Changes in Quality Attributes During Storage of Litchi Juice Treated With Dimethyl Dicarbonate (DMDC) and Nisin

The aim of this work was to evaluate the change in the quality of litchi juice treated by DMDC combined with Nisin during storage of 4 °C. Results found that addition of 250 mg/L of DMDC combined with 100 IU/mL of Nisin can ensure the microbiological safe of litchi juice during storage at 4 °C. Compare with heat treatment (95°C, 1 min), the treatment of DMDC combined with Nisin can retain a more value of sensory attributes, but a more loss in the content of total phenolics, ascorbic acid, and antioxidant capacity was observed during storage at 4 °C because of the ineffectiveness of DMDC and Nisin to the oxidase of litchi juice. Moreover, no significant change (P > 0.05) was observed in the value of L∗, a∗, b∗, and △E in the heat-treated litchi juice, and yet the litchi juice treated by DMDC and Nisin gradually turned into light red at the end of storage because of the oxidation of phenolics by residual POD in the litchi juice, which resulted in a significant changes (P < 0.05) in the value of L∗, a∗, b∗, and △E in the litchi juice. This study would provide technical support for commercial application of DMDC combined with Nisin in litchi juice processing.


Introduction
Litchi (Litchi chinensis Sonn.) is a non-climacteric subtropical fruit with high commercial value for sweet and juicy flesh and attractive bright red pericarp.Apart from being consumed freshly, litchi fruit is also processed into juice, canned litchi and dried fruits.Litchi juice is enriched with sugar, minerals, vitamin, and various antioxidants and widely appreciated flavor, and thus it is able to compete in the market of fruit juices (Wu et al., 2007;Zeng et al., 2008;Saxena et al., 2011).But litchi juice is a low-acid food with an approximate pH of 4.5-5.0,so it has a higher risk of microbial contamination than more acidic foods, such as apple juice and orange juice.Some yeast and lactic acid bacteria grow quickly in litchi juice, which can cause spoilage and produce undesirable organoleptic changes in products (Li et al., 2012).Therefore, litchi juices should undergo some type of preservation to ensure product quality and safety.
Thermal processing is the most straightforward means to inactivate the microorganism and enzymes in fruit juices and which are generally at 70-121 °C for 30-120 s (Li et al., 2012).But many juice producers, especially small seasonal operators, are unable to use thermal processing for economic reasons or opposed to thermal processing because of perceived undesirable effects on product quality and acceptability.Therefore, low-temperature alternatives are being sought: particular attention has been given to the use of antimicrobials (Cao et al., 2012;Yu et al., 2013).DMDC (dimethyl dicarbonate), a dicarbonic acid ester, is a powerful antimicrobial agent due to its potential high reaction capacity with nucleophilic groups of enzymes from microorganisms, such as imidazoles, amines, or thiols, and which results in the rapid inactivation of microorganisms (Golden et al., 2005).In 1988, dimethyl dicarbonate (DMDC) (Velcorin) was first approved for use as an inhibitor of yeasts in wine and ready to drink tea beverages; and subsequently in 1996, approved as a yeast inhibitor in spirit drinks and in carbonated or non-carbonated, non-flavored beverages containing added electrolytes, juice sparklers, dilute beverages containing juice, fruit flavor, or both, with juice content not to exceed 50%, at a concentration of 250 mg/L (FDA, 1996;Golden et al., 2005).In addition, as of 2000, there is an effective notification for the use of DMDC as a microbial control agent in noncarbonated juice beverages containing up to and including 100% juice (FDA, 2000).Nisin is a heat-stable antimicrobial peptide produced by certain strains of Lactococcus lactis subsp.lactis (De Vuyst, 1994), and it is the only bacteriocin recognized as safe for the food industry by the World Health Organization (De Arauz et al., 2009;Li et al., 2012).Nisin exhibits antimicrobial activity toward a wide range of Gram-positive bacteria in juices (Li et al., 2012;Zhao et al., 2013).
The efficiency of DMDC against microorganism depends on the strain, initial cell concentration, temperature, pH, and kinds of juice beverages (Fisher et al., 1998;Williams et al., 2005).Some bacteria in juice are very resistant to DMDC, especially Leuconostoc mesenteroides.So individual addition of 250 mg/L DMDC to juice cannot completely kill and control some bacteria of juice, especially the low-acid juice (Yu et al., 2013a).Nisin had been evaluated for its efficacy in enhancing the inactivation of DMDC to bacteria of litchi juice in our previous study, and results showed that the treatment of DMDC combined with nisin offers a useful alternative to conventional heat treatment for controlling microbial growth and significantly extending the shelf-life of litchi juice (Yu et al., 2013a).
The aim of this work was to compare the effects of DMDC combined with nisin, and thermal pasteurization (95 °C /1 min) on the quality attributes of litchi juice during storage of 4 °C.This study would provide technical support for commercial application of DMDC combined with Nisin in litchi juice processing.

