CN104560820A - Enterococcus faecium KQ2.6 and application thereof - Google Patents
Enterococcus faecium KQ2.6 and application thereof Download PDFInfo
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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
(1) technical field
The present invention relates to a kind of enterococcus faecalis and the application as probiotic bacteria thereof.
(2) background technology
Obesity is that body energy is taken in and consumes unbalance result, and namely Energy intaking is greater than energy expenditure, causes energy to store with the form of fat in vivo, and body weight obviously increases and forms obesity.Under normal circumstances, the absorption of body energy remains relative balance with consumption, and the body weight of human body also keeps relative stability.Once be destroyed, the energy of absorption is more than the consumption of energy, then unnecessary energy is stored up with the form of fat in vivo, accumulates over a long period, and finally obesity occurs.The environment such as fat generation is accustomed to family, personal lifestyle, socio-economic development, culture background about and the bad factor such as dietary habit, hypomotility relevant.
Obesity is a kind of chronic disease of human body, it be the mankind face at present the most out in the cold, but a kind of disease that sickness rate is sharply rising.In China, the number suffering from obesity increases year by year.According to estimates, Chinese existing obese people about 8,000 ten thousand people.The fat worry not only bringing its own shape, causes quality of life to decline.Also a lot of disease has obvious relation to obesity with diabetes, hypertension, hyperlipemia, hyperuricemia, ischemic cardiac disease of brain, cancer, osteoarthrisis deformans knee, irregular menstruation, gestation and abnormal labor etc., and can increase dead danger.Obesity has become China's mainly lethal, cause of disease of disabling.For this reason, eliminate fat disease, become the instant task of medical circle.
Probiotic bacteria is a class by the microecological balance improved in host and then the microorganism formulation of work promoting the single of host health or mixing.Probiotic bacteria is for healthy, and especially the health of intestinal tract is most important.The relation of probiotic bacteria and the mankind is very close, can say " if do not have probiotic bacteria, the health of the mankind is just difficult to ensure, existence is just had any problem ".
Probiotic bacteria can be divided into three major types substantially: (1) lactobacillus class (as bacillus acidophilus, lactobacillus casei, Lactobacillus Jensenii, Raman lactobacillus, Lactobacillus brevis, Lactobacillus bulgaricus etc.); (2) bacillus bifidus class (as bifidobacterium longum, bifidobacterium breve, avette bacillus bifidus, bifidobacterium thermophilum, bifidobacterium adolescentis etc.); (3) gram-positive cocci (as streptococcus faecalis, Lactococcus, intermediary streptococcus etc.).In addition, some saccharomyces cerevisiae, Bu Shi yeast, part mycete and non-lethal escherichia coli are also had also can be included into probiotic bacteria category.The method of probiotic bacteria by repeatedly cultivating in suitably abundant culture medium or selective medium, is separated and obtains from the oral cavity of humans and animals, intestinal contents and feces.Deepening continuously in recent years along with probiotic bacteria research, people have screened from various source and have identified multiple probiotic bacteria.It is reported, China just loses weight one every year will spend compatriots 60,000,000,000 yuan.In order to reduce the financial burden of obese people and the demand to health, need some can treating both the principal and secondary aspects of a disease, economical and practical diet products.Therefore this seminar is from fresh peafowl Excreta, ites is desirable to filter out to have probiotic bacteria that is antibacterial, effect of weight reducing, for the exploitation of probiotic bacteria Related product lays the first stone.
(3) summary of the invention
The object of the invention is to provide a strain new strains--enterococcus faecalis (Enterococcus faecium) KQ2.6, and this bacterial strain or its tunning are as the application of probiotic bacteria.This bacterial strain or its tunning are except having sex pheromones such as suppressing staphylococcus aureus, staphylococcus epidermidis, bacillus subtilis, Klebsiella pneumonia, moscow' paratyphi B, bacillus cereus, micrococcus scarlatinae, Pseudomonas aeruginosa, Candida albicans, also there is the effect suppressing Mouse Weight to increase, can be used for preparing probiotics preparation or slimming medicine.
The technical solution used in the present invention is:
The invention provides a strain new strains--enterococcus faecalis (Enterococcus faecium) KQ 2.6, be preserved in China typical culture collection center, preservation date is on 05 12nd, 2014, deposit number is CCTCC NO:M 2014197, preservation address is Wuhan, China Wuhan University, described enterococcus faecalis KQ 2.6 has the toleration of opposing ambient pressure, and described toleration is antibiotic resistance, sour toleration, pepsin, trypsin resistance or salt toleration.
The present invention also provides a kind of probiotics preparation, and described probiotics preparation comprises the supernatant that pharmaceutic adjuvant and described enterococcus faecalis KQ 2.6 obtain through fermentation culture.
Further, preferred described pharmaceutic adjuvant is sodium carboxymethyl cellulose or carboxymethyl starch sodium.
