WO2022033085A1

WO2022033085A1 - Firmicutes strain allied to bacillus and anti-cancer use thereof

Info

Publication number: WO2022033085A1
Application number: PCT/CN2021/091883
Authority: WO
Inventors: Shulin Liu; Guirong LIU; Yang Li
Original assignee: Shulin Liu
Priority date: 2020-08-11
Filing date: 2021-05-06
Publication date: 2022-02-17
Also published as: CN112852682A

Abstract

Provided is a Firmicutes strain with anti-cancer activity and the anti-cancer use thereof. The strain has a preservation number of GMCC No. 17886, which belongs to a newly discovered bacterial taxon kin to the genus Bacillus. Also provided is an anti-cancer bacterial preparation using the strain as its active anti-cancer bacteria, a metabolite made from the strain, a pharmaceutical comprising the metabolite, a method for culturing the strain, and anti-cancer use of the preparation, the metabolite and/or the pharmaceutical.

Description

FIRMICUTES STRAIN ALLIED TO BACILLUS AND ANTI-CANCER USE THEREOF

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to biopharmaceutical technologies, and more particularly to a Firmicutes strain allied to Bacillus species and its anti-cancer use.

2. Description of Related Art

Cancers, as the number-one killer to human health, have long been a to-be-conquered challenge in the medical circles. The unwholesome trend in modern lifestyle and endlessly emerging new risk factors are furthering incidence and development of cancers.

Traditionally, cancers have been treated with radiation therapy and chemotherapy. However, these means are notorious for being physically and mentally harmful to patients and having issues about drug resistance. Immunotherapy is a recently emerging therapy. As it is far from mature, there are many concerns to be addressed and the therapeutic practice is often not satisfying. Hence there is a pressing need for an efficacious alternative anti-cancer approach.

SUMMARY OF THE INVENTION

By referring to many recent studies on the relationship between intestinal microbiota and health, the inventors came to realize that intestinal microbiota play a key role in fighting cancer. In order to discover more bacteria with anti-cancer activity and provide more novel and effective alternative method for treating cancers, the inventors isolate and identify bacteria with potent anti-cancer activities by way of the combination of metagenomic high throughput screening of intestinal microbiota with traditional bacteriology, molecular microbiology and genetics methods. Based on the findings, the following technical solution is disclosed. Based on this discovery, the following technical solutions are provided.

The present invention in a first aspect provides a Firmicutes strain allied to Bacillus species. The strain has a preservation number of CGMCC No. 17886.

Another identification standard for the strain is: it is Gram-positive, aerobic or facultatively anaerobic, has capsule, spores being elliptical, oval, columnar or spherical; by comparing its whole genome sequence with the bacterial sequences published in GenBank, it is most closely related to Bacillus paralicheniformis in terms of genome sequence, yet have two signature sequences shown by SEQ ID NO. 1 and SEQ ID NO. 2.

The disclosed strain is a strain of Firmicutes (Phylum: Firmicutes) with potent anti-cancer activity. It is kin to Bacillus species and is numbered CCPM7643 (also referred to as Tianjian No. 5) , representing a species that has never been isolated before. The disclosed strain has been preserved in China General Microbiological Culture Collection Center, the preservation number is CGMCC No. 17886, the preservation date is June 3, 2019, and the preservation address is No. 3, Courtyard 1, Beichenxi Road, Chaoyang District, Beijing City, China.

CCPM7643 has the following morphologic characteristics: as it is kin to Bacillus species in terms of genome, they share similar morphologic characteristics, including being Gram-positive, aerobic or facultatively anaerobic, and having capsule, having endospores that are elliptical, oval, columnar or spherical, and being resistant to many adverse environments. The colony has a flat round shape on the agar medium, being greyish white and translucent, with irregular edges (as shown in FIG. 1) .

CCPM764 has the following molecular biological characteristics: by comparing its whole genome sequence with the bacterial sequences published in GenBank, CCPM764 was found to be most consistent to Bacillus subtilis in terms of genome sequence, as shown in the evolutionary tree of FIG. 2, yet have distinguishable features, such as the two signature sequences (SEQ ID NO. 1 and SEQ ID NO. 2) . Hence, CCPM764 represents a new, previously unknown bacterial species. The CCPM764 strain has significant and potent anti-cancer activity.

