Fermentation Quality Characteristics and Flavor Formation Changes During the Process of Pickled Wax Gourd in Eastern Zhejiang

Abstract

Using pickled brine as samples of natural pickled wax gourd at different stages of 0, 5, 10, 15, and 20 d, respectively, pH value, reducing sugar content, number of lactic acid bacteria and total bacteria, as well as organic acid content and volatile components, were investigated in this study. As a consequence, the lactic acid and acetic acid concentration increased with the decreasing of pH and sugar exhaustion through the pickling process. A total of 60 kinds of volatile compounds were detected, including 12 alcohols, 6 acids, 11 aldehydes, 12 ketones, 4 esters, 6 silicones, and 9 other compounds. At the end of 20 days fermentation, the abundant volatile compounds were identified including alcohols (47.58%), acids (36.00%), and esters (5.94%), with the highest content of acetic acid (27.97%), followed by ethanol (23.69%). The contributions of these compositions were clarified by electronic nose technology method combined with principal components analysis.

Keywords:

• Wax gourd
• Pickling
• Lactic acid bacteria
• Flavor components

INTRODUCTION

Pickled wax gourd (Benincasa hispida) named as “stinky wax gourd,” has a long history in the east of Zhejiang and is very popular as an appetizer with its strong flavor. The flavor of pickled food includes the taste and smell, which directly affect people’s selection and acceptance of its products. Meanwhile, it contributes to make an evaluation of the product information such as brands, the place of origin, nutrition, and safety.

The previous studies in picked vegetables mainly focused on the pickling process technology,^[1] the quality and safety,^[2] application of fermentation starters,^[3,4] as well as the microbiota,^[5–7] and less attention have been paid on the flavor formation and factors of pickling process. The flavor compounds were mainly produced from the original flavor of vegetables, such as the components of myronate potassium and isothiocyanate.^[8] The interaction among the various compounds in pickling system generated complete flavor of volatile and non-volatile compounds, such as alcohols, acids, esters, aldehydes, ketones, and sulfur compounds. In addition, various amino acids hydrolyzed by protein in vegetable resulted in a delicious taste of the product. The flavor changes in the pickling process of Fuling mustard have been preliminary stated.^[9] The major volatile compounds in kimchi were detected including alcohols, organic acids, and sulfur compounds.^[4] Kim and Chun^[10] have determined the microorganism community structure in kimchi, which affected the final quality of the product, and the results showed that the predominant bacteria were Lactobacillus, Leuconostoc, and Weissella by the method of 16S rRNA clone library. The metabolic pathways of different bacteria during fermentation of kimchi in Korea using the method of metagenomics are further conducted.^[11] A total of 39 strains of Lactobacillus spp. from the fermentation samples of kimchi were isolated,^[12] and 21 strains among them showed higher activity during the fermentation. The relationship between the changes of the microbial communities and the organic acids both non-volatile and volatile components was investigated during the fermentation of kimchi.^[13] Jung et al.^[14] tried to find the relationship between the changes in microbial communities and the various metabolites, by using pyro-sequencing and nuclear magnetic resonance (NMR) to monitor kimchi fermentation.

In our previous studies, the detection methods of aroma compounds,^[15] the bacteria community analysis of fermented wax gourd^[16,17] and mustard tuber^[6,7] were conducted, and the quality changes of raw pickled wax gourd were also investigated.^[16] However, the information of flavor compounds corresponding to the quality of pickled wax gourd is very limited so far. The aims of this study were mainly to investigate the quality characteristics and flavor formation of traditional pickled wax gourd during the processing. Meanwhile the changes of these characters were evaluated by electronic nose detection, combined with the diversity and variation of microbial communities obtained in our previous studies. The results could provide theoretical basis for quality assessment, the establishment of production standards and optimization of pickling process of this local characteristic pickled wax gourd.

MATERIALS AND METHODS

Preparation of Wax Gourd Sample

Fresh wax gourd (Benincasa hispida) was bought from the local market. Edible salt (NaCl) was added to the wax gourd for a final concentration of 5%. During the whole fermentation period for 20 days, the pickled jar was sealed with water and kept at a normal room temperature. Each sample used for various assays was collected from the center of the jar at different periods of 0, 5, 10, 15, and 20 d, respectively, and kept at –20°C for further study.

