Commercial Grape Juice Research Articles

Two-Stage Screening of Metschnikowia spp. Bioprotective Properties: From Grape Juice to Fermented Must by Saccharomyces cerevisiae.

Gluconobacter oxydans (Go) and Brettanomyces bruxellensis (Bb) are detrimental micro-organisms compromising wine quality through the production of acetic acid and undesirable aromas. Non-Saccharomyces yeasts, like Metschnikowia species, offer a bioprotective approach to control spoilage micro-organisms growth. Antagonist effects of forty-six Metschnikowia strains in a co-culture with Go or Bb in commercial grape juice were assessed. Three profiles were observed against Go: no effect, complete growth inhibition, and intermediate bioprotection. In contrast, Metschnikowia strains exhibited two profiles against Bb: no effect and moderate inhibition. These findings indicate a stronger antagonistic capacity against Go compared to Bb. Four promising Metschnikowia strains were selected and their bioprotective impact was investigated at lower temperatures in Chardonnay must. The antagonistic effect against Go was stronger at 16 °C compared to 20 °C, while no significant impact on Bb growth was observed. The bioprotection impact on Saccharomyces cerevisiae fermentation has been assessed. Metschnikowia strains' presence did not affect the fermentation time, but lowered the fermentation rate of S. cerevisiae. An analysis of central carbon metabolism and volatile organic compounds revealed a strain-dependent enhancement in the production of metabolites, including glycerol, acetate esters, medium-chain fatty acids, and ethyl esters. These findings suggest Metschnikowia species' potential for bioprotection in winemaking and wine quality through targeted strain selection.

Provenance discrimination of commercial grape juices from Argentina and Brazil based on elemental composition and chemometric methods

Provenance discrimination of commercial grape juices from Argentina and Brazil based on elemental composition and chemometric methods

Polyphasic identification of a <i>Zygosaccharomyces rouxii</i> isolated from grape juice concentrate and its control using thermal processing

<abstract> <p>A yeast, isolated from grape juice concentrate by a grape juice processing company in central Washington, was subjected to thermal processing in 52° Brix grape juice concentrate, commercial grape juice, and Yeast-Mold (YM) medium to determine <italic>D</italic>-values and <italic>z</italic>-values in the search for an effective means of control. At 50 ℃ in grape juice concentrate the <italic>D</italic>-value was 33.44 minutes, dropping rapidly as the temperature increased to 52.5 ℃, 55 ℃, 57.5 ℃, and 60 ℃. From this data, a <italic>z</italic>-value was determined to be 4.38 ℃. Grape juice and YM broth were similarly tested. Commercial grape juice at 45 ℃, 47 ℃, and 50 ℃ showed <italic>D</italic>-values of 9.80, 7.68, and 1.42, respectively, resulting in a <italic>z</italic>-value of 5.69 ℃. The isolate heat treated in YM broth at 40 ℃, 45 ℃, 46 ℃, 47.5 ℃, and 50 ℃ showed no effect at 40 ℃ but at higher temperatures, with a <italic>D</italic>-value of 2.28 at 45 ℃, 1.61 at 46 ℃, 1.05 at 47.5 ℃, and with a <italic>z</italic>-value of 7.46 ℃. This data suggests that an increase in the concentration of sugars in the environment has a protective effect on the yeast against elevated temperatures. Even so, pasteurization at 90 ℃ for thirty seconds appears to be an effective means of control for the yeast investigated. A yeast with the identical colony and cell morphology was isolated from 52° Brix grape juice concentrate from the same grape juice processing company. Both isolates were subjected to 28S DNA sequencing and FAME analysis for identification and determined to be <italic>Zygosaccharomyces rouxii</italic>, a known problem yeast in the grape juice and wine-making industry, to this point unknown in the central Washington region.</p> </abstract>

