Interaction between bacterial diversity and biogenic amines production in a salted mackerel stored at soft frozen (−7℃–0℃) storage

Abstract The bacterial diversity of salted mackerel “one‐night courtyard” at soft frozen area (−7℃–0℃) storage was studied. The fish samples at 0, 14, 21, 28, and 35 days were analysis for bacterial structure using high‐throughput sequencing technologies (HTS) and biogenic amines using high‐performance liquid chromatography (HPLC). The analysis results of HTS showed that the dominant bacteria species was varied gradually following with storage time. On the 0th, 21st, and 28th days of storage, dominant Vibrionaceae was accounting for 71.70%, 59.16%, and 70.68% of the total sequences analyzed, respectively. On the 14th and 35th days, Shewanellaceae was the dominant bacterial, accounting for 87.53% and 70.95% of the total sequences analyzed, respectively. In addition, 21st and 28th days, an abundance of Operational Taxonomic Units (OTUs) was top. The dominant bacterial of Proteobacteria, Firmicutes, was producer of biogenic amines. Furthermore, the analysis results of HPLC shown the total biogenic amines of maximum amount 363.01 mg/kg in the sample of HY.14 lower than 1000 mg/kg of the FDA regulation. The range ability of cadaverine was obvious following with the storage time. Cadaverine was 87.36 mg/kg on the 0th day, and it was maximum amount of 276.89 mg/kg on the 14th days. Putrescine was 20 mg/kg on the 0th day and maximum amount of 55.04 mg/kg on the 28thdays of storage. The tyramine was smallest amount of production, and the largest amount was 38.99 mg/kg on 28th, and the smallest amount was 11.97 mg/kg on 35th. Nevertheless, the maximum amount of histamine was 55.04 mg/kg on the 0th day and about 23.14 mg/kg of histamine was little change from 14th to 35th days of storage. Dominant bacteria affect the change of biogenic amines. The study can help understand the interaction between microbial flora and biogenic amines in the salted mackerel of one‐night courtyard.


| INTRODUC TI ON
The salted fish is a traditional processed aquatic product consumed in China. It is popular because of its easy preservation, special flavor, and rich nutrition (Dziezak, 1986). Especially, the salted fish of one-night courtyard (salted fish pickled overnight) of coastal area in Guangdong of China has a strong fresh flavor. The salted fish of onenight courtyard belongs to low salt and coarse dry curing products, can eat after 12 hr of pickling time, and maintain the freshness of the fish and keep the fish nutrition, combined with domestic logistics transport developed, the salted fish of one-night courtyard sells well all over the country, so the eat-by freshness date is extended to more than one month. Famous fish pickles include salted mackerel, redfish and so on (Wu et al., 2016). The flavor of salted fish is the resultant of fish characteristics and the activity of the microbial communities involved in the pickling process, the salt concentration, and the pickling conditions. The variety of microorganisms involved in the pickling process affect the quality and the flavor of salted fish. The microorganisms such as Lactic acid bacteria, Micrococcus, and Staphylococci, which have enzyme such as proteases, lipases, and amino acid decarboxylases, can promote the decomposition of proteins into amino acids and amino acids convert into biogenic amines or break up fats into short chains of volatile fatty acids and esters (Liu et al., 2016). The metabolic products of microorganisms not only impart a unique flavor to pickled fish products but can also form harmful products (Bianchi et al., 2011). In addition, some Lactic acid bacteria, Micrococcus, can produce acids and antibiotics, which can inhibit the growth of spoilage bacteria and pathogens such as Staphylococcus aureus, Salmonella, Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus in salted fish products (Papageoriou et al., 2018).
Biogenic amines, which are produced by microbial amino acid, decarboxylase to amino acids under favorable conditions, and the salted fish is rich in amino acids. Such as Escherichia coli can produce cadaverine from lysine and putrescine from ornithine in the fish products (Pessione et al., 2016). Lactobacillus curvatus can also produce histamine from histidine (Singh et al., 2012). The appropriate amount of biogenic amines contributes to normal physiological function within the body, but excessive biogenic amine consumption may lead to death in severe cases due to adverse effects on human health that include hypertension, hypotension, rash, headache, flushing, oral numbness, and digestive problems symptoms of food poisoning (Lee et al., 2012;Wilson et al., 2012).
The formation of useful and harmful substances is closely related to the bacterial structure. However, the structure and composition of microorganisms in crude processing pickled fish of one-night courtyard of South China are not comprehensive at present and in-depth understanding. The ecological composition of bacterial communities is one of the most important factors among those responsible for the properties of one-night pickling salted fishes.
High-throughput sequencing technology (HTS) is being widely applied to obtain a more comprehensive analysis of microbial diversity (Aldrete-Tapia et al., 2014;Ercolini et al., 2012). This technique leads to accurate identification of microbial taxa, including those present in low abundance. HTS technology can be carried out by the amplification of 16S rRNA regions coupled with the generation of multimillion reads, and it has enough potential for obtaining a complete coverage of microbial communities, for in-depth study of the complex interactions between the species present and amount of biogenic amines in a given microbial ecosystem.
The study aimed to analyze the bacteria species involved at different pickling and storing process of salted mackerel until 35 days, and understanding the effect of biological community structure of salted fish on biogenic amines. Biological community structure of salted fish samples was analyzed with HTS analysis conducted by Illumina Miseq of the bacterial 16S rRNA hypervariable regions V3 and V4. The information obtained through HTS approach of microbiology can provide the useful data in understanding the interaction between bacterial flora and biogenic amines, and improving the quality of salted fish.

