基于化学分析和智能感官方法对不同产地川醋特征风味物质的比较分析

乔明锋，蔡雪梅，魏 宇,2，易宇文，刘 阳，邓 静，吴华昌,

（1.烹饪科学四川省高等学校重点实验室，四川旅游学院，四川 成都 610100；2.数字媒体艺术四川省重点实验室，四川音乐学院，四川 成都 610041）

Vinegar is consumed worldwide not only as a condiment, but also as ingredient and a preservation product for a wide range of foods, especially in the Chinese diet, which is produced from alcoholic and subsequent acetic fermentation of any cereals or fruits rich in carbohydrates, containing approximately 5%～20% of acetic acid[1−2]. Chinese vinegar has a history of more than 3000 years, with a strong regional characteristic, for example, Shanxi Province is famous for mature vinegars and Jiangsu Province for aromatic vinegars, and a large amount of vinegar is produced and consumed every year in China[3−4].Due to the diversity commercially available of vinegar types on the market and the increase on demand, the quality assessment of vinegars has been more and more attracting the attention of people and it has been considered necessary to develop reliable analytical methodologies to establish criteria for determining quality and origin.

As known, the quality of food is the result of a harmonious equilibrium among parameters having a different origin: Chemical, physical, biological, and organoleptic. Flavor is the most important parameters in the assessment of vinegar character and quality,which depends on the raw materials (rice, sticky rice,wheat bran, sugar, salt, and so on), the constituents formed during the fermentation, bacteria and environmental climate, especially in natural fermentation[5]. Traditionally, sensory and instrumental techniques are used to describe the flavor of vinegar[6−7]. However, sensory analysis requires panels of trained technicians, and low-cost experimental methods such as destructive mechanical tests and other conventional physicochemical measurements are specific for a particular quality index,instead of providing comprehensive quality information[8]. On the other hand, high-end instrumental techniques such as high performance liquid chromatography (HPLC), gas chromatography with mass spectrometry (GC-MS), could provide information on the chemical compositions of the sample[9−10].Nevertheless, these analytical techniques often require laborious and time-consuming sample preparation, as well as skilled personnel to operate the equipment and interpret the analytical results[11−12].

There were many researches about determination and analysis of components in vinegars. The main organic components of vinegar were identified and quantified by HPLC and high resolution1H NMR spectroscopy[13−14]. Many analytical methods had been used to characterize the aroma profiles and the aromaactive compounds in vinegar samples, such as supercritical fluid extraction (SFE), solvent-assisted,flavor evaporation (SAFE), headspace stir bar sorptive extraction (HSSE) and solid-phase micro-extraction(SPME), the aroma extract dilution assay (AEDA),gas chromatography-olfactometry (GC-O)[15−20].Moreover, sensory analysis and various chemical analysis methods, intelligent sensory evaluation instruments are employed to discriminate many different kinds of food, such as electronic nose (enose) and electronic tongue (e-tongue), which offer a fast, comprehensive, and easy-to-handle alternative to assess food quality[21−22]. Compared to the information from a single sensory organ, simultaneous application of e-noses and e-tongues may increase the amount of information extracted from a sample, which will be a very promising technique for characterizing vinegar.However, according to the knowledge, few studies have been reported about the flavor characterization of Chinese vinegars using e-nose, e-tongue, HPLC,SPME-GC-MS and other analytical instruments simultaneously.

In this work, e-nose, e-tongue, HPLC, solidphase microextraction, and gas chromatography with mass spectrometry (SPME-GC-MS), automatic amino acid analyzer and other analytical instruments were simultaneously applied to identify and quantify the flavor quality of four Chinese vinegars in Sichuan,combined with multivariate analysis tools, such as PCA and cluster analysis (CA), providing method reference for qualitative and quantitative comprehensive analysis of the quality of vinegar.

1 Materials and methods

1.1 Materials and instruments

Four famous commercial Chinese vinegars Sichuan Province, known as the “land of plenty” and the origin for a rich and diverse cuisine, were purchased from a local market in Chengdu, China,and they were all produced by solid state fermentation for three years. Table 1 lists the name, raw materials,and production area which were copied from the vinegar bottle labels; 9 organic acids: Oxalic acid,tartaric acid, pyruvic acid, malic acid, lactic acid,acetic acid, citric acid, succinic acid and n-propionic acid Aladdin Biochemical Technology Co., Ltd.;All the other chemicals used were at least of analytical grade.

