# Effects of Ultrasound Processing on Physicochemical Parameters, Antioxidants, and Color Quality of Bayberry Juice

Authors: Xiamin Cao; Chunfang Cai; Yongling Wang; Xiaojian Zheng
Journal: Journal of Food Quality
 (2019)

---

## Abstract

Effects of ultrasound on physicochemical parameters, ascorbic acid, anthocyanins, polymeric color (PC), 5-hydroxymethylfurfural (HMF), browning degree (BD), color, and superoxide dismutase (SOD) activity of bayberry juice were investigated. Treatments were carried out at amplitude levels from 20 to 100% of total input power (600 W) at 20 kHz for 2–10 min. The results showed that no notable differences in pH, total soluble solids, titratable acidity, and yellownessb∗ values were found in ultrasound-treated samples. The HMF, PC, BD, and L∗ values of bayberry juice obviously increased with enhancing ultrasonic intensity and treatment time. The ascorbic acid exhibited no notable changes after ultrasound treatment at lower intensity levels for short time, while anthocyanins showed an increasing tendency. With increasing ultrasonic intensity and time, antioxidants gradually decreased. Furthermore, the SOD activity apparently increased at short-time treatment and then decreased with ultrasound processing extension.


---

## Body

## 1. Introduction

Bayberry (Myrica rubra Sieb. et Zucc.) is one of the most popular berry fruits in Chinese markets because of its sweet-sour taste, pleasant aroma, and attractive color [1].Bayberry fruits with high content of polyphenols are considered antioxidants and demonstrated to have beneficial health effects on humans [2–6]. However, the fresh bayberry is harvested in hot and rainy seasons (June to July), and the postharvest life is only 1-2 days at ambient temperature and 4-5 days under refrigeration because of its high susceptibility to mechanical injury and microbiological decay [7]. Therefore, the processed products of bayberry for shelf-life extension are of high commercial and economic importance.Nowadays, bayberry fruits are commercially processed into wine, jam, or juice by different thermal technologies for microorganism and endogenous enzyme inactivation [8]. These bayberry products can reach a widespread market and longer shelf-life, but they also suffer from losses of nutritional compounds and changes of sensory quality during thermal processing because of their heat sensitivity [4, 6]. With the increase in minimally processed and fresh food products demanded by consumers, it is necessary to develop innovative techniques which use minimal heat and maximally retain food quality [7].Ultrasound is an innovative preservation technique with a power of 10–1000 W/cm2 and high intensity-low frequency (20–100 kHz) [2, 8, 9]. Several studies using ultrasound treatment on food reported promising results of notable endogenous enzyme and microorganism inactivation [10–14]. Attributed to lower degradation of nutrition and quality [15, 16], ultrasound is increasingly studied for the processing of fruit juice and puree, such as grapefruit juice [17], soursop juice [16], gourd juice [18], pear juice [19], blackberry juice [20], avocado puree [21], raspberry puree [22], orange juice [23, 24], tomato juice [25], apple juice [26], and strawberry juice [27] in recent years.However, little information has been reported on the effects of ultrasound on quality parameters of bayberry juice. The aim of our study was to investigate effects of ultrasound on antioxidants including monomeric anthocyanins, ascorbic acid, polymeric color, antioxidant activity, HMF, BD, color attributes, and SOD activity of bayberry juice.


## 2. Materials and Methods

### 2.1. Bayberry Juice

The juice preparation was done following our previous study [28]. Frozen bayberries (Wuzi) purchased from Hanling Supermarket (Suzhou, China) were squeezed for juice with an extractor (JY-610, Joyoung Co., Ltd., Shandong, China). After centrifuging at 5733 ×g for 10 min (DL6MB, Xiangzhi Centrifuge Instrument Ltd., Hunan, China), the supernatants were filtered through a 0.23 mm pore diameter filter, and the juice was collected, packaged into plastic bags (EVOH), and frozen at −18°C until use.


### 2.2. Ultrasound Treatments

The ultrasound treatment conditions were the same as those in our previous study on the enzyme inactivation in bayberry Juice [28]. The ultrasonic processor (600 W; BILON-600Y, Bilon Co., Ltd., Shanghai, China) used had a 13 mm diameter probe tip, and the experimental setup for ultrasound treatments is shown in Figure 1(a). A constant ultrasound frequency of 20 kHz was selected, and the total energy input was controlled by setting the amplitude at 20%, 40%, 60%, 80%, and 100% with the corresponding ultrasonic intensity levels at 90, 181, 271, 362, and 452 W/cm2, respectively. The treatment time was 2, 4, 6, 8, and 10 min with pulse durations of 5 s on and 5 s off. Bayberry juice was placed in a glass vessel surrounded with an ice bath, and the ultrasound probe was submerged to a depth of 25 mm in the juice. The initial and final temperature of bayberry juice was exactly determined and is displayed in Table 1.Figure 1
Experimental setup for ultrasound treatments (a) and thermal processing (b). 1: ultrasound transducer; 2: ultrasonic generator; 3: ultrasound probe (19 mm); 4: glass vessel; 5: ice bath for a cooling (h) depth of probe into the sample (25 mm); 6: hotplate; 7: boiling water bath; 8: bayberry juice samples; 9: thermometer.
(a)
(b)Table 1
Effects of ultrasound processing on pH, TSS, TA, color, and BD in bayberry juice.
Ultrasound intensity (W/cm2)
Time (min)
Temperature (°C)
pH
TA (%)
TSS (°Brix)
Color attributes
BD
L
∗
a
∗
b
∗
ΔE
Control
25
2.85 ± 0.02a
0.57 ± 0.01a
9.2 ± 0.1a
32.88 ± 0.64a
15.99 ± 0.38c
−2.68 ± 0.06a
—
0.227 ± 0.004a
TP treatment
90
2.84 ± 0.03a
0.57 ± 0.02a
9.4 ± 0.01a
30.63 ± 0.74c
13.44 ± 0.41f
−3.11 ± 0.08b
2.86 ± 0.05
0.276 ± 0.007h
90
2
29.5 ± 0.8
2.85 ± 0.01a
0.56 ± 0.02a
9.2 ± 0.1a
32.69 ± 0.49a
16.06 ± 0.41bc
−2.56 ± 0.01a
0.21 ± 0.05
0.230 ± 0.005a
4
34.3 ± 1.9
2.84 ± 0.01a
0.56 ± 0.01a
9.3 ± 0.1a
32.66 ± 0.41a
15.99 ± 0.84c
−2.64 ± 0.13a
0.22 ± 0.03
0.231 ± 0.003ab
6
40.3 ± 1.5
2.86 ± 0.02a
0.58 ± 0.02a
9.3 ± 0.1a
32.70 ± 0.37a
16.24 ± 0.55b
−2.68 ± 0.06a
0.30 ± 0.01
0.233 ± 0.003b
8
45.6 ± 2.8
2.85 ± 0.03a
0.57 ± 0.01a
9.2 ± 0.1a
32.06 ± 0.88ab
15.96 ± 0.67c
−2.75 ± 0.08a
0.83 ± 0.01
0.236 ± 0.004bc
10
48.4 ± 1.6
2.86 ± 0.02a
0.57 ± 0.02a
9.1 ± 0.1a
31.68 ± 0.44b
15.78 ± 0.18c
−2.74 ± 0.06a
1.23 ± 0.04
0.238 ± 0.004c
180
2
32.5 ± 1.4
2.84 ± 0.01a
0.56 ± 0.02a
9.3 ± 0.2a
32.85 ± 0.43a
16.19 ± 0.39b
−2.62 ± 0.03a
0.21 ± 0.06
0.231 ± 0.004ab
4
36.4 ± 2.2
2.83 ± 0.03a
0.58 ± 0.02a
9.2 ± 0.1a
32.59 ± 0.44a
16.28 ± 0.22b
−2.73 ± 0.01a
0.66 ± 0.02
0.234 ± 0.004b
6
43.1 ± 0.9
2.84 ± 0.02a
0.55 ± 0.01a
9.2 ± 0.1a
32.31 ± 0.59a
16.41 ± 0.64ab
−2.65 ± 0.02a
0.70 ± 0.02
0.239 ± 0.006c
8
47.5 ± 2.5
2.86 ± 0.02a
0.56 ± 0.01a
9.4 ± 0.1a
32.25 ± 1.28a
15.71 ± 0.45c
−2.74 ± 0.03a
0.70 ± 0.04
0.241 ± 0.006cd
10
52.8 ± 1.9
2.85 ± 0.02a
0.58 ± 0.02a
9.3 ± 0.1a
31.98 ± 0.64ab
15.55 ± 0.72d
−2.67 ± 0.04a
0.99 ± 0.02
0.245 ± 0.003de
271
2
34.2 ± 1.1
2.84 ± 0.02a
0.57 ± 0.02a
9.4 ± 0.1a
32.72 ± 0.99a
16.15 ± 0.61bc
−2.67 ± 0.05a
0.23 ± 0.03
0.233 ± 0.008b
4
38.7 ± 1.7
2.84 ± 0.03a
0.57 ± 0.01a
9.3 ± 0.1a
32.35 ± 0.72a
16.28 ± 0.45b
−2.61 ± 0.07a
0.60 ± 0.02
0.236 ± 0.003c
6
46.3 ± 1.9
2.87 ± 0.02a
0.56 ± 0.02a
9.3 ± 0.1a
32.19 ± 1.09a
15.79 ± 0.51c
−2.72 ± 0.03a
0.81 ± 0.04
0.241 ± 0.005 cd
8
50.5 ± 1.2
2.83 ± 0.02a
0.58 ± 0.01a
9.2 ± 0.1a
31.99 ± 0.65ab
15.78 ± 0.83c
−2.71 ± 0.01a
0.92 ± 0.03
0.246 ± 0.004de
10
55.6 ± 2.6
2.85 ± 0.02a
0.57 ± 0.01a
9.1 ± 0.1a
31.67 ± 0.78b
15.45 ± 0.39 cd
−2.74 ± 0.05a
1.34 ± 0.03
0.249 ± 0.007e
362
2
35.3 ± 0.8
2.83 ± 0.03a
0.56 ± 0.02a
9.1 ± 0.1a
32.62 ± 0.42a
16.74 ± 0.44ab
−2.69 ± 0.06a
0.79 ± 0.01
0.234 ± 0.005b
4
41.4 ± 1.4
2.85 ± 0.02a
0.58 ± 0.01a
9.2 ± 0.1a
32.38 ± 1.42a
16.58 ± 0.48b
−2.68 ± 0.02a
0.78 ± 0.02
0.239 ± 0.005c
6
49.3 ± 0.9
2.84 ± 0.01a
0.57 ± 0.01a
9.3 ± 0.1a
32.11 ± 0.66a
15.84 ± 0.37c
−2.74 ± 0.09a
0.79 ± 0.04
0.246 ± 0.010de
8
53.5 ± 1.9
2.85 ± 0.01a
0.58 ± 0.02a
9.1 ± 0.1a
31.89 ± 0.65ab
15.22 ± 0.61d
−2.76 ± 0.02a
1.26 ± 0.03
0.253 ± 0.005f
10
59.4 ± 2.3
2.84 ± 0.03a
0.56 ± 0.01a
9.2 ± 0.1a
31.56 ± 0.63b
14.95 ± 0.59d
−2.76 ± 0.03a
1.69 ± 0.04
0.257 ± 0.005f
452
2
38.5 ± 1.2
2.85 ± 0.02a
0.57 ± 0.01a
9.2 ± 0.1a
32.51 ± 0.62a
17.16 ± 0.74a
−2.72 ± 0.01a
1.22 ± 0.04
0.238 ± 0.001c
4
45.6 ± 0.8
2.84 ± 0.02a
0.57 ± 0.01a
9.3 ± 0.1a
32.25 ± 0.86a
16.81 ± 0.65a
−2.72 ± 0.04a
1.03 ± 0.03
0.249 ± 0.006ee
6
51.4 ± 2.2
2.85 ± 0.02a
0.58 ± 0.02a
9.2 ± 0.1a
31.79 ± 0.46ab
16.26 ± 0.47b
−2.71 ± 0.01a
1.12 ± 0.02
0.255 ± 0.003f
8
57.5 ± 1.6
2.86 ± 0.02a
0.56 ± 0.02a
9.3 ± 0.1a
31.57 ± 0.70b
15.38 ± 0.42d
−2.69 ± 0.03a
1.44 ± 0.03
0.259 ± 0.005f
10
61.2 ± 2.1
2.87 ± 0.01a
0.57 ± 0.01a
9.2 ± 0.1a
31.19 ± 0.68b
14.42 ± 0.54e
−2.70 ± 0.02a
2.33 ± 0.06
0.264 ± 0.005f
All data are expressed as mean ± SD (n = 6). Values with different letters within one column are significantly different (p<0.05).


