Use of barley malt in inhibiting growth and pathogenicity of bacillus cereus by regulating its quorum sensing system

Abstract

The application discloses application of barley malt alkali in inhibition of growth and pathogenicity of Bacillus cereus by regulating a quorum sensing system of the Bacillus cereus. The barley malt alkali is a green natural product, is safer for the environment and human bodies than traditional chemical antibacterial agents such as antibiotics, and reduces the risk of drug residue and drug-resistant strain generation. Research results show that the barley malt alkali can play a role at a low concentration (1 / 8 MIC), so that the use cost is low. The application can target regulation of expression of multiple key genes (such as Spo0A, CodY, rpoN and the like), so as to not only inhibit pathogenicity, but also effectively control proliferation of the Bacillus cereus, reduce the risk of food contamination and foodborne disease infection, and has important application prospects in the field of food safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of malt extract in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus. BACKGROUND

[0002] Bacillus cereus is a gram-positive bacterium of the genus Bacillus, facultative anaerobic, widely distributed in nature. Pathogenic Bacillus cereus is often found in food such as rice products, which can produce a variety of toxins and threaten food safety. It is a very important foodborne pathogen and opportunistic pathogen, ranking first in the number of food poisoning events and the total number of poisoning. Therefore, it is necessary to study the process of the harm of Bacillus cereus in food, and it is of great significance to inhibit it.

[0003] The proliferation and pathogenicity of Bacillus cereus are regulated by signal pathways such as quorum sensing system (QS) and pleiotropic transcriptional regulators. The production of many bacterial degradation enzymes, virulence factors and biofilm formation processes depend on QS, which can effectively control bacterial growth and virulence through QS pathway. However, traditional methods lack targeting and are difficult to achieve precise regulation. Existing prevention and control measures such as chemical antibacterial agents and high-temperature sterilization have problems such as drug residues, increasing the risk of drug resistance, and affecting the taste of food, so there is an urgent need for a safer and more effective green antibacterial means.

[0004] Green natural products are attracting attention due to their safety and environmental friendliness, but how to precisely regulate the quorum sensing signal pathway of pathogenic bacteria still needs further study. Malt extract, as a natural product, has good biocompatibility and can target regulate the signal pathway of Bacillus cereus at low concentration, thereby inhibiting the quorum sensing signal and virulence gene expression of Bacillus cereus, reducing pathogenicity and not easily causing bacterial resistance. Natural products can regulate gene expression by targeting PlcR-PapR quorum sensing system, Spo0A transcriptional regulator, etc., to achieve antibacterial effect, have high safety and low cost advantage, and are increasingly valued in today's antibiotic epidemic. Bacillus cereus is a drug-resistant bacterium, and the development of its quorum sensing signal inhibitors is of great significance. The development of new anti-Bacillus cereus drugs and the control of Bacillus cereus hazards are of great help. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide an application of malt extract in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The application of barley malt in inhibiting the growth and pathogenicity of Bacillus cereus by regulating the quorum sensing system of Bacillus cereus, and the growth and pathogenicity of various Bacillus cereus can be inhibited at a low concentration of barley malt.

[0008] The application of barley malt in preparing a Bacillus cereus bacteriostatic agent.

[0009] The application of barley malt in preparing a preparation for inhibiting the pathogenicity of Bacillus cereus.

[0010] The application of barley malt in preparing a preparation for inhibiting the biofilm formation of Bacillus cereus.

[0011] The application of barley malt in preparing a preparation for inhibiting the quorum sensing system of Bacillus cereus.

[0012] The inhibition of the quorum sensing system of Bacillus cereus refers to the inhibition of the expression level of the quorum sensing system related genes.

[0013] The quorum sensing system related genes include at least one of plcR, papR and LuxS.

[0014] A method for regulating the quorum sensing system of Bacillus cereus by using barley malt to inhibit the growth and pathogenicity of Bacillus cereus, comprising the following steps:

[0015] Barley malt is added to the system to inhibit the growth and pathogenicity of Bacillus cereus.

[0016] The addition amount of barley malt is 0.08-5 mg / mL; preferably 0.3125 mg / mL.

[0017] The Bacillus cereus includes at least one of Bacillus cereus ATCC 14579, Bacillus cereus IAM 12605, Bacillus cereus CCM 2010, Bacillus cereus Bcer A9 and Bacillus cereus Bcer L1.

