CN112940960B

CN112940960B - Bacillus subtilis BS-15, preparation method thereof and application thereof in inducing synthesis of phytolactobacillin EF

Info

Publication number: CN112940960B
Application number: CN202011613413.9A
Authority: CN
Inventors: 刘国荣; 聂蓉; 李雪
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-02-08
Anticipated expiration: 2040-12-30
Also published as: CN112940960A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis BS-15, a preparation method thereof and application thereof in inducing the synthesis of phytolactobacillin plantaricin EF. The invention provides a bacillus subtilis BS-15, wherein the preservation number of the bacillus subtilis BS-15 is CGMCC No. 20851. The lactobacillus plantarum BS-15 and lactobacillus plantarum provided by the invention can be used for co-culturing to effectively improve the lactobacillus plantarum plantaricin EF secreted by the lactobacillus plantarum.

Description

Bacillus subtilis BS-15, preparation method thereof and application thereof in inducing synthesis of phytolactobacillin EF

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis BS-15 strain, a preparation method thereof and application thereof in inducing plant lactobacillin plantaricin EF synthesis.

Background

The lactobacillus bacteriocin is protein or polypeptide with antibacterial activity which is processed and modified by ribosome after being synthesized in order to adapt to growth conditions or environmental changes, and becomes a hotspot for researching and developing active metabolites of probiotics because the protein or polypeptide can be degraded by human gastrointestinal protease and has the excellent characteristics of no drug resistance, safety, no toxicity, no residue and the like. The plant lactobacillus plantarum EF belongs to a class IIb bacteriocin, consists of two peptide chains, has heat resistance, pH tolerance and broad-spectrum antibacterial property, and can inhibit various pathogenic bacteria such as Listeria monocytogenes, Bacillus cereus, Escherichia coli and Bacillus subtilis; can inhibit other fermentation strains, such as Saccharomyces cerevisiae, Lactobacillus fermentum, Lactobacillus plantarum, and Leuconostoc mesenteroides.

Due to the influence of culture conditions, the growth period of bacteria and a self-regulation system, the production level of the phytolactobacillin plantaricin EF is very low, and the industrial production and application of the phytolactobacillin plantaricin EF are severely restricted. Under pure culture conditions, the yield of the phytolactobacillin plantaricin EF is low, which limits the large-scale application of the phytolactobacillin EF.

The current methods for improving the yield of the plant lactobacillus plantaricin EF include optimizing a culture medium and fermentation conditions, efficiently expressing and synthesizing related genes, adding an inducer or applying environmental condition stimulation and the like. The effect of improving the yield of the plant lactobacillus plantaricin EF by adopting the optimization of fermentation conditions is limited, and the cost of most of the used culture media is not economical; the plant lactobacillus plantaricin EF heterologous expression system constructed by utilizing the genetic engineering technology has the problems that the activity of the plant lactobacillus plantaricin EF is low, the safety needs to be verified and the like; the addition of an inducer or the application of environmental condition stimulation can improve the yield of the plant lactocin plantaricin EF by utilizing induction regulation and stress response, does not relate to safety problems, has low cost, and is considered to be the most promising technical means. The prior art still has certain limitation on the yield of the phytolactobacillin plantaricin EF obtained by co-culture.

Disclosure of Invention

In order to solve the problems, the invention provides a Bacillus subtilis BS-15 strain, a preparation method thereof and application thereof in inducing the synthesis of phytolactobacillin plantaricin EF. The Bacillus subtilis BS-15 provided by the invention can effectively improve the secretion of the plantaricin EF by the lactobacillus plantarum.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a Bacillus subtilis BS-15, wherein the preservation number of the Bacillus subtilis BS-15 is CGMCC No. 20851.

The invention also provides a culture method of the Bacillus subtilis BS-15, which comprises the following steps:

inoculating the Bacillus subtilis BS-15 into a culture medium for culturing to obtain the Bacillus subtilis BS-15.

Preferably, the medium comprises tryptone soy broth; the inoculation amount of the inoculation is 1-5% of the volume of the culture medium; the culture temperature is 30-37 ℃, and the culture time is 18-36 h.

The invention provides application of the Bacillus subtilis BS-15, bacterial suspension of the Bacillus subtilis BS-15, fermentation liquor of the Bacillus subtilis BS-15 or secretion of the Bacillus subtilis BS-15 in promoting lactobacillus plantarum to secrete the plantaricin EF.

