The microbial community, biogenic amines content of soybean paste, and the degradation of biogenic amines by Lactobacillus plantarum HM24

Abstract Soybean paste was a traditional fermented product in Northeast China, mainly fermented by molds, yeast, Bacillus, and lactic acid bacteria. This study investigated the dynamic changes of the microbial community and biogenic amine content during the fermentation of the traditional soybean paste. The microbial diversity of soybean paste in different regions was analyzed by MiSeq sequencing technology. The results showed that Penicillium and Tetragenococcus were the dominant microorganisms responsible for the fermentation of soybean paste. Biogenic amine was found in the traditional soybean paste at different fermentation stages, putrescine, and tyramine were the mainly biogenic amines and their content increased with the extension of fermentation time. Serratia in the soybean paste was positively correlated with the formation of spermine, cadaverine (p < .01), and β‐phenethylamine (p < .05), Leuconostoc was negatively correlated with tyramine formation (p < .05), and Enterococcus was positively correlated with the formation of histamine, tryptamine and cadaverine (p < .01). Lactobacillus fermentum HM22, Lactobacillus plantarum HM24, and Enterococcus faecalis YF10042 with strong biogenic amine degrading capacity were inoculated into the koji. After 20 days of fermentation, the degradation rates of tryptamine, β‐phenylethylamine, putrescine, cadaverine, histamine, and tyramine in soybean paste inoculated with L. plantarum HM24 were 35.31%, 43.14%, 30.18%, 33.44%, 32.74%, and 39.91%, respectively, indicating that the use of L. plantarum HM24 as a starter culture in soybean paste fermentation might be a good strategy for biogenic amines reduction.


| INTRODUC TI ON
The traditional fermented soybean paste in the northeast of China was made from soybeans and fermented naturally in the open air by natural microorganisms, and microbial community composition was an important factor in determining the flavor of soybean paste (Peng-Fei et al., 2019;Yu & Sun, 2019). Yong et al. (2016) found that Bacillus and Enterococcus were the dominant microbes in the Korean soybean paste. Li et al. (2017) found that Tetragenococcus, Lactobacillus, Staphylococcus, Acinetobacter, Pseudomonas, and Streptococcus were the dominant bacteria responsible for the fermentation of doubanjiang. However, there were few reports on the dynamic changes of microorganisms and dominant fermenting bacteria in the whole fermentation stage of soybean paste. Therefore, it was of great significance to deeply understand the changes of microbial communities at different fermentation stages of traditional soybean paste. In recent years, MiSeq sequencing technology has been widely used, given its accuracy and sequencing depths, to determine the accurate microbial community and dynamic change in the microbial community during fermentation of soybean paste (Jeong et al., 2016;Nam et al., 2012).
Fermented soybean products contained abundant proteins and biogenic amine precursor amino acids. If microorganisms with decarboxylase activity were present simultaneously in the fermentation process, biogenic amine would be produced in the natural fermentation process of soybean paste. It was reported that tyramine content was generally higher in protein-rich fermented products (Awan et al., 2008;Fausto et al., 2016;Mayer & Gregor, 2018;Mah et al., 2019). Typical biogenic amines included tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine (Awan et al., 2008;Lu et al., 2018;Shukla et al., 2010), among which histamine and tyramine had relatively high toxicity Taylor et al., 1978).
Intake of high concentration biogenic amine may cause headache, palpitation, spasm, vomiting, and pupil dilation in some people (Hazards, 2011;Rice et al., 1976;Lehane & Olley, 2000). Biogenic amines have been found in fermented soybean products such as sufu (Guan et al., 2013), soy sauce (Lu et al., 2019), Japanese miso (Shukla et al., 2011), and natto . However, there were few systematic studies on the content and variation of biogenic amines in traditional soybean paste in northeast China.
Due to the unique flavor, traditional fermented soybean paste was very popular in northeast China. However, few studies were conducted on the correlation between dynamic changes of microorganisms and biogenic amine content during the soybean paste fermentation. In this study, the microbial community changes during the soybean paste fermentation, made by families in Heilongjiang Province and Liaoning Province, were studied by high-throughput sequencing method and the content of biogenic amines at different fermentation stages was detected by high-performance liquid chromatography, to clarify the correlation between dynamic changes of microorganisms and the content of biogenic amines. The strains with the ability to degrade biogenic amine were screened and applied in lab-scale soybean paste fermentation.

