CN104560820A - Enterococcus faecium KQ2.6 and application thereof - Google Patents

Abstract

The invention discloses a new strain-enterococcus faecium KQ2.6 and an application of the strain or a fermented product of the strain as probiotics or preparation of weight-losing drugs. The new strain-enterococcus faecium KQ2.6 is separated from fresh excrements of a peacock for the first time and has good tolerance on environmental pressure, and the strain or the fermented product of the strain has an inhibition effect on common pathogenic microorganisms; in addition, a mouse safety experiment shows that the strain is safe; the strain also has an effect of inhibiting body weight of a mouse from being increased. Therefore, the strain has a good application prospect in the fields of foods and the like.

Description

Enterococcus faecium KQ2.6 and its application

(1) Technical field

The invention relates to an enterococcus faecium and its application as a probiotic.

(2) Background technology

Obesity is the result of an imbalance between energy intake and consumption in the human body, that is, energy intake is greater than energy consumption, resulting in energy being stored in the form of fat in the body, resulting in significant weight gain and obesity. Under normal circumstances, the energy intake and consumption of the human body maintain a relative balance, and the body weight remains relatively stable. Once it is destroyed and the energy intake is more than the energy consumption, the excess energy will be stored in the form of fat in the body, which will accumulate over time and eventually become obese. The occurrence of obesity is related to family, personal living habits, social and economic development, cultural background and other factors, as well as factors such as bad eating habits and lack of exercise.

Obesity is a chronic disease of the human body. It is the most easily overlooked disease faced by human beings, but the incidence rate is rising sharply. In my country, the number of people suffering from obesity is increasing year by year. It is estimated that there are about 80 million obese people in China. Obesity not only brings troubles to people's own shape, but also leads to a decline in the quality of life. Obesity is also significantly related to many diseases such as diabetes, hypertension, hyperlipidemia, hyperuricemia, ischemic heart and brain disease, cancer, rheumatoid arthritis, abnormal menstruation, abnormal pregnancy and childbirth, and can be Increased risk of death. Obesity has become the main cause of death and disability in my country. For this reason, eliminating obesity diseases has become an urgent task in the medical field.

Probiotics are a class of single or mixed live microbial preparations that improve the host's microecological balance and then promote the health of the host. Probiotics are essential to good health, especially the health of the intestinal system. The relationship between probiotics and human beings is very close. It can be said that "without the existence of probiotics, human health will be difficult to guarantee and survival will be difficult."

Probiotics can be roughly divided into three categories: (1) Lactobacillus (such as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus jansii, Lactobacillus ramans, Lactobacillus brevis, Lactobacillus bulgaricus, etc.); (2) Bifidobacteria (such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium ovale, Bifidobacterium thermophile, Bifidobacterium adolescent, etc.); (3) Gram-positive cocci (such as Streptococcus faecalis, milk cocci, intermediary streptococci, etc.). In addition, some Saccharomyces cerevisiae, Brucella, some molds and non-lethal Escherichia coli can also be classified as probiotics. Probiotics can be isolated from the oral cavity, intestinal contents and feces of humans and animals by repeatedly culturing on appropriately rich or selective media. In recent years, with the deepening of probiotic research, people have screened and identified a variety of probiotics from various sources. According to reports, my country will spend 60 billion yuan per year on weight loss. In order to reduce the economic burden and health needs of obese people, some affordable weight loss products that can treat both symptoms and root causes are needed. Therefore, starting from fresh peacock excrement, our research group hopes to screen out probiotics with antibacterial and weight-loss effects, laying a foundation for the development of probiotic-related products.

(3) Contents of the invention

The purpose of the present invention is to provide a new bacterial strain—Enterococcus faecium (Enterococcus faecium) KQ2.6, and the application of the bacterial strain or its fermented product as probiotics. In addition to inhibiting Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae, Salmonella paratyphi B, Bacillus cereus, Streptococcus pyogenes, Pseudomonas aeruginosa , Candida albicans and other pathogenic microorganisms also have the effect of inhibiting the weight gain of mice, and can be used to prepare probiotic preparations or weight loss drugs.

The technical scheme adopted in the present invention is:

The present invention provides a new bacterial strain—Enterococcus faecium (Enterococcus faecium) KQ 2.6, which is preserved in the China Typical Culture Collection Center, with a preservation date of May 12, 2014, a preservation number of CCTCC NO: M 2014197, and a preservation address of Wuhan University, Wuhan, China, said Enterococcus faecium KQ 2.6 has tolerance against environmental stress, said tolerance being antibiotic tolerance, acid tolerance, pepsin, trypsin tolerance or salt tolerance .

The present invention also provides a probiotic preparation, the probiotic preparation comprises pharmaceutical excipients and the supernatant obtained through fermentation and cultivation of the Enterococcus faecium KQ 2.6.

Further, preferably, the pharmaceutical excipient is sodium carboxymethyl cellulose or sodium carboxymethyl starch.

