Accelerating vinegar aging by combination of ultrasonic and magnetic field assistance.

Most fermented foods need a natural aging process to enrich desired flavours. This process is normally the bottleneck for cost-effective production. Therefore, it is desirable to accelerate the process and obtain products with the same flavour profile. Here, we used physical interventions (ultrasonic field, alternating magnetic field, or combination of both) to assist the aging process with naturally brewed vinegar as a case example. Flavour profiles of different physical-assisted aging process were compared with that of the naturally aged vinegar by using gas-chromatography mass-spectrometry (GC-MS) and electronic nose. Principal component analysis (PCA) and Pearson correlation analyses show that ultrasonic and alternating magnetic fields treatment could accelerate the aging process of vinegar. The highest accelerating aging effect was combination of ultrasonic and magnetic field followed by individual ultrasonic or magnetic field and natural process (combination of ultrasonic and magnetic field > ultrasonic or magnetic field individual > natural process). These results suggest that physical field intervention could potentially be used for acceleration of aging of fermented products without affecting flavour quality.

Introduction

Most fermented foods need a natural aging process to enrich desired flavours. This process is normally the bottleneck for cost-effective production. Therefore, it is desirable to accelerate the process and obtain products with the same flavour profile. Up to date, several physical and chemical strategies have been used to accelerate aging of fermented foods. For example, hydrostatic high pressure, ultrasound energy and electric field were used to accelerate aging of wines, brandies and vinegars, with positive results [1], [2], [3]. In food production, wood shavings are widely employed in vinegar making to reduce aging time, while stem bromelain can accelerate the ripening of sufu [4]. Some methods, an elevated ripening temperature, modified starters, exogenous enzymes and cheese slurries have been proposed for acceleration of cheese ripening [5].

As one of the most popular fermented foods worldwide, vinegar has been historically utilized since 3,000 BCE. Most fresh vinegar needs to be aged for a period of time before selling and cooking because of its light odor, bitter taste and some potential harmful side effects [6]. The aging process contributes to the formation of esters, alcohols, and acids. During natural aging or after-ripening, a series of chemical reactions will greatly enrich and enhance the flavor of vinegar. Ancient Chinese vinegar brewing utilized long-term aging to “insulate in summer and taking out ice in the winter”. Due to the disadvantages of traditional aging technology such as lengthy processing time and high cost, innovative aging technologies need to be developed [7]. The use of ultrasonic field for wine aging has extensively progressed and may be related to acoustic cavitation, including bubble formation, growth, and implosion collapse [8]. Ultrasonic treatment of yellow rice wine has been proven to be effective in wine aging, with similar taste as that using the traditional methods [9]. Magnetic fields, due to their positive biological effects, have a wide range of applications in the food processing industry. Magnetic fields treatment can interfere with reaction kinetics by increasing the rate of collision between chemical substrates, or by increasing the rate of diffusion [10]. Magnetic fields treatment may lead to the breakdown of proteins into smaller peptides [11]. However, whether or not magnetic fields are capable of accelerating the aging of fermented food is not known. The aim of our study was to investigate the effect of ultrasound and magnetic fields either alone or in combination on flavour compounds in accelerating the aging process of vinegar. We hope to take vinegar as an example to explore a new method accelerating aging of fermented products and obtaining equal flavor quality.

Materials and methods

Vinegar samples and materials

Samples of natural aged vinegar with different storage time (fresh, 24, 36, 60, 72, and 120 months) were obtained from Cellar Vinegar of Qianhe Flavour Industry (Qianhe Condiment and Food Co., Ltd., Meishan, China). Fresh refers to the fresh vinegar (the new vinegar after a traditional one-year aging). The chemicals used in this study are all analytical grade materials.

