The first is an on-farm loss caused by cluster thinning to reduce berry variability during ripening, a practice presumed to improve wine quality. Cluster thinning at veraison is a common practice performed in California wine grape cultivation, and the timing is dependent on variety, vineyard location, and cultivation practices. The second by-product is grape marc, which consists of grape skins, stems, seeds, and pulp accumulated either after extracting the juice to ferment white wine or after fermentation and pressing for red wine . Several studies on red and white wine grape varieties have shown that concentrations of total phenolic content, -catechin, -epicatechin, procyanidin dimers and trimers, and proanthocyanidins of wine grapes would generally decrease and then stabilize between veraison and harvests . Thus, thinned at veraison are considered rich sources of flavanols. Nonetheless, thinned clusters are traditionally made into verjus, sour grape sauce, and unripe grape syrup in European and Middle East countries or simply left behind in many vineyards as soil fertilizers in spite of the increased production costs and lost yields . The health-promoting properties of flavanol monomers, an important subgroup of the dietary polyphenols, particularly -catechin and -epicatechin, and their oligomeric procyanidins, have been extensively studied in cocoa and chocolate . Epidemiology studies have suggested that the consumption of flavanol-rich diets and plant extracts is associated with reducing risk of cardiovascular disease , lowering blood pressure, especially in pre-hypertensive and hypertensive individuals, and enhancing their physical activity . Cocoa oligomeric procyanidins were found to be effective at preventing weight gain, development of glucose intolerance, and insulin resistance during a long-term high-fat feeding study by Dorenkott et al. . Lotito and Fraga demonstrated in an in vitro study, dutch bucket for tomatoes that the addition of -catechin and -epicatechin would delay lipid oxidation, and α-tocopherol and β-carotene depletion in human oxidized plasma induced by a radical generator.

The biological activities of -epicatechin and -epicatechin-containing foods are of particular interests in recent years because -epicatechin causes multiple actions that may provide beneficial synergy for cardiovascular and neuropsychological health . It is also worth mentioning that a predominance of -catechin, rather than the more bio available -catechin , was found in commercial chocolate samples. -Catechin is the naturally occurring form in foods such as berries and cacao beans, however, the Dutching process converts cocoa flavanols to the -catechin enantiomer . Grapes are known for their rich polyphenols including abundant -catechin, -epicatechin, and procyanidins. In an evaluation of anti-platelet activity of grape marc extracts, the ethanolic extract with catechin, epicatechin, and quercetin being the most abundant phenolic compounds was capable of inhibiting platelets aggregation in a wide range of agonist concentrations . Another recent study on the digested fractions of seedless red and white wine grape marc revealed that all digested fractions prevented the hyperglycemic actions in the cell viability and nitric oxide /reactive oxygen species balance, suggesting the protective effects of grape marc products on the vascular endothelial barrier function . Holt et al. also conducted a comprehensive literature review on using chardonnay marc, a plant-based natural product, as a new model for upcycled co-products in food applications and comparing with considerable clinical data generated from cocoa products on human cardiometabolic health in the context of healthy dietary patterns. Thus, it is logical to explore upcycling solutions for flavanol-rich thinned clusters in food applications, especially in cocoa-based products that have been perceived as flavanol-rich by consumers. The upcycling of wine-making by-products in food applications has been generating more interest in recent years with the main focus on using dried grape marc as a bulking agent to change textural and rheological properties in foodstuffs such as cookies , soft candies , and chocolate spread . In a chocolate ice cream feasibility study conducted by Soukoulis and Tzia , grape molasses was used as a potential sucrose substitution that exhibited the best chocolate color and mouth-coating ability in the final product.

