Isolation and identification of the spoilage microorganisms in Sichuan homemade Paocai and their impact on quality and safety

Abstract The spoilage microbiology in Paocai (fermented vegetables) affects not only the quality of this popular traditional Chinese food but also its safety. This study aimed to isolate and identify the microorganisms commonly leading to spoilage in homemade Paocai from the Sichuan region and, further, to scientifically assess the impact of these microorganisms on product quality and safety. Seven putrid Paocai samples were collected from 7 families in different Sichuan cities. From these, 45 strains were isolated by means of a nutrient agar medium and rose bengal agar. All of the 22 strains (5 fungi and 17 bacteria) with different colonial morphologies and corruption phenomenon in Paocai were determined 16S rDNA/18S rDNA gene sequences. Bacteria were identified as Bacillus spp. (16 strains) and Staphylococcus saprophyticus (1 strain), while the 5 fungi were identified as Candida spp. (3 strains), Kodamaea ohmeri (1 strain), and Geotrichum candidum (1 strain). Based on the results of identification, 7 representative strains of different species were determined as the spoilage characteristics in paocai. All the representative strains metabolized to produce nitrite. Strain SPF21 (K. ohmeri) has a particularly serious impact on the crunchiness of Paocai; however, strains SPF19 (Bacillus subtilis) and SPF21 have the greatest influence on its color. All five of the fungi were seen to form a film on the surface of the Paocai, with SPF5 (G. candidum) exerting the most extreme influence. The growth characteristics in the broth showed that all the representative strains investigated metabolized most of the carbohydrates and were able to tolerate the salinity and acidity of the ordinary homemade Paocai. Moreover, these strains were found to have obvious impacts on the volatile components of Paocai, especially SPF2 and SPF8, with higher dimethyl disulfide and dimethyl trisulfide, which were found to have a pungent odor when highly concentrated.


| INTRODUC TI ON
Paocai, or fermented vegetable, has a history of thousands of years in China, and Sichuan Paocai is considered to be the most famous of these traditional recipes . Paocai is a brine-salted pickle, fermented mainly by naturally adherent microorganisms on the vegetable, especially lactic acid bacteria (LAB). The raw material in Paocai is crisp and tender vegetables, such as radish, cabbage, mustard, and cowpea. Paocai is often served as an appetizer in the traditional Chinese diet. Its uniquely appetizing features include sourness, a distinct aroma, freshness, and crispness, while its nutritional advantages include strengthening the spleen, promoting digestion, antioxidation, antibacteria, lowering cholesterol, prevention hypertension, and tumors, all of which explains the popularity of this dish (Seong-Eun et al., 2019;Xia et al., 2017). However, Paocai is usually homemade by Sichuan families all year round, so the success of the fermentation process tends to be dependent on the indigenous microflora adhering to the vegetables. Moreover, the quality is affected by factors such as the environment and the changing seasons, which results in numerous uncertainties in the process and outcome (Xiong, Guan, Song, Hao, & Xie, 2012). When either the temperature or the aerobic environment is unfit for the growth of LAB, spoilage microorganisms flourish, causing the corruption of Paocai in terms of flavor, biofilms on the surface, color deterioration, and harmful compositions, which severely affect the quality, edibility, and safety of the Paocai.
Previous research has reported Bacillus spp., yeast, and aerogenic LAB as the dominant spoilage microorganisms in fermented vegetables (Wang, Zhang, Chen, & Zhang, 2018;Wang et al., 2015;Zhang et al., 2018). The reproduction and metabolism of the spoilage microorganisms greatly influence the quality of Paocai for the following three reasons: first, their metabolites negatively affect the flavor and sensory quality; second, some microorganisms can produce toxins in the metabolic process that seriously affect the safety of products; and, finally, some microorganisms can convert nitrate into nitrite through metabolic processes, thereby increasing the risk of cancer. Therefore, the purpose of this study is to provide theoretical basis for improving the quality and safety control of homemade Paocai by determining the species of spoilage microorganisms and their influences on the quality of Paocai.

| Sample collection
Samples of Paocai were collected from seven families in seven different cities in Sichuan, in July, 2017, the season in which Paocai is most prone to corruption. The samples were numbered A-G, respectively, and transported to the laboratory in an icebox for immediate analysis. The spoilage phenomena and collection information with respect to the Paocai samples were shown in Table 1.

| Isolation of spoilage microorganisms from samples
The 7 Paocai samples were diluted in different gradients and cultivated on nutrient agar media (peptone 10 g, beef extract 3 g, NaCl 5 g, agar 15-20 g, distilled water 1,000 ml) and rose bengal agar (peptone 5 g, glucose 10 g, KH 2 PO 4 1 g, MgSO 4 ·7H 2 O 0.5 g, agar 20 g, 1/3,000 bengal red solution 100 ml, distilled water 1,000 ml, chloramphenicol 0.1 g) to isolate bacteria at 37°C for 24 hr and fungi at 25°C for 48 hr, respectively. The isolated strains were further purified and stored in 15% glycerol solution at −70°C for subsequent experiments.