### 2.3. Thermal Processing

Bayberry juice was treated in boiling water (Figure1(b)) until reaching a core temperature of 90°C and was held at this temperature longer than 1 min [29]. Then, the juice was immediately cooled down to room temperature with an ice water bath and stored at 4°C.


### 2.4. Measurement of TSS, pH, and TA

TSS was determined as °Brix using a WAY-2S Digital Abbe refractometer (Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China) at 25 ± 1°C.The pH value measurements were carried out by a pH meter (MP511, Sanxin Co., Ltd, Shanghai, China) with a combined pH electrode at 25 ± 1°C.The TA was determined via titration by an automatic titrimeter (G20, Mettler-Toledo, Shanghai, China). The TA value expressed as citric acid equivalents was analyzed and calculated using the following formula:(1)TA%=C×V2×KV1×V0m×2×100%,where C is the NaOH concentration (0.1 M); m is the weight of juice; V2, V1, and V0 (mL) are the volume of NaOH used and juice used and the total volume of juice, respectively; and K is the conversion factor of citric acid (0.07).


### 2.5. Measurement of Ascorbic Acid

The determination of ascorbic acid was performed referring to our previous study [30]. 2.5% of metaphosphoric acid was used as extraction solution, and the ratio was 1 : 5 (v : v). After 2 h extraction at 4°C, the mixture was centrifuged at 6880 ×g at 4°C for 10 min, and the supernatant was collected with final volume measurement.Ascorbic acid was measured using the liquid chromatograph method (Shimadzu Co., Japan) with an Alltech AlltimaTM C18 column (4.6 × 250 mm i.d.; 5 μm particle size) from Waters. The mobile phase consisted of 95% monopotassium phosphate (50 mM; pH = 3.0) and 5% acetonitrile, and the flow rate was 1 mL/min at 25 ± 1°C. The detection was carried out at 245 nm in the absorbance mode. The content was calculated by using the following formula:(2)m=m020×10−3×V1×100V0,where m0 is the value drawn in the standard curve of ascorbic acid (mg/L) and V1 and V0 are the volume of the final extract and initial bayberry juice, respectively.


### 2.6. Measurement of Monomeric Anthocyanins

The anhydrous methanol with 0.1% HCl was used for anthocyanin extraction, and the material-to-liquid ratio was 1 : 2. After extraction for 1 h at 4°C, the mixture was centrifuged (6880 ×g/10 min/4°C), and the supernatant was collected [29].A Venusil C18 column (250 mm × 4.6 mm i.d.; 5 μm particle size) equipped with a 5 μm C18 guard column both from Agela Technologies Co., Ltd. (Tianjin, China) was used for anthocyanin determination, and methanol (A) and formic acid/water (B) (5 : 95, v/v) were the mobile phases with a flow rate of 1 mL/min at 520 nm. The gradient elution consisted of 15% A for 0–5 min, 15%–30% A for 5–8 min, 30%–50% A for 8–12 min, 50%–80% A for 12–15 min, 80%∼15% A for 15–18 min, and 15% A for 18∼20 min. Cy-3-glu was quantified as monomeric anthocyanins by external standards. The results were calculated by using the following formula:(3)m=m020×10−3×V1×100V0,where m0 is the value drawn in the standard curve of anthocyanins (mg/L) and V1 and V0 are the volume of the final extract and initial bayberry juice, respectively.


### 2.7. Measurement of Polymeric Anthocyanins

Polymeric color was determined within 24 h referring to the previous study [29]. The bayberry juice was diluted with water until its absorbance at 520 nm reached 0.5∼1.0. 1 mL of 0.90 M potassium metabisulfite; 3 mL of the diluted juice was mixed and equilibrated for 30 min, and then the samples were determined at 700, 520, and 420 nm. Color density (CD), polymeric color (PC), and percent polymeric color (PPC) were calculated using the following formulas:(4)CD=A420−A700+A512−A700×dilution factor,PC=A420−A700+A512−A700×dilution factor,PPC=polymeric colorcolor density×100%.


### 2.8. Measurement of Antioxidant Activity

The free-radical-scavenging effect on DPPH radicals was determined as antioxidant activity [30]. Fivefold diluted bayberry juice (500 μL) and 0.14 mM methanolic DPPH solution (4 mL) were mixed and equilibrated for 45 min at 25 ± 1°C protected from light. Then, the samples were measured at 517 nm by a spectrophotometer (UV-726, Shimadzu, Shanghai, China). Standard Trolox dissolved in methanol (10–100 mg/L) was used for plotting calibration curves. The results (expressed as milligrams of Trolox equivalents per 100 mL of bayberry juice) were calculated using the following formula:(5)DPPH%=1−A0A1×100%,where A0 and A1 are the absorbance at 517 nm of the control and juice.


### 2.9. Measurement of HMF

Chromatographic separation and determination of HMF referring to the study of Liu et al. [31] were performed on a Venusil XBP (L) C18 column (250 mm × 4.6 mm i.d.; 5 μm particle size) from Agela Technologies Co., Ltd. (Tianjin, China). The mobile phase was methanol/water (10/90, v/v) with a flow rate of 1 mL/min at 30°C. The levels and results (milligrams per 100 mL of bayberry juice) were calculated using HMF as the external standard.


### 2.10. Measurement of BD

BD was studied by a spectrophotometric method at 420 nm [30]. The samples were firstly centrifuged (6880 ×g/4°C/10 min) and passed through a 0.45 μm cellulose nitrate membrane.


### 2.11. Color Assessment

Color was determined in the reflectance mode at 20 ± 1°C by a color difference meter (SBDY-3, Yuefeng Co., Shanghai, China) and expressed asL∗, a∗, and b∗ values. ΔE was calculated using the following equation, where L∗0, a∗0, and b∗0 are the control values for untreated juice:(6)ΔE=L∗−L∗02+a∗−a∗02+b∗−b∗021/2.