Assay of pH and Reduced Sugar Content

The pH of wax gourd brine sample was measured with a pH meter (PHS-3C, LeiZhi, Shanghai). The content of reduced sugar was analyzed by 3,5-Dinitrosalicylic acid (DNS) method.

Assay of colony forming unit (CFU) of lactic acid bacterias (LABs), Bacteria, and Yeast

The supernatant (20 mL) was serially diluted in saline to assay the CFU. Serially diluted pickled wax gourd samples were inoculated on de Man Rogosa Sharpe (MRS) medium and plate count agar (PCA medium, Land Bridge, Beijing, China) and the number of colonies appeared on the media were counted after 48 h incubation at 37°C. Numeration of yeast populations in samples was performed on malt extract agar (MEA, Land Bridge, Beijing, China) according to the method above, incubated aerobically at 28°C for 48 h.

Determination of Organic Acid

Samples of wax gourd 2 g and pickled brine (2 mL) were collected as described above, respectively. The brine was mixed with 400 μL H₂SO₄ (50%), then centrifuged at 8000 g for 5 min. The supernatant (200 μL) was diluted to 1 mL with ultrapure water, filtered with 0.30 μm membrane (Jet, Guangzhou, China) for further analysis. Organic acids concentration of the samples was measured by high performance liquid chromatography (HPLC) methods (Agilent 1260 Infinity). The filtrate was a vacuum filtration system (Jinteng, Hubei, China). The sample was injected into an Agilent HPLC system equipped with a UV detector at 215 nm (0–20 min), 254 nm (20–25 min), 215 nm (26–27 min), with a TC-C18 column (4.6 × 250 mm, 5 μm, Agilent, California, USA) and 0.7 mL/min flow rate was used for quantitative analysis of various organic acids of pickled wax gourd. The mobile phase was CH₃OH (Juhua, Zhejiang, China) and 0.01 mol/L NaH₂PO₄ (pH 2.8, National Medicines Corporation Ltd., Beijing, China) with gradient elution, 100% NaH₂PO₄ (0–6 min), adding the CH₃OH to 5% (6–15 min), increasing to 45% (15–20 min, kept for 5 min), 5% CH₃OH (25–26 min), and 100% NaH₂PO₄ (27 min). The standard solutions of various organic acids were prepared with ultrapure water are as follows, ascorbic acid (0.5 g/L), oxalic acid (2.0 g/L), tartaric acid (5.0 g/L), citric acid (10.0 g/L), malic acid (12.5 g/L), lactic acid (15.0 g/L), acetic acid (15.0 g/L), and butanedioic acid (25.0 g/L), respectively.

Gas Chromatography–Mass Spectrometry Analysis (GC–MS)

The wax gourd or samples described above were put into the sample bottle of headspace with a cover, and extracted at 50°C for 45 min, then balanced in water bath at 20°C for 10 min. The samples were inserted into the GC instrument, desorped for 5 min. All samples were measured using QP 2010 GC–MS (attached with a mass quadrupole detector, USA). The separation was conducted on a non-polar Vocol capillary column (60 m × 0.32 mm i.d. × 1.8 μm film thickness) from J&W, Agilent Technologies (USA). The carrier gas was ultra-purified helium at a flow rate of 0.8 mL/min. The injection temperature was 210°C. The column temperature procedure was from 35°C (kept for 3 min), 3°C/min to 40°C (kept for 1 min), and 5°C/min to 210°C (kept for 16 min). The mass spectra were acquired with a source temperature of 200°C, under a detector voltage of 800 V. Scanning mass range (m/z) of mass spectrometry was 30–650 u. Identification of volatile compounds were performed by comparing their mass spectra with those contained in the National Institute for Standards and Technology (NIST; Search Version 1.6) and Wiley (NY, 320 k compounds, Version 6.0.) mass spectral library.

