KR20230156296A - Bacillus strains having suppression and degradation activities of biogenic amine and use thereof - Google Patents

Abstract

The present invention relates to Bacillus subtilis HJ0-6, Bacillus subtilis D'J53-4 or Bacillus idriensis RD13-10 strains with biogenic amine decomposition activity, and their Regarding a composition containing an active ingredient, the strain does not produce the decarboxylase gene, tdc or hdc , but increases the production of the amine oxidase gene, yobN , and biogenic amine. By confirming that it not only suppresses the expression of histamine and tyramine but also does not produce them, it can be usefully used in the production of safe, non-toxic food.

Description

Bacillus strains having suppression and degradation activities of biogenic amine and use thereof}

The present invention relates to a strain of the genus Bacillus that has biogenic amine decomposition activity and its use. More specifically, it relates to tyramine, a biogenic amine, from the Bacillus subtilis HJ0-6 strain. and a strain having the ability to decompose histamine and a composition containing its active ingredient.

Biogenic amines (BAs) are nitrogen compounds that are mainly produced by the decarboxylation of amino acids, amination of aldehydes and ketones, and transamination reactions. They have low molecular weight and are synthesized during the metabolic process of microorganisms, plants, and animals, so they are used in these cells. It is a component commonly found in Biogenic amines in foods and beverages are produced through the enzyme action of raw materials and the amino acid decarboxylation action of microorganisms.

Biogenic amines are formed by the action of amino acid decarboxylase in microorganisms, including Acromonas, Bacillus, Citrobacter, Clostridium, and Escherichia. Escherichia, Klebsiela, Lactobacillus, Pediococcus, Proteus, and Pseudomonas genera are known to have amino acid decarboxylase (Taylor et al. , 1983 Mar. Fish. Rev. 45, 35-39). Representative biogenic amines include putrescine, histamine, tyramine, cadaverine, spermidine, and serotonin. Since biogenic amines act directly or indirectly as neurotransmitters in the body and also affect cardiovascular diseases such as blood pressure control and blood flow, the consumption of foods containing biogenic amines may cause various pharmacological phenomena. If you have a disease in your small intestine, even small amounts of biogenic amines may cause harmful symptoms to your body.

The most widely known substance among biogenic amines is histamine, which causes ‘scombrotoxicosis’ when fish species such as mackerel, saury, sardines, and tuna are treated unsanitarily and decomposition occurs. Additionally, tyramine is involved in vasoconstriction, causing hypertensive crises such as the ‘cheese reaction’ and also triggering migraines.

When histamine and tyramine, which exceed the human body's decomposition limit, are consumed in food, it causes symptoms such as rash, local skin inflammation, allergies, vomiting, nausea, and diarrhea. In addition, biogenic amines increase blood vessel constriction and heart rate activity, causing an increase in blood pressure, dilating the pupil and eyelid tissue to promote tear secretion, hypersecretion of saliva, increased breathing, and increased blood sugar levels, thereby migraine headaches. It has also been reported to cause Biogenic amines are contained in a variety of foods, including fish products, meat products, dairy products, wine, beer, vegetables, fruits, nuts, and chocolate (Askar and Treptow, 1986; Brink et al., 1990).

Since the production of biogenic amines depends not only on the hygienic aspects of food but also on manufacturing methods such as fermentation technology, recent domestic and foreign research is being conducted in the direction of minimizing these ingredients along with investigating their content in various foods.

Korean soybean paste foods such as cheonggukjang, soybean paste, and soy sauce have been consumed by our ancestors for hundreds of years. They are foods with no particular side effects, and contain various beneficial bioactive substances that cause antioxidant effects, blood clot dissolving effects, and blood pressure lowering effects. However, according to a recent report, high levels of biogenic amines were detected in Korea's conventional or improved soybean paste and cheonggukjang (Ministry of Food and Drug Safety). Therefore, methods to reduce biogenic amines that cause harm to the human body are being explored in various ways (Ministry of Food and Drug Safety Administrative Publication Registration No. 11-1470000-001742-14).

