Wine grape pomace, composed of skins, seeds, stems, and pulp, is the major byproduct of the winemaking industry. While this byproduct can be diverted towards other agricultural uses, such as compost or animal feed, the environmental burden and poor animal digestibility of the material hinders the sustainability and effectiveness of these low-value mitigation strategies. Since wine grape pomace contains valuable bioactive compounds, especially phenolics, there remains an opportunity to valorize this material to benefit human health with the development of innovative food, beverage, cosmetic, and supplement applications. The extraction of wine grape pomace phenolics typically relies on the use of hazardous solvents like ethanol or methanol, which requires subsequent downstream processing steps for use in food-grade products. To present an eco-friendly alternative, this thesis focuses on the design of green extraction methods that improve phenolic extractability and support the in vitro antioxidant activity of grape pomace extracts while using water as the only solvent under optimized extraction conditions.

In Chapter 1, an overview of the recent advances and research gaps in green extraction technologies is discussed with a focus on the large-scale feasibility of these methods to support the potential for commercial adoption. In addition, the compositions of various winemaking byproduct fractions (pomace, leaves, lees, vinasse, and wastewater) are highlighted to showcase the diversity of components available for the development of value-added products.

In Chapter 2, the aqueous extraction process (AEP) is explored as an environmentally-friendly strategy for enabling the release of phenolics from the grape cell-matrix without the use of conventional solvents. A series of experiments, using a central composite rotatable design paired with a kinetic study, was used to identify the concurrent effects of pH, solids-to-liquid ratio, temperature, and time on the total phenolic content (TPC) of the grape pomace extracts. To reduce water consumption without compromising phenolic extractability, a two-stage countercurrent method was employed, which recirculates the extraction processing fractions and increases the concentration gradient to drive phenolic diffusion. This technique provides an economically-viable and environmentally-friendly alternative to conventional solvent extraction methods.

The findings from Chapter 2 (i.e., the role of alkaline conditions in phenolic extraction) guided the design of enzyme- and microwave-assisted extractions as outlined in Chapter 3. Enzyme specificity and alkaline conditions enable the degradation of ester- and ether-linkages between phenolics, structural proteins, and cell-wall carbohydrates within the grape cell-matrix, while microwave radiation causes rapid temperature and pressure changes that can rupture the cell. Multiple stepwise screening experiments were performed to select the optimal conditions for maximizing TPC while reducing total enzyme concentration, water usage, and extraction time. The structural changes to the grape cell-matrix, as illustrated by scanning electron microscopy, represent the successful cellular disintegration caused by the synergistic effects of enzymatic hydrolysis, microwave heating, and intracellular pressure on promoting phenolic extractability.

The series of studies herein provides new approaches to extracting phenolics from wine grape pomace. Notably, the starting material used in these studies represents a non-conventional winemaking process using red wine grapes (Vitis vinifera L. cv. Cabernet Sauvignon) to produce white wine. The use of red wine grape pomace collected prior to fermentation ultimately affected the phenolic composition of the grape pomace extracts, as detailed in Chapters 2 and 3 with the abundance of flavonol glycosides, which represent high residual sugars in the matrix. This finding emphasizes the influence of upstream winemaking conditions on the downstream phenolic profiles and potential biological properties of the extracts. In addition, this study offers support for the use of alkaline treatments for the extraction of phenolics from wine grape pomace, which is not yet widely reported due to the use of acidic conditions in conventional ethanol and methanol extractions. These insights, along with further work on scaling-up the microwave and enzyme-assisted extraction process, illuminate the possibilities for green extraction methods to revitalize agricultural byproducts while boosting human health, supporting a circular economy, and promoting environmental sustainability.