Quantitative genetic analysis of attractiveness of yeast products to Drosophila

The interaction between two model organisms, the fruit fly Drosophila melanogaster, and the budding yeast Saccharomyces cerevisiae, is widely perceived as mutually beneficial. The bridge of this biological connection is the volatile compounds from yeast fermentation, which can be attractive to the flies. The generation of volatile molecules derives from complex metabolism and different yeast will generate different combinations of volatiles. The attractiveness to fruit flies can therefore be considered a complex trait. In this study, six strains from SGRP were selected for fermenting commercial grape juice. The attractiveness of these ferments to a wild fruit fly line was then tested. Flies prefer the ferments of the Wine European (WE) strain over the North America (NA) strain. The preference assay was then applied to ferments of genotyped F1 progeny of WE crossed to NA for QTL mapping of genetic variation in attractiveness. GC-MS profiling of all ferments demonstrated that NA could be easily distinguished from other strains. 15 compounds were further quantified and the concentrations in F1 progeny ferments were correlated with genotypes using our r/QTL pipeline. Candidate genes from QTL mapping were tested by reciprocal hemizygosity analysis, validating the impact of two genes, PTC6 and YFL040W. The alleles of these validated genes from NA has better effect on the preference assay than the allele from WE, which is can be considered as antagonistic QTLs. The identification variation in these two genes underpinning metabolic differences, demonstrates how WE and NA have different attraction to one wild fruit fly line.

PTR-ToF-MS for the Online Monitoring of Alcoholic Fermentation in Wine: Assessment of VOCs Variability Associated with Different Combinations of Saccharomyces/Non-Saccharomyces as a Case-Study

The management of the alcoholic fermentation (AF) in wine is crucial to shaping product quality. Numerous variables (e.g., grape varieties, yeast species/strains, technological parameters) can affect the performances of this fermentative bioprocess. The fact that these variables are often interdependent, with a high degree of interaction, leads to a huge ‘oenological space’ associated with AF that scientists and professionals have explored to obtain the desired quality standards in wine and to promote innovation. This challenge explains the high interest in approaches tested to monitor this bioprocess including those using volatile organic compounds (VOCs) as target molecules. Among direct injection mass spectrometry approaches, no study has proposed an untargeted online investigation of the diversity of volatiles associated with the wine headspace. This communication proposed the first application of proton-transfer reaction-mass spectrometry coupled to a time-of-flight mass analyzer (PTR-ToF-MS) to follow the progress of AF and evaluate the impact of the different variables of wine quality. As a case study, the assessment of VOC variability associated with different combinations of Saccharomyces/non-Saccharomyces was selected. The different combinations of microbial resources in wine are among the main factors susceptible to influencing the content of VOCs associated with the wine headspaces. In particular, this investigation explored the effect of multiple combinations of two Saccharomyces strains and two non-Saccharomyces strains (belonging to the species Metschnikowia pulcherrima and Torulaspora delbrueckii) on the content of VOCs in wine, inoculated both in commercial grape juice and fresh grape must. The results demonstrated the possible exploitation of non-invasive PTR-ToF-MS monitoring to explore, using VOCs as biomarkers, (i) the huge number of variables influencing AF in wine, and (ii) applications of single/mixed starter cultures in wine. Reported preliminary findings underlined the presence of different behaviors on grape juice and on must, respectively, and confirmed differences among the single yeast strains ‘volatomes’. It was one of the first studies to include the simultaneous inoculation on two non-Saccharomyces species together with a S. cerevisiae strain in terms of VOC contribution. Among the other outcomes, evidence suggests that the addition of M. pulcherrima to the coupled S. cerevisiae/T. delbrueckii can modify the global release of volatiles as a function of the characteristics of the fermented matrix.

Nutritional and total phenolic analyses of unfermented and fermented red grape (Vitis vinifera) beverages

Grape juices-fresh, homemade fermented and commercial non-alcoholic were evaluated for their nutritional, Physico-chemical, phenolic and anthocyanin content. The grape juices obtained were clear, purple, and pH ranging between 3.3-3.5. The titrable acidity increased in the commercial grape juice (8.65%) whereas total solids (22.67%) was higher in the homemade fermented samples. The nutritional parameters such as carbohydrate, protein, dietary fibres etc. increased in the commercial sample except for vitamin C, which was higher in the fresh juice sample. The total phenolic content (2650 mg/lit) and total anthocyanin content (310 mg/lit) of commercial grape juice was higher compared to the homemade fermented sample. The increase in various parameters in the commercial sample could be attributed to manufacturing and processing techniques used during the production of grape juice.