### 2.12. SOD Assay

The extraction and determination of SOD activity were performed by Pang et al. [32]. 20 mL of samples and 40 mL of phosphate buffer (0.05 M; pH 7.8) were mixed and extracted (1 h/4°C) and then centrifuged (6880 ×g/10 min/4°C), and the supernatants were collected.SOD activity was determined by pyrogallol autoxidation [32]. 3 mL of 0.07 M catechol (o-diphenol) in 50 mmol/L Tris-HCL buffer solution (pH 8.0) and 0.5 mL of SOD extracts were mixed, and absorbance changes at 325 nm/25 ± 0.1°C were immediately detected at intervals of 0.1 s−1 by a UV spectrophotometer (U-3010, Hitachi, Ltd., Tokyo, Japan). The enzymes in per milliliter reaction solution which inhibited 50% of the autocidal rate of pyrogallol were defined as one enzyme unit (U).


### 2.13. Statistical Analysis

The data were analyzed using the SPSS 12.0 software for analysis of variance and Duncan’s test. The significance was determined atP<0.05.


## 2.1. Bayberry Juice

The juice preparation was done following our previous study [28]. Frozen bayberries (Wuzi) purchased from Hanling Supermarket (Suzhou, China) were squeezed for juice with an extractor (JY-610, Joyoung Co., Ltd., Shandong, China). After centrifuging at 5733 ×g for 10 min (DL6MB, Xiangzhi Centrifuge Instrument Ltd., Hunan, China), the supernatants were filtered through a 0.23 mm pore diameter filter, and the juice was collected, packaged into plastic bags (EVOH), and frozen at −18°C until use.


## 2.2. Ultrasound Treatments

The ultrasound treatment conditions were the same as those in our previous study on the enzyme inactivation in bayberry Juice [28]. The ultrasonic processor (600 W; BILON-600Y, Bilon Co., Ltd., Shanghai, China) used had a 13 mm diameter probe tip, and the experimental setup for ultrasound treatments is shown in Figure 1(a). A constant ultrasound frequency of 20 kHz was selected, and the total energy input was controlled by setting the amplitude at 20%, 40%, 60%, 80%, and 100% with the corresponding ultrasonic intensity levels at 90, 181, 271, 362, and 452 W/cm2, respectively. The treatment time was 2, 4, 6, 8, and 10 min with pulse durations of 5 s on and 5 s off. Bayberry juice was placed in a glass vessel surrounded with an ice bath, and the ultrasound probe was submerged to a depth of 25 mm in the juice. The initial and final temperature of bayberry juice was exactly determined and is displayed in Table 1.Figure 1
Experimental setup for ultrasound treatments (a) and thermal processing (b). 1: ultrasound transducer; 2: ultrasonic generator; 3: ultrasound probe (19 mm); 4: glass vessel; 5: ice bath for a cooling (h) depth of probe into the sample (25 mm); 6: hotplate; 7: boiling water bath; 8: bayberry juice samples; 9: thermometer.
(a)
(b)Table 1
Effects of ultrasound processing on pH, TSS, TA, color, and BD in bayberry juice.
Ultrasound intensity (W/cm2)
Time (min)
Temperature (°C)
pH
TA (%)
TSS (°Brix)
Color attributes
BD
L
∗
a
∗
b
∗
ΔE
Control
25
2.85 ± 0.02a
0.57 ± 0.01a
9.2 ± 0.1a
32.88 ± 0.64a
15.99 ± 0.38c
−2.68 ± 0.06a
—
0.227 ± 0.004a
TP treatment
90
2.84 ± 0.03a
0.57 ± 0.02a
9.4 ± 0.01a
30.63 ± 0.74c
13.44 ± 0.41f
−3.11 ± 0.08b
2.86 ± 0.05
0.276 ± 0.007h
90
2
29.5 ± 0.8
2.85 ± 0.01a
0.56 ± 0.02a
9.2 ± 0.1a
32.69 ± 0.49a
16.06 ± 0.41bc
−2.56 ± 0.01a
0.21 ± 0.05
0.230 ± 0.005a
4
34.3 ± 1.9
2.84 ± 0.01a
0.56 ± 0.01a
9.3 ± 0.1a
32.66 ± 0.41a
15.99 ± 0.84c
−2.64 ± 0.13a
0.22 ± 0.03
0.231 ± 0.003ab
6
40.3 ± 1.5
2.86 ± 0.02a
0.58 ± 0.02a
9.3 ± 0.1a
32.70 ± 0.37a
16.24 ± 0.55b
−2.68 ± 0.06a
0.30 ± 0.01
0.233 ± 0.003b
8
45.6 ± 2.8
2.85 ± 0.03a
0.57 ± 0.01a
9.2 ± 0.1a
32.06 ± 0.88ab
15.96 ± 0.67c
−2.75 ± 0.08a
0.83 ± 0.01
0.236 ± 0.004bc
10
48.4 ± 1.6
2.86 ± 0.02a
0.57 ± 0.02a
9.1 ± 0.1a
31.68 ± 0.44b
15.78 ± 0.18c
−2.74 ± 0.06a
1.23 ± 0.04
0.238 ± 0.004c
180
2
32.5 ± 1.4
2.84 ± 0.01a
0.56 ± 0.02a
9.3 ± 0.2a
32.85 ± 0.43a
16.19 ± 0.39b
−2.62 ± 0.03a
0.21 ± 0.06
0.231 ± 0.004ab
4
36.4 ± 2.2
2.83 ± 0.03a
0.58 ± 0.02a
9.2 ± 0.1a
32.59 ± 0.44a
16.28 ± 0.22b
−2.73 ± 0.01a
0.66 ± 0.02
0.234 ± 0.004b
6
43.1 ± 0.9
2.84 ± 0.02a
0.55 ± 0.01a
9.2 ± 0.1a
32.31 ± 0.59a
16.41 ± 0.64ab
−2.65 ± 0.02a
0.70 ± 0.02
0.239 ± 0.006c
8
47.5 ± 2.5
2.86 ± 0.02a
0.56 ± 0.01a
9.4 ± 0.1a
32.25 ± 1.28a
15.71 ± 0.45c
−2.74 ± 0.03a
0.70 ± 0.04
0.241 ± 0.006cd
10
52.8 ± 1.9
2.85 ± 0.02a
0.58 ± 0.02a
9.3 ± 0.1a
31.98 ± 0.64ab
15.55 ± 0.72d
−2.67 ± 0.04a
0.99 ± 0.02
0.245 ± 0.003de
271
2
34.2 ± 1.1
2.84 ± 0.02a
0.57 ± 0.02a
9.4 ± 0.1a
32.72 ± 0.99a
16.15 ± 0.61bc
−2.67 ± 0.05a
0.23 ± 0.03
0.233 ± 0.008b
4
38.7 ± 1.7
2.84 ± 0.03a
0.57 ± 0.01a
9.3 ± 0.1a
32.35 ± 0.72a
16.28 ± 0.45b
−2.61 ± 0.07a
0.60 ± 0.02
0.236 ± 0.003c
6
46.3 ± 1.9
2.87 ± 0.02a
0.56 ± 0.02a
9.3 ± 0.1a
32.19 ± 1.09a
15.79 ± 0.51c
−2.72 ± 0.03a
0.81 ± 0.04
0.241 ± 0.005 cd
8
50.5 ± 1.2
2.83 ± 0.02a
0.58 ± 0.01a
9.2 ± 0.1a
31.99 ± 0.65ab
15.78 ± 0.83c
−2.71 ± 0.01a
0.92 ± 0.03
0.246 ± 0.004de
10
55.6 ± 2.6
2.85 ± 0.02a
0.57 ± 0.01a
9.1 ± 0.1a
31.67 ± 0.78b
15.45 ± 0.39 cd
−2.74 ± 0.05a
1.34 ± 0.03
0.249 ± 0.007e
362
2
35.3 ± 0.8
2.83 ± 0.03a
0.56 ± 0.02a
9.1 ± 0.1a
32.62 ± 0.42a
16.74 ± 0.44ab
−2.69 ± 0.06a
0.79 ± 0.01
0.234 ± 0.005b
4
41.4 ± 1.4
2.85 ± 0.02a
0.58 ± 0.01a
9.2 ± 0.1a
32.38 ± 1.42a
16.58 ± 0.48b
−2.68 ± 0.02a
0.78 ± 0.02
0.239 ± 0.005c
6
49.3 ± 0.9
2.84 ± 0.01a
0.57 ± 0.01a
9.3 ± 0.1a
32.11 ± 0.66a
15.84 ± 0.37c
−2.74 ± 0.09a
0.79 ± 0.04
0.246 ± 0.010de
8
53.5 ± 1.9
2.85 ± 0.01a
0.58 ± 0.02a
9.1 ± 0.1a
31.89 ± 0.65ab
15.22 ± 0.61d
−2.76 ± 0.02a
1.26 ± 0.03
0.253 ± 0.005f
10
59.4 ± 2.3
2.84 ± 0.03a
0.56 ± 0.01a
9.2 ± 0.1a
31.56 ± 0.63b
14.95 ± 0.59d
−2.76 ± 0.03a
1.69 ± 0.04
0.257 ± 0.005f
452
2
38.5 ± 1.2
2.85 ± 0.02a
0.57 ± 0.01a
9.2 ± 0.1a
32.51 ± 0.62a
17.16 ± 0.74a
−2.72 ± 0.01a
1.22 ± 0.04
0.238 ± 0.001c
4
45.6 ± 0.8
2.84 ± 0.02a
0.57 ± 0.01a
9.3 ± 0.1a
32.25 ± 0.86a
16.81 ± 0.65a
−2.72 ± 0.04a
1.03 ± 0.03
0.249 ± 0.006ee
6
51.4 ± 2.2
2.85 ± 0.02a
0.58 ± 0.02a
9.2 ± 0.1a
31.79 ± 0.46ab
16.26 ± 0.47b
−2.71 ± 0.01a
1.12 ± 0.02
0.255 ± 0.003f
8
57.5 ± 1.6
2.86 ± 0.02a
0.56 ± 0.02a
9.3 ± 0.1a
31.57 ± 0.70b
15.38 ± 0.42d
−2.69 ± 0.03a
1.44 ± 0.03
0.259 ± 0.005f
10
61.2 ± 2.1
2.87 ± 0.01a
0.57 ± 0.01a
9.2 ± 0.1a
31.19 ± 0.68b
14.42 ± 0.54e
−2.70 ± 0.02a
2.33 ± 0.06
0.264 ± 0.005f
All data are expressed as mean ± SD (n = 6). Values with different letters within one column are significantly different (p<0.05).


