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Sweet cherry pits, date seeds, and grape seeds are abundant fruit by-products in the Mediterranean region. Assessing their antioxidant capacity is crucial for their valorization. Grape and date seeds exhibited higher concentrations of total phenolic and flavonoid contents, and significant antioxidant capacity. Epicatechin was the main flavonoid in sweet cherry pits and date seeds (29-85 mg/g), while vanillic acid was the predominant phenolic acid across all by-products (5-23 mg/g). However, some sweet cherry pit varieties exceeded Maximum Residue Levels (MRL) for five pesticides, while grape seeds contained thirteen fungicide residues, all below MRL. Ochratoxin A was detected in one date seed but below the limit of quantification. Additionally, grape seeds showed an Al content of approximately 130 mg/kg, along with levels of As, Cd, and Pb. Date seeds exhibited high potential for food and pharmaceutical applications, pending evaluation for chemical contaminants.

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The clear juice fermentation technique for white wines suggests that white grape seeds, rich in flavan-3-ols and proanthocyanidins, are not effectively utilized in the winemaking process. This study incorporated 'Gewürztraminer' grape seeds into 'Cabernet Sauvignon' must before cold soak to investigate how the resultant red wines' phenolic compound profiles, color, and astringency were affected. The results showed that adding seeds primarily inhibited the leaching of flavan-3-ols from both skins and seeds. A significant increase in the levels of flavan-3-ols, tannins, and phenolic acids, as well as direct and aldehyde-bridged flavan-3-ol-anthocyanin polymers, were observed in the wines with additional seeds. This led to the improvement in the wine' red hue and its resistance to SO2 bleaching. Furthermore, the wine added with seeds exhibited stronger astringency compared to those without. The findings provide a promising winemaking strategy to improve color stability and intensify the astringency of red wines through the utilization of grape seeds.

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Grape seeds (GS), wine lees (WL), and grape pomace (GP) are common winery by-products, used as bio-fillers in this research with two distinct biopolymer matrices-poly(butylene adipate-co-terephthalate) (PBAT) and polybutylene succinate (PBS)-to create fully bio-based composite materials. Each composite included at least 30 v% bio-filler, with a sample reaching 40 v%, as we sought to determine a composition that could be economically and environmentally effective as a substitute for a pure biopolymer matrix. The compounding process employed a twin-screw extruder followed by an injection molding procedure to fabricate the specimens. An acetylation treatment assessed the specimen's efficacy in enhancing matrix-bio-filler affinity, particularly for WL and GS. The fabricated bio-composites underwent an accurate characterization, revealing no alteration in thermal properties after compounding with bio-fillers. Moreover, hygroscopic measurements indicated increased water-affinity in bio-composites compared to neat biopolymer, most significantly with GP, which exhibited a 7-fold increase. Both tensile and dynamic mechanical tests demonstrated that bio-fillers not only preserved, but significantly enhanced, the stiffness of the neat biopolymer across all samples. In this regard, the most promising results were achieved with the PBAT and acetylated GS sample, showing a 162% relative increase in Young's modulus, and the PBS and WL sample, which exhibited the highest absolute values of Young's modulus and storage modulus, even at high temperatures. These findings underscore the scientific importance of exploring the interaction between bio-fillers derived from winery by-products and three different biopolymer matrices, showcasing their potential for sustainable material development, and advancing polymer science and bio-sourced material processing. From a practical standpoint, the study highlighted the tangible benefits of using by-product bio-fillers, including cost savings, waste reduction, and environmental advantages, thus paving the way for greener and more economically viable material production practices.

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Phenolic compounds, especially tannins, are important for red wine quality. Wines made from cold-hardy hybrid grape cultivars have much lower tannin concentrations than wines from Vitis vinifera grape cultivars. This study assessed the phenolics content of berry tissues of three red cold-hardy hybrid cultivars in comparison to V. vinifera cv. 'Pinot noir' throughout development and ripening. Basic chemical properties, iron-reactive phenolics content, and tannin content were evaluated in the juice, skins, and seeds of Vitis spp. cvs. 'Crimson Pearl', 'Marquette', and 'Petite Pearl' and 'Pinot noir' at six time points from one week post-fruit set to harvest in 2021 and 2022. 'Crimson Pearl' displayed similar iron-reactive phenolics and tannin contents in juice, skins (22.6-25.4 mg/g dry skin and 8.0-12.2 mg/g dry skin, respectively), and seeds (12.8-29.8 mg/g dry seed and 4.2-22.0 mg/g dry seed, respectively) as 'Petite Pearl' and 'Marquette' at harvest in 2022. The hybrid cultivars showed a similar trend of phenolic accumulation as 'Pinot noir' but resulted in overall lower content in skins and seeds. Despite differences in developmental trends, the three hybrid grape cultivars displayed similar phenolic content at harvest ripeness. This is the first study examining the phenolic content of 'Crimson Pearl' and 'Petite Pearl' throughout berry development and ripening. This study provides important information for the wine industry to make informed decisions on making wine with these cultivars.

