Enhancing Anthocyanin Extraction from Wine Lees: A Comprehensive Ultrasound-Assisted Optimization Study.

Wine lees, an important by-product of the wine industry, pose a major environmental problem due to the enormous quantities of solid-liquid waste that are discarded annually without defined applications. In this study, the optimization of a method based on a Box-Behnken design with surface response has been carried out to obtain extracts with high anthocyanin content and potent antioxidant activity. Six variables have been considered: %EtOH, temperature, amplitude, cycle, pH, and ratio. The developed method exhibited important repeatability properties and intermediate precision, with less than 5% CV being achieved. Furthermore, these novel methods were successfully applied to diverse wine lees samples sourced from Cabernet Sauvignon and Syrah varieties (Vitis vinifera), resulting in extracts enriched with significant anthocyanin content and noteworthy antioxidant activity. Additionally, this study evaluated the influence of grape variety, fermentation type (alcoholic or malolactic), and sample treatment on anthocyanin content and antioxidant activity, providing valuable insights for further research and application in various sectors. The potential applications of these high-quality extracts extend beyond the winemaking industry, holding promise for fields like medicine, pharmaceuticals, and nutraceuticals, thus promoting a circular economy and mitigating environmental contamination.

On the Valorization of Arbutus unedo L. Pomace: Polyphenol Extraction and Development of Novel Functional Cookies.

The fruits of Arbutus unedo L. have a crimson colour and are enriched with remarkable concentrations of bioactive compounds such as anthocyanins and polyphenols. These fruits are commonly used in the production of a Portuguese Protected Geographical Indication distillate called "Aguardente de Medronho". During this process, a solid pomace is generated and presently discarded without valuable applications. In this work, two strategies have been developed for the valorisation of A. unedo pomace. The first approach considers the extraction of polyphenols from this by-product through the optimization of an ultrasound-assisted method using a Box-Behnken design coupled with response surface methodology. The results indicate that the temperature and the percentage of methanol, along with their interaction, significantly influence the total concentration of polyphenols and the antioxidant activity of the extracts obtained. The optimal conditions identified consider the extraction of 0.5 g of sample with 20 mL of a solvent containing 74% MeOH (aq), at a pH of 4.8, maintained at 70 °C for 15 min. On the other hand, the second valorisation strategy considered the use of A. unedo pomace in the development of functional cookies. The incorporation of 15-20% pomace in the cookie formulation was well-received by consumers. This incorporation results in an intake of ca. 6.55 mg of polyphenols per gram of cookie consumed, accompanied by an antioxidant activity of 4.54 mg Trolox equivalents per gram of cookie consumed. Overall, these results encourage the employment of A. unedo pomace either as a reliable source of extracts enriched in polyphenols or as a nutraceutical active ingredient in functional cookies, thereby positively impacting human health.

Ultrasound-Assisted Anthocyanins Extraction from Pigmented Corn: Optimization Using Response Surface Methodology.

This study aimed to determine the optimal UAE conditions for extracting anthocyanins from pigmented corn using the Box-Behnken design (BBD). Six anthocyanins were identified in the samples and were used as response variables to evaluate the effects of the following working variables: extraction solvent pH (2-7), temperature (10-70 °C), solvent composition (0-50% methanol in water), and ultrasound power (20-80%). The extraction time (5-25 min) was evaluated for complete recovery. Response surface methodology suggested optimal conditions, specifically 36% methanol in water with pH 7 at 70 °C using 73% ultrasound power for 10 min. The method was validated with a high level of accuracy (>90% of recovery) and high precision (CV < 5% for both repeatability and intermediate precision). Finally, the proposed analytical extraction method was successfully applied to determine anthocyanins that covered a wide concentration range (36.47-551.92 mg kg-1) in several pigmented corn samples revealing potential varieties providing more health benefits.

Determination of Caffeoylquinic Acids Content by UHPLC in Scolymus hispanicus Extracts Obtained through Ultrasound-Assisted Extraction.