## 2.3. Thermal Processing

Bayberry juice was treated in boiling water (Figure1(b)) until reaching a core temperature of 90°C and was held at this temperature longer than 1 min [29]. Then, the juice was immediately cooled down to room temperature with an ice water bath and stored at 4°C.


## 2.4. Measurement of TSS, pH, and TA

TSS was determined as °Brix using a WAY-2S Digital Abbe refractometer (Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China) at 25 ± 1°C.The pH value measurements were carried out by a pH meter (MP511, Sanxin Co., Ltd, Shanghai, China) with a combined pH electrode at 25 ± 1°C.The TA was determined via titration by an automatic titrimeter (G20, Mettler-Toledo, Shanghai, China). The TA value expressed as citric acid equivalents was analyzed and calculated using the following formula:(1)TA%=C×V2×KV1×V0m×2×100%,where C is the NaOH concentration (0.1 M); m is the weight of juice; V2, V1, and V0 (mL) are the volume of NaOH used and juice used and the total volume of juice, respectively; and K is the conversion factor of citric acid (0.07).


## 2.5. Measurement of Ascorbic Acid

The determination of ascorbic acid was performed referring to our previous study [30]. 2.5% of metaphosphoric acid was used as extraction solution, and the ratio was 1 : 5 (v : v). After 2 h extraction at 4°C, the mixture was centrifuged at 6880 ×g at 4°C for 10 min, and the supernatant was collected with final volume measurement.Ascorbic acid was measured using the liquid chromatograph method (Shimadzu Co., Japan) with an Alltech AlltimaTM C18 column (4.6 × 250 mm i.d.; 5 μm particle size) from Waters. The mobile phase consisted of 95% monopotassium phosphate (50 mM; pH = 3.0) and 5% acetonitrile, and the flow rate was 1 mL/min at 25 ± 1°C. The detection was carried out at 245 nm in the absorbance mode. The content was calculated by using the following formula:(2)m=m020×10−3×V1×100V0,where m0 is the value drawn in the standard curve of ascorbic acid (mg/L) and V1 and V0 are the volume of the final extract and initial bayberry juice, respectively.


## 2.6. Measurement of Monomeric Anthocyanins

The anhydrous methanol with 0.1% HCl was used for anthocyanin extraction, and the material-to-liquid ratio was 1 : 2. After extraction for 1 h at 4°C, the mixture was centrifuged (6880 ×g/10 min/4°C), and the supernatant was collected [29].A Venusil C18 column (250 mm × 4.6 mm i.d.; 5 μm particle size) equipped with a 5 μm C18 guard column both from Agela Technologies Co., Ltd. (Tianjin, China) was used for anthocyanin determination, and methanol (A) and formic acid/water (B) (5 : 95, v/v) were the mobile phases with a flow rate of 1 mL/min at 520 nm. The gradient elution consisted of 15% A for 0–5 min, 15%–30% A for 5–8 min, 30%–50% A for 8–12 min, 50%–80% A for 12–15 min, 80%∼15% A for 15–18 min, and 15% A for 18∼20 min. Cy-3-glu was quantified as monomeric anthocyanins by external standards. The results were calculated by using the following formula:(3)m=m020×10−3×V1×100V0,where m0 is the value drawn in the standard curve of anthocyanins (mg/L) and V1 and V0 are the volume of the final extract and initial bayberry juice, respectively.


## 2.7. Measurement of Polymeric Anthocyanins

Polymeric color was determined within 24 h referring to the previous study [29]. The bayberry juice was diluted with water until its absorbance at 520 nm reached 0.5∼1.0. 1 mL of 0.90 M potassium metabisulfite; 3 mL of the diluted juice was mixed and equilibrated for 30 min, and then the samples were determined at 700, 520, and 420 nm. Color density (CD), polymeric color (PC), and percent polymeric color (PPC) were calculated using the following formulas:(4)CD=A420−A700+A512−A700×dilution factor,PC=A420−A700+A512−A700×dilution factor,PPC=polymeric colorcolor density×100%.


## 2.8. Measurement of Antioxidant Activity

The free-radical-scavenging effect on DPPH radicals was determined as antioxidant activity [30]. Fivefold diluted bayberry juice (500 μL) and 0.14 mM methanolic DPPH solution (4 mL) were mixed and equilibrated for 45 min at 25 ± 1°C protected from light. Then, the samples were measured at 517 nm by a spectrophotometer (UV-726, Shimadzu, Shanghai, China). Standard Trolox dissolved in methanol (10–100 mg/L) was used for plotting calibration curves. The results (expressed as milligrams of Trolox equivalents per 100 mL of bayberry juice) were calculated using the following formula:(5)DPPH%=1−A0A1×100%,where A0 and A1 are the absorbance at 517 nm of the control and juice.


## 2.9. Measurement of HMF

Chromatographic separation and determination of HMF referring to the study of Liu et al. [31] were performed on a Venusil XBP (L) C18 column (250 mm × 4.6 mm i.d.; 5 μm particle size) from Agela Technologies Co., Ltd. (Tianjin, China). The mobile phase was methanol/water (10/90, v/v) with a flow rate of 1 mL/min at 30°C. The levels and results (milligrams per 100 mL of bayberry juice) were calculated using HMF as the external standard.


## 2.10. Measurement of BD

BD was studied by a spectrophotometric method at 420 nm [30]. The samples were firstly centrifuged (6880 ×g/4°C/10 min) and passed through a 0.45 μm cellulose nitrate membrane.


## 2.11. Color Assessment

Color was determined in the reflectance mode at 20 ± 1°C by a color difference meter (SBDY-3, Yuefeng Co., Shanghai, China) and expressed asL∗, a∗, and b∗ values. ΔE was calculated using the following equation, where L∗0, a∗0, and b∗0 are the control values for untreated juice:(6)ΔE=L∗−L∗02+a∗−a∗02+b∗−b∗021/2.


## 2.12. SOD Assay

The extraction and determination of SOD activity were performed by Pang et al. [32]. 20 mL of samples and 40 mL of phosphate buffer (0.05 M; pH 7.8) were mixed and extracted (1 h/4°C) and then centrifuged (6880 ×g/10 min/4°C), and the supernatants were collected.SOD activity was determined by pyrogallol autoxidation [32]. 3 mL of 0.07 M catechol (o-diphenol) in 50 mmol/L Tris-HCL buffer solution (pH 8.0) and 0.5 mL of SOD extracts were mixed, and absorbance changes at 325 nm/25 ± 0.1°C were immediately detected at intervals of 0.1 s−1 by a UV spectrophotometer (U-3010, Hitachi, Ltd., Tokyo, Japan). The enzymes in per milliliter reaction solution which inhibited 50% of the autocidal rate of pyrogallol were defined as one enzyme unit (U).


## 2.13. Statistical Analysis

The data were analyzed using the SPSS 12.0 software for analysis of variance and Duncan’s test. The significance was determined atP<0.05.


## 3. Results and Discussion

### 3.1. Effects of Ultrasound on TSS, TA, and pH

Effects of ultrasound on TSS, TA, and pH are displayed in Table1. Ultrasound did not significantly result in any change in the TSS, TA, and pH of bayberry juice, irrespective of ultrasonic intensity (W/cm2) or time. Similar results regarding pH, TA, and TSS were also observed in pear, grapefruit juice, orange, tomato, and apple juice treated with ultrasound [18, 19, 25–27].