Further, in described probiotics preparation, the content of enterococcus faecalis KQ 2.6 is 5 × 10
7~ 5 × 10
12cFU/mL.In described probiotics preparation, pharmaceutic adjuvant adds with the form of mass concentration 1% aqueous solution, and more preferably probiotics preparation is: mass concentration 1% sodium carboxymethyl cellulose solution and enterococcus faecalis KQ 2.6 fermented supernatant fluid are mixed into 5 × 10
7~ 5 × 10
12cFU/mL bacteria suspension (preferably 5 × 10
12cFU/mL).
The use amount of probiotics preparation of the present invention is 10
7~ 10
12cFU/kg body weight.
The supernatant that enterococcus faecalis KQ 2.6 of the present invention obtains through fermentation culture is prepared as follows:
(1) slant culture
Enterococcus faecalis KQ 2.6 is seeded to slant medium, cultivates 1 day for 37 DEG C, obtain inclined-plane thalline; Often liter of slant medium final concentration composition: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, agar 20g, distilled water 1000mL, pH 6.2;
(2) fermentation culture
Be seeded to fermentation medium from inclined-plane thalline picking one inoculating loop thalline, cultivate 12 ~ 24 hours for 37 DEG C, obtain fermentation culture, fermentation culture is centrifugal, get supernatant, be enterococcus faecalis KQ2.6 bacterium liquid; Described fermentation medium final concentration consists of: peptone 10g/L, Carnis Bovis seu Bubali cream 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, glucose 20g/L, magnesium sulfate 0.20g/L, manganese sulfate 0.050g/L, yeast leaching powder 4.0g/L, Triammonium citrate 2.0g/L, sodium acetate 5.0g/L, tween 80 1.0g/L, solvent is distilled water, pH 6.2.
The invention still further relates to a kind of described enterococcus faecalis KQ 2.6 and preparing the application in slimming medicine, described slimming medicine comprises the supernatant or the dried mycopowder of supernatant concentration that pharmaceutically acceptable auxiliaries and described enterococcus faecalis KQ 2.6 obtain through fermentation culture.
Described pharmaceutically acceptable auxiliaries comprises sodium carboxymethyl cellulose or carboxymethyl starch sodium.
In described slimming medicine, enterococcus faecalis KQ 2.6 content is 5 × 10
7~ 5 × 10
12cFU/g.
The preferred described slimming medicine of the present invention is: the supernatant that enterococcus faecalis KQ 2.6 obtains through fermentation culture is evaporated to concentration 5.0 × 10
8~ 5.0 × 10
13the concentrated solution of CFU/mL (preferably 5.0 × 10
10cFU/ml), then pharmaceutically acceptable auxiliaries (preferably carboxymethyl cellulose sodium) is mixed homogeneously with concentrated solution, put into freezer dryer-40 DEG C of dryings, prepare bacteria containing amount 5.0 × 10
7~ 5.0 × 10
12the slimming medicine of CFU/g (preferably 5.0 × 10
11cFU/g).
The use amount of slimming medicine of the present invention is 10
7~ 10
12cFU/kg body weight, uses 2 every day, and taking 60 ~ 90 days is a course for the treatment of, takes the 3 ~ 10kg that loses weight for 1 ~ 2 course for the treatment of.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: be separated from peafowl fresh excreta first and obtain a strain new strains--enterococcus faecalis KQ 2.6, this bacterial strain not only has good toleration to ambient pressure, and this bacterial strain or its tunning inhibited to encountered pathogenic microorganism.In addition, mice safety experiment shows that this bacterial strain is safety.This bacterial strain also has the effect suppressing Mouse Weight to increase.The present invention demonstrates this bacterial strain and has a good application prospect in fields such as food.
(4) accompanying drawing explanation
Fig. 1 enterococcus faecalis KQ 2.6 acid resistance.
Fig. 2 enterococcus faecalis KQ 2.6 high-salt tolerance.
Fig. 3 enterococcus faecalis KQ 2.6 pepsin toleration.
Fig. 4 enterococcus faecalis KQ 2.6 trypsin resistance.
Fig. 5 enterococcus faecalis KQ 2.6 adhesion property.
Fig. 6 enterococcus faecalis KQ 2.6 fermented supernatant fluid is to the bacteriostasis of micrococcus scarlatinae.
Fig. 7 mice acute toxicity test organs and tissues is cut into slices: A is liver, and B is kidney, and C is spleen.
The sub-acute toxicity test organs and tissues section of Fig. 8 mice: A is liver, and B is kidney, and C is spleen.
