Inhibitory Effects of Red Wine on Lipid Oxidation in Fish Oil Emulsion and Angiogenesis in Zebrafish Embryo

The capabilities of red wine against lipid oxidation and angiogenesis were evaluated by using a fish oil emulsion system and an in vivo zebrafish embryos model, respectively. The red wine contained 12 different antioxidant phenolics which levels were led by anthocyanins (140.46 mg/L), catechin (55.08 mg/L), and gallic acid (46.76 mg/L). The diversity of the phenolics in red wine was greater than the tea, coffee, or white wine selected as a peer control in this study. The total phenolics concentration of red wine was 305.53 mg/L, although the levels of tea, coffee, and white wine were 85.59, 76.85, and 26.57 mg/L, respectively. The activity of red wine in scavenging DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) free radicals was approximately 4 times higher than the tea and 8 times than the coffee or white wine. The red wine showed the highest capability in preventing long chain PUFA oxidation in the fish oil emulsion. Because of the outstanding antioxidant activity of red wine, the red wine dried extract was used to monitor its inhibitory effect against angiogenesis by using transgenic zebrafish embryos (Tg[fli1:egfp]^y1) with fluorescent blood vessels. After incubated in 100 μg/mL of the extract solution for 26 h pf, each of the embryos had a lower number of intersegmental vessel than the control embryo. The inhibition rate of red wine extract against growing of angiogenic blood vessel reached 100%.     

ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of Alternaria spp. in foodstuffs. The method uses Alternaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 190 commercial food samples, including 80 fresh fruit and vegetable samples and 110 processed foodstuffs. The assay demonstrated the presence of Alternaria spp. DNA in 46 out of the 80 raw samples (57.5%) and in 66 out of the 110 processed samples (60%), enabling quantitative detection of Alternaria spp. DNA at levels as low as 1 CFU/g. The estimated Alternaria counts obtained by real-time PCR showed a good relationship (R² = 0.9006, P < 0.01) with the Alternaria counts obtained by plating on Potato Carrot Agar (PCA). The developed real-time PCR assay provides a useful tool for early detection of Alternaria spp. and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.     

Analysis of phenolic compounds including tannins,gallotannins and flavanols of Uapaca kirkiana fruit

Tannins and phenolic acids were extracted from Uapaca kirkiana unripe pulp, seed coat, peel and embryo, as well as from ripe pulp, seed coat, embryo and peel portions of the fruit. Extraction was done using 50% methanol. More tannins were detected in the embryos of both ripe and raw fruit. Both ripe and raw seed coat contained the least amounts of tannins. The flavanoids and gallotannins also followed the same trend, with most of them in the embryos of both, the ripe and raw, fruit portions whilst the least were found in the seed coat. Tannins were, however lost during sun-drying compared to oven-drying, even though the differences were not very large. Also, raw fruits showed high concentrations of tannins compared to the ripe fruits. Hydroxybenzoic acid was found in the peel and pulp but none was detected in the seed coat or in the embryo. Similar phenolic acids were detected in the seed coat, except for caffeic acid and protocatechuic acid, detected in the embryo by both the UV florescence and Folin spray.     

Carotenoid content and expression of phytoene synthase and phytoene desaturase genes in bitter melon (Momordica charantia)

Momordica charantia, a tropical plant, produces a fruit that has a β-carotene concentration five times higher than that of carrot. To elucidate the molecular basis of β-carotene accumulation in M. charantia, the gene expression levels of phytoene synthase (McPSY) and phytoene desaturase (McPDS) were determined. These levels were particularly high in the flowers of M. charantia. During fruit maturation, the expression levels of McPSY and McPDS decreased during the mid-stages but increased in the fully mature fruit. In addition, carotenoids accumulated as the peel changed from green to orange. Thus, McPSY and McPDS expression correlated with carotenoid accumulation during fruit maturation. Principal component analysis (PCA) also was used to evaluate the differences among the profiles of seven carotenoids identified in the fruit at several maturation stages. Riper fruits had higher carotenoid concentrations than less ripe fruits.     