Acid Lactic Bacteria as a Bio-Preservant for Grape Pomace Beverage

A probiotized juice may be an advantage as it provides an alternative to probiotic dairy products. In addition, the use of agricultural residue to supplement a probiotic juice may be a solution for overproduction of this organic material. Many studies have been developed with fruit residues; however a definitive solution to this environmental problem has not yet been established. Therefore the objective of this study was to propose a probiotized juice using lactic bacteria that, besides functioning as starter culture, has a potential biopreservative action and provide suitable stability for the final product. A fermented beverage was formulated using commercial grape juice, Vitis vinifera Pinot noir grape pomace and lactic bacteria. Pathogenic strains were used to simulate a microbial contamination. The procedures were carried out according to the American Public Health Association and the results were expressed in media with standard deviation using statistical analyses performed by Prism. Lactic bacteria showed a cell growth around 4 log CFU/ml. There was a significant decrease in pH values (p < 0.05) when pure grape juice was fermented. The grape juice supplemented with grape pomace from white winemaking was able to induce a higher growth of lactic bacteria population during fermentation, around 1 log cycle CFU/mL, comparing to juice without supplementation. The beverage containing grape juice, water and pomace also presented growth on lactic bacteria population but Lactobacillus rhamnosus reached a higher concentration, of approximately 108 CFU/ml after 12 hour of fermentation. This was also observed when beverages were stored, only L. rhamnosus remained viable for ten days at 10oC. For all beverage samples contaminated with pathogens, the lactic bacteria population remained around 2 log CFU/g above the added pathogen population, and the final probiotic population was above 108 CFU/mL, but L. rhamnosus wasn`t able to reduce significantly pathogenic strains, however a bacteriostatic effect was observed. A probiotic grape juice supplemented with pomace was produced with viable probiotic cell during 12 hours fermentation and stored for 10 days under refrigeration temperature. And a potential bacteriostatic effect against food pathogens was observed.

Trans-Stilbenes in Commercial Grape Juices: Quantification Using HPLC Approaches.

Trans-stilbenes belong to the group of polyphenolic phytoalexins, and occur in many plant foods. These compounds have received great attention by researchers due to their well-known beneficial health effects. In the present study a chromatographic method that comprises the use of variable wavelength (VWD) and fluorescence (FLD) detectors in series for the analysis of trans-stilbenes is presented. The relation of peak-area obtained with both detectors is proposed as an alternative and complementary approach for the rapid identification of these phenolic compounds. The proposed method was applied to determine trans-stilbenes in commercial fruit juices. Trans-piceid was the most common trans-stilbene found in the samples analyzed. The method was validated in terms of linearity, sensitivity and repeatability. Appropriate sensitivity and good linearity (r2 > 0.9991) were achieved.

Detection of addition of apple juice in purple grape juice

Due to increasing grape juice consumption, quality control is a reality in all countries which produce and consume this product. The most common form of adulteration is by substituting grape juice with apple juice. The adulterated samples can be identified by specific analysis, since apple juice has some compounds that grape has not. There are many studies about the phlorizin and sorbitol content in wines, but in grape juice they are scarce. Therefore, analytical methods to identify the composition of grape juices and determine their authenticity are needed to ensure quality control. The present study aimed to the validation and application of analytical methods for the determination of phlorizin and sorbitol, to detect the addition of apple juice in purple grape juice, by high-performance liquid chromatography. Initially, the methods were validated and tests were conducted by additions of percentages of apple juices in grape juices. Finally, experimental and commercial grape and apple juices were analyzed. Our study found that both methods are effective in detecting the addition of apple juice in grape juice. Four of the 39 commercial grape juices analyzed showed adulteration by the addition of apple juice in grape juice.

