Enzyme Activity and Nutritional Quality of Peach (Prunus persica) Juice: Effect of High Hydrostatic Pressure

Abstract

The inactivation of polyphenol oxidase and pectin methylesterase in peach juice was investigated after high hydrostatic pressure processing at 400–600 MPa and 25°C for 5–25 min, respectively. At 400 MPa, polyphenol oxidase and pectin methylesterase were activated by 7.3 and 2.6%. At 500 and 600 MPa, polyphenol oxidase and pectin methylesterase were inactivated significantly with increasing the pressure and time, and the inactivation kinetics was fitted by the first order model. Moreover, some physio-chemical properties were studied. The results revealed that high hydrostatic pressure treatment preserved more L-ascorbic acid and maintained the color and sensory quality better than thermal treatment.

Keywords:

• Peach juice
• High hydrostatic pressure processing
• Enzymes inactivation
• Nutritional quality

INTRODUCTION

Polyphenol oxidase (PPO) and pectin methylesterase (PME) are endoenzymes widespread in fruits and vegetables, which contribute to the quality of fruit products, especially juices. In the juice industry, the catalytic action of PPO causes enzymatic browning, and the catalytic action of PME results in cloud loss in fruit juices.[1, 2] Enzymatic browning is the result of PPO catalyzed oxidation of mono- and diphenols to form o-quinones, which in turn polymerize to form brown melanins (brown pigments).[3] Degree of enzymatic browning depends on phenol contents and PPO activity.[4] PME is a de-esterifying enzyme, which catalyzes the demethylation of carboxylate groups on pectin substances to form low methoxy pectin. The demethylated pectin can cross-link with polyvalent cations, such as Ca²⁺, to form insoluble calcium pectates precipitates or become a target for pectin-degrading polygalacturonases (PG).[2, 5, 6]

In order to avoid the undesirable results caused by the two endoenzymes, it is necessary to inactivate them. Most foods, such as fruit juice, are thermally processed to sterilize and inactivate enzymes. However, with the growing concerns regarding quality and the nutritional aspects of foods, consumers demand foods which are natural, nutritious, free of chemical preservatives and microbiologically safe with extended shelf-life.[7] However, due to high temperatures during processing, thermal treatment can lower the sensory and nutritional quality of foods, especially for thermosensitive fruit products. To overcome these problems, several non-thermal processing (NTP) techniques, including high hydrostatic pressure (HHP) technology, have been developed.

HHP processing (100–1000 MPa) is a NTP techniques applied to food products, often at room temperature, which has shown great potential in producing microbiologically-safe products while maintaining the natural characteristics of the food items.[8 ⁻ 10] Many studies on HHP effects on the inactivation of PPO and PME, obtained from different fruit products, report that pressures exhibit different effects on inactivation of enzymes.[2, 6, 11 ⁻ 14] The nutritional qualities of many fruit products processed by HHP also have been studied.[15 ⁻ 19] However, there are currently no studies on effect of HHP processing on the nutritional quality and enzyme activity of peach juice. The purpose of this work was to investigate the effectiveness of HHP processing of peach juice on its PPO and PME activity. Concurrently, the change of color, L-ascorbic acid content, viscosity, and sensory quality of the juice was studied. In addition, the change of color and L-ascorbic acid content during storage at 4°C was also studied.

MATERIAL AND METHODS

Materials

Mature peaches (Longquanyi peach, Sichuan Province, China) were obtained from a local retailer in Mingguangcun market of Beijing, China. The peaches were washed, pitted, diced, and squeezed with a screw juice extractor (T6G7, Zhejiang Light Industry Machinery Plant, Zhejiang, China). To prevent the browning of the juice, 0.02% L-ascorbic acid was added to the freshly squeezed juice. All juice was filtered with two layers of cheesecloth, and then transferred to polyethylene-polyamide pouches. Each pouch was filled with 25 mL juice. The pouches were vacuum sealed at appropriate pressure (0.04 MPa) immediately, and then stored at 4°C until the experiment began.

