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Cultivar and growing region determine the antioxidant polyphenolic concentration and composition of apples grown in New Zealand.

Evidence suggests that increasing consumption of fruit and vegetables contributes to improved health and well-being by providing protection from diseases including various cancers and cardiovascular disease. Although there is uncertainty about which components generate this effect, an attractive hypothesis is that the antioxidants are at least partly responsible. We measured the polyphenolic concentrations in 10 different apple cultivars grown commercially in New Zealand, each sourced from three different geographic regions. Our results showed that the concentration of polyphenolics varied among the apple cultivars, with Pacific Queen containing 2.7 times the amount of polyphenolics found in Cox's Orange. Furthermore, there were significant differences in polyphenolic concentrations in fruit from different regions for some cultivars but not for others. We also measured the polyphenolic concentrations in apple skin and flesh and found that on average 46% of the polyphenolics in whole apples were in the skin. Essentially all of the flavonols (quercetin derivatives) were present in the skin. To maximize the intake of apple polyphenols, it is necessary to consume apples of cultivars with high polyphenolic concentrations such as Pacific Queen and include the skin. Our results also showed that there is potential for promoting apple fruit from specific geographical regions because they contained elevated concentrations of antioxidant polyphenolic compounds.

Antioxidants↗

Apples prevent mammary tumors in rats.

Regular consumption of fruits and vegetables has been consistently shown to be associated with reduced risk of developing chronic diseases such as cancer and cardiovascular disease. Apples are commonly consumed and are the major contributors of phytochemicals in human diets. It was previously reported that apple extracts exhibit strong antioxidant and antiproliferative activities and that the major part of total antioxidant activity is from the combination of phytochemicals. Phytochemicals, including phenolics and flavonoids, are suggested to be the bioactive compounds contributing to the health benefits of apples. Here it is shown that whole apple extracts prevent mammary cancer in a rat model in a dose-dependent manner at doses comparable to human consumption of one, three, and six apples a day. This study demonstrated that whole apple extracts effectively inhibited mammary cancer growth in the rat model; thus, consumption of apples may be an effective strategy for cancer protection.

9,10-Dimethyl-1,2-benzanthracene↗

Modulation of key elements of the Wnt pathway by apple polyphenols.

Glycogen synthase kinase-3beta (GSK3beta) is one of the key elements of the Wnt pathway involved in the regulation of beta-catenin homeostasis. The inhibition of GSK3beta kinase activity might lead to the onset of beta-catenin/TCF/LEF-mediated gene transcription, representing a potentially mitogenic stimulus. Apple polyphenols have been shown to mediate several biological effects that might be of interest with respect to chemoprevention. The objective of the study was to elucidate whether apple polyphenols also modulate key elements of the Wnt pathway, an effect that might limit the usefulness of these compounds for the prevention of carcinogenesis. A polyphenol-rich apple juice extract (AE02) was found to effectively inhibit the kinase activity of GSK3beta, immunoprecipitated from HT29 cells. Treatment of HT29 cells with AE02 for 24 h resulted in a concentration-dependent decrease of the cellular GSK3beta kinase activity. The inhibition of the kinase activity in HT29 cells was observed at polyphenol concentrations corresponding to the concentration of the constituents in the original apple juice. The apple characteristic dihydrochalcone glycoside phloridzin was found to be inactive up to 500 muM. The free aglycon phloretin as well as the flavonol quercetin effectively inhibited isolated GSK3beta, but did not affect the respective kinase activity within HT29 cells. In accordance with the inhibition of GSK3beta kinase activity by AE02, treatment of HT29 cells resulted in a significant decrease of phosphorylated beta-catenin. However, the total intracellular beta-catenin level was also found to be diminished, indicating that the interference of the apple constituents with GSK3beta was not associated with a stabilization of beta-catenin in HT29 cells. In line with these results, TCF/LEF-mediated gene transcription remained unaffected by treatment with AE02 as shown in a reporter gene approach. We can assume from the results that at consumer-relevant concentrations apple polyphenols do not mediate growth-stimulating effects in HT29 cells via the Wnt pathway.

