Purification of citrus peel juice and molasses

Citrus peel juice and molasses are extremely bitter and unpalatable byproducts of orange and grapefruit juice production. Major components of interest are soluble sugars, glucose, fructose, and sucrose, which account for 60-70% of the dry solids. Analyses indicate that the remaining components are suspended tissue fragments, proteins, organic acids, mineral ions, phenolic compounds, and polyols. A purification sequence that removed a majority of bitter limonoids and phenolic compounds by adsorption on nonionic, macroporous resins was tested. Residual phenolic compounds were removed by adsorption on activated carbon or anion-exchange resin, which also removed anions of organic and inorganic acids. Taste panel results suggested that debittered products could be acceptable for food uses.     

Hypolipidemic effects and absorption of citrus polymethoxylated flavones in hamsters with diet-induced hypercholesterolemia

Formulations containing citrus polymethoxylated flavones (PMFs), mainly tangeretin, or citrus flavanone glucosides, hesperidin and naringin, were evaluated for cholesterol-lowering potential in hamsters with diet-induced hypercholesterolemia. PMF metabolites were also investigated. Diets containing 1% PMFs significantly reduced serum total and very low-density lipoprotein (VLDL) + LDL cholesterol (by 19-27 and 32-40%, respectively) and either reduced or tended to reduce serum triacylglycerols. Comparable reductions were achieved by feeding a 3% mixture of hesperidin and naringin (1:1, w/w), implying lower hypolipidemic potency of the hesperidin/naringin vs PMFs. HPLC-MS analysis identified high serum, liver, and urine concentrations of tangeretin metabolites including dihydroxytrimethoxyflavone and monohydroxytetramethoxyflavone glucuronides and aglycones. Total liver concentrations of tangeretin derivatives corresponded to hypolipidemic concentrations of intact tangeretin in earlier experiments in vitro. This suggests that PMFs are novel flavonoids with cholesterol- and triacylglycerol-lowering potential and that elevated levels of PMF metabolites in the liver might be directly responsible for their hypolipidemic effects in vivo.     

Structure-activity relationship of citrus polymethoxylated flavones and their inhibitory effects on Aspergillus niger

Citrus peels are rich in polymethoxylated flavones (PMFs) and are potential sources of natural preservatives. Six PMFs extracts, isolated and purified from the peels of three mandarins (Citrus reticulata) and three sweet oranges (Citrus sinensis), were identified and quantitated. Their inhibitory effects on Aspergillus niger were evaluated using a microbroth dilution assay. The Red tangerine variety exhibited the greatest antifungal activity (MIC = 0.2 mg/mL), while Jincheng showed the lowest activity (MIC = 1.8 mg/mL). An analysis of principal components was applied to the results in order to elucidate the structure-activity relationships of the citrus PMFs. The structure-activity relationship analysis revealed that, for good inhibitory effect, the 5-OH, 3-OCH?, and 8-OCH? functionalities were essential, while the presence of 3-OH and 3'-OCH? greatly reduced inhibition. The findings of this study provide important information for the exploitation and utilization of citrus PMFs as natural biopreservatives.     

Selective recovery of anthocyanins and hydroxycinnamates from a byproduct of citrus processing

The adsorption-desorption performances of commercial resins (two polystyrene-divinylbenzene copolymers and one methacrylic) were tested in column studies for the selective recovery of anthocyanins and hydroxycinnamates from pigmented pulp wash (PW), a byproduct of blood orange juice processing. Methanol, ethanol, and their mixtures with different percentages of water as eluents were tested in order to investigate the selective desorption of these natural antioxidants with the goal of minimizing the presence of other compounds, mostly flavanones and limonoids, in the concentrated eluates. The results indicated that polystyrene-divinylbenzene resins were able to adsorb a major amount of anthocyanins and hydroxycinnamates and to desorb them in more-concentrated fractions. No selectivity was observed using pure methanol and ethanol which resulted in concentrates with no further separation of components from the starting raw PW. The ethanol/water concentrates showed higher yields than the fractions attained using methanol/water; however, a lower selectivity toward anthocyanin pigments was observed. The best performing resin, EXA-118, together with the mixture methanol/water 50:50 (v/v) as best eluent, appears to be the most suitable system to obtain highly concentrated extracts. Thus, it was chosen to perform a larger experiment, to analyze the selectivity of the removal upon an increase in elution volume.     

Extraction parameters and capillary electrophoresis analysis of limonin glucoside and phlorin in citrus byproducts.

