Cold storage of blueberry (Vaccinium spp.) fruits and juice: Anthocyanin stability and antioxidant activity

Blueberries (Vaccinium spp.) may be considered one of the best potential sources of antioxidants in the diet. This characteristic results from the presence of flavonoids (especially anthocyanins), tannins and phenolic acids in the fruits. The objective of this work was to analyze the anthocyanin stability and antioxidant activity of refrigerated whole blueberry juice stored at 4 °C for 10 d, and of fruits stored frozen (−18 °C) for 6 months, in order to determine the changes occurring during storage. The frozen fruits showed a significant increase (p < 0.05) in antioxidant activity during the 3rd month of frozen storage, followed by a decrease up to the end of the 6-month period. The juice was analyzed every other day, and the antioxidant activity changed on the 8th d of refrigerated storage, remaining stable up to the 10th d. There were significant losses of anthocyanins both in the frozen fruits (59%) and refrigerated juice (83%). The antioxidant capacity was shown to be stable during cold storage both in the case of the fruits and whole juice, whereas the anthocyanins were degraded, possibly due to oxidations and/or condensation reactions with other phenolic compounds. More studies are required to optimize the storage time and temperature of these products with respect to nutrient stability.     

The effect of fruit size and fruit colour on chemical composition in ‘Kordia’ sweet cherry (Prunus avium L.)

The effect of fruit size (<26 mm, 26–28 mm, >28 mm) and fruit colour (Ctifl 4 and Ctifl 6) of ‘Kordia’ sweet cherry on composition of nutritive (sugars, organic acids) and bioactive (phenolics) compounds, on content of vitamin C, total phenolic content (TPC) and total antioxidant activity (TAA) was evaluated. The compositional data analysis was used for comparing relative contents of chemical compounds. The differences in average composition were examined on the basis of multivariate analysis of variance and further analysis of variance for each isometric log-ratio transformed variable. Five compounds have been identified for the first time: dicaffeoylquinic acid ([M−H]⁻ at m/z 515), procyanidin trimer ([M−H]⁻ at m/z 865), quercetin-3-(2G-glucosyl-rutinoside) ([M−H]⁻ at m/z 771), quercetin-3-galactoside ([M−H]⁻ at m/z 463) and kaempferol-3-glucoside ([M−H]⁻ at m/z 447). In the composition of sweet cherry ‘Kordia’ fruit bioactive compounds represented from 0.6% to 0.8%. The fruit colour modifies the chemical composition of phytochemicals more than fruit size; the two variables were correlated. More coloured fruit were larger, with better pomological traits and changed composition of nutritive compounds and phenolics.     

Color and chemical stability of spray-dried blueberry extract using mesquite gum as wall material

Blueberry is an important source of anthocyanins, which are highly colored substances recognized for their antioxidant activity. One of the drawbacks of using anthocyanins as food colorant is their low stability. The objective of this study was to evaluate the variations found in color and concentration of the compounds (which produce the color) on spray-dried powders, obtained from blueberry extracts with added mesquite gum. Ethanolic blueberry extracts were concentrated until reaching 35% of soluble solids. They were then spray-dried using mesquite gum as an encapsulating agent at 140 and 160 °C of air inlet temperature and 8.5, 9.1 and 9.6 mL/min of feeding rates. The lowest losses in the content of total phenolics, total anthocyanins, and color of the samples were found in samples dried at 140 °C and 9.1 mL/min. The microencapsulates that were stored for 4 weeks at 4 °C in the absence of light presented low degradation of phenolics (10%), anthocyanins (7%) and antioxidant activity (15%). Final color values were L = 39.87, C = 47.83 and H° = 28.59, with a total color difference ΔE = 5.     