The Mechanisms Underlying the Anthracnose Disease Reduction by Rice Hull as a Silicon Source in Capsicum (Capsicum Annuum L.) Grown in Simplified Hydroponics

Silicon has proven to be effective in controlling many diseases in plants and could be used as an alternative strategy against chemical control of diseases. Rice hull is an environmental friendly natural source of silicon. This study was conducted to investigate the effect of rice hull as a Si source on anthracnose disease and also on fruit cuticle thickness (CT), total soluble and cell wall-bound phenolic compounds (TSP and CWBP) in fruits and formation of fungal appressoria on fruits as possible mechanisms of disease reduction in capsicum (Capsicum annuum L.). In this study a simplified hydroponics system (SHS) with rice hull as an inert media and nutrients supplied with either NF (New Formula) or Albert's solution was used. A liquid hydroponic system (LHS) was also maintained with same nutrient solutions as controls. Disease development was assessed by challenge inoculation with Colletotrichum gloeosporioides on fruits. CT was measured using stage and ocular micrometer. TSP and CWBP in fruits were analysed by Folin-Ciocalteu method during first 5 days after inoculation (DAI). Appressoria formation by fungal conidia on fruit peels at inoculated spots was observed through micrometer daily after inoculation. More than 83% disease reduction was observed in fruits harvested from SHS compared to that of LHS supplied with both nutrient solutions. There were significantly higher values of CT and CWBP (about 45% and 30% respectively) in fruits from SHS compared to that of LHS (Si-free). However, TSP was not significantly affected by Si treatment. A higher percentage of appressoria was prevailed on fruits harvested from SHS thus the disease initiation was delayed compared to that of LHS. There may be a possibility that germination of appressoria was hindered by thicker cuticle or biochemical reaction involved with induced CWBP in fruits from Si treated plants.     

Variations in essential oil and fatty acid composition during Myrtus communis var. italica fruit maturation

The essential oil and fatty acid composition of Myrtus communis var. italica fruit during its ripening was determined. The effect of the harvesting time on some physical properties of Myrtus fruits, fruit weight and moisture content, were significant. The increase of fruit weight (from 2.54 to 8.79 g% fruits) during ripeness was correlated positively with that of moisture content (from 28% to 72%). Fruit essential oil yields varied from 0.003% to 0.01% and showed a remarkable increase at 60 days after flowering to reach a maximum of 0.11%. Forty-seven volatile compounds were identified in fruit essential oils; 1,8-cineole (7.31–40.99%), geranyl acetate (1.83–20.54%), linalool (0.74–18.92%) and α-pinene (1.24–12.64%) were the main monoterpene compounds. Total fatty acid contents varied from 0.81% to 3.10% during fruit maturation and the predominant fatty acids were linoleic (12.21–71.34%), palmitic (13.58–37.07%) and oleic (6.49–21.89%) acids. The linoleic acid proportions correlated inversely with palmitic and oleic acids during all the stages of ripening.     

Volatile compounds from pitanga fruit (Eugenia uniflora L.)

Extracts from pitanga leaves are considered to be effective against many diseases, and are therefore used in popular Brazilian medicines. In this study, the volatile constituents of pitanga fruits (Eugenia uniflora L.) were trapped on to Porapak-Q and eluted with ethyl acetate, and the chemical composition of the extract was analyzed by gas chromatography and gas chromatography/mass spectrometry. Fifty-four compounds were detected, and twenty-nine of those were identified by close matches with standard MS spectra. Monoterpenes (75.3% in mass) were found to comprise the largest class of the pitanga fruit volatiles, including trans-β-ocimene (36.2%), cis-ocimene (13.4%), the isomeric β-ocimene (15.4%) and β-pinene (10.3%). Several known therapeutic constituents of pitanga leaf extract, such as selina-1,3,7(11)-trien-8-one (the major constituent) were also found to be present in the fruit volatile extract, suggesting that the fruit may display therapeutic properties similar to those of the leaf extract.     

