradient represents the contribution of each variable (warmer colors indicate higher contributions). The analysis showed that the blood samples corresponding to the start of the study are dispersed throughout the upper quadrants of the graph, mostly represented in the upper right quadrant, and show high correlations with GOT, GGT, GPT, CK and lower correlations with ORAC and IL-6 variables (Fig. 6.5. and 6.6.). Notably, the samples after placebo intake show a tendency to cluster in the origin of the graph, slightly displaced towards the upper right quadrant, and correlates with the same variables as the start group. The samples after the intake of the supplement cluster in the origin, suggesting that no correlations with proinflammatory or antioxidant variables were found. After the washout period, (Fig. 6.6.), samples were represented in the opposite quadrant (lower left quadrant), but dispersed mostly in the lower quadrants, and correlated negatively with the same variables. Figure 6.6. Principal Component Analysis (PCA) Biplot of Individuals per Inflammatory, Antioxidant Status, and Muscle Damage Variables (16). The PCA biplot displays the projection of individuals on the two main principal components. Each point represents an individual, colored and shaped according to their treatment group: Start (blue squares), H30BIO or supplement (red Chapter 6 — Supplementation on Volunteers 261 circles), Malto (maltodextrin) or placebo (green triangles), and Wash (purple crosses). The ellipses represent the 95% confidence intervals for each group. 6.5.9. General Decision Tree Analysis (DTA) The general DTA analysis (Fig. 6.7.), which includes both male and female volunteers, examines the variable "Treatment", divided into four groups: Start, Supplement, Placebo, and Washout. The behavior of each group across the different nodes formed in the analysis model is explained below. In the initial stage of the study (Start), the IL-6, and GOT variables predominantly explained the initial behavior. The first subset was determined by IL-6 levels, with splits at IL-6 < 27.94 (Node 1) or IL-6 > 27.94 (Node 2). Node 2 primarily explained the behavior of the Start samples, where IL-6 > 27.94 (N=35), GOT > 25.50 (N=20). For the “supplement” and “placebo” groups, more variability was observed in the initial classification. Both the “placebo” and “supplement” groups split subjects almost evenly between Node 1 and Node 2. Notably, in Node 1, IL-6 concentrations less than 27.94 pg/mL for those taking the supplement (20) and placebo (16) and higher variability was observed in this node including variables like myoglobin, IL-8 and GGT. In Node 2, subjects taking the supplement had IL-6 from 27.94 to 62.75 pg/mL (N=16), showing GOT < 25.50 (N=18) and myoglobin < 26.15 (N=16), while those on placebo showed higher variability. After the washout period, the behavior of these samples was primarily explained by Node 1 (N=75). The samples were characterized by IL-6 > 27.94 and myoglobin < 31.45 (N=61). For those subjects that showed higher myoglobin an improvement of IL-8 < 62.75 (N=11) was observed. Development of Functional Foods for Application to Sport Nutrition 262 Figure 6.7. General decision tree diagram illustrating per general population. Fig. 6.7. continues in the next page. Chapter 6 — Supplementation on Volunteers 263 Figure 6.7. Continuation. General decision tree diagram illustrating per general population continuation. Development of Functional Foods for Application to Sport Nutrition 270 Table 6.11. Importance of Independent Variables for DTA models. Variable General (%) Male (%) Female (%) IL-6 100.00 100.00 91.80 Myoglobin 43.20 59.80 48.20 LDH 36.30 35.70 100.00 GOT 35.50 56.80 15.70 GPT 24.30 35.70 - CK 24.00 19.80 42.30 AlkP 22.90 22.50 15.70 GGT 21.80 16.10 18.60 IL-8 21.40 8.60 15.70 ORAC 16.50 24.30 - GPx 16.40 22.30 40.90 GR 15.90 41.10 53.90 ABTS 15.60 41.10 14.60 CRP 14.10 54.60 23.80 CREAT 12.70 10.50 16.00 FRAP 12.60 11.20 - Growth method: CRT; Dependent variable: Treatment 6.6. Discussion In this chapter, we evaluated the adequacy of the study design, assessed the food safety of the CPH supplement as well as explored the trends and more influential biomarker considering the CPH intake and taking account the gender. 