Physicochemical profiles,minerals and bioactive compounds of stingless bee honey (Meliponinae)

This study investigates the physicochemical properties, majority minerals, total phenolic compounds and antioxidant activity of stingless bee honey. Thirty-three samples from ten species were analyzed. The results show moisture as 23.1–43.5% (w/w), the electrical conductivity as 0.150–1.34 mS cm⁻¹, the free acid as 16.2–139 mEq kg⁻¹, the pH as 3.33–6.56, the diastase activity as 4.34–49.6 in Göthe units, the insoluble solids as 55.2–76.1°Brix, the carbohydrates as 48.6–70.5% (w/w) sucrose and the 5-hydroxymethylfurfural (5-HMF) was below the limit of quantitation (LOQ). The most abundant element in the samples was potassium followed by calcium sodium, magnesium and manganese. Stingless bee honeys possess relevant amounts of phenolic compounds and antioxidant activity, suggesting a source of natural antioxidants. The results show that there is great variability in the composition of honey from stingless bees of different species, predominantly flowering and geographical origin. However, it was not possible to identify the real factor influencing its features.     

The effect of frying on anthocyanin stability and antioxidant activity of crisps from red- and purple-fleshed potatoes (Solanum tuberosum L.)

Red- and purple-fleshed potatoes (Solanum tuberosum L.) are a good source of polyphenols, mainly anthocyanins as one of the most important components. The chemical composition with reducing sugars and the total polyphenol contents were analyzed in four purple potato varieties (Salad Blue, Vitelotte, Valfi, Blue Congo) and three red-fleshed potato varieties (Rosalinde, Herbie 26, Highland Burgundy Red). The anthocyanin composition of raw potato and potato crisps was characterized by high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method. Moreover, the antioxidant activity was measured with the radical scavenging assays using 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as ferric reducing ability of plasma assay (FRAP). In addition, colour parameters in prepared potato crisps were evaluated. Both red and purple-fleshed potato varieties contained high levels of total polyphenols (227–845 mg 100 g⁻¹ dry weight) and anthocyanins (21–109 mg 100 g⁻¹ dry weight). The process of frying caused degradation of anthocyanin compounds (38–70%). The HPLC–MS/MS analysis showed that pelargonidin and malvidin derivates were more stable during frying than petunidin derivatives. Although frying process affected the anthocyanin and polyphenol levels, obtained potato crisps exhibited bright intensive colour and good antioxidant activity.     

Flow-injection spectrophotometric method with on-line photodegradation for determination of ascorbic acid and total sugars in fruit juices

This article reports a flow-injection spectrophotometric determination (FIA) with a photodegradation step to determine ascorbic acid and total sugars. The flow-injection system includes a simple ultraviolet photoreactor for the on-line photodegradation. The method is based on the determination of ascorbic acid at 300 nm before the photodegradation step, followed by UV irradiation and measurement of total sugars at 268 nm. The proposed method was used to determine ascorbic acid and total sugars in commercial and natural fruit juice samples. The method was validated by using spiked samples with recoveries in the range 96.4-108.3% for ascorbic acid, and 91.0-113.2% for total sugars.     

Quantification of bioactive constituents and antioxidant activity of Chinese yellow wine

Rice wines have been consumed in China for more than 9000 years. Chinese yellow wine (CYW) is one of the traditional fermented alcoholic beverages of rice. Studies have shown that CYW improves atherosclerotic plaque in LDL-receptor-knockout mice. Rice wine consumption at a level of alcohol >12.8 g/day in Chinese women has been associated with a 34% lower risk of developing metabolic syndrome compared to non-drinkers. However, evidence for the health benefits of CYW is still very limited. In the present study, phenolic compounds, amino acids, γ-aminobutyric acid and hydroxymethylfurfural in 5-year-old Guyuelongshan CYW were characterized and quantitated as a first step in launching the studies of potential health benefits of CYW. Our results show that three of the nine phenolic compounds characterized by HPLC-DAD-ESI-MS/MS were not previously documented in the CYW. Fifteen free amino acids and γ-aminobutyric acid quantitated by the HPLC-fluorescence method and the protein assay demonstrate that CYW is a good source of amino acids (2923 μg/mL), γ-aminobutyric acid (10.1 ± 0.3 μg/mL) and peptides/proteins (4210 ± 430 μg/mL). The Trolox equivalent antioxidant capacity of CYW (1.76 ± 0.07 mM) is higher than white wines but lower than red wines.     