Electronic Nose Analysis of Flavor Components

Analysis of flavor for each sample by electronic nose methods (portable electronic nose [PEN3], Airsense company, Germany) was carried out according to the manufacture’s instruction. Each 2 g sample was added into the headspace sampling bottle, temporarily kept in the refrigerator at 4°C. The sampling and gas injection rate were set at 300 mL/min, and measuring time is 300 s. The cleaning time was set between 300 to 1500 s according to the actual situation of the samples involved. The data were analyzed by principal components analysis (PCA) method according to the manufacture’s instruction of PENs instrument.

Statistical Analysis

The data obtained were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Any significant difference was evaluated by one-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons considering difference statistically at p < 0.05.

RESULTS AND DISCUSSION

Changes of Reducing Sugar (RS) Content and pH Value of Wax Gourd Brine Samples in the Pickling Process

Figure 1 presented the RS content and pH values through the 20 days processing of pickled wax gourd. A maximal value of RS of 14.70 mg/mL was found at the beginning of the pickling (0 day), with a maximal pH value of 5.22. The pH declined rapidly in the first 10 days, and then showed a slow decrease. The pH value decreased to a minimum of 3.45 at the 10th day and maintained a stable value after the 10th day of fermentation.

FIGURE 1 Changes in reducing sugar content and pH value during wax gourd fermentation.

The data of RS and pH value changed significantly in the early stage of pickling process. In this period, most of the contaminating microorganism growth was inhibited by the high salinity on the surface of wax gourd. Some of salt-tolerance LABs attached to the wax gourd gradually became the main fermentation strains. The growth of LABs accelerated the production of various organic acids and rapidly reduced the pH value of pickled system. Accordingly, lactic acid was metabolized by some special species of bacteria and pH value led to a slow increase until to maintain a stable level at the middle and late fermentation stages.

Microbial Counts of LAB and Bacteria in the Storage of Pickled Wax Gourd

Total amounts of LABs and bacteria in wax gourd brine samples are presented in Table 1 at different pickling period. The populations of LAB and bacteria increased through fermentation of wax gourd, the LAB amounts reached the maximum of 7,15 (logCFU/mL) after 20 days of fermentation. The ratio of total amounts of LABs and bacteria increased until the 10th day, it reached a high level of 53.3%. The initial population of LABs and bacteria were low, due to the cooked procedure of wax gourd with boiling water, most of thermolabile microorganisms were killed and only a minority of heat-resistant bacillus and spore survived. It led to the total amounts of LABs and bacteria less than 4,00 (logCFU/mL) at the beginning of pickling. With the steady accumulation of lactic acid, the system of pH value continued to decline. Low pH value causes the inhabitation of partial bacteria, and some acid resistant microorganisms including yeast and LABs grew well. The proportion of LABs in total bacteria changed to a high value and reached a state of dynamic balance in the late fermentation.

TABLE 1 Changes of total amounts of lactic acid bacteria, total bacteria and yeast (log CFU/mL) during fermentation of wax gourd

	Fermentation time (d)
	0	5	10	15	20
The total amounts of lactic acid bacteria	3, 68	5, 69	6, 24	6, 97	7, 15
The total amounts of bacteria	3, 30	6, 18	6, 45	7, 23	7, 79
The total amounts of yeasts	3, 54	7, 43	8, 14	8, 11	7, 90

Changes of Organic Acids in Pickled Processing

The organic acid content in brine samples at different pickling periods of 0, 5, 10, 15, and 20 days was calculated by various organic acid standard curve (Table 2). As a consequence, the ascorbic acid content in the whole process of pickling was not detected until it was finished at the 20th day. Oxalic acid had high concentration in pickled wax gourd, and gradually increased with the pickling time. It appeared to have a growing in fluctuations between the 10th and 20th day. Tartaric acid content was a low level, and there is a significant difference among the 10th, 15th, and 20th day. Citric acid content increased significantly in the early stage of pickling, then decreased gradually and kept a stable level of around 0.077 g/L. Malic acid content increased significantly in the early period of pickling (0–5 days), reached the maximum of 0.076 g/L, then declined significantly to a low level around 0.005 g/L. The content of lactic acid and acetic acid increased through the pickling process. The succinic acid showed an increase in the early stage, and reached the maximum of 0.065 g/L at the 10th day, then decreased to a lower level. Change of malic acid content is due to the fermentation of LABs, it is converted gradually into acetic acid and lactic acid under the action of LABs.^[13] It also caused the increase of lactic acid and acetic acid through pickled processing of wax gourd. The concentration of organic acids and its kinds affect the community diversity and the growth of spoilage microorganisms in pickled system of wax gourd.