Preferably, the application comprises: bacillus subtilis BS-15 and Lactobacillus plantarum are co-cultured.

Preferably, during the co-culture, the ratio of the volume of the fermentation liquid of the Bacillus subtilis BS-15 to the volume of the lactobacillus plantarum is (1-10): (1-10).

Preferably, during the co-culture, the concentration of the Bacillus subtilis BS-15 in the fermentation liquor of the Bacillus subtilis BS-15 is 10⁵～10⁸CFU/ml。

Preferably, the Lactobacillus plantarum comprises Lactobacillus plantarum (Lactobacillus plantarum) RX-8, and the preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) RX-8 is CGMCC No. 20852.

The invention provides a Bacillus subtilis BS-15, wherein the preservation number of the Bacillus subtilis BS-15 is CGMCC No. 20851. The lactobacillus subtilis BS-15 provided by the invention can be used for effectively improving the lactobacillus plantarum plantaricin EF secreted by the lactobacillus plantarum by co-culturing with the lactobacillus plantarum. From the examples, it can be seen that the synthesis amount of the plant lactobacillin plantaricin EF in the co-culture system is about 20 hours and stable when the Bacillus subtilis BS-15 and the Lactobacillus plantarum (Lactobacillus plantarii) RX-8 are co-cultured, and the synthesis amount of the plant lactobacillin plantaricin EF in the co-culture system is about 3 times of that of the pure cultured Lactobacillus plantaricin (Lactobacillus plantarium) RX-8 when the synthesis amount reaches the stable period. The results show that the co-culture of the Bacillus subtilis BS-15 and the Lactobacillus plantarum RX-8 not only can greatly improve the yield of the Lactobacillus plantarum EF, but also can shorten the production time of the Lactobacillus plantarum EF by 4 h.

Biological preservation Instructions

Bacillus subtilis BS-15 is preserved in China general microbiological culture Collection center (CGMCC) within 10 months and 12 days of 2020, and the preservation place is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 20851.

Lactobacillus plantarum RX-8 (Lactobacillus plantarum) is preserved in China general microbiological culture Collection center (CGMCC) 10, 10 and 12 days 2020, and the preservation place is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 20852.

Drawings

FIG. 1 shows the colony morphology of Bacillus subtilis BS-15;

FIG. 2 shows the somatic cells of Bacillus subtilis BS-15;

FIG. 3 is an electrophoretogram of plnE and plnF genes;

FIG. 4 shows the amount of phytolactobacillin plantaricin EF synthesized in pure culture and co-culture;

FIG. 5 shows the variation of the amount of phytolactobacillin plantaricin EF synthesized during pure culture and co-culture;

FIG. 6 shows the detection of the induction ability of Bacillus subtilis BS-15 under different treatment conditions;

FIG. 7 shows the variation of the expression level of the EF gene plnE of the plant lactobacillus plantericin during pure culture and co-culture;

FIG. 8 shows the variation of the expression level of the phytolactobacillin plantaricin EF gene plnF during pure and coculture;

FIG. 9 is a study of the substances responsible for induction in Bacillus subtilis BS-15;

FIG. 10 shows the bacteriostatic activity of the fractions separated by ultrafiltration;

FIG. 11 shows the induction activity of the fraction obtained by ultrafiltration;

FIG. 12 shows gel chromatography;

FIG. 13 is the induction activity of component A;

FIG. 14 is the induction activity of component B;

FIG. 15 shows the amount of plantaricin EF synthesized by Lactobacillus plantarum WCFS1 in pure culture and co-culture.

Detailed Description

The invention provides a Bacillus subtilis BS-15, wherein the preservation number of the Bacillus subtilis BS-15 is CGMCC No. 20851. The sequence information of the Bacillus subtilis BS-15 is as follows:

CATGGGGGTGCTATACATGCAAGTCGAGCGGACAGATGGG AGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGT GGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGG GCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAA AGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTA GCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAG CCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAA TGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAG GTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGC CGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGC CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGG CAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCG GTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAG GGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGT GGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA ACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTG AGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTC CGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG GAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGG CCCGCACAGCGGTGGAGCATGTGGTTTATTCGAGCACGCGAGA CCTTACCAGGTCTTGACATCCTCTGACATCTAGAGATAGGACGT CCCTTCGGGGCAGAGTGACAGTGTGCATGTGTCGTCAGCTCGT GTCGTGAGATGTGGGTAGTCCCGCACGAGCGCAACCTGATCTA GTTGCAGCATCAGTGGCACTCTTAAGGTGACTTCCGGTGCCAA CCGGAGGAAGGTGGGGGATGGACGTCAATCATCAATGTCCTTAT GACTTGGCCTAACACTGCTTCATATGGGACGAACATAAGGCACG GAACCGCTAGT, SEQ ID NO: 1; the colony morphology of the Bacillus subtilis BS-15 is shown in figure 1, and the cell photograph of the Bacillus subtilis is shown in figure 2. The Bacillus subtilis BS-15 provided by the invention can effectively improve the secretion of the plantaricin EF by the lactobacillus plantarum.

In the present invention, the medium preferably includes Tryptone Soy Broth (TSB); the inoculation amount of the inoculation is preferably 1-5% of the volume of the culture medium, and is further preferably 1%; the temperature of the culture is preferably 30-37 ℃, the further preference is 37 ℃, and the time of the culture is preferably 18-36 h, the further preference is 24 h.

The invention provides application of the Bacillus subtilis BS-15, bacterial suspension of the Bacillus subtilis BS-15, fermentation liquor of the Bacillus subtilis BS-15 or secretion of the Bacillus subtilis BS-15 in promoting lactobacillus plantarum to secrete the plantaricin EF. In the present invention, the Lactobacillus plantarum is preferably Lactobacillus plantarum RX-8. In the invention, the preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) RX-8 is CGMCC No. 20852.

In the invention, when the bacterial suspension of the Bacillus subtilis BS-15 and the Lactobacillus plantarum are co-cultured, the mass ratio of the volume of the bacterial suspension of the Bacillus subtilis BS-15 to the Lactobacillus plantarum is preferably (1-10): 1-10, and more preferably 1: 10; what is needed isThe concentration of the Bacillus subtilis BS-15 in the Bacillus subtilis BS-15 bacterial suspension is preferably 10⁵～10⁹CFU/ml, more preferably 10⁷CFU/ml。

In the invention, when the fermentation liquor of the Bacillus subtilis BS-15 and the lactobacillus plantarum are co-cultured, the ratio of the volume of the fermentation liquor of the Bacillus subtilis BS-15 to the volume of the lactobacillus plantarum is preferably (1-10): 1-10, and more preferably 10: 1; the concentration of the Bacillus subtilis BS-15 in the fermentation liquor of the Bacillus subtilis BS-15 is preferably 10⁵～10⁸CFU/ml, more preferably 10⁸CFU/ml。

The Bacillus subtilis BS-15, the fermentation liquor and the secretion can effectively improve the yield of the lactobacillus plantarum secreting the plantaricin EF and can also accelerate the production time of the plantaricin EF.

To further illustrate the present invention, the following detailed description of a strain of Bacillus subtilis BS-15, its preparation method and its application in inducing the synthesis of phytolactobacillin plantaricin EF are provided in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Identification of bacteriocin produced by lactobacillus plantarum RX-8 as lactobacillus plantarum plantaricin EF

Lactobacillus plantarum RX-8 was spread on MRS solid medium and cultured at 37 ℃ for 24 hours. And (3) selecting a single colony, inoculating the single colony to 10ml of MRS liquid culture medium, and culturing for 9h at 37 ℃ to obtain the lactobacillus plantarum RX-8 bacterial liquid. Lactobacillus plantarum RX-8 genomic DNA was extracted according to the protocol of the bacterial genomic DNA extraction kit (DP302) from Tiangen Biochemical technology Ltd. Primers were designed based on the sequences of the phytolactobacillin plantaricin EF genes (plnE and plnF) published in GenBank by Lactobacillus plantaricin C11(X94434) and Lactobacillus plantaricin WCFS1(AL935253), and the sequences of the primers are shown in Table 1.

TABLE 1 PCR primers

The target fragment is amplified by using the genomic DNA of lactobacillus plantarum RX-8 as a template and adopting a PCR amplification reaction kit, wherein the PCR reaction system is shown in Table 2. The PCR reaction condition is pre-denaturation at 95 ℃ for 5 min; 30 times of circulation at 94 ℃ for 30s, at 53 ℃ for 30s and at 72 ℃ for 10 s; extension at 72 ℃ for 10min, followed by storage at 4 ℃. Taking 4 μ l of PCR product, using DL2000 as DNA molecular marker, methyl bromophenol blue as dye, and electrophoresis of 1% agarose gel to detect the PCR product, the electrophoresis result is shown in FIG. 3. And sending the products with the same amplification size to Huada gene company for sequencing analysis, and comparing the sequencing result with the NCBI database.