The present invention in a second aspect provides a metabolite made from the strain as indicated in the first aspect of the present invention.

The present invention in a third aspect provides an anti-cancer bacterial preparation, which is obtained by amplification and culturing using the strain as indicated in the first aspect of the present invention as active anti-cancer bacteria.

Preferably, the bacterial preparation is:

(i) . solid culture or liquid culture of the strain;

(ii) . liquid portion obtained after solid-liquid separation of the liquid culture of (i) ; or

(iii) . product obtained by further purification of the liquid portion of (ii) .

The disclosed preparation may be a liquid preparation and may be used to balance gastrointestinal microbiota, or to provide anti-cancer effects by balancing gastrointestinal microbiota. As used herein, the term “anti-cancer” may refer to prevention, inhibition or elimination of cancer. The term “inhibition” may refer to retarding development of cancers or mitigating symptoms of cancers. The term “elimination” may refer to eliminating a part or all of symptoms of cancers, such as curing cancers.

Preferably, the culture medium used for amplification is a starch culture medium; preferably, the starch in the starch culture medium is from grain, or is from at least one selected from the group consisting of potato, maize, soybean and millet.

In a fourth aspect of the present disclosure, a preparation method for the metabolite or bacterial preparation is provided, which share the common feature that the method comprises a step of inoculating the strain to a culture medium and amplifying in an environment between 14 ℃to 39 ℃.

In some preferred embodiments, the culture medium is a liquid culture medium, the metabolite or the bacterial preparation is prepared using a method comprising the following steps:

(1) culturing the strain in a liquid culture medium at 14 ℃ to 39 ℃ (for example, 15 ℃, 20 ℃, 25 ℃, 30 ℃ or 35 ℃) and 100 to 150 rpm (for example, 120 rpm) for 2 to 10 days (for example, 5 or 8 days) , thereby obtaining a cultured bacterial liquid; and

(2) performing solid-liquid separation on the cultured bacterial liquid, thereby obtaining a liquid portion as the metabolite.

In some further preferred embodiments, the solid-liquid separation is achieved by centrifuging, which is preferably performed at a centrifugation speed of 8000 rpm for a centrifugation time of 10 minutes.

In some further preferred embodiments, the liquid culture medium is a GRC001 liquid culture medium, which is formulated with: starch, 20 parts by weight; KNO ₃, 1 part by weight; K ₂HPO ₄, 0.5 part by weight; MgSO ₄·7H ₂O, 0.5 part by weight; NaCl, 0.5 part by weight; FeSO ₄·7H ₂O, 0.01 part by weight; and potassium dichromate 0.05 part by weight.

In some further preferred embodiments, the starch in the starch culture is from grain, or is from at least one selected from the group consisting of potato, maize, soybean and millet.

In some more specific implementations, the metabolite or bacterial preparation is prepared using a method comprising the following steps:

(1) transferring a single colony from a plate-cultured culture medium to 5 mL of the GRC001 liquid culture medium, and amplifying the colony at 14℃ to 39℃ and 120 rpm for 2 to 10 days, so as to obtain a to-be-amplified cultured bacterial liquid;

(2) transferring 5 mL of the to-be-amplified cultured bacterial liquid to 100 mL of the GRC001 liquid culture medium, and amplifying it at 14℃ to 39℃ and 120 rpm for 2 to 10 days, so as to obtain an amplified, cultured bacterial liquid; and

(3) centrifuging the amplified, cultured bacterial liquid (for example, at 8000 rpm for 10 min. ) , and taking the resulting supernatant as the metabolite.

The present disclosure in a fifth aspect provides an anti-cancer pharmaceutical, the anti-cancer pharmaceutical comprises the metabolite provided in the second aspect of the present disclosure, or the bacterial preparation provided in the third aspect of the present disclosure.

In a sixth aspect, the present disclosure provides use of the strain of the first aspect of the present disclosure, of the metabolite of the second aspect of the present disclosure, of the bacterial preparation of the third aspect of the present disclosure, and of the pharmaceutical of the fifth aspect of the present disclosure for treating a cancer.