Ultrasonic and alternating magnetic fields treatment

Ultrasonic field treatment (UF): Each time, around 100 mL of fresh vinegar was filled into a 250-mL flask with a lid to prevent the evaporation of volatile compounds. The flask was then placed at the centre of the ultrasonic chamber (Bionoon-950 ultrasonic Crusher, Bonuo Biotechnology Co., Ltd., Shanghai, China). An ice water bath, with 2 cm higher than the vinegar surface, was used as medium for ultrasonic treatment. The ice water was changed after each treatment to maintain the same ultrasonic effect. The vinegar samples were finally treated for total processing time of 0.5 h (UF-0.5 h group) and1 h (UF-1 h group) with the following conditions: ultrasonic power: 90 W, maximum limit temperature: 40 °C.

Alternating magnetic field treatment (MF): A magnetic field-assisted incubator shaker (MFOI-L1, Induc Scientific Co., Ltd., Wuxi, China) was used for the treatment. MFOI-L1 included a magnetic field generator, a shaking platform, a control panel, a thermostatic chamber (or sample chamber), and a temperature-control unit. The magnetic field generator consisted of a pair of Helmholtz coils (80 cm × 80 cm square; 400 turns) and a power supply and produced a uniform magnetic field at an excitation current of 8 A. The sample chamber measured 38 cm × 38 cm × 38 cm, and the temperature ranged from 4 °C to 60 °C. A BLD-1030 Gaussian meter (Bolandun Corporation, Ltd., Beijing, China) and a micro-displacement platform were used to measure the field strength and its homogeneity in the chamber. At these settings, the maximum measured magnetic field intensity was 5 mT (AC or DC), and the homogeneity of the magnetic field was 99%. Fresh vinegar (100 mL) was first filled into a flask (250 mL) with lid to avoid the evaporation of samples, and it was then placed in MFOI-L1. The treatment conditions were as follows: temperature: 40 °C, agitation speed: 110 rpm, alternating magnetic field: 0–5 mT, and processing time: 1 h (MF-1 h group) and 3 h (MF-3 h group).

Ultrasonic and alternating magnetic fields co-treatment (UF + MF): The flask (250 mL) with vinegar (100 mL) was first treated by ultrasonic field for 0.5 h and then by alternating magnetic field for 1 h (UF + MF group), time interval: 2 min. The treatment condition for UF and MF was the same as described earlier.

Determination of volatile flavours by GC–MS

The volatile properties of vinegar samples were analyzed by headspace solid phase microextraction (HS-SPME) combined with GC–MS. GC–MS analysis was conducted on an Agilent 7000D gas chromatograph equipped with an Agilent HP-Innowax (60 m × 0.25 mm i.d., 0.25-μm film thickness). The injection was conducted in split mode (1:10) at 250 °C. The GC operation conditions were as follows: the initial temperature of 40 °C was held for 3 min and then raised to 150 °C with a ramp of 5 °C/min. Then, the temperature was increased to 250 °C with a ramp of 10 °C/min. The final temperature of 250 °C was held for 3 min, resulting in a total run time of 38 min.

Electronic nose

Electronic nose (iNose; Ruifen Trading Co., Shanghai, China) was used to characterize the aroma information of vinegar samples. The electronic nose consisted of 14 metal oxide semiconductor gas sensors (S1–S14). Before testing, the gas path of the electronic nose was cleaned with reference air of 1 L/min gas flow for 30 min for sensor signal calibration [12]. For the single test, 5 g of the sample was placed into an airtight bottle (20 mL). The bottle was then incubated at 25 °C for 20 min under agitation (500 rpm). The determination time for sensors was 150 s, and the recovery time was 300 s. Each sample was analysed thrice.

Statistical analysis

All data were expressed as mean ± SD for at least three replicates. Origin 8.0 and Excel 2007 were used to complete the data analysis. All data obtained were statistically analysed by SPSS Statistics 17.0. Principal component analysis and LDA were used to detect volatile organic compounds in different vinegar libraries [13]. The peak areas of the selected identified compounds were obtained through a one-way ANOVA with significance at P < 0.05. A heat map and PCA were used for sample cluster analysis. The heat map and PCA plots were generated using the following R software packages: heatmap for heat maps and factoextra for the PCA plots [14]. Visualization of a correlation matrix in R was performed using the package corrplot.

