Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction,using micellar electrokinetic chromatography and reverse-phase liquid chromatography

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.     

Effect of processing techniques at industrial scale on orange juice antioxidant and beneficial health compounds

Phenolic compounds, vitamin C (L-ascorbic acid and L-dehydroascorbic acid), and antioxidant capacity were evaluated in orange juices manufactured by different techniques. Five processes at industrial scale (squeezing, mild pasteurization, standard pasteurization, concentration, and freezing) used in commercial orange juice manufacturing were studied. In addition, domestic squeezing (a hand processing technique) was compared with commercial squeezing (an industrial FMC single-strength extraction) to evaluate their influences on health components of orange juice. Whole orange juice was divided into soluble and cloud fractions after centrifugation. Total and individual phenolics were analyzed in both fractions by HPLC. Commercial squeezing extracted 22% more phenolics than hand squeezing. The freezing process caused a dramatic decrease in phenolics, whereas the concentration process caused a mild precipitation of these compounds to the juice cloud. In pulp, pasteurization led to degradation of several phenolic compounds, that is, caffeic acid derivatives, vicenin 2 (apigenin 6,8-di-C-glucoside), and narirutin (5,7,4'-trihydroxyflavanone-7-rutinoside) with losses of 34.5, 30.7, and 28%, respectively. Regarding vitamin C, orange juice produced by commercial squeezing contained 25% more of this compound than domestic squeezing. Mild and standard pasteurization slightly increased the total vitamin C content as the contribution from the orange solids parts, whereas concentration and freezing did not show significant changes. The content of L-ascorbic acid provided 77-96% of the total antioxidant capacity of orange juice. Mild pasteurization, standard pasteurization, concentration, and freezing did not affect the total antioxidant capacity of juice, but they did, however, in pulp, where it was reduced by 47%.     

Countercurrent supercritical fluid extraction and fractionation of alcoholic beverages

A procedure for the recovery of aromatic extracts from distilled alcoholic beverages by means of a countercurrent supercritical fluid extraction (CC-SFE) on a pilot plant scale is studied. The beverage is directly in contact with the carbon dioxide current in a packed column, and the extracts are recovered in two different fractionation cells, where the depressurization occurs. The proposed method allows the selective extraction of aromatic components of the brandy flavor, rendering a high-value concentrated extract and a colored residue without brandy aroma. The content in ethanol of the aromatic extract can be modified by tuning the extraction/fractionation conditions, rendering from 15 to 95% recovery. The effect of the main variables, including extraction pressure and quality of extracting CO(2), has been tested.     

New phenolic grape skin products from Vitis vinifera cv. Pinot Noir

Anthocyanins and their related compounds were extracted from grape skins of Pinot noir, using 50% aqueous methanol, and purified by solid phase extraction chromatography using XAD-7 resin to obtain a pigment-rich fraction. This fraction was subjected to multilayer coil countercurrent chromatography (MLCCC) using a quaternary solvent system consisting of tert-butyl methyl ether/n-butanol/acetonitrile/water acidified with 0.01% trifluoroacetic acid (2:2:0.1-1.8:5) (v/v/v/v) in a step gradient elution to separate anthocyanin oligomers from grape anthocyanins. In the process of the characterization of the MLCCC fractions by electrospray mass spectrometry, two noncolored anthocyanin derivatives were found and characterized on the basis of their mass spectral data. As a result, these compounds have been tentatively identified as coupling products between both hydrated malvidin-3-glucoside and peonidin-3-glucoside, with 2-S-glutathionyl caffeoyl tartaric acid (GRP). It is therefore proposed that grape skins contain this new class of coupling product, and a possible chemical pathway for their formation is suggested.     

Liquid chromatographic methodology for the characterization of orange juice

Liquid chromatographic (LC) methodology potentially useful for the characterization of orange juice, with particular regard to detecting adulteration of orange juice by computer pattern recognition analysis, has been developed. After dilution with methanol the juice is extracted with hexane to remove the carotenoids, which are chromatographed on a C18 column with an acetonitrile-methanol-methylene chloride mobile phase and detection at 450 nm. Further extraction of the juice with methylene chloride isolates the methoxylated flavones, which are chromatographed by reverse phase LC with an acetonitrile-methanol-water mobile phase and detection at 280 nm. The flavanone glycosides remaining in solution are chromatographed on a C18 column with an acetonitrile-water mobile phase and detection at 280 nm. The precisions of the heights of the 32 LC peaks selected for pattern recognition analysis were determined from 5 replicate analyses of a single juice. Coefficients of variation of the replicates ranged from 0.3 to 4.5%, with an average of 2.1%. Adulteration of products with sodium benzoate-fortified pulpwash or grapefruit juice can be detected by this method. Pattern recognition analysis of the data obtained for 80 authentic and 19 adulterated orange juices showed that the method is potentially useful for distinguishing between authentic and adulterated products.     