In some preferred embodiments, the cancer is selected from the group consisting of lung cancer, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer and leukemia, and is preferably ovarian cancer.

In a seventh aspect, the present disclosure provides a culturing method of the strain of the first aspect of the present disclosure. The method is conducted using a GRC001 solid culture medium. Preferably, the GRC001 solid culture medium is formulated with: starch, 20 parts by weight; KNO ₃, 1 part by weight; K ₂HPO ₄, 0.5 part by weight; MgSO ₄·7H ₂O, 0.5 part by weight; NaCl, 0.5 part by weight; FeSO ₄·7H ₂O, 0.01 part by weight; and potassium dichromate 0.05 part by weight; agar, 20 parts by weight. In some preferred embodiments, the starch in the starch culture is from grain. More preferably, the starch is from at least one selected from the group consisting of potato, maize, soybean and millet.

In an eighth aspect, the present disclosure provides an amplification method of the strain of the first aspect of the present disclosure. The amplification is achieved by using a GRC001 liquid culture medium. Preferably, the GRC001 solid culture medium is formulated with: starch, 20 parts by weight; KNO ₃, 1 part by weight; K ₂HPO ₄, 0.5 part by weight; MgSO ₄·7H ₂O, 0.5 part by weight; NaCl, 0.5 part by weight; FeSO ₄·7H ₂O, 0.01 part by weight; and potassium dichromate 0.05 part by weight. In some preferred embodiments, the starch in the starch culture is from grain. Preferably, the starch is from at least one selected from the group consisting of potato, maize, soybean and millet.

In a ninth aspect, the present disclosure provides a preparation method of the metabolite of the third aspect of the present disclosure. The method comprises the following steps:

(1) culturing the strain in the liquid culture medium, at 14 ℃ to 39 ℃, with a speed of 100 to 150 rpm, for 2 to 10 days, thereby obtaining a cultured bacterial liquid;

(2) performing solid-liquid separation on the cultured bacterial liquid and obtaining a liquid portion as the metabolite.

In some further preferred embodiments, the solid-liquid separation is achieved by centrifugation, which is preferably performed at a speed of 8000 rpm for 10 minutes.

In some further preferred embodiments, the liquid culture medium is a GRC001 liquid culture medium, which is formulated with: starch, 20 parts by weight; KNO ₃, 1 part by weight; K ₂HPO ₄, 0.5 part by weight; MgSO ₄·7H ₂O, 0.5 part by weight; NaCl, 0.5 part by weight; FeSO ₄·7H ₂O, 0.01 part by weight; and potassium dichromate 0.05 part by weight. In some further preferred embodiments, the starch in the starch culture is from grain, more preferably, the starch is from at least one selected from the group consisting of potato, maize, soybean and millet.

In some more detailed embodiments, the metabolite is prepared by the following steps:

(1) transferring a single colony from a plate-cultured culture medium to 5 mL of the GRC001 liquid culture medium, and amplifying the colony at 14 ℃ to 39 ℃ and 120 rpm for 2 to 10 days, so as to obtain a to-be-amplified cultured bacterial liquid; (2) transferring 5 mL of the to-be-amplified cultured bacterial liquid to 100 mL of the GRC001 liquid culture medium, and amplifying it at 14 ℃ to 39 ℃ and 120 rpm for 2 to 10 days, so as to obtain an amplified, cultured bacterial liquid; and (3) centrifuging the amplified, cultured bacterial liquid (for example, at 8000 rpm for 10 min) , and taking the resulting supernatant as the metabolite.

The present invention has the following benefits compared to the prior art:

(1) The disclosed CCPM7643 strain is allied to bacteria of the Bacillus genus and originates from the intestinal tract of a healthy human adult. It is a Bacillus strain from a new source, and possesses properties not seen in Bacillus strains from other sources (such as ocean or soil) , experiment data show that the metabolite that the strain generates has potent anti-cancer activity.

(2) Being found in human intestinal tract means that the strain has little to none side effect on human body, so the adaptability to patients can be good.

(3) By adjusting the population structure of the microorganisms in the human intestinal tract in terms of functional states or relative abundance among them, cancer prevention is possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the colony morphology of CCPM7643 after three-region streak culture.