Consumption of raw vegetables and fruits: a risk factor for Campylobacter infections

The purpose of this study was to determine the prevalence of Campylobacter in fresh vegetables and fruits at retail level in the Netherlands, and to estimate its implications on the importance of vegetables and fruits as risk factor for campylobacteriosis.Thirteen of the 5640 vegetable and fruit samples were Campylobacter positive, resulting in a prevalence of 0.23% (95% confidence interval (Cl): 0.12-0.39%). The prevalence of packaged products (0.36%, 95% Cl: 0.17-0.66) was significantly higher than of unpackaged products (0.07; 95% Cl: 0.01-0.27). No statistical differences were found between seasons.Combining the mean prevalence found in this study with data on the consumption of vegetables and fruits, an exposure of 0.0048 campylobacters ingested per person per day in the Netherlands by transmission via vegetables and fruits, was calculated. This exposure, as input in a Beta-Poisson dose-response model, resulted in an estimated number of 5.3 × 10⁵ cases of infection with Campylobacter per year for the whole Dutch population. This constitutes the consumption of raw vegetables and fruits, especially when packaged, to be a risk factor for Campylobacter infections.     

Influence of strawberry tree (Arbutus unedo L.) fruit ripening stage on chemical composition and antioxidant activity

Arbutus unedo is a widespread shrub with economic importance, derived from the use of its berries in the production of alcoholic beverages and in folk medicine. This work intends to evaluate for the first time the effect of fruit ripening stage on antioxidant activity, total phenolic content, fatty acid profile and tocopherol composition. Ripe fruits shown higher extraction yield (45.04 ± 2.23%) when compared to other fruit ripening stages. By contrast, total phenol contents were higher in the unripe and intermediate stage of ripeness (108 and 111 mg GAES/g dry fruit, respectively, against 60 mg/g dry fruit when ripe). Ripe and intermediate fruits shown the lower EC₅₀ values on the DPPH radicals (0.25 ± 0.02 mg/mL) and reducing power assay (1.09 ± 0.05 mg/mL), respectively. A significant correlation was established between antioxidant activity and fruits ripening stage. Fatty acid profiles were very similar between the ripening stages, being alfa-linolenic, linoleic and oleic, the three major ones. Polyunsaturated fatty acids (PUFA) represent as much as 60% of the total fatty acids, with a highly favorable omega 3/omega 6 ratio. From the analysis of the vitamin E vitamers, the most important was ??-tocotrienol, with a clear reduction in the total free vitamin E content with ripening.These results highlight that the fruits of intermediate ripeness can be regarded important sources of biologically active compounds with a fatty acid profile rich in omega-3 PUFA, properly supplemented with high vitamin E amounts.     

Changes on essential oil composition of coriander (Coriandrum sativum L.) fruits during three stages of maturity

The essential oils composition of coriander (Coriandrum sativum L.) fruits obtained by hydrodistillation was studied at three stages of maturity by GC–FID and GC–MS. Essential oil yields showed marked increase during maturation process and forty one compounds were identified. Geranyl acetate (46.27%), linalool (10.96%), nerol (1.53%) and neral (1.42%) were the main compounds at the first stage of maturity (immature fruits). At the middle stage, linalool (76.33%), cis-dihydrocarvone (3.21%) and geranyl acetate (2.85%) were reported as the main constituents. Essential oils at the final stage of maturity (mature fruits) consist mainly on linalool (87.54%) and cis-dihydrocarvone (2.36%). Additionally, accumulation of monoterpene alcohols and ketones was observed during maturation process of coriander fruit.     

Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA

Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.     