Scolymus hispanicus L., also known as golden thistle, Spanish oyster thistle or, more commonly, as tagarnina is a plant that belongs to the Asteraceae family. It is collected from the wild for human consumption in Mediterranean countries. It is a relevant ingredient in Andalusian culinary culture, where the midribs of young plants are harvested for consumption. Scolymus hispanicus L. contains a wide variety of phenolic compounds such as caffeoylquinic acids (CQAs), among others. In the present work, the major phenolic compounds present in tagarnina have been identified, with 5-caffeoylquinic acid (5-CQA) and 3,5-dicaffeoylquinic acid (3,5-diCQA) being the main ones. A method based on ultrasound-assisted extraction (UAE) has been developed for the extraction of these compounds, with the percentage of methanol, sample-to-solvent ratio and the pH being the most influential factors. The developed method has been validated and employed to determine the concentration of 5-CQA and 3,5-diCQA in the midribs of Scolymus hispanicus, collected in six different places in the south of Spain. The antioxidant activity of the samples has also been determined, and a direct correlation with their caffeoylquinic compounds content has been established, showing an antioxidant effect.

Effects of Different Freezing Treatments during the Winemaking of a Varietal White Wine with Regard to Its Phenolic Components.

In white wine production, the technique consisting of freezing whole or crushed grapes usually increases the levels of aroma-related compounds in the final wine products. However, this technique may affect phenolic compounds, among other chemical compounds. Phenolic compounds are crucial to white wines because of their susceptibility to oxidation and their role with regard to color stability. In this study, white wines made from Muscat of Alexandria grapes were subjected to two different freezing techniques: whole-bunch freezing and crushed-grape freezing. In addition, a pre-fermentative maceration was applied to each experiment in order to determine if the effects of freezing were comparable to those of maceration. The phenolic compounds studied were gallic acid, protocatechuic acid, caffeic acid, trans-coutaric acid, and epicatechin, which are the key compounds from the point of view of wine stability. The freezing of crushed grapes enhanced the extraction of phenolic compounds in comparison to the freezing of whole bunches of grapes without pre-fermentative maceration. On the other hand, the effect of pre-fermentative maceration was comparable to that resulting from freezing crushed grapes. This step made the must from whole frozen grapes having even larger levels of phenolic compounds. Without pre-fermentative maceration, freezing whole bunches of grapes only allowed a moderate extraction of phenolic compounds and produced wines with lower individual phenolic contents than those obtained through traditional winemaking procedures.

Application of Direct Thermal Desorption-Gas Chromatography-Mass Spectrometry for Determination of Volatile and Semi-Volatile Organosulfur Compounds in Onions: A Novel Analytical Approach.

The population is now more aware of their diets due to the connection between food and general health. Onions (Allium cepa L.), common vegetables that are minimally processed and grown locally, are known for their health-promoting properties. The organosulfur compounds present in onions have powerful antioxidant properties and may decrease the likelihood of developing certain disorders. It is vital to employ an optimum approach with the best qualities for studying the target compounds to undertake a thorough analysis of these compounds. In this study, the use of a direct thermal desorption-gas chromatography-mass spectrometry method with a Box-Behnken design and multi-response optimization is proposed. Direct thermal desorption is an environmentally friendly technique that eliminates the use of solvents and requires no prior preparation of the sample. To the author's knowledge, this methodology has not been previously used to study the organosulfur compounds in onions. Likewise, the optimal conditions for pre-extraction and post-analysis of organosulfur compounds were as follows: 46 mg of onion in the tube, a desorption heat of 205 °C for 960 s, and a trap heat of 267 °C for 180 s. The repeatability and intermediate precision of the method were evaluated by conducting 27 tests over three consecutive days. The results obtained for all compounds studied revealed CV values ranging from 1.8% to 9.9%. The major compound reported in onions was 2,4-dimethyl-thiophene, representing 19.4% of the total area of sulfur compounds. The propanethial S-oxide, the principal compound responsible for the tear factor, accounted for 4.5% of the total area.

Optimization of a Microwave Assisted Extraction Method for Maximum Flavonols and Antioxidant Activity of Onion Extracts.

