Unripe Apples Research Articles

γδ T cells characteristic of the gut mucosa are stimulated by oligomeric tannins (52.11)

Abstract γδ T cells function, in part, to regulate the innate immune response and maintain tissue homeostasis at mucosal surfaces. Increasing the activity of the γδ T cells may be effective for treating a variety of pathogens that target the gut mucosa including rotavirus and Salmonella. Treatment with the phosphoantigen family of agonists stimulates only the Vδ2 subset of human γδ T cells and does not stimulate Vδ1 T cells, the primary γδ T cell population at the sites of pathogen entry. Here we define the biological activity of an agonist that stimulates both the Vδ2 and importantly, the Vδ1 subsets of human γδ T cell population. Initial examination of the effects in vivo suggests that the agonist may induce expansion of mouse intestinal γδ T cells after oral administration. The agonist is part of an unripe apple peel extract that contains a mixture of phenolics including monomeric and oligomeric tannins. Preliminary characterization of the extract suggests that the γδ T cell stimulation is due to the specific interaction of an oligomeric tannin.

Plant polyphenols induce differential regulation of CD11b expression and function on monocytes and γδ T cells (97.11)

Abstract Leukocyte adhesion and migration is partially mediated by CD11b/CD18 (MAC-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on monocytes and CD8+ T cells. We have recently shown a stimulatory effect of unripe apple polyphenols (APP) on γδ T cells. Thus, we compared the effect of APP on γδ T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted peripheral blood lymphocytes were cultured with APP for 24 hours. CD11b levels decreased on monocytes in response to APP. In contrast, a subset of γδ T cells responded to APP by up-regulating CD11b. Consistant with the changes in surface CD11b expression, APP treated γδ T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. The affects of APP on monocytes could be inhibited by anti-CD11b mAb, whereas the mAb had no effect on the APP response in γδ T cells, suggesting the two cell types respond to APP via different mechanisms. Polyphenols are typically thought of as anti-inflammatory agents, however, these data, as well as other ongoing studies, indicate they have a pro-inflammatory effect on γδ T cells. In vivo, plant polyphenols may enhance γδ T cell migration to sites of inflammation, where they can enact rapid immunoregulatory and innate-like responses. Funded by: NIH –NIAID DAIT-BAA-03 -41

Absorption of Dietary Cholesterol Oxidation Products and Their Downstream Metabolic Effects Are Reduced by Dietary Apple Polyphenols

Exogenous and endogenous cholesterol oxidation products (COPs) perturb various metabolic processes, and thereby they may induce various homeostasis-related disorders. Here, we observed that procyanidin-rich dietary apple polyphenol (APP) from unripe apples alleviates the perturbation of lipid metabolism by decreasing the exogenous COP levels in rats. Dietary COPs may be the greatest source of COPs found in the human body. Rats (4 weeks of age) were fed AIN-purified diets containing 0.3% COPs supplemented with 0.5 or 2.5% APP for 3 weeks. Dietary APP alleviated the growth inhibition action of the exogenous COPs. The modulations of the liver lipid profile by COPs remained unchanged. However, serum total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels increased following the intake of dietary APP. Further, dietary APP inhibited the increase in lipid peroxide levels in the liver and serum by COPs. The activity of hepatic Delta6 desaturase was lowered by dietary APP in a dose-dependent manner, although exogenous COPs generally increased the activity of this enzyme. In keeping with this observation, Delta6 desaturation indices in the phospholipids and cholesteryl esters of the liver and serum lipids were lower in the APP-fed groups than those in the control group. Dietary APP also promoted the excretion of exogenous COPs, cholesterol, and acidic steroids in feces. Therefore, the inhibition of intestinal absorption of COPs may partly contribute to the alleviation of the perturbation of lipid metabolism and lipid peroxidation levels. Thus, APP may be an important removal agent of exogenous toxic material such as COPs contained in processed or fast foods.