Antioxidant capacity and hydrophilic phytochemicals in commercially grown native Australian fruits

Hydrophilic phytochemicals and antioxidant capacities of eight commercially grown native Australian fruits were determined. Kakadu plum (Terminalia ferdinandiana) contained a 6-fold higher level of total phenolic compounds and quandong (Santalum acuminatum) a 1.9-fold higher level of total phenolic compounds (TP, Folin–Ciocalteu assay) than blueberry (Vaccinum sp., cv. Biloxi). Both fruits displayed superior oxygen radical-scavenging capacity (ORAC-H assay) that was, respectively 4.1-fold and 6.5-fold of that of blueberry. The total reducing capacity (TRC; ferric reducing antioxidant power (FRAP) assay) of Kakadu plum and quandong exceeded the TRC of blueberry, respectively, 13.1- and 2.3-times. The primary sources of antioxidant capacities in the evaluated fruits were phenolic acids (benzoic and cinnamic) and flavonoids (flavonols, flavanones and anthocyanins) tentatively detected by liquid chromatography–mass spectrometry (LC–PDA–MS/MS). A high level of vitamin C was recorded for Kakadu plum and Australian citrus fruits. The major organic acids detected were citric and malic acid.     

Nutritional constituents and antioxidant properties of indigenous kembayau (Dacryodes rostrata (Blume) H. J. Lam) fruits

The nutritional and antioxidant properties of peels, pulp and seeds of kembayau (Dacryodes rostrata) fruits were evaluated. Kembayau seeds and pulp were rich in fat, while peels had the highest ash contents. Potassium was the most prevalent mineral in peels (380.72-1112.00 mg/100 g). In kembayau fruits, total flavonoid content (1012.74-28,022.28 mg rutin equivalent/100 g) was higher than total phenolic and total monomeric anthocyanin contents. Kembayau seeds exhibited high flavonoid and phenolic contents compared to the contents in peels and pulp. Antioxidant capacities were also higher in seeds as typified by trolox equivalent antioxidant capacity assay (51.39-74.59 mmol TE/100 g), ferric reducing antioxidant power assay (530.05-556.98 mmol Fe²⁺/100 g) and by 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity (92.18-92.19%) when compared to peels and pulp. Pulp and peels of kembayau fruit may be an important source of energy and minerals for human consumption, while seeds have a good potential as antioxidants.     

Organic acids,antioxidant capacity,phenolic content and lipid characterisation of Georgia-grown underutilized fruit crops

Four underutilized Georgia-grown fruit crops, namely loquat (Eriobotrya japonica), mayhaw (Crataegus sp.), fig (Ficus carica), and pawpaw (Asimina triloba), and their leaves were analysed for total polyphenols by Folin–Ciocalteau method, and antioxidant capacity by ferric-reducing antioxidant power (FRAP) and Trolox-equivalent antioxidant capacity (TEAC) assays. Organic acids and phenolic compounds were identified by RP-HPLC. For lipid profile, fruits were separated into two fractions – seed and fruit (i.e., without seed); lipid was extracted using the Folch method and analysed for fatty acids, phytosterols, tocopherols, and phospholipids. The major organic acid identified in all samples was malic acid (177–1918 mg/100 g FW). The predominant phenolic acids in all the fruits were gallic (1.5–6.4 mg/100 g FW) and ellagic (0.2–33.8 mg/100 g FW), and the most abundant flavonoid was catechin (12.2–37.8 mg/100 g FW). Total lipid content varied from 0.1% in mayhaw fruit to 21.5% in pawpaw seed. Linoleic acid was the predominant fatty acid in all of the samples (28.2–55.7%).     

Prevalence, concentration, spoilage, and mitigation of Alicyclobacillus spp. in tropical and subtropical fruit juice concentrates