Blotting, Western↗

Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development.

beta-Amyloid precursor protein binding protein 1 (APP-BP1) was previously identified based on its binding to the carboxyl terminal of beta-amyloid precursor protein. In this report, we have discovered that a mutation of dAPP-BP1 (Drosophila ortholog of APP-BP1) hinders tissue development, causes apoptosis in imaginal disc cells, and blocks the NEDD8 conjugation pathway. We show that dAPP-BP1 specifically binds the intracellular domain of APP-like protein (APPL). The dAPP-BP1 mutation partially suppresses the abnormal macrochaete phenotype of Appl(d), while overexpression of dAPP-BP1 causes abnormal macrochaetes. When APPL is overexpressed, the normal bristle pattern in the fly thorax is disturbed and apoptosis is induced in wing imaginal discs. APPL overexpression phenotypes are enhanced by reducing the level of dAPP-BP1. APPL overexpression is shown to inhibit the NEDD8 conjugation pathway. APPL-induced apoptosis is rescued by overexpression of dAPP-BP1. Our data suggest that APPL and dAPP-BP1 interact antagonistically during Drosophila development.

Amino Acid Sequence↗

Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers.

The genetic variation within and between wild apple samples (Malus sylvestris) and cultivated apple trees was investigated with amplified fragment length polymorphisms (AFLP) and microsatellite markers to develop a conservation genetics programme for the endangered wild apple in Belgium. In total, 76 putative wild apples (originating from Belgium and Germany), six presumed hybrids and 39 cultivars were typed at 12 simple sequence repeats (SSR) and 139 amplified fragment length polymorphism (AFLP) loci. Principal co-ordinate analysis and a model-based clustering method classified the apples into three major gene pools: wild Malus sylvestris genotypes, edible cultivars and ornamental cultivars. All presumed hybrids and two individuals (one Belgian, one German) sampled as M. sylvestris were assigned completely to the edible cultivar gene pool, revealing that cultivated genotypes are present in the wild. However, gene flow between wild and cultivated gene pools is shown to be almost absent, with only three genotypes that showed evidence of admixture between the wild and edible cultivar gene pools. Wild apples sampled in Belgium and Germany constitute gene pools that are clearly differentiated from cultivars and although some geographical pattern of genetic differentiation among wild apple populations exists, most variation is concentrated within samples. Concordant conclusions were obtained from AFLP and SSR markers, which showed highly significant correlations in both among-genotypes and among-samples genetic distances.

Belgium↗

Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly.

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (Malus pumila)- and hawthorn (Crataegus spp.)-infesting races of Rhagoletis pomonella (Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect's life cycle with the phenology of its host plant.

Journal Article↗

Effects of dietary fibre-rich juice colloids from apple pomace extraction juices on intestinal fermentation products and microbiota in rats.

Effects of colloids isolated from apple pomace extraction juices (so-called B-juices) produced by enzymic liquefaction on food intake, body and faecal weights, short-chain fatty acid (SCFA) profile and selected intestinal microbiota were investigated in rats. Ten male Wistar rats per group were fed diets without any apple dietary fibre (DF) (control) or supplement with 5 % B-juice colloids or an alcohol-insoluble substance (AIS) from apples for 6 weeks. Rats fed with apple DF (5 % B-juice colloids or AIS) gained less weight than control rats (P<0.05). B-juice colloids did not affect food intake, whereas feeding AIS resulted in a 10 % higher food consumption than in control rats. Both juice colloids and AIS increased the weight of caecal contents in rats and lowered luminal pH values (P<0.05). In addition, SCFA concentrations and total yields were also raised (P<0.05) in caecum of these rats indicating good fermentability of apple substrates by gut microflora. Distinctly higher concentrations of acetate and propionate were found in intestinal contents of juice colloid-fed rats (P<0.05), whereas AIS also increased butyrate yield. Changes in microbiota due to apple DF in diets were restricted in the caecum to the Eubacterium rectale cluster (AIS; P<0.05) and in faeces to the Bacteroidaceae (juice colloids and AIS; P<0.05). The present study shows the physiological effects of apple DF isolated from pomace extraction juices produced by enzymic liquefaction on intestinal fermentation. Results may be helpful for the development of such innovative juice products that are rich in DF of fruit origin.

Animals↗

Patulin in apple-based foods: occurrence and safety evaluation.

Patulin is a mycotoxin produced by certain species of Penicillium and Aspergillus, often detectable in mouldy fruits and their derivatives. On the basis of a PMTDI of 0.4 microgram/kg bw, limit values of 50 micrograms/kg or 50 micrograms/l of patulin have been set in fruit derivatives. To estimate the quantity of patulin that can be taken in with the diet, we analysed by HPLC samples of apples and apple derivatives which are most likely to be contaminated with patulin. In apple juices and in homogenized baby-foods, the mycotoxin concentration was always below the established limits, while in some samples of juice with pulp the mycotoxin content exceeded the safe levels. In rotten apples, not only was the amount of patulin extraordinarily high in the rotten area, but the mycotoxin had also spread to the part unaffected by mould. The data presented in this study indicate that the intake of patulin with apple derivatives is usually below the tolerable level of 0.4 microgram/kg bw/day, but since the patulin content in apples can vary considerably, the quality of fruits used in the production of apple derivatives should be strictly controlled in order not to exceed the safe limits.