When consuming fermented foods such as soybeans with high protein content, it is essential to select fermentation strains that do not produce biogenic amines and can decompose at the same time.

Accordingly, the present inventors made efforts to select strains with excellent decomposition activity of biogenic amines including tyramine and histamine, and as a result, Bacillus subtilis HJ0-6, Bacillus subtilis D'J53-4, and By confirming that the Bacillus idriensis RD13-10 strain reduces the production of histamine and tyramine, increases the expression of the amine oxidase gene, and suppresses the expression of the decarboxylase gene. , completed the present invention.

The purpose of the present invention is to provide a strain with biogenic amine decomposition activity from the Bacillus subtilis HJ0-6 strain.

Another object of the present invention is to provide a composition for reducing biogenic amines containing the above strain or its culture medium as an active ingredient.

Another object of the present invention is to provide a method for reducing biogenic amines comprising the step of treating the strain or composition.

In order to achieve the above object, the present invention is a biogenic amine containing as an active ingredient a strain with biogenic amine decomposition activity and its culture medium from Bacillus subtilis HJ0-6 strain. Provides a composition for reducing amine).

Additionally, the present invention provides a method for reducing biogenic amines comprising the step of treating the strain or composition.

Bacillus subtilis HJ0-6, Bacillus subtilis D'J53-4, and Bacillus idriensis RD13-10 strains reduce the production of histamine and tyramine and contain the amine oxidase gene ( By confirming that it increases the expression of yobN , an amine oxidase gene, and suppresses the expression of tdc and hdc , a decarboxylase gene, it can be useful in producing safe, non-toxic food.

Figure 1 relates to the selection of strains that reduce biogenic amines in decarboxylase broth. During cultivation of bacterial strains producing biogenic amines, color changes are measured on synthetic media containing histidine, tyrosine, and cresol as pH indicators. A negative reaction appears yellow, and a positive reaction appears purple. A, In this study, a Bacillus genus strain (Bacillus certilis H'J53-3 strain) producing biogenic amines was isolated (A). Strains that do not produce Bacillus subtilis HJ0-6 (B), Bacillus subtilis D'J53-4 (C), and Bacillus idriensis RD13-10 strain (D). B, a graph is shown based on the color change for each strain appearing in the medium.
Figure 2 shows tdc (tyrosine decarboxylase), hdc (histidine decarboxylase), and yobN (amine oxidase) mRNA expression measured by RT-PCR and qPCR. A, mRNA of each gene was detected and measured using RT-PCR, band intensity was quantified by densitometry, and comparative analysis was performed based on 16SrRNA. The graph shows tdc ( tyrosine decarboxylase ), hdc ( histidine decarboxylase ) and yobN ( amine oxidase ) Indicates the relative mRNA level for the gene. Each value is the mean ± SD of three replicate analyses, P < 0.05 (one way ANOVA followed by Duncan's multiple comparison test). B, The expression level of the mRNA of each gene was measured using qPCR and displayed in a graph.
Figure 3 is a diagram showing the protein expression levels of histamine and taramine produced by selected strains. A, Histamine expression levels in total cells obtained from all strains were determined by Western blot using histamine 3 receptor antibody (anti-histamine H3 receptor antibody). The above data is a representative figure among the results of an experiment performed three times. B, This is a diagram showing the levels of histamine and tyramine in the supernatant of cells normalized by ELISA analysis. Histamine and tyramine concentrations were increased in Bacillus certilis H'J53-3 compared to other strains. All experiments were performed at least three times and bars correspond to the mean ± SD. The average value of the samples was obtained by one-way ANOVA followed by Dincan's multiple comparison test, and the average values of different samples labeled with different letters were statistically significantly different (P<0.005).

Hereinafter, the present invention will be described in detail.

The present invention provides a strain having biogenic amine decomposition activity from the Bacillus subtilis HJ0-6 strain.

The biogenic amine may be histamine, tyramine, putrescine, or cadaverine, but is not limited thereto.

In the strain according to an embodiment of the present invention, the strain is characterized in that it does not produce biogenic amines, but is not limited thereto.