EFFECT OF PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY

The mechanisms governing the enzymatic clarification of apple juice were studied by electron microscopy techniques. Full ripe and unripe apple juice samples (Granny Smith) were treated with commercial pectinase (Solvay 5XLHA) and amylase (Röhalase HT) enzymes, respectively. Scanning electron microscopy studies revealed that commercial amylolytic enzymes quickly reduced starch content in unripe apple juice to undetectable values. It was also observed that after pasteurization of this juice (90C, 5 min) all starch granules gelatinized. Using transmission electron microscopy, it was possible to observe pectin bonded to ripe apple juice particles. This protective colloid is known to be responsible for cloudy juice stability. The effect of pectic enzyme to destroy the protective pectin colloid was also detected with this technique. As a result of the enzymatic treatment, average particle size initially increased from 1000 to 1500 nm and decreased thereafter to ≈1100 nm, and Z-potential increased in absolute values from −9.6 to −11.4 mV. It was speculated that the destruction of the weak pectin net by the action of the specific enzyme caused particle aggregation, followed by the collapse of aggregates, increasing the number of particles <500 nm.

Dose‐dependent hypocholesterolemic actions of dietary apple polyphenol in rats fed cholesterol

The dose-dependent hypocholesterolemic and antiatherogenic effects of dietary apple polyphenol (AP) from unripe apple, which contains approximately 85% catechin oligomers (procyanidins), were examined in male Sprague-Dawley rats (4 wk of age) given a purified diet containing 0.5% cholesterol. Dietary AP at 0.5 and 1.0% levels significantly decreased the liver cholesterol level compared with that in the control (AP-free diet-fed) group. Dietary AP also significantly lowered the serum cholesterol level compared with that in the control group. However, the HDL cholesterol level was significantly higher in the 1.0% AP-fed group than in the control group. Accordingly, the ratio of HDL-cholesterol/total cholesterol was significantly higher in the 0.5% AP-fed group and 1.0% AP-fed group than in the control group. Moreover, the atherogenic indices in the 0.5 and 1.0% AP-fed groups were significantly lower than those in the control group. The activity of hepatic cholesterol 7alpha-hydroxylase tended to be increased by dietary AP in a dose-dependent manner. In accord with this observation, dietary AP increased the excretion of acidic steroids in feces. Dietary AP also significantly promoted the fecal excretion of neutral steroids in a dose-dependent manner. These observations suggest that dietary AP at a 0.5 or 1.0% level exerts hypocholesterolemic and antiatherogenic effects through the promotion of cholesterol catabolism and inhibition of intestinal absorption of cholesterol.

Morphological, Structural, Thermal, and Rheological Characteristics of Starches Separated from Apples of Different Cultivars

The starches were separated from unripe apples of five cultivars (Criterion, Ruspippum, Red Spur, Skyline Supreme, and Granny Smith) and evaluated using scanning electron microscopy (SEM), gel permeation chromatography (GPC), X-ray diffraction, differential scanning calorimetry (DSC), and dynamic viscoelasticity. SEM showed the presence of round granules as well as granules that had been partially degraded, probably by amylases. The starch granules in different apple starches ranged between 4.1 and 12.0 mum. Debranching of starch with isoamylase and subsequent fractionation of debranched materials by GPC revealed the presence of an apparent amylose, an intermediate fraction (mixture of amylose and amylopectin), long side chains of amylopectin, and short side chains of amylopectin in the range of 28-35.2, 3.6-4.4, 20-21.3, and 39.9-47.1%, respectively. The swelling power of starches ranged between 14.4 and 21.3 g/g. X-ray diffraction of apple starches showed a mixture of A- and B-type patterns. All apple starches showed peak intensities lower than that observed for normal corn and potato starch, indicating the lower crystallinity. The transition temperatures (onset temperature, T(o); peak temperature, T(p); and conclusion temperature, T(c)) and enthalpy of gelatinization (deltaH(gel)) determined using DSC ranged between 54.7 and 56.2 degrees C, between 57.1 and 59.1 degrees C, between 60.2 and 63.5 degrees C, and between 3.3 and 4.2 J/g, respectively. The viscoelastic properties of starch from different cultivars measured during heating and cooling using a rheometer differed significantly. Red Spur and Criterion starches with larger granule size showed higher G' and G' ' values, whereas those containing smaller size and amylolytically degraded granules showed lower G' and G' '.