[0018] The inhibition of the pathogenicity of Bacillus cereus refers to the inhibition of the expression level of the virulence gene.

[0019] The virulence gene includes at least one of hblB, cytK, nheB and clo.

[0020] The present application has the following advantages and effects relative to the prior art:

[0021] The present application uses green natural product hordeine, which is safer to the environment and human body than traditional chemical antibacterial agents such as antibiotics, reduces the risk of drug residues and drug-resistant strains, and the research results show that hordeine can play a role at a low concentration (1 / 8 MIC), which makes the use cost low, the present application can target regulate the expression of multiple key genes (such as Spo0A, CodY, rpoN, etc.), not only inhibit pathogenicity, but also effectively control the proliferation of Bacillus cereus, reduce the risk of food contamination and foodborne disease infection, and has important application prospect in the field of food safety. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC.

[0023] Figure 2 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC.

[0024] Figure 3 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC.

[0025] Figure 4 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC.

[0026] Figure 5 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC.

[0027] Figure 6 Figure 1 is a growth curve of Bacillus cereus in a free state at 1 / 8 MIC. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below in conjunction with the embodiments and drawings, but the embodiments of the present application are not limited thereto.

[0029] In the following embodiments, if the specific test conditions are not specified, the conventional test conditions or the test conditions recommended by the reagent company are generally used. If not specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0030] Example 1 Determination of the minimum inhibitory concentration (MIC) of hordeine on Bacillus cereus

[0031] (1) Glycerol stock of B. cereus ATCC 14579, B. cereus IAM 12605, B. cereus CCM 2010, Bcer A9 (purchased from Guangzhou Peptide Valley Co., Ltd.) and Bcer L1 (purchased from Guangzhou Peptide Valley Co., Ltd.) were streaked onto TSA solid medium and incubated at 37°C for 24 h. Single colonies were picked and inoculated into 2 mL of TSB liquid medium and incubated at 37°C, 200 rpm for 12 h.

[0032] (2) 10 mg of Humuline was dissolved in 1 mL of DMSO, filtered to remove bacteria, and prepared into a 10 mg / mL humuline solution.

[0033] (3) The overnight culture of the five strains of B. cereus was diluted to an initial concentration of 10 5 CFU / mL. In the first seven columns of each of the first five rows of a sterile 96-well plate, 180 μL of the diluted bacterial solution was added to each well, and 20 μL of the humuline solution diluted by a factor of 2 was added to each well in order of increasing concentration. The final concentrations of the humuline solution in the first seven columns were 5, 2.5, 1.25, 0.625, 0.313, 0.106, and 0.053 mg / mL, respectively. The plate was incubated at 37°C, 200 rpm for 24 h, and the OD 600nm was measured using a full-wavelength microplate reader.

[0034] The results are shown in Table 1. MIC (minimum inhibitory concentration) is an important indicator of the antibacterial activity of a drug, and refers to the minimum concentration of a drug that can inhibit the growth and reproduction of pathogenic bacteria in a culture medium after 18-24 h of incubation. In this experiment, the OD 600nm of the culture medium was consistent with that of the blank control when the humuline concentration was 2.5 mg / mL, indicating that this concentration inhibited the growth of B. cereus. Therefore, the MIC of humuline for B. cereus was 2.5 mg / mL.

[0035] Table 1 OD600nm after 24 h of incubation with humuline

[0036]

[0037] Example 2 Determination of the growth curve of free B. cereus

[0038] The overnight culture of the five strains of B. cereus was diluted to an initial concentration of 10 5CFU / mL, according to the conclusion of reference example 1, 1 / 8 MIC, i.e. 0.3125 mg / mL of green natural product maltol solution was added, and the culture was carried out at 37℃, 200 rpm on a shaking table. Every 2 hours, 200 μL of bacterial solution was sampled in a 96-well plate, and the optical density value thereof at OD 600nm was measured, and the bacterial solution without the added liquid was set as a control.

[0039] The results are shown in Table 1. Figure 1 As shown in Table 1, 1 / 8 MIC concentration of maltol has no effect on the growth of Bacillus cereus, and this concentration can be used for the research of Bacillus cereus quorum sensing signal inhibitors.