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BACKGROUND: To manage industrial waste in accordance with the circular bioeconomy concept it is sometimes necessary to handle grape seeds, an abundant by-product of the wine-making process. This study presents a process based on ultrasound technology for the extraction of grape-seed proteins, due to their nutritional and techno-functional properties. The protein content of extracts obtained under silent and lab-scale conditions was compared with that obtained under semi-industrial ultrasound conditions, and the chemical composition (carbohydrates, total phenols, and lipids) and the elemental profiles of the final, up-scaled downstream extracts were characterized. RESULTS: This work found that the maximum amount of protein in the final product was 378.31 g.kg-1 of the extract. Chemical characterization revealed that each 1 kg of extract had an average content of 326.19 g gallic acid equivalent as total phenols, 162.57 g glucose equivalent as carbohydrates, and 382.76 g of lipophilic compounds. Furthermore, when the extract was checked for hazardous elements, none were found in levels that could be considered a risk for human health. CONCLUSION: The proposed semi-industrial strategy has the potential to contribute greatly to the valorization of grape seeds through the preparation of a protein-rich extract that can be used as an alternative to synthetic wine stabilizers and for the development of novel food and nutraceutical products. © 2024 Society of Chemical Industry.

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Grape seeds are a valuable source of natural phenolic compounds, particularly flavan-3-ol derivatives such as condensed tannins. Recent studies have shown that grape seed powder can be applied to reduce the undesirable effects of protein instability in wine. One pretreatment method applied to grape seeds is roasting. Roasting causes the heavier proanthocyanidins (PAC) oligomers to break down, thereby increasing the concentration of smaller oligomers available for interaction with proteins. In addition, roasting can prolong grape seed storage. Among the subclasses of proanthocyanidins, oligomeric macrocyclic proanthocyanidins have also shown potential effects in terms of wine stabilization, particularly by presenting selective interactions with metal cations such as potassium and calcium. However, their composition in grape seed extracts has never been studied. Here, the characterization of condensed tannins according to the degree of polymerization in grape seeds, the profile of cyclic proanthocyanidins and the total polyphenol content were characterized in relation to different grape varieties and the application of roasting. Roasting greatly influenced the distribution of PAC according to the degree of polymerization, increasing the abundance of almost all classes of PAC. However, the overall effect of roasting was highly dependent on grape variety. PAC were analyzed according to the degree of polymerization. Grape seed roasting of red varieties (Croatina and Sangiovese) showed an increase in all classes of PAC except trimers. The white variety (Ortrugo) and the mix of Nebbiolo and Barbera varieties (80% and 20% w/w, respectively) showed no clear effect on the profile of PAC upon roasting. Notably, cyclic procyanidins were identified for the first time in grape seeds: a cyclic tetrameric procyanidin (ESI + m/z 1153) and cyclic pentameric procyanidin (ESI + m/z 1441) were found. The abundances of these cyclic PAC were found to be completely stable upon roasting, also in agreement with the already known stability of these compounds against depolymerizing conditions. Interestingly, the cyclic pentameric procyanidin was significantly more abundant in Ortrugo (white variety), than in Sangiovese and Croatina (red varieties). Besides, no effect of roasting occurred on the profile of cyclic procyanidins in grape seed powder. Finally, the total polyphenol content was evaluated, showing that roasting caused an increase of polyphenolic molecular species potentially available for protein stabilization, but only in GSP of red varieties. Overall, the grape variety was found to be a significant factor in determining how much the roasting would change the PAC profile, providing valuable information for future applications of GSP in enology.

