CN113528367A

CN113528367A - Bacillus coagulans with functions of preventing diarrhea and degrading cholesterol

Info

Publication number: CN113528367A
Application number: CN202010293304.7A
Authority: CN
Inventors: 段治; 崔洪昌; 张景燕; 郭超群; 张陆霞
Original assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2021-10-22
Anticipated expiration: 2040-04-15
Also published as: CN113528367B

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus coagulans strainBacillus coagulans) And applications thereof. The bacillus coagulans is selected from a Chinese cabbage sample, and the preservation number is CCTCC NO: m2019738 has strong oxidation resistance and cholesterol degradation capability, can effectively regulate intestinal peristalsis, prevent diarrhea, recover the microbial barrier function of the intestinal tract, shorten the course of disease, and has very obvious effect.

Description

Bacillus coagulans with functions of preventing diarrhea and degrading cholesterol

Technical Field

The invention relates to the technical field of probiotic screening and application, in particular to bacillus coagulans with functions of preventing diarrhea and degrading cholesterol and application thereof.

Background

The term probiotic (probiotics) originates from greek, and with the deep understanding of people, keywords for regulating intestinal flora, endogenous microorganisms, living microorganisms, viable bacteria number and the like are also integrated into the concept of probiotics. At present, it is widely accepted by the united nations food and agriculture organization/world health organization definition that active microorganisms have a beneficial effect on the host by improving the balance of the flora in the gut. In a broad sense, all strains that can satisfy the basic conditions of probiotics can become probiotics. Probiotics thus encompass many species of microorganisms, and probiotics currently being investigated include: bifidobacteria, lactic acid bacteria, yeasts, bacilli, and the like.

Probiotics were first discovered by russian microbiologists at the beginning of the 20 th century and developed for over a century to date. The concept and efficacy of probiotics has been recognized by scientists and consumers for over the century. 1) Strengthen the mucosal barrier. As probiotics capable of being normally planted in the intestinal tract, the probiotic can not only form dominant flora in the intestinal tract of a human body, but also be tightly combined on the intestinal mucosa to form a natural biological barrier, so that the whole intestinal tract of the human body is in a healthy state. 2) And (4) an antibacterial effect. As non-pathogenic bacteria present in the human or animal intestine, probiotics play a very important role in the health and balance of the intestine. Probiotics regulate gut and body health primarily through bacterial antagonism, immunomodulation or alterations in the organization of intestinal epithelial cells. 3) Affecting the immune system. The influence of probiotics on the immunity of the organism is mainly realized by regulating the secretion of a series of cell factors involved in phagocytic function. When probiotics are present in the intestinal tract, the activity of monocytes and macrophages (phagocytes), neutrophils and Natural Killer (NK) cells (polymorphonuclear leukocytes) in the human body is activated. Under the action of the cells, a large amount of immune factors such as active oxygen, lysosomal enzyme, Interleukin (IL) and Interferon (IFN) are produced, thereby enhancing the immunity of the human body. 4) Inhibiting intestinal diseases. The probiotics inhibit the intestinal diseases by mainly inhibiting the growth of harmful microorganisms through an antagonistic function, thereby changing the microbial flora structure of the intestinal tract to play a role in inhibiting the intestinal diseases. Most of the available clinical studies show that probiotics as placebo have a significant effect on specific conditions, such as prevention of antibiotic-mediated diarrhea, treatment of acute gastritis, irritable bowel inflammation and helicobacter pylori infection. 5) Inhibiting antibiotic-mediated diarrhea. Antibiotic-mediated diarrhea is a ubiquitous side effect of antibacterial therapy. Many researchers believe that antibiotic-mediated diarrhea is due to changes in the intestinal microflora, however, there are also researchers who believe that clostridia is the predominant cause of this diarrhea. 6) Inhibiting postoperative complications. RaYESN et al, after a liver transplantation surgery, performed double-blind experiments on 95 and 66 patients, respectively, with probiotics as a therapeutic agent, found that the incidence of postoperative infection complications decreased from 48% to 13% and 3%, respectively, in patients who were continuously taking probiotics on day 1 after surgery (P ═ 0.017). 7) Inhibiting infection complications. The study on the effect of probiotics on the inhibition of complications of patients suffering from severe trauma shows that the probiotics can inhibit the occurrence of the complications by regulating the barrier function of the intestinal tract and changing the immunoregulation function. 8) Inhibiting tumor growth. Studies have shown that the stimulus for colon cancer production is primarily the inflammatory environment and the microbial composition in the gut. This is mainly because microorganisms in the intestinal tract can cause a number of physiological and biochemical reactions that increase the risk of cancer. CAPURSOG et al found that Lactobacillus acidophilus GG can reduce polyamines in HGC-27 and DLD-1 cancer cells by in vitro experiments, thereby inhibiting division and proliferation of cancer cells, and the like.

Bacillus has been used as a probiotic for at least 50 years, with the main species studied being bacillus subtilis, bacillus cereus, bacillus crohn, bacillus coagulans and bacillus licheniformis. Compared to other non-spore preparations such as lactic acid bacteria, thermostable bacilli have many advantages: can be stored in dry form at room temperature without any detrimental effect on viability; spores are able to survive the low pH conditions of the gastric barrier, which is not possible with all kinds of lactic acid bacteria. In 2016, Bacillus coagulans was officially listed by the national institute of health, "list of strains available for food", and is the only species of Bacillus listed so far. The bacillus coagulans has the functions of improving the intestinal flora structure, adjusting intestinal dysfunction and maintaining the flora balance in the intestinal tract by probiotics, and has the characteristic of stress resistance which other probiotics such as high temperature resistance, acid resistance, cholate resistance and the like do not have. In view of the characteristics, the bacillus coagulans can be applied to food industries needing heat treatment, such as baking industry, feed processing enterprises and the like, and can resist gastric acid and bile salt to successfully reach the intestinal tract to play a role.

The difference of the application effect and the stability of the current commercial probiotic products is large, the quality of the products is uneven, and the increasing demand of people on the probiotic products cannot be met, so that the screening of high-quality probiotic resources, especially bacillus resources, is still the research focus in the field.

Disclosure of Invention

The invention aims to provide a novel Bacillus coagulans strain and application thereof. The bacillus coagulans is screened from the Chinese cabbage sample, has strong oxidation resistance and cholesterol degradation capability, can effectively regulate intestinal peristalsis, prevent diarrhea, recover the microbial barrier function of the intestinal tract, shorten the disease course, and has very obvious effect.