Nowadays, consumers demand bioactive foods that have the potential to limit the risk of suffering from several medical conditions. Onions present these desirable capabilities owing to its high content in antioxidant bioactive compounds. This work has used a Box-Behnken design with a response surface methodology to determine the best conditions in which to extract the polyphenols that are found in onions. Two extraction methods-one for the extraction of total flavonols and another one intended to obtain extracts with the highest possible antioxidant activity-have been developed and optimized. The following factors have been studied: temperature, %methanol in water, solvent pH, and sample-solvent volumetric ratio. The optimal conditions for the extraction of flavonols were 93.8% methanol in water, pH 2, 50 °C extraction temperature and 0.2:17.9 g:mL sample-solvent ratio. The best antioxidant activity levels were registered when using 74.2% methanol in water, pH 2, 99.9 °C extraction temperature and 0.2:18.2 g:mL sample-solvent ratio. Both optimized methods used short extraction times, and presented good precision levels and successful results when used with an assortment of onion varieties. According to total flavonols and antioxidant activity data, with 7.557 ± 0.3261 and 12.08 ± 0.0379 mg g-1, respectively, the developed methods achieved comparable or even superior results to those obtained by other authors.

Extraction of Antioxidant Compounds from Onion Bulb (Allium cepa L.) Using Individual and Simultaneous Microwave-Assisted Extraction Methods.

Despite the excellent beneficial properties that anthocyanins and total phenolic compounds give to the red onion bulbs, few articles have investigated modern extraction techniques or experimental designs in this field. For this reason, the present study proposes the development and optimization of alternative methods for the extraction of these compounds based on microwave-assisted extraction and the Box-Behnken experiment design. The optimal values for the extraction of total anthocyanins have been established at 62% methanol composition as a solvent, pH 2, 56 °C temperature, and 0.2:13 g:mL sample-solvent ratio. Regarding the extraction of total phenolic compounds, the optimal conditions have been established at 100% pure methanol as a solvent with pH 2, 57 °C temperature, and 0.2:8.8 g:mL sample-solvent ratio. Short extraction times (min), good recoveries (mg of bioactive compound g-1 of dry onion), and high repeatability and intermediate precision (coefficient of variation (%)) have been confirmed for both methods. Regarding total anthocyanins, the following results have been obtained: 2 min, 2.64 ± 0.093 mg of total anthocyanins g-1 of dry onion, and 2.51% and 3.12% for precision. Regarding phenolic compounds, the following results have been obtained: 15 min, 7.95 ± 0.084 mg of total phenolic compound g-1 of dry onion, and 3.62% and 4.56% for precision. Comparing these results with those of other authors and with those obtained in a previous study of ultrasound-assisted extraction, it can be confirmed that microwave-assisted extraction is a quantitative, repeatable, and very promising method for the extraction of phenolic compounds and anthocyanins, which offers similar and even superior results with little solvent expense, time, and costs.

A comparison study between ultrasound-assisted and enzyme-assisted extraction of anthocyanins from blackcurrant (Ribes nigrum L.).

Blackcurrant (Ribes nigrum L.) is a fruit rich in vitamins, fatty acids, minerals, essential oils and phenolic compounds, including anthocyanins. In the present work, two anthocyanin extraction methods from blackcurrant samples based on Ultrasound-Assisted Extraction (UAE) and Enzyme-Assisted Extraction (EAE) have been developed. A Plackett-Burman design with seven variables has been preliminary used for both UAE and EAE in order to determine the most influential variables in each methodology. After that, a Box-Behnken design was employed to optimize the extraction methods. The composition of the extraction solvent (% EtOH in water) has been the most influential variable for both UAE and EAE. The optimal extraction times have been 5 min for UAE and 10 min for EAE. No differences have been observed in anthocyanin extraction with both methodologies. Both methods have been applied to blackcurrant-derived products and proven their suitability for quality control analysis.

Development of Optimized Ultrasound-Assisted Extraction Methods for the Recovery of Total Phenolic Compounds and Anthocyanins from Onion Bulbs.

Allium cepa L. is one of the most abundant vegetable crops worldwide. In addition to its versatile culinary uses, onion also exhibits quite interesting medicinal uses. Bulbs have a high content of bioactive compounds that are beneficial for human health. This study intends to develop and optimize two appropriate ultrasound-assisted methods for the extraction of the phenolic compounds and anthocyanins present in red onion. A response surface methodology was employed and, specifically, a Box-Behnken design, for the optimization of the methods. The optimal conditions for the extraction of the phenolic compounds were the follows: 53% MeOH as solvent, pH 2.6, 60 °C temperature, 30.1% amplitude, 0.43 s cycle, and 0.2:11 g sample/mL solvent ratio. On the other hand, the optimal conditions for the anthocyanins were as follows: 57% MeOH as solvent, pH 2, 60 °C temperature, 90% amplitude, 0.64 s cycle, and 0.2:15 g sample/mL solvent ratio. Both methods presented high repeatability and intermediate precision, as well as short extraction times with good recovery yields. These results illustrate that the use of ultrasound-assisted extraction, when properly optimized, is suitable for the extraction and quantification of the compounds of interest to determine and improve the quality of the raw material and its subproducts for consumers.