Litchi Juice Preparation
Litchi fruit (cv.Fei zi xiao) at 95% maturation was harvested from a commercial orchard in Guangzhou, China.The peeled and destined litchi fruits were processed with a pulper (Midea Co., Guangdong), and passed through filter cloth (100 mesh).And then the pulps were centrifuged at 3000 × g for 5 min, and the supernatant was collected as fresh litchi juice.

Preparation of Nisin Solution
Nisin stock solution (20 000 IU/ml) was prepared by dissolving commercial nisin powder (10 6 IU/g, Zhejiang Silver-Elephant Bio-engineering Co., Zhejiang, China) in a 0.05 M of citric acid solution.Nisin stock solution was sterilized by immersed in boiling water (100 °C) for 5 min, and stored in the refrigerator of 4 °C after cooling (no more than 6 days).

Thermal Pasteurization of Litchi Juice
Fresh litchi juice was thermally processed (95 °C, 1 min) in a tubular heat exchanger (Shanghai pilotech Equipment Co., Ltd., China).After heating, the juice was manually filled under hygienic conditions into aseptic PET bottles, and immediately cooled down to 15 ± 2 °C by a cooled water bath.

DMDC and Nisin Treatment of Litchi Juice
After fresh litchi juice was cooled to 4 ± 1 °C, 100 IU/mL of Nisin and 250 mg/L of DMDC was added to the fresh litchi juice, immediately mixed vigorously, and then the litchi juice above was divided into aseptic PET bottles under hygienic conditions.

Storage and Sampling
The PET bottles were stored in the refrigerator of 4 ± 2 °C, and taken out at a regular interval for further analysis.

Microbial Analysis
Each sample was serially diluted with sterile 0.85% NaCl solution, and then the dilution was used for microbial enumeration by pour plate methods.The viable cells of total aerobic bacteria were enumerated on the Plate Count Agar (PCA, Guangzhou HuanKai Microbiological Technology Co. Ltd., China), and the incubation was performed at 37 °C for 2 days.The Rose Bengal Chloramphenicol Agar (Guangzhou HuanKai Microbiological Technology Co. Ltd., Guangdong, China) was used for detecting the viable cells of yeast and molds, and the incubation was performed at 30 °C for 3 days.The total viable cells of lactic acid bacteria were detected using MRS agar (Guangzhou HuanKai Microbiological Technology Co. Ltd.).The MRS agar plates were incubated at 30 °C for 2 to 3 days.Each test was performed in duplicate and results were expressed as colony-forming units (CFU) per milliliter.Rose bengal agar (Guangzhou HuanKai Microbiological Technology Co. Ltd., China) was used for counting the molds and yeasts after incubating at 28 °C for 72-120 h.

Determination of pH, Titratable Acidity and Total Soluble Solids (TSS)
TSS were determined with a refractometer (Model RP-101, Atago Co. Ltd, Tokyo, Japan) at 20 °C and expressed in degree Brix (°Brix).The pH was determined using a pH meter (Metrohm744, Netherland) with a glasselectrode.Titratable acidity values were obtained by titrating 10 mL of juice with 0.1 mol/L NaOH to pH 8.1, expressing the results as g of citric acid per 100 mL.