Antioxidant capacity of underutilized Malaysian Canarium odontophyllum (dabai) Miq. fruit

This study aimed to evaluate the antioxidant capacities of various parts of the dabai (Canarium odontophyllum Miq.) fruit. The antioxidant capacities of C. odontophyllum were evaluated using a β-carotene bleaching assay, ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays. Total phenolic content of the dabai fruit was estimated using the Folin–Ciocalteu method. The antioxidant capacities ranked in the following order: skin > flesh + skin > flesh > kernel. Our findings show that a high correlation exists between total phenolic content and total antioxidant activity. This indicates that phenolic compounds could be the major contributors to the antioxidant capacity of C. odontophyllum fruit. Therefore, dabai fruit, especially the skin, can be recommended as a major source of natural antioxidants.     

Antioxidant activity,quality parameters and mineral content of Portuguese monofloral honeys

Physicochemical properties (pH, free, lactonic and total acidity, electrical conductivity and moisture), main mineral content (potassium, sodium, calcium, magnesium and iron) and antioxidant activity (total phenolics, 1,1diphenyl-2-picrylhydrazyl radical scavenging and ferric reduction antioxidant power) were determined for 39 Portuguese monofloral honeys (rosemary, orange, thyme, arbutus, locust podshrub and heather) with different geographical origins. Portuguese honeys showed good chemical and nutritional qualities fulfilling the criteria described in Directive 2001/110/CE. Potassium was the main mineral component representing 85% of total minerals. The mineral composition of sodium (4.63–200.60 mg/kg), calcium (0.43–72.30 mg/kg), magnesium (3.05–82.20 mg/kg) and iron (below 7.06 mg/kg) were higher for heather, eucalypt and arbutus honeys. Monofloral honeys of arbutus (Arbutus unedo), locust podshrub (Ceratonia siliqua L.) and heather (Erica umbellata) showed higher antioxidant activity with phenolic contents higher than 600 mg gallic acid equivalents (GAE)/kg, a DPPH scavenging activity higher than 50% and a ferric reduction antioxidant power above 600 μM Fe²⁺. Furthermore, the chemometric analysis revealed that total phenolic content was a crucial variable explaining the antioxidant activity of arbutus and locust podshrub honeys characteristic of the south of Portugal.     

Antioxidant characteristics of honey from Mozambique based on specific flavonoids and phenolic acid compounds

The most recent guidelines of IUPAC and AOAC recommend the analysis of specific compounds present in antioxidant fractions. For the first time, honey from different provinces of North (Nampula) and Central Mozambique (Sofala, Manica and Zambezia) was analysed considering specific flavonoids and phenolic acid profiles. Seven phenolic acids (chlorogenic, caffeic, ellagic, ferulic, gallic, p-coumaric and sinapic) and eight flavonoids (catechin, chrysin, kaempferol, luteolin, naringenin, pinocembrin, quercetin and rutin) were screened in the samples. Nampula honey had a higher content of most of these compounds and the total antioxidant activity (even reaching up to 40 mg TE/100 g) compared honey from the other provinces. Unlike in other African honeys, luteolin had the greatest impact in the flavonoid content (in some cases up to 72 mg/100 g), representing alone more than 50% of this family. Resulting from a discriminant analysis, specific flavonoids (pinocembrin, kaempferol, rutin and catechin) followed by the chlorogenic phenolic acid were the most important variables that distinguishes Nampula from the other provinces. This work underlines the importance of Mozambiquean honey as a source of natural antioxidants both of which concern the health benefits and its exploitation as a viable and sustainable income for the local population.     