6.6.1. Protocol Evaluation The study design protocol was chosen with a focus on both scientific robustness and practical feasibility. Randomized, blinded, and crossover nutritional intervention minimizes potential biases, thereby ensuring the validity of the results. Additionally, the inclusion of a placebo group allowed for effective control of external variables, ensuring that observed changes in blood parameters could be more confidently attributed to the H30BIO supplement. Chapter 6 — Supplementation on Volunteers 271 6.6.1.1. Adequacy of Placebo-Control Maltodextrin was chosen as the placebo because it maintains a texture similar to the CPH in the reconstituted beverage, although the protein beverage was slightly more bitter and had a milky appearance. On the other hand, maltodextrin is a rapidly digestible carbohydrate that provides an energy source without contributing proteins or other bioactive nutrients. Additionally, by including maltodextrin also in the protein formulation, we ensured that observed differences in results could be more directly attributed to the protein of the supplement, facilitating comparison between the supplement and the placebo results (Baker et al., 2015; Hartley et al., 2022). As existing scientific literature shows, maltodextrin is widely used in sports supplementation studies. Thus, the results would be apparently uncomplicated to compare and validate with other studies (Candow et al., 2006; Moreno-Pérez et al., 2018). Other studies, such as (Samsamikor et al., 2020), suggest using the base whole protein as placebo instead of maltodextrin. However, in our case, concentrated chickpea protein had poor solubility and tended to sediment at the bottom, making it unwieldy to prepare and consume. 6.6.1.2. Volunteers Adherence to the Study Of the 48 initial eligible volunteers, 81% completed the first phase and 75% completed the entire study. These facts suggest that the study protocol was generally well-tolerated by the participants. Moreover, the causes of discontinuation were not directly related to the intake of the supplement or potential adverse effects. During the execution of the study, the action that ensured participant adherence included weekly visits. In these visits, numerous questions from the volunteers were addressed, demonstrating their interest in the study. Questions regarding sports nutrition were also answered, emphasizing the importance of maintaining good dietary habits for Development of Functional Foods for Application to Sport Nutrition 272 general health, as well as for improving athletic performance and recovery. This approach allowed for close monitoring of the participants and ensured consistency in the administration of treatments throughout the study duration. 6.6.2. Anthropometric Characteristics The descriptive analysis revealed distinct anthropometric profiles for male and female participants as expected with typical gender-specific physiological variations (Lewis et al., 1986). In the case of body fat% we obtained a mean of 24.5 ± 4.5 per female and 12.4 ± 2.6 per male subjects. In comparison with values studied in elite football players, body fat percentage was in range for both genders, 25.9 ± 2.0% and 12.2 ± 3.1%, in male and female respectively (Milanovic et al., 2012; Wittich et al., 2001). Variability in values may be due to playing position. The body fat% varied from 6.1 to 19.5% in professional male football players considering the player position (Wittich et al., 2001). The female BMI calculated had a mean value (23.36 ± 3.25 kg/m2) somewhat higher than in elite female players (21.6 ± 0.2 kg/m2) while the median was 22.51 kg/m2, closely value to the observed in the elite players (Milanovic et al., 2012), deducing that a few subjects have light overweight. However, BMI is not a completely adequate marker for athletes (Jeukendrup & Gleeson, 2019). On the other hand, the male BMI calculated had a similar mean value (23.35 ± 1.48 kg/m2) to that observed in elite male football players 24.4 ± 1.3 kg/m2 (Wittich et al., 2001). 6.6.3. Dietary Evaluation The collection of dietary data through food frequency questionnaires and 24-hour recalls throughout the study represents an innovative approach that enhances the significance of this study, as most studies in Chapter 6 — Supplementation on Volunteers 273 athletes do not accurately collect this information (Capling et al., 2017). Including dietary data in the analysis algorithm for future analyses can help adjust the model, allowing for a more rigorous analysis. In this sense, dietary patterns can influence the final results of the study. The results from the food frequency questionnaire and 24-hour recalls indicate that amateur football players should improve their diet. This population had an average caloric intake of 2,343 kcals for males and 2,228 kcals for females. However, due to the intense physical activity, their requirements were 3,385 kcals and 2,625 kcals, respectively, which was higher than their actual intake. these results should be taken into account with caution due to