Development and validation of a decigram-scale method for the separation of limonin from limonin glucoside by C-18 flash chromatography

A preparative method for decigram-scale polishing of limonin glucoside (1) with regard to removing traces of limonin (2), the main bitter principle in Citrus, is reported. During the method development and up-scaling stages, sample purity, sample size, solvent amounts, and drying conditions were varied. The resulting polishing method consists of a simple step gradient that utilises food grade solvents, ethanol and water at 15% EtOH and 50% EtOH steps. Using a 75 × 300 mm C-18 column, this method is capable of processing 20 g of material per run in less than 3 h. Recovery of the purified limonin glucoside following evaporation of the solvent was 93.5% (±2.8, n = 6) and the limonin concentrations in the resulting materials were found to be reduced 10- to 15-fold.     

柑桔中类柠檬苦素含量及分布研究

对几种柑桔品种果皮、种子、果汁中类柠檬苦素的含量进行了测定,结果表明,柑桔果实中不同部位类柠檬苦素含量不同,以种子中含量最高,果皮次之,而果汁中含量最低。不同品种间类柠檬苦素含量依次为:邓肯葡萄柚>琯溪蜜柚>南充实生甜橙>锦橙>大红袍红桔。     

Pesticide residues in orange fruit from citrus orchards in Nuevo Leon State,Mexico

ABSTRACT

Some international organisations established maximum residue limits (MRLs) in food to protect human health. Mexico lacks regulations in this matter, affecting national and international trade from agroindustry. The aim of this study was to diagnose pesticide residues in oranges from Nuevo Leon, México, in citrus orchards. In May 2014, 100 orange fruit samples were taken randomly from orchards and subjected to analysis for 93 pesticides at residual level by GC/QQQ-MS and LCQ-TOF-MS. Results showed the presence of 15 pesticide residues in the samples. The comparison of the residual levels of pesticides found in orange samples among the MRLs allowed by USA, EU and Japanese regulations demonstrated that all samples were below MRLs issued by USA and Japan. Some orange samples were above MRLs issued by the EU. This provides a basis to establish strategies in order to satisfy International Standards to protect human health and encourage Food Safety in Mexico.     

Pesticide residues survey in citrus fruits

The use of pesticides is widespread in citrus fruits production for pre- and post-harvest protection and many chemical substances may be applied in order to control undesirable moulds or insects. A survey was carried out to evaluate levels of pesticide residues in citrus fruits. Two multiresidue analytical methods were used to screen samples for more than 200 different fungicides, insecticides and acaricides. A total of 240 samples of citrus fruits including lemon, orange, mandarin, grapefruit, lime, pomelo and kumquat were taken in various markets in the Geneva area during the year 2003. Ninety-five percent of the 164 samples issued from classical agriculture contained pesticides and 38 different compounds have been identified. This high percentage of positive samples was mainly due to the presence of two post-harvest fungicides, imazalil and thiabendazole, detected in 70% and 36% of samples respectively. Only three samples exceeded the Swiss maximum residue limits (MRLs). Fifty-three samples sold with the written indication “without post-harvest treatment” were also controlled. Among theses samples, three exceeded the Swiss MRLs for penconazole or chlorpyrifos and 18 (34%) did not respect the written indication since we found large amounts of post-harvest fungicides. Finally, 23 samples coming from certified organic production were analysed. Among theses samples, three contained small amounts of pesticides and the others were pesticides free.     

Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against Penicillium digitatum

Citrus peel is rich in flavanone glycosides and polymethoxyflavones. In view of their importance for industrial application as well as for their pharmacological properties, their content was analyzed in the mature fruits of several Citrus paradisi (grapefruit) and Citrus sinensis (orange) varieties, with a view to select the most interesting for isolation. The results shows that the Star Ruby grapefruit and the Sanguinelli orange stand out for their high contents of naringin and hesperidin, respectively. The presence of the polymethoxyflavones nobiletin, heptamethoxyflavone and tangeretin, could be ascertained in all the grapefruit varieties analysed. Higher polymethoxyflavone levels were recorded in orange, with Valencia Late showing the greatest nobiletin, sinensetin and tangeretin contents and Navelate the highest heptamethoxyflavone levels. An in vitro study revealed that these compounds acted as antifungal agents against Penicillium digitatum, the polymethoxyflavones being more active than the flavanones in this respect. The possible participation of these phenolic compounds in the defence mechanism of Citrus against P. digitatum is discussed.     