Fig. 9 mice subacute toxicity test shoot formation.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1 strains separation Isolation and characterization
(1) operate
1, strains separation purification
Adopt sterile working, get the test tube that healthy peafowl fresh excreta (1.0g) is placed in physiological saline solution, fully concussion evenly, forms suspension.Get 100 μ L suspensions in 10mL cholate liquid medium, be placed in 37 DEG C and cultivate 24h; Then draw 0.50mL culture fluid in the test tube filling 4.5mL physiological saline solution, this dilution factor is 10
-1, repeat above process and do 10 doubling dilutions, to 10
-5diluted concentration, under drawing each dilution factor, 0.10mL bacterium drop is in 2.0%CaCO
3on-MRS solid medium flat board, after coating evenly, flat board is placed in 37 DEG C and cultivates 24h.The bacterium colony that picking white is circular regular, MRS solid medium carries out line separation and Culture, obtains primary election bacterial strain, be placed in 4 DEG C of Refrigerator stores for subsequent use.
By primary election inoculation in MRS solid medium, after 37 DEG C of cultivation 24h, carry out catalase test.Screening Gram’s staining is positive, the bacterial strain of catalase test feminine gender, and to put up a resistance the evaluation of ambient pressure toleration to the bacterial strain by above-mentioned test, and screening obtains a strain the strongest active bacterial strain, called after bacterial strain KQ 2.6.Bacterial strain KQ 2.6 is transferred in MRS fluid medium, after being put in 37 DEG C of cultivation 24h, get 600 μ L bacterium liquid add be added with 400 μ L, volumetric concentration is mix in the cryopreservation tube of 50% glycerol (sterilizing), cryopreservation tube is put into-20 DEG C of Refrigerator stores, carries out fungi preservation.The present invention will introduce the screening of bacterial strain KQ 2.6, qualification and performance evaluation.
In the method for above-mentioned screening, relate to culture medium as follows:
Often liter of MRS solid medium composition: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, agar 20g, distilled water 1000mL, pH 6.2.
Often liter of MRS fluid medium composition: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, distilled water 1000mL, pH 6.2.
Often liter of 2.0%CaCO
3-MRS solid medium forms: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, CaCO
320g, agar 20g, distilled water 1000mL, pH 6.2.
Often liter of cholate fluid medium composition: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, cholate 5.0g, distilled water 1000mL, pH 3.0.
2, thalline qualification
2.1 thalline Gram stain test
Get clean slide, get a ring sterilized water on slide with inoculating loop, then a small amount of bacterial strain KQ 2.6 of picking, coating is evenly, dry fixing.Drip violet staining liquid, dye one minute, washing, then drip the mordant dyeing of Lushi's iodine liquid, act on one minute, washing, then drip volumetric concentration 95% ethanol water and decolour 30 seconds, finally drip sarranine counterstain liquid and redye 2-3 minute, washing, dries.Under ordinary optical microscope, observe strain morphology and color, if thalline is dyed to bluish violet, be gram positive bacteria, and being dyed to redness is then gram negative bacteria.
Gram’s staining preparation of reagents:
1) preparation of ammonium oxalate crystal violet dyeing liquor
A liquid crystal violet 2.0g, 95% ethanol 20mL; B liquid ammonium oxalate 0.80g, distilled water 80mL mixing A, B liquid, uses after leaving standstill 48h.
2) Lushi's iodine liquid
Iodine tablet 1.0g, potassium iodide 2.0g, distilled water 300mL
First potassium iodide is dissolved in a small amount of water, then iodine tablet is dissolved in liquor kalii iodide, after iodine dissolves completely, add distilled water.
3) sarranine redyes liquid
Sarranine 2.5g, 95% ethanol 100mL
Get the above-mentioned sarranine ethanol 10mL for preparing and 80mL distilled water to mix and form.
2.2 catalase tests: being applied in by a little ring bacterial strain KQ 2.6 to drip has volumetric concentration to be on the slide glass of 3.0% hydrogen peroxide, observes whether alveolate generation.If there is bubble to produce, be positive, it is then negative that bubble-free produces.
2.3 bacterial strain physiological and biochemical tests
2.3.1 hydrogen sulfide production test: picking bacterial strain KQ 2.6 is inoculated in hydrogen sulfide biochemical tube, is put in 37 DEG C and cultivates 24h, if there is black precipitate to produce in biochemical tube, is then positive, otherwise is negative.
2.3.2 nitrate reduction test: get bacterial strain KQ 2.6 and be inoculated in nitrate reduction reaction biochemical tube, cultivate 3 ~ 5 days for 37 DEG C, often prop up in biochemical tube and all drip a Griess reagent A liquid and Griess reagent B liquid, observe culture fluid and whether become red, orange or brown, if there is above-mentioned reaction, illustrate in culture fluid to there is nitrite; If without above-mentioned reaction, then instill 1 ~ 2 diphenylamines reagent, whether observe culture fluid in blue reaction.If in blue reaction, represent in culture fluid also there is nitrate; If not in blue reaction, represent that nitrate and newly-generated nitrite have been reduced into other material all.
