Effects of thermal and nonthermal processing technology on the quality of red sour soup after storage

Abstract In the present study, we investigated the effects of thermal preservation, such as pasteurization, and nonthermal preservation, including irradiation, sodium dehydroacetate (SDHA), and nisin, on the quality of red sour soup after storage. Single‐factor experiments were used to optimize the parameters of different processing technologies, and the best irradiation dose and heating temperature were 4 kGy and 85℃, respectively. The optimal additive amounts of SDHA and nisin were 150 mg/500 g. During the shelf storage experiment, prepared red sour soup was stored at room temperature in the glass bottles, and further analyses were carried out up to 5 weeks of storage. The quality of red sour soup was evaluated by microflora and sensory analysis. The results showed that Lactobacillus, Streptomyces, Pediococcus, Pichia, Kazachstania, and Candida were the main microorganisms in all samples, and there were no harmful microorganisms. The sensorial attributes were observed, including different parameters, such as odor, organic acid content, color, taste, texture, apparent viscosity, and thixotropy. All of the data showed that the irradiated groups were more dramatically changed compared with the other groups, while these changes did not directly affect the sensory quality of the products. Consequently, irradiation could be used as an ideal quality preservation method for the red sour soup to reduce the impact of heat treatment and chemical additives on the quality of characteristic food.

At present, many methods are available to prolong the shelf life of food, including heat treatment, drying, freezing, high pressure, coating, and pickling (Zim et al., 2017). Among them, in the salt or vinegar environment, fermentation pickling with lactic acid bacteria as functional bacteria is a more commonly used preservation method for fruits and vegetables (Mani, 2018). Therefore, if processed properly, the shelf life of red sour soup will be effectively extended (Nout, 1994). Pasteurization is a traditional and relatively mild thermal sterilization method, which can kill pathogenic bacteria and most spoilage bacteria, showing a good effect on inhibiting enzyme activity. To further ameliorate the defect of uneven heating of traditional pasteurization, won II Cho et al. (2016) have changed conduction heating to ohmic heating, and such procedure has been successfully applied in Korean fermented chili sauce. The spore inactivation rate of Bacillus in fermentation broth reaches 99.7%, which effectively improves the shelf-life of chili sauce. In the field of physical sterilization, in addition to pasteurization, there are also irradiation sterilization technologies using 60 Co, 137 Cs, or electron accelerators as irradiation sources. It destroys the DNA of microorganisms in food by high-energy radiation instead of heating, which is also known as "cold" pasteurization (Ahn et al., 2013;Inabo, 2006). Currently, irradiation sterilization has been widely used in vegetables, fruits, nuts, cereals, meat products, and other agricultural and livestock raw materials because of its terminal sterilization, nonheating, and nonadditive characteristics (Ajibola, 2020). Chemical preservatives (benzoate and sorbate) are used as a type of cost-efficient storage technology for fermented food. Sodium benzoate and sodium dehydroacetate (SDHA) are often used for the preservation of pickled pepper, and the results show that the expected target can be achieved when the addition of SDHA is 0.03% (Xie et al., 2008). However, it is urgently necessary to develop more reliable methods for food preservation due to the potential microbial resistance and food safety of chemical preservatives (Russell, 1991;Yamazaki et al., 1998). Relatively, the exploitation and application of natural preservatives, such as chitosan, polyphenols, and bacteriocin, have become a new trend in the preservation of fermented food (Delavar & Sedaghat, 2020;Gutiérrez-del-Río et al., 2018;Pal, 2018;Santos et al., 2018). Among them, a Lactobacillus metabolite named nisin has been widely used in dairy products, meat products, and seafood because of its excellent safety and significant inhibitory effect on most Gram-positive food pathogens and spoilage bacteria (Druggan & Iversen, 2014). As one of the characteristic ecological food, the shelf quality of red sour soup has an important impact on the maintenance of brand value. It is of great significance to choose the appropriate technology among various preservation methods to avoid shelf-life deterioration issues. Based on the abovementioned facts, we aimed to compare the effects of pasteurization, irradiation sterilization, SDHA, and nisin on the microflora and sensory quality of red sour soup after storage. Collectively, our findings provided technical support for shelf-life quality optimization of red sour soup.