Bolenz and Glöde evaluated the processing aspects and the impact on chocolate properties like particle size, total phenolic content, and sensory perception of milk chocolate enriched with grape marc and grape seed flour. However, concentration data on bio active flavanols such as -epicatechin are beyond the study scope. When cocoa-based products are analyzed for their flavanol content, individual ingredients, such as thinned clusters in various forms , are generally not separated from the matrix to gain insights on the individual contribution from each ingredient. Our study utilized improved ultra-performance liquid chromatography-fluorescence detector methods that provided new reference information on health-promoting flavanol monomers -catechin and -epicatechin, and procyanidins of thinned clusters alongside cocoa flavanols. The idea of incorporating grape thinned grape clusters into cocoa-based food products as functional ingredients opens new doors to upcycle underutilized grapevine by-products and to improve cocoa-based products with potentially more health-promoting and positive sensory properties with fewer calories.Fresh Chardonnay and Pinot noir thinned clusters were collected at veraison from the Los Carneros American Viticultural Areas in Sonoma County in mid-August 2019. The clusters were then categorized into light and dark for Chardonnay, and red and green for Pinot noir, respectively, based on berry colors. To gain more in-depth understanding of the flavanol composition of the thinned clusters, the seed and seedless fractions were manually separated, freeze-dried, and milled as described in Sinrod et al. .Flavanols and procyanidins of grape thinned grape clusters and cocoa powders were analyzed and reported following a single laboratory validated UPLC-FLD method by Bussy, Hewitt, et al. and Bussy, Ottaviani, and Kwik-Uribe with modifications. Cocoa powders were defatted three times by sonicating 5 g powder in ~45 mL hexane at 50°C for 5 min each time and completely dried in a fumehood prior to extraction. Defatting was skipped for grape thinned clusters due to low-fat content of seedless samples and concerns of phenolic degradation during extensive hot hexane extraction for seed fractions.

For subsequent extraction of flavanols and procyanidins, 0.05 g defatted Acticoa™ cocoa powder, 0.2 g defatted Mullica™ cocoa powder, and 0.2 to 0.25 g thinned clusters were dissolved in 5–10 mL acetone:water:acetic acid , sonicated at 50°C for 5 min, and centrifuged at 5000 rcf for 10 min, respectively. Supernatants were further cleaned on Oasis PRiME MCX cartridges , diluted, and filtered through PTFE filters for UHPLC-FLD analysis. A Waters™ Torus diol column kept at 50°C was used for separation on an Agilent 1290 Infinity UPLC coupled with a 1260 Infinity FLD. Mobile phase A was acetonitrile: acetic acid and mobile phase B was methanol:water:acetic acid . The gradient was following 0% B for 0.37 min to 45% B in 10.03 min to 95% B in 0.25 min and held for 2.35 min. The flow rate was 1 mL min−1 with a sample injection volume of 2 μL. The FLD condition was optimized at a photo multiplier tube gain at 11 with an excitation wavelength of 230 nm and an emission wavelength of 321 nm. The quantification was determined using NIST RM 8403 as external standards.Flavanol enantiomers – and -epicatechin as well as – and -catechin were characterized using a modified method from Machonis et al. For cocoa powders, 0.05 and 0.1 g of defatted Acticoa™ and Mullica™ samples were dissolved in 5 mL methanol:water:acetic acid , respectively,blueberry grow pot while for grape thinned clusters, 0.1 to 0.15 g freeze-dried samples were dissolved in 5 mL MWAA. All mixtures underwent sonication at 50°C for 5 min and centrifuged at 5000 rcf for 10 min to obtain supernatants. Supernatants were further diluted and filtered for UPLC-FLD analysis. An Astec® Cyclobond® I2000 RSP kept at 35°C was used for separation on an Agilent 1290 Infinity UPLC-FLD. The isocratic mobile phase was 20 mM ammonium acetate buffer :methanol . The flow rate was 1 mL min−1 for a total run time of 30 min with a sample injection volume of 10 μL. The FLD was set at an excitation wavelength of 276 nm and an emission wavelength of 316 nm. Five-point calibration curves of each enantiomer were plotted for quantification.The moisture content of freeze-dried thinned grape clusters was determined according to Thakur et al. for reporting TPC, and flavanols and procyanidins of thinned clusters in DW.The total phenolic content was measured in triplicate for thinned cluster fractions. All data are expressed as mean ± standard deviation . One-way analysis of variance followed by Tukey & Dunnett multiple comparisons were applied using XLSTAT to evaluate significant differences among thinned clusters and cocoa powders flavanol content. For all comparisons, differences were considered statistically significant at a value of p< .05.The two wine grape varieties, Chardonnay and Pinot noir, were categorized into four different thinned cluster fractions based on berry color. Each variety that was analyzed in the current study included: Chardonnay seed and seedless dark , Chardonnay seed and seedless light , Pinot noir seed and seedless red , and Pinot noir seed and seedless green . To help illustrate the potential of using Chardonnay and Pinot noir grape thinned clusters as functional ingredients in cocoa-based products, two commercial cocoa powder samples were also analyzed and used as reference samples for their monomeric and oligomeric flavanols and procyanidins content. The total phenolic content determination was the starting point for the thinned grape cluster characterization because it provided important information, although unspecific, about the relative composition of the thinned grape cluster sample .