### 3.2. Effects of Ultrasound on Ascorbic Acid

The initial content of ascorbic acid in fresh juice was 22.82 mg/100 mL. 25.16% of ascorbic acid decreased in the samples after TP treatment, indicating that ascorbic acid was highly thermosensitive.Results regarding effects of ultrasound treatments on ascorbic acid of bayberry juice are exhibited in Figure2, showing the ascorbic acid retention ranged from 80.39% to 101.82% in all ultrasound-treated juice as compared with control juice. No significant changes of ascorbic acid were found in bayberry juice treated at different ultrasonic intensity within 4 minutes. With increasing ultrasonic intensity and extending treatment time, ascorbic acid gradually decreased, and the highest loss was 19.61% at 450 W/cm2 for 10 min. The results were in agreement with those of previous studies. Adekunte et al. reported that ascorbic acid of tomato juice decreased by 96.9% to 60.7% when treated with ultrasound at amplitude levels of 24.4–61 μm for 2–10 min [25]. Chaikham et al. found that ultrasound treatment at 40% and 80% power using 20 kHz (750 W) for 30 min caused 27.24% and 60.61% losses of ascorbic acid in honey [33]. The decreases of ascorbic acid by ultrasound in strawberry (frequency 33 kHz/power 60 W) were 36.68, 35.57, and 32.20% for a treatment time of 10, 20, and 30 min, respectively [34]. The degradation of ascorbic acid was mainly attributed to the sonochemical reactions and the extreme physical conditions which occurred during ultrasound. It has been demonstrated that hydrogen ions (H+), free radicals (O−, OH−, and HO2−), and hydrogen peroxide (H2O2) formed during the sonolysis of water molecules present in juice [11, 25, 35]. The degradation could also be related to oxidation reactions promoted by the interaction of hydroxyl radicals produced by cavitation during ultrasonic processing [25]. Another factor that affected the stability of ascorbic acid was the thermal effect attributed to the enormous microzone temperatures achieved during cavitation. As shown in Table 1, the final temperature of samples after ultrasound treatment increased to 29.5–61.2°C. Previous study on sonicated pear juice also found the temperature reached 38.60–68.50°C after ultrasound treatment [36].Figure 2
Effects of ultrasound processing on ascorbic acid of bayberry juice.However, different results were observed in some ultrasound-treated fruit juices. Zenker et al. [37] and Martínez-Flores et al. [38] found that ultrasound had no significant effect on ascorbic acid in orange and carrot juice. Moreover, increases of ascorbic acid content were found in ultrasound-treated apple juice, soursop juice, grapefruit juice, and kasturi lime juice [16, 17, 39, 40]. This increase of ascorbic acid could be directly due to the removal of entrapped oxygen because of cavitation [16]. The explanation of differences in effects of ultrasound treatment on ascorbic acid could lie in a competition (different reaction rates) between hydroxyl groups and all the other oxidizable substrates available such as proteins and sugars [16].


### 3.3. Effects of Ultrasound on Monomeric Anthocyanins

Cyanidin-3-glucoside (Cy-3-glu) was identified as the most abundant monomeric anthocyanin in bayberries, representing more than 95% of total anthocyanins [7]. The content of Cy-3-glu in control samples was 27.65 mg/100 mL, and it decreased by 16.61% after TP treatment (Table 2). The obvious degradation and structure stability of fruit anthocyanins during thermal processing have been reported clearly, as well as their degradation mechanisms [41].Table 2
Effects of TP treatment on the quality of bayberry juice.
Key parameter
Control
TP
Monomeric anthocyanins
27.65 ± 0.51a
23.06 ± 0.68b
Polymeric color
CD
2.56 ± 0.12a
2.41 ± 0.04a
PC
0.27 ± 0.01a
0.38 ± 0.01b
PPC
10.55 ± 0.12a
15.77 ± 0.05b
Ascorbic acid
22.82 ± 0.57a
17.08 ± 0.24b
Antioxidant activity
193.19 ± 4.56a
174.78 ± 5.65b
HMF
0.016 ± 0.001a
0.032 ± 0.001b
SOD activity
237.73 ± 8.41a
102.75 ± 2.88b
All data are expressed as mean ± SD (n = 6). Monomeric anthocyanins, ascorbic acid, and HMF are expressed as mg/100 mL. Total phenols are expressed as mg GAE/100 mL. Antioxidant activity is expressed as mg TE/100 mL. SOD activity is expressed as U·mL−1. Values with different letters are significantly different (p<0.05).Effects of ultrasound intensity and time on retention of monomeric anthocyanins of barberry juice are shown in Figure3. The Cy-3-glu exhibited no significant changes after ultrasound treatment at lower amplitude levels (90, 181, and 271 W/cm2) within 8 min and higher amplitude levels (362 and 452 W/cm2) within 6 min, indicating that monomeric anthocyanins were well retained. Moreover, Cy-3-glu of bayberry juice treated within 4 min even showed an increasing tendency by 0.71–4.91%, which might be attributed to the extraction of bound anthocyanins from the suspended juice. Similarly, Tiwari et al. also found a slight increase in pelargonidin-3-glucoside content in strawberry juice at lower amplitude levels and treatment times [27]. The results indicated that ultrasound could promote extraction of anthocyanins and other bioactive compounds from fruit juice or pulp. Ultrasound-assisted extraction of anthocyanins from sweet lime juice was recently researched by Chanukya and Rastogi, and the extraction yield was improved by a value between 6 and 35% compared to conventional processing [42]. Chen et al. also reported improved extraction efficiency of cyanidin-3-glucoside from raspberries [43]. The extractability of bioactive compounds in food components was enhanced by increasing the frequency and power [44]. Anthocyanins are located in the vacuoles in plants, and ultrasound treatment is known to destroy the membranes in vegetable cells, as well as the vacuole membrane, and enhance cell permeability because of its ability to deprotonate charged groups and disrupt salt bridges and hydrophobic bonds in cell membranes [29].Figure 3
Effects of ultrasound processing on monomeric anthocyanins and polymeric color of bayberry juice.
(a)
(b)
(c)
(d)However, prolonged treatment power levels and time might induce chemical decomposition of anthocyanins. Figure3 shows higher amplitude levels and longer times had adverse effects on anthocyanins of bayberry juice. The decreases of Cy-3-glu ranged from 3.27% to 9.95%, which is consistent with that reported in earlier literatures. Tiwari et al. reported that a 5% loss of total anthocyanins was observed at the maximum treatment conditions of 100% amplitude for 10 min in blackberry juice [20]. Cy-3-glu, malvidin-3-O-glucosides, and delphinidin-3-O-glucosides in sonicated red grape juice decreased by 2.5%, 51.8%, and 19.1%, respectively [45]. The observed degradation of anthocyanins was mainly due to three main mechanisms: the effect of cavitation which generated high temperature, pressure, or mechanical action between solid and liquid interfaces [46]. Another effect was resulted by the presence of organic acid or ascorbic acid and could be related to oxidation reactions promoted by the interaction of free radicals formed during ultrasound as shown for ascorbic acid [47]. The third effect was caused by the mechanical forces and shear forces, created by microstreaming and shock [8]. The rise of temperature during ultrasound treatment might also promote the increase of hydroxyl radicals which were involved in the degradation of anthocyanins by the opening of rings and formation of chalcone [46].


### 3.4. Effects of Ultrasound on Polymeric Color

The color of bayberry is primarily attributed to its anthocyanins. However, the monomeric anthocyanins might convert to polymeric ones by some intrinsic factors, including temperature, pressure, and pH during processing or storage [48]. Anthocyanin condensation reactions occurred through the covalent association of anthocyanins with other flavanols through ethyl bridges or with other small molecules, such as pyruvic acid, vinylphenol, and glyoxylic acid [41]. Since polymerization is an important reaction occurring during anthocyanin degradation, PPC is considered a good indicator of anthocyanin degradation [48].The PPC of bayberry juice increased from 10.55 to 15.77% after TP treatment, indicating the condensation reactions between anthocyanins and other phenolics which form colored polymer pigments [49]. Hager et al. reported that TP processing caused 12.1% and 13.4% increases of the PPC in caned blackberry syrup and puree, respectively [50]. An increase in PC was also observed in pasteurized blueberry juice compared with frozen fruit [51].Results regarding effects of ultrasound treatment on PC of bayberry juice are shown in Figure3. The CD of bayberry juice exhibited no significant differences after ultrasound treatment, while PC progressively increased by 6.93–31.73% with ultrasonic intensity and time, which were accompanied by the decreases in the monomeric anthocyanins. Compared with control bayberry juice, the PPC of ultrasound-treated samples improved and ranged from 11.99 to 14.19%. The formation of polymeric color might be due to the formation of chalcone, an intermediate product of anthocyanin degradation [52]. The chalcone was unstable and would be quickly degraded to brown-colored products, leading to notable increases of polymeric color [53]. Previous studies also found a strong negative correlation between the anthocyanin content and PPC in hydrothermodynamic-processed blueberries and black carrot juice concentrates during storage [48, 53].