TABLE 2 PCR reaction System

The results of alignment of sequencing results in FIG. 3 show that the coding regions of plnE and plnF genes are 158bp and 171bp, and the homology with the published Lactobacillus plantarum C11 is 99.60% and 98.67%, and the bacteriocin produced by the Lactobacillus plantarum RX-8 can be determined to be the Lactobacillus plantarum EF.

Example 2

Screening co-culture inducing strains under different phytolactobacillin plantaricin EF culture systems

(1) Preparing pure culture and co-culture fermentation supernatant

Inoculating lactobacillus plantarum RX-8 to an MRS liquid culture medium, and culturing at 30 ℃ for 24h to obtain pure culture fermentation liquor. Inoculating Bacillus subtilis to be screened into TSB liquid culture medium from slant, culturing to logarithmic phase, inoculating 1% inoculum size to first generation, and measuring OD every 1h₆₀₀Absorbance. Continuously culturing for two generations, and taking the third generation strain for later use. Adding 10 co-culture test strains into lactobacillus plantarum RX-8 fermentation liquor for co-fermentation under a normal (culture temperature of 37 ℃, culture medium 1MRS, initial pH of 6.8 and culture time of 24h), low-yield phytolactobacillin plantaricin EF (culture temperature of 37 ℃, culture medium initial pH5, culture medium 1/10MRS, inoculum size of 1% and culture time of 24h) and non-yield (culture temperature of 42 ℃, culture medium initial pH5, culture medium 1/10MRS, inoculum size of 1% and culture time of 24h) culture model systems, wherein the inoculation volume ratio is 1:1 (the thallus density is equal), the inoculation density is determined according to the OD value when each thallus reaches the stable initial stage, and culturing for 24h to obtain the co-culture fermentation liquor.

The pure culture (Lactobacillus plantarum RX-8), the Lactobacillus plantarum RX-8 and the different Bacillus subtilis co-culture fermentation broth are centrifuged at 10000r/min to obtain the supernatant, and the pH is adjusted to 6.5 by 2.5mol/L NaOH. Adding catalase dissolved in phosphate buffer solution into the fermentation supernatant with adjusted pH to a final concentration of 5mg/ml, carrying out water bath at 37 ℃ for 2h, and filtering with a 0.22 mu m filter membrane to obtain cell-free fermentation supernatant (CFS).

(2) Preparation of plant lactobacillin plantaricin EF crude extract

The ammonium sulfate precipitation method is adopted to prepare the pure cultured plant lactobacillus plantaricin EF crude extract from the supernatant of the cell-free fermentation. The pH of the cell-free fermentation supernatant (CFS) was adjusted to 7 with NaOH, and ammonium sulfate, which was ground to a powder, was slowly added and stirred continuously with a magnetic stirrer until the final saturation of ammonium sulfate was 80%. Standing the precipitate in a refrigerator at 4 deg.C overnight, freezing and centrifuging at 12000r/min at 4 deg.C for 10min, and collecting the precipitate. Redissolving the precipitate in 0.02M Na₂HPO₄-NaH₂PO₄(pH7.0) to obtain coarse extractive solution of phytolactobacillin plantaricin EF.

(3) Determination of antibacterial activity of crude extract of phytolactobacillin plantaricin EF

First, 1ml of the solution is diluted to 10 by pipetting with a pipette^-4(1×10⁶CFU/mL) of Listeria monocytogenes ATCC 35152(4b) diluted bacterial solution in a dish having a diameter of 9cm, and 10mL of molten bacterial solution was added theretoThe MRS solid culture medium is mixed evenly and placed on a horizontal table board to form an even thickness, and a plate is blown for half an hour near an alcohol lamp by opening a plate cover. Placing oxford cups on the solidified plates by using sterile tweezers, adding 100 mu l of pure culture and co-culture phytolactocin EF crude extract, placing the plates in a refrigerator at 4 ℃ for overnight diffusion, taking out the plates, placing the plates in an incubator at 30 ℃ for culture for 6h, observing whether a bacteriostatic circle appears, measuring the bacteriostatic diameter by using a vernier caliper, and accurately reading to 0.01 mm. Each treatment was set to 3 replicates and the average was taken as the result for calculation. The results are shown in Table 3.

