abstract: Physical methods were applied for objective monitoring of apple freshness on the basis of measurements of the activity of the pathway of enzymatic oxidation with phenolics participation, activated by mechanical bruising. Spectrophotometric analysis of apple flesh browning and measurements in apple flesh by means of the redox electrode were applied as objective measures of the biological activity of tissue after bruising. The created model of browning was verified on apples in consumption maturity. The description of changes in flesh freshness was perfor¬med for apples of over thirty cultivars originating from a cultivar experiment, after two long-term periods of normal cold storage at 3-6 C, 89-95% RH.
Tested material was characterized by considerable variability, e.g. flesh firm¬ness: 10-40 N (by flat penetrometer Ø 7.9 mm and standard deviation for cultivar s = 2-6 N,), total soluble solids 10-16 Brix, and intercellular spaces 17-50%, content in the flesh (per 100 g) of chlorogenic acid 3-25 mg, epicatechine 7-45 mg, and phloretin-xyloglucoside 0.5-7.5 mg. The new parameters may find appli¬cation in quality assessment of stored apples and of fresh cut apple products.
Changes caused by enzymatic browning in UV-Vis spectra (250-450 nm) and increase in the content of brown pigments in the flesh (440 nm band) were expres¬sed by differences in diffuse absorbance A = log(1/R), where R is the reflectance of apple flesh. Chemometric analysis showed a close relation between the content of HPLC determined phenolics in the flesh, time-dependent UV-Vis reflectance spec¬tra and the potential of redox electrode inserted in the flesh, all collected at the flesh-air phase boundary. The analysis also showed a time-course hierarchy in substrate transformations at the non-enzymatic stage of the browning reaction, and – in the spectra – bands characteristic for phenolics.
The possibility of creating freshness indexes on the basis of analysis of bruised apple flesh was demonstrated - concentrations of polyphenols in apple flesh were estimated on the basis of time-dependent spectrophotometric spectra.
The effects of enzymatic browning, with participation of oxygen from the inter¬cellular spaces, were registered with the kinetic method from the first 1-3 minutes in UV (approx. 275 nm), and after 6-12 minutes the strong effects were observed in visible violet (365-390 nm). A close relation was shown between the potential of redox electrode inserted in apple flesh (E0 = 420-580 mV) and kinetic measurements after 3-4 min in bands of 260 nm and 275 nm, and of the saturation potential on redox electrode (Ef = 510-650 mV) in visible violet. A model of browning of mechanically bruised fresh apple tissue was proposed. The first stage is a reaction controlled by excess content of the PPO enzyme in the flesh, and the second is non-enzymatic oxidation of polyphenols, coupled e.g. with chlorogenic acid, dependent on oxygen from the intercellular spaces. For the practical estimation of changes in freshness and over-ripening of apples in storage, the scheme of browning was simplified and a first-order kinetics model was applied relative to melanin pigments absorbing in the band of 440 nm. The model includes the effect of varied ripeness of the fruits and of the period of storage.
Different periods of storage and various levels of fruit ripeness (within con¬sumption ripeness) had a bearing on the activity of biological mechanisms and on the response of fruits to bruising. What remained constant was the half-life of browning (t½ = 5-45 min), characteristic for cultivars, which increased only in the case of apple flesh slicing under water. The biological activity of substrates decreased in over-ripening apples, described as a decrease in the browning rate of the wedges sliced in air (υ = 0.003-0.035 Abs min-1, s = 0.002-0.017 Abs min 1) and also of wedges sliced under water (υ = 0.001-0.02 Abs min-1). The flesh of over-ripening apples was characterized by higher diffuse absorbance, determined in freshly sliced apple flesh (A0 = 0.2-0.5 Abs, s = 0.01-0.3 Abs), and by lower increase in diffuse absorbance (Af = 0.05-0.35 Abs, s = 0.03-0.1 Abs) till reaching the final equilibrium of browning.
It was observed that in fresh tissue browning developed in the direction of recreating protective barriers against organic radicals and oxygen. Another characteristic feature of fresh apple flesh is reduced browning rate by half, after slicing under water and rinsing of cell contents. Browning continues and there is only a slight decrease in the cultivar-characteristic value of flesh absorbance attained at sill browning – A0 + Af. This does not change the kinetic constant of enzymatic browning of apple flesh (KM = 0.23 Abs under normal environmental conditions). Likewise, reduced temperature of apple flesh and surface-effect PPO inhibitors cause only a reduction in the browning rate and extend the colour stability of sliced wedges (colour stability t0,08 = 2-80 min, defined as time of absorbance increase to 0.08 Abs under normal conditions, when sliced in air).
The studies show that apple flesh browning can be treated as a varietal feature. Analysis indicates that estimation of apple freshness is related with over-ripening and as well as with enzymatic activity of phenolics in the tissue, with repair mechanisms in apple flesh, and with authentic nutritional value of apples. Physical measures of biological activity of tissue as a response to bruising due to slicing or puncture can be used as freshness indices of apples and of sliced apples.
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