Unlocking the health enhancing potential of grape marc through chemical and microbial analysis of its oligosaccharides and phenolic compounds

Grape marc, or pomace, is the largest waste product of wine production with over 1.2 tons produced in the 2019 crush alone. The marc is the combination of grape skins, seeds, and stems removed from the juice with pressing. Although grape marc is currently underutilized primarily as compost and animal feed, initial studies identified its potential to act as a functional food ingredient to improve the gut microbiome and overall health. This thesis compiles the work of a multi-lab approach to identify the potentially bioactive oligosaccharides and phenolics in chardonnay marc and its individual components, how to isolate chardonnay marc oligosaccharides and phenolic compounds for individual analysis, and the potential bioactivity and health benefits of chardonnay marc.Chapter 1 describes the chemical characterization of chardonnay marc, its seed and seedless fractions, a seed extract, and unripe chardonnay grapes. This included determining their gross compositions (i.e., protein, lignin, fat, carbohydrates, polysaccharides), phenolic contents, and oligosaccharide profiles. The gross compositions were as expected with the seeds containing more protein, fat, and polysaccharides than the seedless marc which had more carbohydrates and sugars. Phenolic compounds were abundant throughout the samples with the seed extract possessing the highest concentration. The individual phenolics varied with each fraction with most abundant phenolics being (-)-gallocatechin in the marc and seedless marc and (-)- epicatechin in the seeds and seed extract. Thirty-six distinct oligosaccharides with three to nine degrees of polymerization and eleven distinct monosaccharide subunits were discovered within the chardonnay marc samples. Each fraction had a unique oligosaccharide profile however overlap occurred such as with some hexose and hexose-pentose oligosaccharides present in all samples. Chardonnay marc oligosaccharide and polyphenol separation and purification is essential to assess their individual potential health benefits. Chapter 2 evaluates six separation methodologies for chardonnay marc oligosaccharide and phenolics purification: C18 and PGC SPE, C18 and PGC SPE with dialysis, C18 and DPA-6S SPE, C18 and HLB SPE, PVPP absorption, and PVPP absorption and C18 SPE. Oligosaccharide purification with PVPP and C18 SPE produced the most distinct confirmable oligosaccharides through NanoChip QToF mass spectrometry and had minimal phenolic contamination. Applying C18 and HLB SPE enabled the confirmation of the largest oligosaccharides (DP 8) and had the lowest phenolic content. Chardonnay marc phenolics were best isolated by washing loaded C18 with 40% methanol and PVPP with 70% acetone. Using C18 with 40% methanol generated the highest yields of (-)-epigallocatechin gallate, vanillic acid, (-)-gallocatechin gallate, and (-)-epicatechin but almost no gallic acid. Desorbing phenolic compounds from PVPP with 70% acetone produced high yields of gallic acid, (-)-epigallocatechin, (-)-gallocatechin, and (+)-catechin. Further improvement to chardonnay marc oligosaccharide identification was made through pairing a Dionex HPIC system with Q Exactive HF-X hybrid quadrupole-Orbitrap MS. This new analysis identified three new oligosaccharides, increased isomer separation, and corrected three oligosaccharides confirmed using LC NanoChip QToF MS. Several published studies have begun analyzing the potential beneficial health effects of consuming grape marc. Chapter 3 performs a literature review of these in vitro, GI tract simulation, small animal, livestock, and human studies in addition to investigating the potential functionalities of isolated chardonnay marc oligosaccharides and phenolics. In vitro studies illustrate grape marc’s prebiotic potential as it increased the growth of several commensal Bifidobacterium and Lactobacillus strains and prevented the growth of pathogenic strains including some in the Enterobacteriaceae family. When livestock consumed grape marc, they experienced increased growth of commensal bacteria and decreased populations of pathogenic bacteria in their intestines. Additionally, the livestock had improved health and meat quality through decreased lipid and protein oxidation. Furthermore, mice and rats who consumed grape marc had increased gut microbiome complexity and decreased obesity related illnesses. A human clinical trial, however, did not find chardonnay seed phenolics to have cardioprotective effects. We performed initial in vitro analysis of isolated chardonnay marc oligosaccharides and phenolics and found the oligosaccharides acted as a carbon source for commensal bacteria but not pathogenic bacteria and that the phenolics suppressed Gram positive pathogen growth but not Gram negative pathogen growth

Uncovering the promising role of grape pomace as a modulator of the gut microbiome: An in-depth review.

Grape pomace is the primary wine coproduct consisting primarily of grape seeds and skins. Grape pomace holds immense potential as a functional ingredient to improve human health while its valorization can be beneficial for industrial sustainability. Pomace contains bioactive compounds, including phenols and oligosaccharides, most of which reach the colon intact, enabling interaction with the gut microbiome. Microbial analysis found that grape pomace selectively promotes the growth of many commensal bacteria strains, while other types of bacteria, including various pathogens, are highly sensitive to the pomace and its components and are inactivated. In vitro studies showed that grape pomace and its extracts inhibit the growth of pathogenic bacteria in Enterobacteriaceae family while increasing the growth and survival of some beneficial bacteria, including Bifidobacterium spp. and Lactobacillus spp. Grape pomace supplementation in mice and rats improves their gut microbiome complexity and decreases diet-induced obesity as well as related illnesses, including insulin resistance, indicating grape pomace could improve human health. A human clinical trial found that pomace, regardless of its phenolic content, had cardioprotective effects, suggesting that dietary fiber induced those health benefits. To shed light on the active components, this review explores the potential prebiotic capacity of select bioactive compounds in grape pomace