Chromatography, High Pressure Liquid↗

Distribution of multiple pesticide residues in apple segments after home processing.

The effects of washing, storing, boiling, peeling, coring and juicing on pesticide residue were investigated for field-sprayed Discovery and Jonagold apples. Residues of chlorpyrifos, cypermethrin, deltamethrin, diazinon, endosulfan, endosulfan sulfate, fenitrothion, fenpropathrin, iprodione, kresoxim-methyl, lambda-cyhalothrin, quinalphos, tolylfluanid and vinclozolin in the processed apples were analysed by gas chromatography. Statistical analysis showed that reductions of 18-38% were required to obtain significant effects of processing practices, depending on pesticide and apple variety. Juicing and peeling the apples significantly reduced all pesticide residues. In the case of detectable pesticide residues, 1-24% were distributed in the juice and in the peeled apple. None of the pesticide residues was significantly reduced when the apples were subject to simple washing or coring. Storing significantly reduced five of the pesticide residues: diazinon, chlorpyrifos, fenitrothion, kresoxim-methyl and tolylfluanid, by 25-69%. Residues of the metabolite endosulfan sulfate were increased by 34% during storage. Boiling significantly reduced residues of fenitrothion and tolylfluanid by 32 and 81%, respectively. Only a few of the observed effects of processing could be explained by the physical or chemical characteristics of the pesticides. No differences in effect of processing due to apple variety were identified.

Chromatography, Gas↗

Patulin distribution in Fuji and Golden apples contaminated with Penicillium expansum.

This work assesses the extent of patulin contamination in Penicillium expansum-infected apples stored at room temperature for short periods of time and its relationship with apple variety (Golden or Fuji), degree of ripeness and size of lesions. Inoculated apples were incubated at 20 degrees C. Patulin was determined in both sound and decayed tissue from cylindrical samples taken around the lesions and cut into 0.5-cm thick sections. Higher accumulation of patulin occurred in Golden apples, with less ripened apples showing higher concentrations. Total accumulated patulin was similar or higher in 4-cm compared to 2-cm lesioned apples, although a decrease in patulin concentration was observed in older lesion sections. Patulin accumulation occurred over a short period of time at room temperature, thus the stand-by period before processing should be minimised. Of total patulin, 2-6% migrated to the surrounding sound tissue, thus trimming tissue around the rotten part may be a good preventive practice for apple derivative production.

Food Analysis↗

Lyophilized apple counteracts the development of hypercholesterolemia, oxidative stress, and renal dysfunction in obese Zucker rats.

Apples may have selective effects on abnormalities associated with the plurimetabolic syndrome. Therefore, the effects of 20% lyophilized apple supplementation on plasma and tissue lipids and on protection against susceptibility to oxidative stress and renal dysfunction were investigated in Zucker lean (Fa/-) or obese (fa/fa) rats. The experimental diets were equilibrated for sugar supply, contained 0.25 g/100 g cholesterol and provided only one third of the vitamin E requirement. Obese Zucker rats were hypercholesterolemic with cholesterol accumulation in LDL and HDL fractions. The apple diet lowered plasma and LDL cholesterol (-22 and -70%, respectively, P < 0.01) in obese Zucker rats and, in parallel, reduced triglyceride accumulation in heart and liver. Zucker rats fed the apple diet also had a larger intestinal pool and greater fecal excretion of bile acids. The heart concentration and urinary excretion of malondialdehyde were reduced by apple consumption in obese Zucker rats, suggesting better protection against peroxidation. Glucosuria and proteinuria in obese Zucker rats were also suppressed by the apple diet. In conclusion, despite their moderate fiber content, apples improve substantially the lipid status and peroxidative parameters in obese Zucker rats, suggesting that other plant constituents such as polyphenols are involved in these effects.

Animals↗

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity.

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.

Alternaria↗

Detection of apple juice adulteration using near-infrared transflectance spectroscopy.