Limonin glucoside (LG) and phlorin were extracted from citrus fruit tissues and assayed by capillary electrophoresis (CE). LG was determined in dried [1.20 +/- 0.10 mg of dry weight (dw)] and wet peel residues (1.16 +/- 0.04 mg of dw), orange juice finisher pulp (0.58 +/- 0.03 mg of dw), dried grapefruit seeds (2.70 +/- 0.15 mg of dw), and 50 degrees Brix molasses (2225 +/- 68 mg/L). Phlorin was purified from orange peel residue and grapefruit albedo, and concentrations were determined in some citrus products. Phlorin and LG were extracted from residues with water/pectinase or with water solutions of methanol and ethanol. Efficient LG extraction from grapefruit seeds (2.40 +/- 0.15 mg/g) was achieved with 50-65% methanol, solvent polarity P' approximately equal to 7-8. Extracts were purified and concentrated by adsorptive resins and HPLC to obtain 95% pure compounds of LG and phlorin. CE analysis did not require extract purification beyond filtration. LG and phlorin migrated as anions in electropherograms containing peaks representing other citrus flavonoids and limonoid glucosides.     

Lipolytic effects of citrus peel oils and their components

This study was conducted to determine the lipolytic effects of eight kinds of citrus peel oils and their components. All of the citrus peel oils revealed lipolytic effects on olive oil model solution ranging from 10.9 to 73.8%. Hakyul (Citrus natsudaidai Hayata) showed the highest lipolytic effect (73.8%), followed by yuza (Citrus junos Sieb. ex Tanaka, 68.1%) and lemon (Citrus limonium, 63.4%), and their effects were comparable with or stronger than that of 5 mM raspberry ketone (p < 0.05). Among 17 authentic compounds relating to citrus peel oils, octanal (78.6%) showed the highest lipolytic effect, followed by gamma-terpinene (76.3%), limonene (75%), terpinen-4-ol (70.7%), nerol (69.9%), p-cymene (67.7%), and geranyl acetate (67.2%), and their effects were stronger than that of 5 mM raspberry ketone (p < 0.05). Ethyl acetate, alpha-pinene, myrcene, citronellal, linallyl acetate, and citronellol exhibited poor lipolytic effect in the model solution. Lipolytic effect was found to be high when the oils included a higher content of gamma-terpinene and p-cymene. Limonene showed potential lipolytic effect, and its effect is likely to be enhanced by the presence of gamma-terpinene and p-cymene. It is considered that monoterpene hydrocarbons consisting of one or two double bonds would have stronger lipolytic effect than those having three double bonds.     

宽皮柑橘移动夹持剥皮力学特性与果皮分离特性试验研究

为了解决目前柑橘剥皮设备,果皮一次剥净率较低,且损伤率较高。需要人工二次剥皮等问题,探索宽皮柑橘剥皮方法,该文以温州蜜柑为研究对象,利用自制的试验平台开展柑橘剥皮试验,对比了不同加载条件(不同剥皮方向、剥皮宽度及剥皮速度)对温州蜜柑的果皮分离过程中拉力值以及果皮分离位移的影响规律。试验结果表明:1)柑橘的剥皮特性具有各向异性,剥皮方向对剥皮特性具有显著性影响(P0.05),沿柑橘轴向方向剥皮,剥离的果皮长度与径向相比长约15%,且剥皮过程果皮能够均匀剥离;2)柑橘果皮分离过程中大致存在3种果皮断裂形态,其中剥离的果皮形态呈对称状,剥皮过程较为平稳;而剥离的果皮形态呈偏移或是果皮边缘带锯齿状,剥皮过程均存在波动过程,果皮呈斜向撕裂,剥离的果皮长度小于正向撕裂的对称状果皮长度;3)剥皮宽度、剥皮速度对柑橘果皮分离的最大拉力值有显著影响(P0.05),对果皮分离位移影响不显著(P0.05),其对剥皮长度影响较小,根据试验指标及自身剥皮设备参数,夹持较宽(采用环割划皮)的果皮,较高的剥皮速度(300 mm/min以上),利于提高剥皮效率。该研究可为宽皮柑橘的机械剥皮加工设备提供一定的理论依据。     

Liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance as complementary analytical techniques for unambiguous identification of polymethoxylated flavones in residues from molecular distillation of orange peel oils (Citrus sinensis)

Liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance techniques with ultraviolet/diode array detection were used as complementary analytical tools for the reliable identification of polymethoxylated flavones in residues from molecular distillation of cold-pressed peel oils of Citrus sinensis. After development of a liquid chromatographic separation procedure, the presence of several polymethoxy flavones such as sinensetin, nobiletin, tangeretin, quercetogetin, heptamethoxyflavone, and other derivatives was unambiguously confirmed. In addition, proceranone, an acetylated tetranortriterpenoid with limonoid structure, was identified for the first time in citrus.     