TABLE 3 screening of Co-culture induced strains under EF culture system of different produced plant lactobacillin plantaricin

As shown in Table 3, under the culture system without producing the phytolactobacillin plantaricin EF, only 1 strain of the strain can induce the lactobacillin RX-8 to produce the phytolactobacillin plantaricin EF, but the yield of the phytolactobacillin plantaricin EF is not realized from the non-production to the normal production, and the nutrient supply of the strain can not be met due to the fact that the nutrient substances of the non-production culture system are few; under a culture system of the low-yield plant lactobacillus plantaricin EF, 4 strains of bacteria remarkably promote the synthesis of the lactobacillus RX-8 plant lactobacillus plantaricin EF, wherein the promotion effect of the bacillus subtilis BS-15 is most remarkable, and the bacteriostatic diameter difference reaches more than 12mm, so that the yield of the plant lactobacillus plantaricin EF is from low yield to high yield; in the normal plant-produced lactobacillus plantaricin EF culture system, although the 4 test strains all have the promotion effect, the promotion effect is lower than that of the low-yield culture system. The bacillus subtilis BS-15 has obvious promotion effect under the system, and the bacteriostatic diameter difference reaches 10.55 mm. According to the experiment, the conditions of different culture systems with different bacteriostatic diameters are combined, and finally the bacillus subtilis BS-15 is selected as the best co-culture strain and the low-yield culture system is used as the culture condition for subsequent research.

Example 3

Detection of the induction capability of Bacillus subtilis BS-15

The lactobacillus plantarum RX-8 is purely cultured under the condition of a low-yield culture system, the lactobacillus plantarum RX-8 and the bacillus subtilis BS-15 are co-cultured according to the inoculation ratio of 1:1, the lactobacillus plantarum BS-15 induced lactobacillus plantarum plantaricin EF synthesis capacity is respectively detected according to the method for detecting the antibacterial activity in the example 2, and the detection result picture is shown in a figure 4.

The effect of the bacillus subtilis BS-15 in inducing the high-efficiency synthesis of the phytolactobacillin plantaricin EF is shown in figure 4, and as can be seen from figure 4, the inhibition zone of the co-culture is the largest, the inhibition zone does not appear in the pure-cultured bacillus subtilis BS-15, and the lactobacillus plantarum RX-8 has the inhibition zone but the diameter is far smaller than the diameter of the co-culture inhibition zone. The bacillus subtilis BS-15 has no inhibition effect on the indicator bacterium listeria monocytogenes, the lactobacillus plantarum RX-8 has certain inhibition effect on the indicator bacterium, and after the bacillus subtilis and the indicator bacterium are co-cultured, the diameter of an inhibition zone is increased, and the inhibition effect is obviously enhanced.

Pure culture and co-culture fermentation broth in a low-yield culture model system are taken every 4h, the synthesis capacity of the bacillus subtilis BS-15 for inducing the phytolactobacillin plantaricin EF is detected by the method in the example 2, and the detection results are shown in a figure 5 and a table 4.

TABLE 4 Bacillus subtilis BS-15 ability to induce the synthesis of phytolactobacillin plantaricin EF

As shown in FIG. 5 and Table 4, a small amount of phytolactobacillin plantaricin EF was produced in the co-culture system at 4h, but the phytolactobacillin plantaricin EF had not been synthesized in the pure culture system. Under two culture systems, the change trend of the synthesis amount of the phytolactobacillin plantaricin EF is approximately same, the synthesis amount is stable within about 20 hours, and begins to decrease within about 32 hours. However, when stationary phase was reached, the amount of phytolactobacillin plantaricin EF synthesized in the co-culture system was about 3 times that in the pure culture. The induced bacteria can not only greatly improve the yield of the plant lactobacillus plantaricin EF, but also advance the time for producing the plant lactobacillus plantaricin EF by 4 hours.

Example 4

Detection of the induction capability of Bacillus subtilis BS-15 under different treatment conditions

Bacillus subtilis BS-15 is treated as follows:

a. centrifuging the Bacillus subtilis BS-15 pure culture fermentation broth at 10000r/min, removing the supernatant, and then cleaning with normal saline for 3 times to obtain a bacterial suspension;

b. treating the fermentation supernatant with a 0.22 μm bacterial filtration membrane to obtain sterile fermentation supernatant;

c. the two bacteria are separated by Trans-well, so that the two bacteria can not directly contact but can freely exchange metabolic substances, and the two bacteria are indirectly co-cultured in a low-yield culture system.