The invention relates to a Bacillus coagulans (Bacillus coagulans) named as Bacillus coagulans VHProbi C08(Bacillus coagulans VHProbi C08), which is preserved in China center for type culture collection of Wuhan university in Wuhan, China in 2019, 9 and 23 days, and the preservation number is CCTCC NO: and M2019738.

One aspect of the present invention relates to the use of said bacillus coagulans for preventing diarrhea.

The diarrhea includes, but is not limited to, cold diarrhea, alcoholic diarrhea, antibiotic-associated diarrhea, or viral diarrhea.

One aspect of the invention relates to the use of the bacillus coagulans for degrading cholesterol.

The invention also relates to a probiotic preparation, which comprises the bacillus coagulans.

The probiotic preparation also comprises one or the combination of two or more of bifidobacterium, lactobacillus, streptococcus, lactococcus, leuconostoc, propionibacterium, saccharomycete, pediococcus and staphylococcus.

The Bifidobacterium is preferably Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, and Bifidobacterium bifidum.

The lactobacillus is preferably Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus bulgaricus.

The streptococcus is preferably streptococcus thermophilus.

The lactococcus is preferably lactococcus lactis subspecies lactis, lactococcus lactis subspecies cremoris and lactococcus lactis diacetyl subspecies lactis.

The propionibacterium is preferably propionibacterium freudenreichii subsp.

The Leuconostoc preferably is Leuconostoc mesenteroides.

The yeast is preferably Kluyveromyces marxianus.

The Pediococcus is preferably Pediococcus acidilactici or Pediococcus pentosaceus.

The staphylococcus is preferably staphylococcus parvum, staphylococcus xylosus and staphylococcus carnosus.

The bacillus coagulans in the probiotic preparationHas a viable bacteria count of at least 10⁸CFU/g。

The invention also relates to the application of the probiotic preparation in preventing diarrhea.

The invention also relates to application of the probiotic preparation in degrading cholesterol.

The bacillus coagulans VHProbi C08 does not produce hemolysin, does not dissolve blood cells, is sensitive to common antibiotics and has good biological safety; can tolerate higher salinity, and the maximum tolerant salt concentration is 7 percent; can grow in a wider temperature range, can produce spores with strong heat resistance, and is convenient for the later-stage industrial processing of the probiotic strain; the hydrophobicity of the cell surface in vitro is 8.58%, and the cell surface has certain adhesiveness to intestinal cells.

The bacillus coagulans VHProbi C08 has a strong antioxidant function, wherein the clearance rate of DPPH free radicals is 87.80%, and the clearance rate of HRS free radicals is 47.79%; the lipid peroxidation inhibition rate of the supernatant was 84.46%, and the lipid peroxidation inhibition rate of the cells was 44.88%.

The bacillus coagulans VHProbi C08 has strong capacity of degrading cholesterol, wherein the degradation rate of the cholesterol in vitro reaches 39.21%; in a hyperlipemia mouse model, after the bacillus coagulans VHProbi C08 provided by the invention is fed to a model mouse for 30 days, the blood cholesterol level of a pretreatment group is reduced by about 34 percent, and the blood cholesterol level of a post-treatment group is reduced by about 10 percent; after 40 days, the blood cholesterol level of the bacillus coagulans VHProbi C08 pretreatment group is reduced by about 39 percent, the blood cholesterol level of the post-treatment group is reduced by about 30 percent, and the effect is very obvious.

The bacillus coagulans VHProbi C08 can effectively prevent and relieve diarrhea and inhibit intestinal peristalsis. After the bacillus coagulans VHProbi C087 days are continuously taken by rats, the diarrhea time can be effectively delayed, and the excrement amount is reduced. Metabolites such as organic acids generated by the metabolism of bacillus coagulans VHProbi C08 can inhibit the growth of harmful bacteria, promote the digestion and absorption of nutrients by intestinal tracts, recover the microbial barrier function of the intestinal tracts, stimulate the immune function of organisms and shorten the disease course.

The Bacillus coagulans VHProbi C08 can be added into most kinds of food to prepare functional food with functions of preventing diarrhea and degrading cholesterol. The strain has the advantages of simple fermentation condition, low industrialization cost and wide market prospect.

Drawings

FIG. 1 is a Riboprinter fingerprint;

FIG. 2 is a RAPD fingerprint;

FIG. 3 is a rep-PCR fingerprint;

FIG. 4 is a comparison of water intake for 12h rats;

FIG. 5 is a comparison of feces amount in rats observed for 4, 8 and 12 hours.

Detailed Description

The examples are merely illustrative of the present invention and do not limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 isolation and screening of strains

1. Primary screen for bacillus

Mashing 1g fermented pickled Chinese cabbage with a mashing machine, soaking in 10ml 0.85% physiological salt for 10min, beating and mixing with a homogenizer, pouring into a test tube, heating in a water bath at 70 deg.C for 10min, diluting the suspension with gradient, and respectively taking 10^-1、10^-2、10^-3The three dilution gradients are coated on an MRS culture medium in a gradient of 100ul, cultured in an incubator at 37 ℃ for 48h under aerobic condition, and subjected to microscopic examination respectively after single strains grow out on the plate. According to the microscopic examination result, the applicant screens 10 strains of bacillus, which are named as NH-1, NH-2, … … and NH-10 respectively.

2. Bacillus rescreening

Liquid fermentation medium: weighing yeast powder 3.0g, peptone 5.0g, beef extract 2.0g, and K₂HPO₄ 3g、MnSO₄0.005g、MgSO₄0.02g and NaCl 2g, mixing, adding 1000ml distilled water to dissolve, and autoclaving at 121 ℃ for 20 minutes.

Bacterial count medium: yeast powder 5.0g, peptone 5.0g, glucose 5.0g, K₂HPO₄ 0.5g KH₂PO₄0.5g、MgSO₄0.3g, 1.0ml of trace element solution and 15.0g of agar powder, adding 1000ml of distilled water for dissolving, and autoclaving at 115 ℃ for 30 minutes. Wherein the formula (1L) of the trace element concentrated solution is as follows: 10g of sodium chloride, 18g of iron sulfate heptahydrate, 16g of manganese sulfate monohydrate, 1.6g of zinc sulfate heptahydrate, 1.6g of copper sulfate pentahydrate and 1.6g of cobalt sulfate heptahydrate.

