Preliminary Study on Ultrasonic Ageing Zhenjiang Vinegar Mechanism Based on Maillard Simulation System

Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China SinoUnison Technology Co. Ltd., Shandong Branch, No. 8 Building, No. 168 Zhuzhou Road Laoshan, Qingdao, Shandong Province, China School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China .e Laboratory Animal Research Center, Jiangsu University, Zhenjiang 212013, China


Introduction
Fermented vinegar has widely been used in China and other Asian countries as a food condiment for hundreds of years [1,2]. Zhenjiang Vinegar, which is one of the most famous traditional fermented vinegar types in China, is world-famous for its special elegant and complex aroma [3]. It is widely accepted like wine because of the harsh taste, pungent smell, and some possible harmful side-effects; fresh vinegar needs a specific process known as ageing in which a specific time period was used to produce high-quality vinegar [4]. Traditionally, ageing vinegar in barrel ageing systems is a common practice used for improving vinegar quality and stability. However, considering the disadvantages of natural ageing such as high production cost and long time period, it is critical and essential to study innovative ageing technology.
Recently, scientists have put great attempts into promoting brand new ageing techniques and enhancing present techniques. Present studies showed that there are diverse physical methods displaying considerable potential for accelerating the ageing process, including ultrasonic wave, electric fields, ultra-high pressure, and gamma irradiation [5][6][7][8][9][10]. It was found that the ultrasound (16-60 kHz) was adept to expedite oxidation, condensation, and polymerization of ethanol, aldehydes, esters, and olefins in wines [11]. Zheng et al. [10] found an optimum treatment for speeding up fresh red wines ageing, which enhanced wines with excellent taste and flavor. Reference [12] found that the high-pressure intervention in the scope from 80 MPa to 120 MPa not beyond 2 hours was shown to considerably improve the wine taste. Better apprehension of each divisor that impacts the quality of vinegar ageing process combined the economic and operational reasons; ultrasonic is a potential physical method to accelerate the vinegar ageing process [13].
Ultrasound, especially with high power and low frequency, has the advantage of pollution-free and being speedy. Over the last decades, abundant works have concentrated on the utilization of ultrasound for food processing, like extraction, freezing, oxidization, sterilisation, and desiccation [14,15]. Evidence from different research [16][17][18] showed that the influence of ultrasound on vinegar ageing processing was possibly associated with the acoustic cavitation [19,20], which includes the generation, expansion, and breakdown of microbubbles. e violent breakdown of bubbles can generate exceedingly steep heat and pressure. Many scientists have studied that during ageing processing, the vinegar undergoes many complicated changes, including Maillard reaction, oxidation reaction, and esterification reaction. Pripis-Nicolau et al. [21] revealed that, under the appropriate wine conditions, the production of odoriferous compositions or strong-smelling by-products would enhance Strecker and Maillard reactions. Casale et al. Reference [22] found that the reaction of oxidization occurs and oxygen boosts a series of chemical and enzymatic reactions that change the vinegar; thus they found an affinity between spectral variables and store time-changes in vinegar. Maillard reaction is defined as the interaction of carbonyl compounds (e.g., reducing sugars) with free amino groups (usually amino acid) leading to flavor compounds and melanoidins [23]. Maillard reaction is of great importance in food quality, especially in heat-process foods, affecting not only the flavor but also the color and nutritional values. Previous work [9] in our laboratory showed that vinegar treated with ultrasound was concluded to be tantamount to 2-3 years aged traditionally Zhenjiang vinegar under the optimum experimental conditions (ultrasonic power 50 W/100 mL, time 75 min, and ethanol addition 0.75% (V/V)). Nevertheless, little work has been done with Zhenjiang vinegar, especially the mechanism of the chemical reactions during the ageing process.
In view of the wide variety of substance in vinegar, Maillard reactions develop complex intermediates and final reaction products, in order to clarify each substance in the ultrasound induced by the specific changes and the mechanisms is very difficult or even almost impossible. erefore, the complex vinegar system was replaced for the simulation system [21,24,25], to study the mechanism of ultrasound to accelerate the ageing process. us, the aim of this study was to investigate the relevant physicochemical indicators and the ageing mechanism based on the Zhenjiang vinegar simulation system. is study is of practicable interest since it provides beneficial information for vinegar industry in order to optimize the process of vinegar.

Chemicals.
Chemicals used in this study were furfural, n-Octane, fructose, glycine, methylene blue, vanillin, anhydrous ethanol, and aniline which were purchased from Sinopharm Group Chemical Co., Ltd. In this study, all chemicals and solvents used were of analytical grade.