The biogenic amine of the present invention is formed from amino acids by the action of microbial amino acid decarboxylase, and when biogenic amines exceeding the decomposition limit of the human body are consumed in food, rashes, local skin inflammation, allergies, It causes symptoms such as vomiting, nausea, and diarrhea.

The present invention also provides a composition for reducing biogenic amines containing the present invention's Bacillus subtilis HJ0-6, strain, or culture medium thereof as an active ingredient.

It may be a composition for reducing tyramine and histamine in soybean paste containing the above strain or its culture medium as an active ingredient. The above sauce may be cheonggukjang, soybean paste, soy sauce, or red pepper paste, but is not limited thereto.

In addition, a method for reducing biogenic amines comprising the step of treating the strain or the composition is provided.

Those skilled in the art will recognize that methods known in the art can be used to cultivate the Bacillus genus strain of the present invention.

In a specific example of the present invention, as a result of screening strains that reduce biogenic amines, the Bacillus subtilis HJ0-6 strain was selected (see Figure 1), and the biogenic amines of the selected strain were selected. As a result of confirming the quantitative production and inhibition of , strain H'J53-3, a biogenic amine producing strain, expressed the decarboxylase genes tdc and hdc , but did not contain the amine oxidase gene yobN . The expression level was confirmed to be relatively low compared to other strains (see Figure 2). As a result of confirming the reduced histamine and tyramine expression levels by the selected strains, it was confirmed that the concentrations were reduced by the HJ0-6, D'J53-4 strains, and RD13-10 strains compared to the H'J53-3 strain, a biogenic amine producing strain. (see Figure 3B).

Therefore, Bacillus subtilis HJ0-6 strain reduces the production of histamine and tyramine, increases the expression of yobN , an amine oxidase gene, and tdc , a decarboxylase gene. And by confirming that it inhibits hdc expression, the strain or composition capable of reducing biogenic amines and a food additive containing its active ingredients can be usefully used in the production of safe, non-toxic food.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

<Example 1> Screening of strains that reduce biogenic amines

This strain is derived from buckwheat seedlings and was isolated by plating on Nutrient agar.

The most efficient way to select strains for reducing biogenic amines is to find bacteria that decompose biogenic amines without producing biogenic amines. In order to select strains that reduce biogenic amines, the following experiment was performed.

Specifically, 5 ㎕ of Bacillus subtilis HJ0-6 strain was instilled on a medium containing histidine, tyrosine, and cresol red (pH indicator), and then 30 Cultured for 24 hours at ℃ (microorganism accession number: KACC92093P).

If biogenic amine reduction occurs, histidine and tyrosine cannot be converted to histamine and tyramine, so there is no color change around the colony, so yellow colonies are generated as a negative reaction. If biogenic amine reduction does not occur, it is a positive reaction. Purple colonies are created.

As a result, as shown in Figure 1, Bacillus subtilis HJ0-6, Bacillus subtilis D'J53-4, and Bacillus idriensis RD13-10 strains were the control group (biogenic amine When compared with the producing strain, Bacillus certilis H'J53-3 strain, each showed a negative reaction. Therefore, it was confirmed that Bacillus certilis HJ0-6, Bacillus certilis D'J53-4, and Bacillus idriensis RD13-10 strains were strains that caused reduction of biogenic amines (see Figures 1A and B).

<Example 2> Confirmation of strains that do not produce biogenic amines

<2-1> Confirmation of genes producing biogenic amines from selected strains

In order to determine whether the strains producing biogenic amines, hdc and tdc , and the gene yobN , which reduces biogenic amines, are present in the strain that reduces biogenic amines obtained from <Example 1>, RT-PCR and qPCR analysis were performed. It was measured using

Specifically, mRNA was extracted from cultures grown in nutrient broth supplemented with 0.005% pyridoxal-5-phosphate, 0.5% histidine, and 0.5% tyrosine using the RNeasy plus mini kit (Qiagen). cDNA was synthesized from the extracted mRNA using amfiRivert Platinum cDNA synthesis Master Mix (GenDEPOT), and RT-PCR and qPCR (quantitative real-time PCR) analysis were performed. After confirming the expression levels of the amine oxidase gene, yobN gene, and the decarboxylase gene, hdc and tdc genes, by RT-PCR, they were detected using the CFX96 Touch™ Real-Time PCR Detection System using iQ™ SYBR ^® Green Supermix. The signals were quantified and compared and measured quantitatively. Cycling conditions were 52°C for 1 minute, 95°C for 3 minutes, followed by 45 cycles of 55°C for 30 seconds, and 72°C for 30 seconds. 16S rRNA was used as a normalization control. The primers used for RT-PCR and qPCR are shown below.