[0040] Example 3 Effect of 1 / 8 MIC concentration of maltol on the expression of free state Bacillus cereus genes

[0041] 3.1 Selection of detection genes

[0042] (1) Quorum sensing system is a mechanism of intercellular information exchange. When the secreted signal molecules (auto-inducers, AIs) of microorganisms accumulate to a certain concentration during the growth process, the QS system is activated to regulate the expression of specific genes so that the cells can adapt and survive through group behavior. PlcR-PapR is a specific quorum sensing system of Bacillus cereus. The activity of PlcR requires the activation of signal molecules encoded by the directly downstream gene papR of plcR. After processing and transport, 2 PapR molecules combine with 2 PlcR molecules to form a tetramer, which binds to the specific site PlcR box on the DNA, and finally starts the transcription of the PlcR regulon. The PlcR regulon regulates the transcription of more than 100 genes, which are involved in the formation of biofilm, the synthesis of toxins and proteases, etc. The LuxS / AI-2 system is determined to exist in Bacillus cereus. The signal molecule AI-2 is synthesized by the luxS gene and involves multiple enzymatic reactions. The initial metabolic substrate undergoes methylation reaction, hydrolysis reaction and cleavage reaction to generate a precursor. Then it will spontaneously cyclize through its own chemical properties to form the active chemical form of AI-2, which will participate in the bacterial quorum sensing process. In Bacillus cereus, the LuxS / AI-2 quorum sensing system is involved in the formation of biofilm and may be related to the synthesis of emetic toxin, i.e. vomitoxin. Therefore, the Bacillus cereus quorum sensing system genes plcR, papR and LuxS are selected as detection genes.

[0043] (2) The common diarrhea enterotoxins in B. cereus include hemolytic enterotoxin (Hbl), non-hemolytic enterotoxin (Nhe) and cytotoxin K (CytK), which are all secreted by the Sec pathway. The virulence genes related to Hbl include hblA, hblB, hblC and hblD, among which hblA, hblC and hblD encode three protein subunits of Hbl complex, i.e. Hbl B, L2 and L1, respectively. The protein Hbl B' encoded by hblB can balance the amount of Hbl B-L1 complex and the corresponding free subunit, which is a way for B. cereus to regulate its own toxicity. The genes encoding three subunits of Nhe are nheA, nheB and nheC, which encode protein subunits of the corresponding name, respectively. Similarly, CytK, Clo and other toxins are encoded by cytK, clo and other genes, respectively. Therefore, the virulence genes hblB, cytK, nheB and clo of B. cereus are selected as the detection genes.

[0044] (3) The NprB protease encoded by the NprB gene of B. cereus is an important protease involved in the catalysis of the maturation of the quorum sensing signal molecule PapR. The protease encoded by the ColA gene can cut the three-helix collagen structure, and promote the pathogenicity of bacteria and the shedding of biofilm by hydrolyzing the extracellular matrix. The ClpP protease encoded by the ClpP gene maintains the stability of intracellular proteins and regulates cell growth by controlling the amount of intracellular proteins, which is confirmed to be related to biofilm formation, spore production and motility. The protein encoded by the Sfp gene is a typical subtilase family protease, which is mainly related to the synthesis of surfactants in B. cereus. Therefore, the protease genes nprB, colA, clpP and sfp of B. cereus are selected as the detection genes.

[0045] (4) The pleiotropic transcriptional regulator genes are involved in the regulation of a large number of genes and affect various biological behaviors. CodY not only participates in the motility and virulence gene expression of B. cereus, but also affects the formation of biofilm by inhibiting the production of an unknown protease. Spo0A is the main regulatory factor for the sporulation of B. cereus, and the genes affected by spo0A reach hundreds, which further affect the transcription of more genes. Therefore, spo0A is related to various biological behaviors of B. cereus, such as surface motility and biofilm formation. Therefore, the pleiotropic transcriptional regulator genes codY and spo0A are selected as the detection genes.

[0046] (5) There are many other key genes in B. cereus, each with its own function and role, which together regulate the biological behavior of B. cereus. For example, tlpA encoding the methylotaxis transducer protein, sigH, a sporulation regulator, RpoN related to growth metabolism, motility, biofilm formation and toxin production. They may play a very key role in B. cereus, so they are also selected as detection genes.