Interest in plant-based food products has risen significantly due to strong health, environmental, and social arguments. To supply this demand, cost-effective and efficient extraction processes need to be developed for plant-based substrates. Being a high source of proteins, starch, and fiber, chickpeas can be subjected to sustainable processing strategies to generate added value compounds for food, feed, and fuel applications. To date, there is limited information about the impact of key extraction parameters on extraction efficiency and functionality of chickpea proteins. In addition, current chickpea research has been conducted primarily with the use of flammable/neurotoxic hexane to produce defatted chickpea flour for subsequent extraction of proteins, which has raised health, environmental, and consumer concerns. The goal of this study was to develop aqueous (AEP) and enzyme-assisted extraction processes (EAEP) without flammable solvents that rely on the use of mechanical treatments, water, and specific enzymes to improve the extractability and functionality of oil, proteins, and carbohydrates from chickpea flour. Enzyme-based processes were developed to efficiently extract lipids, proteins, and carbohydrates from chickpea flour, enhance the solubility and digestibility of the extracted protein, reduce flatulence-related oligosaccharides, and release a more diverse pool of oligosaccharides in the extracts. Because high water usage is a major drawback, a two-stage countercurrent extraction process was developed, resulting in the reduction of nearly 60% of the water used in the extraction process and increase in the overall process extractability. The results presented herein signifies a starting point for the development of a mechanistic understanding of the impact of key extraction parameters on structural protein and carbohydrate modifications that can impact key functional and biological properties of chickpea extracts.

The rising global population, coupled with environmental stresses induced by climate change, is exerting extreme pressure on production systems globally, driving the quest for sustainable sources of macro-and micronutrients intended for industrial applications. Macroalgae, or seaweeds, are macroscopic marine algae that hold promise as a sustainable biomass resource with a wide range of potential industrial applications. Their composition, wide availability, and growth-associated environmental benefits (i.e., carbon sequestration, support to fishery habitats, and reduced dependence on freshwater, arable land, and fertilizer) make them of particular interest. 

These marine organisms contain a range of desirable compounds with the potential for use in human nutrition, animal feed, production of bio-stimulants or fertilizers for plant growth, biotherapeutic, cosmetic, and food applications. Included in these compounds are carbohydrates (laminarin, fucoidans, alginate), lipids, proteins, and phenolics, which exhibit functional attributes such as emulsification and gel formation properties, as well as bioactive properties such as antioxidant, antihypertensive, and antidiabetic effects. Full utilization of macroalgae’s potential hinges on the development of sustainable bio-guided downstream processing strategies which make use of structure and functionality as the benchmark to develop processes capable of both maximizing the extractability of its diverse compounds and safeguarding the functional and biological attributes inherent in the algae extracts. The major goal of this research project was to uncover the effects of pivotal processing conditions (i.e., biomass-to-water ratio, temperature, pH, time) and various extraction methods (i.e., aqueous, enzymatic, and microwave-based extractions) on the extractability, structural composition, and functional/biological properties of the major compounds of the giant kelp species Macrocystis pyrifera, typically found in the Pacific Ocean. The overarching aim was to develop effective and sustainable extraction methods founded on the interplay between structure and functionality to produce algae compounds with the desired properties.

Chapter 1 delves into the current state of the brown macroalgae industry and prevailing extraction methods. It encompasses discussions on environmentally sustainable practices employed in cultivation, harvesting, and industrial integration. This chapter also delves into the intricate structural makeup of brown macroalgae and the multifaceted functional and biological attributes inherent in their compounds. The final section of the chapter offers an in-depth overview of the cutting-edge methodologies employed in the extraction of compounds from macroalgae. 

In Chapter 2, the aqueous extraction process (AEP) and enzyme-assisted aqueous extraction process (EAEP) are explored as sustainable, environmentally friendly strategies for extracting the diverse compounds within the macroalgal matrix. A series of experiments beginning with the AEP investigating pH, biomass-to-water ratio (BWR), time, and temperature were used to guide the EAEP. Based upon yield of various components of interest (laminarin, fucoidan, alginate, protein, phenolics), extraction conditions were selected for more in depth analysis looking into their structure (amino acid composition, phenolic profile, monosaccharide and oligosaccharide analysis, alginate characterization by FTIR) and bioactivity (antioxidant, antidiabetic, antihypertensive). These analyses revealed the mechanisms by which enzymes enhance the degradation of the macroalgal matrix, thereby increasing the extractability of intracellular components (i.e., laminarin, intracellular proteins and peptides), and resulting in the production of extracts with higher antihypertensive activity. 