some lim-itations in energy intake estimation. Some authors have suggested that energy intake is often underestimated and it would actually be higher than reported (Black et al., 2019). In the dietary record inaccurate estimations of the actual amount of food consumed and the full list of ingredi-ents occur frequently (Martín-Moreno and Gorgojo, 2007). On the other hand, the consumption of carbohydrates was low. Carbohydrates are crucial in the diet of athletes as they provide the primary source of energy needed for high-intensity training and competition (Amawi et al., 2024; Kanter, 2018). Poor dietary patterns are related to overtraining syndrome (Cadegiani & Kater, 2019; Cheng et al., 2020; San-Millán, 2019). In this sense, aberrant creatine kinase levels have been also associated with poor nutrition in overtrained athletes, reinforcing the hypothesis that the possible muscle dysfunction is due to a maladaptation in those athletes consuming lower amounts of carbohydrates compared to healthy athletes, as demonstrated by the EROS-PROFILE study (Cadegiani & Kater, 2019). In our study not only PCA showed higher correlation with this parameter associated with the Start group, but also it was observed to have highmedium importance (42.30%) in the female model of DTA, suggesting that poor nutrition may lead to overtraining syndrome more Development of Functional Foods for Application to Sport Nutrition 274 pronounced in the female subset population (which also consume the least amount of carbohydrates). Notably, volunteers often follow a nutritional pattern that poses cardiovascular risks. Their dietary patterns typically include high saturated fat, low unsaturated fat, and high cholesterol, which are associated with the consumption of ultra-processed foods. These types of foods displace the intake of nutrient-dense foods rich in vitamins, minerals, and fiber, such as legumes, fruits, vegetables, and whole grains. Professional athletes must fully satisfy their nutritional needs through high-quality foods in the correct amounts (Amawi et al., 2024; Holzman, 2016). Thus, these findings highlight the necessity for appropriate nutritional counseling for amateur football players throughout the season. It has been seen that the level of knowledge in nutrition influences the quality of the diet (Amawi et al., 2024; Holzman, 2016). Nonetheless, the dietary pattern did not show significant differences throughout the study, thereby eliminating the influence of changes in dietary patterns on the obtained results. 6.6.4. Hematological, Biochemical Parameters, and Food Safety The hematological analysis indicated normal ranges of red blood cells, hemoglobin, hematocrit and leukocytes during all stages of treatment, thus confirming the safety of both formulations placebo (maltodextrin) and protein supplement (H30BIO). Additionally, the adherence of the participants confirmed that the supplement is well tolerated by all subjects, which do not report to have stomach upset, diarrhea, nausea, or other symptom that may be associated with the supplement consumption. Chapter 6 — Supplementation on Volunteers 275 6.6.5. Inflammation, Antioxidant and Muscle Damage Markers In total population, the biomarkers GOT/ALT, CK, LDH, and myoglobin, related with muscle damage, were more associated with the “Start” group as we observed in the PCA. On the other hand, the general DTA model showed that IL-6 and myoglobin were the most contributing parameters defining the variable “treatment”, and more related to the “Start” group. All these parameters are associated to inflammation and muscular damage deducing that the subjects initiate the study in a proinflammatory stage (Cadegiani & Kater, 2019; Lee et al., 2017; San- Millán, 2019). Moreover, these parameters were lower when the placebo or the supplement were administered. Therefore, future data analyzes will be focused on detailing and establishing possible differences between the benefits of H30BIO supplement with respect to the intake of maltodextrin. Additionally, two different subsets of populations were observed with supplement intake, one with lower levels of IL-6 but higher levels of myoglobin, and the other with higher levels of IL-6, but a notable lowering of myoglobin levels. In contrast, the placebo intake, as presented with higher variability, might be affected by carry-over effect, or some subjects will benefit from the maltodextrin/water intake in physiological recovery (Shi & Ye, 2024). The carry-over effect will be analyzed to discard the order of beverage consumption: placebosupplement or supplement-placebo. A similar trend was observed in male subset. The elevated importance