(1) Screening for spore formation rate

Respectively inoculating the screened 10 strains of bacillus into the liquid fermentation culture medium according to the inoculation amount of 6%, culturing in a shaking table under the aerobic condition of 40 ℃, and culturing for 48 hours at 210 rpm/min. Taking the fermentation liquor, counting the bacterial quantity N1, putting the fermentation liquor at 70 ℃ for 10 minutes to inactivate the bacteria, counting again N2, and calculating the spore forming rate of each strain of bacillus.

The sporulation rate is N2/N1 × 100%.

The results show that: the spore forming rates of the screened bacillus NH-1, NH-2, … … and NH-10 are respectively 58%, 70%, 60%, 60%, 62%, 67%, 70%, 90%, 83% and 78%, wherein the spore forming rate of the NH-8 strain is the highest and reaches 90%.

(2) Survival rate screening at 95 deg.C

Respectively inoculating bacillus NH-1, NH-2, … … and NH-10 into the liquid fermentation culture medium according to the inoculation amount of 6 percent, culturing in a shaking table under the aerobic condition of 40 ℃, culturing for 48 hours at 210rpm/min, putting the fermentation liquor at 70 ℃, inactivating the thalli for 10 minutes, and calculating the spore concentration; diluting the fermentation liquor by using a liquid fermentation culture medium to keep the spore amount of 10 strains of bacillus consistent, wherein the logarithmic value of the spore amount is 9.0 logCFU/ml; after the diluted fermentation liquor is treated for 20 minutes at the temperature of 95 ℃, the bacterial quantities are respectively measured, and the logarithmic value of the bacterial quantities is respectively 5.38, 5.92, 6.37, 6.23, 5.84, 5.95, 6.53, 6.86, 5.92 and 5.83logCFU/ml, wherein the spore survival rate of the NH-8 strain is the maximum, and the logarithmic value reaches 6.86 logCFU/ml. Therefore, NH-8 strain in 10 strains of bacillus screened by the invention has the strongest heat resistance and remarkable effect.

In conclusion, NH-8 strains in 10 strains of bacillus screened by the invention have the highest sporulation rate and the strongest heat resistance, and are beneficial to wide application of the bacillus.

Example 2 Strain identification

2.1 colony morphology identification

The single colony of the NH-8 strain is milky white and glossy, the surface of the colony is flat and wet, the edge is neat, and the diameter of the colony is 2-3 mm. The bacillus is rod-shaped under a microscope, and is arranged singly, in pairs or in a chain shape, and spores are grown at the ends.

2.2 characterization of physiological and biochemical characteristics

The inoculation solution in this example was prepared as follows: under the aseptic condition, taking a proper amount of fresh bacterial liquid, centrifuging at 5000rpm for 5min, washing with PBS buffer for 2 times, then resuspending with the same volume of PBS buffer, and diluting by 50 times to obtain inoculation liquid.

1. Temperature growth range experiment

Under aseptic conditions, inoculating the inoculum solution into 10mL of MRS liquid culture medium according to the inoculation amount of 10%, using 10mL of MRS liquid culture medium without inoculation as a control, respectively placing the MRS liquid culture medium in a 15 ℃ constant temperature shaking incubator for culturing for 7 days, and placing the MRS liquid culture medium in 51 ℃ and 61 ℃ constant temperature shaking incubators for culturing for 2 days, and observing whether the culture solution becomes turbid.

The results show that: after culturing for 7 days at constant temperature of 15 ℃, the culture medium is still clear; after 2 days of incubation at 57 ℃, the medium became turbid, and after 2 days of incubation at 61 ℃, the medium remained clear. Thus, it was demonstrated that the NH-8 strain did not grow at 15 ℃ and 61 ℃ and grew normally at 57 ℃.

2. Catalase assay experiment

And (3) dripping fresh bacterial liquid on a glass slide, and then dripping 3-15% of hydrogen peroxide.

The results show that: NH-8 strain did not produce bubbles and the catalase reaction was negative.

3. Carbon source metabolism experiment

The formulation of the phenol red-containing basal medium used in this example was as follows:

peptone 1.5 g; yeast extract 0.6 g; tween 800.1 g; 0.5ml of salt solution; 18mg of phenol red; 100ml of distilled water; pH 7.4. + -. 0.2. Salt solution composition: MgSO (MgSO)₄·7H₂O 11.5g，MnSO₄·4H₂O₂8g, distilled water 100 mL.

A10 g/mL solution of the sugar, alcohol and carbohydrate glycoside was prepared and filtered through a 0.22 μm sterile filter. Under sterile conditions, 20. mu.l of sterilized carbohydrate solution, 4 replicates of each carbohydrate, was added to a 96-well plate, followed by 170. mu.l of sterilized phenol red-containing basal medium, followed by 10ul of inoculum, and no inoculum wells served as controls. 50 μ l of liquid paraffin was added to each well to prevent evaporation of water during the culture. The culture was carried out at 37 ℃ and the color change of the medium was observed using phenol red as an indicator, the results are shown in Table 1.

TABLE 1 results of carbon source metabolism by NH-8 Strain

Note: a "+" positive reaction; "-" negative reaction

4. Glucose acid and gas evolution test

The media formulations used in this example were as follows:

peptone 0.5 g; yeast extract 0.3 g; tween 800.1 mL; 0.5ml of salt solution A; 0.5ml of saline solution B; 0.5g of sodium acetate; 2.5g of glucose; 0.05mL of 2% bromocresol green (w/v); 100ml of distilled water; the pH value is 6.8-7.0.

The prepared culture medium is subpackaged into large test tubes containing inverted small test tubes, 3 mL/tube, and autoclaved for 15min at 121 ℃.

Salt solution composition A: KH (Perkin Elmer)₂PO₄ 10g、K₂HPO₄1.0g, dissolved in distilled water and fixed to 100 mL.

Saline solution B composition: MgSO (MgSO)₄·7H₂O 11.5g、MnSO₄·2H₂O 2.4g、FeSO₄·7H₂0.68g of O, dissolving in distilled water, and fixing the volume to 100 mL.

Under aseptic conditions, the inoculum was inoculated into a medium at 10% inoculum size, the non-inoculated medium was used as a control, and then the top was sealed with 2mL of sterile liquid paraffin and incubated at 37 ℃. The culture was continued for 6 days, and the color of the medium was observed every day.

The results show that: after culturing for 6 days at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the small inverted tube, which indicates that the NH-8 bacterial strain ferments glucose to produce acid and does not produce gas.