Results and discussion

Effects of ultrasonic and alternating magnetic fields treatment on flavour compounds of vinegar

HS-SPME-GC–MS is an effective tool to characterize the volatile compounds. The GC–MS results of vinegar samples with different treatments are shown in Supplement Table 1. About 33 volatile compounds were detected in vinegar (Table 1), including alcohols, acids, aldehydes, ketones, esters, heterocycles, and others. The content of acetic acid in different treatment groups was relatively high, from 25.7% to 57.7%. Furfuryl formate was only detected in 60- to 120-month-old vinegar and MF-3 h vinegar, which could be used as a marker of vinegar.

Table 1

Information on the identified compounds in vinegar by HS-SPME-GC–MS.

No.	RT (min)	Compound	Formula	CAS	Score	Classification
1	3.309	Methyltartronic acid	C4H6O5	595–48-2	84.91	Acids
2	3.435	2,4,5-Trimethyl-1,3-dioxolane	C6H12O2	3299–32-9	92.01	Heterocycles
3	4.073	Acetoxyacetic acid	C4H6O4	13831–30-6	75.33	Acids
4	4.719	Isobutyl acetate	C6H12O2	110–19-0	96.49	Esters
5	5.446	2-Ketoglutaric acid	C5H6O5	328–50-7	80.16	Acids
6	5.937	Ethyl isovalerate	C7H14O2	108–64-5	71.28	Esters
7	6.619	2-Heptyl-1,3-dioxepane	C12H24O2	61732–92-1	77.19	Heterocycles
8	7.198	2-Methylbutyl acetate	C7H14O2	624–41-9	96.22	Esters
9	7.914	2-t-Butyl-5-methyl[1,3]dioxolan-4-one	C8H14O3	146528–25-8	76.37	Ketones
10	8.124	Acetoxyacetic ccid	C9H16O4	13831–30-6	71.67	Esters
11	9.799	2-Methyl-1-butanol	C5H12O	137–32-6	92.69	Alcohols
12	10.306	Ethyl caproate	C8H16O2	123–66-0	66.75	Esters
13	11.918	Acetoin	C4H8O2	513–86-0	89.37	Ketones
14	11.945	Furfuryl formate	C6H6O3	13493–97-5	78.52	Esters
15	13.501	Ethyl lactate	C5H10O3	97–64-3	89.45	Esters
16	14.613	Acetoin acetate	C6H10O3	4906–24-5	66.65	Ketones
17	15.138	2,3,5-Trimethylpyrazine	C7H10N2	14667–55-1	80.52	Heterocycles
18	15.877	Acetic acid	C2H4O2	64–19-7	98.52	Acids
19	16.636	3-Furaldehyde	C5H4O2	498–60-2	98.68	Heterocycles
20	16.863	2,3,5,6-Tetramethylpyrazine	C8H12N2	1124–11-4	83.31	Heterocycles
21	18.047	Benzaldehyde	C7H6O	100–52-7	94.68	Aldehydes
22	18.477	Furfuryl acetate	C7H8O3	623–17-6	91.32	Esters
23	18.617	Ethyl 2-hydroxy-4-methylvalerate	C8H16O3	10348–47-7	61.8	Esters
24	19.158	Isobutyric acid	C4H8O2	79–31-2	90.25	Acids
25	19.462	Ethyl acetate	C4H8O2	141–78-6	74.79	Esters
26	20.594	Butyric acid	C4H8O2	107–92-6	60.94	Acids
27	21.547	Isovaleric acid	C5H10O2	503–74-2	91.78	Acids
28	24.19	Ethyl phenylacetate	C10H12O2	101–97-3	65.63	Esters
29	24.832	Phenethyl acetate	C10H12O2	103–45-7	90.78	Esters
30	25.409	Hexanoic acid	C6H12O2	142–62-1	81.23	Acids
31	26.645	2-Phenylethanol	C8H10O	60–12-8	96.13	Alcohols
32	27.361	2-Methoxy-3-methylphenol	C8H10O2	18102–31-3	92.96	Others
33	28.424	4-Ethyl-2-methoxyphenol	C9H12O2	2785–89-9	71.07	Others