| Materials and chemicals
Red sour soup was collected from Jinsha Guan Xiang Fang Seasoning Food Co., Ltd. (Guizhou, China). SDHA, nisin, L-lactic acid (standard product), and glacial acetic acid (standard product) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Activated carbon, ammonium dihydrogen phosphate, and phosphoric acid were obtained from Chengdu Jinshan Chemical Reagents Co., Ltd.
The radiation dose of the sample was determined by silver dichromate, and the dose absorption rate was less than 7.5 kGy·S -1 . The additive dose in the RN and RD groups was 10, 50, 100, 150, and 200 mg/500 g. The shelf life of samples treated by the optimized conditions was tested for 5 weeks. During the experimental period, the samples were placed in glass sample bottles and stored at room temperature. The colony count in samples was detected according to GB 4,789.2 (CFDA, 2016).

| Illumina miseq sequencing
Microbial DNA extraction and PCR amplification were carried out as previously described with minor modifications (Xie et al., 2018). Briefly, 100 ml sample was centrifuged at 4,000 rpm for 5 min, and the precipitate was collected. Total DNA of the samples was isolated using E.Z.N.A™ Mag-Bind Soil DNA Kit (Omega Bio-Tek, America) and then sent to Sangon Biotech (Shanghai) Co., Ltd., for sequencing. During the experiment, the PCR amplification was completed in two rounds. In the first round of amplification, after the genomic DNA was accurately quantified using Qubit3.0 dsDNA detection kit (Life Technologies, America), the amount of DNA that should be added to the PCR was determined. The primers used in PCR were fused with part of the linker sequence of the sequencing platform, and the target amplification regions were the V3-V4 and ITS1-ITS2 regions of the 16S rRNA gene. Among them, the amplification primers for the V3-V4 region were 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC); and the ITS region primers were ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC). The PCR product was subjected to 2% agarose gel electrophoresis to confirm whether the length of the amplicons was correct. Then, Agencourt AMPure XP magnetic beads (Aisijin Biotechnology, Hangzhou) were used to recover the target strips. Qubit3.0 dsDNA detection kit was used for detection and quantification, and the libraries were mixed at an equimolar ratio. The library was sequenced using the Illumina MiSeq® platform (Illumina, America), and the sequencing mode was 300 bp at both ends.

| Electronic nose analysis
An electronic nose (Airsence PEN3, Germany) was used to analyze the odor difference of samples preserved under different conditions. Briefly, 1.0 g sample was accurately weighed and placed in a headspace bottle, followed by incubation in a water bath at 60 ℃ for 20 min. Odor fingerprint analysis conditions were set as follows: the sampling interval was 1 s, the sensor self-cleaning time was 40 s, the sensor zeroing time was 10 s, the sample preparation time was 5 s, the sampling analysis time was 120 s, and the injection flow rate was 400 ml/min.

| Total acid and organic acid analysis
The analysis of total acid in the red sour soup was carried out according to T/CNFIA 117 (CNFIA, 2020) and GB/T 12,456 (SAC, 2008).
The analysis of organic acid was conducted using liquid chromatography as previously proposed (Xiong et al., 2012).

| Sensory evaluation
The color, smell, taste, and tissue state of red sour soup were investigated according to the requirements of "sour soup seasoning" (CNFIA, 2020). Briefly, 10 experimental personnel were invited to participate in the sensory evaluation, and all participants were trained according to the relevant provisions of GB/T 16,291.1 (SAC, 2012). Sensory evaluation was conducted by a scoring system.
The full score range of color was 30, and a score of 0-10, 10-20, and 20-30 indicated poor, fair, and well, respectively. The full score range of smell and taste was 40, and a score of 0-20, 20-30, and 30-40 indicated poor, fair, and well, respectively. The full score range of tissue state was 30, and a score of 0-10, 10-20, and 20-30 indicated poor, fair, and well, respectively.