HHP and Thermal Processing Treatments

Samples were subjected to HHPs of 400, 500, and 600 MPa for 5, 10, 15, 20, and 25 min respectively at room temperature (25°C) by a HHP apparatus (Baotou Kefa High Pressure Technology Co., Ltd., Inner Mongolia, China). Thermal processing was carried out in a thermostatic water bath (LY-9A, Qingyuan Science & Technology Development Co. Ltd., Beijing, China). Samples were subjected to temperatures of 90 ± 2°C for 1 min and then cooled to room temperature (25°C).[14,20]

Analysis

Untreated peach juice and all HHP-treated peach juice were tested for enzyme activity of PPO and PME. All types of peach juice were tested for color, viscosity, and ascorbic acid content. All the analyses were carried out in triplicate unless otherwise stated.

PPO enzyme activity

Enzyme activity of PPO was assessed according to procedure described by Xu et al.[21] with small modifications. Samples (2 mL) were homogenized in 6 mL 0.2 mol L⁻¹ sodium phosphate buffer, pH 6.8, containing 1% polyvinyl polypyrrolidone (PVPP), then extracted for 4 h at 4°C. The homogenates were centrifuged at 10,000 r min⁻¹ for 15 min at 4°C, and the supernatant was collected to analyze the enzyme activity. Enzyme activity was determined by measuring the rate of linear increase in absorbance at 420 nm at 25°C using a spectrophotometer (Varian Co., Ltd., Santa Clara, CA, USA). Reaction material contained 500 μL extract supernatant and 2.5 mL of 0.1 mol L⁻¹ catechol solution, The reference cuvette contained 500 μL distilled water and 2.5 mL of 0.1 mol L⁻¹ catechol solution. Enzyme activity was defined as the change in absorbance under conditions of the assay (Δ absorbance min⁻¹ mL⁻¹). A_t /A₀ indicates the residual activity (RA) of PPO (A₀ is the PPO activity before HHP processing, and A_t is the PPO activity after HHP processing for a period time t).

PME enzyme activity

Enzyme activity of PME was assessed at pH 7.5 and 30°C according to the procedure of Sampedro et al.[20] with small modifications. A 5 mL portion of juice was added to 40 mL of 1% apple pectin solution (DE 75%) in 0.1mol L⁻¹ NaCl. The pectin-juice mixture was adjusted to pH 7.5 with 0.5 mol L⁻¹ NaOH. During hydrolysis at 30°C, pH was maintained at 7.5 by adding 0.05 mol L⁻¹ NaOH with an automatic pH-stat titrator (Metrohm, Switzerland). The consumption of NaOH was recorded every 30 s over a 10 min reaction period. The PME activity (meq min⁻¹ mL⁻¹) was calculated using the milliequivalent (meq) of sodium hydroxide (V_NaOH* N_NaOH), sample volume (mL) and the reaction time (min). A_t /A₀ indicates the RA of PME. A₀ is the PME activity before HHP processing, and A_t is the PME activity after HHP processing for a period time t.

Measurement of Color

Color assessment was conducted at room temperature (25°C). Parameters a*, b,* and L* were determined with a color difference meter (SC-80, Kang guang Co., Ltd., Beijing, China) in the reflection mode.[22] The net color difference (ΔE) was calculated from the a*, b*, and L* parameter, using the equation:

(1)

Where L*, a*, b* indicate lightness, redness, yellowness, respectively. Subscript “0” indicates initial color.

Viscosity

The viscosity measurement is based on the methods reported by Zhou et al.[23] with small modifications. The viscosity of peach juice was determined using an AR550 rheometer (TA Instruments, New Castle, USA) with a conical end concentric cylinder (stator radius = 15.00 mm, rotor radius = 14.00 mm, immersed height = 42.00 mm, gap = 5920 μm). 13.5 mL juice was applied at each measurement. The temperature was kept at 25 ± 0.1°C by circulating water in a thermostatic system. Flow curves were obtained at shear rate from 10 to 150 s⁻¹. Shear rate versus apparent viscosity was fitted to a Power Law model expressed as following.