| Preparation of fermented soybean paste and sample collection
The soybean paste in this study was homemade by some families in northeast China. The soybean paste was prepared just as follows.
Raw soybeans were soaked in water at room temperature for 24 h and then cooked in boiling water for one hour. After cooling, drained and mashed soybeans into soybean cake and placed in a position with sufficient sunlight and good ventilation at room temperature for 10-15 days. When fungi mycelium appeared in the soybean cake, the cake was smashed and immerged into 12% brine, which was agitated daily.
Twelve soybean paste samples prepared by families in the northeast of China were collected. The soybean paste samples were collected in four different fermentation phase (a, b, and c of the soybean paste with fermentation time of 0, 20, 40, and 60 days). Approximately 5 g of each sample was collected in an aseptic pipe, and each sample was collected in three replicates and stored at −20°C for further analysis. The samples were assigned as koji a, 20a, 40a, and 60a, koji b, 20b, 40b, and 60b, and koji c, 20c, 40c, and 60c. The twelve samples were used for the determination of microbial diversity and the content of biogenic amines.

| Gene sequencing
The genomic DNA was extracted from the soybean paste samples with the Illumina Truseq DNA kit following the manufacturer's instructions. The purity of the genomic DNA was confirmed by 1% agarose gel electrophoresis. Primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) were designed according to the V3-V4 region of bacterial 16S rRNA gene.

Primers
ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC) were designed based on the ITS1F-ITS2R region of the fungal internal transcribed spacer (Li, Tang, et al., 2019;Li, Zou, et al., 2019) and used the following reaction conditions: an initial denaturation at 95℃ for 3 min, followed by 35 cycles at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 45 s and a final 10 min extension at 72℃. The PCR product was sequenced by Shanghai Majorbio Bio-pharm Technology Co., Ltd.

| Sequencing and data analysis
After PCR and purification, a DNA library was constructed and run on the Miseq Illumina platform (Illumina, San Diego) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd.

| Evaluation of BA-degradation Ability
To evaluate the BAs-degradation ability, three strains of bacteria selected from laboratory-collected strains were inoculated into MRS broth with 100 mg/L of tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine and incubated at 37℃ for 48 h (Kung et al., 2017). Cultures of each strain were harvested by centrifugation at 4000 g for 10 min, the MRS without Lactobacillus inoculation was used as the control, determined the concentration of BAs in the culture medium by HPLC after 48 hr of incubation at 37℃. The value of BA-degradation rate was determined based on an equation: X=[(c 1 -c 2 )/c 1 ] ×100%, among them, X: degradation ability of strains, %; C 1 : BAs concentration of control group, mg/L; C 2 : BAs concentration of experimental group, mg/L.

| Identification of BA-degradation bacterial strains
The strains with BA-degradation ability were identified through 16 S ribosomal DNA sequencing analysis. The 16 S ribosomal DNA was obtained through PCR amplification using the following primers: 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTAC HVGGGTWTCTA A T). The PCR product was sequenced by Shanghai Majorbio Bio-pharm Technology Co., Ltd.

| Soybean paste fermentation with BAs degradation strains
Soybeans were crushed after high-pressure steaming for 20 min, and koji was made naturally in the laboratory environment for two months. The koji was ground and put into water containing 8% sea salt, and mixed koji and brine in the ratio of 1:1 (Xu et al., 2020).
To test the effect of isolated bacteria strains on the degradation of BAs in soybean paste, added 4% bacterial solution into the soybean paste. The soybean paste without inoculation of the above bacteria solution was adopted as control. Sealed the bottle with six layers of gauze and fermented in natural environment. During soybean paste fermentation, samples were collected on different fermentation days. BAs concentrations, pH, total acid, amino acid nitrogen, and reducing sugar of the collected samples were analyzed and compared.