| Salting method for fishes based on traditional of one-night courtyard of South China
The 15 fresh mackerels (about 25 cm, 225 g) were purchased in the same booth from the market of Zhongcun, Panyu, Guangzhou, Guangdong province, China, and send the samples with ice bag to lab in Guangdong University of Technology right now, and put the fresh fishes into refrigerator until use. Then, the fresh mackerels were salted, salting method for fishes based on traditional of one-night pickling salted fish of South China. All mackerels were washed clean in the laboratory, and some salts (10% of the wet weight of fish) were spread on them. The fishes were kept in a capped, sterilized bottle at room temperature. After 12 hr, the pickled fishes were kept in the soft frozen area (−7℃-0℃) of the refrigerator. Then, samples were collected from day 14 and taken every 7 days, and the microorganisms of the day 0 were used as the base control, so microbial community analyses were carried out after 0 (HY.00), 14 (HY.14), 21 (HY.21), 28 (HY.28), and 35 (HY.35) days of storage.

| Microbiological analyses
For the accuracy of the experimental data, 10 g fish meat samples were taken from three parts of the salted mackerel sample, respectively, and crushed and blended together of 30 g sample aseptically, then taking the mixed sample of 10 g, and diluted in 90 ml of sterile peptone water (8.5 g/L NaCl and 1.0 g/L peptone solution at pH 7.0), and crushed to make uniform solutions. Then, 1.0 ml homogeneous liquids were diluted according to the ten-fold dilution method.

| PCR amplification and sequencing
The DNA extracted as described above was used to study the bacterial diversity by high-throughput sequencing of 16S rDNA genes. In detail, PCR amplifications of the bacterial V3-V4 16S rRNA regions were carried out using the TaKaRa LA Taq ® (Takara Biotechnology Co., Ltd) and the primer pairs 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). Each PCR mixture was performed using 2.5 µl 10×Ex Taq Buffer, 1 µl dNTP (2.5 mM), 1 µl each of primer, 0.5 µl rTaq enzyme, 2 µl DNA template, and 17 µl ddH 2 O to make the final volume 25 µl. The PCR amplification program was designed as follows: initial denaturation at 94℃ for 5 min; 34 cycles of denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 2 min; and a final extension at 72℃ for 5 min. The PCR amplicons were visualized on a 1.5% agarose gel.
To reduce the randomness of the PCR reaction, each template was subjected to five parallel PCR reactions, and all the products were mixed for further study. The PCR product portions of all samples were multiplexed into a single pool using equal molecular weights and were concomitantly purified using the solid phase reversible immobilization (SPRI) based method of the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Takara Biotechnology Co., Ltd).
Sequencing libraries were generated using TruSeq ® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina HiSeq 2500 platform and 250 bp-end reads were generated.
Resampling for each sample according to the minimum sequence numbers was performed before the downstream analyses.