(2)

Where τ is the shear stress (Pa); γ is the shear rate (s⁻¹); K is the consistency index (Pa sⁿ) and n is the behaviour index. The fitting goodness of the model was assessed using the regression coefficients (R ²).

L-Ascorbic Acid

The L-ascorbic acid measurement was based on the methods reported by Sancho et al.[24] with small modifications. One mL peach juice was homogenized in 3 mL 2.5% metaphosphoric acid. Then the homogenates were centrifuged at 5000 r min⁻¹ for 20 min at 4°C. HPLC system (RF-10AXL, Shimadzu Co., Japan) consisted of a prominence UV-visible detector (SPD-20AV), a system controller (CBM-20A), an auto sampler (SIL-20A), two pumps (LC-20AT), and a column oven (CTO-20A). The analytical column was a Sunfire TM C18 (4.6 × 250 mm i.d, 5 μm particle size) from Waters. The mobile phase was distilled water adjusted to 95% 0.025 mol L⁻¹ potassium dihydrogen phosphate and 5% acetonitrile. The flow rate was 1.0 mL min⁻¹. The temperature was kept at 30°C and the injection volume was 20 μL. The detection was carried out at 254 nm in absorbance mode. The concentration of L-ascorbic acid was calculated using L-ascorbic acid external standard and expressed as milligram ascorbic acid per 100 mL of peach juice.

Sensory Evaluation

Untreated peach juices as well as those processed by HHP (600 MPa, 10 min, 25°C) and thermally treated were given to panellists just after processing for sensory evaluation. The procedure performed for this evaluation was according to Mosqueda-Melgar et al.[25] with small modifications. A total of 10 trained panellists participated in the sensory tests. Twenty mL of each sample was served into cup labelled with a random 3-digit number. A glass containing potable water and a piece of non-salted cracker were provided to panellists for eliminating the residual taste between samples. The panellists were asked to score their preference of color, turbidity, aroma, flavor, and comprehensive impression in a hedonic scale from 1 (dislike extremely) to 5 (like extremely).

MATHEMATICAL AND STATISTICAL ANALYSIS

The inactivation kinetics of PPO and PME were analyzed by a conventional first-order kinetic model.[26]

(3)

A_t /A₀ indicates the RA of enzyme after HHP processing for a period time t, k (min⁻¹) indicates the inactivation rate constant.

The decimal reduction time D-value means time to inactivate 90% of the enzyme at a given pressure.

(4)

All the experiments were performed in triplicate. Analysis of variance (ANOVA) and significance analysis were carried out by using the software Microcal OriginPro 8.1 (Microcal Software, Inc., Northampton, USA). P < 0.05 was taken as an indicator of statistical differences between the means. All the data was expressed as mean value ± standard deviation (n = 3).

RESULTS AND DISCUSSION

Inactivation of PPO and PME by HHP

The inactivation of PPO and PME by pressures at 400–600 MPa is shown in Fig. 1. As shown in Fig. 1a, PPO in peach juice was effectively inactivated by HHP with increasing pressures and there was significant difference of inactivation effects among different pressures (P < 0.05). The activity of PPO in peach juice was reduced significantly with increasing treatment time at 500 and 600 MPa. When the peach juice was subjected to HHP at 600 MPa for 5 and 25 min, the RA of PPO reduced to 54.9% and 18.8%, respectively. However, the RA of PPO increased by 7.3% after processing at 400 MPa for 5 min. This enhancement of PPO activity after pressurization has been reported in some fruit products, such as strawberry,[27] apple,[14] and raspberry.[28] It was reported that there are two forms of PPO, active and latent forms,[1] and the latent could become active by pressurization.[14] Therefore, it can be inferred that effect of HHP treatment on PPO is a dynamic process: HHP treatment can inactivate some PPO, while activating some latent ones. Presentation of the activity was the result of interaction between the two forms. From Fig. 1a, it can be observed that the RA of PPO is 18.8% at 600 MPa for 25 min, which shows that PPO in peach juice has a strong pressure-resistance. Gomes and Ledward[11] also found that the PPO in mushrooms was not completely inactivated until processing at 800 MPa for 5 min. There are studies reported that reducing the pH was helpful in the inactivation of PPO enzymes. Kingsly et al.[29] found that modest cirtic acid concentration (1.2 and 1.0%) in combination with pressure treatment (> 300 MPa, > 5 min) at 25°C effective in inactivation of PPO enzymes.