A Novel Ultrasound-Assisted Extraction Method for the Analysis of Anthocyanins in Potatoes (Solanum tuberosum L.).

Purple potato is one of the least known and consumed potato varieties. It is as rich in nutrients, amino acids and starches as the rest of the potato varieties, but it also exhibits a high content of anthocyanins, which confer it with some attractive health-related properties, such as antioxidant, pain-relieving, anti-inflammatory and other promising properties regarding the treatment of certain diseases. A novel methodology based on ultrasound-assisted extraction has been optimized to achieve greater yields of anthocyanins. Optimal extraction values have been established at 70 °C using 20 mL of a 60% MeOH:H2O solution, with a pH of 2.90 and a 0.5 s-1 cycle length at 70% of the maximum amplitude for 15 min. The repeatability and intermediate precision of the extraction method have been proven by its relative standard deviation (RSD) below 5%. The method has been tested on Vitelotte, Double Fun, Highland and Violet Queen potatoes and has demonstrated its suitability for the extraction and quantification of the anthocyanins found in these potato varieties, which exhibit notable content differences. Finally, the antioxidant capacity of these potato varieties has been determined by means of 2,2-diphenyl-1-picrylhydrazyl (DDPH) radical scavenging and the values obtained were similar to those previously reported in the literature.

Optimization of an Ultrasound-Assisted Extraction Method for the Analysis of Major Anthocyanin Content in Erica australis Flowers.

Erica australis plants have been used in infusions and folk medicine for years for its diuretic and antiseptic properties and even for the treatment of infections. In addition, a recently published thorough study on this species has demonstrated its antioxidant, antibiotic, anti-inflammatory, anticarcinogenic and even antitumoral activities. These properties have been associated with the high content of anthocyanins in E. australis leaves and flowers. The aim of the present research is to optimize an ultrasound-assisted extraction methodology for the recovery of the anthocyanins present in E. australis flowers. For that purpose, a Box Behnken design with response surface methodology was employed, and the influence of four variables at different values was determined: namely, the composition of the extraction solvents (0-50% MeOH in water), the pH level of those solvents (3-7), the extraction temperature (10-70 °C), and the sample:solvent ratio (0.5 g:10 mL-0.5 g:20 mL). UHPLC-UV-vis has been employed to quantify the two major anthocyanins detected in the samples. The extraction optimum conditions for 0.5 g samples were: 20 mL of solvent (50% MeOH:H2O) at 5 pH, with a 15 min extraction time at 70 °C. A precision study was performed and the intra-day and inter-day relative standard deviations (RSDs) obtained were 3.31% and 3.52%, respectively. The developed methodology has been successfully applied to other Erica species to validate the suitability of the method for anthocyanin extraction.

Development of a Rapid UHPLC-PDA Method for the Simultaneous Quantification of Flavonol Contents in Onions (Allium cepa L.).

Onion, one of the most consumed vegetables in the world, is also known to contain high levels of antioxidant compounds, with protective effects against different degenerative pathologies. Specifically, onion is rich in flavonols, mainly quercetin derivatives, which are compounds with high antioxidant and free radical scavenging power. For this reason, it is of the utmost importance to count on optimal analytical methods that allow for the determination and quantification of these compounds of interest. A rapid ultra-high performance liquid chromatography (UHPLC)-photo-diode array (PDA) method for the separation of the major flavonols in onions was developed using a Box-Behnken design in conjunction with multiresponse optimization on the basis of the desirability function. The conditions that provided a successful separation were 9.9% and 53.2% of phase B at the beginning and at the end of the gradient, respectively; 55 °C column working temperature; and 0.6 mL min-1 flow rate. The complete separation was achieved in less than 2.7 min with excellent chromatographic characteristics. The method was validated, and its high precision, low detection and quantification limits, good linearity, and robustness were confirmed. The correct applicability of the method improves the analysis of the raw material, increasing the quality of onions and its subproducts in terms of bioactive compounds and functional characteristics for consumers.