Chapter 3 builds upon the results of Chapter 2 by investigating the impact of integrating microwave processing with the AEP and EAEP. Through a series of stepwise experiments, favorable microwave-assisted extraction (MAE) and microwave-enzyme-assisted extraction (MEAE) conditions were determined based upon yields. Dipole-interactions caused by the microwave resulted in rapid breakdown of the macroalgal matrix resulting in high yields with extractions as short as 15 minutes. As in chapter 2, these favorable conditions underwent more in-depth analysis. 

Conventional macroalgae processing strategies are deemed unsustainable because they use hazardous solvents. The series of studies detailed in this report outlines the potential of sustainable processing strategies for giant kelp, an important step for maintaining the environmentally friendly status of these marine organisms. Of special note is the ability of the MAE to produce comparable extracts (i.e., similar yield and activity) in 15 minutes, in contrast to the AEP, which requires 6 hours. This highlights the potential for industrial scale use, as it enables multiple extractions to be performed daily. Additionally, these studies shine light on the composition of Macrocystis pyrifera, providing more detail than previously seen in literature. While this giant kelp may be low in phenolics, its other functional and bioactive components render it highly intriguing. Of note is the high antidiabetic activity of all extracts. These insights, along with future work on scaling up the extraction strategies discussed herein, underscore the potential of sustainable and bio-guided downstream processing strategies to introduce this sustainable feedstock to industries worldwide.

