Ultrasound and HPP to Control Fruit Enzymatic Browning

Polyphenoloxidase (PPO) is an endogenous enzyme, which is naturally present in plant tissues, and it has also been referred to as polyphenol oxidase, catechol oxidase, tyrosinase, phenolase, catecholase, and o-diphenol oxidase. It is an oxidoreductase-copper-containing metalloprotein, which catalyses the degradation of phenolic fruit constituents to o-quinones in the presence of oxygen. The combination of ultrasound with temperature, and high pressure processing (HPP) with temperature to inactivate polyphenoloxidase enzyme in three fruits (pear, apple and strawberry) was investigated and compared with PPO inactivation exclusively by a thermal process.

polyphenoloxidase;high pressure processing;fruit puree;clean label;sustainable;sonication;thermosonication

## 1. Enzymatic Browning by Polyphenoloxidase (PPO)

It is well known that fruits, vegetables and some crustaceous seafood (e.g., lobster, shrimp) experience enzymatic browning when tissues are exposed to air. Enzymatic browning by polyphenoloxidase (PPO, EC 1.14.18.1) causes color and flavor degradation of foods [1][2][3][4][5][1,2,3,4,5]. Mechanical injuries to whole fruits/vegetables during postharvest handling and storage, and in processing whole fruits into juices, purees/smoothies, and cut products (e.g., cubes, slices, etc.) promotes the enzymatic reaction, with subsequent economic losses to the producers or manufacturers. The much appreciated fresh-cut minimally processed fruit and vegetable products are a class of foods also very susceptible to PPO browning [6]. The resulting o-quinone will subsequently polymerize with other o-quinone, protein or amino acids, producing undesirable brown compounds [7][8][7,8]. The activity of the enzyme can be reduced in the presence of antioxidants.

## 2. Thermal Pasteurization and Kinetics of PPO Enzyme Inactivation in Foods

Pasteurization is primarily used to reduce pathogenic- and/or spoilage-vegetative-microorganisms in foods and beverages, increasing their safety and shelf life [9][10][11][12][10,11,12,13]. However, as endogenous enzymes can also spoil the food and be more resistant than microorganisms, they are also used as pasteurization targets [13][14][14,15]. The conventional thermal pasteurization and blanching operations in the range of 80 to 100 °C inactivate the PPO enzyme contained inside the fruit and vegetable products, according to a linear pattern of the natural logarithm of PPO residual activity vs. processing time [15][16] (Equation (1)). In Equation (1), A and A0 are enzyme activities after and before processing, respectively. A/A0 is the enzyme residual activity (RA) after processing for a specific time t (min), and kT is the first order inactivation rate at temperature T (min−1).
Higher inactivation rates (lower enzyme resistance) are registered for higher processing temperatures. The Arrhenius equation describes the temperature dependence of the PPO inactivation rates (Equation (2)):
$\mathrm{ln}\left({k}_{T}\right)=\mathrm{ln}\left(C\right)-\frac{{E}_{a}\frac{R}{}\left(\frac{1}{T}\right)}{}$
where T is the temperature (K), Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/(mol·K)) and C is Arrhenius constant.