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Seeds of Vitis vinifera L. with a high content of bioactive compounds are valuable by-products from grape processing. However, little is known about the bioactivity of seeds from other Vitis species. The aim of this study has been to compare the phenolic composition, antimicrobial activity, and antioxidant activity of extracts from seeds of four Vitis species (V. riparia Michx., V. californica Benth., V. amurensis Rupr., and V. vinifera L.). Antioxidant activities were assessed as ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging activity, and oxygen radical absorbance capacity (ORAC). The antimicrobial activity was determined using the microdilution method against some Gram-negative (Escherichia coli, Salmonella enterica ser. Typhimurium, and Enterobacter aerogenes) and Gram-positive (Enterococcus faecalis and Staphylococcus aureus) bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to evaluate the phenolic profile of extracts. Flavan-3-ols, procyanidins, phenolic acids, flavonols, anthocyanins, and stilbenoids were detected. (+)-Catechin and (-)-epicatechin turned out to be the most abundant in the phenolic profile of V. amurensis seed extract. Phenolic acids prevailed in the extract from V. vinifera seeds. The V. riparia and V. californica seed extracts had higher contents of most individual phenolics compared to the other Vitis species. They also showed a higher total phenolic content, DPPH• scavenging activity, ORAC, and overall antibacterial activity. Total phenolic content significantly correlated with antioxidant activity and antimicrobial activity against E. coli. The principal component analysis (PCA) showed discrimination between V. vinifera, V. amurensis, and clustered V. riparia and V. californica with respect to variables. To recapitulate, this research demonstrates that seeds of different Vitis species, especially V. riparia and V. californica, are sources of molecules with antioxidant and antimicrobial activities that can be used in different sectors, such as in the food, cosmetic, and pharmaceutical industries.

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Grape seed of Obeidi, a white Lebanese autochthonous variety, was previously tested in different studies as a valuable source of bioactive molecules such as polyphenols, oils, and proteins by means of extraction procedures for the development of cosmetic and therapeutic products. However, an un-valorized, exhausted grape seed residue remains as "secondary waste" after the extraction processes. In this study, the exhausted seeds have been further exploited to produce cosmetic scrubs capable of releasing antioxidant molecules during the exfoliation process, in accordance with the principles of the circular economy and going toward a zero-waste process. The deep characterization of the exhausted seeds confirmed the presence of antioxidant phenolic molecules including gallic acid, catechins and protocatechuic acid (0.13, 0.126, and 0.089 mg/g of dry matter DM), and a high phenolic content (11.85 mg gallic acid equivalents (GAE)/g of dry matter (DM)). Moreover, these residues were shown to possess a sandy texture (Hausner ratio (HR): 1.154, Carr index (CI): 0.133, and angle of repose: 31.62 (°) degrees), similar to commercial natural exfoliants. In this respect, exhausted Obeidi grape seed residues were incorporated at increasing concentrations (0.5, 1, 1.5, and 2% w/w) in a cosmetic scrub, and stored for 5 weeks at 4, 25, and 50 °C for stability testing. All tested scrub formulations exhibited good spreadability with a spread diameter of 3.6-4.7 cm and excellent physical stability, as no phase separation or color change were observed after four cycles of heat shock at 4 and 50 °C. Finally, an in vivo skin irritation test showed that the scrub enriched with 1.5% of exhausted Obeidi grape seed residues was the most promising formulation, as it possessed a high amount of phenolic molecules (0.042 ± 0.001 mg GAE/mL of scrub) and good stability and could be safely applied to the skin with no irritation phenomena. Overall results underlined that exhausted grape seed residues can be transformed into promising systems for both physical and chemical exfoliation, thus confirming the importance of the effective exploitation of agro-industrial by-products for the development of high value cosmeceutics towards a more sustainable and zero-waste approach.

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Alginate/pectin films supplemented with extracts from cranberry pomace (CE) or grape seeds (GE) were developed and applied to herring fillets that were stored for 18 days at 4 °C. Herring coated with films containing GE and CE inhibited the growth of Listeria monocytogenes and Pseudomonas aeruginosa during the storage period, whereas pure alginate/pectin films did not show an antimicrobial effect against the tested pathogens. The application of alginate/pectin films with CE and GE minimised pH changes and inhibited total volatile basic nitrogen (TVN) and the formation of thiobarbituric acid-reactive substances (TBARS) in the herring fillets. The coating of herring fillets with films with CE or GE resulted in three- and six-fold lower histamine formation and one-and-a-half- and two-fold lower cadaverine formation, respectively, when compared to unwrapped herring samples after 18 days of storage. The incorporation of 5% extracts isolated from cranberry pomace or grape seeds into the alginate/pectin film hindered herring spoilage due to the antimicrobial and antioxidant activity of the extracts.