FIG. 2 is the evolutionary tree of the whole genome of CCPM7643.

FIG. 3 shows the morphological changes of ES2 ovarian cancer cells treated with a metabolite of CCPM7643, wherein FIG. A shows ES2 ovarian cancer cells treated with CCPM7643 while FIG. B is the control (i.e., without metabolite of CCPM7643) .

FIG. 4 graphically shows the results of a CCK8 experiment of ES2 ovarian cancer cells treated with a metabolite of CCPM7643 for 6 hours.

FIG. 5 shows the comparison of anti-cancer activities of CCPM7643 bacterial preparations produced in different culture media to A549 cells;

FIG. 6 shows the comparison of anti-cancer activities of CCPM7643 bacterial preparations produced in different culture media to Hela cells.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described below with reference to some examples for further illustrating the present invention. It is to be understood, however, that these embodiments are for exemplification only and not intended to limit the scope of the present invention.

Embodiment 1: Screening of CCPM7643 Strain

1 Material and Method

1.1 Culture Medium and Related Solutions for Screening

1.1.1 Formula of GRC001 Solid Culture Medium (1L) :

Starch (from potato or maize or soybean or millet or other grain or other plants) , 20 g; KNO ₃, 1g; K ₂HPO ₄, 0.5 g; MgSO ₄·7H ₂O, 0.5 g; NaCl, 0.5 g; FeSO ₄·7H ₂O, 0.01 g; and agar, 20 g. Starch was dissolved or suspended first and then the rest components were added. After autoclaving, the resulting mixture was agitated homogeneous. Preferably, starch can be replaced by other examples suited for carbon source, including but not limited to: sugars, such as glucose, fructose, sucrose, starch hydrolysates, cellulose hydrolysates, and molasses; organic acids, such as acetic acid, propionic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerin. KNO ₃ can also be replaced with other illustrative examples of suitable nitrogen sources, including, but not limited to: ammonia, including ammonia gas and ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium acetate; and other nitrogen-containing substances, including meat extract, peptone, corn steep liquor, casein hydrolysate, soybean cake hydrolysate and yeast extract.

1.1.2 Potassium dichromate: at a concentration of 50 mg/L. Stock solution: 5 g/L (prepared fresh, away from light, filtered and for sterilization; the solution is toxic and so caution is needed not to contact or inhale it) . During preparation of 100 mL of GRC001 solid or liquid culture medium, 1mL of potassium dichromate as stock solution was added (when the culture medium has been autoclaved and cooled to 40-55℃, it should be well mixed) .

1.1.3 FeSO ₄·7H ₂O stock solution: 0.01 g/ml. 1 g of FeSO ₄·7H ₂O was dissolved in 100 mL of H ₂O. In every 1000mL of the GRC001 solid or liquid culture medium, 1mL of ferrous sulfate stock solution was added.

1.1.4 Formula of PBS (1L) : KH ₂PO ₄, 0.27 g; Na ₂HPO ₄, 1.42 g; NaCl, 8 g; and KCl, 0.2 g. 1.1.4 Formula of PBS (1 L) : KH ₂PO ₄, 0.27 g; Na ₂HPO ₄, 1.42 g; NaCl, 8 g; and KCl, 0.2 g. The buffer salt may be any known inorganic salt, but is preferably a salt selected from the following group: alkali metal nitrate, acetate, chloride, bromide, iodide, sulfate, hydroxide, carbonic acid Salt, bicarbonate (also called as bicarbonate) , phosphate, sulfide, and sulfite; ammonium nitrate, ammonium acetate, ammonium chloride, ammonium bromide, ammonium iodide, ammonium sulfate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate (also known as ammonium bicarbonate) , ammonium phosphate, ammonium sulfide and ammonium sulfite; nitrates, chlorides, bromides, iodides, sulfates, sulfides and bicarbonates of alkaline earth metals; nitrate, acetate, chloride, bromide, iodide and sulfate of manganese, iron, copper and zinc; citrate and borate.