Furthermore, the content of Enterococcus faecium KQ 2.6 in the probiotic preparation is 5×10 ⁷ -5×10 ¹² CFU/mL. The pharmaceutical excipients in the probiotic preparation are added in the form of a 1% aqueous solution with a mass concentration, and more preferably the probiotic preparation is: mix a 1% aqueous solution of sodium carboxymethylcellulose with a mass concentration of Enterococcus faecium KQ 2.6 fermentation supernatant to form 5 ×10 ⁷ ~5×10 ¹² CFU/mL bacterial suspension (preferably 5×10 ¹² CFU/mL).

The dosage of the probiotic preparation of the present invention is 10 ⁷ -10 ¹² CFU/kg body weight.

The supernatant that Enterococcus faecium KQ 2.6 of the present invention obtains through fermentation culture is prepared as follows:

(1) Incline cultivation

Enterococcus faecium KQ 2.6 was inoculated into the slant medium, cultured at 37°C for 1 day, and the slant bacteria were obtained; the final concentration of the slant medium per liter consisted of: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, Magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, Tween-801.0g, agar 20g, distilled water 1000mL, pH 6.2;

(2) Fermentation culture

Pick an inoculation loop of bacteria from the slant and inoculate it into the fermentation medium, culture at 37°C for 12-24 hours to obtain the fermentation culture medium, centrifuge the fermentation culture medium, and take the supernatant, which is Enterococcus faecium KQ2.6 liquid; the final concentration of the fermentation medium consists of: peptone 10g/L, beef extract 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, glucose 20g/L, magnesium sulfate 0.20g/L, manganese sulfate 0.050g/L L, yeast extract powder 4.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, Tween-801.0g/L, solvent is distilled water, pH 6.2.

The present invention also relates to an application of the Enterococcus faecium KQ 2.6 in the preparation of weight-loss drugs, wherein the weight-loss drugs include pharmaceutically acceptable excipients and the supernatant or supernatant obtained by fermentation and cultivation of the Enterococcus faecium KQ 2.6 Bacteria powder after liquid concentration and drying.

The pharmaceutically acceptable excipients include sodium carboxymethylcellulose or sodium starch glycolate.

The content of Enterococcus faecium KQ 2.6 in the weight-loss medicine is 5×10 ⁷ -5×10 ¹² CFU/g.

In the present invention, the weight-loss drug is preferably: the supernatant obtained by fermentation and cultivation of Enterococcus faecium KQ 2.6 is concentrated under reduced pressure to a concentration of 5.0×10 ⁸ to 5.0×10 ¹³ CFU/mL (preferably 5.0×10 ¹⁰ CFU /ml), and then mix the pharmaceutically acceptable excipients (preferably sodium carboxymethyl cellulose) with the concentrate evenly, put them into a freeze dryer and dry them at -40°C, and prepare the bacteria with a bacterial content of 5.0×10 ⁷ to 5.0×10 ¹² CFU/g of weight loss drug (preferably 5.0×10 ¹¹ CFU/g).

The dosage of the weight-loss drug of the present invention is 10 ⁷ -10 ¹² CFU/kg body weight, used twice a day, taking 60-90 days as a course of treatment, and taking 1-2 courses of treatment to lose 3-10kg in weight.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that a new bacterial strain, Enterococcus faecium KQ 2.6, is isolated from the fresh excrement of peacocks for the first time. This bacterial strain not only has good tolerance to environmental pressure, Moreover, the bacterial strain or its fermentation product has inhibitory effect on common pathogenic microorganisms. In addition, the mouse safety experiment shows that the strain is safe. The strain also had the effect of inhibiting weight gain in mice. The invention shows that the bacterial strain has good application prospects in the fields of food and the like.

(4) Description of drawings

Figure 1. Acid resistance of Enterococcus faecium KQ 2.6.

Figure 2 High salt tolerance of Enterococcus faecium KQ 2.6.

Figure 3 Pepsin resistance of Enterococcus faecium KQ 2.6.

Figure 4. Trypsin resistance of Enterococcus faecium KQ 2.6.

Figure 5 Adhesion performance of Enterococcus faecium KQ 2.6.

Fig. 6 Antibacterial effect of Enterococcus faecium KQ 2.6 fermentation supernatant on Streptococcus pyogenes.

Fig. 7 Organ tissue sections of mouse acute toxicity test: A is the liver, B is the kidney, and C is the spleen.

Fig. 8 Sections of organ tissues of mouse subacute toxicity test: A is liver, B is kidney, and C is spleen.