Flavonol Composition and Antioxidant Activity of Onions (Allium cepa L.) Based on the Development of New Analytical Ultrasound-Assisted Extraction Methods.

The onion is one of the most cultivated and consumed vegetables, and is a very valuable source of antioxidant substances. Every onion variety is rich in flavonols-mainly quercetin derivatives which makes onions the main dietary source of these compounds. This study intends to develop an ultrasound-assisted extraction method (UAE), an economical, green, and efficient technique, that allows us to determine the quality of onion extracts in terms of flavonol composition and antioxidant activity. For this purpose, an ultrasound-assisted extraction method has been optimized to obtain extracts with a high amount of flavonols, as well as with a high antioxidant activity, not only separately using a Box-Behnken design, but also simultaneously, based on multi-response optimization. Multiple response optimization has not only demonstrated an efficacy level similar to that of the individual ones, but it also represents a considerable reduction in terms of cost, time and work. The optimal conditions for simultaneous extractions were determined as follows: 76.8% methanol as an extraction solvent at pH 2 and 58.5 °C temperature, 85% amplitude, 0.9 s cycle, and 0.2:13 g:mL sample mass/solvent volume ratio. Furthermore, the developed method exhibited a high precision level and great recoveries in a rather short extraction time (2 min). These results, together with the development of a fast and simple UHPLC analysis method, lead us to consider the developed UAE method as a suitable technique for the extraction of bioactive compounds from onion matrices.

Volatile and Semi-Volatile Organic Compounds May Help Reduce Pollinator-Prey Overlap in the Carnivorous Plant Drosophyllum lusitanicum (Drosophyllaceae).

Most carnivorous plants show a conspicuous separation between flowers and leaf-traps, which has been interpreted as an adaptive response to minimize pollinator-prey conflicts which will reduce fitness. Here, we used the carnivorous subshrub Drosophyllum lusitanicum (Drosophyllaceae) to explore if and how carnivorous plants with minimal physical separation of flower and trap avoid or reduce a likely conflict of pollinator and prey. We carried out an extensive field survey in the Aljibe Mountains, at the European side of the Strait of Gibraltar, of pollinating and prey insects of D. lusitanicum. We also performed a detailed analysis of flower and leaf volatile and semi-volatile organic compounds (VOCs and SVOCs, respectively) by direct thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS) to ascertain whether this species shows different VOC/SVOC profiles in flowers and leaf-traps that might attract pollinators and prey, respectively. Our results show a low overlap between pollinator and prey groups as well as clear differences in the relative abundance of VOCs and SVOCs between flowers and leaf-traps. Coleopterans and hymenopterans were the most represented groups of floral visitors, whereas dipterans were the most diverse group of prey insects. Regarding VOCs and SVOCs, while aldehydes and carboxylic acids presented higher relative contents in leaf-traps, alkanes and plumbagin were the main VOC/SVOC compounds detected in flowers. We conclude that D. lusitanicum, despite its minimal flower-trap separation, does not seem to present a marked pollinator-prey conflict. Differences in the VOCs and SVOCs produced by flowers and leaf-traps may help explain the conspicuous differences between pollinator and prey guilds.

Optimization of a Novel Method Based on Ultrasound-Assisted Extraction for the Quantification of Anthocyanins and Total Phenolic Compounds in Blueberry Samples (Vaccinium corymbosum L.).

In recent years, consumers' preference for fruits such as blueberry has increased noticeably. This fact is probably related to their bioactive components such as anthocyanins, phenolic compounds, vitamins, minerals, and tannins that have been found in blueberries by the latest research studies. Both total anthocyanins (TA) and total phenolic compounds (TPC) are known for their multiple beneficial effects on our health, due to their anti-inflammatory, anti-oxidant, and anti-cancer properties. This is the reason why the development of new methodologies for the quality control analysis of raw materials or derived products from blueberry has a great relevance. Two ultrasound-assisted extraction methods (UAE) have been optimized for the quantification of TA and TPC in blueberry samples. The six variables to be optimized were: solvent composition, temperature, amplitude, cycle, extraction solvent pH, and sample/solvent ratio using response surface methodology. The optimized methods have proven to be suitable for the extraction of the TPC and TA with good precision (repeatability and intermediate precision) (coefficient of variation (CV) < 5%) and potentially for application in commercial samples. This fact, together with the multiple advantages of UAE, makes these methods a good alternative to be used in quality control analysis by both industries and laboratories.