Dietary fiber,phytochemical composition and antioxidant activity of Mexican commercial varieties of cactus pear

Cactus pear is one of the most important fruit in Mexican culture; however the phytochemical characterization of the whole cactus pear (pulp, peel and seeds) has not been studied. Therefore, the goal of this research was to evaluate the dietary fiber, vitamin C, total phenolic (TP), flavonoid and betalain concentrations and antioxidant activity (AOX) in the pulp, juice, peel and seeds of four Mexican commercial varieties of cactus pear. Rojo Cenizo and Rojo San Martín pulps presented concentrations of total dietary fiber, vitamin C, TP, betalains, and AOX of 145–166 g, 3–7 g ascorbic acid, 3–6 g gallic acid equivalents (GAE), 500–3444 mg betanin equivalents and 1044–5954 mg indicaxanthin equivalents, and 46–67 mm Trolox equivalents (TE) per kg, respectively. Rojo San Martín peel presented TP content over two times greater than the pulp, whereas AOX levels of Rojo San Martín and Verde Villanueva peels were 4–9 higher than their respective pulp. Cactus pear peel was the most important source of phytochemical compounds and AOX of the fruit, because of that it has to be considered in future applications for the formulation of new health-promoting ingredients and/or foods.     

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. 744 mg gallic acid/L), flavonoids (98 vs. 63 mg catechin/L) and a higher antioxidant activity (9.16 vs. 2.83 meq Trolox/L) in comparison to white grape juices. In comparison to the white wines, white grape juices contained more total phenols (744 vs. 286 mg gallic acid/L) and flavonoids (63 vs. 29 mg catechin/L) and evidenced higher antioxidant activity (p < 0.05). In comparison to the red wines, a lower content of total phenols (286 vs. 2358 mg gallic acid/L) and flavonoids (228 vs. 29 mg catechin/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.     

Antioxidants and other chemical and physical characteristics of two strawberry cultivars at different ripeness stages

The present work evaluated the quality and antioxidant characteristics of ‘Osogrande’ and ‘Camino Real’ strawberries at different ripeness stages. Strawberries (Fragaria x ananassa Duch.) were harvested, selected, graded according to ripeness (green, pink or 3/4 ripe and ripe) and evaluated for pH, total soluble solids, total titratable acidity, sugar/acid ratio, vitamin C, total phenolic compounds, total ellagic acid, total and individual anthocyanins and antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The highest total soluble solid content was found for pink (7.5° Brix) and ripe (7.9° Brix) ‘Osogrande’ strawberries. At pink stage, this cultivar showed higher amounts of total phenolics (2909.48 mg kg⁻¹ FW) and total ellagic acid (454.16 mg kg⁻¹ FW). Pink ‘Camino Real’ strawberries presented the highest content of vitamin C (528.55 mg kg⁻¹ FW). Antioxidant activity was higher for ‘Osogrande’ cultivar, at green stage, according to DPPH (11.91 μmol BHT g⁻¹ FW) and FRAP (36.75 μmol ferrous sulphate  g⁻¹ FW) assays and at ripe stage, only for DPPH assay (12.83 μmol BHT g⁻¹ FW). Anthocyanins increased along ripening, with more elevated concentrations in ripe ‘Camino Real’ strawberries (292.9 mg kg⁻¹ FW). Cyanindin-3-glucoside showed a higher concentration for the same treatment (17.23 mg kg⁻¹ FW), which might contribute to a more redish color. Although ripe berries have a better flavor and are more appreciated, higher antioxidant contents and activities were observed at pink stage in which higher amounts of total phenolics, total ellagic acid and vitamin C were noticed for both cultivars.     

Determination of total phenolic content of raspberry and blackberry cultivars by immobilized horseradish peroxidase bioreactor

Raspberry and blackberry cultivars were assayed for total phenol content by enzymatic method using immobilized horseradish peroxidase (HRP). The HRP was immobilized on the styrene–divinylbenzene–polygluteraldehyde (STY–DVB–PGA) beads. Total phenol content determined by immobilized and free enzymatic methods was compared with those obtained by applying the Folin–Ciocalteu method. The total phenol content by immobilized HRP method ranged from 254 to 973 mg gallic acid equivalent (GAE) per 100 g fw as well as 105 to 435 mg ellagic acid equivalent (EAE) per 100 g fw in raspberry and blackberry cultivars. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical values were correlated with total phenol content in raspberry using immobilized enzymatic method as GAE (R² = 0.7450, P = 0.05) and EAE (R² = 0.7449, P = 0.05). All antioxidant activity values were highly correlated with total phenol content in blackberry using free and immobilized enzymatic methods as GAE (R² = 0.9777 and 0.9223, P = 0.05 respectively) and EAE (R² = 0.9979 and 0.9223, P = 0.05 respectively). A significant additivity was observed using individual phenolic compounds by immobilized enzymatic method as 95% (GAE) and 90% (EAE). These results show that the immobilized enzymatic assay can be applied to determine the total phenol content of berry fruits, which is more specific and not affected by interfering compounds.     