Figure 9 Organ index of mouse subacute toxicity test.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1 strain isolation and purification and identification

(1) Operation

1. Isolation and purification of bacterial strains

Using aseptic operation, take the fresh excrement of healthy peacocks (1.0 g) and place it in a test tube of sterile physiological saline, shake it fully and evenly, and form a suspension. Take 100 μL of the suspension in 10 mL of bile salt liquid culture solution and incubate at 37°C for 24 hours; then pipette 0.50 mL of the culture solution into a test tube filled with 4.5 mL of sterile saline, the dilution is 10 ^-1 , repeat the above In the process, make 10-fold dilution to 10 ^-5 dilution concentration, pipette 0.10mL bacterial solution at each dilution and drop it on the 2.0% CaCO ₃ -MRS solid medium plate. After coating evenly, the plate is incubated at 37°C for 24h. Pick white round and regular colonies, streak and culture them on MRS solid medium to obtain primary strains, and store them in a 4°C refrigerator for later use.

The primary strains were inoculated into MRS solid medium, and after culturing at 37°C for 24 hours, the catalase test was carried out. The strains with positive Gram staining and negative catalase test were screened, and the strains that passed the above tests were evaluated for their tolerance to external pressure, and a strain with the strongest activity was screened, named strain KQ 2.6. Transfer the bacterial strain KQ 2.6 to the MRS liquid medium, place it at 37°C for 24 hours, and then take 600 μL of the bacterial solution and add it to a cryopreservation tube that has been added with 400 μL of 50% glycerol (sterilized) and mix it. The cryopreservation tubes were stored in a -20°C refrigerator for strain preservation. The present invention will focus on the screening, identification and performance evaluation of strain KQ 2.6.

In the method of above-mentioned screening, relate to culture medium as follows:

The composition of MRS solid medium per liter: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, acetic acid Sodium 5.0g, Tween-801.0g, agar 20g, distilled water 1000mL, pH 6.2.

Composition of MRS liquid medium per liter: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, acetic acid Sodium 5.0g, Tween-801.0g, distilled water 1000mL, pH 6.2.

Composition of 2.0% CaCO ₃ -MRS solid medium per liter: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, tricitric acid Ammonium 2.0g, sodium acetate 5.0g, Tween-801.0g, CaCO ₃ 20g, agar 20g, distilled water 1000mL, pH 6.2.

Composition of bile salt liquid medium per liter: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, Sodium acetate 5.0g, Tween-801.0g, bile salt 5.0g, distilled water 1000mL, pH 3.0.

2. Bacteria identification

2.1 Bacterial Gram staining test

Take a clean slide, use an inoculation loop to take a ring of sterile water on the slide, then pick a small amount of strain KQ 2.6, spread it evenly, dry and fix. Add crystal violet staining solution dropwise, stain for one minute, wash with water, then add dropwise mordant staining with Luxe's iodine solution, act for one minute, wash with water, then add dropwise 95% ethanol aqueous solution for decolorization for 30 seconds, and finally add safranin counterstaining solution dropwise for re-staining Dye for 2-3 minutes, wash and dry. Observe the shape and color of the strain under an ordinary optical microscope. If the bacteria are stained blue-purple, they are Gram-positive bacteria, and if they are stained red, they are Gram-negative bacteria.

Gram staining reagent preparation:

1) Preparation of ammonium oxalate crystal violet staining solution

A liquid crystal violet 2.0g, 95% alcohol 20mL; B liquid ammonium oxalate 0.80g, distilled water 80mL, mix A and B liquids, and use after standing for 48 hours.

2) Lushi iodine solution

Iodine tablets 1.0g, potassium iodide 2.0g, distilled water 300mL

Dissolve potassium iodide in a small amount of water first, then dissolve iodine tablets in the potassium iodide solution, and add distilled water after the iodine is completely dissolved.

3) Safranin Counterstaining Solution

Safranin 2.5g, 95% alcohol 100mL

Take the above prepared 10mL of safranin alcohol and mix with 80mL of distilled water.

2.2 Catalase test: smear a small ring strain KQ 2.6 on the slide with a volume concentration of 3.0% hydrogen peroxide, and observe whether there are bubbles. If there are bubbles, it is positive, and if there are no bubbles, it is negative.

2.3 Physiological and biochemical tests of strains

2.3.1 Hydrogen sulfide test: Pick the strain KQ 2.6 and inoculate it in a hydrogen sulfide biochemical tube, and culture it at 37°C for 24 hours. If there is black precipitate in the biochemical tube, it is positive, otherwise it is negative.

2.3.2 Nitrate reduction test: Inoculate strain KQ 2.6 into nitrate reduction reaction biochemical tubes, incubate at 37°C for 3 to 5 days, add a drop of Griess reagent A and Griess reagent B to each biochemical tube, and observe Whether the culture medium turns red, orange or brown, if there is the above reaction, it means that there is nitrite in the culture medium; if there is no such reaction, add 1-2 drops of diphenylamine reagent, and observe whether the culture medium turns blue. If there is a blue reaction, it means that nitrate still exists in the culture medium; if there is no blue reaction, it means that both nitrate and newly formed nitrite have been reduced to other substances.