TABLE 2 Changes of organic acids content (g/L) in pickled wax gourd during fermentation

Name	Fermentation time (d)
	0	5	10	15	20
Ascorbic acid	–	–	–	–	0.002 ± 0.002^a
Oxalic acid	0.312 ± 0.027^a	0.294 ± 0.042^a	0.316 ± 0.015^a	0.331 ± 0.013^b	0.352 ± 0.039^c
Tartaric acid	0.026 ± 0.008^a	0.037 ± 0.011^b	0.029 ± 0.016^a	0.035 ± 0.004^b	0.043 ± 0.018^c
Citric acid	0.036 ± 0.010^a	0.082 ± 0.008^c	0.069 ± 0.013^b	0.077 ± 0.013^b	0.073 ± 0.009^b
Malic acid	0.023 ± 0.006^b	0.076 ± 0.002^d	0.043 ± 0.007^c	0.021 ± 0.001^b	0.005 ± 0.002^a
Lactic acid	0.037 ± 0.003^a	0.132 ± 0.019^b	0.302 ± 0.062^c	0.311 ± 0.048^c	0.346 ± 0.022^d
Acetic acid	–	0.055 ± 0.011^a	0.089 ± 0.016^b	0.104 ± 0.017^c	0.133 ± 0.008^d
Succinic acid	–	0.029 ± 0.004^a	0.065 ± 0.005^d	0.036 ± 0.002^b	0.041 ± 0.009^c

^a-d Different lowercases indicate significant differences (P < 0.05) among different fermentation time.

Changes of Volatile Compounds in Pickling Process

Table 3 showed the results of volatile compounds at different periods of pickling wax gourd. A total of 60 kinds of volatile compounds were detected, including alcohols (12), acids (6), aldehydes (11), ketones (12), esters (4), silicones (6), and other compounds (9). At the beginning of pickling (0 days), dominant volatile compounds were aldehydes (80.03%), alcohols (8.10%), and ketones (5.02%), with the highest content of 62.16% (hexanal), followed by 9.64% (pentanal) and 5.60% (1-pentanol). The ketones were in lower levels of content but make a great contribution to the flavor of pickled wax gourd with its lower threshold. In the 5th day, the major volatile compounds include ketones (47.44%), alcohols (34.55%), aldehydes (6.36%), and acids (5.81%), with the highest content of 39.7% (3-hydroxy-2-butanone), followed by 21.85% (2,3-butanediol), 10.38% (ethanol), and 5.81% (acetic acid). In addition, 2-propanone, hexanal, acetaldehyde, 2,3-butanedione and ethyl acetate were also found in this period. In the 10th day, the major volatile compounds contain alcohols (48.04%), acids (32.83%), ketones (6.97%), and esters (5.57%), characterized by the highest content of 29.23% (acetic acid), followed by 21.98% (2,3-butanediol) and 11.44% (ethanol). At this stage, some alcohols such as 3-methyl-1-butanol, 2-methyl-1-butanol, 1-butanol, 2-methyl-1-propanol and organic acid such as butanoic acid, 3-methyl-butanoic acid, 2-methyl-butanoic acid were found from the pickled wax gourd. These compounds contribute to the flavor of the product. In the 15th day, the major volatile compounds of alcohols (41.24%), acids (36.44%), and esters (7.89%) were found, which was the highest content of acetic acid (30.54%), followed by ethanol (16.79%), 2,3-butanediol (13.89%), and ethyl acetate (7.03%). At the end of 20 days pickling time, the abundant volatile compounds were identified including alcohols (47.58%), acids (36.0%), and esters (5.94%), with the highest content of acetic acid (27.97%), followed by 23.69% (ethanol), 7.74% (3-methyl-1-butanol), 6.94% (2-methyl-1-butanol), 5.49% (2,3-butanediol), and 5.17% (ethyl acetate).