Phlorizin and sorbitol inVitis labruscagrape juices

In Brazil, grape juice is a not fermented beverage, made from Vitis labrusca varieties and their hybrids. The most common form of adulteration is by the addition of apple juice. The adulterated samples can be identified by specific analysis, since apple juice has some compounds that grape has not. A more simplified and assessible way to determine this kind of adulteration is the phlorizin analysis by high performance liquid chromatography (HPLC). Phlorizin is a phenolic compound that has been used to identify adulterations in juices. Besides phlorizin, it can be seen that adulteration by quantifying the levels of sorbitol, present in apples, but absent in healthy grapes. The content of this compounds in grape juices can vary due to the variety of grape and harvest.This study aimed to analyze phlorizin and sorbitol, in 141 experimental samples of Vitis labrusca grape juices, harvest 2016, of 9 varieties and 43 commercial samples (different vintages). The experimental juices from red grapes showed higher sorbitol content than the white grapes. For sorbitol analysis, the juices of Ives differed statistically of the juices of: Isabella, Concord, Niagara Branca and White Muscat. It was detected the presence of apple juice in 5 commercial grape juices.

Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads

Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20μg/kg OTA and after 48h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72–120h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed.

Phenolic compounds and antioxidant activity of Spanish commercial grape juices

Evaluation of the antioxidant activity of components of the diet is important in order to establish healthy consumption patterns. Data are reported here on the antioxidant activity (FRAP and ABTS), of 20 commercial grape juices and 10 typical Spanish wines and on their content of total phenolic compounds, anthocyanins, flavonoids and 10 individual phenolic compounds (flavanols, benzoic acids and cinnamic acids, measured by HPLC-UV). Red grape juices had a significantly higher (p<0.05) concentration of total phenols (1177 vs. 744mggallic acid/L), flavonoids (98 vs. 63mgcatechin/L) and a higher antioxidant activity (9.16 vs. 2.83meqTrolox/L) in comparison to white grape juices. In comparison to the white wines, white grape juices contained more total phenols (744 vs. 286mggallic acid/L) and flavonoids (63 vs. 29mgcatechin/L) and evidenced higher antioxidant activity (p<0.05). In comparison to the red wines, a lower content of total phenols (286 vs. 2358mggallic acid/L) and flavonoids (228 vs. 29mgcatechin/L) and an absence of anthocyanins were observed in the white wines, which are therefore less antioxidant. Although a two-fold higher concentration of antioxidant compounds was found in red wines than in red grape juices, the latter may be a good option for all age groups because of the absence of alcohol and the potentially beneficial health effects of their phenolic composition and elevated antioxidant activity.

Controlled depositing of silver nanoparticles on flexible film and its application in ultrasensitive detection

Surface-enhanced Raman scattering (SERS) has been proven to be a powerful analytical tool for the detection of trace molecules because of its integration of high sensitivity, unique spectroscopic fingerprint, and non-destructive data acquisition. However, the lack of reliable, stable, well-defined, and uniform SERS substrates impedes their further practical applications. Herein, we have developed a SERS-active substrate based on a flexible and stable polymer (PDMS) film loaded with nanoparticles for ultrasensitive Raman signal readout and trace detection of pesticide residues in agricultural products and environments. By controlling the deposition of Ag nanoparticles, tremendous Raman enhancement can be achieved due to numerous hot spots generated among the Ag nanoparticles on the PDMS composite film. The flexible SERS film can be employed to detect rhodamine 6G (R6G) with an enhancement factor (EF) of 3.37 × 1011 and the pesticide thiram in commercial grape juice with a detection limit of as low as 0.1 μm (0.03 ppm), which is considerably lower than the maximum residue limit (MRL) of 7 ppm in fruit prescribed by the U.S. Environmental Protection Agency (EPA). Furthermore, spiked detection indicated that the AgNP–PDMS flexible film can be used to monitor thiram in commercial grape juice and natural lake water without further treatment. Therefore, the AgNP–PDMS flexible film enhanced Raman spectroscopic technique offers great potential for practical applications in the on-site monitoring and assessment of pesticide residues in agricultural products and environments.