In summary, the results of physiological and biochemical identification of the NH-8 strain are as follows: glycerol, D-ribose, D-xylose, D-galactose, D-mannose, L-rhamnose, D-mannitol, amygdalin, salicin, D-cellobiose, maltose, lactose, D-melibiose, sucrose, D-trehalose, inulin, D-raffinose and sodium gluconate can be utilized as carbon sources, but D-sorbitol, D-melezitose and L-fucose can not be utilized as carbon sources; can not grow under the conditions of 15 ℃ and 61 ℃, and can normally grow under the condition of 57 ℃; the catalase reaction is negative; the fermented glucose produces acid and does not produce gas.

2.3 molecular biological identification

A single colony of the NH-8 strain on the plate was picked up and cultured in MRS broth at 37 ℃ for 24 hours, and then 500ul of the fermentation broth was subjected to the operation with reference to the Tiangen genomic DNA extraction kit (catalog No.: DP302) to obtain the genome of the strain, which was used for the subsequent molecular biological identification.

1. 16s rDNA gene sequence identification

16s rDNA Gene amplification

1) The primer sequence is as follows:

27F：AGAGTTTGATCCTGGCTCA

1492R：GGTTACCTTGTTACGACTT

2) reaction System (50. mu.L)

TABLE 216 s rDNA PCR amplification System

3) And (3) the electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.

4) Sequencing of PCR products

The sequencing result showed that the 16s rDNA sequence of the NH-8 strain was SEQ ID NO 1. The sequence was BLAST aligned in the NCBI database and found to have the highest similarity to Bacillus coagulans (Bacillus coagulans). Therefore, the NH-8 strain was preliminarily determined to be Bacillus coagulans (Bacillus coagulousns).

2. Riboprinter fingerprint

And (2) dipping a single bacterial colony of the purified NH-8 bacterial strain from an agar culture medium plate by using a bacterial taking rod, putting the single bacterial colony into a sample tube with a buffer solution, stirring by using a handheld stirrer to enable the bacterial strain to be suspended in the buffer solution, putting a sample rack into a heater to inactivate, putting the sample rack into a Riboprinter system, and carrying out DNA preparation, membrane conversion, imaging detection and data processing on the sample to obtain a bacterial identification result. The identification result shows that the NH-8 strain is Bacillus coagulans (Bacillus coagulans), and the Riboprinter fingerprint spectrum of the strain is shown in figure 1.

By comparison, the highest similarity between the Riboprinter fingerprint of the NH-8 strain and the fingerprints of the tens of thousands of strains attached to the Riboprinter library is only 73%, which indicates that the NH-8 strain is different from all the strains in the strain library. In addition, after inquiring Riboprinter fingerprint of the bacillus coagulans in all published documents in the world, no strain which is consistent with the fingerprint is found, thereby further indicating that the NH-8 strain is a new bacillus coagulans strain.

3. RAPD fingerprint identification

1. RAPD fingerprint identification

1) The primer sequence is as follows: m13 (5'-GAGGGTGGCGGTTCT-3')

2) RAPD reaction system

TABLE 3 RAPD reaction System

3) Electrophoresis

Preparing a 1.5% agarose gel plate, using DL2000 DNA Marker as a result contrast, performing electrophoresis at a constant voltage of 100V for 80min, and finally detecting an electrophoretogram by using a gel imaging system, wherein RAPD fingerprints of the NH-8 strain and a contrast strain Bacillus coagulans GBI-306086 are shown in figure 2.

As can be seen from FIG. 2, the RAPD fingerprint of the NH-8 strain provided by the invention is obviously different from that of the control strain Bacillus coagulans GBI-306086, and no strain which is consistent with the RAPD fingerprint is found by inquiring the RAPD fingerprint of the Bacillus coagulans in all published documents in the world, so that the NH-8 strain is a new Bacillus coagulans strain.

4. rep-PCR fingerprint identification

1) rep-PCR primer

GTGGTGGTGGTGGTG

2) rep-PCR reaction system

TABLE 4 reaction System for rep-PCR

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The voltage is 100V, the electrophoresis time is 80min, and the rep-PCR fingerprints of the NH-8 strain and the control strain Bacillus coagulans GBI-306086 are shown in figure 3.

As can be seen from FIG. 3, the rep-PCR fingerprint of the NH-8 strain provided by the invention is obviously different from that of the control strain Bacillus coagulans GBI-306086, and no strain matched with the rep-PCR fingerprint is found by inquiring the rep-PCR fingerprint of the Bacillus coagulans in all published documents in the world, so that the NH-8 strain is a new Bacillus coagulans strain.

In summary, the results of colony morphology and physiological and biochemical characteristics of the NH-8 strain were uploaded to the website https:// www.tgw1916.net/bacteria _ poison _ desktop. html, and compared with the results published in the document De Clerck E, et al. By combining the identification results of molecular biology, the conclusion can be drawn that the NH-8 strain is a new Bacillus coagulans strain, and is formally named as Bacillus coagulans VHProbi C08(Bacillus coagulans VHProbi C08).

Example 3 Bacillus coagulans VHProbi C08 salinity tolerance test

Under aseptic conditions, inoculating the inoculum solution into 5mL of MRS liquid culture medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% respectively according to the inoculation amount of 10%, using 5mL of MRS liquid culture medium without inoculation as a control, placing the MRS liquid culture medium at the constant temperature of 37 ℃ for shaking culture, and observing whether the culture medium becomes turbid.

The results show that: bacillus coagulans VHProbi C08 grew at 1% -7% salt concentration, did not grow at 8% salt concentration, and bacillus coagulans VHProbi C08 had a maximum tolerated salt concentration of 7%.

Example 4 hemolytic test of Bacillus coagulans VHProbi C08

1. Preparing an inoculation solution: streaking and inoculating the frozen and preserved bacillus coagulans VHProbi C08 strain into an MRS solid culture medium, culturing for 24-48 h at 37 ℃, subculturing for 1 time through an MRS liquid culture medium, inoculating bacillus coagulans VHProbi C08 into a fresh MRS liquid culture medium at 40 ℃ for shaking culture for 24-48 h with the inoculation amount of 5%, and obtaining fresh bacterial liquid serving as an inoculation liquid;

2. blood cell culture medium preparation: weighing the components of TBS basic culture medium, dissolving, autoclaving at 121 deg.C for 15min, adding 5% sterile defibered sheep blood when the culture medium is cooled to 50 deg.C, mixing, and flattening;

3. and (3) streak culture: and streaking the test strain on a prepared blood cell plate, culturing in an incubator at 37 ℃, and observing whether the test strain has hemolysis or not within 24-48 hours.

The results show that: the bacillus coagulans VHProbi C08 grew and the blood cell plate was unchanged, indicating that the bacillus coagulans VHProbi C08 did not produce hemolysin and could not lyse blood cells.