<2-2> Confirmation of quantitative production of biogenic amines in selected strains through UPCL analysis

In order to investigate the production of biogenic amines in the selected strains, the following experiment was performed.

Specifically, a medium containing 0.005% pyridoxal-5-phosphate, 0.5% histidine, and 0.5% tyrosine was used in nutrient broth, and to investigate biogenic amine inhibition, 0.25% histamine and 0.25% tyramine were added to nutrient broth. After adding (tyramine), the medium was used. After adjusting the optical density (O.D.) value of the selected strain to 0.3, 2% of the bacterial culture was inoculated into 5 ml of medium and cultured at 30°C for 1 day. For the measurement of biogenic amines, 5 ml of cultured broth was centrifuged (8,000 rpm, 10 minutes) and 4 ml of supernatant was used. To 4 ml of the supernatant centrifuged from the culture medium, 2 ml of 2N NaOH and 50 ㎕ of benzoyl chloride were added, mixed for 3 minutes, and derivatized at 30°C for 40 minutes. After derivatization, 3 ml of saturated NaCl was added to stop the reaction. Extract by adding 4 ml of diethyl ether and shaking for 3 minutes, centrifuge (3,500 rpm, 10 minutes), collect 2 ml of supernatant, concentrate with nitrogen, add 1 ml of acetonitrile, and filter through 0.2 ㎛ filter paper. It was used as a test solution. UPCL (Ultra performance liquid chromatography) analysis using derivatized samples used the waters system and C18 (2.1 x 75 mm, waters) as the column. The flow rate was adjusted to 0.4 ㎖/min and 0.2 ㎕ of sample was injected and analyzed. The solvent conditions used as the mobile phase at this time are shown in [Table 2].

<Conditions for analysis of biogenic amines by UPLC>

<Experimental Example 1> Identification of biogenic amine-reducing strains through confirmation of amine oxidase gene and decarboxylase gene

In order to confirm the expression level of the amine oxidase gene in the selected strains, the following experiment was performed.

Specifically, RNA was extracted from each selected strain culture medium and RT-PCR (reverse transcription-PCR) and qPCR (quantitative real time PCR) analysis were performed. The amine oxidase gene yobN and the decarboxylase genes tdc and hdc were selected to measure the degree of reduction of biogenic amines.

As a result, as shown in Figure 2, strain H'J53-3 is a biogenic amine-producing strain and expressed the decarboxylase genes tdc and hdc , but the expression level of the amine oxidase gene yobN was different from that of other strains. (HJ0-6, D'J53-4, and RD13-10).

On the other hand, the HJ0-6, D'J53-4, and RD13-10 strains showed no expression of tdc and hdc compared to the H'J53-3 strain, and the expression of the amine oxidase gene yobN The amount increased significantly.

Therefore, strain H'J53-3 is a strain that can be used as a positive control that produces biogenic amines, and HJ0-6, D'J53-4, and RD13-10 do not produce biogenic amines or produce biogenic amines. It was confirmed that it was a strain that could be reduced.

<Experimental Example 2> Confirmation of quantitative production and inhibition of biogenic amines in selected strains

To quantitatively confirm the biogenic amine production and inhibition activities of the selected strains, UPLC analysis was performed.

As a result, as shown in [Table 3] and [Table 4], the highest biogenic amine production was confirmed in the positive control group, H'J53-3, in the biogenic amine production medium, and HJ0-6 and D'J53-4 and RD13-10 strains, histamine was not detected at all, and tyramine was detected in a moderate amount (Table 3).