| Sequence data preparation and analyses
Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH (Magoc & Salzberg, 2011), a very fast and accurate analysis tool, designed to merge paired-end reads when at least some of the reads overlap the read generated from the opposite end of the same DNA fragment, and the splicing sequences were called raw tags.

| Biogenic amines determination
The analysis methods and HPLC systems for biogenic amines determination were described in a previous research paper (Qiao et al., 2020).
All biogenic amine standards, histamine dihydrochloride, tyramine dihydrochloride, putrescine dihydrochloride, and cadaverine dihydrochloride were purchased from Wako (Tokyo, Japan). The standard amines (50 mg) were dissolved in 0.1 M HCl. The standard amine solutions were diluted by 0.1 M HCl (each at 10 μg/ml) and used as the standard stock solution, respectively. Each fish sample of 5 g was homogenized with 50 ml of 20% trichloroacetic acid in centrifuge tube for 10 min at room temperature. The homogenates were centrifuged (8000g, 10 min, 4°C) and filtered with 0.2 μm membrane filter paper (Wako, Japan), and 10 ml supernatant was diluted to 100 ml with distilled water. Standard amine samples 5 ml and fish samples 5 ml were derivatized with dansyl chloride. A derivatized standard solution and a derivatized fish sample solution 10 μl were used for HPLC analysis. Standard biogenic amines and fish samples were analyzed together. A standard solution was also analyzed intermittently between fish samples to check chromatographic consistency. Each sample was analyzed for triplicate. The peak heights of the standard biogenic amine solutions were used to prepare standard curves and then for determination of the amine content in the fish samples.

| Statistical analysis
All experiments were performed in triplicate. All statistical analyses were performed using the SPSS statistical program (Version on 21, IBM Co.). And a value of p < .05 was considered as statistically significant. Data were expressed as mean ± standard deviation (n = 3).

Plate count agar media for microbiology analysis
Bacterial counts were calculated on plate count agar media, and the results are shown in Figure 1. The total number of colonies decreased slowly following with the increase of storage time and ranged from 3.54 to 3.12 log cfu/g from 0 day to 35 days. Staphylococcus and Enterobacteriaceae counts were approximately the same, and Staphylococcus and Enterobacteriaceae appeared in a decreasing trend. Staphylococcus was 1.23log cfu/g at the 0 day and decreasing to 0.56log cfu/g at 35 days, and Enterobacteriaceae was 0.88log cfu/g at the 0 day and decreasing to 0.47logcfu/g at 35 days. While because the Lactobacillus are the dominant bacteria in pickled products, so they showed an increasing trend, but the increasing rate of The rarefaction curve, which is a form of sample dilution curve, was constructed by the extracted sequencing data and the corresponding number of species (OTUs number) (Figure 2). The fivecurve tended to be flat, reflecting the rationality of sequencing data and that more data will only produce a small number of new species (OTUs).
The α-diversity of the bacteria in the salted fishes was analyzed (Table 1). The ACE estimator demonstrated the difference between 56.17 (HY.14) and 402.83 (HY.21) of the potential numbers of OTUs in the samples, and similar analysis data were obtained with the chao richness estimator (54.11-396.8), indicating a significant variation in bacterial richness. And HY.21 had the highest Shannon index (4.31), and the second Shannon index was HY.28 (3.57), and the lowest Shannon index was 1.57, indicating the highest bacteria diversity was HY.21 and the lowest bacteria diversity was HY.14.