Example 5 antibiotic resistance test of Bacillus coagulans VHProbi C08

1. Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into 2048 μ g/mL stock solutions, and stored at-20 deg.C for further use. When in use, the stock solution is serially diluted into a use solution by 2 times of gradient with MRS liquid culture medium, and the gradient dilution concentration is 1-1024 mu g/mL, and the total concentration is 11 gradients.

2. Preparing an inoculation solution: preparing inoculation liquid: taking a proper amount of fresh bacterial liquid (cultured for 24-48 h at 40 ℃), centrifuging for 5min at 5000rpm, washing once with sterile normal saline, and diluting by 50 times after resuspending with the same volume of normal saline to serve as inoculation liquid.

3. The minimum inhibitory concentration MIC of the antibiotic to Bacillus coagulans VHProbi C08 was determined by broth dilution.

(1) Adding MRS liquid culture medium without antibiotics into the 96-well plate in the 1 st row as a negative control, sequentially adding 190 mu L of MRS liquid culture medium containing antibiotics with different concentrations into the 2 nd to 12 th rows, then respectively inoculating 10 mu L of the inoculated liquid, making 3 parallel wells, and taking 1 well without adding bacteria liquid as a blank.

(2) Cover by adding 50 μ L of paraffin oil to prevent evaporation of water.

(3) Culturing 96-well plate at 40 deg.C for 48 hr, taking out, and determining OD₆₀₀Values, MIC values of antibiotics to the strains were counted using the 48h results, and the results are shown in Table 5.

TABLE 5 antibiotic MIC values of B.coagulans VHProbi C08

Note: MIC unit μ g/mL

The results in table 5 show that bacillus coagulans VHProbi C08 provided by the present invention is sensitive to common antibiotics such as erythromycin, and has good biological safety.

Example 6 hydrophobic cell surface testing of Bacillus coagulans VHProbi C08

1. Preparing bacterial liquid to be detected: selecting purified bacillus coagulans VHProbi C08 colony to inoculateAnd (3) carrying out shake culture for 24-48 h at 40 ℃ in a newly prepared MRS liquid culture medium. Inoculating to MRS liquid culture medium according to the inoculation amount of 1% (V/V), continuously performing shake culture at 40 deg.C for 24-48 h, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile normal saline for 2 times, and sterilizing with 0.1M KNO₃1mL of the solution was used as a suspension to be assayed.

2. Surface hydrophobicity measurement: sucking 50 μ L of the above bacterial suspension, adding 2450 μ L of 0.1M KNO₃And recording the OD₆₀₀Is A₀1.5ml of the bacterial suspension was mixed with 500. mu.L of xylene and allowed to stand at room temperature for 10min (a two-phase system was formed). And (3) vortexing and oscillating the two-phase system for 2min, and then standing for 20min to form a water phase and an organic phase again. The absorbance A was measured at 600nm by carefully taking up the aqueous phase (not taking up the organic phase)₁. Cell hydrophobicity according to the formula: hydrophobicity (%) ═ a₀-A₁)/A₁X 100, and taking an average value by measuring three experiments.

The results show that: the cell surface hydrophobicity of the bacillus coagulans VHProbi C08 provided by the invention is 8.58%, and the standard deviation is 0.48%.

Example 7 Bacillus coagulans VHProbi C08 assay for antioxidant function

1. Determination of DPPH and Hydroxyl Radical (HRS) eliminating capability of strain

1) Preparation of PBS bacterial suspension

Inoculating single colony with excellent growth state into 3ml of MRS liquid culture medium, culturing for 18-20h at 37 ℃, taking the culture solution as inoculation solution, inoculating into 50ml of MRS liquid culture medium according to the inoculation amount of 2%, and standing and culturing for 18h to obtain the culture solution of the strain. Sucking 1mL of bacterial liquid, collecting the bacteria, washing the bacteria for 2 times by using 1mLPBS buffer solution, and then adding 2mLPBS solution to resuspend the bacteria for later use.

2) Determination of DPPH free radical scavenging ability of strain

Taking 1ml PBS bacterial suspension of the strain to be detected, adding 1ml of 0.4mM in-situ prepared DPPH free radical solution, mixing uniformly, then placing at room temperature for shading reaction for 30min, and then measuring the absorbance A of the sample at the wavelength of 517nm_Sample(s)And 3 times of paralleling. Control samples were mixed with equal volumes of PBS and DPPH-ethanolAnd (4) carrying out blank zero adjustment by using a mixed solution of the PBS bacterial suspension and the ethanol with equal volumes. The clearance rate is calculated according to the following formula: clearance%_{Sample (I)}-A_{Blank space})/A_Control]×100％。

The results show that: the clearance rate of the bacillus coagulans VHProbi C08 on DPPH free radicals is up to 87.80%, and the standard deviation is 3%.

3) Determination of HRS (hydroxyl radical scavenger) capacity of strain

100ul of 5mM sodium salicylate-ethanol solution, 100ul of 5mM ferrous sulfate, 500ul of deionized water and 200ul of lactic acid bacteria PBS bacterial suspension are mixed uniformly, 100ul of hydrogen peroxide solution (3mM) is added, and the absorbance of the sample is measured at the wavelength of 510nm after water bath at 37 ℃ for 15 min. The hydroxyl radical clearance was calculated according to the following formula: clearance%_{Sample (I)}-A_{Control of})/(A_{Blank space}-A_{Control of}) X 100% where A_{Control of}To replace the sample with deionized water, A_{Blank space}Replacement of sample and H for deionized Water₂O₂。

The results show that: the clearance rate of the bacillus coagulans VHProbi C08 on HRS free radicals is up to 47.79%, and the standard deviation is 2.51%.

2. Determination of anti-lipid peroxidation capability of strain

1) Strain culture and preparation of fermentation supernatant, thallus and intracellular extract:

culturing the strain in MRS liquid culture medium at 37 deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000r/min at 4 deg.C for 10min, and collecting supernatant as fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min in PBS buffer (pH 7.4) and washed 3 times. The cells were resuspended in PBS buffer to adjust the cell concentration to 1.0X 10⁹cells/mL to obtain a bacterial suspension.

2) Preparing a linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.