Co-culturing the treated products of a and b with lactobacillus plantarum RX-8 directly under a low-yield culture system. The lactobacillus plantarum RX-8 pure culture broth is used as a control.

The fermentation liquids of the cultured different treatment groups and the control group were tested for the induction ability of Bacillus subtilis BS-15 under different treatment conditions according to the method of example 2, and the results are shown in FIG. 6 and Table 5.

TABLE 5 Induction Capacity of Bacillus subtilis BS-15 under different treatment conditions

As shown in FIG. 6 and Table 5, the bacterial suspension, fermentation broth and metabolite bacteria of Bacillus subtilis BS-15 can induce the efficient synthesis of phytolactobacillin plantaricin EF.

Example 5

Variation of expression levels of phytolactobacillin plantaricin EF genes plnE and plnF in pure culture and co-culture

Taking pure culture and co-culture fermentation liquor every 4h, centrifuging at 10000r/min, discarding supernatant and retaining thalli. Extracting plant milk according to the instruction of RNAprep Pure Cell/Bacteria Kit RNA extraction Kit purchased from Tiangen Biochemical technology limited companyBacillus RX-8 Total RNA. RNA of Lactobacillus plantarum RX-8 was reverse transcribed into cDNA using the Tiangen FastQuant cDNA first strand synthesis kit. RT-qPCR was performed using a SuperReal fluorescent quantitative premix reagent enhanced (SYBR Green) kit from Tiangen Biochemical technology Ltd, the primers used are shown in Table 6, the reaction system is shown in Table 7, and the reaction conditions are as follows: 2min at 95 ℃; circulating for 39 times at 94 ℃ for 20s and 63 ℃ for 45 s; 5min at 60 ℃; the 16S gene was used as an internal reference, a blank experimental group was used as a control, and 2 was used^-△△CtThe method performs relative quantitative calculation. The detection results are shown in fig. 7 and 8, and table 8 and 9.

TABLE 6 RT-qRCR primers

TABLE 7 RT-qRCR reaction System

TABLE 8 variation of the expression level of the EF gene plnE of the plant Lactobacillus plantericin in pure and coculture

TABLE 9 variation of the expression level of the phytolactobacillin plantaricin EF gene plnF in pure and co-cultures

As shown in FIGS. 7 and 8, and tables 8 and 9, the transcription level of plnEF gene was greatly increased in the co-culture system after 8 hours as compared with the pure culture, and the expression levels of plnE and plnF genes after co-culture were increased by 8.3 times as compared with the pure culture when the stationary phase of fermentation was finally reached. With reference to fig. 5, fig. 7 and fig. 8, the expression levels of the phytolactobacillin plantaricin EF in coculture were consistent with the expression levels of the plnE and plnF genes, while the expression level of the phytolactobacillin plantaricin EF in pure culture hardly changed with the culture time; it is demonstrated that Bacillus subtilis BS-15 can produce inducer, and the yield of plant lactobacillin plantaricin EF can be improved by inducing the expression of plnE and plnF genes.

Example 6

Substance having inducing effect in bacillus subtilis BS-15

Bacillus subtilis BS-15 is treated as follows:

b. inactivating for 15min at 121 ℃ on the basis of the treatment a to obtain an inactivated bacteria suspension;

c. treating the fermentation supernatant with a 0.22 μm bacterial filtration membrane to obtain sterile fermentation supernatant;

d. on the basis of the treatment c, adding 2mg/ml proteinase K into the sterile fermentation supernatant, incubating for 2h at the temperature of 37 ℃ under the pH value of 8.0, and heating for 15min at the temperature of 80 ℃ to inactivate enzyme, thereby obtaining the enzymatic sterile fermentation supernatant;

and (4) directly co-culturing the obtained products obtained by the treatment of a to d and lactobacillus plantarum RX-8 in a low-yield culture system. The lactobacillus plantarum RX-8 pure culture broth is used as a control.

The fermentation liquids of the cultured different treatment groups and the control group were tested for the induction ability of Bacillus subtilis BS-15 under different treatment conditions according to the method of example 2, and the results are shown in FIG. 9 and Table 10.