The presence of Alicyclobacillus in fruit juices and concentrates poses a serious problem for the juice industry. This study was undertaken to determine the (i) prevalence, concentration, and species of Alicyclobacillus in tropical and subtropical concentrates; (ii) efficacy of aqueous chlorine dioxide in reducing Alicyclobacillus spp. spores on tropical and subtropical fruit surfaces; and (iii) fate of and off-flavor production by Alicyclobacillus acidoterrestris in mango and pineapple juices. One hundred and eighty tropical and subtropical juice concentrates were screened for the presence and concentration of Alicyclobacillus spp. If found, the species of Alicyclobacillus was determined by 16S rDNA sequencing and analysis with NCI BLAST. Of these samples, 6.1% were positive for Alicyclobacillus, and nine A. acidoterrestris strains and two Alicyclobacillus acidocaldarius strains were identified. A five-strain cocktail of Alicyclobacillus spp. was inoculated onto the surface of fruits (grapefruit, guava, limes, mangoes, oranges and pineapple), which were then washed with 0, 50, or 100 ppm aqueous chlorine dioxide. Significant reductions due to chlorine dioxide were only seen on citrus fruits. A five-strain cocktail of A. acidoterrestris was inoculated into mango and pineapple juices. Microbial populations were enumerated over a 16-day period. Aroma compounds in the juice were analyzed by GC-olfactometry (GC-O) and confirmed using GC-MS. GC-O of mango juice identified previously reported medicinal/antiseptic compounds. GC-O of pineapple juice revealed an unexpected “cheese” off-aroma associated with 2-methylbutyric acid and 3-methylbutyric acid.     

Bioactivities of açaí (Euterpe precatoria Mart.) fruit pulp,superior antioxidant and anti-inflammatory properties to Euterpe oleracea Mart.

There are two predominant palm tree species producing edible fruit known as “açaí” found widely dispersed through the Amazon: Euterpe oleracea Mart. and Euterpe precatoria Mart. They differ from each other in terms of how the plants grow and their phytochemical composition. E. oleracea (EO) has received considerable attention as a “super fruit” because of its high antioxidant capacity, while studies on E. precatoria (EP) remain rare. In this study, the antioxidant and anti-inflammatory activities of EP fruit pulps were evaluated by different assays including a series of oxygen radical absorbance capacity (ORAC) based assays, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the cell-based antioxidant protection in erythrocyte (CAP-e) assay, as well as the nuclear factor-kappa B (NF-κB) secreted embryonic alkaline phosphatase (SEAP) assay. Total phenolics were also measured as an indication of the total phenol content. For comparative purposes, the EO fruit pulp was included. The antioxidant capacity of the EP fruit pulp was determined to be superior to the EO fruit pulp in every chemical based assay. In the cell-based CAP-e assay, the EP fruit pulp showed a dose-dependent inhibition against oxidative damage with an IC₅₀ of 0.167 g/l. In the SEAP reporter assay, the EP fruit pulp polyphenol-rich extracts inhibited lipopolysaccharide (LPS)-induced NF-κB activation by 23% (p < 0.05) at 20 μg/ml, whereas the extract of the EO fruit pulp did not show a significant inhibitory effect at comparable doses. In addition, carotenoids were quantified for the first time in EP, since EP has high scavenging capacity against singlet oxygen.     

Effect of Lactobacillus plantarum and chitosan in the reduction of browning of pericarp Rambutan (Nephelium lappaceum)

The effects of Lactobacillus plantarum alone or in combination with chitosan were evaluated on quality and color retention in rambutan fruits (Nephelium lappaceum) stored at 25 °C and 10 °C with 75 ± 2.5% of relative humidity for 10 and 15 days, respectively. The development of the microorganisms was evidenced by viability analyses and lactic acid production. The application of L. plantarum significantly improved color retention (a* and L*), and reduced weight losses. The lactobacilli, alone or in combination with chitosan, preserved fruit quality characteristics such as firmness, total soluble solids and titratable acidity. The lactobacilli application on rambutan pericarp produced acidification of pericarp and avoided the browning; thereby desiccation was prevented due to biofilm formation.     