Table 1 The details of the vinegar samples utilized in the experiment

DC-P3 Automatic Chromatic Meter Xingguang Instruments Co., Ltd., China; PHSJ-3F pH Meter Shanghaileici Instruments Co., Ltd., China; L-8900 High-Speed Amino Acid Analyzer Hitachi Instruments Co., Ltd., Japan; Waters 2695 High Performance Liquid Chromatography Waters & Milford Instruments Co., Ltd., USA;57330U Supelco Solid Phase Microextraction Sampler (with Manual Injection Handle), 57318 CAR/PDMS Fibre (75 mm)Supelco Inc., USA; SQ680 Gas Chromatography and Mass Spectrometer PerkinElmer Inc., USA; FOX 4000 Electronic,α-Astree Electronic Tongue Alpha Nose MOS, France; Other commonly used laboratory equipment.

1.2 Experimental methods

1.2.1 Sample pretreatment All sample vinegars were kept in a refrigerator at 4 ℃.

1.2.2 Analysis of physico-chemical properties Total acid: 10 mL of vinegar was mixed with 20 mL of distilled water and the mixture was titrated up to pH8.2 by using 0.1 mol/L NaOH. Total acid was expressed as acetic acid equivalent[23](titratable acid by titration with 0.1 mol/L NaOH to phenolphthalein end point).

Total sugar: The total sugar content was done by anthrone-sulfuric acid colorimetry method according to the method of CHEN et al[24].

pH measurement: The sample pH was measured using a pH meter acoording to GB 5009.237-2016National standard for food safety determination of pH value of food.

Color measurement: Surface color was measured with an automatic chromatic meter and prior to analysis, the instrument calibrated against a standard white reference tile (GSB A67002-86). The color results were expressed in terms of CIELABscale parametersL*,a*andb*representing lightness, redgreen and yellow-blue, respectively. All analyses were carried out in triplicate.

1.2.3 Amino acids analysis The free amino acid(FAA) contents were analyzed using an L-8900 High-Speed Amino Acid Analyzer according to GONG et al[25], with a modification. Briefly，after centrifuging at

10000 r/min for 10 min，all the centrifuged samples of were deproteinized with 10% trichloroacetic acid, and then all of free amino acids were quantitated using Ophthalaldehyde (OPA)/9-fluorenylmethyl chloroformate (FMOC) derivatives with an L-8900 High-Speed Amino Acid Analyzer according to the previous description.

1.2.4 Organic acids analysis HPLC Sample：All vinegar samples were centrifuged at 10000 r/min for 10 min , and then were filtered through a 0.22 μm membrane prior to HPLC analysis[26].

The reference value of 9 organic acids (oxalic acid, tartaric acid, pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid and npropionic acid) was determined by HPLC according to the method of LU, et al[27]. Briefly, appropriate amount of each standard product was weighed and dissolved in water to prepare standard reserve solution.The concentrations were 2.00 mg/mL (lacticacid) ,2.00 mg/mL (n-propionic acid), 2.20 mg/mL (citric acid), 2.00 mg/mL (acetic acid), 2.00 mg/mL (malic acid), 2.00 mg/mL (tartaric acid), 0.85 mg/mL(pyruvate), 0.80 mg/mL (oxalic acid) and 4.00 mg/mL(succinic acid). Stored in a refrigerator at 4 ℃, when used, the standard mixed working solution with appropriate concentration was diluted step by step with the mobile phase. A mixture of different concentrations of standard solution is used to make a standard curve for the determination of actual samples.

The HPLC conditions were as follows: The instrument was equipped with a 717+ automatic injector with 20 μL once, and a 2996 Photodiode Array detector (PDA) UV at 210 nm. Separation was achieved using an Agilent Zorbax SB-C18column(5 μm, 4.6 mm×250 mm) at 25 ℃.