The composition and concentration of phenolics in grapes vary greatly due to variety and degree of ripeness , and environmental and vineyard management factors such as soil type and irrigation strategy . Thus, it is not surprising to see a 2.1-fold and 1.7-fold variations of TPC in Chardonnay and Pinot noir fractions, respectively. As shown in Figure 1, Chard_S_Light, which was considered the least mature among all fractions, had the highest TPC at 70.1 ± 1.1 mg g−1 in dry weight . The highest TPC for Pinot noir was detected in its green seedless fraction at 62.4 ± 1.1 mg g−1 DW. Within the Chardonnay fractions, the less mature fractions had higher TPC compared to the more mature fractions in both seed and seedless forms although no significant difference was found in two seedless fractions . The same trend was also observed in Pinot noir fractions as the less mature green fractions for both seed and seedless had higher TPC compared to the more mature red fractions. Pantelić et al. also found that TPC values were significantly higher in seeds than in skins and pulps in seven red and six white grapevine varieties grown in Serbia; our Chardonnay Dark and Pinot noir Green fractions were on the opposite where the seedless fractions yielded higher TPC for both varieties. Typically, the amount of TPC in the white grape varieties is lower than that of red grapes due to lack of anthocyanins synthesis , yet, previous studies have found that the TPC of grapes mainly depends on the varietal differences, not on grape skin color . Furthermore, in this study, thinned clusters for Pinot noir were collected at the beginning of veraison before anthocyanins accumulation reached its peak. Variations among grape fractional sample preparation , TPC extraction , and data reporting format could result in significant TPC differences from study to study for the same varieties . Flavanol monomers and procyanidins are abundant in cocoa and grapes. In cocoa, procyanidins are the major flavanols which consist of oligomers as well as polymers of catechins and epicatechins commonly bonded through a C4-C6 or C4-C8 linkage . Grapes are also rich sources for oligomeric and polymeric procyanidins with studies showing oligomeric procyanidins such as dimers and trimers being easier to be absorbed in vivo . Although cocoa-based products can be perceived as “healthy” among consumers because cocoa flavanols exhibited numerous health-promoting properties , the amount of bioactive and bioavailable cocoa flavanols, mainly -epicatechin, -catechin, and procyanidin dimers and trimers, vary greatly among cocoa-based products thus are unlikely to achieve the same level of healthpromoting effects in vivo. Therefore, it is crucial to quantify the flavanol monomers and oligomeric procyanidins content in the grape thinned clusters to determine their bioactive potential when compared with cocoa ingredients such as cocoa powder. A new cocoa extract reference NIST RM8403 was used as an external standard in the recently approved 2020.05 AOAC official method which allows more accurate and reliable determination of cocoa flavanols and procyanidins using UPLC-FLD . The current study successfully applied this method to the Chardonnay and Pinot noir thinned cluster fractions as well as two commercial cocoa powders in the context of catechin, epicatechin, and their oligomeric procyanidins . An example of chromatogram is shown in Figure 2.