[0047] 3.2 Primer design

[0048] B. cereus 16s rRNA was selected as the internal reference gene, and several key genes of B. cereus were selected as detection genes, including: quorum sensing system genes plcR, papR and LuxS; virulence genes hblB, cytK, nheB and clo; protease genes nprB, colA, clpP and sfp; pleiotropic transcriptional regulator genes codY and spo0A; other genes tlpA, sigH and rpoN. PCR primers were designed according to the selected genes, and the specific primer design process is as follows:

[0049] (1) Search the complete genome sequence of B. cereus on GenBank, continue to search and obtain the gene sequence of the target detection target;

[0050] (2) Directly search the target detection gene sequence on GenBank, and use Clustaw software for alignment analysis. Through multiple alignment between different sequences, the highest conserved region sequence is obtained;

[0051] (3) Further use Primer Premier and other software to analyze the obtained region sequence, and at the same time, combine the sequencing platform and method used by the sequence to conduct comprehensive fault tolerance analysis. For example, Sanger sequencing method has lower reliability of the first 20-30 and last 20-30 base sequences, which can be excluded by two-end sequencing. For example, PacBio sequencing has base mismatch phenomenon, which can be corrected by Illumina or Sanger sequencing. Comprehensive analysis is made to further accurately judge the sequence fault tolerance, obtain the highest conserved region sequence of the target detection gene, and design primers based on it.

[0052] Primers were synthesized by Guangzhou Aik Biotechnology Co., Ltd. The primer sequences are shown in Table 2.

[0053] Table 2 Primer sequences for RT-qPCR

[0054]

[0055]

[0056] 3.3 Real-time quantitative PCR system establishment

[0057] (1) Bacillus cereus total RNA extraction

[0058] The overnight culture of five Bacillus cereus strains was diluted to an initial concentration of 10 5 CFU / mL in a culture tube using TSB medium, and then 1 / 8 MIC concentration of malt extract solution was added. At 37°C, 200 rpm, samples were taken at 4h, 8h, 12h, and 24h, and RNA was extracted using the Trizol method.

[0059] (2) Reverse transcription

[0060] The gDNA was removed using the Rnase free DNase I kit (Thermol). Reverse transcription of RNA into cDNA was performed using the reverse transcription kit (Biosharp).

[0061] (3) Real-time quantitative PCR

[0062] The cDNA was diluted 10-fold, and the primer concentration was diluted to 10 mM for standby. The real-time quantitative PCR reaction system: 10 mL of 2x Mastermix, 2 mL of cDNA template (after dilution), 0.4 mL of upstream and downstream (F / R) primers (10 mM) each, and finally 20 mL of RNase free water. Amplification program: 94°C pre-denaturation for 3 min; 94°C denaturation for 15 s, 55°C annealing for 15 s, and 72°C extension for 20 s, for 40 cycles.

[0063] (4) Data statistics

[0064] The relative quantitative method was used to determine the changes in the expression of Bacillus cereus target detection genes over time, with 3 replicates for each sample to reduce errors. The 4h culture group was used as the reference, and after normalization with the internal reference gene, the value of 2 –ΔΔCt (Livak method) was used to represent the fold change in the expression of the target gene.

[0065] 3.4 Results and discussion

[0066] Figure 1 The growth curve of Bacillus cereus in the free state at 1 / 8 MIC is shown in the figure. As can be seen, 1 / 8 MIC malt extract inhibited the growth of Bacillus cereus to some extent. For example, ATCC 14579 was cultured for 10-14h, CMCC 2010 was cultured for 6-12h, IAM 12605 was cultured for 8-14h, L1 was cultured for 8-16h, and A9 was cultured for 6-14h, the bacterial concentration was less than that of the control group.

[0067] Figure 2 It is shown that Bacillus cereus under the action of 1 / 8 MIC of malt barley can completely inhibit the expression of PlcR-PapR quorum sensing system when the expression of the system is slightly weak. Under the action of malt barley, the plcR gene of strains ATCC 14579, A9 and L1 is completely inhibited when the strains are cultured for 24 h, and the PapR is inhibited when ATCC 14579 is cultured for 24 h. The LuxS gene is completely inhibited when ATCC 14579 is cultured for 24 h, IAM 12605 is cultured for 8 h, and CCM 2010 is cultured for 4 h and 8 h. It is proved that malt barley can target to inhibit the plcR, PlcR-PapR, LuxS signal pathway and the multiplication of Bacillus cereus to a certain extent.