Table 1 Kinetic parameters of PPO and PME inactivation by HHP

Enzymes	Pressure (MPa)	k (min^−1) × 10^−3	D (min)	R^2
PPO	400	23.96	961.2	0.754
	500	72.94	315.7	0.984
	600	128.4	179.4	0.961
PME	400	0.1829	12,590	0.295
	500	22.02	1046	0.976
	600	54.40	423.4	0.973
k (min^−1) indicates the inactivation rate constant.
D (min) means time to inactivate 90% of the enzyme at a given pressure.
R^2 represents the degree of model fit.

Figure 1 Pressure inactivation of PPO and PME in peach juice at 25°C. (a) Residual activity of PPO, (b) residual activity of PME, (c) pressure inactivation kinetics of PPO, and (d) pressure inactivation kinetics of PME. (▪) 400 MPa, (•) 500 MPa, and (▲) 600 MPa. The solid straight lines represent the model (Eq. (3)) fitting.

As shown in Fig. 1b, the RA of PME was reduced with enhanced pressures, and inactivation effects among varying pressures were also notable (P < 0.05). The RA of PME was reduced significantly with an increasing treatment time at 500 and 600 MPa. When the peach juice was subjected to HHP at 600 MPa for 5 and 25 min, The RA of PME reduced to 81.2% and 49.6%, respectively. However, the RA of PME increased by 2.6% at 400 MPa for 15 min. The activation effect has been reported in some papers.[30, 31] Morild[32] indicated that this PME activation was due to the cohesion of high pressure. Low HHP treatment could destroy the isolation of complete fruit organization, which separated PME from matrix. When the enzyme is in contact with matrix, the enzymatic reaction is accelerated. Ogawa et al.[33] considered that the main reason for this active phenomenon might be the change of configuration of the enzymes and the substrate molecule. From Fig. 1b, it can also be concluded that inactivation of PME by HHP treatment in peach juice was incomplete. The reduction of PME was only 50.4% at 600 MPa for 25 min, indicating that PME had a much stronger pressure-resistance than PPO. According to Boulekou's[2] research, PME in peach pulp could be completely inactivated only at 800 MPa and 70°C for more than 90 s. Baron et al.[34] indicated that effect of HHP treatment on PME mainly depended on different analog systems or food systems and the different enzyme sources.

As shown in Figs. 1c and 1d, inactivation of PPO and PME activity were described by a first order kinetic model. The parameters of the linear relationship are presented in Table 1. R² represents the fitting degree of the curve, with a larger R² -value indicating a better fit. As shown in the Table 1, due to activation of the two enzymes at 400 MPa, inactivation curves were unable to fit first order kinetics model properly (R ² < 0.900). But at 500 and 600 MPa, inactivation curves of the two enzymes fit first order kinetics model precisely (R ² > 0.900). The k value increased with the higher pressure, which indicated that higher pressures resulted in higher inactivation of PPO. At 600 MPa, D-value of PPO and PME were 179.4 min and 423.4 min, respectively. It required a significant amount of time to reduce the RA of two enzymes to lower than 10%. In order to achieve better inactivation effect of PPO and PME in peach juice, higher pressure or other processing methods (such as pressure-assisted thermal processing, PATS) were required.