1.1.5 Formula of LB liquid culture medium (1L) : tryptone 10g, yeast extract 5g and NaCl 10g were added into 900 mL deionized water. The container was shaked to dissolve the solvent, and pH was adjusted to 7.0 with 5mol/L NaOH. Then deionized water was added to make the solution 1L. The solution was steam sterilized under a pressure of 15 psi for 20 minutes.

1.2 Acquisition of CCPM7643 of the Bacillus Genus: the bacterial strain was obtained from human stool specimen.

1.2.1 Introduction of Sampling

Each stool donor, preferably a healthy adult, was provided with a sampling toolkit including the following items: 50 mL centrifuge tube × 1; spoon × 1; PE glove × 2; paper plate × 1; Informed Consent × 1; sampling instruction × 1.

1.2.2 Sampling Instruction: The stool donors were instructed to read the Informed Consent carefully and fill the form with their basic information before putting their signatures. To sample, the stool donor placed the stool on the disposable paper plate and used the spoon, with gloved hands, to remove a ca. 10 g sample from the middle of the stool and placed it to the 50mL centrifuge tube. The centrifuge tube was then well capped and stored in the attached ziplock bag. Throughout the process, the stool donor tried not to touch anything irrelevant. The centrifuge tube containing the sample and the signed Informed Consent were put in the ziplock bag and collected by the lab staff.

2 Isolation, Culture and Storage of Bacteria:

2.1 Sample Handling and Isolation & Culture of Bacteria: each of the samples was numbered and 5g of stool in the centrifuge tube was suspended in PBS ten times of its volume, and then well agitated, mixed, and diluted by 10, 100 and 1000 times. Then 100 μl of the suspension of the diluent diluted 100 and 1000 times were applied to a 90 mm-diameter plate (GRC001 solid culture medium (containing potassium dichromate) ) and cultured at 14-39℃. A single colony was three-region streaked on an agar plate and incubated at 14-39℃; a single colony was placed on a glass slide, mixed with an appropriate amount of water and then covered with a cover glass; Mycelium spores can be seen when magnified 40 times. Results of the single colony after three-region streak cultured at 14 ℃ to 39 ℃ for 5 to 10 days are shown in FIG. 1.

2.2 Storage: the single colony was inoculated in 2 ml GRC001 liquid culture medium (containing potassium dichromate) , agitated and cultured for 8 to 24 hours. 500 μl of 50%sterilized glycerol was added into 500 μl of GRC001 liquid culture medium with a pipette, which were mixed and put in cryostorage at -80 ℃.

2.3 Amplification Culture: The bacteria were transferred to the GRC001 liquid culture medium for amplification culture at 120 rpm and 14-39℃.

2.4 Identification: By comparing its whole genome sequence with the bacterial sequences published in GenBank, CCPM7643 was found to be most closely related to Bacillus subtilis in terms of genome sequence, as shown in the evolutionary tree of FIG. 2, yet have distinguishable features, such as the two signature sequences SEQ ID NO. 1 and SEQ ID NO. 2. Hence, CCPM7643 represents a new, previously unknown bacterial species.

The CCPM7643 strain has significant and potent anti-cancer activity. Its whole genome sequence information clearly shows its uniqueness in terms of genetic background and phylogeny, which has not been reported previously in the Bacillus genus or other genera belonging to the Firmicutes phylum, making it meaningful in the regard of bacterial species delimitation. The inventors have suggested updated criteria for natural species delimitation of bacteria (see Le Tang and Shu-Lin Liu. 2012. The 3Cs provide a novel concept of bacterial species: messages from the genome as illustrated by Salmonella. Antonie van Leeuwenhoek 101 (1) : 67-72; and Le Tang, Yang Li, Xia Deng, Ang Li, Randal N. Johnston, Gui-Rong Liu, Shu-Lin Liu. 2013. Defining natural species of bacteria: clear-cut genomic boundaries revealed by a turning point in nucleotide sequence divergence. BMC Genomics. 14: 489) . The inventors thus identify the disclosed bacterial strain as representing a new species of the Firmicutes phylum kin to the Bacillus species.