2.3.3 Indole test: Inoculate the strain KQ 2.6 into peptone water medium, after culturing for 48 hours, add 3-4 drops of diethyl ether dropwise, shake it several times, and let it stand for 1 min. After the diethyl ether rises, slowly add it along the wall of the test tube 2 drops of indole reagent. A red ring between the ether and the culture is positive, and the absence of a red ring is negative.

The composition of each liter of peptone water medium is: tryptone 10g, sodium chloride 5.0g, distilled water 1000mL, pH 7.4.

2.3.4 Sugar fermentation test: Inoculate the strain KQ 2.6 into the culture medium of the sugar fermentation tube and culture at 37°C for 24 hours. After 24 hours, if the indicator of the bacterial solution turns yellow, it is positive, and if the indicator remains unchanged, it is negative. Lactose, maltose, soluble starch, D-fructose, L-rhamnose, D-mannose, D-xylose, D-galactose, D-cellobiose, glucose, D-mannitol, sucrose were used in the experiment , Raffinose sugar fermentation test.

The medium composition of each liter of sugar fermentation tube is: beef extract 5.0g, peptone 10g, sodium chloride 3.0g, disodium hydrogen phosphate 2.0g, 0.20% bromothymol blue solution 12mL, test sugar 50g, distilled water 1000mL, pH 7.4.

2.3.5 v-p test: Inoculate the strain KQ 2.6 in a biochemical tube of glucose phosphate peptone water, incubate at 37°C for 24-48 hours, and the red reaction is positive.

2.3.6 Citrate utilization test: inoculate the strain KQ 2.6 in a citrate biochemical tube, culture at 37°C for 1-4 days, if the medium turns blue, it is positive, and if it turns green, it is negative .

2.3.7 Methyl red test: Inoculate the strain KQ 2.6 in glucose-peptone medium and culture at 30°C. From the next day (that is, after 24 hours), take 1.0 mL of the culture solution every day and add methyl red indicator 1 dropwise. ~2 drops, bright red is positive, light red is weakly positive, yellow is negative. The result can be judged until the positive result is found or the result is still negative on the 5th day.

Per liter of glucose-peptone medium consists of: beef extract 5.0g, peptone 10g, sodium chloride 3.0g, disodium hydrogen phosphate 2.0g, 0.20% bromothymol blue solution 12mL, glucose 50g, distilled water 1000mL, pH 7.4.

2.4 Identification of strain 16S rDNA sequence

The DNA of the extracted bacterial strain KQ 2.6 is used as a PCR amplification template, PCR amplification, and the primers used for 16SrDNA PCR amplification are: upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-'3, downstream primer: 5'-GGTTACCTTGTTACGACTT-'3, obtained 16S rDNA gene sequence, PCR reaction system is 10×buffer 5.0μL, template DNA 5.0μL, Taq DNA polymerase 1.5μL, 2.5mM dNTPs 4.0μL, upstream primer (15pmol) 1.0μL, downstream primer (15pmol) 1.0μL, super Pure water 32.5 μL, total 50 μL. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 58°C for 30 s, extension at 72°C for 1 min, 30 cycles in total, and finally extension at 72°C for 10 min. After the reaction, the PCR amplification product was detected by 1.0% agarose gel electrophoresis, and a band of about 1500 bp was obtained after amplification. The PCR amplified bands were recovered from the PCR reaction system and sent to the biological company for DNA sequence determination. The gene sequence obtained by sequencing was compared with the 16S rDNA sequence in the GenBank database for Blastn comparison and homology comparison.

(2) Conclusion

1. Identification of strains

1.1 Morphological characteristics: The screened strain KQ 2.6 showed a round colony on the MRS solid medium, white and opaque, with a smooth and moist surface and neat edges. Gram staining is performed on the bacteria, the bacteria are spherical, arranged in pairs or chains, and the Gram staining is positive.

1.2 Physiological and biochemical characteristics: The physiological and biochemical characteristics of strain KQ 2.6 are shown in Table 1.

Table 1 The results of physiological and biochemical characteristics of the strains

Note: "+" is positive, "-" is negative

1.3 Identification of strain 16S rDNA: After sequencing, the homology of the 16S rDNA of strain KQ 2.6 and the 16S rDNA sequence of Enterococcus faecium reached 100%.

Comprehensive strain morphological observation, physiological and biochemical identification and 16S rDNA sequencing results, it can be determined that the strain KQ 2.6 is Enterococcus faecium (Enterococcus faecium), named Enterococcus faecium (Enterococcus faecium) KQ 2.6, preserved in the China Center for Type Culture Collection , the deposit date is 2014-05-12, the deposit number is CCTCC NO: M 2014197, and the deposit address is Wuhan University, Wuhan, China.