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Condensed tannins (CT) in wine are derived from the seeds and skins of grapes, and their composition and content contribute to the bitterness/astringency characteristics and ageing potential of the wine. Global warming has accelerated the ripening process of grape berries, making them out of sync with seed ripening. To understand the influence of berry ripening on the seed CT composition and content, we analyzed the changes in the soluble and insoluble CT in the seeds of 'Cabernet Sauvignon' grapes from two vineyards over two years. The results showed that the seed-soluble CT presented a slight downward trend in fluctuation during grape berry development, while the insoluble CT increased continuously before the véraison and remained at a high level afterwards. Relatively speaking, a lower sugar increment in developing grape berries favored the conversion of seed CT towards a higher degree of polymerization. The terminal unit of soluble CT was dominated by epigallocatechin gallate, the content of which decreased as the seeds matured. It is suggested that the seeds should be fully matured to reduce this bitter component in tannins. This study provides a reference for us to control the grape ripening process and produce high-quality grapes for wine making.

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Among the waste materials of wine production, grape seeds constitute an important fraction of the pomace, from which the precious edible oil is extracted. The residual mass from oil extraction, the defatted grape seeds (DGS), can be destined for composting or valorized according to the circular economy rules to produce pyrolytic biochar by gasification or pellets for integral energy recovery. Only a small quantity is used for subsequent extraction of polyphenols and tannins. In this study, we performed a chemical characterization of the DGS, by applying spectroscopic techniques (ICP-OES) to determine the metal content, separation techniques (HS-SPME-GC-MS) to evaluate the volatile fraction, and thermal methods of analysis (TGA-MS-EGA) to identify different matrix constituents. Our main goal is to obtain information about the composition of DGS and identify some bioactive compounds constituting the matrix in view of possible future applications. The results suggest that DGS can be further exploited as a dietary supplement, or as an enriching ingredient in foods, for example, in baked goods. Defatted grape seed flour can be used for both human and animal consumption, as it is a source of functional macro- and micronutrients that help in maintaining optimal health and well-being conditions.

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The aim of this study was to evaluate the total phenolic content, composition, and antioxidant and antibacterial activities of four grape seed extracts (Cabernet Sauvignon, Marselan, Pinot Noir, and Tamyanka). The total phenolic content (TPC) and flavonoid, anthocyanin, procyanidin, ascorbic acid, DPPH, and ABTS antioxidant capacities of the grape seed extracts (GSEs) were determined. The extracts showed high TPC values (79.06-111.22 mg GAE/g). The individual components in the GSEs were determined using HPLC. High contents of catechin, epicatechin, and procyanidin B1 were found in the extracts. The antimicrobial activity of the obtained GSEs against Staphylococcus aureus, Bacillus cereus, and Escherichia coli was evaluated using the agar diffusion test and a test to determine the minimum inhibitory concentration (MIC). According to the effect on the growth of pathogens, the extracts were ranked in the following order: Pinot Noir > Marselan > Cabernet Sauvignon > Tamyanka. The tested bacteria showed high sensitivity to the extracts (MIC = 0.12-0.50 mg/mL). According to the MIC values, the bacteria were in the following order: S. aureus > B. cereus > E. coli. A correlation was found between the phenolic content of the GSEs and their antibacterial potential. The obtained results show that the studied GSEs have good potential as antioxidant and antimicrobial agents.

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Seeds' abundant biologically active compounds make them a suitable primary platform for the production of natural extracts, innovative foods, medicines, and cosmetics. High levels of industrial and agricultural residues and byproducts are generated during the processing of grapes, although some parts can also be repurposed. This paper examines the phytochemical composition, manufacturing processes, and health-improving attributes of many varieties of grape oil derived using various extraction methods. Since the results are influenced by a range of factors, they are expressed differently among studies, and the researchers employ a variety of measuring units, making it difficult to convey the results. The primary topics covered in most papers are grape seed oil's lipophilic fatty acids, tocopherols, and phytosterols. In addition, new methods for extracting grape seed oil should therefore be designed; these methods must be affordable, energy-efficient, and environmentally friendly in order to increase the oil's quality by extracting bioactive components and thereby increasing its biological activity in order to become part of the overall management of multiple diseases.