Citrus flavonoids: Molecular structure,biological activity and nutritional properties: A review

Epidemiological studies have shown an inverse relationship between dietary flavonoid intakes and cardiovascular diseases. Citrus fruits are the main winter fruits consumed in the Mediterranean diet, so they are the main source of dietary flavonoids. The possible beneficial effects are due, not only to the high amounts of vitamins and minerals, but also to the antioxidant properties of their flavonoids. Dietary flavonoids may help to supplement the body antioxidant defences against free radicals. These compounds’ possible beneficial effects are due to their antioxidant activity, which is related to the development of atherosclerosis and cancer, and to anti-inflammatory and antimicrobial activity. The present review summarizes the existing bibliography on biological and pharmacological studies of Citrus flavonoids, both in vitro and in vivo.     

Estimating volume and mass of citrus fruits by image processing technique

An image processing based technique was developed to measure volume and mass of citrus fruits such as lemons, limes, oranges, and tangerines. The technique uses two cameras to give perpendicular views of the fruit. An efficient algorithm was designed and implemented in Visual Basic (VB) language. The product volume was calculated by dividing the fruit image into a number of elementary elliptical frustums. The volume is calculated as the sum of the volumes of individual frustums using VB. The volumes computed showed good agreement with the actual volumes determined by water displacement method. The coefficient of determination (R²) for lemon, lime, orange, and tangerine were 0.962, 0.970, 0.985, and 0.959, respectively. The Bland–Altman 95% limits of agreement for comparison of volumes with the two methods were (−1.62; 1.74), (−7.20; 7.57), (−6.54; 6.84), and (−4.83; 6.15), respectively. The results indicated citrus fruit’s size has no effect on the accuracy of computed volume. The characterization results for various citrus fruits showed that the volume and mass are highly correlated. Hence, a simple procedure based on computed volume of assumed ellipsoidal shape was also proposed for estimating mass of citrus fruits. This information can be used to design and develop sizing systems.     

Behaviour of spirotetramat residues and its four metabolites in citrus marmalade during home processing

The effect of home processing on the residues of spirotetramat and its four metabolites (B-enol, B-glu, B-mono and B-keto) in citrus marmalade is comprehensively investigated in this paper by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A five-fold recommended dose of spirotetramat was applied to citrus fruit under field conditions and the processing included five steps: washing, peeling, pre-treatment for peel, mixing and boiling. The results showed that spirotetramat was the predominant component detected in unprocessed citrus, accounting for 64%. All the detected residues were primarily deposited on citrus peel, except for B-enol which was also present in the citrus pulp. Washing reduced spirotetramat, B-enol, B-glu and B-keto by 83%, 56%, 41% and 16%, respectively, and pre-treatment of the peel removed between 42% and 68% of the residues. Four compounds were all below the limit of detection after the mixing step. In the final product, only B-keto was detected at the concentration of 0.010 mg kg^–1. After the whole process, the processing factors for spirotetramat, B-enol, B-glu and B-keto were < 0.041, < 0.125, < 0.294 and 0.313, respectively, which indicated that home processing can significantly reduce residues of spirotetramat and its metabolites in citrus marmalade.     

Effect of harvesting time and fruit size on titratable acidity, soluble solid and distribution of limonin in Thai tangerine juice

The effect of fruit size and harvesting of Thai tangerine fruit ( Citrus reticulata , Blanco) at different growth stages, 8–12 months after fruit set, on the distribution of limonin in whole fruit as well as in the individual fruit parts and extracted juice was investigated. The highest limonin concentration was observed in seed, followed by albedo, flavedo, segment membrane and juice sacs in decreasing order. The limonin concentration in juice as well as in whole fruit was decreased when fruit was harvested 8–12 months of fruit set. Increasing harvesting time from 8 to 12 months showed corresponding decreases in the amount of limonin in flavedo, albedo and seed. There was a decrease in titrable acidity and increase in total soluble solid and total soluble solid/titrable acidity ratio of juice with a later than normal harvesting time. However, fruit size showed no effect on limonin content and other properties of juice extracted from tangerine.     