TABLE 3 The content of volatile compounds (percentage of peak area) of pickled wax gourd during fermentation

No.	Compounds	Fermentation time (d)
		0	5	10	15	20
	Alcohols
1	1,2,3-Propanetriol	0.16	-	-	-	-
2	1-Pentanol	5.60	-	-	-	-
3	1-Hexanol	2.21	-	0.26	-	0.50
4	1-Nonanol	0.13	-	-	-	-
5	Ethanol	-	10.38	11.44	16.79	23.69
6	2-Methyl-1-propanol	-	0.74	1.48	0.70	2.16
7	1-Butanol	-	0.26	4.18	2.64	0.90
8	2,3-Butanediol	-	21.85	21.98	13.89	5.49
9	2-Propanol	-	-	0.66	0.42	0.12
10	2-Methyl-1-butanol	-	-	2.84	2.16	6.94
11	3-Methyl-1-butanol	-	1.32	5.20	4.46	7.74
12	2-Ethanolamine nitrate	-	-	-	-	0.24
Subtotal		8.10	34.55	48.04	41.24	47.58
	Acids
1	Acetic acid	-	5.81	29.23	30.54	27.97
2	Butanoic acid	-	-	2.38	4.75	1.54
3	3-Methyl-butanoic acid	-	-	0.57	0.60	1.16
4	2-Methyl-butanoic acid	-	-	0.65	0.55	1.74
5	Propiolic acid	-	-	-	-	3.30
6	Propanedioic acid	-	-	-	-	0.29
Subtotal		0	5.81	32.83	36.44	36.00
	Aldehydes
1	Hexanal	62.16	3.26	1.61	0.88	0.41
2	Acetaldehyde	-	3.10	1.72	1.16	0.83
3	2-Methyl-butanal	0.31	-	-	-	-
4	Butanal	0.40	-	-	-	-
5	Pentanal	9.64	-	-	-	-
6	(E)-2-pentenal	0.47	-	-	-	0.19
7	2-Ethyl-3-methyl-butanal	2.20	-	-	-	-
8	(E)-2-hexenal	0.35	-	-	-	-
9	(Z)-2-heptenal	2.27	-	-	0.48	0.76
10	Nonanal	2.27	-	-	-	-
11	2-Butenal	0.66	-	-	-	0.34
Subtotal		80.03	6.36	3.33	2.52	2.53
	Ketones
1	1-Penten-one	1.23	-	-	0.34	0.28
2	2,3-Pentanedione	0.29	-	-	-	0.13
3	2-Methyl-3-pentanone	1.84	-	-	-	-
4	3-Penten-2-one	0.42	-	-	-	-
5	2,3-Octanedione	0.13	-	-	-	-
6	1-Octen-3-one	0.55	0.15	0.11	0.26	0.22
7	4-Methyl-2-pentanone	0.20	-	-	-	-
8	3-Hepten-2-one	0.36	-	-	-	-
9	2-Propanone	-	5.65	2.97	1.42	2.03
10	2,3-Butanedione	-	1.94	-	-	-
11	3-Hydroxy-2-butanone	-	39.70	3.89	1.78	0.56
12	2-Butanone	-	-	-	0.40	-
Subtotal		5.02	47.44	6.97	4.20	3.22
	Esters
1	Ethyl acetate	0.10	1.23	5.57	7.03	5.17
2	Methyl acetate	-	0.11	-	0.69	0.35
3	Methyl propionate	-	-	-	0.17	0.12
4	Amyl formate	-	-	-	-	0.30
Subtotal		0.10	1.34	5.57	7.89	5.94
	Siloxane
1	Hexamethyl-cyclotrisiloxane	-	0.62	-	0.78	-
2	Octamethyl-cyclotetrasiloxane	1.07	0.67	0.50	1.49	0.37
3	Decamethyl-tetrasiloxane	-	-	-	0.45	-
4	Decamethyl-cyclopentasiloxane	2.68	2.31	1.74	2.79	1.24
5	Dodecamethyl-pentasiloxane	-	-	-	0.11	-
6	Dodecamethyl-cyclohexasiloxane	0.91	0.73	0.64	0.88	0.55
Subtotal		4.66	4.33	2.88	6.5	2.16
	Others
1	Methyl-oxirane,	1.00	-	-	-	-
2	Dimethyl-sulfide	-	0.17	0.26	0.88	0.67
3	Dimethyl disulfide	-	-	0.12	-	0.51
4	1-Bromo-2-methyl-butane	0.39	-	-	-	-
5	1,3,5,7-Cyclooctatetraene	-	-	-	0.33	-
6	Fluoro-ethyne	-	-	-	-	0.64
7	1,2-Dimethyl-hydrazine	-	-	-	-	0.13
8	Acethydrazide	-	-	-	-	0.20
9	Diheptyl-disulfide	-	-	-	-	0.24
Subtotal		1.39	0.17	0.38	1.21	2.57