| Rheological property analysis
To study the rheological properties of samples preserved under different conditions, the rheologic behavior of red sour soup was analyzed by rheometer (DHR-1, TA Instruments, America), including static shear test and rheological behavior in oscillatory mode. For the static shear test, the apparent viscosity and shear stress of red sour soup were measured at 25 ℃ with a shear rate ranging from 0.1 s -1 to 100 s -1 . The Ostwald model was used to assess shear rate (x) and shear stress (y), and flow index (n), consistency coefficient (k), and complex correlation coefficient R 2 were recorded. Ostwald equation: y = kx n (1). Rheological behavior was tested in the oscillatory mode. A plate mold with a diameter of 60 mm was selected, the gap was 1.0 mm, the angular frequency was 1 Hz, and the stress was 5.00 Pa. The red sour soup was placed on the measuring platform, and the cover plate was pressed down. The storage modulus of red sour soup was tested at the temperatures programmed from 25 ℃ to 90 ℃ and then from 90 ℃ to 25 ℃ for 1 min at a rate of 5 ℃/ min.

| Data analysis
The data were presented as mean ±standard deviation (SD), and the

| Single-factor optimization for different preservation methods
Before we investigated the effects of pasteurization, irradiation, SDHA, and nisin on the shelf quality of red sour soup, the heating temperature, irradiation dose, and dosage of preservatives were optimized by single-factor experiments, and the results were shown in Figure 1. The colony count was 1.2 × 10 6 CFU/g in the red sour soup without preservation treatment, while the total bacteria changed dramatically after thermal, such as pasteurization, and nonthermal (irradiation, SDHA, nisin) processing technology. For pasteurization, the sterilization temperature was negatively correlated with the colony count. When the temperature reached 85 ℃, the colony count in the sample was only 200 ± 10 CFU/g and there was no significant change in the sensory quality of the samples. The results of the single-factor experiment also showed that the colony count of the red sour soup was sharply decreased when the irradiation dose was more than 2 kGy. At 4 kGy, the colony count was less than 1,000 CFU/g, reaching 900 ± 43.59 CFU/g. Our results were consistent with previous studies that most pathogenic microorganisms in food are killed when the irradiation dose is 1-10 kGy (Morehouse & Komolprasert, 2004). BPOM (2006) has also pointed out that the radiation dose below 10 kGy is harmless, and there is no need to carry out relevant toxicological experiments. The associations between the total colony count and the dose of SDHA or nisin showed the same trend, while the antibacterial effect of the latter was significantly better compared with the former. When the dose was 150 mg/500 g, the colony count in the RD group was 1.6 × 10 5 CFU/g, and the colony count in the RN group was 1.8 × 10 4 CFU/g. As the dose was increased, the total colony count changed insignificantly. Similarly, the dose and effect of SDHA and nisin have also been proved in the studies of Xie et al. (2008) and Chang et al. (2019). Therefore, the temperature of the RP group was set to 85℃, the irradiation dose of the RI group was limited to 4 kGy, and the additive dose of SDHA and nisin of the RP and RD groups was 150 mg/500 g in the experiment. Subsequently, a 5-week shelflife experiment was carried out on the red sour soup samples with different treatments. The results showed that the colony count in all treated groups was much lower compared with the RCK group during the experimental period. The RD group and RCK group showed an upward trend in the total colony count at the 3rd week, reaching 8.2 × 10 5 CFU/g and 7.4 × 10 6 CFU/g at the 5th week, respectively.