Evaluation of potential side activities of commercial enzyme preparations used in winemaking

SummaryTwenty‐one commercial enzyme preparations used in winemaking were characterised for the α‐L‐rhamnosidase, α‐L‐arabinosidase, β‐D‐xylosidase, β‐D‐galactosidase, β‐D‐glucosidase, esterase, protease, cinnamoyl esterase and laccase activities. A new rapid fluorimetric method to assay esterase activity was developed. Enzyme preparations differed for the level of each enzyme activity assayed rather than for the type of enzymatic activity detected. High levels of protease, glycosidase, esterase and cinnamoyl esterase activity were found among enzyme preparations for different technological applications. A drastic reduction in the level of cinnamoyl esterase was observed in commercial grape juice, pH 3.6, total acidity 5.3 g L−1, sugar 170 g L−1. Protease activity was only weakly reduced, from 10 to 20%, in commercial grape juice. β‐glucosidase activity levels are reduced in the presence of increasing concentration of glucose but are still present at the higher glucose concentration (100 g L−1). The extent of the reduction observed was dependent on the enzymatic preparations tested.

GRAPE JUICE QUALITY CONTROL BY MEANS OF1H NMR SPECTROSCOPY AND CHEMOMETRIC ANALYSES

This work shows the application of 1H NMR spectroscopy and chemometrics for quality control of grape juice. A wide range of quality assurance parameters were assessed by single 1H NMR experiments acquired directly from juice. The investigation revealed that conditions and time of storage should be revised and indicated on all labels. The sterilization process of homemade grape juices was efficient, making it possible to store them for long periods without additives. Furthermore, chemometric analysis classified the best commercial grape juices to be similar to homemade grape juices, indicating that this approach can be used to determine the authenticity after adulteration.

Dispersive Liquid–Liquid Microextraction Followed by Capillary High-Performance Liquid Chromatography for the Determination of Six Sulfonylurea Herbicides in Fruit Juices

In this study a simple, rapid, and efficient method has been developed for the determination of six sulfonylurea herbicides (SUHs): triasulfuron, metsulfuron-methyl, chlorsulfuron, flazasulfuron, chlorimuron-ethyl, and primisulfuron-methyl in commercial grape and apple juice samples, using dispersive liquid–liquid microextraction coupled with capillary high-performance liquid chromatography with diode array detection. Various parameters that influence the extraction efficiency, such as the type and volume of extraction and disperser solvents, sample pH, and salt addition, were investigated and optimized. Under the optimum conditions, limits of detection and quantification of the method were in the ranges of 2–9 and 8–29 μg L−1, respectively, lower than the maximum residue limits set by the European Union for the raw fruits, such as grape and apple. The intra- and inter-day relative standard deviations varied from 1.0 to 8.2 and 1.8 to 9.8 %, respectively, with recoveries between 72.0 and 109.5 % for commercial grape (both white and red) and apple juice samples, showing satisfactory accuracy for the determination of SUHs in fruit juices.

Preparation of resveratrol-enriched grape juice from ultrasonication treated grape fruits

Grape (Vitis spp.) is a major source of resveratrol that can be eaten directly or after making jam, jelly, wine and juice. Resveratrol (3,5,4′-trihydroxystilbene) has a profound positive influence on human health, including anti-carcinogenic, anti-cancer, anti-inflammatory, and anti-ageing effects and the ability to lower blood sugar. During industrial production of grape juice, resveratrol is lost because of the use of clarifying agents and filtration; therefore, commercial grape juice contains very low amounts of resveratrol. In this study, we investigated the accumulation of resveratrol in grape juice prepared from three varieties of grape, viz. Campbell Early, Muscat Bailey A (MBA) and Kyoho, following post-harvest ultrasonication cleaning for 5min and 6h of incubation in the dark at 25°C. This process resulted in the amounts of resveratrol increasing by 1.53, 1.15 and 1.24times in juice prepared from Campbell Early, MBA and Kyoho, respectively, without changing the amounts of total soluble solids. Overall, our results indicate that ultrasonication treatment of post-harvested grape fruits can be an effective method for producing resveratrol-enriched grape juice as well as cleaning grapes thoroughly.