Embodiment 2: Preparation of CCPM7643 metabolite (i.e., the supernatant of the culture of the bacterium, containing the metabolite)

LB solid culture medium: tryptone 10g, yeast extract 5g and NaCl 10g were added into 900 mL deionized water. The container was shaked to dissolve the solvent, and pH was adjusted to 7.0 with 5mol/L NaOH. Then deionized water was added to make the solution 1L. Agar 15g was added and shaked homogeneous, then steam sterilized under a pressure of 15 psi for 20 minutes, poured onto a plate with a thickness of the culture medium being 4 mm.

LB liquid culture medium: tryptone 10g, yeast extract 5g and NaCl 10g were added into 900 mL deionized water. The container was shaked to dissolve the solvent, and pH was adjusted to 7.0 with 5mol/L NaOH. Then deionized water was added to make the solution 1L. The solution was steam sterilized under a pressure of 15 psi for 20 minutes.

Experimental bacterial preparation: the liquid culture obtained from the amplification culture of Embodiment 1 was plate-cultured on the GRC001 solid culture medium (GRC001 liquid culture medium + agar) . Then a single colony was transferred from the plate to 5 mL of liquid GRC001 for culture at 14-39℃ and 120 rpm. After amplification, the liquid culture was centrifuged at 8000 rpm for 10 min. The metabolite was obtained as the supernatant for later use in the anti-cancer cell experiment.

Control bacterial preparation: the GRC001 solid culture medium was replaced with LB solid culture medium and GRC001 liquid culture medium was replaced with LB liquid culture medium, thus control bacterial preparation was obtained as subsequent for later use in the anti-cancer cell experiment.

Embodiment 3: Anti-Cancer Activity Experiment

Human ovarian cancer cell line ES2: Epithelial ovarian cancer cell line ES2 was purchased from Wuhan Procell Life Science & Technology Co Ltd.

CCK8: Cell Counting Kit-8, Beyotime Biotechnology.

3.1 Culture of ES2 Cell: the process was started with plating, wherein each well was filled with 100 μl of the ES2 cancer cells at a density of 1×10 ⁵/ml. After adhesion, the ES2 cancer cells were added with 5 μl of the metabolite obtained from Embodiment 2. The control group was another GRC001 liquid culture medium of the same volume, which was cultured for 6 more hours before microscopy.

The result was shown in FIG. 3, wherein the picture on the left shows the ES2 ovarian cancer cell after treatment of CCPM7643, while the picture on the right is control. After treatment of the CCPM7643 bacterial preparation, the ES2 cells became aggregated, spherical, detached and died.

3.2 CCK8 Experiment: ES2 cells were plated, wherein each well was filled with 100 μl with the cells at a density of 1×10 ⁵/ml. After adhesion, the ES2 cells were added with 5 μl, 2.5 μl, 1.25 μl, 0.625 μl, 0.313 μl or 0.156 μl of the metabolite obtained from Embodiment 2, while the control group added no such metabolite (i.e. without the strain but with the supernatant obtained after culture and centrifuging) . Each had 4 duplicates. Culture was continued for 6-24 hours before microscopy. In each well, 10 μl of CCK8 solution was added and culture was continued for 0.5-1 more hour. Afterward, an enzyme-linked immunity analyzer was used to measure the absorbance of each well at OD450nm.

The results are shown in FIG. 4. Therein, *P<0.05 indicates that the difference is statistically significant, and *P<0.01 indicates that the difference is statistically highly significant. As can be seen from FIG. 4, the inhibiting activity of the CCPM7643 metabolite to ES2 cells showed a gradient changing with its concentration, and did inhibit the activity of ES2 cells.

Embodiment 4: Comparison of influence of different culture media to the anti-cancer activities of the CCPM7643 bacterial preparations

A549 cell: lung cancer cell, purchased from Wuhan Procell Life Science & Technology Co Ltd.

HeLa cell: cervical cancer epithelial cell HeLa, purchased from Wuhan Procell Life Science & Technology Co Ltd.

Experiment reagent: experiment bacterial preparation obtained in Embodiment 2.

Blank control: GRC001 liquid culture medium (bacteria-free) .

Cell culture: plating on a 96-well culture plate, wherein each well was filled with 100 μl of the cells at a density of 1x10 ⁵/ml. After adhesion, the ES2 cells were added with 5 μl of the experiment bacterial preparation. The control plate was added with control bacterial preparation (GRC001 liquid culture medium, bacteria-free) . They were observed after being cultured for 6, 12, 18, and 24 hours. Cell morphology was observed under microscope.