### 3.5. Effects of Ultrasound on Antioxidant Activity

The antioxidant capacity of control juice was 193.19 mg TE/100 mL (Table2). TP treatment caused a significant decrease in DPPH free-radical-scavenging activity of bayberry juice, which strongly correlated with the changes of ascorbic acid and monomeric anthocyanin content.Results regarding effects of ultrasound treatment on antioxidant capacity of bayberry juice are shown in Figure4. The·DPPH did not change significantly when bayberry juice was treated with low ultrasound intensity at 90.41 and 180.82 W/cm2 regardless of treatment time or higher intensity for short time (within 4 min). Moreover, there was a notable drop in antioxidant activity after 6–10 min treatment of sonication at 271.23, 361.64, and 452.05 W/cm2 intensity. A similar increasing or decreasing trend of antioxidant capacity of red raspberry puree and ascorbic acid solution treated with ultrasound at different frequency and intensity was reported by Golmohamadi et al. [22]. The changes of antioxidant capacity were well related to the presence of concentration of antioxidants, and the correlation coefficients between DPPH and ascorbic acid/anthocyanins were 0.864/0.768. In previous studies, there was also a positive relation between phenolic compounds and antioxidant activity [54–56]. Different results had also been reported that significant increases existed in antioxidant capacity of sonicated grapefruit juice [17] and sonicated kasturi lime juice [40]. The increase might be induced by increases of phenols during sonication [17].Figure 4
Effects of ultrasound processing on antioxidant activity of bayberry juice.


### 3.6. Effects of Ultrasound on BD

BD is an important parameter that indicates the nonenzymatic browning and quality deterioration of foods [24]. As listed in Table 1, BD values of barberry juice significantly increased with ultrasound treatment intensity and time. The BD increased by 102.65% at the lowest treatment intensity and time (90 W/cm2 and 2 min) and by 116.47% at the highest condition (450 W/cm2 and 10 min), respectively. Similar changes were reported by Guerrouja et al. for ultrasound-treated orange juice [57], by Aadil et al. for grapefruit juice [17], and by Ugarte-Romero et al. for sonicated apple cider [58]. The changes of BD followed zero-order reaction kinetics (R2 ranged from 0.933 to 0.989) with increasing ultrasonic intensity and time, and the reaction rate constant k0 (0.0011–0.00373) of the zero-order kinetic model was closely dependent on ultrasonic intensity. Increasing ultrasonic intensity resulted in higher k0 values. Previous kinetics studies on BD reactions based on A420 in orange juice [24] and apple juice [26] also reported zero-order reaction kinetics.The nonenzymatic browning of fruit products during processing and storage might be resulted by the formation of brown pigments: the reactions between amino acids and reducing sugars (Maillard reactions), the degradation of ascorbic acid, and the intramolecular condensation of polyphenols or intermolecular reactions with amino acids [59]. The main type of the nonenzymatic browning mechanism is not alike in juice because of the difference of composition and processing condition. As shown in Table 3, the rapid increase of the BD value of ultrasound-treated bayberry juice highly correlated with degradation of ascorbic acid (R2 = 0.918) and formation of HMF (R2 = 0.934). Fustier et al. [59] also found that the BD value and ascorbic acid presented a high negative correlation in orange juice. However, the correlation coefficients between BD and monomeric or polymeric anthocyanins were only 0.626 and 0.779. The results indicated that degradation of ascorbic acid could provide reactive carbonyls groups that acted as precursors playing a major role in the nonenzymatic browning of bayberry juice during ultrasound treatment.Table 3
Linear correlation coefficients between BD and nonenzymatic browning-related compositions.
Compositions
Correlation coefficients
BD
Ascorbic acid
0.918
Monomeric anthocyanins
0.626
Polymeric anthocyanins
0.779
HMF
0.934
L
∗
0.909


### 3.7. Effects of Ultrasound on HMF

HMF is a product of the Maillard reaction and ascorbic acid degradation, and its formation is remarkably influenced by the processing and storage conditions [60]. HMF in fruit-processed products has gained interest for a long time because of the effect on color quality as well as its potential toxicological risk [31]. The HMF in control bayberry juice was 0.016 mg/100 mL, and after thermal processing, the increase of HMF to 0.032 mg/100 mL was observed (Table 1). Liu et al. also reported that HMF in TP-treated mango nectar increased from 0.017 to 0.029 mg/100 mL [31]. As shown in Figure 5, ultrasound treatment significantly improved the content of HMF in bayberry juice except for the samples treated at lower power levels (90, 181, and 271 W/cm2) for 2 min. Moreover, the levels of HMF obviously accumulated when the ultrasound treatment intensity and processing time increased. A highest HMF content of 0.019 mg/100 mL was found in the sample processed at 450 W/cm2 for 10 min. The formation and subsequent degradation of Amadori rearrangement products might be accelerated by ultrasound treatment, resulting in increasing intermediate and advanced Maillard reaction products [61], and this could explain the increases of HMF in ultrasound-processed bayberry juice. Similarly, Chaikham et al. found that HMF in honey noticeably enhanced after ultrasound treatment at 40% and 80% amplitude [33].Figure 5
Effects of ultrasound processing on HMF of bayberry juice.


### 3.8. Effects of Ultrasound on Color

Color is a visual indicator for evaluating the quality of food and affects the consumer’s satisfaction as well [28]. TP treatment induced significant decreases of L∗, a∗, and b∗ values of bayberry juice, and the ΔE value of TP-treated samples was 2.86, revealing a marked browning reaction that occurred during thermal treatment (Table 1). The degradation of polyphenols and formation of Maillard reaction products by intensive thermal processing caused the obvious changes in color [28]. In addition, the degradation of ascorbic acid was also remarkably related to color browning [28].A slightly decrease inL∗ values could be observed for almost all ultrasound-treated bayberry juice, which was highly correlated (R2 = 0.909) with the increases in BD values of treated bayberry juice (Table 3). Samples treated at 450 W/cm2/10 min showed the lowest L∗ values. The observations were in accordance with those of ultrasound-processed orange juice [24] and grapefruit juice [17], where the decrease in L∗ values was attributed to oxidative darkening. However, an increase of L∗ values in watermelon juice [62] and soursop juice [16] had been found, which might be explained by the homogenization effect of sonication. The a∗ values of bayberry juice exhibited no significant changes when treated at lower power levels (90, 181, and 271 W/cm2) within 8 min. However, higher ultrasound power with short-time treatment noticeably enhanced the a∗ values of samples. The a∗ values of samples are generally affected by the existence of natural anthocyanins. Changes of a∗ values affected by ultrasound were highly consistent with those of monomeric anthocyanins, as well as those of CD. The b∗ values of all samples were well retained.TheΔE values of ultrasound-treated bayberry juice ranged from 0.21 to 2.33 (Table 1), and they increased with the ultrasound intensity or treatment time enhancement, which was in accordance with the anthocyanin and ascorbic acid changes [16, 28]. As described by da Rocha Cordeiro Dias et al., changes of color in ultrasound-processed products might be caused by the accelerated chromogenic substance degradation, and the increases of oxidation reactions were induced by free radicals generated during cavitation [16]. Furthermore, it is noteworthy that, besides bayberry juice processed at 452 W/cm/10 min, the ΔE values of samples were all lower than 2, revealing that the color change of ultrasound-treated bayberry juice could not be found by the consumer’s naked eyes.


### 3.9. Effects of Ultrasound on SOD Activity

TP treatment caused a 56.78% inactivation of SOD activity of bayberry juice (Table2), and the ultrasound treatment also significantly affected the SOD activity, as displayed in Figure 6. There was a notable increase of SOD activity for the first 2–6 min treatment regardless of ultrasound intensity. The detected SOD activity increased by 21.39%∼28.02% in bayberry juice treated at 90.41∼452.05 W/cm2 intensity for 4 min, which was probably because of the activation of SOD during ultrasound treatment, as well as higher extractability of enzymes caused by the mechanical disruption of the cell wall in bayberry juice [63, 64]. The application of ultrasound in the extraction of SOD from plant materials was previously studied. Wang and Xu [63] reported that ultrasound treatment improved the SOD activity of Rosa roxburghii, and the highest enzyme activity was observed in samples treated at 200 W/3 min. Similarly, Pan et al. [64] also found that the SOD activity of sweet potato leaves increased from 240.16 to 359.54 U·g−1 after ultrasound treatment (500 W/4 min). However, with the further extension of processing time, the decreases of SOD activity of bayberry occurred, and the inactivation effects were obviously related to the ultrasound intensity and time. When the treatment was applied for 10 min at 90, 180, 271, 362, and 452 W/cm2 ultrasonic intensity, the SOD residual activity was 93.46%, 95.25%, 94.83%, 85.99%, and 73.91%, respectively. The inactivation of SOD was mainly due to physical and chemical principles [14, 17, 28]. Cavitation effects and temperature increases during ultrasound processing could result in the changes of the secondary and tertiary structures of enzymes and losses in their biological activity [14, 17, 28]. Furthermore, sonication also promoted the water decomposition to H+ and OH− free radicals, which would connect with amino acids on the enzyme structure, thereby affecting the SOD activity [14, 17, 28].Figure 6
Effects of ultrasound processing on the activity of SOD in bayberry juice.


## 3.1. Effects of Ultrasound on TSS, TA, and pH

Effects of ultrasound on TSS, TA, and pH are displayed in Table1. Ultrasound did not significantly result in any change in the TSS, TA, and pH of bayberry juice, irrespective of ultrasonic intensity (W/cm2) or time. Similar results regarding pH, TA, and TSS were also observed in pear, grapefruit juice, orange, tomato, and apple juice treated with ultrasound [18, 19, 25–27].