- indicate not detected

The major volatile components of aldehydes were found at the beginning of the fermentation and it is an initial flavor of fresh wax gourd, and hexaldehyde showed the highest content of 62.16% in them. Hexaldehyde is a volatile component in green leaf, and is naturally presented in various fruits and vegetables. This compound exhibits a high content in fresh raw materials, which is formed from C18 or C16 unsaturated linoleic acid by the specific catalysis of lipoxygenase (LOX) or hydroperoxide lyase (HPL) in the plant.^[18]

Flavor Analysis of Pickled Wax Gourd by Electronic Nose Technology (ENT)

Dominant aroma components involved the flavor characters of the product were detected by ENT, and the data were conducted by the PCA analysis. As a consequence (Fig. 2), the contribution rate of the first principal component is 76.76%, followed by 22.33% by second principal components. The volatile components showed a significant difference between 0 and 5 days, or 5 and 10 days of pickling periods (p < 0.05). No significant difference of volatile compounds could be observed after the 10th day of fermentation. The formation of flavor compositions is closely related to the presence of microorganisms. In our previous study,^[16] it was shown that the flavor components of uncooked pickled wax gourd was formed by fermentation of LABs and other microorganisms, and the variety of yeasts was relatively single. Combined with the microbial diversity of pickled product, the flavor formation process can be divided into three stages. First, the heterofermentative LABs have an active role in flavor formation, such as Leuconostoc mesenteroides. The second stage is the fermentation of homofermentative LABs, including Lactobacillus. The third stage is mainly caused by the interactions between various metabolites and decomposition or synthesis reaction of compositions in wax gourds. According to previous reports,^[6,11] LABs detected during the pickled processing of vegetables, are mainly Leuconostoc, Lactobacillus, and Weissella, of which Leuconostoc and Lactobacillus play a major role in the fermentation. Shim et al.^[13] also reported similar conclusions. In addition, Chang et al.^[5] detected the presence of yeast in Korean kimchi, the total number of which stayed stable and showed no significant effect on the fermentation. It is in consistent with our results.

FIGURE 2 PCA analysis of the volatile components in wax gourd during fermentation.

With the start of fermentation, Leuconostoc mesenteroides gradually became the dominant microbes for their characters of rapid growth and acid production.^[2] Due to the lack of glycolytic enzymes, Leuconostoc spp cannot process in Embden-Meyerhof-Parnas (EMP) pathway, but it can convert sugar into 6-phosphate glucose, and generate a variety of metabolites including lactic acid, acetaldehyde, acetic acid, and ethanol, by phosphoketolase (PK) pathway to consume the glucose. In addition, Leuconostoc mesenteroides can also conduct citric acid fermentation, and produce acetic acid, diacetyl, acetoin, 2,3-butanediol, and other flavor components.^[2] At the 5th day of fermentation, the volatile components in pickled wax gourd are mainly acetoin (39.7%), 2,3-butanediol (21.85%), ethanol (10.38%), and acetic acid (5.81%), which are closely dependent on the fermentation of Leuconostoc.