of IL-6, CRP, and myoglobin parameters in the DTA indicates an acute inflammatory and muscle damage response to intense exercise associated to overtraining syndrome as previously reported, mostly for the “Start” group (Cadegiani & Kater, 2019; Lee et al., 2017; San-Millán, 2019). In contrast to the general DTA model, the male DTA showed two subgroups in the comportment of the parameters after the H30BIO supplement intake introducing three new variables (CRP, GR, ABTS). The Development of Functional Foods for Application to Sport Nutrition 276 first one showed lower levels of IL-6, and higher levels of myoglobin and GR. On the other hand, the subset with higher IL-6 was related to obtaining lower ABTS values, but lower myoglobin and CRP. Thus, decreasing inflammation but having a controversial value in IL-6. In the case of female subjects, a few differences were observed. This group was highly defined by LDH and IL-6 in the DTA, in line with the same inflammatory responses (Lee et al., 2017; San-Millán, 2019). However, the high internal variability among the supplement, placebo, and start groups does not reveal any different trend. In contrast, the washout group indicated that subjects consuming either the placebo or supplement after the washout period experienced an improvement in the IL-6 and LDH. Despite some authors indicated that female cyclists can improve training intensity and fast recovery using carbohydraterestricted supplement with proteins (Oosthuyse et al., 2021), our findings indicate that the female footballers may have more benefits from the alternating use of placebo and supplement. This highlights the necessity to study specific supplements for this population (Tarnopolsky, 2000). However, the size of the female subset is a limiting factor, and carry-over effects are still there, which need to be discarded in future analysis. 6.6.6. The Signaling Regulation Through IL-6 The soluble cytokine IL-6 becomes interest due to the pleiotropic effect in inflammation, immune response, and hematopoiesis (Tanaka et al., 2014). IL-6 also promotes healing of skeletal muscle, can increase protein synthesis and lead inflammation recovery (Howard et al., 2020). This cytokine classified as myokine, have a role in the short-term energy allocation during physical activity and few studies elucidates that IL-6 deficiency may have an impact in muscular recovery and adaptation (Kistner et al., 2022). In this sense, other study about a plant-based beverage (NPN_1), observed an improvement in the stench recovery and muscle markers, and a transient increase in IL-6 expression levels Chapter 6 — Supplementation on Volunteers 277 after the NPN_1 intake and a decrease expression after 2, 48 and 72 hours of the intake (Kerr et al., 2023). The interest in our supplement is that the IL-6 measure taken at the final stage of the study, it was defined by lowers levels of IL-6. On the other hand, myoglobin is a muscle hemoprotein that allows the storage and transport of oxygen to the mitochondria during muscle contraction. This function is crucial for ATP production through oxidative phosphorylation under aerobic conditions. It is considered an important marker of skeletal muscle stress and damage. In this sense, some studies have shown that this marker experiences an increase after physical activity and returns within 24 hours after recovery due to its rapid kinetics (Haller et al., 2023; Saita et al., 2023). Interestingly, myoglobin is the second parameter in importance in the DTA model maintaining the opposite trend with IL-6. As IL-6 is also a major regulator of myogenesis and are related to energy allocation during physical activity in response to stress (Howard et al., 2020; Kistner et al., 2022), it may also relate to muscle repair (Kistner et al., 2022). Hence, it is possible to observe myoglobin allocation associated with IL-6 expression, which could be a new line of research to evaluate. Due to the IL-6 role in energy production during physical activity. It is mainly produced by skeletal muscle and is dependent on the lactate production during exercise, with an increase of LDH for the increased energy requirements (de Melo Madureira et al., 2022). Therefore, in the case of female subjects, our results suggest an increase in the energy production due to the modulation of energy allocation towards the muscle which is important in athlete recovery (Kistner et al., 2022). Regarding results of cellular experiments in chapter 5, IL-6 signaling pathway is closely related to TNF-α. It has been observed an inhibitory effect of monocyte production of TNF-α, particularly modulating TNF receptor (Kistner et al., 2022). This effect has been observed in several studies indicating the importance of muscular induced IL-6 and TNF-α Development of Functional Foods for Application to Sport Nutrition 278 regulation to protect against insulin resistance (Pedersen et al., 2001). Moreover, due to the energy allocation mitochondria may have important implications in mechanisms activated by IL-6. Hence, through data obtained from chapters 5 and 6 we can suggest that the consumption of the chickpea protein hydrolysate improves energy allocation during physical activity via IL-6. However, more experiments will need to confirm this hypothesis. 