The mobile phase was 20 mmol/L NaH2PO4(0.01 mol/L potassium dihydrogen phosphate solution was used with pH of 2.60), with a flow rate of 1.00 mL/min. The running time was set as 8 min. The settings were based on several trials and previous similar studies[28]. All organic acids were recorded on a computer-based data system with standard substance. Each organic acid was quantified by the calibration curve of the authentic standards.

1.2.5 SPME-GC-MS analysis The SPME conditions: Added 2 mL vinegar sample each time and put it into 15 mL sample bottle. After that, 2.5 g sodium chloride was added, the cap was tightened, and placed in a constant temperature water bath at 50 ℃ for balancing for 10 min. The fiber head was pushed out about 1.5 cm away from the liquid level, and the headspace adsorption lasted for 40 min. The stirring speed was 250 r/min. After the extraction operation was completed, the fiber head should be rotated first,then the extraction head should be slowly removed,and then the extraction head should be carefully placed into the sampling port of the gas chromatography equipment, and the fiber head should be pushed out to the specified length for desorption, and the desorption should be completed within 5 min[29].

The GC conditions were as follows: Chromatographic column: Elite-5 MS (30 m×0.25 mm×0.25 μm); Inlet temperature: 250 ℃; Heating procedure: Hold at 40 ℃ for 3 min, rise to 140 ℃ at 6 ℃/min,hold for 4 min, rise to 250 ℃ at 20 ℃/min, hold for 2 min; Carrier helium (99.999%), flow rate 1 mL/min,split ratio was 5:1.

The MS conditions were as follows: Electron ionization with ion source, 70 eV of electron energy,full-scan scanning mode, a scanning mass (m/z) range of 35～400 amu, the scanning delay was 1.1 min; ion source temperatures of 200 ℃ and transmission line temperatures of 250 ℃.

Identifications were achieved by matching both the retention indices and the mass spectra of reference standards. For the compounds with which no reference standard was available, tentative identification was made through comparing the mass spectra with those of the known compounds from the standard NIST 2011 library and comparing the retention indices sourced from NIST Standard Reference Database.

1.2.6 E-nose analysis Flavor analysis was performed with a Fox 4000 electronic nose with three metal oxide sensors chambers equipped with 18 sensors(LY2/AA, LY2/G, LY2/g CT, LY2/g CTl, LY2/Gh,LY2/LG, P10/1, P10/2, P30/1,P30/2, P40/1, P40/2,PA2,T30/1, T40/2, T70/2, T40/1, TA2) , and the carrier gas was TOC grade synthetic air (P=5 psi).Prior to detection, each sample (5 g) was placed in a 10 mL airtight glass vial for 5 min at 50 ℃(headspace-generation time and temperature), and the measurement phase lasted for 120 s, which was long enough for the sensors to reach stable signal values,and after each measurement, zero gas (air filtered by active carbon) was pumped into the sample gas path from the other port of the instrument for 180 s (flush time). The signal data from the sensors were collected by the computer once per second during the measurements, and when the measurement process completed, the acquired data were stored. Each test was repeated 5 times.

1.2.7 E-tongue analysis Taste analysis was performed withα-Astree e-tongue consisting of an array of seven liquid cross-sensitive electrodes or sensors named ZZ, BA, BB, CA, GA, HA, and JB,respectively, a 16-position autosampler, and associated interface electronic module. 80 mL of each sample was injected into a 120 mL beaker for detection. The measuring time was set to 120 s for each sample, and the sensors were rinsed for 10 s using ultrapure water to reach stable potential readings before detecting the next sample. Four replicated measurements were run on each sample. The first three measurement cycles were discarded due to instability, and only the fourth stable sensor responses were obtained to be the original data from the sample.

1.2.8 Sensory evaluation Table 2 was a reference brewing vinegar (GB/T 18187-2000 Brewed vinegar)of the vinegar on the sensory index of the vinegar sensory scoring criteria, with modification. Briefly,sensory evaluation of the vinegar was conducted by a panel of 5 judges with rich experiences on vinegar descriptive analysis. A total of 9 descriptors of appearance (color and clarity), aroma (ester, burnt and vinegar), taste/flavor (sweet, sour and umami) and mouth-feel (astringency) were selected by the panel.Intensity ratings were scored on a scale from 0 to 10.