Countercurrent supercritical fluid extraction and fractionation of high-added-value compounds from a hexane extract of olive leaves

Countercurrent supercritical fluid extraction (CC-SFE) at a pilot scale plant was used for fractionation of high-added-value products from a raw extract of olive leaves in hexane. Compounds found in the raw extract were waxes, hydrocarbons, squalene, beta-carotene, triglycerides, alpha-tocopherol, beta-sitosterol, and alcohols. The CC-SFE extraction process was investigated according to a 2(3) full factorial experimental design using the following variables and ranges: extraction pressure, 75-200 bar; extraction temperature, 35-50 degrees C; and ethanol as modifier, 0-10%. Data were analyzed in terms of extraction yield, enrichment, recovery, and selectivity. Higher extraction yields were attained at 200 bar. For most of the compounds analyzed enrichment was attained at the same conditions, that is, 75 bar, 35 degrees C, and 10% ethanol. Hydrocarbons were usually recovered in the separators, whereas waxes and alpha-tocopherol remain in the raffinate. Selectivity data reveal that alpha-tocopherol is the most easily separable compound. The influence of the experimental factors on the recovery of all the compounds was studied by means of regression models. The best fitted model was attained for beta-sitosterol, with R2 = 99.25%.     

Identification of isolutein (lutein epoxide) as cis-antheraxanthin in orange juice

The carotenoid profile of orange juice is very complex, a common characteristic for citrus products in general. This fact, along with the inherent acidity of the product, which promotes the isomerization of some carotenoids, makes the correct identification of some of these pigments quite difficult. Thus, one of the carotenoids occurring in orange juice has been traditionally identified as isolutein, a term used to refer to lutein epoxide, although enough evidence to support that identification has not been given. In this study, the carotenoid previously identified as isolutein/lutein epoxide in orange juice has been isolated and identified as a 9 or 9'-cis isomer of antheraxanthin as a result of different tests. To support this identification, a mixture of geometrical isomers of lutein epoxide isolated from petals of dandelions was analyzed under the same conditions used for orange juice carotenoids to check that neither their retention times nor their spectroscopic features matched with those of the orange juice carotenoid now identified as a cis isomer of antheraxanthin.     

Subcritical water extraction of antioxidant compounds from rosemary plants

Subcritical water extraction at several temperatures ranging from 25 to 200 degrees C has been studied to selectively extract antioxidant compounds from rosemary leaves. An exhaustive characterization of the fractions obtained using subcritical water at different temperatures has been carried out by LC-MS, and the antioxidant activities of the extracts have been measured by a free radical method (DPPH). Results indicate high selectivity of the subcritical water toward the most active compounds of rosemary such as carnosol, rosmanol, carnosic acid, methyl carnosate, and some flavonoids such as cirsimaritin and genkwanin. The antioxidant activity of the fractions obtained by extraction at different water temperatures was very high, with values around 11.3 microg/mL, comparable to those achieved by SFE of rosemary leaves. A study of the effect of the temperature on the extraction efficiency of the most typical rosemary antioxidant compounds has been performed.     

Characterization of anthocyanins and pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice

Anthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juices were isolated and purified by means of high-speed countercurrent chromatography and preparative high-performance liquid chromatography. Structures of the pigments were then elucidated by electrospray ionization multiple mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The major anthocyanins of the juice were characterized as cyanidin 3-glucoside and cyanidin 3-(6"-malonylglucoside). Furthermore, six minor anthocyanins were detected and identified as cyanidin 3,5-diglucoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, delphinidin 3-(6"-malonylglucoside), peonidin 3-(6"-malonylglucoside), and cyanidin 3-(6"-dioxalylglucoside). The occurrence of the latter compound in blood oranges is reported here for the first time, together with full NMR spectroscopic data. Further investigations revealed the presence of four anthocyanin-derived pigments, which are formed through a direct reaction between anthocyanins and hydroxycinnamic acids during prolonged storage of the juice. These novel pyranoanthocyanins were identified as the 4-vinylphenol, 4-vinylcatechol, 4-vinylguaiacol, and 4-vinylsyringol adducts of cyanidin 3-glucoside through comparison of their mass spectrometric and chromatographic properties with those of synthesized reference compounds.     

Use of 2-furoylmethyl derivatives of GABA and arginine as indicators of the initial steps of maillard reaction in orange juice

The formation of 2-furoylmethyl derivatives of GABA (2-FM-GABA) and arginine (2-FM-Arg) as early indicators of nonenzymatic browning in different types of orange juice was studied. In dehydrated orange juice, the presence of 2-FM-GABA and 2-FM-Arg was detected from the first day of storage at 30 degrees C. In this type of juice, the content of these two compounds increased with temperature (30, 50 degrees C) and time (1-7 days) of storage. A noticeable increase in 5-hydroxymethylfurfural was only observed after 4 days of storage at 50 degrees C. No formation of 2-FM-GABA and 2-FM-Arg was detected in liquid orange juice heated under conditions similar to those used in the industry. These furoylmethyl derivatives were also found in commercial orange juice made from concentrates. A slight increase in their concentration was observed in the two samples stored during 8 months at room temperature. According to the results obtained, 2-FM-GABA and 2-FM-Arg contents could be suitable indicators to assess the main modifications due to Maillard reaction produced during the manufacture and/or storage of orange juice concentrates.     