## 3.2. Effects of Ultrasound on Ascorbic Acid

The initial content of ascorbic acid in fresh juice was 22.82 mg/100 mL. 25.16% of ascorbic acid decreased in the samples after TP treatment, indicating that ascorbic acid was highly thermosensitive.Results regarding effects of ultrasound treatments on ascorbic acid of bayberry juice are exhibited in Figure2, showing the ascorbic acid retention ranged from 80.39% to 101.82% in all ultrasound-treated juice as compared with control juice. No significant changes of ascorbic acid were found in bayberry juice treated at different ultrasonic intensity within 4 minutes. With increasing ultrasonic intensity and extending treatment time, ascorbic acid gradually decreased, and the highest loss was 19.61% at 450 W/cm2 for 10 min. The results were in agreement with those of previous studies. Adekunte et al. reported that ascorbic acid of tomato juice decreased by 96.9% to 60.7% when treated with ultrasound at amplitude levels of 24.4–61 μm for 2–10 min [25]. Chaikham et al. found that ultrasound treatment at 40% and 80% power using 20 kHz (750 W) for 30 min caused 27.24% and 60.61% losses of ascorbic acid in honey [33]. The decreases of ascorbic acid by ultrasound in strawberry (frequency 33 kHz/power 60 W) were 36.68, 35.57, and 32.20% for a treatment time of 10, 20, and 30 min, respectively [34]. The degradation of ascorbic acid was mainly attributed to the sonochemical reactions and the extreme physical conditions which occurred during ultrasound. It has been demonstrated that hydrogen ions (H+), free radicals (O−, OH−, and HO2−), and hydrogen peroxide (H2O2) formed during the sonolysis of water molecules present in juice [11, 25, 35]. The degradation could also be related to oxidation reactions promoted by the interaction of hydroxyl radicals produced by cavitation during ultrasonic processing [25]. Another factor that affected the stability of ascorbic acid was the thermal effect attributed to the enormous microzone temperatures achieved during cavitation. As shown in Table 1, the final temperature of samples after ultrasound treatment increased to 29.5–61.2°C. Previous study on sonicated pear juice also found the temperature reached 38.60–68.50°C after ultrasound treatment [36].Figure 2
Effects of ultrasound processing on ascorbic acid of bayberry juice.However, different results were observed in some ultrasound-treated fruit juices. Zenker et al. [37] and Martínez-Flores et al. [38] found that ultrasound had no significant effect on ascorbic acid in orange and carrot juice. Moreover, increases of ascorbic acid content were found in ultrasound-treated apple juice, soursop juice, grapefruit juice, and kasturi lime juice [16, 17, 39, 40]. This increase of ascorbic acid could be directly due to the removal of entrapped oxygen because of cavitation [16]. The explanation of differences in effects of ultrasound treatment on ascorbic acid could lie in a competition (different reaction rates) between hydroxyl groups and all the other oxidizable substrates available such as proteins and sugars [16].


## 3.3. Effects of Ultrasound on Monomeric Anthocyanins

Cyanidin-3-glucoside (Cy-3-glu) was identified as the most abundant monomeric anthocyanin in bayberries, representing more than 95% of total anthocyanins [7]. The content of Cy-3-glu in control samples was 27.65 mg/100 mL, and it decreased by 16.61% after TP treatment (Table 2). The obvious degradation and structure stability of fruit anthocyanins during thermal processing have been reported clearly, as well as their degradation mechanisms [41].Table 2
Effects of TP treatment on the quality of bayberry juice.
Key parameter
Control
TP
Monomeric anthocyanins
27.65 ± 0.51a
23.06 ± 0.68b
Polymeric color
CD
2.56 ± 0.12a
2.41 ± 0.04a
PC
0.27 ± 0.01a
0.38 ± 0.01b
PPC
10.55 ± 0.12a
15.77 ± 0.05b
Ascorbic acid
22.82 ± 0.57a
17.08 ± 0.24b
Antioxidant activity
193.19 ± 4.56a
174.78 ± 5.65b
HMF
0.016 ± 0.001a
0.032 ± 0.001b
SOD activity
237.73 ± 8.41a
102.75 ± 2.88b
All data are expressed as mean ± SD (n = 6). Monomeric anthocyanins, ascorbic acid, and HMF are expressed as mg/100 mL. Total phenols are expressed as mg GAE/100 mL. Antioxidant activity is expressed as mg TE/100 mL. SOD activity is expressed as U·mL−1. Values with different letters are significantly different (p<0.05).Effects of ultrasound intensity and time on retention of monomeric anthocyanins of barberry juice are shown in Figure3. The Cy-3-glu exhibited no significant changes after ultrasound treatment at lower amplitude levels (90, 181, and 271 W/cm2) within 8 min and higher amplitude levels (362 and 452 W/cm2) within 6 min, indicating that monomeric anthocyanins were well retained. Moreover, Cy-3-glu of bayberry juice treated within 4 min even showed an increasing tendency by 0.71–4.91%, which might be attributed to the extraction of bound anthocyanins from the suspended juice. Similarly, Tiwari et al. also found a slight increase in pelargonidin-3-glucoside content in strawberry juice at lower amplitude levels and treatment times [27]. The results indicated that ultrasound could promote extraction of anthocyanins and other bioactive compounds from fruit juice or pulp. Ultrasound-assisted extraction of anthocyanins from sweet lime juice was recently researched by Chanukya and Rastogi, and the extraction yield was improved by a value between 6 and 35% compared to conventional processing [42]. Chen et al. also reported improved extraction efficiency of cyanidin-3-glucoside from raspberries [43]. The extractability of bioactive compounds in food components was enhanced by increasing the frequency and power [44]. Anthocyanins are located in the vacuoles in plants, and ultrasound treatment is known to destroy the membranes in vegetable cells, as well as the vacuole membrane, and enhance cell permeability because of its ability to deprotonate charged groups and disrupt salt bridges and hydrophobic bonds in cell membranes [29].Figure 3
Effects of ultrasound processing on monomeric anthocyanins and polymeric color of bayberry juice.
(a)
(b)
(c)
(d)However, prolonged treatment power levels and time might induce chemical decomposition of anthocyanins. Figure3 shows higher amplitude levels and longer times had adverse effects on anthocyanins of bayberry juice. The decreases of Cy-3-glu ranged from 3.27% to 9.95%, which is consistent with that reported in earlier literatures. Tiwari et al. reported that a 5% loss of total anthocyanins was observed at the maximum treatment conditions of 100% amplitude for 10 min in blackberry juice [20]. Cy-3-glu, malvidin-3-O-glucosides, and delphinidin-3-O-glucosides in sonicated red grape juice decreased by 2.5%, 51.8%, and 19.1%, respectively [45]. The observed degradation of anthocyanins was mainly due to three main mechanisms: the effect of cavitation which generated high temperature, pressure, or mechanical action between solid and liquid interfaces [46]. Another effect was resulted by the presence of organic acid or ascorbic acid and could be related to oxidation reactions promoted by the interaction of free radicals formed during ultrasound as shown for ascorbic acid [47]. The third effect was caused by the mechanical forces and shear forces, created by microstreaming and shock [8]. The rise of temperature during ultrasound treatment might also promote the increase of hydroxyl radicals which were involved in the degradation of anthocyanins by the opening of rings and formation of chalcone [46].


## 3.4. Effects of Ultrasound on Polymeric Color

The color of bayberry is primarily attributed to its anthocyanins. However, the monomeric anthocyanins might convert to polymeric ones by some intrinsic factors, including temperature, pressure, and pH during processing or storage [48]. Anthocyanin condensation reactions occurred through the covalent association of anthocyanins with other flavanols through ethyl bridges or with other small molecules, such as pyruvic acid, vinylphenol, and glyoxylic acid [41]. Since polymerization is an important reaction occurring during anthocyanin degradation, PPC is considered a good indicator of anthocyanin degradation [48].The PPC of bayberry juice increased from 10.55 to 15.77% after TP treatment, indicating the condensation reactions between anthocyanins and other phenolics which form colored polymer pigments [49]. Hager et al. reported that TP processing caused 12.1% and 13.4% increases of the PPC in caned blackberry syrup and puree, respectively [50]. An increase in PC was also observed in pasteurized blueberry juice compared with frozen fruit [51].Results regarding effects of ultrasound treatment on PC of bayberry juice are shown in Figure3. The CD of bayberry juice exhibited no significant differences after ultrasound treatment, while PC progressively increased by 6.93–31.73% with ultrasonic intensity and time, which were accompanied by the decreases in the monomeric anthocyanins. Compared with control bayberry juice, the PPC of ultrasound-treated samples improved and ranged from 11.99 to 14.19%. The formation of polymeric color might be due to the formation of chalcone, an intermediate product of anthocyanin degradation [52]. The chalcone was unstable and would be quickly degraded to brown-colored products, leading to notable increases of polymeric color [53]. Previous studies also found a strong negative correlation between the anthocyanin content and PPC in hydrothermodynamic-processed blueberries and black carrot juice concentrates during storage [48, 53].