2.3.3 indole test: be inoculated in peptone water medium by bacterial strain KQ 2.6, after cultivating 48h, drip 3-4 and drip ether, shake for several times, leaves standstill 1min, after ether rises, slowly adds 2 indole reagent along test tube wall.Between ether and culture, produce red ring for positive, redfree ring is negative.
Often liter of peptone water medium consists of: tryptone 10g, sodium chloride 5.0g, distilled water 1000mL, pH 7.4.
2.3.4 sugar fermentating test: be inoculated into by bacterial strain KQ 2.6 in sugar fermentation pipe culture medium, cultivates 24h for 37 DEG C.What after 24h, bacterium liquid indicator turned yellow is positive, and what indicator was constant is negative.Lactose, maltose, soluble starch, D-Fructose, L-rhamnose, D-MANNOSE, D-xylose, D-galactose, D-cellobiose, glucose, PEARLITOL 25C, sucrose, cottonseed sugar is used to carry out sugar fermentating test in test respectively.
Every glycemic fermentation tube culture medium consists of: Carnis Bovis seu Bubali cream 5.0g, peptone 10g, sodium chloride 3.0g, sodium hydrogen phosphate 2.0g, and 0.20% bromothymol blue solution 12mL, tests and use sugared 50g, distilled water 1000mL, pH 7.4.
2.3.5v-p test: be inoculated in by bacterial strain KQ 2.6 in glucose phosphate salt peptone water biochemical tube, cultivate 24-48h for 37 DEG C, the reaction that takes on a red color is for positive.
2.3.6 citrate utilization test: be inoculated in by bacterial strain KQ 2.6 in citrate biochemical tube, cultivates 1-4 days for 37 DEG C, if culture medium becomes blueness, being positive, becoming green, is then negative.
2.3.7 methyl red test: bacterial strain KQ 2.6 is inoculated in dextrose peptone medium, 30 DEG C of cultivations, from second day (namely after 24h), get culture fluid 1.0mL every day, drip methyl red indicator 1 ~ 2, cerise is positive, pale red is the weak positive, and yellow is negative.Up to finding positive or being still negative to the 5th day, get final product result of determination.
Often liter of dextrose peptone medium consists of: Carnis Bovis seu Bubali cream 5.0g, peptone 10g, sodium chloride 3.0g, sodium hydrogen phosphate 2.0g, 0.20% bromothymol blue solution 12mL, glucose 50g, distilled water 1000mL, pH 7.4.
2.4 bacterial strain 16S rDNA Sequence Identification
The DNA of extracting bacterial strain KQ 2.6 is as pcr amplification template, pcr amplification, primer for 16SrDNA pcr amplification is: forward primer: 5 '-AGAGTTTGATCCTGGCTCAG-' 3, downstream primer: 5 '-GGTTACCTTGTTACGACTT-' 3, obtain 16S rDNA gene order, PCR reaction system is 10 × buffer 5.0 μ L, template DNA 5.0 μ L, Taq archaeal dna polymerase 1.5 μ L, 2.5mM dNTPs 4.0 μ L, forward primer (15pmol) 1.0 μ L, downstream primer (15pmol) 1.0 μ L, ultra-pure water 32.5 μ L, total 50 μ L.PCR reaction condition is: 94 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 58 DEG C of annealing 30s, and 72 DEG C extend 1min, circulate 30 times altogether, and last 72 DEG C extend 10min.After reaction terminates, agarose gel electrophoresis pcr amplification product being carried out to 1.0% detects, and result obtains the band of about 1500bp through amplification.From PCR reactant system, reclaim pcr amplification band, deliver to biotech firm and carry out determined dna sequence.16S rDNA sequence in the gene order and GenBank data base that obtain checking order carries out Blastn comparison and tetraploid rice.
(2) conclusion
1, the qualification of bacterial strain
1.1 morphological features: screen the bacterial strain KQ 2.6 rounded bacterium colony on MRS solid medium obtained, White-opalescent, smooth surface is moistening, neat in edge.Thalline carries out Gram’s staining, thalline globulate, paired or catenation, Gram-positive.
1.2 physio-biochemical characteristics: by as shown in table 1 for bacterial strain KQ 2.6 physio-biochemical characteristics.
Table 1 bacterial strain physio-biochemical characteristics result
Note: "+" is positive, "-" is negative
1.3 bacterial strain 16S rDNA identify: through order-checking, the homology of the 16S rDNA of bacterial strain KQ 2.6 and the 16S rDNA sequence of Enterococcus faecium reaches 100%.
Comprehensive strain morphology is observed, Physiology and biochemistry is identified and 16S rDNA sequencing result, can determine that this bacterial strain KQ 2.6 is for enterococcus faecalis (Enterococcus faecium), called after enterococcus faecalis (Enterococcus faecium) KQ 2.6, be preserved in China typical culture collection center, preservation date is 2014-05-12, deposit number is CCTCC NO:M 2014197, and preservation address is Wuhan, China Wuhan University.