1.3 Statistical analyses

All tests were conducted according to a completely randomized design and the experiments were performed at least in three replicates. Analysis of variance (ANOVA) was used to search for significant differences between mean values of the different results, and the results were presented as means±SD. Furthermore, principal component analysis(PCA) and cluster analysis (CA) were carried out with the aim of highlighting the main contributors to the variance amongst samples. Statistical analyzes were carried out using the software Statistical Package for the Social Sciences (SPSS), version 18.0 (SPSS Inc.,Chicago, Illinois, USA) and Origin 9.1 (Origin Lab Corporation, Northampton, MA, USA).

2 Results and discussion

2.1 Physicochemical properties

Table 3 showed physicochemical properties of the four vinegar samples, and a wide variability was observed in the values, indicating remarkable differences (P<0.05) in vinegar qualities. In this study, total sugar values of the vinegars varied in a wide range (from 0.40 to 0.92 g/100 g), representing a high variability. Compared with Shanxi aged vinegar,the total acid of fresh vinegar could reach 0.76 g/100 g,and that of eight-years vinegar could even reach 5.72 g/100 g[26]. Therefore, the main reasons for the change of total acid content may be raw materials,fermentation microorganisms and storage time[31].

In addition, total acid levels of the vinegar samples were generally correlated with their pH,varying from 16.40% to 18.07%, in conformity with Codex Alimentarius Commission (CAC). The pH of the vinegar samples varied from 3.08 to 3.29, in accordance with the previous studies[4]. In general,other traditional vinegars had higher pH, for example,Shanxi aged vinegar from 3.67 to 3.88[26]and that of Zhenjiang vinegar from 3.35 to 3.97[32]. Color was an important factor affecting consumer perception when buying vinegar, and color properties (L*,a*andb*) of the vinegar samples were shown in Table 3, too.L*,indicating the brightness, ranged from −12.08 to−15.51. Botha*andb*of the samples were almost variable among the samples, indicating that color properties of the vinegar were very different from each other.

Table 2 Sensory evaluation rules of the vinegar

2.2 Amino acids and organic acids assay

In addition to acetic acid, vinegar is also rich in organic acids, amino acids, mineral substances, and so on, playing a role on its organoleptic properties. Table 4 showed amino acids contents of the vinegar samples.In general, Ala, Glu, Ser, Gly, Val, Leu, Pro and Lys were the most abundant amino acids present in the vinegars. While, Cys and His, of which quantities were always lower than 20.00 mg/100 mL, were the amino acids with the lowest amounts among the other amino acids tested. It attracted attention grabbing that the sample 1 was the richest one in terms of the amounts of a group of amino acids, namely Asp, Gly,Ala, Val, Lys, Arg, and Pro. In addition, it could note that the content of Glu, Lys, Ala, Val, Met and Leu in the four samples exceeded the known sensory threshold, which would give the vinegar umami,sweet, bitter and other tastes. The contents of total AA and essential AA (ranged from 969.9 mg/100 mL to 2126.35 mg/100 mL, from 412.43 mg/100 mL to 805.53 mg/100 mL, respectively) in four samples of Sichuan vinegar were significantly higher than sample in Zhenjiang vinegar (ranged from 871.04 to 1082.26 mg/100 mL, from 39.38 to 40.65 mg/100 mL,respectively)[30]. Many amino acids are the intermediates of some volatile compounds that can influence vinegar quality, while others such as L-arginine and L-histidine are precursors of harmful substances such as ethyl carbamate and histamine, respectively.

Table 3 Physicochemical properties of four Chinese vinegars in Sichuan

Table 4 The amino acids content of four Chinese vinegars in Sichuan (mg/100 mL)

