A model of fruit microstructure

Researchers are using advanced medical technologies to unravel the relationship between storage disorders and internal structure in pome fruit. By Anna Mouton.

Commercial controlled-atmosphere storage has been around for nearly a century and helped fresh apples become a year-round supermarket staple. But optimising storage conditions remains challenging, with mismatches between fruit physiology and gas concentrations contributing to storage disorders such as internal browning.

To improve our understanding of the relationship between oxygen (O₂) and carbon dioxide (CO₂) levels and browning disorders, doctoral student Siphumle Jama is looking inside pome fruit with the help of technologies usually found in hospitals.

His research is part of a Hortgro-funded project led by Dr Elke Crouch, Postharvest Physiology Research Chair in Deciduous Fruit in the Department of Horticultural Science at Stellenbosch University. Jama is co-supervised by Dr Alemayehu Tsige, Senior Researcher in the Packaging and Cold Chain Research Group in the Department of Horticultural Science.

The project forms part of a larger investigation on fruit respiration and microstructure in collaboration with Prof. Bart Nicolaï and Dr Pieter Verboven of KU Leuven.

When cells suffocate

Apples and pears are alive. Their cells respire — they burn O₂ and produce CO₂ while generating the energy that sustains metabolic processes. Ripening is associated with a spike in respiration rate and ethylene production. Low temperatures and O₂ levels suppress respiration and delay ripening.

Low O₂ levels in controlled-atmosphere storage help maintain fruit quality. But if O₂ levels drop too low, cells switch from aerobic respiration to anaerobic fermentation. Ethanol production, which can cause off-flavours in the fruit, is one consequence of fermentation, and internal browning is another.

Anaerobic fermentation is less efficient than aerobic respiration. Oxygen-starved cells run into an energy deficit, leaving them unable to repair their membranes. Leaky membranes allow polyphenol-oxidase enzymes to mingle with phenols inside cells. Polyphenol oxidases convert phenols to melanin, the pigment responsible for browning.

Browning isn’t only caused by unsuitable O₂ and CO₂ concentrations. For example, chilling injuries and senescent breakdown also damage membranes and lead to browning.

“We’ve studied browning in Forelle, Fuji, and Cripps Pink quite extensively,” says Crouch. “So, we know Forelle, Rosy Glow and Fuji are CO₂ sensitive, and we know what low O₂ damage looks like in Fuji and Rosy Glow. The internal disorders and drivers for Cripps Pink and Rosy Glow are the same.”

Cultivar variation is one reason why optimising controlled-atmosphere storage is hard. “Each cultivar has a different physiology and different gas concentration thresholds,” says Jama. “Our aim was to show the relationship between the fruit microstructure, the gas pathway dynamics inside the fruit, and storage disorders.”

Pores and pathways

Pome-fruit flesh consists of cells and the air-filled spaces or pores between them. Inside the fruit, gases such as O₂ and CO₂ diffuse passively along a concentration gradient by moving through connected pores. Along the way, respiring cells absorb some O₂ and release some CO₂.

“Our hypothesis is that the internal microstructure will affect the gas distribution and exchange between the fruit and the ambient air,” says Tsige, “and that things like porosity and tortuosity are responsible for internal defects.”

Porosity is the total pore volume as a percentage of the fruit’s volume. Besides total pore volume, pore size, shape, and connectivity will affect the fruit flesh’s permeability — how easily gas can move through it. Tortuosity describes the complexity of the pore network, which also affects permeability.

Previous Hortgro-funded research led by Crouch and conducted by Dr Kenias Chigwaya for his PhD, demonstrated that microstructure was related to browning in Fuji. Chigwaya examined normal and brown flesh in the same fruit, finding that brown tissues had significantly lower porosity and pore connectivity than normal tissues.

He speculated that Fuji may be more CO₂-sensitive than, for example, Golden Delicious, because Fuji’s smaller pores hinder gas movement, leading to internal O₂ depletion and CO₂ accumulation.

Chigwaya could zoom in on Fuji’s pores by using X-ray CT (computed tomography), a technology usually found in hospitals, where it’s an essential medical imaging tool. Rotating X-ray sources and detectors take a series of measurements that a computer reconstructs as a cross-sectional image.

Jama is using the same protocols as Chigwaya to measure the porosity of Forelle, Fuji, and Rosy Glow after three different storage regimes. He will also draw on previous X-ray micro- and nano-CT data to understand fruit microstructure. The higher resolution of nano-CT allows visualisation of individual cells and microstructural features such as pores, whereas micro-CT only indicates relative density as shades of grey.

“It’s clear how O₂ and CO₂ will travel through empty space,” says Tsige. “But from the X-ray CT data, we can model how O₂ and CO₂ will travel through the tortuous paths in fruit, and how much time it will take. We will use the information obtained from this scale to model gas exchange in an entire fruit.”

From model to reality

Knowing how gases move through the fruit is only part of the puzzle. A gas-exchange model must also account for cellular O₂ consumption and CO₂ production. Jama has been conducting a series of experiments to obtain this data.

He placed Forelle, Fuji, and Rosy Glow fruit in jars with known O₂ and CO₂ concentrations and measured their maximum O₂ consumption and CO₂ production rates at different temperatures. He also determined the respiratory quotient (the ratio of CO₂ to O₂), which indicates the threshold where fruit switches from primarily aerobic respiration to primarily anaerobic fermentation.

“The fruit is currently in controlled-atmosphere storage,” says Jama. “When we take them out, we will do the exact same experiments again.”

Measurements on fruit from the inner and outer canopies, at harvest and after three different storage regimes, will allow the research team to investigate the role of these variables on fruit respiration.

Jama is also collecting maturity indexing data at harvest, after storage, and shelf life and recording storage disorders. Some of the storage protocols in this project are specifically designed to induce internal browning.

All this data will eventually be used to construct a model of gas exchange between the fruit and its environment, as well as gas concentrations within the fruit. “The model will give us a time profile of the gas concentrations and respiratory quotient inside the fruit, which we believe is highly related to internal browning,” says Tsige.

The research team will validate the model by comparing its predictions of internal browning to the actual occurrence of browning. The long-term vision is to scale the model for application to fruit bins and eventually entire cold rooms.

“What we’re trying to add with this project is using mathematical modelling to make predictions about the physiological disorders in fruit stored in controlled atmosphere,” says Jama. “We want to find ways to optimise gas concentrations.”

What does industry say about this research?

“In the case of Cripps Pink, you have bigger and smaller fruit, and less and more dense fruit, but you’re putting them all in the same DCA [dynamic controlled atmosphere] room. The fruit that’s denser or has a bigger diffusion distance has completely different gas exchange, and that’s why you get internal browning.

“When we talk about internal browning in Cripps Pink, maturity is only half the answer. The other half is understanding the structure and texture, and how gas exchange happens. The more you think about this, the more you realise the importance of what we do in the orchard before harvest, because it affects the fruit structure and texture.

“The same is true for bitter pit and postharvest problems in pears. So this research is very relevant.”

Willie Kotze. Technical Manager at Dutoit Agri.