In addition, as a result of observing the reduction rate of histamine and tyramine, the positive control H'J53-3 showed the lowest reduction rate. On the other hand, HJ0-6 reduced histamine by about 47.6% and tyramine by about 33.5%, which showed the highest reduction rate. Additionally, strains D'J53-4 and RD13-10 showed higher reduction rates compared to H'J53-3 (Table 4). Therefore, it was confirmed that HJ0-6, D'J53-4, and RD13-10 strains were strains capable of reducing biogenic amines.

¹⁾ Each value is the mean ± SD of three replicate analyses, and each row with a different superscript is statistically P < 0.005 (one-way ANOVA followed by Duncan's multiple comparison test).

²⁾ Not detected.

¹⁾ Express the degradation rate as a percentage of the strain-free control. Each value is the mean ± SD of three replicate analyses, and each row with a different superscript is statistically P < 0.005 (one-way ANOVA followed by Duncan's multiple comparison test).

<Experimental Example 3> Confirmation of histamine and tyramine expression levels reduced by selected strains

In order to visually confirm whether the expression levels of histamine and tyramine were reduced by the HJ0-6, D'J53-4, and RD13-10 strains, the following experiment was performed.

Specifically, Western blot was performed using an anti-histamine H3 receptor antibody.

The co-culture was centrifuged at 10,000 The gel was transferred to a nitrocellulose membrane. After blocking the membrane for 1 hour, anti-H3 receptor primary antibody (polyclonal anti-histamine H3 receptor (1:1,000; Signalway Antibody LLC, College Park, ML, USA) was added and reacted for 1 hour, followed by TBS. -T (Tris-buffered saline with 0.1% Tween-20) was washed three times at 10-minute intervals. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat IgG secondary. antibody (Bio-Rad, USA) solution and reacted for 1 hour. Afterwards, the membrane was washed three times at 10-minute intervals with TBS-T solution and blotted with BM chemiluminescence blotting substrate (POD). ) (Roche, Mannheim, Germany) was used to sensitize and develop X-ray film.

As a result, as shown in Figure 3, the expression level of H3 receptor, which indicates the expression of histamine and tyramine, was hardly expressed by the HJ0-6 and D'J53-4 strains compared to the control H'J53-3 strain, A clear decrease was observed by the RD13-10 strain (Figure 3A). Additionally, as a result of measuring the levels of histamine and taramine using ELISA analysis, it was observed that the concentrations were reduced by the HJ0-6, D'J53-4, and RD13-10 strains compared to the H'J53-3 strain ( Figure 3B).

Therefore, it was confirmed that strains HJ0-6, D'J53-4, and RD13-10 were strains capable of reducing biogenic amines.

Rural Development Administration National Academy of Agricultural Sciences Microbial Bank (KACC) KACC92093P 20150923

<110> Foundation of Agri. Tech. Commercialization and Transfer <120> Bacillus strains having suppression and degradation activities of biogenic amine and its use <130> 2023P-11-002 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>tdc-L <400> 1 acatagtcaa ccatrttgaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>tdc-R <400> 2 caaaatggaag aagaagtagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>hdc-L <400> 3 gatggtattg tttcktatga 20 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223>hdc-R <400> 4 caaacaccag catcttc 17 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> yobN-L <400> 5 ccgtgtctgc ackttgagat c 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> yobN-R <400> 6 acatccgcca atttcttkcw gg 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16s-rRNA-L <400> 7 agagtttgat cctggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16s-rRNA-R <400> 8 ggctaccttg ttacgactt 19

Claims (3)

Accession number: Bacillus subtilis HJ0-6 strain deposited as KACC92093P,
Histamine or tyramine decomposition activity through the expression of the amine oxidase gene yobN , while the decarboxylase gene, tdc ( tyrosine decarboxylase ) or hdc ( histidine decarboxylase ), is not expressed. A strain characterized in that it does not produce histamine or tyramine. A composition for reducing histamine or tyramine comprising the strain of claim 1 or its culture medium as an active ingredient. A method for reducing histamine or tyramine comprising the step of treating the strain of claim 1 or the composition of claim 2.