Organic acids are vital compounds of vinegars because they greatly contribute to sensory quality and functional activity, and the content of organic acids is variable and depends on several factors, mainly raw materials, techniques of processing and microbiological growth. The organic acids content of samples were determined by HPLC as the same method of 9 authentic standards (Fig.1), and the statistical values were shown in Table 5. First, it was evident that almost all of the measured organic acids were significantly above known sensory thresholds.Different organic acids had different flavor descriptions, which could prove that Sichuan vinegar had rich flavor levels. In vinegars, the most abundant organic acids were acetic acid and lactic acid, which aggregately accounted for more than 50% of the total acids. Additionally, tartaric acid, malic acid, citric acid and propionic acid were major organic acids in the vinegars samples, comprising 28～49 g/L of the total quantified acids. The total organic acid content in different samples of vinegar was very different.Quantities of total organic acids of the vinegars ranged from 22.44 mg/mL to 70.06 mg/mL, and it was attention grabbing that the sample1 was the richest one in terms of the amounts of a group of all organic acids. In fact, even the same place of vinegar,organic acid content would be very different. For example, in 16 samples of Chishui vinegar, the content of total organic acids varied greatly from 34.69 mg/mL to 108.03 mg/mL[28].

Table 5 The organic acids content ,flavor profile and flavor threshold of four Chinese vinegars in Sichuan (mg/mL)

Fig.1 HPLC chromatogram of different organicacids standard solution

2.3 Volatile organic compounds (VOCs) assay

Aroma is important quality criteria for vinegar samples, and the volatile organic compounds (VOCs)identification responsible for their aroma is considered to be a key factor for quality and authentication control. These compounds were grouped according to their chemical structure as esters, alcohols, acids,aldehydes, ketones, heterocyclic, benzodiazepines and others. For all the compounds detected, the mean relative areas percentages and the number of the volatiles in each class of different vinegar samples were demonstrated in Fig.2.

Fig.2 Substance classification statistics of flavor counpounds（a） and relative content（b） in vinegar samples

In total, more than 150 VOCs were isolated and identified in the four commercial Chinese vinegars in Sichuan. And most of these compounds have been found in vinegars previously[20,25]. Table 6 showed the identification results of 26 main volatile flavor components in four kinds of vinegar by SPME-GCMS.

Table 6 The relative content of volatile flavor compounds (>1.00%) in sample vinegar

Acids and esters still had the most abundant volatile flavor compounds in the four samples,accounting for 21.56%～44.34% and 4.64%～29.47%,respectively. In terms of specific volatile compounds,glacial acetic acid (19.65%～38.00%), 2-methyl-3-heptanone (4.18%～8.98%), 2-pentadecyl-1,3-dioxepane (1.44%～9.35%), the three compounds, had high relative content and were all contained in the four samples of vinegar, which may be the important components of aromatic substances in Sichuan vinegar.

In terms of the unique volatile flavors that make up the samples, butanedioic acid, monopropargyl ester (5.38%), 2,4,5-trimethyl-1,3-oxazole (1.01%) in sample1 were unique in terms of their unique volatile flavors. And the relative content was more than 1.00%, it was very likely to constitute the unique flavor of aromatic substances. Similarly, four compounds in Sample2 (3,5-dimethyl-1H-Pyrazole-1-methanol, 3-chloro-2, 2-dimethyl-Propanoic acid, 3-methyl-4-oxo-Pentanoic acid, 1,2,3,4-tetramethylbenzene) and Sample3 contained isobutylbenzene(9.4%), isoamyl acetate (4.73%), 3-methylbutyl 2-hydroxypropanoate (3.83%), (R,R)-2,3-butanediol(1.69%) were likely to be unique aromatic substances.

2.4 E-nose and e-tongue assay

The characterization and discrimination of food have been widely investigated with chemometric tools, such as principal component analysis (PCA)and cluster analysis (CA)[34]. The PCA results of the vinegars data obtained from e-nose and e-tongue were shown in Fig.3 (b) and Fig.4 (b) .The first two principal components were kept because they totaled more than 98% of the variance in the data set (PC1 and PC2 accounted for 99.933 and 98.155 of e-nose and e-tongue, respectively). The PCA results were redrawn in two or three groups, and it was evident that sample 1 and 2 obviously differed from the other two vinegars detected by e-nose and e-tongue.

Fig.3 CA （a） and PCA （b） results of the vinegars data obtained from e-nose

As mentioned above, vinegars contained a large amount of components, such as esters, alcohols,aldehydes, phenol, glucose and aminophenol, though main ingredients were water and acetic acid, and all the components contributed to smell, taste and the responses of the e-nose and e-tongue. Traditionally,Chinese vinegars had a strong regional characteristic.Although the production areas of the four vinegars samples were in the same province, good separation could still be seen by CA in Fig.3 (a) and Fig.4 (a),although there were more or less overlaps.