Cluster heat map analysis of the compounds was used to better understand the differences and similarities between different treatment groups. Cluster analysis was conducted for the naturally aged samples (24, 36, 60, 72, and 120 months) and UF- or MF-treated samples to determine their similarities (Fig. 1). Vinegar aged for 120 months was significantly different from that of other treatments. The compound composition of vinegar treated with ultrasonic or alternating magnetic field was similar to that of fresh and 24-month-old vinegar. The compound composition of vinegar co-treated with ultrasonic and alternating magnetic fields was closer to that of 36-, 60-, and 72-month aged vinegar. The results showed that the composition of material vinegar was significantly different at various aging times. After alternating magnetic field treatment for 1 h or ultrasonic field treatment (0.5 h or 1 h), the chemical composition of fresh vinegar was close to that of 24 months. Ultrasonic and alternating magnetic fields treatment can accelerate the aging of vinegar to a certain extent.

Fig. 1

Hierarchical cluster analysis based on flavor characteristics of 11 vinegar samples. Red: high concentration compounds; blue: low concentration compounds. UF: ultrasonic field treatment; MF: alternating magnetic field treatment; UF + MF: ultrasonic and alternating magnetic fields co-treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

To further analyze the data, PCA was used to emphasize variations and to visualize patterns within the dataset. Fig. 2A shows the results of PCA with respect to principal component 1 (PC1) and PC2. The PC1 and PC2 accounted for 97.37% of the total variance in the dataset. PC1 accounted for 95.1% of the variance, while PC2 accounted for 2.27% of the variance in the data.

Fig. 2

PCA analysis of volatile compounds in different groups of vinegar samples. (A) Score plot of the first two principal components; (B) Loading plot of different variances. UF: ultrasonic field treatment; MF: alternating magnetic field treatment; UF + MF: ultrasonic and alternating magnetic fields co-treatment.

The distribution of parallel samples in each group is close, which indicates a good parallelism. It was evident that three distinct regions could be identified within the PCA profile (Fig. 2). Region A included fresh group, 24 month-aged vinegar, and UF- or MF-treated samples, region B included 36–72 months and UF + MF group vinegar, while region C included 120 months vinegar. These results further support the clustering analysis shown in the heat map (Fig. 1). Loading diagrams were used to determine the volatile compounds that contributed the most to the sample clustering. As shown in Fig. 2B, the direction and length of the vector indicated the contribution of the variables towards the two principal factors. For example, acetic acid, as the main characteristic flavors of vinegar, primarily contributed to the variance in PC1. The top 10 characteristic compounds with contribution rate were as follows: acetic acid, 3-furaldehyde, 2-t-butyl-5-methyl[1,3]dioxolan-4-one, acetoxyacetic acid, 2-ketoglutaric acid, furfuryl formate, 2-methylbutyl acetate, isovaleric acid, furfuryl formate, and isobutyl acetate.