Samples.
e simulated Maillard reaction system was constructed to be 100 mL of 0.1 mol/L fructose and glycine aqueous solution. All the vinegar samples of different storing times used in this study were collected from Jiangsu Hengshun Vinegar Co., Ltd. Samples of vinegar were divided into 2 kinds of different treatments. One is the naturally aged vinegar samples, which were aged traditionally for different periods such as 12, 18, 32, and 44 months and kept in glasses. e samples of fresh and aged vinegar were stored at 4°C in a refrigerator. Samples with ultrasonic were treated according to the method of Wang et al. [9].

Color Evaluation.
Color is one significant sensory characteristic of vinegar, which affects the consumers' overall acceptableness. e change of color can well reflect the process of Maillard reaction. Color measurement is measured by Hunterlab Spectrophotometer. e L * , a * , and b * values (CIELAB parameters) were monitored by using the software CromaLab [26], which better test the color of wines and permit better differentiation [27][28][29][30]. In this study, the CIE Lab system was used to define the color of vinegar. Record the color difference value of the vinegar L * , a * , and b * , measured three times, and take the average. Value of L * indicates the brightness, the value of 0-100; the greater the value the greater the brightness; a * represents the values of red/green, positive partial red, and negative partial green; b * represents the yellow/blue value, positive yellow, and negative blue. e value ΔE * is measured as the Euclidean distance between two points in the three-dimensional space defined by L * , a * , and b * and tells from color differences: (1)

Determination of Reducing
Sugar. e content of reducing sugar in the samples was analyzed as stated in the GB/T 5009.7-2016 method including different ages of traditional aged vinegar and optimum ultrasonic experimental conditions treated vinegar (ultrasonic power 50 W/100 mL, time 75 min, and ethanol addition 0.75% (V/V)). All vinegar samples were determined in triplicate.

Determination of Free Amino
Acid. Automatic amino acid analyzer was exploited to measure the content of free amino acids in the vinegar samples. e vinegar sample to be tested was diluted 20 times with 1% sulfosalicylic acid solution, centrifuged at 10000 rpm for 15 min, and the supernatant was clarified through 0.22 μmmicroporous membrane filter for the determination of the use of the machine.

Determination of Hydroxyl Radicals.
e hydroxyl radical evoked by the cavitation of ultrasound was assessed indirectly by using methylene blue as the radical scavenger and ultraviolet-visible spectrophotometer as the testing method. [31,32] e methylene blue was handled with dissimilar ultrasonic treatments and the maximum absorption wavelength was measured by UV-visible spectrophotometer full-wavelength scanning. e hydroxyl radical produced by ultrasound was also measured by the decrease of absorbance at the maximum absorption wavelength.

Determination of Superoxide Radicals.
Vanillin aniline fluorescent reagent synthesis method: 1.5 g vanillin was dissolved in 20 mL of anhydrous ethanol; 0.9 g of aniline was added dropwise, refluxed at 80°C, for 5 h; most of the solvent was distilled off; and a pale yellow solid was obtained after cooling. Recrystallize from anhydrous ethanol and vacuum dry. 2 × 10 −4 mol/L vanillin aniline ethanol solution was configured.
In a 10 mL colorimetric tube, add 2 mL of vanillin aniline ethanol solution, 2 mL of sonicated aqueous solution, and 1.5 mL of Tris-HCL buffer solution and bring the volume to the mark. e fluorescence intensity was measured under the excitation wavelength of 282 nm, and the emission wavelength range was 250-400 nm. Excitation and emission slit widths were measured at 5 nm and 10 nm, respectively.