6. 7. Limitations and Future Directions While the preliminary results are promising, there are several limitations to consider. The sample size, although calculated to be adequate, may still be insufficient to detect significant changes in some variables, such as ABTS/TEAC and CRP. On the other hand, the study's crossover design, while robust, may introduce carryover effects related to, for example, the length of the washout period, that need to be carefully managed and examined in future analyses. The 4-week duration in intervention phases also might be insufficient. On the other hand, there may be variability in protocol adherence by participants, which could have influenced the results. Future studies should also aim to explore the molecular mechanisms underlying the observed changes in inflammatory and antioxidant markers, potentially incorporating metabolomic approaches (San- Millán, 2019). The data analysis techniques used suggest that organizing the values into quartiles could be highly beneficial in being able to obtain different levels of the measured biomarkers and identify overtrained subjects. This would help detect subgroups that might benefit more significantly from the supplement. However, adaptations and careful validation will be necessary to ensure the robustness and reliability of the results due to the small size of population. Chapter 6 — Supplementation on Volunteers 279 Additionally, incorporating dietary data into the analysis could be crucial since the nutrients and dietetic bioactive compounds have a profound impact on the metabolism and could influence the effectiveness of the supplementation. In addition to it, we can control for potential confounding variables and better understand the relationship between diet, supplementation, and health/overtraining outcomes. As indicated by the preliminary results, there are significant differences in how male and female participants respond to the supplement. Gender differences should also be taken into account and evaluated in greater depth. Gender-specific analyses will provide more tailored insights and could lead to the development of more effective, genderspecific nutritional interventions. 6.8. Conclusion The preliminary findings obtained in this study support the hypothesis that chickpea protein hydrolysate (H30BIO) supplementation could serve as a strategy to mitigate exercise-induced oxidative stress and inflammation and muscle damage in male football players. This effect probably is conducted by IL-6 modulation. However, in the case of females, responses might be long term after the intake of supplement or placebo indicating the necessity to perform different nutritional strategies in female athletes. Future studies might confirm these findings. Hence, the CHP supplement intake may act as an adaptive food which modulates IL-6 activity controlling energy allocation through myoglobin and the antioxidative system depending on individual characteristics. These results suggest that the hydrolysate also maintain ergogenic activity. Development of Functional Foods for Application to Sport Nutrition 286 Tavano, O. L. (2013). Protein hydrolysis using proteases: An important tool for food biotechnology. Journal of Molecular Catalysis B: Enzymatic, 90, 1– 11. https://doi.org/10.1016/j.molcatb.2013.01.011 Vioque, J., & Millán, F. (2005). Los hidrolizados proteicos en alimentación: Suplementos alimenticios de gran calidad funcional y nutricional. Centro Tecnológico Nacional de La Conserva y Alimentación, 26(96–102). Wittich, A., Oliveri, M. B., Rotemberg, E., & Mautalen, C. (2001). Body Composition of Professional Football (Soccer) Players Determined by Dual X-Ray Absorptiometry. Journal of Clinical Densitometry, 4(1), 51–55. https://doi.org/10.1385/JCD:4:1:51 Xue, Z., Wen, H., Zhai, L., Yu, Y., Li, Y., Yu, W., Cheng, A., Wang, C., & Kou, X. (2015). Antioxidant activity and anti-proliferative effect of a bioactive peptide from chickpea (Cicer arietinum L.). Food Research International, 77, 75–81. https://doi.org/10.1016/j.foodres.2015.09.027 