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Proanthocyanidins (PACs) are a group of bioactive molecules found in a variety of plants and foods. Their bioavailability depends on their molecular size, with monomers and dimers being more bioavailable than those that have a higher polymerization degree. This study aimed to develop a method to convert high-molecular-weight PACs to low-molecular-weight ones in a grape seed extract (GSE) from Vitis vinifera L. Therefore, GSE was subjected to alkaline treatment (ATGSE), and its difference in chemical composition, compared to GSE, was evaluated using a molecular networking (MN) approach based on results obtained from HPLC-ESI HRMS/MS characterization analysis. The network analysis mainly noted the PAC cluster with about 142 PAC compounds identified. In particular, the obtained results showed a higher content of monomeric and dimeric PACs in ATGSE compared to GSE, with 58% and 49% monomers and 31% and 24% dimers, respectively. Conversely, trimeric (9%), polymeric (4%), and galloylated PACs (14%) were more abundant in GSE than in ATGSE (6%, 1%, and 4%, respectively). Moreover, in vitro antioxidant and anti-inflammatory activities were investigated, showing the high beneficial potential of both extracts. In conclusion, ATGSE could represent an innovative natural matrix rich in bioavailable and bioaccessible PACs for nutraceutical applications with potential beneficial properties.

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Grape seeds are agro-industrial by-products, which if improperly managed, may be responsible for socioeconomic and environmental problems. Nevertheless, it is possible to effectively valorize them by means of extraction of the bioactive compounds, especially the antioxidant phenolic molecules, using a safe, green, and environmentally-friendly extractive medium (i.e., hydro-glyceric solution). In the present study, the extraction was performed using seeds from two Lebanese varieties, Obeidi and Asswad Karech, and three international varieties, Marselan, Syrah, and Cabernet Franc. The type and amount of phenolic compounds were identified by High-Performance Liquid Chromatography (HPLC). Marselan was the extract richer in catechins (132.99 ± 9.81 µg/g of dried matter), and it also contained a higher amount of phenolic compounds (49.08 ± 0.03 mg of gallic acid equivalent/g of dry matter and 10.02 ± 0.24 mg of proanthocyanidin content/g of dry matter). The antioxidant capacity of the extracts was assessed using three different colorimetric assays including 2,2-DiPhenyl-1-PicrylHydrazyl (DPPH), CUPRIC ion Reducing Antioxidant Capacity (CUPRAC), and Ferric Reducing Antioxidant Power (FRAP). As expected, Marselan exhibited the highest antioxidant activity; as well, the total phenolic and proanthocyanidin content were the highest. The stability of the Marselan extract incorporated into a commercial cream, was performed at three different temperatures (4, 25, and 50 °C), and four different concentrations (5, 4, 3, 2%), over a period of 4 months, using different methods such as centrifugation, Heat-Shock Cycles, pH, and viscosity. All Marselan hydro-glyceric extract formulations were proven to be stable over the entire 4 months, where the highest stability was achieved at 4 °C and the least at 50 °C. This study supports the suitability of the incorporation of phenolic extracts into commercial creams to enrich the cosmetic industry with effective, natural, and safe skincare products.

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Staphylococcus aureus is an important etiological agent for causing food poisoning leading to high mortality in the world. The sea gene is encoded in a polymorphic family of temperate bacteriophage chromosomes and became a prophage, and the transcription of this gene is associated with the life cycle of this prophage. It has been suggested that the grape polyphenols can eradicate the enterotoxin production of food-borne bacteria. This study aimed to evaluate the activity of the aqueous and alcoholic extracts of the grape seeds in inhibiting the expression of the sea gene encoding staphylococcal enterotoxin type A in S. aureus isolated from different sources. This study used five enterotoxin A producing isolates belonging to S. aureus. The results showed that minimum inhibition concentration and sub-minimum inhibition concentration of the aqueous extract were 32 and 16 µg/mL for all isolates, respectively. However, in the case of the alcoholic extract, these concentrations were 16 and 8 µg/mL for all isolates, respectively, and the results of the chemical analysis of the aqueous and alcoholic extracts confirmed that they contain active chemical compounds, such as flavonoids, alkaloids, tannins, and glycosides; moreover, they contain many functional groups according to the analysis of the infrared spectrum. Both extracts were shown to be active in inhibiting the expression of the sea gene in the isolates under study. As the results indicated, the gene expression of these isolates was inhibited by approximately 0.31-0.63 fold, and all pathogenic and environmental isolates showed a decrease in the expression of this gene. These results practically open the door to the possibility of using these extracts to inhibit the ability of S. aureus to produce these dangerous enterotoxins; thereby decreasing or preventing their pathogenicity, especially their food poisoning infections.