3)0.5mL of PBS solution (pH 7.4) was added 1mL of an emulsion of linoleic acid, 1mL of LFeSO₄(1%), adding 0.5mL of sample, water bath at 37 ℃ for 1.5h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), water bath at 100 ℃ for 30min, rapidly cooling,centrifuging at 4000r/min for 15min, collecting supernatant at OD_532nmThe absorbance measured is A; the control group uses 0.5mL of distilled water to replace the sample to obtain A₀. Inhibition rate/% (A)₀－A)/A₀×100。

The results show that: the supernatant liquid anti-lipid peroxidation inhibition rate of the bacillus coagulans VHProbi C08 provided by the invention is 84.46%, and the standard deviation is 0.28%; the inhibition rate of lipid peroxidation of the bacterial cells is 44.88 percent, and the standard deviation is 0.43 percent.

Example 8 Bacillus coagulans VHProbi C08 in vitro cholesterol degradation assay

1. Preparation of cholesterol micelle solution: accurately weighing 1g of cholesterol, dissolving in absolute ethyl alcohol, diluting to 100mL, and filtering and sterilizing with a 0.22-micron microporous filter membrane under aseptic condition.

2. 10.0g of peptone and 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate and 20.0g of glucose are weighed, 801.0 mL of Tween, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate and 1000mL of distilled water are weighed, the pH value is adjusted to be 7.3, sterilization is carried out at 115 ℃ for 30min, and then cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%. Inoculating fresh bacterial liquid according to the inoculation amount of 0.1%, statically culturing at 37 ℃ for 48 hours, then taking 0.2ml of bacterial liquid, adding 1.8ml of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 rpm for 5 minutes, and taking supernatant for measuring the cholesterol content. The method for measuring cholesterol was carried out in accordance with GB/T5009.128-2003, "determination of cholesterol in food".

The results show that: the degradation rate of the bacillus coagulans VHProbi C08 on cholesterol is up to 39.21%.

Example 9 use of Bacillus coagulans VHProbi C08 for prevention of diarrhea in rats

1. Experimental Material

(1) Laboratory animal

Wistar rats were SPF grade, 30, half male and female, weight 150-. Supplied by experimental animal breeding, Inc. of Jinanpunyue, produces license number SCXK (Shandong) 20190003.

Animal quarantine and identification: after all animals arrive, the adaptation period is at least one week, quarantine observation is carried out, the performance of the animals such as activity, diet and the like is observed, the animals need to be checked to be qualified before the test, and the qualified animals can be used for the test. After animal quarantine was approved, each animal was assigned a single animal number and the animal tail was marked. The cage card of the quarantine observation period is marked with a special number, an animal number, a cage number, a sex, an animal receiving date and a special person in charge; after grouping, special serial numbers, animal numbers, cage numbers, sexes, groups, test start and stop dates and special responsible persons are marked on the cage cards.

Environmental conditions for experimental animal feeding management: the temperature of the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%, and the light and shade alternation time is 12/12 hours. Animals were housed in standard rat cages, 10 animals per cage.

Animal feed and drinking water: free ingestion and drinking. The feed is SPF-level rat growth breeding feed provided by Jinanpunyue laboratory animal breeding company Limited (batch number: 20190905). The drinking water is urban tap water sterilized at high temperature.

(2) Reagent consumable

Loperamide (batch No. 190106588), Saian Ponsen pharmaceutical Co., Ltd; castor oil (batch No. 20190426), national pharmaceutical group Chemicals, Inc.

2. Experimental methods

(1) Preparation of bacterial liquid

Dissolving Bacillus coagulans VHProbi C08 lyophilized powder in physiological saline to obtain a solution with a concentration of 10⁹CFU/ml bacterial fluid.

(2) Modeling, grouping and administering drugs

The grouping and administration of SPF Wistar rats are started after adaptive feeding for 5 days. The qualified rats are randomly divided into 5 groups, namely a blank control group, a positive control group, a negative control group, a probiotic pretreatment group and a probiotic post-treatment group. After the grouping, probiotic pretreatment groups were started to administer 1ml of bacillus coagulans VHProbi C08 bacterial solution for 7 days, and other groups were left untreated. After day 7, rats were fasted for 18h with free access to water. On the 8 th day of the experiment, the blank control group was not treated, the negative control group was administered with physiological saline of the same volume, the positive control group was administered with loperamide at a weight of 1mg/kg, and the probiotic pretreatment group and the probiotic post-treatment group were administered with Bacillus coagulans VHProbi C08 bacterial solution at a dose of 1 ml/ml. After 4h of administration, the placebo group was left untreated, and the rats in the remaining groups were each administered 1ml of castor oil for diarrhea induction, and after the diarrhea induction, the time for primary diarrhea was observed, and the feces weight and the water intake of the rats were weighed for 4h, 8h and 12h of diarrhea, and the results are shown in Table 6.

3. Data statistical processing method

All experimental data are expressed as mean ± standard deviation "± SD", and data statistics and plots were performed using Microsoft EXCEL, and significant differences were judged as P <0.05 using t-test for comparison between the two sets of data.

4. Results and analysis of the experiments

TABLE 6 comparison of index of prevention of diarrhea in rats

Comparison with negative control group: p <0.05

As can be seen from the data in Table 6, the rats in the negative control group and the rats in the administration group both showed diarrhea when compared with the blank control group, which proves the success of molding.

As shown in fig. 4, the water intake of rats in each group within 12h was not statistically different, so the water intake was not related to the defecation amount, and the defecation amount was not large due to the water intake.

As shown in fig. 5, 4h of continuous observation, compared with the negative control group, the positive control group had significantly reduced frequency of loose stools and significantly different weight reduction of stools (P < 0.05); the weight reduction of the probiotic pretreated feces has a significant difference (P < 0.05); the post-treatment group was not reduced with no significant difference. Continuously observing for 8h, and comparing with a negative control group, the positive control group has reduced frequency of loose stools and has significant difference in the reduction of the stool amount (P < 0.05); there was a significant difference in the reduction of fecal mass with probiotic pretreatment (P <0.05), and no reduction in post-treatment occurred. The decrease of the stool quantity of the positive control group is obviously different after continuous observation for 12h (P < 0.05); neither pretreatment nor post-treatment of the probiotic appeared to be reduced. The positive control, probiotic pretreatment and post-treatment groups all showed a different degree of reduction compared to the negative control group, but no significant difference.

5. Conclusion of the experiment

According to the indexes, the rats in the bacillus coagulans VHProbi C08 pretreatment group are observed to delay diarrhea time, and the excrement amount reduction has significant difference; whereas no therapeutic effect was observed in B.coagulans VHProbi C08 post-treated rats. Therefore, the bacillus coagulans VHProbi C08 provided by the invention can effectively prevent rat diarrhea, but has an insignificant treatment effect on the rat diarrhea.