Determination of Polyphenol Oxidase (PPO), and Peroxidase (POD) Activity
The activity of PPO was assayed by a spectrophotometric method (Yingsanga et al., 2008) with some modifications.Catechol was chosen as the substrate.The reaction mixture contained 20 μl sample juice and 2.5 ml substrate solution (10 mM catechol in 50 mM phosphate buffer, pH 7.0).The absorbance of the mixture was measured at 420 nm for 3 min at 1 min intervals at 30 °C immediately after incubation in a spectrophotometer (UV-2450, Shimadzu Co., Japan) with a peltier thermostatic cell holder.
The activity of POD was also assayed by a spectrophotometric method (Yingsanga et al., 2008).The reaction mixture contained 50 μl of sample juice and 2.95 ml of substrate solution (0.1 mL 4.0% guaiacol solution, dissolved with 50% ethanol; 0.1 mL 0.46% H 2 O 2 ; 2.75 mL 0.1M phosphate buffer, pH 5.5).The absorbance of the mixture was measured at 470 nm for 5 min at 1 min intervals at 30 °C immediately after incubation in a spectrophotometer (UV-2450, Shimadzu Co., Japan) with a peltier thermostatic cell holder.
One unit of PPO or POD activity was defined as a change in absorbance of 0.001 OD/min of each microlitre of litchi juice.The relative activities of PPO or POD were obtained with the following formula: Specific activity of PPO or POD in the treated litchi juice Residual activity 100% Specific activity of PPO or POD in fresh litchi juice  

Determination of Ascorbic Acid, Total Polyphenols, and Antioxidant Capacity
Ascorbic acid was determined by HPLC method using an Agilent system.The litchi juice (1 mL) was mixed with 1 mL of metaphosphoric acid (6%, v/v) aqueous solution, centrifuged at 10000g (5 min), and then the supernatant was used for further HPLC analysis (Hernandez, Lobo, & Gonzalez, 2006).Ascorbic acid was separated on an Agilent ZORBAX SB-Aq (4.6 × 250 mm) column using 0.02 mol/L (NH 4 ) 2 HPO 4 aqueous solution (pH 2.7) as the mobile phase at a flow rate 1 mL/min and 30 °C.Its content was detected using DAD detector at 254 nm and reported using external standards (L-ascorbic acid).
Total polyphenols were determined using the Folin-Ciocalteu method with some modifications (Singleton et al., 1965).In a 30 mL of test tube, 1mL of litchi juice diluents (litchi juices diluted 20-50 folds with distilled water), and 2 mL of Folin-Ciocalteu reagent was added and mixed.After exactly 1 min, 2 ml of sodium carbonate (10 g/100ml) was added and mixed, and allowed to stand at room temperature for 1 h.The absorbance was read at 760 nm by a spectrophotometer (UV-1800, Shimadzu, Japan), and the total polyphenol concentration was calculated from a calibration curve (R 2 =0.999), using pyrogallic acid as standard (0-50 mg/L).
The antioxidant capacity of litchi juice sample was evaluated by oxygen radical absorbance capacity (ORAC).
ORAC assay was performed according to Ou et al. (2001) using the Infinite M200 microplate reader (Tecan Group Ltd., Switzerland).The 80μL of freshly prepared sodium fluorescein solution (1.25 μM/L in 75 mM/L of phosphate buffer, pH 7.4) and 20 μL litchi juice diluted with water was added into microplate wells for 5 min.
Then, 100 μL of freshly prepared 2, 2'-Azobis (2-methylpropionamidine) dihydrochloride (140 mM/L in 75 mM/L phosphate buffer, pH 7.4) was also added in wells.Fluorescence was collected at 485 nm on excitation at 520 nm on emission, taking measurements every 150 s for 1.5 h at 37 °C.The standard curve was linear between 100 and 500 μM/L Trolox.The result was expressed as mM Trolox equivalent (TE)/L.

