Exploring Alternative Methods for Extracting Bioactive Phenolics from Winemaking Byproducts

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

Conversion of Food Waste into 2,3-Butanediol via Thermophilic Fermentation: Effects of Carbohydrate Content and Nutrient Supplementation

Fermentation of food waste into 2,3-butanediol (2,3-BDO), a high-value chemical, is environmentally sustainable and an inexpensive method to recycle waste. Compared to traditional mesophilic fermentation, thermophilic fermentation can inhibit the growth of contaminant bacteria, thereby improving the success of food waste fermentation. However, the effects of sugar and nutrient concentrations in thermophilic food waste fermentations are currently unclear. Here, we investigated the effects of sugar and nutrients (yeast extract (YE) and peptone) concentrations on 2,3-BDO production from fermenting glucose and food waste media using the newly isolated thermophilic Bacillus licheniformis YNP5-TSU. When glucose media was used, fermentation was greatly affected by sugar and nutrient concentrations: excessive glucose (\u3e70 g/L) slowed down the fermentation and low nutrients (2 g/L YE and 1 g/L peptone) caused fermentation failure. However, when food waste media were used with low nutrient addition, the bacteria consumed all 57.8 g/L sugars within 24 h and produced 24.2 g/L 2,3-BDO, equivalent to a fermentation yield of 0.42 g/g. An increase in initial sugar content (72.9 g/L) led to a higher 2,3-BDO titer of 36.7 g/L with a nearly theoretical yield of 0.47 g/g. These findings may provide fundamental knowledge for designing cost-effective food waste fermentation to produce 2,3-BDO

Revitalizing Unfermented Cabernet Sauvignon Pomace Using an Eco-Friendly, Two-Stage Countercurrent Process: Role of pH on the Extractability of Bioactive Phenolics

As the major byproduct of the winemaking industry, grape pomace remains an untapped source of valuable bioactive phenolic compounds. This study elucidated the optimal aqueous extraction parameters for maximizing phenolic extractability, while avoiding the use of harsh conventional solvents and limiting water usage, from Cabernet Sauvignon grape pomace in which the red grape was processed for white wine. In the single-stage aqueous extraction process (AEP), the concurrent impact of pH (2.64–9.36), solids-to-liquid ratio (SLR, g pomace/mL water) (1:50–1:5), and temperature (41.6–58.4 °C) on the total phenolic content (TPC) of Cabernet Sauvignon pomace was evaluated alongside a kinetic study (15–90 min). Optimal single-stage extraction conditions (pH 9.36, 1:50 SLR, 50 °C, 75 min) guided the development of a two-stage countercurrent extraction process (pH 9.36, 1:10 SLR, 50 °C, 75 min) to further reduce water consumption without compromising overall extractability. The countercurrent process reduced fresh water usage by 80%, increased the TPC of the extracts by 18%, and improved the in vitro antioxidant activities (ABTS and ORAC) of the extracts. Untargeted metabolomics enabled the identification of a diverse pool of phenolics, especially flavonol glycosides, associated with grape pomace, while further phenolic quantitation detected improvements in the release of commonly bound phenolics such as ferulic acid, p-coumaric acid, syringic acid, and protocatechuic acid in alkaline extracts compared to the ethanolic extract. This investigation provides an efficient, eco-friendly extraction strategy suitable for applications in functional food, beverage, nutraceutical, and cosmetic industries

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field