Colostrum is a nutrient-dense secretion produced by the mammary glands for several days after parturition. In the dairy industry, colostrum is often produced surpassing the calf’s needs and is subsequently considered a surplus, representing an opportunity for further utilization. Because of its high protein concentration, which includes immunoglobulins, colostrum has a higher viscosity than mature milk, posing challenges to downstream processing. While excess liquid colostrum can be pasteurized and dried into colostrum powder for both animal-rearing and human consumption, these heat-dependent processing methods reduce the nutritional potential of the bioactive components. Further, colostrum is prone to bacterial contamination due to its high nutrient content, underscoring the importance of proper processing to salvage this valuable resource. While colostrum has long been recognized as essential for the health and development of newborn calves, there has been growing interest in its application to human health. Immunoglobulins have received the most extensive research attention among the components of bovine colostrum. Nevertheless, there are other small bioactive components like oligosaccharides (OS) and peptides that have gained recognition. To date, most commercial processing of bovine colostrum has primarily focused on whole colostrum preservation. However, these existing techniques rarely consider the loss of bioactivity in the whole colostrum or the potential enhancement of bioactivity through purification strategies or alternative processing methods. Therefore, this thesis presents the development of scalable processing strategies to improve the recovery of bioactive OS and peptides for enhanced in vitro biological activity from bovine colostrum and its byproducts.Chapter 1 provides an overview of current commercial-scale processing technologies for bovine colostrum. It also introduces scalable strategies to recover and enrich for various bioactive components. In addition, this chapter delves into the nutrient-rich composition of colostrum, encompassing lipids, protein, carbohydrates, and peptides, to underscore the diversity of components with bioactive potential as well as their human health applications. Chapter 2 evaluates the use of a hollow fiber ultrafiltration membrane to efficiently recover small bioactive components (e.g., OS and peptides) from bovine colostrum whey. Hollow fiber membranes can be utilized in applications with viscous material, like colostrum whey, and are generally operated in the laminar region, thereby reducing energy consumption compared to other configurations. A series of experiments, using sequential design, was utilized to identify the effects of feed flow, transmembrane pressure, and method of diafiltration on the permeate flux and selective permeation of OS and peptides. The goal was to maximize the recovery of OS and peptides while minimizing protein permeation and reducing water use. The optimal filtration conditions were utilized to produce whey permeate with high peptide and OS content for subsequent concentration, fractionation, and characterization of their composition and biological properties in the proceeding chapters (Chapters 3 and 4). Chapter 3 explores the use of pilot-scale nanofiltration membranes as a scalable, environmentally friendly strategy for concentrating small bioactive components, OS and peptides, from the bovine colostrum whey permeate produced by the conditions defined in Chapter 2. A central composite rotatable design (CCRD) was utilized to identify the impact of membrane pore size and material, feed flow, and transmembrane pressure on the permeate flux and recovery of OS and peptides by two nanofiltration membranes. The optimal filtration conditions for each membrane were utilized to concentrate whey permeate, and the efficiency of the filtration was evaluated through resistance calculations and overall retention of the target molecules. The recovered materials (rich pools of OS and peptides) were then evaluated for in vitro prebiotic potential and antioxidant, antihypertensive, and antidiabetic properties. This work provides an efficient and effective method for concentrating small bioactive components from colostrum whey permeate. Furthermore, it demonstrates promising health applications for these compounds when used in combination. The methods optimized for colostrum whey permeate in this work can also be applied to whey permeate derived from mature milk, a substantial stream produced during the whey protein concentration process, commonly disposed of as animal feed. The impact of fractionation by membrane filtration to selectively enrich for OS or peptides from bovine colostrum whey permeate is detailed in Chapter 4. Two tight ultrafiltration membranes were individually optimized by using CCRDs to understand the role of membrane chemistry, pore size, feed flow, and transmembrane pressure, similar to Chapter 3. However, this work focused on developing filtration conditions that could fractionate OS from peptides rather than recovering both components in the same pool as described above. The differences in in vitro prebiotic activity and antioxidant, antihypertensive, and antidiabetic properties of the recovered fractions elucidated the distinct biological role of each component (OS or peptides) and paves the way for future development of commercially scalable technologies to recover OS or peptide fractions for targeted health applications. Chapter 5 explores the use of another industrially viable processing strategy, enzymatic hydrolysis, on bovine colostrum and mature milk protein fractions to generate bioactive peptides. While proteins can be bioactive, the utilization of proteases can generate peptides that may exhibit greater bioactivity. Through a series of sequential experiments, the impact of milking time (colostrum vs. mature milk), substrate (curd vs. whey), enzyme type, and hydrolysis time on degree of hydrolysis and in vitro bioactivity (antihypertensive and antioxidant capabilities) was evaluated. To assess the role of peptide size on bioactivity, select hydrolysates were fractionated using a 3 kDa centrifugal filter and each fraction (permeate and retentate) was re-evaluated for antihypertensive and antioxidant capacity, confirming the distinct impact of peptide size and purity on bioactivity. In this collection of studies, we present the applications of commercially scalable processing approaches to isolate small bioactive components from dairy streams, specifically bovine colostrum and its byproducts. The starting materials utilized in these studies, bovine colostrum and mature milk, possess the same components yet in different proportions. The differences in component concentration, in addition to batch variation, are discussed in Chapters 4 and 5, and confirm the importance of studying different matrices while developing strategies with broad applicability. Additionally, this work underscores the value of harnessing bovine colostrum and developing industrially viable processing strategies to enrich for and utilize individual components for human health benefits, reframing colostrum from an excess stream with limited animal applications to a rich starting material for producing nutraceutical ingredients for human health applications. These insights, along with further investigation into scalable purification and extraction strategies, highlight the opportunities to repurpose food byproducts and industrial streams as alternatives to conventional pharmaceuticals. This approach holds potential to improve human health in an environmentally sustainable manner.