The preparation of embodiment 2 enterococcus faecalis KQ 2.6 fermentation liquid
Enterococcus faecalis KQ 2.6 is seeded to slant medium, cultivates 1 day for 37 DEG C, obtain inclined-plane thalline.Be seeded to fermentation medium from inclined-plane thalline picking thalline KQ 2.6, cultivate 12 ~ 24 hours for 37 DEG C, obtain fermentation culture, fermentation culture is centrifugal, get supernatant, be enterococcus faecalis KQ 2.6 bacterium liquid (concentration 1 ~ 2 × 10
8cFU/mL).Described often liter of fermentation medium final concentration consists of: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, distilled water 1000mL, pH 6.2.
Embodiment 3 enterococcus faecalis KQ 2.6 antibiotic-resistant performance
Example 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid 0.10mL (2 × 10
7cFU/mL) MRS solid plate culture medium is coated, after planar surface is slightly dry, place common antibiotics (tetracycline, vancomycin, gentamycin, kanamycin, neomycin, chloromycetin, erythromycin, to Colistin B, streptomycin, ciprofloxacin, midecamycin, cefazolin sodium, rifampicin, penicillin, ofloxacin) drug sensitive test paper (purchased from Beijing match for thinking biotechnology research and development centre) respectively, be adjacent to media surface, cultivate 24h for 37 DEG C, measure antibacterial circle diameter, obtain this bacterial strain to above-mentioned antibiotic drug resistance.Antibiotic resistance criterion is: be insensitive without inhibition zone, and antibacterial circle diameter <10mm is hypoallergenic, and antibacterial circle diameter 10 ~ 15mm is quick in being, antibacterial circle diameter >15mm is Gao Min, the results are shown in Table shown in 2.
Table 2 enterococcus faecalis KQ 2.6 antibiotic resistance
Note: without inhibition zone be-, antibacterial circle diameter <10mm is+, antibacterial circle diameter 10-15mm is ++, antibacterial circle diameter >15mm is +++.
As shown in Table 2, enterococcus faecalis KQ 2.6 pairs of polymyxin Bs have resistance, to all the other antibiotic non-resistants.Different antibiotic is different on the impact of enterococcus faecalis KQ 2.6, and wherein, tetracycline has the greatest impact to enterococcus faecalis KQ's 2.6, and midecamycin takes second place, and gentamycin is minimum.
Embodiment 4 enterococcus faecalis KQ 2.6 acid resistance
Get clean tube some, add 10mL MRS fluid medium respectively, regulate the pH of culture medium to be 6.2,5.0,4.0,3.0,2.5,2.0,1.0 respectively with 0.10M HCl and 0.10M NaOH, sterilizing.After cooling, access embodiment 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid 100 μ L, fully mixes on the oscillator, cultivates 24h, gets culture fluid and carry out Concentraton gradient dilution, choose 10 for 37 DEG C
-3~ 10
-11the dilution bacterium liquid coating MRS culture medium of dilution factor (10 times of gradients).Take out after MRS culture medium after coating being put into 37 DEG C of cultivations 24h, 24h and carry out colony counting, the results are shown in Figure shown in 1.As shown in Figure 1, enterococcus faecalis KQ 2.6 can under lower pH condition (pH 1.8-3.0) growth and breeding, along with the increase of pH, strain growth ability is more and more stronger.It can thus be appreciated that enterococcus faecalis KQ 2.6 has good acid resistance.
Embodiment 5 enterococcus faecalis KQ 2.6 high-salt tolerance
Get clean tube some, adding sodium chloride mass concentration is respectively 0.90%, 2.0%, 3.0%, 5.0%, 8.0%MRS fluid medium, sterilizing.After cooling, access embodiment 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid 100 μ L, fully mixes on the oscillator, puts into 37 DEG C and cultivates 24h, gets culture fluid and carries out 10 times of Concentraton gradient dilutions, choose 10
-4~ 10
-10dilution bacterium liquid coating MRS solid medium, takes out after 37 DEG C of cultivations 24h, 24h and carries out colony counting, the results are shown in Figure shown in 2.As shown in Figure 2, when salinity is 0.90%, enterococcus faecalis KQ 2.6 is survived the about 10LogCFU/mL of number, and when salinity is 8.0%, this Strain survival number can also maintain 5.0LogCFU/mL.It can thus be appreciated that enterococcus faecalis KQ 2.6 has good salt tolerance.