Structure and abundance analysis of the flora
Venn diagram can be used to count the number of OTUs that are common and unique to multiple samples. The OTU with a similarity F I G U R E 1 The number of total bacteria, staphylococci, lactic acid bacteria, Escherichia coli following with storage time    Vibrionaceae belonged to Proteobacteria.

The content of biogenic amines
The contents of biogenic amines are shown in the Table 2 and Putrescine was 20. 00, 40.05, 36.44, 55.04, 20.74 mg/kg on the 0th, 14th, 21st and 28th, 35th days of storage. The tyramine was smallest amount of production, and the largest amount was 38.99 mg/ kg on 28th days, and the smallest amount was 11.97 mg/kg on 35th days. The variation trend of the content of putrescine and tyramine was basically the same. Nevertheless, the amounts of histamine decreased gradually following with storage time, the largest amount was 55.04 mg/kg on the 0th day and the smallest amount was 23.14 mg/kg on 28th days of storage, and there was little change in histamine on 21st and 28th, 35th days of storage.

| D ISCUSS I ON
The analysis of bacterial communities involved in the salted fish was carried out by the microbiological and molecular method of HTS, and pickling (Kim et al., 2009). The amount of biogenic amines was related to dominant bacteria of Prevotellaceae in the sample of HY.14, although no paper has reported the ability of Prevotellaceae to produce biogenic amines, and screened Prevotellaceae in this study has the ability to producing biogenic amines, mainly cadaverine and tyrosine (data not shown). And dominant bacteria of Shewanella, Proteus, in the sample of HY.21 can produce the biogenic amines (Ge et al., 2017;Helinck et al., 2013;Wang et al., 2019). And Lactobacillus, Vibrionaceae, and Staphylococcus dominated in the sample of HY.28, and there were also dominant amine-producing strains in Vibrionaceae (Landete et al., 2007). And on the one hand, lactic acid bacteria have the ability to produce amine; on the other hand, lactic acid bacteria can degrades biogenic amines, mainly degrades putrescine and cadaverine. And Staphylococcus also degrades biogenic amines. Resulting in the gradual decrease of amount of biogenic amines (Zaman et al., 2010), because of the presence of dominant lactic acid bacteria from 28th day, lactic acid bacteria and its metabolites can inhibit the growth of other microorganisms, moreover, because of the influence of low temperature and low salt, so the microbial population and microbial metabolites decrease gradually, and the content of biogenic amines decrease gradually. The F I G U R E 5 Tree species classification of the salted fish samples. Different colors of circles indicated different levels of classification, the size of the circle represented the relative abundance of the classification, two digital classification below were indicated relative abundance percentage, the percentage of all the former said the classification and classification of the species in the sample, while the latter represented the percentage of the species classification accounted for the selected samples change of bacterial flora leads to the increase and then decrease of amount of biogenic amines following with the storage time, the complex bacteria were affected by low temperature, salt concentration, and metabolic products of the bacteria, the large numbers of experiments are needed to further study the interactions between bacterial flora and biogenic amines. And further analyze the influence of microbial structure on the quality of salted fish.

| CON CLUS ION
The change rule of microbial diversity and BAs content were studied during duration of storage period in salted fishes of one-night courtyard in China. Two factors including salinity and structure of flora affect the safety and quality on salted fish product. Most of biogenic amines decreased after 14 days following with the changes in flora and salt penetration, but the biogenic amines in salted fish are not ignored. Microbes give special flavor for salted fish, but form the biogenic amines at the same time. The interaction was preliminary analysis between bacterial diversity and biogenic amines production in a salted mackerel in the study. So microbes should be controlled in the process of processing salted fishes. Future studies will focus on interactions between bacterial flora and biogenic amines by detail experiment.

ACK N OWLED G M ENTS
The research was financially supported by the National Natural Note: "-": Not detected.
Each result was measured three times to calculate the average value.
The values are expressed as averages of the average value ± SD (p < .05).

E TH I C A L S TATEM ENT
This article does not contain any studies with human participants or animals performed by any of the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.