[0068] Figure 3 It is shown that under the action of malt barley, the expression of nheB gene of the other four strains is completely inhibited after being cultured for 8 h, and some even stop expressing at 4 h. Therefore, it is considered that the inhibitory effect of malt barley on the nheB virulence gene of Bacillus cereus is very obvious, which can effectively reduce the Nhe toxicity of Bacillus cereus, and to a certain extent, it can be considered that the harm caused by the multiplication of Bacillus cereus is reduced.

[0069] Figure 4 It is shown that under the action of malt barley, the expression of Spo0A gene at each time point is basically adjusted. The expression of CodY gene is inhibited when the other four strains are cultured for 24 h except strain L1. Malt barley plays an obvious promoting role in the expression of the pleiotropic transcriptional regulator gene Spo0A in most cases studied, which can promote the sporulation and biofilm formation of Bacillus cereus, so that the bacteria tend to protect themselves rather than invade the host when the survival pressure is larger, and the inhibition of malt barley on the expression of CodY gene can also slow down the growth rate of bacteria.

[0070] Figure 5 It is shown that the influence of malt barley on the expression of clpP, nprB and sfp genes is quite different. It is shown that malt barley has no obvious effect on the expression of protease genes. The expression of protease genes of strain CCM 2010 is inhibited by malt barley, and the growth of strain CCM 2010 is still not affected under this condition, which shows that these proteases are not essential for the growth of Bacillus cereus.

[0071] Figure 6 It is shown that under the action of malt barley, the rpoN gene is completely inhibited at multiple time points. The inhibition of malt barley on the expression of rpoN gene may reduce the nitrogen metabolism efficiency of Bacillus cereus, thereby inhibiting its colonization and pathogenic potential.

[0072] In summary, Bacillus cereus with 1 / 8 MIC of maltobacillin can completely inhibit the expression of PlcR-PapR quorum sensing system when the expression of the system is slightly weak. Maltobacillin can basically completely inhibit the expression of nheB gene after a period of time, effectively reduce the Nhe toxicity of Bacillus cereus, and indirectly inhibit the proliferation. The promotion of maltobacillin on the expression of pleiotropic transcriptional regulator gene Spo0A and the inhibition of maltobacillin on the expression of CodY gene can also slow down the growth rate of Bacillus cereus. The inhibition of maltobacillin on the expression of rpoN gene can inhibit the colonization and pathogenic potential of Bacillus cereus.

[0073] The selected green natural product maltobacillin of the present application has wide application prospects, and its influence on the expression of signal pathway genes is not only suitable for Bacillus cereus, but also can be extended to other strains and even the field of food. It has important value for the growth detection of Bacillus cereus and the in-depth understanding of the proliferation mechanism of Bacillus cereus, and will be expected to improve food safety management, and provide beneficial reference and reference for microbiology research and other fields of application.

[0074] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of hordeum malt extract in the preparation of bacillus cereus bacteriostatic agent.

2. Use of hordeum malt extract in the preparation of bacillus cereus pathogenicity inhibiting agent.

3. The use according to claim 1 or 2, wherein the hordeum malt extract inhibits the growth and pathogenicity of bacillus cereus by regulating the quorum sensing system of bacillus cereus.

4. The use according to claim 1 or 2, wherein the hordeum malt extract inhibits the growth and pathogenicity of bacillus cereus by inhibiting the formation of biofilm of bacillus cereus.

5. The use according to claim 3, wherein the regulation of the quorum sensing system of bacillus cereus is to inhibit the expression level of the quorum sensing system related genes of bacillus cereus. plcR papR LuxS The quorum-sensing system-related gene includes at least one of 6. The use according to claim 1 or 2, wherein the bacillus cereus comprises at least one of bacillus cereus ATCC 14579, bacillus cereus IAM 12605, bacillus cereus CCM 2010, bacillus cereus Bcer A9 and bacillus cereus Bcer L1. , 7. The use according to claim 2, wherein the inhibition of the pathogenicity of bacillus cereus is to inhibit the expression level of the virulence genes of bacillus cereus. and hblB . cytK nheB clo ​ The virulence genes include at least one of ​ , ​ , ​ and ​ .

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