It noticed that there was a slight discrepancy between the e-nose and e-tongue in the dendrogram,the difference was mainly due to the differences in the samples of different Sichuan vinegars. From Fig.3 (a),it was known that the difference in the CA analysis of the e-nose was mainly due to the great difference between sample1 and sample 3. Through the analysis of Fig.3 (a) and Fig.5, it could be known that it may be related to the astringent in the sensory evaluation and the number of ketones, heterocycli, and other in VOCs. Similarly, as shown in Fig.4(a), the difference of electronic tongue CA analysis mainly lied in the great difference between sample 1 and sample 3 and sample 4, which may related to the pH of the sample,total amino acids, essential amino acids, organic acid,and total acid.

Fig.5 Results of sensory evaluation for 4 vinegar samples

2.5 Results and analysis of sensory evaluation

A quantitative description analysis was carried on the indicators of vinegar based on Table 2, in order to express the sensory results of all vinegar groups more clearly. Based on the sensory quantitative analysis results of the 4 samples, a radar graph was made, as shown in Fig.5.

As shown in Fig.5, the color score of the four sample vinegars showed a correlation withL*. The smallerL*, the higher the color score. It could be that the sensory color score was related to the brightness of the vinegar color. Up to a point, the darker the vinegar, the higher the score. Burnt score of vinegar were basically similar, but there were distinct differences in tastes (sweet, sour, umami) and aroma(vinegar and ester). Sweet score were as follows:Sample 1 (9.80±0.45)>sample 2 (8.20±0.55)>sample 3(7.6±0.45)>sample 4 (6.8±0.45), which were consistent with the content of total sugar and sweet amino acid.Similarly, the order of umami score was consistent with MSG-like amino acid. In contrast, Sour score was not entirely determined by the total amount of organic acids. This may be caused by different sensory thresholds for different acids[35]. In terms of flavor, there was no obvious trend in the score of esters and vinegar. This may be because it depends not only on the amount of species and relative content in the flavor of the sample vinegar, but also on the effect of the threshold and combined effect of other compounds such as different esters and acids[33].

3 Conclusions

There were significant differences in the technical formula and testing and evaluation indicators of traditional vinegar in China. In this paper, for the four Sichuan vinegar samples produced in Sichuan, physical and chemical testing, chromatographic analysis, sensory evaluation, intelligent sensory, chemometrics and other analytical methods are used to determine the total sugar, total acidity, pH,L*,a*,b*, amino acids, organic acids, and volatile flavor substances of the four Sichuan vinegar samples were analyzed and compared.

The experimental results showed that compared with Shanxi aged vinegar and Zhenjiang vinegar, the color index and total sugar of the four Sichuan vinegar samples did not change much. The content of total acid, total amino acid and essential amino acid was generally higher, while pH was just the opposite.Compared with Chishui vinegar, the total organic acid content of Sichuan vinegar samples was smaller.Regarding the content of VOCs, 26 major compounds(relative content>1.00%) were identified from 151 VOCs. Among samples, three organic compounds may be considered as common components that constitute the aroma of Sichuan vinegar. Samples 1,2, and 3 had 2, 4, and 4 VOCs, respectively, which could be considered as their unique flavor substances.Sensory evaluation method and intelligent sensory combined with chemometric method could be used to characterize the correlation of four Sichuan vinegar samples due to differences in raw materials, processes, formulas, etc. This article would provide a practical reference for establishing a reliable characterization of Chinese vinegar, provide an effective tool for the evaluation of vinegar quality and authenticity, and help to understand the relationship between the internal and external quality characteristics of vinegar.

The differences in the characteristic flavors of vinegar from in Sichuan were compared, but this article did not make a specific and in-depth study on the reasons for the differences. In the study of volatile flavors, the qualitative, quantitative, and description of flavors at the molecular level have not been able to conduct a comprehensive and in-depth analysis of the flavors of vinegar. In future research, systematic molecular sensory technology can be used to conduct detailed research on the specific factors that cause the difference in characteristic flavor substances of Sichuan vinegar.