Embodiment 6 enterococcus faecalis KQ 2.6 pepsin toleration
Getting clean tube some, add 20mL normal saline respectively, is that its pH is adjusted to 2.5, sterilizing by 36 ~ 38% concentrated hydrochloric acid by mass concentration.Pepsin (purchased from Li Rui bio tech ltd, Shanghai, 2500U/mg, final concentration 5.0mg/mL) is added after cooling, access embodiment 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid 100 μ L, fully mixes on the oscillator, cultivates 0 respectively for 37 DEG C, 1,2,3,4,5,6h, gets the cultivation after cultivation and carries out 10 times of Concentraton gradient dilutions, choose 10 night
-7~ 10
-11dilution bacterium liquid coating MRS solid medium, takes out after 37 DEG C of cultivations 24h, 24h and carries out colony counting, the results are shown in Figure shown in 3.As shown in Figure 3, pepsin solution is not very large to the growth effect of enterococcus faecalis KQ 2.6, illustrates that strain has good toleration to pepsin.
Embodiment 7 enterococcus faecalis KQ 2.6 trypsin resistance
Get clean tube some, add 20mL normal saline respectively, sterilizing.Trypsin is added purchased from Dalian Mei Lun Bioisystech Co., Ltd, 2500U/mg, final concentration 10mg/mL) after cooling, access embodiment 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid 100 μ L, fully mixes on the oscillator, cultivates 0 respectively for 37 DEG C, 1,2,3,4,5,6h, gets culture fluid respectively and carries out 10 times of Concentraton gradient dilutions, choose 10
-7~ 10
-11dilution bacterium liquid coating MRS solid medium, takes out after 37 DEG C of cultivations 24h, 24h and carries out colony counting, the results are shown in Figure shown in 4.As shown in Figure 4, enterococcus faecalis KQ 2.6 is containing after cultivating 6h in tryptic culture fluid, and clump count declines about 4.0Log CFU/mL, shows that enterococcus faecalis KQ 2.6 has certain trypsin resistance.
Embodiment 8 enterococcus faecalis KQ 2.6 adhesiveness
Hep-2 cell (purchased from Shanghai Yu Chen Bioisystech Co., Ltd) is placed in the DMEM culture medium (purchased from Bo Sheng bio tech ltd, Shanghai) containing 10% hot deactivation new-born calf serum and dual anti-(penicillin, streptomycin concentration are 100U/mL), in 37 DEG C, 10%CO
2, 95% humidity CO2 gas incubator in hatch, change 1 culture fluid every day, within 3 ~ 4 days, go down to posterity 1 time, be inoculated in by cell after 15 ~ 20 days and include in 6 well culture plates of coverslip, inoculum density is about 4 × 10
4individual/mL, grows to after monolayer until cell and carries out adhesion assay.To grow up to cell Plondrel acid buffer pH 7.4 rinsing 2 times of monolayer, every hole adds 1.0mL embodiment 2 and obtains enterococcus faecalis KQ 2.6 bacterium liquid (concentration 1 ~ 2 × 10
8and the fresh DMEM culture fluid of 1.0mL CFU/mL).Put 37 DEG C, 10%CO
2, 95% humidity CO2 gas incubator in hatch 1h, with phosphate buffer (pH 7.4) the rinsing cell 5 times of sterilizing, then fix with methanol, Gram’s staining, the adhesion situation of basis of microscopic observation thalline, the results are shown in Figure shown in 5.As shown in Figure 5, cell peripheral sticks circle enterococcus faecalis KQ 2.6 (the transparent comparatively fine granularity material namely in figure), this shows that enterococcus faecalis KQ 2.6 has certain adhesiveness, also can be attached on Intestinal epithelial cells in vivo, thus plays its physiological action.
Embodiment 9 enterococcus faecalis KQ 2.6 biocidal property
LB solid plate is coated with the pathogen (specifying information is as shown in table 3) such as staphylococcus aureus, moscow' paratyphi B and bacillus subtilis respectively, after planar surface is slightly dry, punch with the card punch of 3mm, then with a little LB solid medium back cover melted, in each hole, add embodiment 2 respectively obtain enterococcus faecalis KQ 2.6 bacterium liquid, cultivate 20h, observe with or without inhibition zone, the results are shown in Figure shown in 6 and table 4 for 37 DEG C.
Often liter of LB solid medium consists of: yeast extract 10g, tryptone 4.0g, sodium chloride 1.0g, agar 20g, distilled water 1000mL, and pH value is 6.2.
Table 3 the present invention relates to pathogen relevant information
Table 4 enterococcus faecalis KQ 2.6 biocidal property result of the test
Note: to be 0-5mm be antibacterial circle diameter+, to be 6-10mm be antibacterial circle diameter ++, to be 11-15mm be antibacterial circle diameter +++, to be > 16mm be antibacterial circle diameter ++++.
From table 3 and Fig. 6, in enterococcus faecalis KQ 2.6 his-and-hers watches 3,13 kinds of indicator bacterias all have bacteriostasis in various degree, wherein particularly evident to the bacteriostasis of Bacillus cereus, staphylococcus epidermidis, moscow' paratyphi B, micrococcus scarlatinae.