Comparison of the contents of bioactive compounds and the level of antioxidant activity in different kiwifruit cultivars

The aim of this investigation was to find the best among four different cultivars of kiwifruit (‘Hayward’, ‘Daeheung’, ‘Haenam’ and ‘Bidan’) for human consumption. The bioactive compounds and the level of antioxidant activity of these cultivars were determined and compared. The presence of polyphenols (flavonoids and phenolic acids) in the investigated samples was studied by Fourier transform infrared (FT-IR) spectroscopy. By far the highest levels (P < 0.05) of polyphenols and ascorbic acids were found in ‘Bidan’ (25.9 ± 1.3 mg GAE/g and 152 ± 10.4 mg/g DW, respectively), which also contained the highest levels of protocatechuic and vanillic acids. In addition, the level of antioxidant activity (μM TE/g DW) was significantly higher (P < 0.05) in ‘Bidan’ (121 ± 5.8, 109 ± 11.2, 102 ± 6.6 and 94 ± 4.7 for CUPRAC, ABTS, DPPH and FRAP radical scavenging assays, respectively). Pattern-recognition techniques (cluster analysis, principal component analysis, factor analysis, and canonical discriminant analysis) were used to compare the cultivars. A high correlation was found among the polyphenols (R² = 0.99), ascorbic acid (R² = 0.99+) and the antioxidant activity in the studied cultivars. In conclusion, the overall bioactivity of the cultivars was: ‘Bidan’ > ‘Haenam’ > ‘Daeheung’ = ‘Hayward’. ‘Bidan’, a relatively new cultivar, can be recommended for consumption.     

Variation of nitrate content among randomly selected taro (Colocasia esculenta (L.) Schott) genotypes and the distribution of nitrate within a corm

Taro (Colocasia esculenta) is an important staple crop distributed throughout many tropical and subtropical regions. Very little information is available about the nitrate content and its distribution within taro corms. The aim of this study, based on ion chromatography, was to analyze the concentration and distribution of nitrate within four crucial parts (upper, central, marginal, basal) of taro corms of 8 representative cultivars. The results revealed that the levels of nitrate in taro corms were variable depending on the cultivar and corm parts. The highest levels were determined in the upper part of the corm which is used for the multiplication and establishment of a new crop. Therefore, the size of this part is closely associated with the quality of the planting material. As there are no roots, the initial growth of the new taro plant depends entirely on the nutrients accumulated in the upper part of the corm. During the growth season, this part of the corm gradually becomes the lower (basal) part containing the oldest and the least biochemically active tissue, which has the lowest concentration of nitrate ions. The nitrate content in the central part, which is the most important for human nutrition, was relatively low and varied between 29 and 313 mg/kg fresh weight.     

Influence of storage and in vitro gastrointestinal digestion on total antioxidant capacity of fruit beverages

Eight fruit beverages containing grape, orange and apricot, with/without iron and/or zinc and with/without milk added were analyzed for total antioxidant capacity (ORAC and TEAC methods), ascorbic acid content, and total polyphenols. The influence of cold storage (2-4 °C) during the product shelf-life (135 days) and antioxidant capacity after an in vitro gastrointestinal digestion were evaluated. Antioxidant capacity for all beverages together increased significantly (p < 0.05) at the end of storage (16.4% and 12.8% for ORAC and TEAC, respectively), whereas ascorbic acid remained stable. Regarding in vitro digestion, antioxidant values of bioaccessible fractions of fruit beverages increased (p < 0.05) 59% and 20% for ORAC and TEAC, respectively, while ascorbic acid and polyphenol content fell 36% and 16%, respectively (p < 0.05). Supplementation of fruit beverages with milk and/or Fe/Zn did not modify total antioxidant capacity of samples during either cold storage or during in vitro digestion.     