Determination of Browning Degree and Color Analysis
The browning degree (BD) of litchi juice was analyzed using a spectrophotometric method (Roig et al., 1999).
Juice was centrifuged with a refrigerated Centrifuge (GL-166-A, Shanghai Anting Scientific Equipment Factory, Shanghai, China) at 10000 rpm/min at 4 °C for 5 min, then passed through a 0.45μm cellulose nitrate membrane (Beijing Bomex Co., Beijing, China).The BD was determined by measuring the absorbance value at 420 nm using a spectrophotometer (UV-726 Shimadzu, Shanghai, China) at room temperature.
Color assessment was conducted using a color measurement spectrophotometer (HunterLab ColorQuest XE, Hunter Associates Laboratory, Inc., Virginia, USA) in the reflectance (transmission) mode.Color was expressed as L* (lightness; 0=black, 100=white), a*(-a*=greenness, +a*=redness) and b* (-b*=blueness, +b*=yellowness).Three measurements were performed, and the results were averaged.These values were then used to calculate the total color difference (ΔE) (Yu et al., 2013b).The calculated equations were as follows, where L* 0 , a* 0 and b* 0 are the control values for fresh litchi juice samples:

Sensory Analysis
Sensory analysis of litchi juice was performed by a semi-trained panel composed by 15 panelists (12 and 39 year of age, equal distribution between male and female), using a 9-point hedonic scale for scoring odor, sweetness, acidity, color and overall acceptability (9 like very much, 1 dislike very much, and 5 as rejection point) (YeH et al., 1998;Walkling-Ribeiro et al., 2009;Lim 2011)

Statistical Analysis
For all kinds of samples, two different batches were considered and analyzed separately.All experiments were conducted in triplicate.Analysis of one-way ANOVA was accomplished with the software SPSS Statistics 19.0 (IBM Co., USA).Duncan's multiple range tests were used to determine statistically significant differences of variables at 95% confidence.

Changes in the Indigenous Microorganism of Litchi Juice Treated With DMDC and Nisin During Storage
After added 250 mg/L of DMDC and 100 IU/mL of Nisin to the fresh litchi juice at 4 °C, the counts of total aerobic bacteria, lactic acid bacteria, yeast and mold in litchi juice linearly declined with increase of time during initial 6 h, and the total aerobic bacteria, lactic acid bacteria, yeast and mold in litchi juice were not detected after 12 h and during 3-month storage, indicating that the juices were microbiologically safe in this study (Table 1).This study focused on the changes in quality of the juices during storage at 4 °C, and the analyses concerning the changes were presented as the following.Moreover, the litchi juice processed by the thermal pasteurization (95 °C, 1 min), which was equivalent effects on the inactivation of microorganisms with the litchi juice treated with 250 mg/L DMDC and 100 IU/mL of Nisin, was as the contrast for quality analysis.N.D.: Colony counts below the detection limit (1 CFU/mL).

Changes of pH, Titratable Acidity and Total Soluble Solids
The pH, titratable acidity and total soluble solids of fresh litchi juice were 4.48 ± 0.08, 0.28 ± 0.03 g of citric acid per 100 mL, and 17.22 ± 0.24 °Brix, respectively.Compared with untreated fresh litchi juice, no significant changes (P > 0.05) of pH, titratable acidity and total soluble solids were observed as treated by DMDC combined with Nisin, and heat (95 °C, 1 min).And also, during storage of 90 d at 4 °C, the pH, titratable acidity and total soluble solids did not show significant changes (P > 0.05) in the litchi juice treated by DMDC combined with Nisin, and heat (95 °C, 1 min), which may related to the inhibition of growth of indigenous microorganism in the litchi juice. www.ccsen

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The POD which was efficiently heat-treate as added 2 Nisin cann showed sig observed a protein (D Figure 1.

Table 1 .
Changes of microbial counts (Lg CFU/mL) in the litchi juice treated with 250 mg/L of DMDC combined with 100 IU/mL of Nisin, and thermal pasteurization (95 °C, 1 min) during storage at 4