The preparation of embodiment 2 Enterococcus faecium KQ 2.6 fermented liquid

Enterococcus faecium KQ 2.6 was inoculated into the slant culture medium and cultured at 37°C for 1 day to obtain slant bacteria. Pick the bacterium KQ 2.6 from the slant and inoculate it into the fermentation medium, cultivate it at 37°C for 12 to 24 hours to obtain the fermentation medium, centrifuge the fermentation medium, and take the supernatant, which is Enterococcus faecium KQ 2.6 bacterium ( Concentration 1～2×10 ⁸ CFU/mL). The final concentration of each liter of fermentation medium consists of: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, Tween-801.0g, distilled water 1000mL, pH 6.2.

Embodiment 3 Enterococcus faecium KQ 2.6 antibiotic resistance performance

Get 0.10 mL (2×10 ⁷ CFU/mL) of the Enterococcus faecium KQ 2.6 bacterium solution obtained in Example 2 and spread it on the MRS solid plate medium. After the surface of the plate is slightly dry, place common antibiotics (tetracycline, vancomycin, Gentamicin, kanamycin, neomycin, chloramphenicol, erythromycin, p-colistin B, streptomycin, ciprofloxacin, midecamycin, cefazolin, rifampicin, penicillin , ofloxacin) drug-sensitive paper sheet (purchased from Beijing Saiweisi Biotechnology Research and Development Center), adhered to the surface of the medium, cultured at 37°C for 24 hours, measured the diameter of the inhibition zone, and obtained the drug resistance of the strain to the above antibiotics . The criteria for judging antibiotic resistance are: no zone of inhibition is insensitive, zone diameter <10 mm is low sensitivity, zone diameter 10-15 mm is moderately sensitive, zone diameter >15 mm is high sensitivity, and the results are shown in Table 2. shown.

Table 2 Antibiotic resistance of Enterococcus faecium KQ 2.6

Note: No zone of inhibition is -, the diameter of the zone of inhibition <10mm is +, the diameter of the zone of inhibition is 10-15mm is ++, the diameter of the zone of inhibition >15mm is +++.

It can be seen from Table 2 that Enterococcus faecium KQ 2.6 is resistant to polymyxin B and has no resistance to other antibiotics. Different antibiotics had different effects on E. faecium KQ 2.6, among which tetracycline had the greatest effect on E. faecium KQ 2.6, followed by midecamycin and gentamicin the least.

Embodiment 4 Enterococcus faecium KQ 2.6 acid resistance

Take some clean test tubes, add 10mL MRS liquid medium respectively, adjust the pH of the medium to 6.2, 5.0, 4.0, 3.0, 2.5, 2.0, 1.0 with 0.10M HCl and 0.10M NaOH, and sterilize. After cooling, insert it into Example 2 to obtain 100 μL of Enterococcus faecium KQ 2.6 bacterium solution, fully mix it on a shaker, cultivate it at 37°C for 24 hours, take the culture solution and carry out concentration gradient dilution, and select a dilution ratio of 10 ⁻³ to 10 ⁻¹¹ (10 times Gradient) diluted bacterial solution to coat MRS medium. The coated MRS medium was placed in 37°C for 24 hours, and after 24 hours, it was taken out for colony counting. The results are shown in Figure 1. It can be seen from Figure 1 that Enterococcus faecium KQ 2.6 can grow and reproduce at a lower pH (pH 1.8-3.0), and the growth ability of the strain becomes stronger as the pH increases. It can be seen that Enterococcus faecium KQ 2.6 has better acid resistance.

Example 5 Enterococcus faecium KQ 2.6 high salt tolerance

Take several clean test tubes, add sodium chloride mass concentration of 0.90%, 2.0%, 3.0%, 5.0%, 8.0% MRS liquid medium respectively, and sterilize. After cooling, insert it into Example 2 to obtain 100 μL of Enterococcus faecium KQ 2.6 bacteria solution, mix it well on a shaker, put it in 37 ° C for 24 hours, take the culture solution and carry out 10-fold concentration gradient dilution, choose 10 ^-4 ~ 10 ^-10 dilution 24 h after culture at 37°C, take out and count the colonies, the results are shown in Figure 2. It can be seen from Figure 2 that when the salt concentration is 0.90%, the survival number of Enterococcus faecium KQ 2.6 is about 10 LogCFU/mL, and when the salt concentration is 8.0%, the survival number of this strain can still maintain 5.0 LogCFU/mL. It can be seen that Enterococcus faecium KQ 2.6 has good salt tolerance.

Example 6 Enterococcus faecium KQ 2.6 pepsin resistance

Take some clean test tubes, add 20mL normal saline respectively, adjust the pH to 2.5 with concentrated hydrochloric acid with a mass concentration of 36-38%, and sterilize. After cooling, add pepsin (purchased from Shanghai Lirui Biotechnology Co., Ltd., 2500U/mg, final concentration 5.0mg/mL), insert into Example 2 to obtain 100 μL of Enterococcus faecium KQ 2.6 bacterial solution, mix thoroughly on a shaker, Cultivate at 37°C for 0, 1, 2, 3, 4, 5, and 6 hours respectively, take the cultured nights and perform 10-fold concentration gradient dilution, and select 10 ^-7 ~ 10 ^-11 dilutions of bacterial liquid to coat MRS solid medium , cultivated at 37°C for 24h, and took out after 24h for colony counting, the results are shown in Figure 3. It can be seen from Figure 3 that the pepsin solution has little effect on the growth of Enterococcus faecium KQ 2.6, indicating that the strain has good tolerance to pepsin.