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Proanthocyanidins (PACs) have been proven to possess a wide range of biological activities, but complex structures limit their study of structure-function relationships. Therefore, an efficient and general method using hydrophilic interaction high-performance liquid chromatography coupled with high-resolution quadrupole time-of-flight tandem mass spectrometry (HILIC-QTOF-MS) was established to analyze PACs from different plant materials. This method was successfully applied to characterize PACs from Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves (BLPs), sorghum testa (STPs) and grape seeds (GSPs). BLPs with the degree of polymerization (DP) from 1 to 8 were separated. BLPs are mainly B-type prodelphinidins and A-type BLPs were first found in this study. STPs and GSPs belonging to procyanidins showed DP from 3 to 11 and 2 to 12, respectively. A-type linkages were found for every DP of STPs and GSPs, which were first found. These results showed that HILIC-QTOF-MS can be successfully applied for analyzing PACs from different plant materials, which is necessary for the prediction of their potential health benefits.

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A novel thiolysis-high-performance liquid chromatography method for the quantitative determination of total proanthocyanidins and the mean degree of polymerization in grape seeds has been developed. Following thiolysis with formic acid and benzyl mercaptan, reaction products were separated and purified. Three proanthocyanidin monomers and three derivatives were obtained and their structures were identified by liquid chromatography-mass spectrometry, FTIR spectroscopy, and NMR spectroscopy. A decomposition model of the thiolysis products and a correction formula for proanthocyanidins concentration were established. This thiolysis-high-performance liquid chromatography method displayed good calibration linearity (R2  > 0.999 over the concentration range 0.01 to 10 mg/mL), excellent accuracy (recoveries of 97.9-99.6%), and precision (repeatability relative standard deviations of 0.45-0.75%). This method is suitable for the quantitative analysis of proanthocyanidins in grape seed products.

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The chemical composition of wine grapes changes qualitatively and quantitatively during the ripening process. In addition to the sugar content, which determines the alcohol content of the wine, it is necessary to consider the phenolic composition of the grape skins and seeds to obtain quality red wines. In this work, some imaging techniques have been used for the comprehensive characterisation of the chemical composition of red grapes (cv. Tempranillo and cv. Syrah) grown in a warm-climate region during two seasons. In addition, and for the first time, mathematical models trained with laboratory images have been extrapolated for using in field images, obtaining interesting results. Determination coefficients of 0.90 for sugars, 0.73 for total phenols, and 0.73 for individual anthocyanins in grape skins have been achieved with a portable hyperspectral camera between 400 and 1000 nm, and 0.83 for total and individual phenols in grape seeds with a desktop hyperspectral camera between 900 and 1700 nm.

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Intraspecific recurrent selection in V. vinifera is an effective method for breeding of high quality, disease-, cold-, and drought-resistance grapes. Exploring the optimal treatment methods for grape (V. vinifera) seeds can help to accelerate the process of intraspecific recurrent selection and improve breeding efficiency. In this study, seeds of six V. vinifera varieties were used as experimental materials, and the germination and seedling formation characteristics were studied by single factor treatment and orthogonal compound treatment, respectively. To do this, stratification, chemical substances, beak cutting, and pre-germination treatments were tested, and the optimal treatment combination was determined for each variety. The results indicated that the optimal conditions obtained in the orthogonal experiments were not completely consistent with those in the single-factor experiments. Single factor experiment results demonstrated that two stratification methods (chilling gauze-storage and chilling sand-storage) and two pre-germination methods (pre-germination in petri dishes and pre-germination in a bean sprouter) vary in effectiveness for different varieties. gibberellin acid (GA3) soaking and beak-cutting promote the germination and seedling rate of the tested varieties. Orthogonal test results demonstrate that, for Dunkelfelder and Cabernet Sauvignon, the optimal treatment combination was chilling sand-storage + GA3 soaking seed + beak cutting + pre-germination in petri dishes. For Meili, the optimal treatment combination was chilling sand-storage + acetic acid (HAc) soaking seed + beak cutting + pre-germination in petri dishes. For Ecolly, the optimal treatment combination was chilling sand-storage + GA3 soaking seed + beak cutting + pre-germination in a bean sprouter. For Garanior, the optimal treatment combination was chilling sand-storage + HAc soaking seed + no beak cutting + pre-germination in petri dishes. For Marselan, the optimal treatment combination was chilling gauze-storage + GA3 soaking seed + beak cutting + pre-germination in a bean sprouter. This study identified the optimal conditions for seed germination and seedling formation of six grape varieties, which will facilitate future work to characterize the seed germination and seedling formation of seeds obtained by intraspecific hybridization of these varieties. This work also provides a reference for addressing problems of low seed germination rate and suboptimal seedling formation for better utilization of grape germplasms.