Gas chromatographic determination of fatty acids and sterols in orange juice

A gas chromatographic (GC) method has been developed for the simultaneous quantitation of fatty acids and sterols in orange juice, using a bonded phase fused silica capillary column of intermediate polarity, splitless automatic injection, and flame ionization detection. Sample preparation has been simplified by using 1 g C-18 adsorbent in a disposable minicolumn to extract 2 mL orange juice. Methylation of fatty acids and silylation of the sterols were carried out in the eluted extract (low polarity lipid fraction). The method precision was 7%; recoveries ranged from 83 to 113%. The precision of the injection technique was 2%. Seven major fatty acids and 5 sterols in orange juice were quantitated by the GC method and identified by GC/mass spectrometry. Quantitative data for several orange juice samples indicated that the levels of the compounds of interest were in the 1.3-72.0 mg/L range. The results demonstrate that bonded phase fused silica capillary GC has great versatility and potential for the quantitative determination of fatty acids and sterols.     

锦橙汁辐照和巴氏灭菌处理后相关品质的分析

为探讨辐照和巴氏杀菌对橙汁品质的影响,对锦橙汁分别进行1.4、2.8、5.6kGy3种不同剂量辐照及巴氏灭菌处理,使用固相微萃取-气质联用技术对挥发性成分进行分析,测定橙汁色度、pH值和Vc含量,并对橙汁香气进行感官评定。在鲜橙汁、巴氏灭菌汁和3种辐照样品中分别检测到了54、47、57、55和53种成分,2.8kGy剂量辐照处理后橙汁挥发性成分总峰面积最高,β-月桂烯、柠檬烯和γ-松油烯等橙汁特征香气物质经辐照后保持率高于巴氏灭菌。各种处理后Vc含量均有所下降,橙汁橙香均减弱。1.4kGy辐照后的橙汁感官评价结果最好,因此可以对橙汁进行低剂量辐照灭菌。     

Characterization of paprika (Capsicum annuum) extract in orange juices by liquid chromatography of carotenoid profiles.

The carotenoid pigment profiles of authentic pure orange juices from Spain and Florida and an industrial paprika (Capsicum annuum) extract used for food coloring were obtained using reversed-phase liquid chromatography with a C18 packed column and an acetone/methanol/water eluent system. The procedure involving the carotenoid extraction is described. Both retention times and spectral properties using photodiode array detection for characterization of the major carotenoids at 430 and 519 nm are given. The influence of external addition of tangerine juice and/or paprika extract on orange juice color is described using the U.S. Department of Agriculture scale and adulterated orange juice. The procedure for quantitation of externally added paprika extract to orange juice is investigated, and the limit of quantitation, coefficient of variation, and recoveries are determined.     

Effect of ultrahigh-temperature continuous ohmic heating treatment on fresh orange juice

The scope of this study is the effect of ohmic heating thermal treatment on liquid fruit juice made of oranges. Effects of ohmic heating on the quality of orange juice were examined and compared to those of heat pasteurization at 90 degrees C for 50 s. Orange juice was treated at temperatures of 90, 120, and 150 degrees C for 1.13, 0.85, and 0.68 s in an ohmic heating system. Microbial counts showed complete inactivation of bacteria, yeast, and mold during ohmic and conventional treatments. The ohmic heating treatment reduced pectin esterase activity by 98%. The reduction in vitamin C was 15%. Ohmic-heated orange juice maintained higher amounts of the five representative flavor compounds than did heat-pasteurized juice. Sensory evaluation tests showed no difference between fresh and ohmic-heated orange juice. Thus, high-temperature ohmic-heating treatment can be effectively used to pasteurize fresh orange juice with minimal sensory deterioration.     

Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.     

Sensitive micellar electrokinetic chromatography-laser-induced fluorescence method to analyze chiral amino acids in orange juices

In this work a new method to detect the existence of chiral amino acids in orange juice is presented. The method employs beta-cyclodextrins and micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) to separate and detect L- and D-amino acids (L-aa and D-aa) previously derivatized with fluorescein isothiocianate (FITC). A systematic optimization of the chiral-MEKC conditions is done bringing about in less than 20 min a good separation of the main amino acids found in orange juice (i.e., Pro, Asp, Ser, Asn, Glu, Ala, Arg, and the nonchiral GABA, i.e., gamma-aminobutyric acid). Using this procedure, the analysis time reproducibility for the 15 standard compounds (L-aa, D-aa, and GABA) has been determined to be better than 0.2% (n = 5) for the same day and better than 0.7% (n = 15) for three different days. Corrected peak area reproducibility is somewhat lower, providing values better than 3.3% (n = 5) for the same day and 6.9% (n = 15) for three different days. The limit of detection using this procedure was determined to be 0.86 attomoles for L-Arg. The optimized FITC derivatization method allows the easy and straightforward detection of amino acids in orange concentrates and juices (i.e., only centrifugation of diluted samples for 5 min is needed prior to their derivatization). D-Ala, D-Asp, D-Arg, and D-Glu were determined in orange juices and orange concentrates from different geographical origins using this new method. Moreover, the effect of different temperature treatments (50, 92, and 150 degrees C) on the content of D-aa in orange juice was evaluated.