Bioactive compounds and antioxidant activities of different watermelon (Citrullus lanatus (Thunb.) Mansfeld) cultivars as affected by fruit sampling area

This investigation reports the variability of antioxidant components and antioxidant activities of six watermelon cultivars (cvs) (four commercial cvs Aramis, Crimson Sweet, Dumara, Giza, and two new selections P503 and P403 produced by the National Agricultural Research Institute of Tunisia) as influenced by sampling area. All cvs were simultaneously grown in an open-field and subjected to identical horticultural practices in order to minimize the effects of environmental conditions and maximize those related to genotype. Significant differences were found between watermelon cvs for lycopene, phenolics, flavonoids, ascorbic acid (AsA), dehydroascorbic acid (DHA) and total vitamin C (AsA + DHA) contents, as well as in the antioxidant activity of their hydrophilic and lipophilic fractions. P503 cv showed the highest lycopene and flavonoid contents. Crimson Sweet and Giza cvs showed the highest HAA and LAA when TEAC was used as assay method, while the highest HAA and LAA were detected in Giza and Dumara cvs and in P503 cv, respectively, when FRAP assay was used. This study demonstrates that the amount of each specific antioxidant, as well as the HAA and LAA, were both influenced by genotype and sampling area, emphasizing the need to evaluate watermelon biodiversity in order to improve its nutritional value.     

Identification and quantification of phenolic compounds responsible for the antioxidant activity of sweet potatoes with different flesh colours using high performance thin layer chromatography (HPTLC)

The objectives of the present study were to develop a simple high performance thin layer chromatography (HPTLC)-based protocol: (i) to allow high-throughput profiling of phenolic compounds of microwaved roots from 295 sweet potato varieties and breeding lines, (ii) to quantify the content of anthocyanins and caffeoylquinic acid (CQA) derivatives, and (iii) to determine their respective contributions to the antioxidant activity of sweet potato methanolic extracts using the DPPH test. Analysed accessions were separated into three groups: white-fleshed (n = 100), orange-fleshed (n = 64) and purple-fleshed (n = 131). Purple-fleshed accessions presented the highest mean CQA content. After DPPH treatment and transmittance scanning of the plate at 517 nm, the most active free radical scavengers were found to be the four CQAs (CGA, 3,4-, 4,5- and 3,5-diCQA) while the anthocyanins were found to be less active. The total antioxidant capacity of the sweet potato methanolic extracts was mostly linked to total CQAs content. This method can now be used for fast routine analysis and selection of sweet potato breeding clones.     

Compositional characteristics of commercial beetroot products and beetroot juice prepared from seven beetroot varieties grown in Upper Austria

Beta vulgaris L. (beetroot) contains high amounts of biologically active substances including betalains and inorganic nitrate. We determined the amounts of various compounds (minerals, betalains, oxalic acid, phenolic acids, and sugars) in juice prepared from seven different beetroot varieties cultivated in Upper Austria. Large differences were found between the varieties for some substances (such as nitrate), whereas others showed only minor variation (certain minerals and sugars). The total betalain content was found to range between 0.8 and 1.3 g/L fresh juice (about 60% betacyanins and 40% betaxanthins) that accounted for 70–100% of the total phenolics content. Other detected phenolics were hydroxycinnamic acids, which accounted for up to 2.6% of total phenolics. Nitrate content varied 10-fold between single varieties. Sugar composition was similar in all varieties with an average total content of about 7.7%, consisting of 95% sucrose. Only minor differences in the concentration of oxalic acid (0.3–0.5 g/L fresh juice) were found between the varieties. In addition, 16 commercial juices and four powders were analyzed for their nitrate contents, as its metabolic product nitric oxide has been reported to provide cardiovascular benefits. Large variations of the nitrate levels, ranging from 0.01 to 2.4 g/L, were found.