Example 10 use of Bacillus coagulans VHProbi C08 for regulating gastrointestinal motility in rats

1. Experimental Material

(1) Laboratory animal

Wistar rats were SPF rated, 24, male and female halves, weight 150-. Supplied by experimental animal breeding, Inc. of Jinanpunyue, produces license number SCXK (Shandong) 20190003.

(2) Reagent consumable

Atropine sulfate (batch No. 1902161), Tianjin Jinyao pharmaceutical Co., Ltd; acacia (batch No. 20190502), length of Beichen Fangzheng reagent in Tianjin; activated carbon powder (batch number: 7440-44-0), Solibao.

2. Experimental methods

(1) Preparation of the solution

Preparing a bacterial liquid: dissolving Bacillus coagulans VHProbi C08 lyophilized powder in physiological saline to obtain a solution with a concentration of 10⁹CFU/ml bacterial fluid.

Preparation of 3% of deactivated carbon: dissolving arabic gum in high-purity water to prepare 2% arabic gum water solution; the carbon powder was suspended in 2% gum arabic aqueous solution to make 3% inactivated carbon.

(2) Modeling, grouping and administering drugs

The grouping and administration of SPF Wistar rats are started after adaptive feeding for 4 days. Selecting qualified rats, randomly dividing into 4 groups, respectively as negative control group (equal volume of normal saline), positive control group (atropine sulfate, 4mg/kg body weight), and probiotic pretreatment group (10)⁹CFU/only), probiotic post-treatment group (10)⁹CFU/only). After the grouping, probiotic pretreatment groups were started to administer 1ml of bacillus coagulans VHProbi C08 bacterial solution for 7 days, and other groups were left untreated. After day 7, rats were fasted for 18h with free access to water. On the 8 th day of the experiment, the positive control group was injected with atropine sulfate at a dose of 1ml/100g, 2ml of 3% inactivated carbon was administered to each rat by gavage after 2 hours, and CO was added after 1 hour₂Suffocation and sacrifice; respectively feeding bacillus coagulans VHProbi C08 bacterial liquid to a probiotic pretreatment group and a probiotic post-treatment group according to a gavage rate of 1 ml/one, feeding equal volume of normal saline to a negative control group, feeding 3% inactivated carbon 2ml to each rat after 4h, and feeding CO after 1h₂Suffocation and sacrifice; the small intestine was quickly divided and pulled into a straight line, placed on a sterile sheet, the length of the intestinal canal (pylorus to ileocecal part) was measured as the total length of the small intestine, the distance from the leading edge of the carbon powder to the pylorus was the advancing distance of the carbon powder in the small intestine, and the advancing rate of the carbon powder and the inhibiting rate of the carbon powder advancing were calculated, and the results are shown in table 7.

The carbon powder propulsion rate (%) — the propulsion distance (cm) of carbon powder in the intestine/the total length (cm) of the small intestine × 100%.

3. Data statistical processing method

All experimental data are in mean ± sd

Showing, using Microsoft EXCEL software for data statistics and mapping, and comparing the two data sets using t-test, with P<0.05 was judged to have significant differences.

4. Results and analysis of the experiments

TABLE 7 comparison of small intestine carbon powder propulsion rates of rats in each group

As can be seen from the data in Table 7, the propulsion rate of carbon powder in the small intestine of the rat in the positive control group is reduced and has significant difference compared with that in the negative control group; the propelling rate of the probiotic pretreatment carbon powder is reduced, and the obvious difference exists; and the reduction of the carbon powder advancing rate of the probiotic post-treatment group has no significant difference.

5. Conclusion of the experiment

According to the indexes, the propulsion rate of carbon powder in small intestines of rats in the bacillus coagulans VHProbi C08 pretreatment group is reduced, and the significant difference exists, but the post-treatment group has no significant difference compared with the negative group, so that the bacillus coagulans VHProbi C08 can be taken in advance, and the enterokinesia can be effectively inhibited.

The results of two application experiments in example 10 and example 11 of the present invention show that: after the bacillus coagulans VHProbi C087 days are taken in advance, the effects of slowing down the excessive peristalsis of intestinal tracts and relieving diarrhea can be achieved, and the bacillus coagulans VHProbi C087 has a good function of preventing diarrhea. However, after 4 hours of taking the bacillus coagulans VHProbi C08 only once, the effects of slowing down the intestinal peristalsis and diarrhea are not obvious. Therefore, the regulation capacity of the bacillus coagulans VHProbi C08 on the intestinal peristalsis can be realized within a certain time, and the bacillus coagulans VHProbi C08 can grow and fix in the intestinal tract within a sufficient time, secrete metabolites beneficial to the organism, regulate intestinal flora, and exert the effect of relieving diarrhea. If the colonization time of the bacillus coagulans VHProbi C08 in the intestinal tract is too short, the beneficial effect on the organism cannot be shown.

Example 11 use of Bacillus coagulans VHProbi C08 for reducing cholesterol levels in mice

1. Experimental Material

(1) Laboratory animal

Female SPF-grade BALB/c mice aged 8 weeks, 90, weighing 150-200 g. Supplied by experimental animal breeding, Inc. of Jinanpunyue, produces license number SCXK (Shandong) 20190003.

Environmental conditions for experimental animal feeding management: the temperature of the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%, and the light and shade alternation time is 12/12 hours. Animals were housed in standard mouse cages, 6 animals per cage.

Animal feed and drinking water: free ingestion and drinking. The feed is SPF-level mouse growth breeding feed and comprises the following components: 23.9% protein, 4.6% fat, 5.5% fiber, 75.5% total digestible nutrient was provided by jinanpunyue laboratory animal breeding limited. The drinking water is urban tap water sterilized at high temperature.

(2) Reagent consumable

Cholesterol was purchased from Beijing Sorleibao technologies, Inc.; cholesterol detection kits were purchased from Merck Sigma-Aldrich.

2. Experimental methods

(1) Preparation of bacterial liquid

Dissolving Bacillus coagulans VHProbi C08 lyophilized powder in physiological saline to obtain a solution with a concentration of 10⁹CFU/mL of bacterial liquid.