The preparation of embodiment 10 enterococcus faecalis KQ 2.6 slimming medicine
Example 2 obtains enterococcus faecalis KQ 2.6 bacterium liquid and is evaporated to concentration 5.0 × 10
11the concentrated solution of CFU/mL, then mixs homogeneously pharmaceutically acceptable auxiliaries carboxymethyl cellulose (10g) with concentrated solution, puts into freezer dryer-40 DEG C of dryings, prepares enterococcus faecalis KQ 2.6 slimming medicine (bacteria containing amount 5 × 10
10cFU/g).
Embodiment 11 enterococcus faecalis KQ 2.6 safety evaluatio
(1) 50 healthy mices (purchased from Hangzhou Pedagogic University's animal experimental center) random packet is divided into 10 groups, male and female half and half, adaptability is raised after 3 days and is carried out safety evaluatio test.Animal housing light 12h throws light on, and heating ventilation and air-conditioning equipment is good, and room temperature controls at 20 ~ 25 DEG C, and relative humidity is 45 ~ 50%, the results are shown in Figure shown in 7.
As shown in Figure 7, during feeding, experimental mice does not all have obvious abnormal symptom, and the internal organs such as Mouse Liver, kidney and spleen find change all without exception, and each internal organs of pathologic finding are change all without exception also.Illustrate that this enterococcus faecalis KQ 2.6 pairs of male and female mices do not have toxicity.
(2) acute toxicity test in mice: choosing is healthy, adult, about body weight 25g mice 40, male and female half and half.Random packet, often organizes 20, male and female half and half, and the bacteria suspension that test group feeds enterococcus faecalis KQ 2.6 (according to the enterococcus faecalis KQ 2.6 bacterium liquid of embodiment 2 preparation, is mixed into 5.0 × 10 with mass concentration 1.0% carboxymethyl cellulose aqueous solution
12the bacteria suspension of CFU/mL), 1.0% carboxymethyl cellulose aqueous solution of matched group equivalent substitutes bacteria suspension, and feeding volume is 2 × 10
11cFU.Gavage at twice in 24h, Continuous Observation 10 days, the body weight change of every day entry group mice, the mental status, growth and breath state.Put to death mice after 10 days, observe the morphological characteristic of the organs such as Mouse Liver, kidney and spleen.The results are shown in Figure shown in 8.As shown in Figure 8, during feeding, observe the internal organs such as the liver of mice, kidney and spleen and find change all without exception, each internal organs of pathologic finding are change all without exception also.Prove that enterococcus faecalis KQ 2.6 does not have toxicity.
(3) mice subacute toxicity test: choosing is healthy, adult, about body weight 25g mice 40, male and female half and half.(the enterococcus faecalis KQ 2.6 bacterium liquid prepared according to embodiment 2 and mass concentration 1.0% carboxymethyl cellulose aqueous solution are mixed into 1 × 10 to be divided into high dose group at random
11the bacteria suspension of CFU/mL), middle dosage group (according to embodiment 2 prepare enterococcus faecalis KQ 2.6 bacterium liquid and mass concentration 1.0% carboxymethyl cellulose aqueous solution be mixed into 5.0 × 10
10the bacteria suspension of CFU/mL), low dose group (according to embodiment 2 prepare enterococcus faecalis KQ 2.6 bacterium liquid and mass concentration 1.0% carboxymethyl cellulose aqueous solution be mixed into 1.0 × 10
10the bacteria suspension of CFU/mL) and matched group.Often organize 10, male and female half and half.The other gavage 0.10mL of high, medium and low dosage component, every day gavage once, continuous gavage 14 days.Matched group does not process.The body weight change of every day entry group mice, the mental status, growth and breath state.Put to death mice after 14 days, observe the morphological characteristic of the organs such as Mouse Liver, kidney and spleen.The results are shown in Figure shown in 9.12h fasting before mice sudden death, then weighs Mouse Weight.Mice is got liver,spleen,kidney immediately after dissecting and weighs, and calculates each organ index (organ weight/Mouse Weight × 100%).By each group of mice respectively sterile working's eyeball get blood, anticoagulant is diluted, with routine blood test detector, the projects such as Erythrocytes (RBC), total white blood cells (WBC), hemoglobin sum (Hb) are measured, the results are shown in Table shown in 5.
As shown in Figure 9, the enterococcus faecalis KQ 2.6 gavage mice of various dose, its organ index all without appearance obviously fluctuation, illustrates that enterococcus faecalis KQ 2.6 pairs of liver,kidney,spleens have no adverse effects.
Table 5 Mouse Blood is conventional
As shown in Table 5, hemoglobin (Hb) between various dose group, Erythrocytes (RBC), total white blood cells (WBC), and granulocyte, lymphocyte, mononuclear cell percentage ratio there are no significant difference.Routine blood test situation is without exception, shows enterococcus faecalis KQ 2.6 to mouse tissue organ without impact.