超高效液相色谱-串联质谱法检测柑橘中的氟唑菌酰胺残留量

目的 建立了超高效液相色谱-四级杆串联质谱(UPLC-MS/MS)检测柑橘中氟唑菌酰胺的方法。方法 选取Acquity UPLC BEH C₁₈(2.1×100 mm,1.7 μm)色谱柱对氟唑菌酰胺进行分离,梯度洗脱选用0.1%甲酸-乙腈为流动相。使用电喷雾正离子电离模式进行数据收集,采用基质匹配外标法对氟唑菌酰胺进行定量分析。样品采用乙腈提取,经N-丙基乙二胺(primary secondary amine, PSA)固相吸附剂净化后,用0.1%甲酸溶液定容。本文在对样品前处理实验方式优化的基础上,先对乙腈-甲酸和乙腈-乙酸铵2种流动相体系进行比对;再比较PSA用量对全果和果肉基质的净化作用。结果 选取乙腈-0.1%甲酸溶液作为流动相, 使用量为50 mgPSA和300mg无水硫酸镁时净化效果最佳。氟唑菌酰胺在柑橘全果中的加标回收率为84%~104%,RSD≤4%。氟唑菌酰胺在果肉中的加标回收率为100%~106%, RSD≤2%。消解试验结果表明：氟唑菌酰胺在全果中的消解半衰期为13.56天,在果肉中的消解半衰期为10.56 天。最终残留试验表明：在采样时间分别为距离最后一次施药后第14天和第21天时,氟唑菌酰胺在柑橘全果中的残留量为0.206 mg/kg、0.178 mg/kg;在柑橘果肉中的残留量为0.028 mg/kg、0.020 mg/kg。结论 该检测方法灵敏度较高、操作简单,准确度与精密度均能达到定量分析要求,适用于柑橘中氟唑菌酰胺的检测;根据氟唑菌酰胺在柑橘中的残留试验结果,推荐施药剂量为不超过稀释2500倍液的制剂用量,且最多施药3次,安全间隔期以14 天为宜。     

Chemical composition of dehulled seeds of selected lupin cultivars in comparison to pea and soya bean

With the objective of promoting the cultivation, usage, and consumption of lupin, in the present study, the chemical and fibre composition of dehulled seeds of seven cultivars of different lupin species (Lupinus angustifolius, Lupinus albus and Lupinus luteus) was determined in comparison to pea (Pisum sativum) and soya bean (Glycine max).     

Loss of selected water-insoluble polysaccharides and component neutral sugars from swede (Brassica napus (cv. danestone)) and cereal bran measured during digestion in the pig caecum

Sources of dietary fibre with a high pentose sugar content have been shown to have the greatest effect in promoting faecal bulking, though no underlying reason for this has been found. Pentose-containing polysaccharides in dietary fibre are, in isolation, as degradable as other polysaccharides, though their known association with lignin could be important in helping to maintain the structural integrity and hence the bulking ability of a fibre source. Using a modified nylon bag technique, the potential digestibility of fibre prepared from a source rich in pentose sugars (cereal bran) and a source poor in pentose sugars (Swede; Brassica napus, cv. Danestone) was estimated in the pig caecum. Swede fibre was effectively completely degraded (within 18 h) but in bran 100% digestion was not achieved even with extended incubation. Rates of fibre component loss, estimated from material recovered from the nylon bags, showed that different fibre components were degraded at different rates and that the rate of degradation was not always linear. In swede xylose was less readily lost than other fibre components and the rate was non-linear whilst in bran xylose was preferentially lost and at a linear rate. In bran arabinose was less readily lost. The results showed that the bulking effects of fibre sources with a high pentose sugar content were unlikely to be due solely to pentose content. Differences in rates of digestion suggested that different linkage groups contained within a component may be able to modify the degree of fibre digestion, most probably by the linkage of specific pentosans to lignin.     