TABLE 10 Induction Capacity of Bacillus subtilis BS-15 under different treatment conditions

As can be seen from FIG. 9 and Table 10, compared with the pure cultured Lactobacillus RX-8, only the supernatant and cells of Bacillus subtilis BS-15 induced the production of phytolactobacillin EF by Lactobacillus RX-8, and the Bacillus subtilis BS-15 after protease treatment and autoclaving had no inducing effect, indicating that the inducing substance is a protein substance produced by living cells.

Example 7

Isolation of the Induction effective fraction of Bacillus subtilis BS-15

Taking the crude extract of the phytolactobacillin plantaricin EF, filtering the crude extract by using a 0.22 mu m filter membrane, adding the filtered extract into a 5000Da ultrafiltration tube, and centrifuging the mixture at 6000r/min for 15 min. Fractions greater than 5000Da were cut off by the ultrafiltration tube and set as fraction 1, and fractions less than 5000Da were passed through the ultrafiltration tube and set as fraction 2. Fractions 1 and 2 were collected and the bacteriostatic and inductive activities of the two fractions were verified by the method of reference example 2. The results are shown in fig. 10, fig. 11 and table 11.

As can be seen from FIG. 10, fraction 1 with a molecular weight greater than 5000Da was able to detect bacteriostatic activity; whereas fraction 2 with a molecular weight of less than 5000Da was only detected without bacteriostatic activity, which is consistent with the results for the two peptides of phytolactobacillin plantaricin EF, the molecular weight of plnE 6191Da, and the molecular weight of plnF 5733 Da.

As can be seen from FIG. 11, both fraction 1 and fraction 2 were detectable for inducing activity, but fraction 2 was much more potent than fraction 1 for inducing activity. FIG. 10 and FIG. 11 together show that the phytolactobacillin plantaricin EF is present in fraction 1 and that the inducing fraction is present in a significant amount in fraction 2 having a molecular weight of less than 5000 Da.

TABLE 11 validation of inducibility of Components 1 and 2

Components	Induction group d/mm	Bacteriostatic group d/mm	Control group d/mm
				Component 1	8.25	23.12	6.70
Component 2	14.63	6.74	6.70

Continuously separating the component 2 by a gel chromatographic column Sephadex G-25, and specifically operating as follows:

1) pretreatment of Sephadex G25: putting a proper amount of Sephadex G-25 powder into a certain amount of ultrapure water, stirring at intervals at the initial stage of soaking to ensure that the Sephadex G25 powder fully absorbs water and swells, and soaking overnight.

2) Column assembling: the specification of the glass beads used was 1.6cm × 60cm, and before filling of the packing, the glass beads were washed with a flow against them and the presence of liquid leakage at the upper and lower ends of the glass column was examined. If the leakage detection is normal, filling of the filler is started. And (3) leading the pipeline at the lower end of the glass column to be in a smooth state by adopting a glass rod, carefully pouring the Sephadex G-25 after full swelling into the glass column, stopping adding liquid when the liquid level is about to overflow, continuously adding the Sephadex G-25 solution after the liquid level in the glass column is reduced, and finally filling the whole glass column with the Sephadex G-25 filler. Care should be taken during filling to avoid the creation of bubbles, which can be slowly removed by the glass rod if they are created, or else only refilled.

3) Balancing: the AKTA pure parameter of the protein purifier is total flow rate of 1mL/min, the flow rate of a pump B is 1mL/min, and the balance is 30 min;

4) removing impurities: after the glass column is balanced, the glass column is cleaned by 0.2M NaOH until the absorption value at 220nm is at the same level value, then the glass column is cleaned by 0.2M NaOH by ultrapure water, and then 20mM phosphate buffer solution is added, and after the glass column is completely filled with the phosphate buffer solution and the ultraviolet absorption value is stable, the sample loading is prepared.

5) Loading: click on protein purification appearance AKTA pure manual sample introduction, then adopt the injector to inject 1mL of component 2, click and accomplish the sample introduction.

6) And (3) elution: the elution flow rate was 1mL/min, and the elution solution was 20mM phosphate buffer, and the elution was linear.

7) Collecting: real-time on-line detection of ultraviolet absorption peaks, and collection of components according to peak shapes.