Dietary unripe apple polyphenol inhibits the development of food allergies in murine models

The incidence of type I allergic disorders has been increasing worldwide, particularly, the hypersensitivity to food. We first showed that apple condensed tannin (ACT) intake would inhibit the development of the oral sensitization and that the inhibition could correlate with the rise in the population of TCRγδ-T cells in the intestinal intraepithelial lymphocytes (IEL) using W/WV mice and B10A mice which were ovalbumin (OVA)-orally sensitized. Serum OVA-specific immunoglobulin E and immunoglobulin G1 titers in the OVA-orally sensitized W/WV and B10A mice ad libitium fed ACT were extremely inhibited compared to those of the control. The ACT intakes of OVA-sensitized W/WV and B10A mice inhibited the immediate reduction of the body temperature or the rise in serum histamine induced by active systemic anaphylaxis. The proportions of the TCRγδ-T cells in the IEL of the OVA-orally sensitized W/WV and B10A mice ad libitium fed ACT were significantly greater than that in the controls. Furthermore, ACT feeding by itself could induce the rise in the percentage of the TCRγδ-T cells among the IEL of the W/WV and B10A mice. This suggests that the ACT intake may prevent the development of food allergies and this effect could be correlated with the rise in the percentage of TCRγδ-T cells among the IEL.

The toxicology and safety of apple polyphenol extract

Apple polyphenol extract has strong antioxidant activity and various physiological functions, and is used in Japan as a food additive and nutritional supplements. Here, we tested the consumption safety of Applephenon®, which is a polyphenol extract produced from unripe apples. The Ames test without S9 mixture revearled that Applephenon®, had slight mutagenicity at a high concentration of 2500 μg/plate; however, both chromosomal aberration test and the micronucleus test found no significant mutagenicity. Furthermore, an acute oral-toxicity test, and a 90-day subchronic-toxicity test showed no significant hematological, clinical, chemical, histopathological, or urinary effects at a dose of 2000 mg/kg. These results confirme that Applephenon® is safe and no toxic at average dietary level.

Characterization of starch in apple juice and its degradation by amylases

Soluble and insoluble starches from apple juice (Granny Smith variety) at different degree of ripeness were separated and quantified by an iodometric method. Non pasteurised unripe apple juice had as much as 8 g/L starch, 2 weeks before usual harvest date. However, juices obtained from unripe apples stored for 3 weeks at room temperature, had undetectable starch contents. Scanning electron micrographs (SEM) showed apple starch granules are particles of regular shape (mean diameter=9.21 μm, standard deviation=2.74). SEM studies also revealed that after pasteurising apple juice (90 °C, 5 min) virtually all the starch granules lost their spherical structure and only gel-like starch fragments dispersed among the other components of haze (pectin, cell wall, etc.) were observed. Röhalase HT (RO) and Tyazyme L300 (TY) amylases normally used in fruit juices clarification, even in doses lower than those industrially recommended, quickly reduced starch contents in pasteurised juices to undetectable values. Microscopy studies also demonstrated that RO enzyme efficiently degraded raw apple starch granules, which showed evidence of centrifugal and centripetal hydrolysis.

Characteristics and physiological functions of polyphenols from apples

Apples contain many kinds of polyphenols, and the main components are oligomeric procyanidins. Applephenon is apple polyphenol extract produced commercially from unripe apples, and has been used as food additive in order to prevent oxidation of components in foods and its application in functional foods is expected. In a lipid metabolism regulation study, administration of Applephenon has the potential to exert strong anti-oxidative activity and to inhibit consumption of vitamin E and anti-oxidative enzymes. Double blind clinical trials of Applephenon on pediatric patients with atopic dermatitis, and tests using type I allergic model mice suggested that Applephenon might regulate allergic reactions. We found the no observed adverse effect level (NOAEL) of Applephenon is greater than 2000 mg/kg in a 90~day consecutive oral administration toxicity test in rats, and Applephenon is safe and acceptable based on mutagenicity tests.

Inhibitory Effects of Apple Polyphenols on Differentiation of 3T3-L1 Cells into Adipocytes.

A crude apple polyphenol (CAP) fraction partially purified from unripe apples was found to inhibit adipose differentiation in mouse fibroblast 3T3-L1 cells. CAP inhibited the increase in intracellular levels of glycerophosphate-3-dehydrogenase (GPDH), one of the most important enzymes in the pathway for triacylglycerol biosynthesis, that occurs upon differentiation of 3T3-L1 cells into adipocytes and decreased the triglyceride content of the cells. In particular, apple condensed tannins (ACT), purified from CAP, inhibited the increase in GPDH activity when these cells differentiated. In contrast, phloridzin, (+)-catechin, and (−)-epicatechin, which were also present in the CAP fraction, decreased the triglyceride content of the cells but did not inhibit the increase in GPDH activity These results suggest that ACT such as procyanidins inhibit the differentiation of preadipocytes into adipocytes.