Table 2 Change of color and L-ascorbic acid content in fresh peach juice and their thermal and HHP processed counterparts over storage at 4℃

Treatment	Storage					L-ascorbic acid
conditions	(days)	L*	a*	b*	ΔE	(mg/100 mL)
Fresh juice	−	48.45 ± 0.33^a	0.12 ± 0.80^abc	3.81 ± 0.21^d	0.00	38.20 ± 0.48^a
600 MPa, 10 min	0	48.85 ± 0.18^a	−0.27 ± 1.98^bc	3.73 ± 0.41^d	0.56	32.62 ± 0.67^b
	15	46.53 ± 0.51^b	0.37 ± 0.58^abc	4.00 ± 0.40^cd	1.95	30.23 ± 0.45^c
	30	44.83 ± 0.42^c	0.80 ± 0.48^abc	4.69 ± 0.27^bcd	3.71	28.88 ± 0.53^d
	60	42.80 ± 0.29^d	1.89 ± 0.36^ab	4.04 ± 0.26^cd	5.93	27.47 ± 0.86^e
	90	41.40 ± 0.92^de	2.36 ± 0.46^a	4.69 ± 0.41^bcd	7.45	26.34 ± 0.95^e
90℃, 1 min	0	48.14 ± 0.20^ab	−1.47 ± 1.04^c	4.94 ± 0.27^abc	1.98	26.23 ± 1.12^e
	15	44.99 ± 0.86^c	0.42 ± 0.75^abc	5.30 ± 0.29^ab	3.78	22.72 ± 0.93^f
	30	42.33 ± 1.12^de	0.83 ± 0.30^abc	5.05 ± 0.32^ab	6.28	19.53 ± 0.48^g
	60	40.52 ± 0.58^ef	1.10 ± 0.40^ab	5.52 ± 0.43^ab	8.17	16.45 ± 0.61^h
	90	38.81 ± 0.66^f	1.99 ± 0.40^ab	5.75 ± 0.43^a	10.01	15.01 ± 0.92^i
^x Means in the same column followed by different letters imply a significant difference (p < 0.05) according to Turkey Test.
All data were the means ± SD, n = 3.

Color

Color directly affects the appearance and the consumer acceptability of fruit juice. As presented in Table 2, compared to fresh juice, L* value increased for HHP-treated juice and decreased for thermally-treated one, which indicated that HHP-treated juice was lighter than thermally-treated one. The a* values of peach juice exhibited no significant difference after HHP and thermal treatment. There were also no significant differences (p > 0.05) between HHP treatment and fresh juice for b*. However, after thermal processing, there was a significant increase for b*, which showed that peach juice could become yellow after thermal treatment. Compared to HHP treatment, the increase of ΔE for thermal treatment was much greater, which indicated that the color of thermally-treated peach juice changed much more obviously than HHP treatment. During storage at 4°C, thermally-treated juice exhibited the larger degree of color change (ΔE) compared to HHP treated juice. Overall, the color of peach juice treated by HHP changed little compared to fresh peach juice. Thermally-treated peach juice became yellow and was darker than HHP-treated juice. HHP treatment has the ability to maintain the color of peach juice better than thermal treatment. Other studies that measure color change of fruit products after HHP treatment have also been completed. According to Keenan's[19] research, no significant effects were observed for L* and color change (ΔE) in fruit smoothies processed by HHP at 600 MPa and 20°C for 10 min. Ferrari et al.[35] found that the high pressure treatment at room temperature increased the intensity of red color of the fresh juice and preserved the content of natural anthocyanins. López-Malo et al.[15] indicated that high pressure maintained the initial color of avocado puree. In his study, pressure-treated avocado puree had a color equivalent to the freshly-prepared avocado puree.

L-Ascorbic Acid Content of Peach Juice

As shown in Table 2, after HHP and thermal treatment, there was significant reduction (p < 0.05) for L-ascorbic acid content. The retention rates for L-ascorbic acid content of HHP-treated and thermally-treated juice were 85.4% and 69.2% respectively. Oey et al.[36] indicated that during HHP treatment, outside oxygen was passed into the food system which accelerating the oxidation of ascorbic acid. During storage at 4°C, L-ascorbic acid content of HHP-treated juice was higher than thermally-treated one. It can be concluded that HHP treatment plays a better role in L-ascorbic acid protection than thermal treatment, which has been reported in other research. Patras et al.[37] found that high pressure treatment (400, 500, and 600 MPa/15 min /10–30°C) significantly retained more ascorbic acid in strawberry purées than thermal treatment (70°C/2 min).