| Paocai manufacture and inoculation of tested strains
Summer radish is the most commonly used ingredient in Paocai. In this assay, 200-g-fresh summer radish was washed and dried on the surface, cut into strips of 10 mm × 10 mm × 80 mm, and placed in a fermentation tank, to which was added the same weight of brine (salt content 8%) (Zhou, Liu, & You, 2018
The 16S rDNA and 18S rDNA genes were sequenced using an ABI 3730 sequencer (Applied Biosystems). The nucleotide sequence data were deposited in the GenBank nucleotide sequence database. The accession numbers are shown in Figures 1-3. Multiple sequences were assembled and aligned with the Clustal W program (Thompson, Cronin, & Martin, 2010). Type strain sequences for comparison were blasted against the GenBank database. The phylogenetic trees were constructed using MEGA 5.1 under the Kimura two-parameter model and bootstrapped with 1,000 replicates.

| Spoilage characteristics of representative strains in pickled summer radish
Based on the results of identification, the representative strains of different species were determined as the spoilage characteristics in paocai. The method of Paocai manufacture and inoculation of representative strains was described in Section 2.3.

| Physical and chemical analysis of Paocai
The chromatic aberration of the samples was measured by means of a colorimeter (Beijing Kang Guang Instrument Co. Ltd.), and the values of L*, a*, and b* were, respectively, recorded. The crunchiness of the samples was determined by means of a TA-XT plus Texture Analyzer (Lotan Science Co. Ltd.). The samples were cut into blocks measuring 10 mm × 10 mm × 4 mm. The probe was P/5, while the test mode was compression test force, the operation started for return, the pretest speed was 5 mm/s, the measured speed was 2.00 mm/s, the post-test speed was 10 mm/s, and the displacement was 5 mm (Kamat, Dash, & Balasubramaniam, 2018). Each Paocai sample (summer radish 5 g + brine 5 g) was crushed and filtered with three layers of gauze, after which the pH value of the sample was measured using a pH meter (Nanjing Century Ark Analytical Instrument Co. Ltd). The content of nitrite in the sample was determined using the N-(1-naphthyl)-ethylenediamine dihydrochloride spectrophotometric method (Ding et al., 2018).

| Analysis of flavor components by SPME-GC-MS
Each Paocai sample (summer radish 2.5 g + brine 2.5 g) was treated at 40°C for 30 min using a water bath after crushing. The volatile compounds were subsequently extracted for 30 min with a manual solid phase microextraction (SPME) device equipped with a 50/30 μm DVB/CAR fiber (Supelco). After extraction, the fiber was inserted into the GC injection port and the analytes desorbed for 5 min at 250°C.
The data were qualitatively analyzed by references and matching results, which compared with the standard mass spectrometry provided by NIST.11. The relative content of the volatile components was determined by the area normalization method.

| Growth and carbohydrate metabolism characteristics of spoilage strains in broth
The growth of the spoilage strains at different temperatures ( 6.0, and 7.0) and salt concentrates (0%, 2%, 4%, 6%, 8%, and 10%) were detected after incubation at 30°C for 7 days (Yang, Cao, Cai, & Terada, 2010). Glucose, fructose, sucrose, and starch were used as the sole carbon sources for testing the metabolism of carbon.

| Data analysis
Data used in physical and chemical analysis were subjected to an analysis of variance, and Tukey's test was used to identify the differences (p < .05) between means using the GLM procedure of SAS (SAS Institute, Inc.).    was the main spoilage strain, especially B. subtilis, which can cause turbidity, rancidity, and film formation ).

| Identification of suspicious spoilage microorganisms
In the current study, the fungi in the spoiled pickles were divided into 3 species (Figure 2). Strains SPF34 and SPF41 were found to be related to Candida tropicalis and identified as C. tropicalis. In addition, strains SPF2 and SPF21 were related to Candida xestobii and Kodamaea ohmeri. In all, the results showed that five fungi, belonging to two genera, along with Candida spp. were the main spoilage microorganisms. Wang et al. also found C. tropicalis to be the main spoilage microorganism responsible for the formation of a film on the surface of Paocai (Wang et al., 2015).
As strain SPF5 was far from the other fungi in the phylogenetic tree, its result was not shown and, therefore, an individual phylogenetic tree was built for its identification (Figure 3)

