Mechanisms of Calcium-induced Firmness in Fruits, Vegetables, and Mushrooms

Six fractions from mushrooms (MFs), as cytoplasmic proteins (F1), cell membrane proteins (F2), alkali-soluble cell wall glucans (F3a), cell wall proteins (F3b), cell wall beta-glucans (F4a), and chitin (F4b) and five fractions from both apples (AFs) and cucumbers (CFs), as cytoplasmic proteins (F1), cell membrane proteins (F2), cell wall proteins (F3), pectic substances (F4), and cellulose (F5) were extracted and analyzed for their neutral carbohydrates, proteins, aminosugar material, soluble phenolics, and pectic substances. The dominant compounds consisted of the following: proteins in MF1, proteins and neutral polysaccharides in MF2, neutral polysaccharides in MF3a, neutral polysaccharides in MF3b, neutral polysaccharides and chitinous material in MF4a, neutral polysaccharides and chitinous material in MF4b; pectic substances in AF1, AF2 and AF4, proteins in AF3, neutral polysaccharides in AF5; proteins in CF1, CF2, and CF3; pectic substances in CF4, and neutral polysaccharides in CF5.

Experiments for binding calcium (CaCl2) to fraction solids were performed in 10 mM Tris-HCl buffer (pH 7.0). The calcium-binding capacity was obtained by both Ion Selective Electrode (ISE) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measurements. The results indicated that calcium-binding capacity of fractions differed significantly in the order of MF1>MF2>MF3a ≥ MF3b≥MF4b~MF4a for mushroom fractions. AF2 and AF4 showed higher binding capacity than AF1 and AF5 for apple fractions. For cucumber, CF1, CF2, and CF4 showed higher binding capacity than CF3 and CF5.

The results revealed that calcium was bound by all fractions, but those fractions dominantly composed of cytoplasmic proteins (MF1) and cell membrane proteins (MF2) in mushroom fractions, pectic substances (AF4) and cell membrane proteins (AF2) in apple fractions, cytoplasmic proteins (CF1), cell membrane proteins (CF2), and pectic substances (CF4) in cucumber fractions possessed higher capacities. The binding of calcium to the biopolymers was further investigated by Isothermal Titration Calorimetry (ITC) using pectin and bovine serum albumin (BSA). The results confirmed that interaction of calcium with pectic substances plays a primary role in firmness improvement. However, data also indicated that interactions between calcium and cytoplasmic and/ or membrane proteins may contribute to the firmness improvement in calcium-treated mushrooms, apples, and cucumbers.