## 3. Ultrasound and High Pressure Processing Pasteurization Technologies and Kinetics of PPO Enzyme Inactivation

### 3.1. Power Ultrasound

Power ultrasound (US) consists of sonic waves with frequencies higher than human ear audible sound (20–24 kHz). Power ultrasound or high-intensity ultrasound uses lower frequencies than medical applications, being characterized by sound intensity or acoustic intensity ranging between 10 to 1000 W/cm2 [16][17]. The sonication generates bubbles in liquid food as the wave energy propagates, a phenomenon known as acoustic cavitation. The formation and collapse of tiny bubbles can occur in few microseconds, and the cavitation increases with acoustic energy(power). The specific acoustic energy/power (W/g) or volumetric acoustic energy/power density (AED/AEP, in W/mL of processed liquid sample) quantify the energy requirements of the process for a given volume of treated food. Higher sound intensity/energy/power also increases the mechanical and sonochemical effects [14][15]. Ultrasound is known to break up proteins, starches and other large biopolymers such as enzymes, and can affect protein and enzyme functionality [17][18]. The damage to protein structure by ultrasound is desirable since it results in enzyme inactivation. Changes in enzyme biological activity are due to changes in the folding of the proteins, the secondary and tertiary structures of the enzyme. Under these extreme changes, hydrogen bonds and van der Waals bonding in the enzyme polypeptide chains can be broken down [18][19], resulting in the loss of enzyme activity [19][20]. The sonication processing conditions can cause partial or total inactivation of enzyme activity, depending on the type and the fruit/vegetable source of the enzyme. Thermosonication (TS) or heat-assisted ultrasound—the simultaneous application of US with heat—can be used to increase the efficiency of the process. The effect of ultrasound treatment on PPO depends on ultrasonic intensity (acoustic energy), duration of process (t), temperature (T), food/beverage matrix pH and ionic strength. The kinetics of PPO inactivation by thermosonication also follows a first order kinetics, similar to thermal inactivation alone.

### 3.2. High Pressure Processing

High pressure processing (HPP) or high hydrostatic pressure (HHP) is a cold pasteurization technology by which vacuum packed food products are introduced to a vessel containing a pressure transmitting liquid (usually water), and submitted to a high level of isostatic pressure (300–600 MPa). The nutritional and sensory characteristics of the original fresh fruit/vegetables, thus are better retained [20][22]. Several studies demonstrated that room temperature HPP has limited effectiveness towards the inactivation of the PPO enzyme [5][21][5,23]. Therefore, the combination of HPP with mild heat (60–90 °C), also referred to as high pressure thermal processing (HPTP), heat-assisted HPP or HPP-thermal, has been investigated for more effective enzyme inactivation [22][24]. HPP typically breaks non-covalent bonds (e.g., hydrogen bonds within the enzymes), but does not break covalent bonds (e.g., peptide bonds within the enzymes) [23][25]. Generally, it is expected that higher enzyme inactivation or lower enzyme residual activity (RA) will result when higher T (temperature for HPTP), P (pressure) and longer processes (t, time) are used for processing. Non-linear inactivation using a first order biphasic model with two rates (kL and kS) was previously used to model PPO inactivation by HPTP (Sulaiman et al., 2015) [22][24] (Equation (3)). AL and AS are activities of the labile and stable fractions, respectively, and kL and kS are the inactivation rate constants of labile and stable fractions of the enzyme, respectively. Other HPP-thermal studies carried out with lychee, sapodilla and Packham pear used the first order kinetic model (Equations (1) and (2)) [24][26].

## 4. Comparison of Three Pasteurization Technologies for the Inactivation of PPO in Three Fruits' Purees

Table 1 shows the inactivation rates (k) used to generate the charts of PPO Residual Activity (RA) vs. time presented in the Figures 1 to 3. In general, TS rates were the highest, indicating a quicker inactivation by TS compared with thermal and HPP-thermal processes. HPP-thermal presented the lowest inactivation rates (stable fraction of the enzyme), meaning more time is needed for the same enzyme inactivation.
Figure 1. Comparing the effect of thermosonication (TS, 1.3 W/g—71 °C), heat assisted high pressure processing (HPTP, 600 MPa—71 °C) and thermal process alone (71 °C) on polyphenoloxidase enzyme residual activity during treatment time for ‘Taylor’s Gold’ pear puree.
Figure 2. Comparing the effect of thermosonication (TS, 1.3 W/g—71 °C), heat-assisted high pressure processing (HPTP, 600 MPa—71 °C) and thermal process alone (71 °C) on polyphenoloxidase enzyme residual activity during treatment time for ‘Royal Gala’ apple puree.
Figure 3. ‘Camarosa’ strawberry polyphenoloxidase residual activity after thermosonication (TS, 1.3 W/g), heat assisted high pressure processing (HPTP, 600 MPa) and thermal treatment alone at 71 °C (A) and 57 °C (B).

Table 1. First order enzyme inactivation rates for thermosonication (TS) and thermal inactivation, and first order biphasic enzyme inactivation rates for heat assisted high pressure processing (HPP-thermal) of Taylor’s pear, Royal Gala apple and Camarosa strawberry purees *.
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