Fractionation of orange peel phenols in ultrafiltered molasses and mass balance studies of their antioxidant levels

Orange peel molasses, a byproduct of juice production, contains high concentrations of phenols, including numerous flavanone and flavone glycosides, polymethoxylated flavones, hydroxycinnamates, and other miscellaneous phenolic glycosides and amines. Extensive fractionation of these phenols was achieved by adsorption, ion exchange, and size exclusion chromatography. Size exclusion chromatography effectively separated the different classes of flavonoids in ultrafiltered molasses, including the polymethoxylated flavones, flavanone-O-trisaccharides, flavanone- and flavone-O-disaccharides, and, finally, flavone-C-glycosides. Mass spectral analysis of the early-eluting flavonoid fractions off the size exclusion column revealed a broad collection of minor-occurring flavone glycosides, which included, in part, glycosides of limocitrin, limocitrol, and chrysoeriol. Most hydroxycinnamates in the molasses were recovered by ion exchange chromatography, which also facilitated the recovery of fractions containing many other miscellaneous phenols. Total antioxidant levels and total phenolic contents were measured for the separate categories of phenols in the molasses. Inhibition of the superoxide anion reduction of nitroblue tetrazolium showed that a significant amount of the total antioxidant activity in orange peel molasses was attributable to minor-occurring flavones. The miscellaneous phenolic-containing fractions, in which a large portion of the total phenolic content in molasses occurred, also constituted a major portion of the total antioxidants in ultrafiltered molasses.     

毛霉诱导脐橙产抗病物质对指状青霉和酸腐菌的抑制

绿霉和酸腐是柑橘果实采后主要的病害,生产上迫切需要化学杀菌剂的替代方法。该研究对毛霉诱导脐橙果皮产生的具有抗病活性的红色物质(Citru Peel Red-substance Extract,CPRE)进行部分分离纯化,评价了CPRE对指状青霉和酸腐菌的抑菌活性,分析活性成分对两病原真菌菌丝细胞壁与膜功能的影响。结果表明,在50～200μg/mL的质量浓度下,CPRE对指状青霉和酸腐菌的孢子萌发,芽管伸长,菌丝生长有强烈的抑制作用,并且呈现剂量效应,最低抑菌浓度均为200μg/mL。进一步研究表明,与空白对照比较,50、100μg/mL抗菌活性成分处理明显改变指状青霉和酸腐菌菌丝细胞壁中几丁质分布,降低了细胞壁中几丁质含量、促进了质外体碱性磷酸酶的释放,表明CPRE严重损伤两致病真菌菌丝的细胞壁功能;CPRE处理还明显损伤两病菌菌丝的细胞膜通透性(P<0.05),刺激菌丝膜外电导率显著上升(P<0.05),加剧细胞内蛋白和核酸的丢失,降低了菌丝细胞膜总脂质含量,影响了细胞膜的正常功能。上述结果表明,红色抗菌成分在防控柑橘采后绿霉病和酸腐病中有潜在应用前景。     

Volatile constituents of wild citrus Mangshanyegan (Citrus nobilis Lauriro) peel oil

Volatiles of a wild mandarin, Mangshanyegan (Citrus nobilis Lauriro), were characterized by GC-MS, and their aroma active compounds were identified by aroma extract dilution analysis (AEDA) and gas chromatography-olfactometry (GC-O). The volatile profile of Mangshanyegan was compared with those of other four citrus species, Kaopan pummelo (Citrus grandis), Eureka lemon (Citrus limon), Huangyanbendizao tangerine (Citrus reticulata), and Seike navel orange (Citrus sinensis). Monoterpene hydrocarbons predominated in Mangshanyegan, in particular d-limonene and β-myrcene, which accounted for 85.75 and 10.89% of total volatiles, respectively. Among the 12 compounds with flavor dilution factors (FD) = 27, 8 oxygenated compounds, including (Z)- and (E)-linalool oxides, were present only in Mangshanyegan. The combined results of GC-O, quantitative analysis, odor activity values (OAVs), and omission tests revealed that β-myrcene and (Z)- and (E)-linalool oxides were the characteristic aroma compounds of Mangshanyegan, contributing to the balsamic and floral notes of its aroma.     