ELECTRICAL PROPERTIES OF APPLE CULTIVARS DURING RIPENING AND STORAGE

Apple cultivars belonging to conc. Jonathan (Jonnee, Jonagold, Jonathan M41) and conc. Golden (Goldenspur, Mutsu) were used to study their electrical properties during ripening and storage. Based on ripening observations and maturity changes in September, the apples were harvested at three different times and were stored for 5, 6 or 7 months. Fruit bioelectric parameters were ascertained in the collenchyma and parenchyma cells under the epidermis. The theoretical basis of determining bioelectric parameters is the Hayden's low frequency RC model of the biological material. The |Z| values in the frequency range of 10 to 20 kHz can be fitted to a straight line with probability higher than 95%. The slope of this line - the differential coefficient - has a high value for unripe (early harvested) apples and shows a decreasing tendency during ripening (for apples later harvested). The Hayden's model was tested for the different apple varieties by t-distribution of two samples. The Hayden's model can also be used for characterization of the apple cultivars during storage. Significant differences were seen in 'Jonathan M41' apples harvested at different dates. In the case of 'Jonagold' ('Golden Delicious' hybrid) and 'Gloster' ('Delicious' hybrid) there was no significant difference in apples harvested at different dates. It is important to emphasize that our results, together with maturity indices, the cultivation and storage technological experiments can be applied to choose optimal harvest and storage times. Using the simple Hayden's model the resistance of cytoplasm and the cell wall was also determined from the complex impedance. There was no significant change in the resistance of the cytoplasm during ripening and storage, but there was a significant decrease in the resistance of the cell wall. At the end of the 7-month storage, in the resistance of cell wall tended to equalize for 'Jonagold' and 'Jonathan' apples.

Identification of Catechin Oligomers from Apple (Malus pumila cv. Fuji) in Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry and Fast-atom Bombardment Mass Spectrometry

Molecular size information for polymerized catechin larger than the decamer in unripe apple was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by fast-atom bombardment mass spectrometry. Matrix-assisted laser desorption/ionization time of flight mass spectrometry provided evidence for the pentadecamer using trans-3-indoleacrylic acid as the matrix in the presence of silver ion. Even in the absence of silver ion, the dodecamer and undecamer were observed in the positive- and negative-ion modes, respectively. Fast-atom bombardment mass spectrometry also afforded evidence for the undecamer in both positive- and negative-ion modes.

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on apple processing wastes

Sequential cultures of the yeasts Saccharomycopsis fibuliger and Candida utilis were grown on selected wastes from the processing of apples. Effluent from cider manufacture supported the growth of 45.4 g cells/100 g substrate and C. utilis formed 96% of the viable cells in the harvested biomass. Whole, unripe apples yielded 44 g cells/100 g substrate with a reduction in the substrate viscosity of 84%. C. utilis formed 56% of the viable cells in the harvested biomass. Effluent from pectin manufacture contained a substantial proportion of reducing compounds and supported the growth of C. utilis without prehydrolysis by S. fibuliger, to yield 33 g cells/100 g substrate.

Acetic and propionic acids, inducers of ripening in pre-climacteric golden delicious apples

When treated with acetic or propionic acid vapours, intact pre-climacteric Golden Delicious apples started ripening prematurely. Experiments with [2− 14C]propionic acid showed that a small part of the added acid is transformed into [ 14C]ethylene, and that ripening probably begins in these circumstances as a result of the artificial increase in the endogenous ethylene level. It may be that in unripe apples the small amount of evolved ethylene is mostly derived from simple organic acids, so that the moment at which its triggering concentration is reached depends on the available supply of acids. As this, in turn, is a function of the state of maturity of the fruit, there must be a direct relationship between the start of ripening and the degree of maturity. Thus, even in very unripe apples, catabolic processes are at work or potentially present because of the modified β–oxidation of [1− 14C]propionic acid observed.