8) Component verification: the eluted peak was concentrated using a vacuum concentrator, and the induction verification test was performed according to example 2 (in which the control group used lactobacillus plantarum RX-8 pure culture broth), and the results are shown in fig. 12, fig. 13, fig. 14, and table 12. Wherein FIG. 12 shows the case of gel chromatography. As can be seen from FIG. 12, two peaks appear at a wavelength of 220nm, representing two different components, designated component A and component B, respectively.

FIG. 13 is the induction activity of component A and FIG. 14 is the induction activity of component B.

TABLE 12 validation of inductivity of Components A and B

Components	Induction group d/mm	Bacteriostatic group d/mm	Control group d/mm
				Component A	6.81	6.79	6.77
Component B	12.11	6.83	6.67

As can be seen from fig. 13 and table 12, component a has no induction ability; as can be seen from fig. 14 and table 12, component B has a significant induction capacity, indicating that the component that plays an induction effect is located in component B.

Example 8

Inoculating lactobacillus plantarum WCFS1 to MRS liquid culture medium, and culturing at 30 ℃ for 24h to obtain pure culture fermentation broth. Under a normal culture model system, the component B obtained in the example 7 is added into the lactobacillus plantarum WCFS1 fermentation liquor for co-fermentation, the inoculation volume ratio is 1:10, and the co-culture fermentation liquor is obtained after 24h of culture. The bacteriostatic activity was measured according to the method of example 2, and the results are shown in fig. 15 and table 14.

TABLE 14 ability of Bacillus subtilis BS-15 to induce the production of Lactobacillus plantarum EF by Lactobacillus plantarum WCFS1

As can be seen from FIG. 15 and Table 14, the secretion of Bacillus subtilis BS-15 can induce the synthesis of secreted plantaricin EF not only by Lactobacillus plantarum RX-8, but also by Lactobacillus plantarum WCFS 1. The lactobacillus plantarum WCFS1 is a widely researched typical strain for producing the lactobacillus plantarum EF, comes from American Type Culture Collection (ATCC) and is numbered as ATCC BAA-793, is a reference strain of other lactobacillus plantarum for producing the lactobacillus plantarum EF, and has good representativeness. From the above, the Bacillus subtilis BS-15 provided by the invention has the ability of inducing the Lactobacillus plantarum to synthesize and secrete the phytolactocin plantaricin EF.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

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caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

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taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacagcg gtggagcatg tggtttattc gagcacgcga gaccttacca ggtcttgaca 960

tcctctgaca tctagagata ggacgtccct tcggggcaga gtgacagtgt gcatgtgtcg 1020

tcagctcgtg tcgtgagatg tgggtagtcc cgcacgagcg caacctgatc tagttgcagc 1080

atcagtggca ctcttaaggt gacttccggt gccaaccgga ggaaggtggg ggatggacgt 1140

caatcatcaa tgtccttatg acttggccta acactgcttc atatgggacg aacataaggc 1200

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Claims

1. The Bacillus subtilis BS-15 is characterized in that the preservation number of the Bacillus subtilis BS-15 is CGMCC No. 20851.

2. The method for culturing Bacillus subtilis BS-15 according to claim 1, comprising the steps of:

inoculating the Bacillus subtilis BS-15 of claim 1 into a culture medium for culturing to obtain the Bacillus subtilis BS-15.

3. The culture method according to claim 2, wherein the medium comprises tryptone soy broth; the inoculation amount of the inoculation is 1-5% of the volume of the culture medium; the culture temperature is 30-37 ℃, and the culture time is 18-36 h.

4. Use of Bacillus subtilis BS-15, a bacterial suspension, a fermentation broth or a secretion thereof according to claim 1 for promoting secretion of lactobacillus plantarum EF;

the lactobacillus plantarum is RX-8 and WCFS 1;

the secretion is protein matter.

5. The application according to claim 4, wherein the application comprises: bacillus subtilis BS-15 and Lactobacillus plantarum are co-cultured.

6. The use of claim 5, wherein the ratio of the volume of the fermentation broth of Bacillus subtilis BS-15 to the volume of Lactobacillus plantarum in the co-cultivation is (1-10): 1-10.

7. The use according to claim 5, wherein the concentration of Bacillus subtilis BS-15 in the fermentation broth of Bacillus subtilis BS-15 during the co-cultivation is 10⁵～10⁸CFU/ml。

8. The use according to any one of claims 4 to 7, wherein the Lactobacillus plantarum comprises Lactobacillus plantarum (Lactobacillus plantarum) RX-8, which has a accession number CGMCC No. 20852.