Analysis of alterations in compounds during the vinegar aging process

The 33 compounds detected in vinegar were divided into six categories for further analysis. The content changes of alcohols, acids, aldehydes, ketones, esters, heterocycles, and benzaldehyde in each treatment group are shown in Fig. 3. With the aging of vinegar, the total acid content significantly decreased. The total acid content of 60-month-old vinegar decreased to 63% of fresh vinegar, while that of 120-month-old vinegar was only 56% of fresh vinegar. The change of total acid content may be related to the volatilization of some acids during aging. In addition, there are slow esterification and oxidation reactions between esters, aldehydes, and alcohols in vinegar during aging. Acid as a reaction substrate was reduced accordingly. However, the contents of total esters, heterocycles, ketones, and benzaldehyde increased over time. Compared with fresh vinegar, the total ester content of 60-month-old vinegar increased by 80%, the heterocyclic content of 120-month-old vinegar increased by 184%, the alcohol content of 120-month-old vinegar increased by 206%, and the benzaldehyde content of 120-month-old vinegar increased by 91%. Except for fresh vinegar, there was no significant difference in the content of benzaldehyde in other treatment groups. The change trend of six kinds of substances in vinegar treated with ultrasonic and alternating magnetic fields coincided with that of natural aging. Compared with fresh vinegar, acids, ketones and benzaldehyde of samples treated with three physical interventions changed significantly. In addition, there were significant differences in acids, heterocycles, ketones and alcohols between UF-MF combined treatment group and separate treatment (Fig. 3). These findings indicate that ultrasound and alternating magnetic field accelerate the process of vinegar aging.

Fig. 3

Effects of the ultrasonic or alternating magnetic field treatments on the chemical indicators of the Qianhe vinegar aging process. UF: ultrasonic field treatment; MF: alternating magnetic field treatment; UF + MF: ultrasonic and alternating magnetic fields co-treatment.

Thermographic cluster analysis of acids, alcohols, aldehydes, ketones, esters, and heterocycles in vinegar of different treatment groups was conducted. As shown in Fig. 4A, vinegar subjected to different treatments could be grouped into two categories. Vinegar from MF, UF, fresh and 24-month was clearly grouped into one cluster. In addition, vinegar co-treated with ultrasound and alternating magnetic fields was closer to that of 36- to 120-month-old vinegar. We analysed the correlation between time and the content of these substances under different treatment conditions. Fig. 4B–D show the correlation coefficients of compounds and time under different aging processes, including natural aging, ultrasound and alternating magnetic field. During natural aging, the correlation coefficient between acids and time was −0.91. The contents of heterocycles and ketones were positively correlated with time, and the correlation coefficients were 0.95 and 0.9, respectively (Fig. 4B). With ultrasonic field treatment, the correlation coefficient of acids with time was −0.91. The contents of esters and ketones were positively correlated with time, and the correlation coefficients were 0.86 and 0.82, respectively (Fig. 4C). Under the condition of alternating magnetic field, the contents of acids were also negatively correlated with time (the correlation coefficient was −0.97). The contents of esters, alcohols, and heterocycles were positively correlated with time, and the correlation coefficients were 0.87, 0.75, and 0.75, respectively (Fig. 4D). Particularly concerning was that the change trend of alcohols under alternating magnetic field treatment is positively correlated with time, which is different from natural aging and ultrasonic treatment. It also shows that ultrasonic treatment and alternating magnetic treatment may have different mechanisms of action. In conclusion, the observed changes in substances in vinegar were similar to that of natural aging under ultrasonic and alternating magnetic fields treatment. In addition, ultrasonic field treatment and alternating magnetic field treatment accelerated the aging of vinegar.

Fig. 4

Correlation analysis of volatile flavor compounds in vinegar samples under different treatment conditions. (A) Heat map cluster analysis of vinegar; (B) Correlation analysis between time and volatile flavor compounds under natural aging conditions; (C) Correlation analysis between time and volatile flavor compounds under ultrasonic treatment conditions; (C) Correlation analysis between time and volatile flavor compounds under alternating magnetic field treatment. UF: ultrasonic field treatment; MF: alternating magnetic field treatment; UF + MF: ultrasonic and alternating magnetic fields co-treatment.