Example 7 Enterococcus faecium KQ 2.6 trypsin tolerance

Take some clean test tubes, add 20mL normal saline respectively, and sterilize. After cooling, add trypsin (purchased from Dalian Meilun Biotechnology Co., Ltd., 2500U/mg, final concentration 10mg/mL), insert into Example 2 to obtain 100 μL of Enterococcus faecium KQ 2.6 bacterial solution, mix thoroughly on a shaker, 37 Cultivate for 0, 1, 2, 3, 4, 5, and 6 hours respectively at ℃, take the culture solution and carry out 10-fold concentration gradient dilution, and select the bacterial solution with a dilution of 10 ^-7 ~ 10 ^-11 to coat the MRS solid medium, at 37 °C Cultivate for 24 hours, take out after 24 hours and count the colonies, the results are shown in Figure 4. It can be seen from Figure 4 that after the Enterococcus faecium KQ 2.6 was cultured in the culture medium containing trypsin for 6 hours, the colony count decreased by about 4.0 Log CFU/mL, indicating that the Enterococcus faecium KQ 2.6 had a certain resistance to trypsin.

Example 8 Adhesion of Enterococcus faecium KQ 2.6

Hep-2 cells (purchased from Shanghai Yuchen Biotechnology Co., Ltd.) were placed in DMEM medium (purchased from Shanghai Bosheng Biotechnology Co., Ltd.), incubate in a carbon dioxide incubator at 37°C, 10% CO ₂ , and 95% humidity, change the culture medium once a day, passage once every 3 to 4 days, and culture the cells after 15 to 20 days. Inoculate in a 6-well culture plate containing a cover glass at a seeding density of about 4× ¹⁰⁴ cells/mL, and conduct an adhesion test after the cells grow to a single layer. The cells that have grown into a monolayer were rinsed twice with sterilized phosphate buffer solution pH 7.4, and 1.0 mL of Example 2 was added to each well to obtain Enterococcus faecium KQ 2.6 bacteria solution (concentration 1～2×10 ⁸ CFU/mL) and 1.0 mL of fresh DMEM medium. Incubate in a carbon dioxide incubator at 37°C, 10% CO ₂ , and 95% humidity for 1 hour, rinse the cells with sterilized phosphate buffer (pH 7.4) for 5 times, fix with methanol, stain with Gram, and observe the bacteria under a microscope The adhesion of the body, the results are shown in Figure 5. It can be seen from Figure 5 that there is a circle of Enterococcus faecium KQ 2.6 adhered around the cells (that is, the transparent and small granular substance in the figure), which indicates that Enterococcus faecium KQ 2.6 has a certain degree of adhesion and can also adhere to the intestinal tract in vivo. On the mucosal epithelial cells, thereby exerting its physiological functions.

Embodiment 9 Enterococcus faecium KQ 2.6 bacteriostasis

Pathogens such as Staphylococcus aureus, Salmonella paratyphi B, and Bacillus subtilis were respectively coated on the LB solid plate (specific information is shown in Table 3), and after the surface of the plate was slightly dry, holes were punched with a 3mm hole puncher. Then cover the back with a little melted LB solid medium, add the Enterococcus faecium KQ 2.6 bacteria solution obtained in Example 2 to each well, culture at 37°C for 20 hours, and observe whether there is a zone of inhibition, the results are shown in Figure 6 and Table 4 .

The composition of each liter of LB solid medium is: 10g of yeast extract, 4.0g of tryptone, 1.0g of sodium chloride, 20g of agar, 1000mL of distilled water, and the pH value is 6.2.

Table 3 The present invention relates to relevant information of pathogenic bacteria

Table 4 Enterococcus faecium KQ 2.6 bacteriostatic test results

Note: The diameter of the antibacterial zone is 0-5mm, it is +, the diameter of the antibacterial zone is 6-10mm, it is ++, the diameter of the antibacterial zone is 11-15mm, it is +++, and the diameter of the antibacterial zone is >16mm, it is ++++ .

It can be seen from Table 3 and Figure 6 that Enterococcus faecium KQ 2.6 has different degrees of antibacterial effects on the 13 indicator bacteria in Table 3, among them, it has antibacterial effects on Bacillus cereus, Staphylococcus epidermidis, Salmonella paratyphi B, Streptococcus pyogenes The antibacterial effect of coccus is particularly obvious.