Zhang, T., Li, Y., Miao, M., & Jiang, B. (2011). Purification and characterisation of a new antioxidant peptide from chickpea (Cicer arietium L.) protein hydrolysates. Food Chemistry, 128(1), 28–33. https://doi.org/10.1016/j.foodchem.2011.02.072 Zhu, Y., Lao, F., Pan, X., & Wu, J. (2022). Food Protein-Derived Antioxidant Peptides: Molecular Mechanism, Stability and Bioavailability. Biomolecules, 12(11), 1622. https://doi.org/10.3390/biom12111622 287 Chapter 7 – General Conclusions Development of Functional Foods for Application to Sport Nutrition 288 Chapter 7 — Conclusions 289 Conclusiones El garbanzo es un alimento versátil, nutritivo y sostenible, por lo que puede contribuir a la economía circular. Se considera una fuente proteica elevada y de calidad. La hidrólisis de la harina de sus subproductos permite la obtención de péptidos bioactivos para ser utilizados en alimentos funcionales generando un nuevo producto de alto valor. Por otra parte, la revisión de literatura existente sobre hidrolizados de proteína de garbanzo puso en énfasis las principales propiedades biológicas de los hidrolizados proteicos de garbanzo: actividad hipolipidémica, antioxidante, antiinflamatoria, antibiótica/antifúngica, anticancerígena e hipotensiva. Teniendo en cuenta estos antecedentes, se ha evaluado en el presente trabajo la optimización del proceso de hidrólisis proteica y el potencial efecto inmunomodulador, antiinflamatorio y antioxidante de los hidrolizados en un modelo de cultivo celular y en un ensayo en humanos, llegando a las siguientes conclusiones generales en concordancia con los objetivos planteados. El análisis de componentes principales (ACP) de los parámetros medidos de 36 hidrolizados (rendimiento en peso, rendimiento en proteína, porcentaje proteico, grado de hidrólisis y actividad antioxidante) identificó al hidrolizado H30BIO (tiempo de hidrólisis 30 minutos utilizando la enzima bioproteasa) como uno de los mejores a todos los niveles destacando en los valores FRAP y TEAC. El escalado a planta piloto replicó no solo el perfil molecular del hidrolizado, sino también la actividad antioxidante, evaluada con los ensayos FRAP y TEAC, aunque el valor ORAC se redujo. Por otro lado, se observó una alta solubilidad y una elevada digestibilidad in vitro del hidrolizado obtenido, determinando que posiblemente pueda permear la barrera intestinal y llegar al órgano diana para ejercer su actividad antioxidante. 1 2 Development of Functional Foods for Application to Sport Nutrition 290 El H30BIO indujo una producción de ROS y NO en el modelo celular. No obstante, el conjunto de los resultados obtenidos sugiere que el hidrolizado dota de protección a la mitocondria, además de tener un efecto inmunomodulador dirigido tanto por NF-kB como por el inflamasoma NLRP3, concluyendo que la generación de ROS y NO es debida a procesos celulares adaptativos que podrían estar relacionados con TNF-α. El ensayo en deportistas reveló que gran parte de la población de estudio presenta una inflamación de base, lo cual podría estar relacionado con la intensidad del ejercicio y con la inadecuada alimentación que mantenían de forma general. El suplemento basado en H30BIO no solo demostró ser seguro e inocuo, sino que su consumo puede estar relacionado con procesos adaptativos a nivel celular modulado por la citoquina IL-6. Estos datos correlacionan con una resolución de la inflamación y la posible movilización de energía en la célula. No obstante, se necesitan análisis complementarios para confirmarlo. 3 4 Chapter 7 — Conclusions 291 Conclusions The chickpea is a versatile, nutritious, and sustainable food, thus contributing to the circular economy. It is considered a high-quality protein source. The hydrolysis of its by-product flour enables the production of bioactive peptides for use in functional foods, generating a high-value new product. Furthermore, the literature review on chickpea protein hydrolysates emphasized the main biological properties of chickpea protein hydrolysates: hypolipidemic, antioxidant, anti-inflammatory, antibiotic/antifungal, anticancer, and antihypertensive activities. Based on this background, the present study evaluated the optimization of the protein hydrolysis process and the potential immunomodulatory, anti-inflammatory, and antioxidant effects of the hydrolysates in a cell culture model and a human trial, reaching the following general conclusions in accordance with the objectives set. Principal Component Analysis (PCA) of the parameters measured from 36 hydrolysates (weight yield, protein yield, protein percentage, degree of hydrolysis, and antioxidant activity) identified hydrolysate H30BIO (30 minutes hydrolysis time using bioprotease enzyme) as one of the best at all levels, highly correlated with FRAP and TEAC activities. The pilot plant scale-up not only replicated the molecular profile of the hydrolysate but also the antioxidant activity, evaluated with FRAP and TEAC assays, although the ORAC value was reduced. Additionally, high solubility and high in vitro digestibility of the obtained hydrolysate were observed, indicating that it might permeate the intestinal barrier and reach the target organ to exert its antioxidant activity. 