| Statistical analysis
All analysis experiments were conducted at least three replicates.
The difference significance was analyzed by one-way ANOVA method using the statistical software SPSS 20.0, and mean values were compared by Tukey's HSD test at 5%.

| Contents of biogenic amines in soybean paste samples in different fermentation phase
Through the elution procedure, each BAs was detected in turn. The representative high performance liquid chromatography chromatogram and peak identification recorded for mixer of standard BAs and a fermented soybean paste samples were shown in Figure 5.
The biogenic amines contents in the soybean paste samples from northeast China were determined by high-performance liquid chromatography. The biogenic amines content in 12 soybean paste samples was shown in Table 2. In the koji-making stage, the contents of the eight biogenic amines and the total biogenic amines were significantly lower than those in other fermentation phases. The content of total biogenic amines in sample a and sample b after fermented for 60 days was significantly higher than that in sample c (p < .05).
In sample a and sample b, the contents of Put, His, and Tyr were the highest. The highest content of Try was found in sample c, and the other biogenic amines were lower. European Food Safety Authority (Hazards, 2011) recommended that the intake of His and Tyr should not surpass 50 and 600mg per meal, respectively. Brink et al. (1990) recommended that the levels of His, Tyr, and Phe in food should be lower than 100, 800, and 30 mg/kg, respectively.
Excessive intake of biogenic amines may be harmful to human being health, including blood pressure changes, vomiting, palpitations, breathing disorders, headaches, and other symptoms (Hazards, 2011;Rice et al., 1976;Shukla et al., 2010). In this study, biogenic amine was commonly found in the traditional soybean paste. Sample a and sample b were produced by families in Heilongjiang Province, and sample c was produced by families in Liaoning Province. Different fermentation technology and environments may result in some differences in the biogenic amine content among these soybean paste samples. The formation of biogenic amines depended primarily on fermentation conditions and microbes (Linares et al., 2011;Kalac & Krausova, 2005;Spano et al., 2010). When the microbes with amino acid decarboxylase propagate in food rich in protein and free amino acids, biogenic amines could accumulate in the foods. Some kinds of biogenic amines were detected in the traditional fermented soybean paste from northeast China, which was naturally fermented by Aspergillus oryzae, yeast, Lactobacillus, and other microorganism Yu & Sun, 2019). Traditional fermented soybean paste had a complex and diverse microbial community, rich free amino acids and was easy to accumulate biogenic amines (Alvarez & Moreno-Arribas, 2014). Therefore, it was important to detect the type and content of biogenic amines and develop effective methods to reduce biogenic amines in soybean paste (Gong et al., 2014;Santos, 1996;Yu & Sun, 2019).

| Correlation analysis of microbiota and biogenic amine content in samples
The amount of biogenic amines formed in fermented food products was greatly influenced by the food composition, microflora, and other factors (Carelli et al., 2008). Peng-Fei et al. (2019) found that bacteria had a stronger effect on biogenic amines than fungi in da-jiang. We investigated the correlation between bacterial community composition and biogenic amine content in different fermentation periods of the soybean paste from northeast China.
As shown in Figure 6, Serratia was positively correlated with the formation of Spm in the koji-making stage (p < .01), and also positively correlated with the formation of Spm, Cad (p < .01), and β-Phe (p < .05) in the samples fermented for 20 days. There was a negative correlation between Leuconostoc and the formation of Tyr (p < .05), and Enterococcus was positively correlated with His, Try, and Cad (p < .01).
In this study, the total biogenic amine content of sample a was  stages contained Leuconostoc. Spearman correlation analysis showed that Leuconostoc was negatively correlated with the contents of tyramine (Rollan et al., 1995), which may explain the lowest content of biogenic amine in sample c.