Results are shown in FIG. 5: after treatment of the bacterial liquid for 24 hours, A549 cells treated with culture product of starch culture medium show significant aggregation, detachment and mortality. Experiment on HeLa, as shown in FIG. 6, shows similar results.

Thus it can be seen: the CCPM7643 strain provided by the present disclosure, compared with blank control, and those with LB culture medium, the culture product of the strain cultured with starch culture medium shows better anti-cancer activity.

As shown in the pictures, after 24 hours treatment of bacterial liquid, compared with those with LB culture medium, the strain cultured with starch culture medium shows greater anti-cancer activity. Starch is an essential part of human’s daily diet. Though the carbon source of common culture medium of bacillus is mainly sucrose and glucose, in order to simulate the decomposition environment of intestinal bacteria to polysaccharose in food, GRC001 culture medium was selected. The commonly used carbon sources of the culture medium of Bacillus are sucrose and glucose. The selection of starch increases the steps of metabolizing polysaccharides into monosaccharides by the bacteria, and enriches the types of primary and secondary metabolites. Although LB is rich in nutrients, the chemical composition of the medium is not constant; the composition of the starch medium is accurate, and the inorganic salt in the composition can provide a large number of elements and trace elements.

The LB medium is extremely nutrient-rich, while the starch medium is poorly nutrient. What is shocking is that these two bacteria have no anti-cancer effect when grown from LB medium, but their anti-cancer effects are outstanding when grown from starch medium.

In conclusion, the CCPM7643 strain selected from the intestines of healthy adults in the present invention has significant inhibitory activity on tumor cells. In particular, the bacterial preparation obtained by culturing with starch medium has more significant anti-cancer activity.

Embodiment 5: Isolation and purification of anti-cancer components

5.1 Performing high performance liquid chromatography (HPLC) on the supernatant to achieve isolation and purification

HPLC: the sample was filtered through a 0.22 μm microfiltration membrane, the mobile phase is: acetonitrile: water (v: v, volume ratio) = 3: 7, the chromatographic column is zorboxXDB-C18 (4.6mm×150mm, 5 μm, i.e., column diameter is 4.6 mm, column length is 150 mm, diameter of the fillers is 5 μm) , detected at ultraviolet 268 nm, column temperature is 25 ℃, flow rate is 1.0mL·min ^-1, and sample load is 10 μl.

5.2 Identification of types of metabolite

The CCPM7643 strain was streaked from the amplification medium onto the LB solid culture medium and grown overnight at 14-39℃. The colonies were inoculated into a 14 mL sterile culture tube added with 5 mL of LB liquid culture medium, and cultured at 14-39℃ at 120 rpm for 12-13 hours. The cultured bacterial solution was inoculated into 550 mL LB liquid culture medium to an initial optical density (OD600nm) of 0.01 at 600nm. The culture was distributed into six sterile deep-well 96-well plates (0.9 mL per well) using the MultiFlo microplate dispenser (BioTek) . Subsequently, a CyBi-Well automatic liquid transfer robot (CyBio) was used to add elicitors from a commercially available natural product library of 502 members (Enzo Scientific, catalog number BML-2865) . Each well receives 2.5 μl of the elicitors (from a stock solution concentration of 10 mM) . Seal the plate with a gas-permeable membrane, and incubate at 14-39℃, 120 rpm, for 44 hours, centrifuge the plate slightly, load the supernatant on 96-well Strata C8 resin (Phenomenex) , and use 600 μl of 50 %and 600 μL of 100%MeCN to elute substances into a new 96-well plate. The eluate was then dried in Speedvac (vacuum centrifugal concentrator) and resuspended in 30 μl of 40%MeCN (in water) .

Each well was imaged using laser ablation electrospray ionization mass spectrometry (LAESI-MS) , and the difference between the image and the image from the control well was identified. The test compound associated with one or more differences is then identified.

The present invention has been described with reference to the preferred embodiments and it is understood that the embodiments are not intended to limit the scope of the present invention. Moreover, as the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all equivalent changes or modifications which do not depart from the concept of the present invention should be encompassed by the appended claims.