Viscosity

Apparent viscosity in HHP-treated peach juice as a function of shear rate is show in Fig. 2a. Rheological behaviour of peach juice was described by power law model. The rheological parameters are present in Table 3. As shown in Fig. 2a, apparent viscosity of peach juice decreased with increasing shear rate (10 ˜ 150 s⁻¹) after all treatments, suggesting that the juices exhibited non-Newtonian characteristics. Both thermal and HHP treatment increased the apparent viscosity, but the thermally-treated juice had higher apparent viscosity than juice treated by HHP. After HHP and thermal treatment, the consistency index (K) increased significantly while flow behavior index (n) decreased (Table 3), suggesting a product with higher viscosity and a trend towards pseudoplastic flow behavior. There is existing data that shows reasoning behind HHP treatment increasing the viscosity of some fruit juices. Sandei et al.[38] indicated that inactivation for PG, compressibility effect from high pressure, and coagulation of protein organization during the HHP treatment could explain this phenomenon. Wu et al.[39] discussed that the viscosity increase of tomato pulp after HHP treatment was due to inactivation of PME and increase of small particles.

Table 3 Rheological parameters of peach juice

Treatment conditions	K(mPa^.S^n)	n	R ^2
Fresh juice	6.93	0.99	0.83
600 MPa,10 min	7.44	0.98	0.94
90°C, 1min	8.53	0.96	0.96
K is the consistency index; n is the flow behaviour index; R ^2 is the regression coefficient by Power law model.

Figure 2 Effects of HHP and thermal treatment on viscosity (a) and sensory properties (b) of peach juice. (▲) 90°C, 1 min; (▪) 600 MPa, 25°C, 10 min; (◆) untreated. The solid lines represent the model (Eq. (2)) fitting.

Sensory Evaluation

As shown in Figure 2b, there was no significant difference between HHP-treated and untreated juice in terms of color and turbidity. HHP-treated juice had greater typical fresh peach aromas than thermally-treated and fresh juice. It is because the content of the characteristic aroma components of peach juice, such as gamma-caprolactone and gamma-decalactone, increased dramatically after HHP treatment (unpublished data). Thermally-treated peach juice displayed the lowest rating in color and aroma. Likewise, the overall impression of thermally-treated juice received the lowest score. All attributes of HHP-treated juice exhibited similar scores to untreated juice. Therefore, the HHP treatment can maintain the sensory quality of peach juice more effectively than thermal treatment. Many other studies have investigated the effect of HHP on sensory quality of fruit products. Yen et al. found that the guava puree treated at 600 MPa and 25°C for 15 min retained good quality similar to the freshly extracted puree after storage at 4°C for 40 days.[40] According to Ferrari's[35] study, high pressure treatment at room temperature improved the quality of pomegranate juice. Laboissiére et al.[16] indicated that HHP may be successfully used to preserve yellow passion fruit pulp, yielding a ready to drink juice with improved sensory quality, as compared to existing commercially-available juices in Brazil. Keenan et al.[19] found that fruit smoothies processed by HHP (450 MPa/20°C/5 min) were similar to unprocessed controls and appeared to retain more fresh-like characteristics.

CONCLUSION

The inactivation of PPO and PME in peach juice is clearly enhanced with increasing pressure treatment and time under HHP treatment. However, it can be observed activation of PPO and PME under pressure of 400 MPa. The inactivation curve fits the first order models under 500 MPa and 600 MPa. The color parameters (including L*, a*, b*, and ΔE) are not significantly changed after HHP treatment. Compared to thermally-treated peach juice, HHP treatment preserves more ascorbic acid. Compared to fresh peach juice, HHP and thermal treatment can lead to increases in the viscosity of peach juice, but the change of HHP-treated juice is not detectable. In the sensory evaluation, HHP treatment maintains the sensory quality of peach juice. Therefore, HHP treatment preserves the quality of peach juice significantly over thermal treatment.

ACKNOWLEDGEMENTS

This work was supported by Project No. 101105046610000 of Beijing Municipal Commission of Science and Technology of China, and the “Novel Technologies and Equipments of Food Non-thermal Processing” (No. 2011AA100801) of 863 High-Tech Plan of China.