A review of limonin in grapefruit (Citrus paradisi) juice,its relationship to flavour,and efforts to reduce it

Ever since limonin was first discovered in grapefruit (Citrus paradisi Macf) juice in 1965 and found responsible for significantly contributing to bitterness in the juice, a great deal of research has been directed towards understanding its role therein. Information is presented relative to limonin's chemical determination, taste threshold and content in grapefruit juice, and to relevant legislation. Also considered are (1) several factors affecting the limonin content of grapefruit juice (2) the effect of limonin content on sensory flavour in grapefruit juice (3) the interaction between the Brix: acid ratio and limonin content in affecting grapefruit juice flavour and (4) methods for reducing limonin in citrus juices generally. Finally, recommendations for future studies on limonin in grapefruit juice are reviewed.     

Quantitation of limonin in Iranian orange juice concentrates using high-performance liquid chromatography and spectrophotometric methods

Bitterness is a major problem in the citrus industry worldwide, and is mostly due to the presence of limonoid compounds, especially limonin. In this study, the limonin content of orange juice concentrates from three active factories in west Mazandaran province, after reconstitution, was assayed using high-performance liquid chromatography (HPLC) and spectrophotometric methods. With HPLC, employing acetonitrile/water (v/v) (32:68) with a flow rate of 0.9 ml/min at =207 nm, the limonin content was in the range 11.7–23.3 g/ml with a mean ± standard deviation (SD) of 18.5±3.6 g/ml. Spectrophotometric measurement of limonin at 503 nm with Burnham reagent gave a range of 9.9–21.8 g/ml with a mean ± SD of 17.16±4.1 g/ml. The correlation coefficients of the detector linear response for the limonin standard (0–50 g/ml) for HPLC and spectrophotometry were 0.995 and 0.989, respectively. The percentages of limonin recovery with and without sample addition were 101.02–111.40 and 93.0–100.8, respectively, for HPLC. The percentages of limonin recovery for the spectrophotometric method resulted in 95.6–101.8 for limonin-added samples. The precision factors for the two assay methods were ±2.10% and ±1.69, respectively. The correlation coefficient of the two methods was 0.84. Sensory evaluation of the reconstituted samples from the view point of bitterness confirmed 85% of the results of the analytical methods. This study recommends spectrophotometric analysis as a useful and suitable method for quantitation of limonin in the quality control laboratories of orange juice plants.     

Effective suppression of azoxymethane‐induced aberrant crypt foci formation in mice with citrus peel flavonoids

Citrus peel or its extract has been reported to exhibit a broad spectrum of biological activity. Herein, we report the first investigation of inhibitory effects of a formulated product from citrus peel extract, gold lotion (GL), on azoxymethane‐induced colonic tumorigenesis. We have demonstrated that oral feeding of GL decreased the number of aberrant crypt foci (ACF), particularly large size of ACF in colonic tissues of mice. Both gene and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) were suppressed by GL treatment. The in vivo data have revealed for the first time that the citrus peel extract–GL–is an effective antitumor agent mechanistically downregulating the protein levels of iNOS, COX‐2, ornithine decarboxylase, vascular endothelial growth factor, and matrix metallopeptidase 9 in colonic tissues of mice, suggesting that GL is a novel functional natural product capable of preventing inflammation‐associated colon tumorigenesis.     

Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps

The pulp extracts of twenty-one varieties of citrus fruits (oranges, satsumah, clementine, mandarins, tangor, bergamot, lemon, tangelos, kumquat, calamondin and pamplemousses), commonly grown in Mauritius, were characterised in terms of their total soluble solids (TSS), titratable acidity (TA), polyphenol composition and vitamin C contents. Total phenolics ranged from 406.3 ± 14 to 1694 ± 19 ??g g^− 1 fresh weight (FW). Total flavonoids varied between 133 ± 6 and 965 ± 7 ??g g^− 1 FW and vitamin C contents were from 166 ± 19 ??g/mL to 677 ± 22 ??g/mL. The pulp of a pamplemousse variety had the highest TSS/TA ratio whereas lemon pulps had lowest TSS/TA ratios. The antioxidant activities of the pulp extracts were assessed and total phenolics correlated strongly with the trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant capacity (FRAP) and hypochlorous acid (HOCl) scavenging activity assays. Based on their antioxidant activities, nine citrus fruits namely, one orange, tangor, kumquat, calamondin and pamplemousse variety and two mandarin and tangelo varieties were further characterised for their flavanone, flavonol and flavone levels by HPLC. Hesperidin (6.89 ± 0.06 to 26.98 ± 0.07 mg/g FW) and narirutin (0.27 ± 0.01 and 20.91 ± 0.10 mg/g FW) were present at high concentrations compared to the other flavonoid glycosides in the pulp extracts. Naringin was detected only in pulp extracts of pamplemousses. In the light of the data obtained, citrus fruit pulps represent an important source of phytochemicals with potent antioxidant capacity.     

Lc/Esi/Ms/Ms determination of postharvest fungicide residues in citrus juices

An LC-ESI-MS/MS method was developed for the quantitative detection of postharvest fungicide residues in citrus juices and reported in this paper. The analyses of thiabendazole (TBZ), carbendazim (MBC), thiophanate methyl (TPM), imazalil (IMZ) and prochloraz (PCZ) residues were performed by using a gradient elution in conjunction with positive ionization mode electrospray ionization tandem mass spectrometry. Fungicides were extracted from citrus juices with recoveries ranging from 79.8 to 101.2% and relative standard deviation better than 15%. The quantification limits ranged from 0.01 μg/kg IMZ to 0.06 μg/kg for MBC. The LC-MS-MS method was applied to commercial citrus juices, detecting MBC, TBZ and IMZ residues in the 90% of the samples. Prochloraz residues were detected only in one of the multifruit juice (orange, lemon and carrot) samples.     

Effect of added citrus fibre and spice essential oils on quality characteristics and shelf-life of mortadella

The objective of this work was to study the effect of adding orange dietary fibre (1%), rosemary essential oil (0.02%) or thyme essential oil (0.02%) and the storage conditions on the quality characteristics and the shelf-life of mortadella, a bologna-type sausage. The moisture, fat, ash content and colour coordinates lightness (L∗) and yellowness (b∗) were affected by the fibre content. The treatments analysed lowered the levels of residual nitrite (57.56% and 57.61%) and the extent of lipid oxidation, while analysis of the samples revealed the presence of the flavonoids, hesperidin and narirutin. No enterobacteria or psychotropic bacteria were found in any of the treatments. The treated samples stored in vacuum packaging showed the lowest aerobic and lactic acid bacteria counts. Sensorially, the most appreciated sample was the one containing orange dietary fibre and rosemary essential oil, stored in vacuum packaging. Orange dietary fibre and spice essential oils could find a use in the food industry to improve the shelf-life of meat products.     

柚子皮中柠檬苦素提取工艺及其抑菌活性研究

采用正交实验法对柚子皮柠檬苦素的提取工艺进行优化,并用所获得的柠檬苦素对几种农作物病害病原菌进行了抑菌实验。结果表明,柚子皮柠檬苦素的最佳提取工艺路线是采用二氯甲烷为提取溶剂,回流提取2次,每次回流提取2h,提取温度为50℃,料液比为1:12。抑菌实验结果表明,按上述工艺获得的柠檬苦素对小麦纹枯病原菌、水稻纹枯病原菌、花椒落叶病原菌、小麦赤霉病原菌和玉米大斑病原菌的生长有一定抑制作用。     

柑橘果汁中的类柠檬苦素及其脱苦方法

介绍了柑橘果汁中的类柠檬苦素苦味，类柠檬苦素苦味物质的结构，由类柠檬苦素引起的后苦味的形成机理．以及目前研究的类柠檬苦素苦味物质的脱除方法。