The preparation of embodiment 10 Enterococcus faecium KQ 2.6 weight loss medicine

Get the concentrated solution of Enterococcus faecium KQ 2.6 bacterium liquid obtained in Example 2 and concentrate it to a concentration of 5.0×10 ¹¹ CFU/mL, then mix the pharmaceutically acceptable auxiliary material carboxymethylcellulose (10g) with the concentrated solution evenly, put in Dry in a freeze dryer at -40°C to prepare Enterococcus faecium KQ 2.6 weight loss medicine (bacteria content 5×10 ¹⁰ CFU/g).

Example 11 Safety Evaluation of Enterococcus Faecium KQ 2.6

(1) 50 healthy mice (purchased from the Animal Experiment Center of Hangzhou Normal University) were randomly divided into 10 groups, half male and half male, and subjected to safety evaluation test after adaptive feeding for 3 days. The animal room was illuminated for 12 hours, the ventilation and air conditioning equipment was good, the room temperature was controlled at 20-25°C, and the relative humidity was 45-50%. The results are shown in Figure 7.

It can be seen from Figure 7 that during the feeding period, the mice in the experimental group had no obvious abnormal symptoms, no abnormal changes were found in the liver, kidney, spleen and other organs of the mice, and no abnormal changes were found in the organs of the pathological examination. Illustrate that this Enterococcus faecium KQ 2.6 has no toxicity to male and female mice.

(2) Acute toxicity test in mice: Select 40 healthy, adult mice with a body weight of about 25 g, half male and half male. Randomly grouped, every group of 20, half male and half male, the test group was fed the bacterial suspension of Enterococcus faecium KQ 2.6 (the bacterial liquid of Enterococcus faecium KQ 2.6 prepared according to Example 2, mixed with mass concentration 1.0% carboxymethyl cellulose aqueous solution mixed into 5.0×10 ¹² CFU/mL bacterial suspension), the control group replaced the bacterial suspension with an equal amount of 1.0% carboxymethyl cellulose aqueous solution, and the feeding amount was 2×10 ¹¹ CFU. Gastrointestinal administration twice within 24 hours, continuous observation for 10 days, daily record of body weight changes, mental state, growth and respiratory conditions of mice in each group. After 10 days, the mice were sacrificed, and the morphological characteristics of the mouse liver, kidney, spleen and other organs were observed. The results are shown in Figure 8. It can be seen from Figure 8 that during the feeding period, no abnormal changes were found in the liver, kidney, spleen and other organs of the mice, and no abnormal changes were found in the organs of the pathological examination. It was proved that Enterococcus faecium KQ 2.6 is not toxic.

(3) Subacute toxicity test in mice: Select 40 healthy, adult mice with a body weight of about 25 g, half male and half male. Randomly divided into a high-dose group (the Enterococcus faecium KQ 2.6 bacterium liquid prepared according to Example 2 and a mass concentration of 1.0% carboxymethyl cellulose aqueous solution were mixed into a bacterium suspension of 1×10 ¹¹ CFU/mL), a middle-dose group ( The Enterococcus faecium KQ 2.6 bacterium liquid prepared according to Example 2 was mixed with a mass concentration of 1.0% carboxymethyl cellulose aqueous solution to form a bacterial suspension of 5.0×10 ¹⁰ CFU/mL), the low dose group (the feces prepared according to Example 2 Enterococcus KQ 2.6 bacteria solution was mixed with a mass concentration of 1.0% carboxymethyl cellulose aqueous solution to form a bacterial suspension of 1.0×10 ¹⁰ CFU/mL) and a control group. 10 in each group, half male and half male. The high, medium and low dose groups were given 0.10 mL of intragastric administration, once a day, for 14 consecutive days. The control group was not treated. The body weight change, mental state, growth and respiratory status of the mice in each group were recorded daily. After 14 days, the mice were killed, and the morphological characteristics of the mouse liver, kidney, spleen and other organs were observed. The results are shown in Figure 9. The mice were fasted for 12 hours before sudden death, and then the mice were weighed. Immediately after dissection, the liver, spleen and kidney were weighed, and the index of each organ was calculated (organ weight/mouse body weight×100%). The mice in each group were aseptically operated eyeballs to take blood, anticoagulated and diluted, and the total number of red blood cells (RBC), total number of white blood cells (WBC), total number of hemoglobin (Hb) and other items were measured with a blood routine detector, and the results are shown in Table 5. Show.

It can be seen from Figure 9 that the organ indexes of mice fed with different doses of Enterococcus faecium KQ 2.6 did not fluctuate significantly, indicating that Enterococcus faecium KQ 2.6 had no adverse effects on the liver, kidney, and spleen.

Table 5 Blood routine of mice

As can be seen from Table 5, there were no significant differences in hemoglobin (Hb), total red blood cells (RBC), total white blood cells (WBC), and percentages of granulocytes, lymphocytes, and monocytes between different dose groups. The blood routine was normal, indicating that Enterococcus faecium KQ 2.6 had no effect on the tissues and organs of mice.