Deconstructing a Fruit Serving: Comparing the Antioxidant Density of Select Whole Fruit and 100% Fruit Juices

Research suggests phytonutrients, specifically phenolic compounds, within fruit may be responsible for the putatively positive antioxidant benefits derived from fruit. Given the prominence of fruit juice in the American diet, the purpose of this research was to assess the antioxidant density of fresh fruit and 100% fruit juice for five commonly consumed fruits and juices and to compare the adequacy of 100% juice as a dietary equivalent to whole fruit in providing beneficial antioxidants. Antioxidant density was measured using an oxygen radical absorbance capacity method on six samples assayed in triplicate for each fruit (grape, apple, orange, grapefruit, pineapple), name-brand 100% juice, and store-brand 100% juice. One-way analysis of variance and Tukey’s honestly significant difference or Student t test were used to assess significance (P<0.05). Antioxidant density (mmol TE/100 g) of apple, orange, and grapefruit was 23% to 54% higher than the mean antioxidant density of name-brand and store-brand juices for each fruit; however, only apple and grapefruit exhibited significantly greater (P<0.05) antioxidant density than either of their name-brand or store-brand juices. In contrast, the mean antioxidant density of name-brand grape and pineapple juice was higher than fresh grape or pineapple fruit; however, both fresh grapes and commercial grape juice contained significantly more (P<0.05) antioxidants than store-brand grape juice. Regardless of the convenience of fruit juice, results support the recommendations of the 2010 Dietary Guidelines for Americans for increasing fruit servings in the whole fruit form due to their provision of beneficial antioxidants and fiber with approximately 35% less sugar.

Graphene oxide embedded sandwich nanostructures for enhanced Raman readout and their applications in pesticide monitoring

Analytical techniques based on surface-enhanced Raman scattering (SERS) suffer from a lack of reproducibility and reliability, thus hampering their practical applications. Herein, we have developed a SERS-active substrate based on a graphene oxide embedded sandwich nanostructure for ultrasensitive Raman signal readout. By using this novel Au@Ag NPs/GO/Au@Ag NPs sandwich nanostructure as a SERS substrate, the Raman signals of analytes were dramatically enhanced due to having plenty of hot spots on their surfaces and the unique structure of the graphene oxide sheets. These features make the sandwich nanostructured film an ideal SERS substrate to improve the sensitivity, reproducibility and reliability of the Raman readout. The sandwich nanostructure film can be applied to detect rhodamine-6G (R6G) with an enhancement factor (EF) of ∼7.0 × 10(7) and the pesticide thiram in commercial grape juice with a detection limit of as low as 0.1 μM (0.03 ppm), which is much lower than the maximal residue limit (MRL) of 7 ppm in fruit prescribed by the U.S. Environmental Protection Agency (EPA). The GO embedded sandwich nanostructure also has the ability to selectively detect dithiocarbamate compounds over other types of agricultural chemical. Furthermore, spiked tests show that the sandwich nanostructure can be used to monitor thiram in natural lake water and commercial grape juice without further treatment. In addition, the GO enhanced Raman spectroscopic technique offers potential practical applications for the on-site monitoring and assessment of pesticide residues in agricultural products and environments.

Analytical characteristics and discrimination of Brazilian commercial grape juice, nectar, and beverage

The production and commercialization of Brazilian grape juice is increasing annually, mainly due to its typicality, quality, and nutritional value. The present research was carried out in view of the great significance of Brazilian grape juice for the grape and wine industry. The purpose of this study, therefore, was to assess its composition as well as the discrimination between grape juice and other beverages. Twenty four samples of whole, sweetened, and reprocessed grape juices, grape nectar, and grape beverage were evaluated. Classical variables were analyzed by means of physicochemical methods; tartaric and malic acids, by HPLC; methanol, by gas chromatography; minerals, by atomic absorption spectrophotometry. These products were discriminated by the Principal Component Analysis (PCA). Results show that whole and sweetened grape juices were discriminated from other grape products because they featured higher values of total soluble solids, tartaric and malic acids, most minerals, phenolic compounds, and K/Na ratio, whereas grape nectar and grape beverage presented higher values of ºBrix/titratable acidity ratio. Reprocessed juice was discriminated due to its higher concentrations of Li and Na and lower hue.