## 3.5. Effects of Ultrasound on Antioxidant Activity

The antioxidant capacity of control juice was 193.19 mg TE/100 mL (Table2). TP treatment caused a significant decrease in DPPH free-radical-scavenging activity of bayberry juice, which strongly correlated with the changes of ascorbic acid and monomeric anthocyanin content.Results regarding effects of ultrasound treatment on antioxidant capacity of bayberry juice are shown in Figure4. The·DPPH did not change significantly when bayberry juice was treated with low ultrasound intensity at 90.41 and 180.82 W/cm2 regardless of treatment time or higher intensity for short time (within 4 min). Moreover, there was a notable drop in antioxidant activity after 6–10 min treatment of sonication at 271.23, 361.64, and 452.05 W/cm2 intensity. A similar increasing or decreasing trend of antioxidant capacity of red raspberry puree and ascorbic acid solution treated with ultrasound at different frequency and intensity was reported by Golmohamadi et al. [22]. The changes of antioxidant capacity were well related to the presence of concentration of antioxidants, and the correlation coefficients between DPPH and ascorbic acid/anthocyanins were 0.864/0.768. In previous studies, there was also a positive relation between phenolic compounds and antioxidant activity [54–56]. Different results had also been reported that significant increases existed in antioxidant capacity of sonicated grapefruit juice [17] and sonicated kasturi lime juice [40]. The increase might be induced by increases of phenols during sonication [17].Figure 4
Effects of ultrasound processing on antioxidant activity of bayberry juice.


## 3.6. Effects of Ultrasound on BD

BD is an important parameter that indicates the nonenzymatic browning and quality deterioration of foods [24]. As listed in Table 1, BD values of barberry juice significantly increased with ultrasound treatment intensity and time. The BD increased by 102.65% at the lowest treatment intensity and time (90 W/cm2 and 2 min) and by 116.47% at the highest condition (450 W/cm2 and 10 min), respectively. Similar changes were reported by Guerrouja et al. for ultrasound-treated orange juice [57], by Aadil et al. for grapefruit juice [17], and by Ugarte-Romero et al. for sonicated apple cider [58]. The changes of BD followed zero-order reaction kinetics (R2 ranged from 0.933 to 0.989) with increasing ultrasonic intensity and time, and the reaction rate constant k0 (0.0011–0.00373) of the zero-order kinetic model was closely dependent on ultrasonic intensity. Increasing ultrasonic intensity resulted in higher k0 values. Previous kinetics studies on BD reactions based on A420 in orange juice [24] and apple juice [26] also reported zero-order reaction kinetics.The nonenzymatic browning of fruit products during processing and storage might be resulted by the formation of brown pigments: the reactions between amino acids and reducing sugars (Maillard reactions), the degradation of ascorbic acid, and the intramolecular condensation of polyphenols or intermolecular reactions with amino acids [59]. The main type of the nonenzymatic browning mechanism is not alike in juice because of the difference of composition and processing condition. As shown in Table 3, the rapid increase of the BD value of ultrasound-treated bayberry juice highly correlated with degradation of ascorbic acid (R2 = 0.918) and formation of HMF (R2 = 0.934). Fustier et al. [59] also found that the BD value and ascorbic acid presented a high negative correlation in orange juice. However, the correlation coefficients between BD and monomeric or polymeric anthocyanins were only 0.626 and 0.779. The results indicated that degradation of ascorbic acid could provide reactive carbonyls groups that acted as precursors playing a major role in the nonenzymatic browning of bayberry juice during ultrasound treatment.Table 3
Linear correlation coefficients between BD and nonenzymatic browning-related compositions.
Compositions
Correlation coefficients
BD
Ascorbic acid
0.918
Monomeric anthocyanins
0.626
Polymeric anthocyanins
0.779
HMF
0.934
L
∗
0.909


## 3.7. Effects of Ultrasound on HMF

HMF is a product of the Maillard reaction and ascorbic acid degradation, and its formation is remarkably influenced by the processing and storage conditions [60]. HMF in fruit-processed products has gained interest for a long time because of the effect on color quality as well as its potential toxicological risk [31]. The HMF in control bayberry juice was 0.016 mg/100 mL, and after thermal processing, the increase of HMF to 0.032 mg/100 mL was observed (Table 1). Liu et al. also reported that HMF in TP-treated mango nectar increased from 0.017 to 0.029 mg/100 mL [31]. As shown in Figure 5, ultrasound treatment significantly improved the content of HMF in bayberry juice except for the samples treated at lower power levels (90, 181, and 271 W/cm2) for 2 min. Moreover, the levels of HMF obviously accumulated when the ultrasound treatment intensity and processing time increased. A highest HMF content of 0.019 mg/100 mL was found in the sample processed at 450 W/cm2 for 10 min. The formation and subsequent degradation of Amadori rearrangement products might be accelerated by ultrasound treatment, resulting in increasing intermediate and advanced Maillard reaction products [61], and this could explain the increases of HMF in ultrasound-processed bayberry juice. Similarly, Chaikham et al. found that HMF in honey noticeably enhanced after ultrasound treatment at 40% and 80% amplitude [33].Figure 5
Effects of ultrasound processing on HMF of bayberry juice.


## 3.8. Effects of Ultrasound on Color

Color is a visual indicator for evaluating the quality of food and affects the consumer’s satisfaction as well [28]. TP treatment induced significant decreases of L∗, a∗, and b∗ values of bayberry juice, and the ΔE value of TP-treated samples was 2.86, revealing a marked browning reaction that occurred during thermal treatment (Table 1). The degradation of polyphenols and formation of Maillard reaction products by intensive thermal processing caused the obvious changes in color [28]. In addition, the degradation of ascorbic acid was also remarkably related to color browning [28].A slightly decrease inL∗ values could be observed for almost all ultrasound-treated bayberry juice, which was highly correlated (R2 = 0.909) with the increases in BD values of treated bayberry juice (Table 3). Samples treated at 450 W/cm2/10 min showed the lowest L∗ values. The observations were in accordance with those of ultrasound-processed orange juice [24] and grapefruit juice [17], where the decrease in L∗ values was attributed to oxidative darkening. However, an increase of L∗ values in watermelon juice [62] and soursop juice [16] had been found, which might be explained by the homogenization effect of sonication. The a∗ values of bayberry juice exhibited no significant changes when treated at lower power levels (90, 181, and 271 W/cm2) within 8 min. However, higher ultrasound power with short-time treatment noticeably enhanced the a∗ values of samples. The a∗ values of samples are generally affected by the existence of natural anthocyanins. Changes of a∗ values affected by ultrasound were highly consistent with those of monomeric anthocyanins, as well as those of CD. The b∗ values of all samples were well retained.TheΔE values of ultrasound-treated bayberry juice ranged from 0.21 to 2.33 (Table 1), and they increased with the ultrasound intensity or treatment time enhancement, which was in accordance with the anthocyanin and ascorbic acid changes [16, 28]. As described by da Rocha Cordeiro Dias et al., changes of color in ultrasound-processed products might be caused by the accelerated chromogenic substance degradation, and the increases of oxidation reactions were induced by free radicals generated during cavitation [16]. Furthermore, it is noteworthy that, besides bayberry juice processed at 452 W/cm/10 min, the ΔE values of samples were all lower than 2, revealing that the color change of ultrasound-treated bayberry juice could not be found by the consumer’s naked eyes.


## 3.9. Effects of Ultrasound on SOD Activity

TP treatment caused a 56.78% inactivation of SOD activity of bayberry juice (Table2), and the ultrasound treatment also significantly affected the SOD activity, as displayed in Figure 6. There was a notable increase of SOD activity for the first 2–6 min treatment regardless of ultrasound intensity. The detected SOD activity increased by 21.39%∼28.02% in bayberry juice treated at 90.41∼452.05 W/cm2 intensity for 4 min, which was probably because of the activation of SOD during ultrasound treatment, as well as higher extractability of enzymes caused by the mechanical disruption of the cell wall in bayberry juice [63, 64]. The application of ultrasound in the extraction of SOD from plant materials was previously studied. Wang and Xu [63] reported that ultrasound treatment improved the SOD activity of Rosa roxburghii, and the highest enzyme activity was observed in samples treated at 200 W/3 min. Similarly, Pan et al. [64] also found that the SOD activity of sweet potato leaves increased from 240.16 to 359.54 U·g−1 after ultrasound treatment (500 W/4 min). However, with the further extension of processing time, the decreases of SOD activity of bayberry occurred, and the inactivation effects were obviously related to the ultrasound intensity and time. When the treatment was applied for 10 min at 90, 180, 271, 362, and 452 W/cm2 ultrasonic intensity, the SOD residual activity was 93.46%, 95.25%, 94.83%, 85.99%, and 73.91%, respectively. The inactivation of SOD was mainly due to physical and chemical principles [14, 17, 28]. Cavitation effects and temperature increases during ultrasound processing could result in the changes of the secondary and tertiary structures of enzymes and losses in their biological activity [14, 17, 28]. Furthermore, sonication also promoted the water decomposition to H+ and OH− free radicals, which would connect with amino acids on the enzyme structure, thereby affecting the SOD activity [14, 17, 28].Figure 6
Effects of ultrasound processing on the activity of SOD in bayberry juice.


## 4. Conclusion

No significant differences in pH, °Brix, TA, andb∗ values were observed in ultrasound-treated bayberry juice. The BD, PC, HMF, and L∗ values of bayberry juice obviously increased after ultrasound treatment. Ultrasound treatment at lower intensity levels and short time showed no notable influences on antioxidant compounds and activity, while these quality parameters progressively decreased with increasing ultrasonic intensity and extending treatment time. It could be revealed that ultrasound treatments at intensity lower than 450 W/cm2 within 8 min were beneficial to retaining the quality of bayberry juice compared with TP treatment.



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*Source: 7917419-2019-09-02.xml*