Example 12 Effect of intragastric administration of probiotic bacterial liquid on mouse body weight

Select 40 healthy, adult mice with a weight of about 25 g, half male and half male. They were randomly divided into high-dose group, middle-dose group, low-dose group and control group. 10 in each group, half male and half male. The high, medium and low dose groups were administered with 0.10mL (low dose), 0.50mL (medium dose), and 1.0mL (high dose) respectively, and the concentration was 5×10 ¹⁰ CFU/mL bacterial suspension (prepared according to Example 2 Enterococcus faecium KQ 2.6 bacterium solution and 1.0% carboxymethyl cellulose aqueous solution were mixed to form 5×10 ¹⁰ CFU/mL bacterial suspension), which was administered orally once a day for 60 consecutive days. The control group was not treated.

The 60-day animal experiment showed that after taking different doses of probiotics, the male and female mice had no abnormal mental state, good growth, stable breathing, and normal defecation. It can be seen from Table 6 that, compared with the control group, the daily body weight change value of the probiotic group was the smallest. It shows that Enterococcus faecium KQ 2.6 bacterial solution can effectively inhibit the increase of mouse body weight. Compared with the control group, each group has no abnormal change rule, and there is no significant dose effect among different dose groups (P>0.05).

Table 6 Changes in body weight of mice

Example 13 Effect of intragastric administration of probiotic powder on body weight of mice

Select 40 healthy, adult mice with a weight of about 25 g, half male and half male. They were randomly divided into high-dose group, middle-dose group, low-dose group and control group. 10 in each group, half male and half male. The high, medium and low dose groups were fed with 0.10g (low dose), 0.50g (middle dose), and 1.0g (high dose) respectively, and the concentrations were 5×10 ¹⁰ CFU/g edible fungus powder (prepared according to Example 10 Faecium KQ 2.6 bacterium powder), gavage once a day, continuous gavage for 90 days. The control group was not treated.

The 90-day animal experiment showed that after ingesting different doses of probiotic powder, the male and female mice had no abnormal mental state, good growth, stable breathing, and normal defecation. The weight changes of the mice fed with different doses for 60 days are shown in Table 7. Show. It can be seen from Table 7 that, compared with the control group, the daily weight change of the probiotic powder group was the smallest. It shows that Enterococcus faecium KQ 2.6 bacteria powder can effectively inhibit the weight gain of mice. Compared with the control group, each group has no abnormal change rule, and there is no significant dose effect among different dose groups (P>0.05).

Table 7 Changes in body weight of mice

Claims (10)

1. Enterococcus faecium (Enterococcus faecium) KQ 2.6, preserved in the China Center for Type Culture Collection, the preservation date is May 12, 2014, the preservation number is CCTCC NO: M 2014197, and the preservation address is Wuhan University, Wuhan, China, postcode 430072 . 2. an application of Enterococcus faecium KQ 2.6 described in claim 1 in the preparation of probiotic preparations. 3. application as claimed in claim 2, it is characterized in that described probiotic preparation comprises pharmaceutical adjuvant and the supernatant that described Enterococcus faecium KQ 2.6 obtains through fermentation culture. 4. application as claimed in claim 3, is characterized in that described pharmaceutical auxiliary material is sodium carboxymethylcellulose or sodium starch glycolate. 5. The application according to claim 3, characterized in that the content of Enterococcus faecium KQ 2.6 in the probiotic preparation is 5×10 ⁷ -5×10 ¹² CFU/mL. 6. The application according to claim 3, wherein the supernatant is prepared according to the following steps: (1) Enterococcus faecium KQ 2.6 is inoculated into the slant culture medium, cultivated at 37°C for 1 day to obtain the slant thalline; The final concentration composition per liter of slant medium: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, Sodium acetate 5.0g, Tween-801.0g, agar 20g, distilled water 1000mL, pH 6.2; (2) Pick the slant bacteria and inoculate it into the fermentation medium, cultivate at 37°C for 12-24 hours to obtain the fermentation medium, and ferment The liquid was centrifuged to obtain the supernatant; the final concentration of the fermentation medium per liter consisted of: peptone 10g, beef extract 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, Tween-801.0g, distilled water 1000mL, pH 6.2. 7. an application of Enterococcus faecium KQ 2.6 described in claim 1 in the preparation of weight-loss drugs. 8. The application according to claim 7, characterized in that the weight loss drug comprises pharmaceutically acceptable adjuvants and the supernatant obtained by fermentation and culture of Enterococcus faecium KQ 2.6 or the bacteria powder after the supernatant is concentrated and dried . 9. The application according to claim 8, characterized in that said pharmaceutically acceptable auxiliary material comprises sodium carboxymethyl cellulose or sodium carboxymethyl starch. 10. The application according to claim 8, characterized in that the content of Enterococcus faecium KQ2.6 in the weight-loss drug is 5×10 ⁷ -5×10 ¹² CFU/g.