Determination of Maillard Reaction Process.
In view of a large variety of components in vinegar, it is difficult or even impossible to find out the specific changes and mechanisms of each substance under ultrasonic induction; therefore, the content of vinegar should be higher when studying the ultrasonic accelerated vinegar ageing mechanism. e representative substances that have an important influence on the quality of vinegar were studied as objects. e results of this study are also easy to compare with the relatively clear mechanism of change in the natural maturation process of these substances. e Maillard reaction is one of the most important reactions in the vinegar ageing process. erefore, it is necessary to construct the Maillard simulation system to study the mechanism of ultrasound on its action. Maillard reaction model system is the 100 mL of 0.1 mol/ L fructose and glycine solution. e degree of browning and the amount of intermediate product of the Maillard reaction were measured according to the way of Gu et al. [33]. According to the literature, absorbance at 294 nm was served to examine the formation of the intermediate compounds of the Maillard reaction and the size of the UV-visible absorption at 420 nm in the Maillard reaction system is related to the final stages of the reactions [34,35]. e browning indices were evaluated by spectrophotometry, recording the absorbance at 420 nm and 294 nm (A 420 nm and A 294 nm ) of each model system against distilled water. e results were recorded on a UV software (Beijing Beifen-Ruili) spectrophotometer using a 1 cm path length quartz cell. All samples were done three times in parallel.
2.9. Data Analysis. All model systems were prepared in duplicate and the analysis was performed in triplicate. Excel 2010, Origin 8.0, and SPSS Statistics 17.0 were applied to complete the data analysis. e significant differences between the different samples were obtained through a oneway ANOVA analysis with a level of P < 0.05. Table 1 shows, the color of the vinegar was significantly affected by the ageing time and different storage times have a significant effect on vinegar L * , a * , b * . For the traditionally natural aged vinegar samples with the extension of storage time, the values showed a downward trend. Lightness is the attribute of a visual sensation according to which a given visual stimulus appears to be more or less light, ranging from "light" to "dark" Gómez-m Guez [26].  [36,37], these minute dissimilarities between the vinegar samples were by reason to the oxidation instead of the enzymatic treatment. However, the previous literature [38,39] reported that the ultrasound can accelerate oxidation, esterification, and other reactions. e values of the ultrasonic treated vinegar (L * � 2.41, a * � 6.37, b * � 5.25) were higher as compared to nonultrasonic treated vinegar. e value of ΔE * was found between the ultrasonic treated vinegar and fresh vinegar (ΔE * � 0.94). ese colorimetric data present that the ultrasonic treated vinegar samples exhibited mainly red and yellow tint, which is also a consumer favor. e result shows that a tester can distinguish the color of these vinegar samples. In addition, the reducing sugar in vinegar declining with the extension of ageing time, combined with the later experimental verification Maillard reaction products, increased after ultrasonic treatment, which is consistent with the results of the color change of vinegar treatment. Figures 1 and  2 show the methylene blue solution UV-visible absorption curve along with the change of ultrasonic time and power. Besides, these results showed that methylene blue solution has the maximum absorption peak at 664 nm and gradually decreases with the increase of ultrasonic time and power. ese results show that the concentration of methylene blue solution gradually decreased to prove that the ultrasonic treatment of hydroxyl radicals generated and hydroxyl radical concentration gradually increased over ultrasonic time and power.

Effect of Ultrasound on Hydroxyl Radicals.
As shown in Figure 3, it is not hard to see that in the initial period of time, the production of hydroxyl radical increased rapidly. As the time extended to 75 min later, the rate of increase slowed down. e reason here might be that the increased time for ultrasonic treatment increases the vinegar temperature too high. e vapor pressure in bubbles increases and consequently bubbles closed to enhance the buffer effect and the cavitation role becomes weakened. erefore, in this study, the ultrasonic time is 75 min. As shown in Figure 4, with the increase of ultrasonic power, the intensity of the ultrasonic wave generated in unit Journal of Food Quality time increases, and the cavitation effect produced is stronger [40]. erefore, when the cavitation bubbles generated by the aqueous solution are crushed, high temperature and high-pressure environment occurs; the number of free radicals generated will be increased sufficiently.
Hydroxyl radical is the most active oxygen free radical [41]. Ultrasound-induced hydroxyl radicals can cause oxidation of sugars, amino acids, proteins, and esters in vinegar [42]. e production of free radicals can speed up the breakage and formation of various substances in vinegar, accelerate the ripening of vinegar, and shorten the ageing time [43]. Figures 5 and 6, it can be seen that superoxide radicals in the solution that has not been sonicated are not detected; however, a large number of free radicals are generated in the aqueous solution after sonication, which proves that the ultrasonic treatment produces superoxide radicals in the solution and has a significant effect. According to Figure 5, the number of superoxide radicals generated increases with the increase of the ultrasound time and reaches the maximum level at 40 min after sonication. Figure 6 shows that the number of superoxide radicals produced by ultrasound gradually decreases with the increase of ultrasonic power of 10 W/100 mL. e generation of superoxide radicals is due to the action of ultrasound on the oxygen molecules in the aqueous solution. e reason for the above research results is that, on the one hand, it is finally converted into hydroxyl radicals through a series of reactions which is consistent with the results of the previous studies on hydroxyl radicals and, on the other hand, superoxide anions can dismutate to produce hydrogen peroxide and oxygen [44].