Near-infrared transflectance spectroscopy was used to detect adulteration of apple juice samples. A total of 150 apple samples from 19 different varieties were collected in two consecutive years from orchards throughout the main cultivation areas in Ireland. Adulterant samples at 10, 20, 30, and 40% w/w were prepared using two types of adulterants: a high fructose corn syrup (HFCS) with 45% fructose and 55% glucose, and a sugars solution (SUGARS) made with 60% fructose, 25% glucose, and 15% sucrose (the average content of these sugars in apple juice). The results show that NIR analysis can be used to predict adulteration of apple juices by added sugars with a detection limit of 9.5% for samples adulterated with HFCS, 18.5% for samples adulterated with SUGARS, and 17% for the combined (HFCS + SUGARS) adulterants. Discriminant partial least squares (PLS) regression can detect authentic apple juice with an accuracy of 86-100% and adulterant apple juice with an accuracy of 91-100% depending on the adulterant type and level of adulteration considered. This method could provide a rapid screening technique for the detection of this type of apple juice adulteration, although further work is required to demonstrate model robustness.

Algorithms↗

Seasonal and cultivar-associated variation in oviposition preference of Oriental fruit moth (Lepidoptera: Tortricidae) adults and feeding behavior of neonate larvae in apples.

The Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) has become a pest of tree fruits since its introduction to the United States in the early twentieth century. Oriental fruit moth has historically been a major pest problem in peach production, and outbreaks in commercial apple (Malus spp.) orchards in the eastern United States were rare until the late 1990s. Recent outbreaks in Mid-Atlantic apple orchards have lead researchers to investigate host-associated effects on oriental fruit moth biology, behavior, and population dynamics. Studies were designed to assess cultivar level effects in apples on oviposition and larval feeding behavior of oriental fruit moth. In a mixed cultivar apple orchard, total oriental fruit moth oviposition and oviposition site preferences varied between cultivars. These preferences also varied over time, when sampling was repeated at various times of the growing season. Although most adult female oriental fruit moth preferentially oviposited in the calyx and stem areas of apple fruit, noticeable numbers of eggs also were laid on the sides of fruit, contradicting some previous reports. Oriental fruit moth females exhibited a strong ovipositional preference for fruit that were previously damaged by oriental fruit moth or codling moth, Cydia ponmonella (L.). The majority of newly hatched oriental fruit moth larvae were observed to spend <24 h on the surface of apple fruit before entry, and this behavior was observed on several apple cultivars. Neonate larvae exhibited a preference for entering fruit at either the stem or calyx ends, regardless of their initial site of placement. Our findings underscore the importance of adequate spray coverage and accurate timing of insecticide applications targeting oriental fruit moth.

Animals↗

Use of hazard analysis critical control point and alternative treatments in the production of apple cider.

The purpose of this study was to evaluate the practices of Maryland cider producers and determine whether implementing hazard analysis critical control point (HACCP) would reduce the microbial contamination of cider. Cider producers (n = 11) were surveyed to determine existing manufacturing practices and sanitation. A training program was then conducted to inform operators of safety issues, including contamination with Escherichia coli O157:H7, and teach HACCP concepts and principles, sanitation procedures, and good manufacturing practice (GMP). Although all operators used a control strategy from one of the model HACCP plans provided, only one developed a written HACCP plan. None developed specific GMP, sanitation standard operating procedures, or sanitation monitoring records. Six operators changed or added production controls, including the exclusion of windfall apples, sanitizing apples chemically and by hot dip, and cider treatment with UV light or pasteurization. Facility inspections indicated improved sanitation and hazard control but identified ongoing problems. Microbiological evaluation of bottled cider before and after training, in-line apples, pomace, cider, and inoculated apples was conducted. E. coli O157:H7, Salmonella, or Staphylococcus aureus were not found in samples of in-line apple, pomace, and cider, or bottled cider. Generic E. coli was not isolated on in-coming apples but was found in 4 of 32 (13%) in-line samples and 3 of 17 (18%) bottled fresh cider samples, suggesting that E. coli was introduced during in-plant processing. To produce pathogen-free cider, operators must strictly conform to GMP and sanitation procedures in addition to HACCP controls. Controls aimed at preventing or eliminating pathogens on source apples are critical but alone may not be sufficient for product safety.

Beverages↗

The survival of Escherichia coli O157:H7 in the presence of Penicillium expansum and Glomerella cingulata in wounds on apple surfaces.