| Effect of the spoilage organisms on the quality of Paocai
The seven strains impacting most negatively on the quality of the Paocai samples were screened, as shown in which all reduce crunchiness. Pectin substances, which can be decomposed by yeast and mold, and degraded sugars and acids, also significantly impact upon the crunchiness of pickles (Hernández, Martín, Aranda, Pérez-Nevado, & Córdoba, 2007). Previous reports have shown that G. candidum has the ability to produce pectinase, which may explain it impact on the crunchiness (Yu, Huang, & Du, 2018); however, K. ohmeri has not been confirmed as a cause of Paocai spoilage.
Vegetable color, such as the red of summer radish, is another indication of Paocai quality. As shown in Table 2, the greater the value L and the smaller the value b, the better the quality of the Paocai.
The results showed that the L values of the samples inoculated with isolates were lower than those in the control, thus indicating that different strains have a certain effect on darkening the Paocai. Strain SPF2 (C. xestobii) was found to significantly affect the brightness of the raw material. The values of a and b indicate that strains SPF8, SPF19, and SPF21 discolored the pickled radish from red to yellow and blue, indicating that these spoilage organisms have a significant effect on the color of Paocai.
Moreover, the formation of a biofilm on the surface of brine is an obvious spoilage phenomenon, always accompanied by a deterioration in taste and smell. From the results, strains SPF5 and SPF21 formed the most serious biofilms, while the biofilm effect of SPF34 was also obvious. If these microorganisms exist in Paocai, a biofilm will occur when LAB is not dominant.

| Physicochemical characteristics of the spoilage organisms
The seven screened strains were cultured in liquid media under different conditions. The results are shown in Table 3. Most strains grew optimally at 25-45°C, with the exception of SPF8, which could not grow at 45°C, while strains SPF5, SPF19, and SPF34 could not grow at 15°C. These results explain why Paocai is more likely to rot in summer and autumn, when daily temperatures average between 25 and 45°C. The results of the pH range indicate that strains SPF5 and SPF2 grow at a relatively narrow pH range of 4-6, which is the acidity at the beginning of Paocai fermentation. Therefore, such strains are generally growing in competition with LAB at the beginning of fermentation, sometimes causing the process to fail. However, the strains SPF34 and SPF42 can grow in a wide pH value range of 3-7, so, even when the paocai ferments normally and the acidity is reduced, the growth of such microorganisms cannot be inhibited when the conditions are suitable. The introduction of oxygen and oil, for example, or a rise in temperature will assist in the rapid multiplication of these microorganisms, causing decomposition of the Paocai.
All the tested strains were found to tolerate salt concentrations ranging from 4% to 6%, which is the same concentration commonly used in homemade Paocai. In order to inhibit the growth of the spoilage microorganism, the salt concentration is sometimes increased to 10%, which makes the paocai taste especially salty and has a harmful effect on health. Strain SPF8 (S. saprophyticus) can grow at a salt TA B L E 2 The effect of the screened strains on the quality of ped summer radish

| Effects of spoilage microorganisms on volatile components of Paocai
Paocai is famous for its sour fragrance and flavor. The most important flavor components in Sichuan Paocai are dimethyl sulfides and aldehydes, among others  Similarly, all samples contained dimethyl disulfide and dimethyl trisulfide; however, the concentrations were higher in the samples inoculated with spoilage bacteria than in the control, especially those with strains SPF2 and SPF8, which are the main flavor substances in many Paocai products. Zheng, Zheng, and Li (2007) and Yoon et al. (2008) determined the flavor substance in Korean pickle and also found sulfur compounds, including dimethyl disulfide and dimethyl trisulfide, to be the main compounds (Yoon et al., 2008;Zheng et al., 2007). The flavor of a sulfur substance can be experienced at a low threshold (0.16 µg/kg), therefore, when the content is too high, it will produce a typical irritant smell, which will seriously affect the overall flavor and taste of Paocai. Moon, Chang, Kim, and Chang (2014) reported that sulfur compounds, including dimethyl disulfide and dimethyl trisulfide, deteriorate the flavor of kimchi (Moon et al., 2014). Furthermore, significantly high dimethyl trisulfide content, which is consistent with this experiment, obviously softens Paocai (Zhao et al., 2016). Notably, strain SPF34 contains 3-thietanol, which becomes foul smelling and toxic when the relative content is high. In this study, the content of 3-thietanol reached 13.91%, thus confirming that contamination with SPF34 will affect the flavor and safety of Paocai.
In addition, all samples were found to contain aldehydes and siloxane, although these substances were less concentrated in the samples inoculated with spoilage bacteria than in the control. The above results indicate that spoilage microorganisms can degrade or inhibit flavor components in Paocai and alter the sensory perception of the food.

| CON CLUS IONS
The 22

ACK N OWLED G M ENTS
This study was supported by the "Chun-hui Project" of China.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest or relationship, financial, or otherwise.