Effect of Ultrasound on Superoxide Radica. From
Superoxide radicals in water can be considered as a base, which can accept H + from a protonated superoxide radical HOO•. In one way, the mechanism is largely to convert it into other more active oxygen ions. e base achieves the promotion of chemical reaction.

Effect of Ultrasound on Browning Degree of Maillard
Reaction. Table 1 shows that after the ultrasonic treatment, the color of the vinegar becomes lighter. e content of  reducing sugar decreased by nearly 50% from 4.06 g/100 mL to 2.24 g/100 mL. Besides, during the traditionally natural ageing process, the changes of vinegar free amino acids are shown in Table 2. It is revealed that as the ageing time becomes longer, the content of free amino acids decreased by 1041.35 mg/100 mL down to 757.87 mg/100 mL. Moreover, after ultrasonic treatment, the content of amino acids in vinegar decreased, consistent with the trend of natural ageing. And ultrasound can produce lots of hydroxyl radicals. Combined with the above experimental results analysis, we speculate that the changes are related to the progress of the Maillard reaction. e monosaccharides in Zhenjiang vinegar is mainly glucose and fructose. And glycine in the vinegar free amino acid mass fraction is relatively high. In particular, fructose-glycine can form a kind of Amadori compounds [45,46]. erefore, in this experiment, the fructose-glycine simulation system was used to verify that ultrasound may speed up the Maillard reaction. As far as we know, the melanoidin produced during the Maillard reaction can make the color of the vinegar darker, so that the degree of browning can be directed to the extent of the Maillard reaction.
As can be seen from Figure 7, the degree of browning of the fructose-glycine simulation system gradually increased with the ultrasonic time rise. And the results showed that    the degree of browning increased slowly after 60 min of sonication. In Figure 8, with the ultrasonic power rise, the degree of browning of the simulated system showed a tendency to increase first and then decrease and reached the maximum when the ultrasonic power density was 50 W/100 mL. is result is consistent with our previous experimental conclusions [9]. e apparent increase in the absorbance indicates that the midterm product of the Maillard reaction is accumulating. is difference could be related to the acoustic cavitation. Since this cavitation activity can be viewed as a dramatic concentration of acoustic energy resulting in localized high stresses, temperatures, and/or fluid velocities, its biological consequences should be understood by those who are trying to either optimize or minimize its effects [47]. Additionally, the violent collapse of bubbles can generate exceedingly steep heat and pressure, which can produce free radicals and so forth [48], and then small molecules can rearrange [4]. e results show that the ultrasonic wave can strengthen the fructose-glycine Maillard reaction, promote the system browning, and induce the middle stage and the formation of advanced stage products.

Effect of Ultrasound on the Amount of the Intermediate Products in Maillard Reaction.
In the Maillard reaction process, there are some noncolor intermediates, such as small molecules of ketones and aldehydes, which are also important indicators of the Maillard reaction [34]. As can be seen from Figures 9 and 10, without ultrasonic processing, the intermediate product of the Maillard reaction has not been detected in the simulation system solution. Applying the ultrasonic vinegar sample, however, the intermediate product of the reaction significantly altered. Results (Figures 9 and 10) showed that accompanied with the ultrasonic time and ultrasonic power rise, the median product of the Maillard reaction increased gradually, reaching the maximum at 75 min and 50 W/100 mL, respectively. e change of Maillard intermediates may largely be due to the fact that ultrasound can induce some oxygen ions and hydroxyl radicals in the aqueous solution [49], and the production and rearrangement of these oxygen ions and hydroxyl radicals can promote the Maillard reaction of the fructoseglycine simulation system, leading to the production of intermediates. e degree of browning and the number of intermediate products in the ultrasonic treatment Journal of Food Quality simulation system can be used to prove the ultrasonic catalytic Maillard reaction.

Conclusion
In this study, under the conditions of ultrasonic processing, the changes of Zhenjiang vinegar physiochemical index, such as color, reducing sugar, and amino acid, are consistent with those of natural ageing. In addition, the sonicated vinegar produces hydroxyl radicals, which generally appear to increase with increasing ultrasound time and ultrasound power. e present study reveals that the mechanism of ultrasonic ageing lies in the cavitation of ultrasound to induce the generation of radicals, such as hydroxyl radicals and superoxide radicals, catalyze the oxidation reaction in vinegar, and verify it by the fructose-glycine Maillard simulation system. e results showed that ultrasonic treatment had positive impacts on the vinegar ageing process. For the future, much research is needed to determine the exact role of ultrasound in vinegar ageing.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
All authors declare no conflicts of interest.