Flavonols, betacyanins content and antioxidant activity of cactus Opuntia macrorhiza fruits

Flavonols, betacyanins content and antioxidant activity of red-purple cactus Opuntia macrorhiza fruits, a promising cactus pear species, were evaluated in comparison to the well known cactus pear fruits of Opuntia ficus-indica. Flavonol profile was evaluated by HPLC-DAD prior to and after enzymatic hydrolysis (glycosides vs. aglycons). In addition betacyanins, responsible for the purple to red color of cactus pear fruits were also estimated and compared to Beta vulgaris ssp. (roots) and red O. ficus-indica fruits. In all cases, cactus fruit pulps showed no flavonols at all. While different derivatives of isorhamnetin glycosides were found in O. ficus-indica fruit's peel, isorhamnetin-3-O-rutinoside was the only compound in O. macrorhiza fruit. Correspondingly, antioxidant activity assays showed a high antioxidant activity of both, O. macrorhiza fruit's peel and pulp. With regard to betacyanins, O. macrorhiza fruit's peel and pulp provide a deep red-purple color, whose average impact is higher compared to red beet (B. vulgaris spp.) and about 8-fold higher than red fruits from O. ficus-indica. Supporting these results, estimation of color attributes (L*, a*, b*, C and H°) showed also a high color impact of both O. macrorhiza fruit's peel and pulp.     

Biochemical monitoring of black raspberry (Rubus coreanus Miquel) fruits according to maturation stage by H NMR using multiple solvent systems

Nuclear magnetic resonance (NMR) techniques coupled with multivariate data analysis were used to conduct monitoring of biochemical changes of black raspberry fruits at different stages of maturation and under various extraction and NMR dissolution solvent conditions: extraction with 50% methanol and D₂O as an NMR dissolution solvent, extraction with 50% methanol and 50% methanol-d₄ as an NMR dissolution solvent, and extraction with 100% ethyl acetate and 100% methanol-d₄ as an NMR dissolution solvent. Partial least-squares discriminant analysis reliably distinguished black raspberry fruits according to the maturation stage, whereby the relative levels of various compounds such as amino acids, organic acids, sugars and phenolic compounds were compared using analysis of variance. Sucrose and most of the amino acids, and organic acids decreased, whereas fructose, glucose, and cyanidins increased in relative concentration according to maturation of black raspberry fruits. The total number and kinds of assigned compounds of the three solvent systems were also compared. This research demonstrates that the metabolic profile of black raspberry fruits changes during maturation, and provides objective criteria for determining the stage of black raspberry fruit maturation via a ¹H NMR-based metabolomics technique using multiple solvent systems.     

Phenolics, betacyanins and antioxidant activity in Opuntia joconostle fruits

Sour prickly pears (xoconostles) are fruits from Opuntia joconostle cactus, which are cultivated in the central Mexico area. Phenolic and pigment content in various parts of O. joconostle fruits were analyzed. The antioxidant activity of a methanolic extraction and different semi-purified fractions were also evaluated by the DPPH⁺ method. Xoconostle fruits were obtained from a commercial orchard in Mexico State. Fruits were analyzed as whole fruit and each fruit part including pericarp, mesocarp and endocarp. Samples were homogenized and kept at 4 °C until sample preparation. Total phenolic content and total flavonoid content varied among the different parts of the fruit. The highest amount of phenolic compounds and total flavonoids content were found in pericarp 2.07 mg gallic acid equivalents (GAE)/g fresh weight (FW) and 0.46 mg(+)-catechin equivalents (CE)/g FW respectively. Seven phenolics were identified as protocatechuic, 4-hydroxybenzoic, caffeic, vanillic and syringic acids, rutin, and quercetin. The color of the fruit parts was mainly due to the presence of betacyanins. The betacyanin concentration was higher in the endocarp (23.03 mg betanin equivalents/100 g fresh weight) than in the pericarp and mesocarp. Betacyanins were identified by HPLC-PDA-MS as betanin, isobetanin, betanidin, isobetanidin, and phyllocactin. Methanolic extracts and semi-purified fractions A (phenolics and flavonols) and B (betacyanins) of xoconostle showed high antioxidant activity mainly in the pericarp. These results suggest that xoconostle is a rich source of antioxidant compounds such as phenolic compounds and betacyanins.