Electronic nose analysis

Electronic nose is a device that can mimic the sense of smell to detect volatile compounds in complex matrices [15]. It can simulate the principle of biological olfactory sensory function and distinguish simple or complex odours [16]. There is a great correlation between electronic nose and sensory evaluation data, which is helpful to objectively evaluate changes in vinegar flavour. PCA was used to analyse the electronic nose signal of vinegar. The total PC 1 (87.1%) and PC 2 (8.5%) were 95.6% (Fig. 5A). Generally, a total value of more than 85% can well reflect the whole flavour information of tested samples [17]. The vinegar treated with ultrasonic and alternating magnetic field was closer to 24 months and farther from other natural aged vinegar. This is similar to the result in Fig. 1. In total, there was a large deviation between natural aged vinegar and vinegar after ultrasonic and alternating magnetic fields treatment. It also showed that ultrasonic and alternating magnetic fields treatment did cause the change of vinegar components.

Fig. 5

PCA-LDA plot and radar plot for electronic nose data of vinegar samples. (A) PCA analysis (loading plot) of vinegar; (B) LDA plot of vinegar; (C) radar plot of the average sensor responses obtained with the gas sensor array for each vinegar sample. UF: ultrasonic field treatment; MF: alternating magnetic field treatment; UF + MF: ultrasonic and alternating magnetic fields co-treatment.

The data were further analysed by linear discriminant analysis (LDA). LDA can maximize the separation between groups. The DI value is small, which indicates that the effect of discrimination is not ideal. There was little difference among the groups, and the vinegar treated with ultrasonic and alternating magnetic fields was closer to 72–120 months (Fig. 5B).

Fig. 5C shows the radar response of 14 sensors to vinegar aroma. The lines of different colours represent vinegar of different treatment groups. The closer the line to the outer ring, the stronger the response of the sensor. The sensor S5 (biosynthetic compounds), which was sensitive to substances produced by biosynthetic compounds such as Maillard reactions, showed the highest value than the other sensors; meanwhile, sensors S1 (aromatic compounds), S9 (dihydrostilbenes), and S13 (ethylene) exhibited higher values. The results meant the volatile compounds in vinegar also included those compounds, such as oxynitride, sulphides, organic acid esters, terpenoids, lenthionine, aliphatic hydrocarbon, amines, hydrocarbon, TVOC, sulphide, and volatile gas produced in cooking. Compared with fresh vinegar sample, ultrasonic and alternating magnetic fields co-treatment showed higher responding signals in sensors S5 (biosynthetic compounds) and S9 (dihydrostilbenes). It is indicated that the odor after ultrasonic and alternating magnetic fields co-treatment had changed, and it was closer to the natural aging flavor in some sensors, which agreed with the results obtained from previous evaluation.

Conclusions

A very long and time-consuming natural aging process is required for most fermented foods. It is important to find a green and low-cost method to accelerate the aging process. Physical fields treatments have attracted increasing attention due to their pollution-free, low-cost, and easy operation. This study explored the accelerating effect of physical field assisted interventions (ultrasonic field, alternating magnetic field, or combination of both) on vinegar aging. It was found that the combination treatment with US and MF could significantly accelerate the aging process, and the accelerating effect followed a decreasing order: combination of ultrasonic and magnetic field > ultrasonic or magnetic field individual > naturally aged. This suggest that the ultrasonic and alternating magnetic fields combination would be an alternative and promising way for acceleration of vinegar aging. This study would provide insights on developing promising aging method with accelerated speed and low lost. However, the mechanism of physical field accelerating aging needs to be further studied.

CRediT authorship contribution statement

Hongbo Li: Conceptualization, Methodology, Software, Funding acquisition. Xujia Ming: Data curation, Writing - original draft. Zhenbin Liu: Visualization, Investigation. Long Xu: Software, Validation. Dan Xu: Data curation. Liangbin Hu: Supervision. Haizhen Mo: Funding acquisition. Xiaohui Zhou: Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.