According to Cogan et al.,^[19] the citric acid fermentation of Leuconostoc was related to pH values, the decrease of pH values may affect the recovery of acetoin. As the fermentation continued, pH began to decrease, the metabolism of Leuconostoc mesenteroides was restrained, and gradually replaced by homofermentation bacteria, including Lactobacillus, and entered the second stage of fermentation. The dominant microorganism at this stage is Lactobacillus plantarum, which belongs to homofermentation bacteria, it can convert glucose to pyruvate by EMP pathway, and further convert to lactic acid. The conversion rate can reach 80–85%. In this process, ethanol, acetic acid, acetoin, and succinic acid can be generated. As it can be seen in Fig. 1, with the proliferation of Lactobacillus, sugar was gradually consumed and almost exhausted in the 10th day, and the pH value also reached the minimum of 3.45. For the flavor compositions in the 10th day, the content of acetic acid increased gradually, ethyl acetate increased with the increasing of acetic acid, and acetoin was gradually reduced to 2,3-butanediol compared to the 5th day. In addition, more isoalcohols, including isobutanol, isopropanol, and isoamyl alcohol (3-methyl-1-butanol and 2-methyl-1-butanol) were found in the 10th day, and studies have shown that these substances were produced by yeasts in pickled vegetables.^[20]

With the exhaustion of sugar, the second stage of fermentation was ended, and the fermentation entered the after-ripening stage. At the late period of pickling, isoalcohols including 3-methyl-1-butanol and 2-methyl-1-butanol gradually increased, esters including methyl acetate, methyl propionate, amyl formate were also formed. The content of volatile components including ketones and aldehydes in pickled wax gourd were relatively less, except 3-hydroxy-2-butanone is formed by the lactic acid fermentation, other ketones or aldehydes such as 1-penten-3-one, 1-octen-3-one, cis-2-heptenal, and trans-2-pentene aldehyde may be derived from enzymatic reaction and chemical oxidation reaction.^[21] In this stage, the fermentation is mainly composed of interactions between various metabolic components in complex microbiota system, including the LABs and yeasts in pickled wax gourd. In addition, formation of small amounts of dimethyl sulfide (dimethyl sulfide and dimethyl disulfide) found in pickled wax gourd, was proven to be related to cysteine and other sulfur-containing amino acids.^[22] The contributing rate of each volatile composition evaluated by aroma analysis with ENT method at different periods of pickling process, showed a similar situation to flavor analysis by GC–MS methods. Therefore, it is a feasible practice for flavor evaluation of pickled vegetables.

CONCLUSIONS

In this study, it was shown that the RS content and pH value varied depending on the microbial amounts in early pickling process of the wax gourd. They reached the lowest level at the 10th day. High content of oxalic acid existed in pickled wax gourd. The citric acid content increased significantly in the first stage of fermentation and decreased after 10 days of fermentation. Lactic and butyric acid content were to maintain the upward trend in the whole pickling process. The content of malic acid and succinic acid fluctuated during fermentation. A total of 63 volatile components contribute to the flavor formation of pickled wax gourd, which include alcohols, acids, aldehydes, ketones, esters, silicones, and others. These components significantly varied at early stage of pickled processing of wax gourd according to the PCA analysis by ENT method. The formation of flavor components and change were dependent on the community diversity in pickled system, including special species of Leuconostoc spp., Lactobacillus spp. and other LABs of heterofermentation, as well as yeasts. To some extent, this article studied the flavors formation mechanism of natural pickled wax gourd, which was the most powerful contributors to the product quality assessing, production standards establishing and process optimizing for the eastern Zhejiang pickled melon.

FUNDING

The authors are grateful for the financial support from National Natural Science Foundation, China (No. 31171735), the support “Sponsored by K. C. Wong Magna Fund in Ningbo University” and the critical program of agriculture field in Ningbo (2012C10016), China.

Additional information

Funding

The authors are grateful for the financial support from National Natural Science Foundation, China (No. 31171735), the support “Sponsored by K. C. Wong Magna Fund in Ningbo University” and the critical program of agriculture field in Ningbo (2012C10016), China.