The survival of Escherichia coli O157:H7 in the presence of one of two plant pathogens, Penicillium expansum and Glomerella cingulata, in wounds on apples was observed during 14 days storage at room temperature (RT) and at 4 degrees C. The aim of this work was to determine if changes in apple physiology caused by the proliferation of fungal decay organisms would foster the survival of E. coli O157:H7. Trials were performed where (A) plant pathogens (4 log10 spores) were added to apple wounds 4 days before the wounds were inoculated with E. coli O157:H7 (3 log10 CFU g(-1) apple) (both RT and 4 degrees C storage), (B) plant pathogens and E. coli O157:H7 were added on the same day (both RT and 4 degrees C storage), and (C) E. coli O157:H7 was added 2 days (RT storage) and 4 days (4 degrees C storage) before plant pathogens. In all trials E. coli O157:H7 levels generally declined to <1 log10 at 4 degrees C storage, and in the presence of P. expansum at 4 degrees C or RT. However, in the presence of G. cingulata at RT E. coli O157:H7 numbers increased from 3.18 to 4.03 log10 CFU g(-1) in the apple wound during trial A, from 3.26 to 6.31 log10 CFU g(-1) during trial B, and from 3.22 to 6.81 log10 CFU g(-1) during trial C. This effect is probably a consequence of the attendant rise in pH from 4.1 to approximately 6.8, observed with the proliferation of G. cingulata rot. Control apples (inoculated with E. coli O157:H7 only) were contaminated with opportunistic decay organisms at RT during trials A and B, leading to E. coli O157:H7 death. However, E. coli O157:H7 in control apples in trial C, where no contamination occurred, increased from 3.22 to 5.97 log10 CFU g(-1). The fact that E. coli O157:H7 can proliferate in areas of decay and/or injury on fruit highlights the hazards associated with the use of such fruit in the production of unpasteurized juice.

Colony Count, Microbial↗

Isolation and characterization of Escherichia coli recovered from Maryland apple cider and the cider production environment.

Contaminated apple cider has been implicated in several Escherichia coli O157:H7 outbreaks. In an attempt to investigate sources and modes of entry of E. coli into apple cider, samples of fresh apple, pomace, and cider and equipment and mill floor swabs were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms (FC), and E. coli. E. coli was isolated from 14 (33%) of 42 samples of bottled fresh cider, from food equipment in 6 (67%) of 9 mills, and from apples, pomace, or cider in 7 (78%) of 9 mills. Seventy-five E. coli isolates were further characterized for Shiga toxin-producing E. coli (STEC)-associated virulence factors, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) type. No E. coli O157:H7 or other STEC was identified. Serotyping and PFGE revealed 64 distinct profiles, suggesting that recovered E. coli arose from multiple independent sources. However, on one occasion, E. coli isolated from the source apple sample was closely related to the E. coli identified in the finished cider sample. E. coli isolates were further tested for antimicrobial susceptibility to 17 antimicrobial agents of human and veterinary importance. Fourteen (19%) of the 75 isolates were resistant to at least one of the antimicrobial agents tested, and 9 (12%) were resistant to at least two of these agents. Of the resistant isolates recovered, 64% were resistant to tetracycline and 57% were resistant to streptomycin. Overall, the level of E. coli contamination in source apple samples did not differ significantly from those in samples of pomace, cider at the press, and cider entering the bottling tank; therefore, source apples cannot be dismissed as a potential contributor of E. coli to the cider-making process.

Anti-Bacterial Agents↗

Effect of pH on the survival of Listeria innocua in calcium ascorbate solutions and on quality of fresh-cut apples.

Fresh-cut apple slices were dipped in calcium ascorbate (CaA) solution at pH values ranging from 2.5 to 7.0 to inhibit browning. After treatment, the cut apples were stored at 4 and 10 degrees C for up to 21 days. Color and texture of the apples were determined on days 1, 14, and 21. In a separate experiement, the pH of CaA solution was adjusted with acetic acid to six different pH levels, and the solution was inoculated with Listeria innocua. The survival of the bacterium and the stability of CaA were determined at 0, 20, and 96 h. The cut apples maintained fresh quality when the pH of the CaA solution was above 4.5, but slight discoloration of apple slices dipped in pH 4.5 solution was observed after 14 days at 10 degrees C. At pH 5.0, the CaA dip maintained the quality of the apples at both temperatures for at least 21 days. The L. innocua population was reduced by 4 to 5 log CFU/ml at pH 4.5 after 96 h. At pH 5, the bacterial population in the CaA solution was reduced by approximately 2 log CFU/ml during the same period. The CaA solution was stable at pH 5 for at least 96 h. Reduction of the pH to between 4.5 and 5.0 might reduce the risk of foodborne illness due to consumption of fresh-cut apples treated with a CaA solution contaminated with Listeria.

Ascorbic Acid↗