| Microflora analysis of samples under different preservation conditions
To clarify the composition of microbial flora in red sour soup after 5 weeks of storage, Illumina MiSeq sequencing with F I G U R E 1 Effects of different preservation methods on the colony count in red sour soup. (a) describes the change of the aerobic plate count in the sample with the increase of pasteurization temperature; (b) describes the change of the aerobic plate count in the sample with the increase of irradiation dose; (c) describes the change of the aerobic plate count in the sample with the increase of SDHA dosage; (d) describes the change of the aerobic plate count in the sample with the increase of nisin dosage high-throughput, parallel, and quantitative characteristics was used to determine and analyze the experimental and blank control samples (Zhai et al., 2016). The Chao and Shannon indices were used to evaluate the microbial diversity in the samples. Table 1 shows that the microbial diversity of the RI group and RP group was the lowest among all samples (Figure 2). On the other hand, the bacterial diversity of the RN group and RD group was similar to the RCK group, while the abundance of fungal species was between the RI group and RP group. Besides, to further verify the distribution of microbial species in the samples, the principal coordinate analysis (PCoA) in the β-diversity analysis was also used for auxiliary explanation, and detailed information was given in Figure 3.
The Venn diagram depicted the common or unique species classification in different treatment groups. The total operational taxonomic unit (OTU) number of bacteria and fungi was 206 and 100, respectively. The number of OTUs shared by bacteria was 63, and that shared by fungi was 36. The histogram of relative abundance of dominant species showed that lactobacillus, streptomyces, and pediococcus were the three most important genera of bacteria in different treatments, and pichia, kazachstania, and candida were the most important fungi affecting the quality of red sour soup (Figures 4-5). The dominant microorganism in our study was similar to previous studies (Lin et al., 2020;Wang et al., 2020). In comparison, the relative abundance of lactobacillus and pichia was higher in the RI group, indicating a stronger killing ability than other microorganisms. However, the excessive reproduction of lactobacillus and pichia will also lead to the occurrence of postacidification, gas production, mildew, and other adverse events. Therefore, it is necessary to further clarify how to properly sterilize the product (Zhang, 2018).  All of the results showed that pasteurization and irradiation treatment affected electronic nose data and organic acid content in red sour soup. The sensory changes of the pasteurization group might be attributed to the loss of volatile components caused by heating, while the differences of the irradiation group might be attributed to sulfur, methyl, and ketaldehyde compounds produced in the process (Yoo et al., 2003). Organic acid is the main contributor to the special flavor of red sour soup. We found that the total acid content in each treatment group was not significantly different. However, the content of lactic acid in each treatment group was lower compared with the RCK group, and the RI group showed the lowest content.

| Effects of different preservation methods on the flavor of red sour soup
This finding might be attributed to the inhibitory effect of preservation technology on the growth of lactic acid bacteria, resulting in the retarded postacidification (Figures 8-9).

| Rheological properties of red sour soup under different preservation conditions
Rheological data play an important role in the analysis of flow conditions in food processing, such as pasteurization and aseptic pro- When the temperature was greater than 60℃, the tanδ value of RCK and RD groups was less than that of RI and RP groups, indicating that the gel properties of RI and RP groups were more seriously weakened at a higher temperature. This might be attributed to the degradation of pectin in chili, tomato, and other raw materials in red sour soup by irradiation and heat treatment (Dogan and Kayacier, 2007;Jiang et al., 2019).
Among them, the preservation effect of irradiation and pasteurization was comparable to chemical and natural additives. Lactic acid bacteria and Pichia were the dominant microorganisms in red sour soup, which might be the important reasons for gas production in the later stage. Besides, irradiation could affect the odor, acidity, and rheological properties of red sour soup. However, these changes had no significant effects on the sensory quality of the product. preservation method for red sour soup seasoning. The preservation effects of physical cold sterilization and natural additives need to be further explored.

ACK N OWLED G M ENTS
The authors thank the department of science and technology of Guizhou Province and Guizhou Academy of Agricultural Sciences for the financial support to conduct this research. Data curation (equal); Investigation (equal); Methodology (equal).