On the origin of internal browning in pears (Pyrus communis L. cv Conference)

<FONT FACE="Arial">Brown discoloration as a result of polyphenoloxidase (PPO) is a well-known problem during food preservation and -processing, which already has been established long ago. Large quantities of fruits and vegetables are lost every year because of loss of taste or because the product looks unattractive to customers. Internal browning or brown core in pears is an example of this. Because affected fruits normally appear healthy from the outside, they may be put on the market, with all the negative consequences that can be expected.This research aimed to explain the mechanisms behind the development of brown core. The research was focussed on pears, however it obviously has a relation with similar disorders in general. The pear functions as a model, and this research has a potential spin-off to other products.<strong>10.1 Polyphenoloxidase</strong>The name PPO is used for a number of enzymes involved in the oxidation of several polyphenols, like chlorogenic acid and (-)-epicatechin. Two groups of PPO's can be distinguished. In this research no laccase activity in pears was established, therefore the PPO activity that was found was attributed to tyrosinase. Browning develops when tyrosinase converts polyphenols into o -quinones. These quinones further polymerise by reacting with a variety of cellular components, and this leads to the brown pigmentation that can be seen with the naked eye. In literature it is often supposed that the activation of tyrosinase initiates the process of brown pigmentation. However, in this research it could not be shown that PPO is activated before or during the initiation of brown core development, or under conditions which cause internal browning (like elevated CO ₂ concentrations).In a healthy cell tyrosinase and phenolic substrates are found at different locations. Tyrosinase can be found in plastids in the cell. In chloroplasts tyrosinase is membrane-bound, while as far as known tyrosinase is not membrane-bound in other plastids. Tyrosinase exists mainly as a latent form in the cell. The ratio active : latent (around 5% : 95%) does not change clearly at the border between healthy and affected tissue, or under storage conditions that have the potential to initiate the development of brown core. In the cortex tissue of the pear tyrosinase is not membrane-bound.Polyphenols are present in the vacuole. A membrane surrounds both, plastids and vacuoles, and PPO and its substrates do not come into contact in healthy tissue. This led to the hypothesis that the initiation of brown core development is caused by (partial) disruption of intracellular membranes and decompartmentation. A new question rose in this investigation, namely: how and under which circumstances are these membranes damaged?<strong>10.2 Vitamin C</strong>One of the hypotheses is that membranes are damaged by oxygen free radicals, which are produced during fruit respiration (mitochondria) and photosynthesis (chloroplast). In healthy tissue these radicals are neutralised by antioxidants. With the appearance of aerobic respiration in evolution, cells developed a protective system against radicals in which antioxidants and several enzymes, like catalase, superoxide dismutase and peroxidase play a role. One of the most abundant antioxidants in fruits (besides GSH and vitamin E), and the main reason why humans consume fruits, is vitamin C or ascorbic acid (AA). A person with 'scorbutic' is suffering from scurvy, a well-known illness in naval history, which can be overcome by the consumption of AA.AA is an efficient quencher of oxygen free radicals. AA can directly neutralise radicals by reacting with them, but AA can also regenerate vitamin E in membranes. Vitamin E protects membranes against oxygen free radicals. Additionally, AA is necessary for ascorbate peroxidase (APX) functioning. APX neutralises peroxide in the chloroplast, where catalase is absent. Furthermore, AA can regenerate o -quinones to form precursor polyphenols in vitro , and so directly avoid browning.AA levels in pears decrease to low levels under conditions that induce brown core. Lowered O ₂ reduces AA levels compared to standard CA conditions. The addition of CO ₂ to the storage atmosphere decreases AA levels even more. By monitoring AA levels in pears it is possible to predict the development of browning. AA levels increase again and brown core is largely avoided when browning-inducing storage conditions (with elevated CO ₂ ) are changed to standard CA conditions (without CO ₂ ) just before the moment brown core is initiated. However, the AA level is not the only factor explaining brown core development. During storage at 21 kPa O ₂ with 5 kPa CO ₂ for example, the AA level is lower than at 0.5 kPa O ₂ without CO ₂ , while only under the latter condition brown core develops. The conclusion is that other factors must be involvedUnder CA conditions fruit respiration is limited to a high extent. This brings along that also the energy production (ATP) is limited. A cell needs a certain energy maintenance level to survive. This maintenance energy is for example necessary for protein turnover, membrane maintenance and antioxidant regeneration. Lowering O ₂ concentrations decreases both ATP levels and the rate of ATP production. Addition of CO ₂ to the storage atmosphere increases the chance that brown core will develop and can inhibit respiration and ATP production. Still, CO ₂ does not inhibit respiration in every case. Directly after harvest of pears CO ₂ does not show an inhibitory effect on pear respiration. Future research should demonstrate if an emerging inhibitory effect on respiration during the beginning of CA storage coincides with the initiation of brown core development.Summarising, it appears that brown core is the result of a combination of factors, like a (temporarily) shortage of available energy and an insufficient capacity to protect the cell against oxygen free radicals caused by decreased antioxidant concentrations.<strong>10.3 Diffusion resistance</strong>Another important subject of the present research is the establishment of the diffusion resistance of the peel of an apple or pear. In literature it is often suggested that that the cause of internal browning can be found in the low porosity of pear tissue. This research showed that this suggestion is unlikely. O ₂ concentrations in the pear are indeed slightly decreased, but not at low external applied O ₂ concentrations under which browning develops. No O ₂ gradients were found in the cortex tissue of a pear, which does not point at an evident O ₂ diffusion resistance. An accumulation of CO ₂ in pears under a variety of conditions was clearly established. The average internal CO ₂ concentration was around 2 kPa higher than the externally applied concentration (at 20°C). There is a strong indication that CO ₂ can accumulate in the cell in the form of bicarbonate.<strong>10.4 Summarising</strong>The relation between elevated CO ₂ concentrations and brown core in pears is not new. However, it is still difficult to indicate the exact mechanisms by which CO ₂ leads to this disorder.CO ₂ has no clear effect on PPO activity. This enzyme does not appear to be activated by CO ₂ or bicarbonate. CO ₂ has a clear effect on AA levels in pear tissue, which are decreased. Yet, this decrease can not be marked as the cause for decompartmentation. CO ₂ can inhibit respiration of pears, but this inhibition is not consistent. ATP levels are not unambiguously decreased by CO ₂ . It is more likely that brown core is caused by a combination of these factors. Furthermore, lowered pH values caused by CO ₂ and direct effects of CO ₂ on membranes and enzymes may play a role.It is clear that apples and pears can not be blindly compared. Apples are often stored at enhanced CO ₂ levels (up to 4 kPa). Furthermore, it was recently found that apples can be commercially stored at extremely low O ₂ concentrations (as low as 0.4 kPa) during so-called Dynamic Control storage (DCS). It seems that this new type of storage is not suitable for storage of Conference pears. Future research should demonstrate the differences between apples and pears, which lead to the large difference in adaptation capacity to CA conditions. A profound knowledge on fruit physiology may lead to decreased risks and losses and may yield improved (interactive) storage systems, like DCS