Anthocyanin accumulation in apple skin during ripening: Regulation by ethylene and phenylalanine ammonia-lyase

In ‘Jonathan’ apples in the field, the concentration of red anthocyanin pigments in the skin increased up to 3-fold before and during commercial harvest. The start of this increase coincided with the start of rapid ethylene production in the apples. Ripening, as measured by increased ethylene production, led to increases in the rate of anthocyanin accumulation and the level of the enzyme phenylalanine ammonia-lyase (PAL) in detached apples held under constant light and temperature conditions. When ethylene was applied to unripe apples, PAL activity and anthocyanin accumulation increased to levels similar to those in ripe apples. Ethylene applied to ripe apples did not increase PAL or the rate of anthocyanin accumulation. These observations suggest that ethylene initiates rapid anthocyanin accumulation during apple ripening by increasing the level of the rate-limiting enzyme, PAL, in the skin.

Temperature Regulation of Anthocyanin Accumulation in Apple Skin

The regulation of anthocyanin accumulation in apple skin (cv. Jonathan) by temperature was studied. In the field the increase in anthocyanin in the skin before harvest coincided with decreasing temperatures and with increasing ethylene production by the fruit. In detached apples held under continuous white light, the optimum temperatures for anthocyanin accumulation were 12 °C in unripe apples and 16–24 °C in ripe apples. These effects were explained by corresponding changes in the level of phenylalanine ammonia-lyase (PAL), a key enzyme of flavonoid synthesis. PAL levels were higher at low than at high temperatures and higher in ripe than in unripe apples. In intermittent light the effects of temperature were similar but levels of PAL and anthocyanin were lower, particularly in unripe apples. It is concluded that temperature, in conjunction with ripening and light, is an important factor regulating anthocyanin accumulation and that its effects are mediated by effects on the level of PAL activity.

Extraction and purification of pectins from Alcohol Insoluble Solids from ripe and unripe apples

Pectins are extracted from Alcohol Insoluble Solids of ripe and unripe apples and fractionated by ion exchange chromatography and gelfiltration. In the extracts mainly pectins with neutral sugar contents of 0·15, 0·24 and 0·53 mol neutral sugar residues/mole galacturonate residues are present. The pectin molecules contain rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid residues. No mannose could be detected. The neutral sugar composition of the glycans bound to the galacturonan was found to be constant, except for the relative amount of galactose. During ripening the neutral sugar composition of the extractable pectin does not change.

The metabolism of alcohols by apple fruit tissue

AbstractThe interconversion of short‐chain aliphatic alcohols, aldehydes and esters by apple fruit has been studied by gas‐liquid chromatographic analysis of the products when substrates (mainly alcohols) were supplied as vapours to fruit slices at 20°C, and to whole fruit during storage at 3°C. In preliminary experiments with fruit from various sources after different periods of storage, cortical tissue slices formed corresponding aldehydes and acetate esters from alcohols; esterification was more rapid with higher alcohols. Esterification by peel tissue slices was more rapid initially but quickly declined. Metabolism was further investigated with a batch of apples during storage under different conditions. Both cortex and peel tissue were capable of acetylating butanol and 2‐methyl propanol at all stages of maturity, from the pre‐climacteric state at normal harvest time, when endogenous ester levels were low, through 58 days ripening at 12°C. The tissues showed constant hydrolytic activity towards butyl acetate throughout this period. Whole apples in air and 2% O2 at 3°C took up ethanol but formed little ethyl acetate and retained very little free ethanol. In both atmospheres whole apples metabolised approximately 40% of added hexanol to hexyl acetate and up to 8% to hexanal. The low levels of esters in apples from low oxygen atmospheres (and the absence of esters in unripe apples) are, therefore, a consequence of low rates of alcohol synthesis.

CORRELATION BETWEEN AROMA AND VOLATILE COMPOSITION OF McINTOSH APPLES

ABSTRACTThe relationship between aroma and volatile composition of McIntosh apples was investigated. Significant correlations were found between aroma qualities determined organoleptically by a trained panel and by GLC. Unripe apples contained low levels of all volatiles and exhibited grassy and green aroma notes. C‐6 aldehydes correlated with overall aroma intensity, ripeness, and fruity and aromatic notes. Esters correlated with a cheesy aroma note. Overripeness correlated with esters and total peaks in unstored but not in stored apples. The GLC peak groups which previously were found to correlate with physical and chemical properties related to maturity in unstored McIntosh apples also correlated with aroma.