Optimisation of an ultrasound-assisted extraction method for the simultaneous determination of phenolics in rice grains.

Fifteen phenolic compounds were determined in rice grains by ultrasound assisted extraction and ultra-performance liquid chromatography with photodiode array detection. Primarily, an UAE method has been developed and validated for the extraction of phenolics from rice grains. For the optimization, a Box-Behnken Design based on six factors including extraction temperature (10-70 °C), solvent composition (0-50% methanol in water), cycle (0.2-0.7 s-1), ultrasound amplitude (30-70%), and solvent to sample ratio (2.5:1 to 5:1) was employed. Multi-response optimization (MRO) was performed to develop a simultaneous extraction method of 15 phenolics. The optimal UAE conditions were: extraction temperature 45 °C, time 25 min, cycle 0.4 s-1, ultrasound amplitude 47%, solvent composition 80% methanol in water at pH 4.25, and sample to solvent ratio 1:5. Linearity, LODs, LOQs, precision and recovery were determined. In addition, the method was successfully applied to analyse a number of rice samples with different varieties.

Tryptophan Levels during Grape Ripening: Effects of Cultural Practices.

Some cultural practices that are carried out during the grape ripening period are associated with vine stress, including leaf removal, grape bunch removal, and vegetable cover crops. Additionally, several nitrogen and sulfur supplements have also been used directly on leaves during the last stage of the ripening period. In the work described here, five different cultural practices and the reference were applied in three replicates in the same vineyard. The evolution of tryptophan levels was evaluated from just after grape veraison until the harvest date. In some cases, certain specific treatments were also evaluated after the regular harvest date. The cultural techniques that involved the application of nitrogen led to higher levels of tryptophan at the harvest day when compared to other cultural techniques. It was also found that the application of nitrogen without sulfur had a faster effect on the level of tryptophan. It was established that a period of around 20 days is needed for the grapes to show clear differences in tryptophan levels after the application of nitrogen.

Multiresponse optimization of a UPLC method for the simultaneous determination of tryptophan and 15 tryptophan-derived compounds using a Box-Behnken design with a desirability function.

A Box-Behnken design was used in conjunction with multiresponse optimization based on the desirability function to carry out the simultaneous separation of tryptophan and 15 derivatives by Ultra Performance Liquid Chromatography. The gradient composition of the mobile phase and the flow rate were optimized with respect to the resolution of severely overlapping chromatographic peaks and the total run time. Two different stationary phases were evaluated (hybrid silica and a solid-core-based C18 column). The methods were validated and a suitable sensitivity was found for all compounds in the concentration range 1-100µgL-1 (R2>0.999). High levels of repeatability and intermediate precision (CV less than 0.25% and 1.7% on average for the retention time and the signal area, respectively) were obtained. The new method was applied to the determination tryptophan and its derivatives in black pigmented glutinous and non-glutinous rice grain samples.

Optimization of the ultrasound-assisted extraction of anthocyanins and total phenolic compounds in mulberry (Morus nigra) pulp.

New ultrasound-assisted extraction methods for the determination of anthocyanins and total phenolic compounds present in mulberries have been developed. Several extraction variables, including methanol composition (50-100%), temperature (10-70°C), ultrasound amplitude (30-70%), cycle (0.2-0.7s), solvent pH (3-7) and solvent-solid ratio (10:1.5-20:1.5) were optimized. A Box-Behnken design in conjunction with a response surface methodology was employed to optimize the conditions for the maximum response based on 54 different experiments. Two response variables were considered: total anthocyanins and total phenolic compounds. Extraction temperature and solvent composition were found to be the most influential parameters for anthocyanins (48°C and 76%) and phenolic compounds (64°C and 61%). The developed methods showed high reproducibility and repeatability (RSD<5%). Finally, the new methods were successfully applied to real samples in order to investigate the presence of anthocyanins and total phenolic compounds in several mulberry jams.