Effect of heat treatment on the phenolic compounds and antioxidant capacity of citrus peel extract

This paper reports the effects of heat treatment on huyou (Citrus paradisi Changshanhuyou) peel in terms of phenolic compounds and antioxidant capacity. High-performance liquid chromatography (HPLC) coupled with a photodiode array (PDA) detector was used in this study for the analysis of phenolic acids (divided into four fractions: free, ester, glycoside, and ester-bound) and flavanone glycosides (FGs) in huyou peel (HP) before and after heat treatment. The results showed that after heat treatment, the free fraction of phenolic acids increased, whereas ester, glycoside, and ester-bound fractions decreased and the content of total FGs declined (P < 0.05). Furthermore, the antioxidant activity of methanol extract of HP increased (P < 0.05), which was evaluated by total phenolics contents (TPC) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS*+) method, and ferric reducing antioxidant power (FRAP) assay. The correlation coefficients among TPC, ABTS, FRAP assay, and total cinnamics and benzoics (TCB) in the free fraction were significantly high (P < 0.05), which meant that the increase of total antioxidant capacity (TAC) of HP extract was due at least in part to the increase of TCB in free fraction. In addition, FGs may be destroyed when heated at higher temperature for a long time (for example, 120 degrees C for 90 min or 150 degrees C for 30 min). Therefore, it is suggested that a proper and reasonable heat treatment could be used to enhance the antioxidant capacity of citrus peel.     

Carbohydrate metabolism as related to high-temperature conditioning and peel disorders occurring during storage of citrus fruit

The aim of this research was to understand the involvement of the carbohydrate metabolism in physiological disorders occurring during the postharvest storage of citrus fruit. These disorders, manifested in the rind, depreciate fruit quality and often originate important losses. There has been increasing interest in the use of nonharmful treatments, such as high-temperature conditioning, to avoid citrus peel damage during fruit storage at low temperature in chilling-sensitive cultivars, but their influence in postharvest disorders occurring at nonchilling temperatures and the mechanisms related to them are poorly understood. The data obtained showed that heat conditioning (3 days/37 degrees C) increases the chilling tolerance of cv. Navelate fruit and favored sucrose, but not hexoses, accumulation and its maintenance after the fruit was transferred to low temperature. This effect was related to heat-induced increase in the activities of the sucrose-synthesizing enzymes sucrose phosphate synthase (SPS) and sucrose synthase (SS). Furthermore, sucrose levels and the activities of both enzymes were higher in cv. Pinalate oranges, a chilling-tolerant spontaneous abscisic acid deficient mutant of Navelate. In contrast, carbohydrates appeared not to be involved in the susceptibility of oranges to rind staining, a physiological disorder different from chilling injury, which mainly occurred at a nonchilling temperature (12 degrees C) and was not reduced by heat conditioning. The effect of low temperature in SS and SPS activities was less than that of high temperature, which might be related to the lower changes occurring in sucrose during fruit storage at 2 degrees C.     

Absorption of hydroxycinnamates in humans after high-bran cereal consumption

Hydroxycinnamic acids are a group of phenolic compounds that exhibit a wide range of in vitro chemoprotective and antioxidant properties. Cereals containing a high proportion of the bran layers are rich in ester-linked hydroxycinnamic acids, such as ferulic and diferulic acids. The present work investigated the absorption in humans of hydroxycinnamic acids from high-bran breakfast cereal (wheat). Plasma and urine samples from six volunteers were collected before and after cereal consumption and analyzed for total hydroxycinnamic acids content after beta-glucuronidase/sulfatase treatment both by HPLC-DAD and by LC-MS (SIM monitoring). High-bran cereal administration resulted in increased plasma ferulic and sinapic acid concentrations (maximum levels detected of approximately 200 and approximately 40 nM, respectively) with absorption peaks between 1 and 3 h. Increases of approximately 4-fold in ferulic acid and approximately 5-fold in feruloylglycine were detected in 24-h urine after consumption of the cereal. Most of the ferulic acid detected in urine and plasma was present as conjugates (feruloylglycine and/or glucuronides). Diferulic acids were undetectable. The data show that ferulic and sinapic acids are taken up in humans from dietary high bran wheat but that absorption is limited and may originate only from the free and soluble portions present in the cereal.     

Polymethoxylated flavones and other phenolic derivates from citrus in their inhibitory effects on P-glycoprotein-mediated transport of talinolol in Caco-2 cells