1 2 Development of Functional Foods for Application to Sport Nutrition 292 The H30BIO demonstrated ROS and NO production in the cell model. However, the overall results suggest that hydrolysate provides mitochondrial protection, as well as an immunomodulatory effect driven by both NF-kB and the NLRP3 inflammasome, concluding that the generation of ROS and NO is due to adaptive cellular processes that might be related to TNF-α. The athlete trial revealed that a significant portion of the study population exhibits baseline inflammation, which could be related to the intensity of exercise and generally inadequate nutrition. The H30BIO-based supplement not only proved to be safe and non-toxic but its consumption might be related to adaptive cellular processes modulated by the cytokine IL-6. These data correlate with inflammation resolution and possible energy mobilization within the cell. However, complementary analyses are needed to confirm it. 3 4 293 294 295 Agradecimientos Me gustaría concluir este trabajo expresando mi gratitud a todas las personas que han estado en mi vida, ya sea de manera permanente o transitoria, ya que a ellas les debo la persona que soy hoy en día. En primer lugar, a aquellos que ya no están con nosotros pero que han sido fundamentales en mi vida. A mis abuelos y a mi tío, quienes siempre me brindaron los mejores consejos, me enseñaron a crecer con felicidad, a terminar todo lo que comienzo y me inculcaron que el esfuerzo es la base de todo. Os echo de menos. A mi prima mayor, porque, aunque me veías como un referente académico, siempre aprendí más de ti que tú de mí. Siempre estaréis en mi corazón. A mis padres, gracias por darme la libertad de explorar todo lo que despertaba mi curiosidad. Agradezco profundamente los valores y la educación que me habéis brindado. Gracias también a mis tíos y tías por su contribución en mi formación, mis valores y mis inquietudes, por ser amigos y, en muchas ocasiones, ejercer como segundos padres. Sabéis que tengo un tarrito de sal de colores para cada uno de vosotros. A mi hermano, porque tu mera existencia te convierte en una de las personas que más quiero en el mundo. Eres una persona maravillosa. A mis primos y primas (todos), porque somos una familia extensa e intensa, siempre muy unida a pesar de los kilómetros. También os quiero mucho, Cris, Toñi, Luis, Vane, Ojito Derecho, Fausto, y las versiones mini, Ingrid y Bruno. A Dani, gracias por tu paciencia infinita. Gracias por ser mi apoyo constante, por entenderme y por compartir conmigo este intenso camino. Tu amor y comprensión han sido fundamentales para alcanzar esta meta. Te quiero. A mi familia no humana, porque sin importar lo que ocurra, siempre están ahí para mí. A mi familia elegida, mis nutris por el mundo, por permanecer siempre presentes a pesar del paso del tiempo. A mis compañeros-amigos de laboratorio, José Carlos, Ana y Rocío, con vosotros hasta el fin del mundo y más allá. Sois personas excepcionales y con un corazón enorme. A mis agregadas Marta y Victoria, ojalá haber empezado el hábito de la “desayunasión” con vosotras mucho antes. A mis compis de despacho, Juanma y Aída, porque tener tanta conexión con alguien es increíble. A los grandes corazones que han pasado por mi despacho: Angy, Reme y Ángela A. A las nuevas adquisiciones, Rosario y Blanca, os queda mucho y poco a la vez, así que haced cuantos más amigos mejor. En general, a todos los predoctorales del Instituto de la Grasa, gracias por los momentos de risas y por participar en mis videos y mis locuras: Marta B., Mónica, Enrique, Alejandro, Fran, Mercedes, Laura, Jesús y José María. A mi “Cork Family”, porque siendo españoles y estando fuera, nos convertimos en familia. No sé qué habría sido de mí sin vosotros. Gracias por todos los momentos, los viajes, las tortillas de patatas y las pintas: Adrián, Ángela, Franco, David, Cristian, Antonio, Inés, Soham, y especialmente a Nieves y María. Sois muy grandes. Development of Functional Foods for Application to Sport Nutrition 302 APPENDIX (Ethics committee) 303 Development of Functional Foods for Application to Sport Nutrition 304