Claims

A Firmicutes strain allied to Bacillus species, being characterized in that the strain has a preservation number of CGMCC No. 17886.
A Firmicutes strain allied to Bacillus species, being characterized in that it is Gram-positive, aerobic or facultatively anaerobic, has capsule, spores being elliptical, oval, columnar or spherical; by comparing its whole genome sequence with the bacterial sequences published in GenBank, the strain is most closely related to Bacillus paralicheniformis in terms of genome sequence, but also includes two signature sequences shown by SEQ ID NO. 1 and SEQ ID NO. 2.
Metabolite produced by the strain of claim 1 or 2.
An anti-cancer bacterial preparation, being characterized in that the preparation is obtained by amplifying culturing the strain of claim 1 or 2.
The bacterial preparation of claim 4, being characterized in that the bacterial preparation is:

(i) . solid culture or liquid culture of the strain;

(ii) . liquid portion obtained after solid-liquid separation of the liquid culture of (i) ; or

(iii) . product obtained by further purification of the liquid portion of (ii) .
The bacterial preparation of claim 4 or 5, being characterized in that the culture medium is a starch culture medium; preferably the starch in the starch culture medium is from grain, or is from at least one selected from the group consisting of potato, maize, soybean and millet.
A preparation method of the metabolite of claim 3 or the bacterial preparation of any of claims 4 to 6, wherein the preparation method comprises inoculating the strain to a culture medium and amplifying culturing in an environment between 14 ℃ to 39 ℃.
The preparation method of claim 7, wherein the culture median is a liquid culture medium, the preparation method comprises the following steps:

(1) inoculating the strain in the liquid culture medium, culturing the strain at 14 ℃ to 39 ℃, such as 15 ℃, 20 ℃, 25 ℃, 30 ℃ or 35 ℃, with a speed of 100 to 150 rpm, preferably 120 rpm, for 2 to 10 days, preferably 5 to 8 days, thereby obtaining a cultured bacterial liquid;

(2) performing solid-liquid separation on the cultured bacterial liquid and obtaining a supernatant; preferably, the solid-liquid separation is achieved by centrifugation, which is preferably performed at a speed of 8000 rpm for 10 minutes.
The preparation method of claim 7 or 8, wherein the preparation method comprises the flowing steps:

(1) transferring a single colony from a plate-cultured culture medium to 5 mL of the GRC001 liquid culture medium, and amplifying the colony at 14 ℃ to 39 ℃ and 120 rpm for 2 to 10 days, so as to obtain a to-be-amplified cultured bacterial liquid;

(2) transferring 5 mL of the to-be-amplified cultured bacterial liquid to 100 mL of the GRC001 liquid culture medium, and amplifying it at 14 ℃ to 39 ℃ and 120 rpm for 2 to 10 days, so as to obtain an amplified, cultured bacterial liquid; and

(3) centrifuging the amplified, cultured bacterial liquid at 8000 rpm for 10 min, and taking the resulting supernatant.
The preparation method of claim 9, wherein the liquid culture medium is a GRC001 liquid culture medium, which is formulated with: starch, 20 parts by weight; KNO ₃, 1 part by weight; K ₂HPO ₄, 0.5 part by weight; MgSO ₄·7H ₂O, 0.5 part by weight; NaCl, 0.5 part by weight; FeSO ₄·7H ₂O, 0.01 part by weight; and potassium dichromate 0.05 part by weight;

preferably, the starch in the starch culture medium is from grain, or is from at least one selected from the group consisting of potato, maize, soybean and millet.
An anti-cancer pharmaceutical, being characterized in that the active ingredient of the anti-cancer pharmaceutical comprises the metabolite of claim 3, or the bacterial preparation of any of claims 4 through 6.
Use of the strain of claim 1 or 2, of the metabolite of claim 3, of the bacterial preparation of any of claims 4 through 6, and of the anti-cancer pharmaceutical of claim 11 for treating a cancer.
The use of claim 12, being characterized in that the cancer is selected from the group consisting of lung cancer, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer and leukemia, and is preferably ovarian cancer.