Many studies investigating drug interactions with citrus compounds focus on the major grapefruit furanocoumarins bergamottin, dihydroxybergamottin, and the flavonoid naringenin. This study evaluated the influence of polymethoxylated flavones (PMFs), tangeretin, nobiletin, 3,5,6,7,8,3,4'-heptamethoxyflavone, and sinensetin, as well as other minor occurring citrus phenols, hesperetin, limettin, 7-OH-coumarin, 7-geranyloxycoumarin, and eriodictyol, on P-glycoprotein-mediated transport of the beta-blocker talinolol using the Caco-2 cell monolayer model and was used to determine the structure-function aspects of the interaction. The transport of talinolol across Caco-2 cells monolayers was determined in the absence and presence of distinct concentrations of the calcium-channel blocker verapamil (a known inhibitor of P-glycoprotein) and citrus compounds. A sigmoid dose-response model was used to fit the data and to estimate the IC50 values of the potential inhibitors. Results from this study show that PMFs significantly decreased talinolol transport from the basolateral to apical side, where tangeretin had the lowest IC50 of 3.2 micromol/L, followed by nobiletin, heptamethoxyflavone, and sinensetin with IC50 values of 3.5, 3.8, and 3.9 micromol/L, respectively. However, the efficacy of the compounds did not appear to be dependent on the number of methoxy groups. Other citrus compounds did not have any significant effect on the transport of talinolol. This study suggests that PMFs have a high potential in the interaction with P-gp-mediated talinolol transport in Caco-2 cells. Based on their relatively low concentrations (< or =3 microg/mL) in citrus, the clinical relevance of these interactions needs to be further elucidated in in vivo studies.     

LC/ES-MS detection of hydroxycinnamates in human plasma and urine

Hydroxycinnamates are components of many fruits and vegetables, being present in particularly high concentrations in prunes. An abundance of phenolic compounds in the diet has been associated with reduced heart disease mortality. However, little is known about the absorption and metabolism of these metabolites after normal foods are consumed. An LC--electrospray--MS method was developed to measure the concentration of caffeic acid in human plasma and urine, but it can also be applied to ferulic acid and chlorogenic acid. The limit of detection was found to be 10.0 nmol/L for caffeic acid and 12.5 nmol/L for ferulic and chlorogenic acids. The method was tested on samples of plasma and urine collected from volunteers who consumed a single dose of 100 g of prunes and increased levels were observed, demonstrating that the method is capable of detecting changes in hydroxycinnamate levels induced by dietary consumption.     

Anti-inflammatory activity of an orange peel polymethoxylated flavone, 3',4',3,5,6,7,8-heptamethoxyflavone, in the rat carrageenan/paw edema and mouse lipopolysaccharide-challenge assays

The anti-inflammatory properties of 3',4',3,5,6,7,8-heptamethoxyflavone (HMF), a citrus polymethoxylated flavone, were studied in the bacterial lipopolysaccharide (LPS)-challenge/tumor necrosis factor-alpha (TNFalpha) response in mice and in the carrageenan/paw edema assay in rats. In each of these trials, HMF administered by intraperitoneal (ip) injection exhibited anti-inflammatory activity, whereas HMF administered orally (po) produced no effects. The inhibition observed in the LPS-challenge/TNFalpha assay correlated with the HMF levels in the blood sera of mice dosed (ip) with either 33 or 100 mg/kg body weight. Low levels of HMF (0.035 +/- 0.024 ppm) were detected in the blood sera of mice dosed orally [100 mg of HMF (suspended in vegetable oil)/kg], whereas ip injection led to higher levels (0.517 +/- 0.051 ppm). This may account for the different levels of anti-inflammatory effects observed in mice following ip vs oral HMF administration. HMF metabolites, including a number of mono- and di-demethylated HMF metabolites and their glucuronic acid conjugates, were also detected, but results of these studies suggest that the glucuronidated metabolites of HMF are inactive in these inflammation models.     

Quantitation of flavonoid constituents in citrus fruits.

Twenty-four flavonoids have been determined in 66 Citrus species and near-citrus relatives, grown in the same field and year, by means of reversed phase high-performance liquid chromatography analysis. Statistical methods have been applied to find relations among the species. The F ratios of 21 flavonoids obtained by applying ANOVA analysis are significant, indicating that a classification of the species using these variables is reasonable to pursue. Principal component analysis revealed that the distributions of Citrus species belonging to different classes were largely in accordance with Tanaka's classification system.     

Composition and stability of herring oil recovered from sorted byproducts as compared to oil from mixed byproducts

Herring oils produced from three different types of byproducts, only heads, mixed, and headless byproducts, were compared. Heads byproducts and its oil presented the highest oxidation levels and the lowest alpha-tocopherol content. Heads contained the lowest polyunsaturated fatty acids content and the highest amount of saturated fatty acids. No significant differences were found between the fatty acid composition of the mixed and the headless either in byproducts or in its oil. The oil was stored at two different temperatures (20 and 50 degrees C). Testing general linear models showed that oxidation was related to the peroxide value with a positive significant effect of the temperature, while the free fatty acids' model was more complex, with significant contribution of all of the effects studied. Fluorescence measurement was the one that correlated best with the oxidation progress.