(2) Modeling, grouping and administering drugs

Grouping and administration are started after 5 days of adaptive feeding of SPF-grade BALB/c mice. Selecting qualified mice, randomly dividing into 3 groups, each group containing 30 mice, and respectively preparing into positive control group (equal volume of normal saline) and probiotic pretreatment group (10)⁹CFU/mL/one), probiotic post-treatment group (10)⁹CFU/mL/only). And feeding the mice with high-cholesterol feed for 14 days after grouping for molding. The probiotic pretreatment group is given with 1 mL/only of bacillus coagulans VHProbi C08 bacterial liquid from the beginning of molding and is continued until the end of the experiment, and the probiotic post-treatment group is given with 1 mL/only of bacillus coagulans VHProbi C08 bacterial liquid from the end of molding and is continued until the end of the experiment. The positive control group was fed with an equal volume of physiological saline from the end of molding. The whole process lasts for 40 days, 6 mice in each group are killed every 10 days, the mice are fasted for 24 hours before detection, water is freely drunk, orbital blood is taken for detection of blood cholesterol level, and the result is shown in table 8.

3. Data statistical processing method

All experimental data are in mean ± sd

Showing that the comparison between the two sets of data is performed by t test and P<0.05 was judged to have significant differences.

4. Results and analysis of the experiments

TABLE 8 comparison of cholesterol lowering indices in mice

Unit mg/dl compared to positive control: p <0.05

As can be seen from the data in table 8, after 30 days of continuous observation, the blood cholesterol level of the bacillus coagulans VHProbi C08 pretreated group was reduced by about 34% and significantly different, and the blood cholesterol level of the bacillus coagulans VHProbi C08 post-treated group was reduced by about 10% and also significantly different, compared to the positive control group; after continuous observation for 40 days, the blood cholesterol level of the bacillus coagulans VHProbi C08 pretreatment group is reduced by about 39 percent, and is significantly different, and the blood cholesterol level of the bacillus coagulans VHProbi C08 post-treatment group is reduced by about 30 percent and is also significantly different.

5. Conclusion of the experiment

According to the indexes, after the bacillus coagulans VHProbi C08 provided by the invention is fed to a mouse model for 30 days, the blood cholesterol level of the mouse with hyperlipidemia can be effectively reduced, so that the bacillus coagulans VHProbi C08 has the effect of reducing the blood cholesterol level.

As one of edible strains in China, the safety of the bacillus coagulans is widely accepted. The bacillus coagulans VHProbi C08 meets the national regulations, can be used as a food raw material, and does not have any side effect or excessive risk after long-term administration. The bacillus coagulans VHProbi C08 is a new bacillus coagulans strain, and is proved to have the functions of preventing diarrhea and degrading cholesterol through in vitro animal experiments. In addition, the bacillus coagulans VHProbi C08 can be added into most kinds of foods to prepare functional foods with functions of preventing diarrhea and degrading cholesterol due to the heat resistance characteristic and the good sporulation rate of the bacillus coagulans VHProbi C08, and the strain is simple in fermentation condition, low in industrialization cost, wide in market prospect and is a novel probiotic strain with huge economic value.

The Bacillus coagulans VHProbi C08(Bacillus coagulans VHProbi C08) has been deposited in China center for type culture Collection with a deposition number of CCTCC NO: M2019738 by the applicant at 23.9.2019.

Sequence listing

<110> Islands Ulva Biometrics Ltd

QINGDAO VLAND BIOTECH GROUP Co.,Ltd.

<120> a bacillus coagulans strain with functions of preventing diarrhea and degrading cholesterol

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1415

<212> DNA

<213> Bacillus coagulans (Bacillus coagulans)

<400> 1

gctggctccg taaggttacc tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg 60

gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120

gattccggct tcatgcaggc gggttgcagc ctgcaatccg aactgggaat ggttttctgg 180

gattggctta acctcgcggt ctcgcagccc tttgtaccat ccattgtagc acgtgtgtag 240

cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc 300

ggcagtcacc ttagagtgcc caactgaatg ctggcaacta aggtcaaggg ttgcgctcgt 360

tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca 420

ctctgtcccc cgaaggggaa ggccctgtct ccagggaggt cagaggatgt caagacctgg 480

taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540

tcaattcctt tgagtttcag ccttgcggcc gtactcccca ggcggagtgc ttaatgcgtt 600

agctgcagca ctaaagggcg gaaaccctct aacacttagc actcatcgtt tacggcgtgg 660

actaccaggg tatctaatcc tgtttgctcc ccacgctttc gcgcctcagc gtcagttaca 720

gaccagagag ccgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac 780

acgtggaatt ccactctcct cttctgcact caagcctccc agtttccaat gaccgcttgc 840

ggttgagccg caagatttca catcagactt aagaagccgc ctgcgcgcgc tttacgccca 900

ataattccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960

gtggctttct ggccgggtac cgtcaaggcg ccgccctgtt cgaacggcac ttgttcttcc 1020

ccggcaacag agttttacga cccgaaggcc ttcttcactc acgcggcgtt gctccgtcag 1080

actttcgtcc attgcggaag attccctact gctgcctccc gtaggagttt gggccgtgtc 1140

tcagtcccaa tgtggccgat caccctctca ggtcggctac gcatcgttgc cttggtgagc 1200

cgttacccca ccaactagct aatgcgccgc gggcccatct gtaagtgaca gcaaaagccg 1260

tctttccttt ttcctccatg cggaggaaaa aactatccgg tattagcccc ggtttcccgg 1320

cgttatcccg atcttacagg caggttgccc acgtgttact cacccgtccg ccgctaacct 1380

tttaaaagca agctttaaaa ggtccgcacg actgc 1415

Claims

1. The bacillus coagulans is characterized in that the preservation number of the bacillus coagulans is CCTCC NO: and M2019738.

2. Use of bacillus coagulans according to claim 1 for preventing diarrhea.

3. Use of bacillus coagulans as claimed in claim 1 for degrading cholesterol.

4. A probiotic formulation comprising the bacillus coagulans of claim 1.

5. The probiotic formulation according to claim 4, characterized in that it further comprises any one or a combination of two or more of bifidobacteria, lactobacilli, streptococci, lactococcus, leuconostoc, propionibacterium, yeasts, pediococci, staphylococci.

6. The probiotic preparation according to claim 5, characterized in that the bifidobacteria are any one or a combination of two or more of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis and Bifidobacterium bifidum.

7. The probiotic formulation according to claim 5, characterized in that the Lactobacillus is any one or a combination of two or more of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus bulgaricus.

8. The probiotic formulation according to claim 4 or 5, characterized in that the probiotic formulation has a live bacillus coagulans count of at least 10⁸CFU/g。

9. Use of a probiotic formulation according to any one of claims 4 to 8 for the prevention of diarrhoea.

10. Use of a probiotic formulation according to any one of claims 4 to 8 for degrading cholesterol.