| Isolation and identification of strains with biogenic amines degrading ability
The biogenic amines degrading ability of the strains HM22, HM24, and YF10042 were shown in Figure 7. The degrading ability of HM24 to spermine and histamine was significantly higher than that of the

| Application of the strains with biogenic amine degrading ability in soybean paste fermentation
The three strains HM22, HM24, and YF10042 with biogenic amines degrading ability were applied in the fermentation of lab-scale soybean paste. As shown in Figure 8a, the content of tryptamine in soybean paste inoculated with the three strains showed reduction after fermentation for 5-20 days, among which the reduction by Lactobacillus plantarum HM24 was the most significant. After fermentation for 20 days, the content of tryptamine in the Lactobacillus plantarum HM24 group was reduced by 35.31% compared to the control. It can be seen from Figure 8b-f that after 20 days of fermentation by Lactobacillus plantarum HM24, the contents of tyramine, β-phenethylamine, putrescine, cadaverine, and histamine in the treated group were significantly lower than those in the control.
As shown in Figure 9a, the pH value of each group showed a trend of decline during the fermentation period, and the pH of the HM24 group was significantly lower than that of the other groups.
The low pH value of fermented soybean paste was related to the growth of lactic acid bacteria and the production of organic acids from free sugars (Chun et al., 2019). The content and variation of F I G U R E 6 Correlation analysis of bacteria and biogenic amine content in northeast soybean paste. **p < .01; *p < .05 F I G U R E 7 Degradation of Lactic Acid Bacteria by Strains total acid and amino acid nitrogen had an important effect on the quality of soybean paste, so the fermentation process of Douchi can be optimized by the change of amino nitrogen content and acidity (Li, Tang, et al., 2019;Li, Zou, et al., 2019). Figure 9b and c showed that the total acid content maintained an increasing trend and then stabilized, the content of amino acid nitrogen increased continuously. The total acid and amino acid nitrogen content of the control was significantly lower than the lactic acid bacteria treated group, which was similar to the trend of total acid and amino acid nitrogen in douchi (Yu & Sun, 2019). Figure 9d showed that the content of reducing sugar firstly increased and then decreased, which was the same as the results of Tang et al. (2013). The reason may be that the amylase, cellulase, and saccharifying enzyme produced by mold in soybean paste hydrolyze carbohydrates to produce reducing sugar. In the later stages of fermentation, the pH and total acid inhibited the activity of amylase, and reducing sugars are consumed by microbial growth and Maillard reaction, which led to the decrease of reducing sugar (Tang et al., 2013). The reducing sugar content of the HM24 treated group was significantly lower than those of the other groups during fermentation for 10 to 20 days, indicating that HM24 could grow well in soybean paste.

| CON CLUS ION
In this study, the microbial diversity of traditional fermented soybean paste in northeast China was investigated in terms of both bacteria and fungi. The fermentation of homemade soybean paste was attributed to the combination of bacteria and fungi.
Tetragenococcus and Penicillium were the dominant bacteria in the fermentation process of soybean paste. With the extension of fermentation time, the content of biogenic amine in the soybean paste gradually increased, the highest value of biogenic amine was 3670.51 ± 34.96 mg/kg, among which tyramine (Tyr) and putrescine (Put) were higher. It was found that spermine (Spm), cadaverine (Cad), and β-phenylethylamine (β-Phe) were positively correlated with Serratia, whereas tyramine (Tyr) was negatively correlated with Leuconostoc. Lactobacillus plantarum HM24 exhibited strong degradation of histamine (His), β-phenethylamine (β-Phe), and tyramine (Tyr) during the fermentation of soybean paste, indicating that the use of Lactobacillus plantarum HM24 as starter culture reduced the production of biogenic amine.

ACK N OWLED G EM ENT
This study was funded by the National Key Research and Development Plan (2018YFC1604102).

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.