Embodiment 12 gavage probiotics bacterial liquid is on the impact of Mouse Weight
Choosing is healthy, adult, about body weight 25g mice 40, male and female half and half.Be divided into high dose group, middle dosage group, low dose group and matched group at random.Often organize 10, male and female half and half.The other gavage 0.10mL (low dosage) of high, medium and low dosage component, 0.50mL (middle dosage), 1.0mL (high dose), concentration is 5 × 10
10(the enterococcus faecalis KQ 2.6 bacterium liquid prepared according to embodiment 2 and mass concentration 1.0% carboxymethyl cellulose aqueous solution are mixed into 5 × 10 to CFU/mL bacteria suspension
10cFU/mL bacteria suspension), every day gavage once, continuous gavage 60 days.Matched group does not process.
Zoopery in 60 days shows, after male and female mice takes in the probiotic bacteria of various dose, the mental status is without exception, upgrowth situation good, it is steady to breathe, defecation is normal, and various dose mice feeds 60 days, and its body weight change feelings are as shown in table 6.As known from Table 6, compared with matched group, probiotic group body weight change every day value is minimum.Illustrate that enterococcus faecalis KQ 2.6 bacterium liquid effectively can suppress the increase of Mouse Weight.Each group does not have abnormal Changing Pattern, without significant dosage effect (P>0.05) between various dose group compared with matched group.
Table 6 Mouse Weight changes
Embodiment 13 gavage probiotic bacteria mycopowder is on the impact of Mouse Weight
Choosing is healthy, adult, about body weight 25g mice 40, male and female half and half.Be divided into high dose group, middle dosage group, low dose group and matched group at random.Often organize 10, male and female half and half.The other gavage 0.10g (low dosage) of high, medium and low dosage component, 0.50g (middle dosage), 1.0g (high dose), concentration is 5 × 10
10cFU/g powder of edible fungus (according to embodiment 10 prepare enterococcus faecalis KQ 2.6 mycopowder), every day gavage once, continuous gavage 90 days.Matched group does not process.
Zoopery in 90 days shows, after male and female mice takes in the probiotic bacteria mycopowder of various dose, the mental status is without exception, upgrowth situation good, it is steady to breathe, defecation is normal, and various dose mice feeds 60 days, and its body weight change feelings are as shown in table 7.As known from Table 7, compared with matched group, probiotic bacteria mycopowder group body weight change every day value is minimum.Illustrate that enterococcus faecalis KQ 2.6 mycopowder effectively can suppress the increase of Mouse Weight.Each group does not have abnormal Changing Pattern, without significant dosage effect (P>0.05) between various dose group compared with matched group.
Table 7 Mouse Weight changes
Claims (10)
1. enterococcus faecalis (Enterococcus faecium) KQ 2.6, be preserved in China typical culture collection center, preservation date is on 05 12nd, 2014, and deposit number is CCTCC NO:M 2014197, preservation address is Wuhan, China Wuhan University, postcode 430072.
2. described in a claim 1, enterococcus faecalis KQ 2.6 is preparing the application in probiotics preparation.
3. apply as claimed in claim 2, it is characterized in that the supernatant that described probiotics preparation comprises pharmaceutic adjuvant and described enterococcus faecalis KQ 2.6 and obtains through fermentation culture.
4. apply as claimed in claim 3, it is characterized in that described pharmaceutic adjuvant is sodium carboxymethyl cellulose or carboxymethyl starch sodium.
5. apply as claimed in claim 3, it is characterized in that the content of enterococcus faecalis KQ 2.6 in described probiotics preparation is 5 × 10
7~ 5 × 10
12cFU/mL.
6. apply as claimed in claim 3, it is characterized in that described supernatant is prepared as follows: enterococcus faecalis KQ 2.6 is seeded to slant medium by (1), cultivate 1 day for 37 DEG C, obtain inclined-plane thalline; Often liter of slant medium final concentration composition: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, agar 20g, distilled water 1000mL, pH 6.2; (2) picking inclined-plane thalline is seeded to fermentation medium, cultivates 12 ~ 24 hours for 37 DEG C, obtains fermentation culture, and fermentation culture is centrifugal, obtains supernatant; Described often liter of fermentation medium final concentration consists of: peptone 10g, Carnis Bovis seu Bubali cream 5.0g, dipotassium hydrogen phosphate 2.0g, glucose 20g, magnesium sulfate 0.20g, manganese sulfate 0.050g, yeast leaching powder 4.0g, Triammonium citrate 2.0g, sodium acetate 5.0g, tween 80 1.0g, distilled water 1000mL, pH 6.2.
7. described in a claim 1, enterococcus faecalis KQ 2.6 is preparing the application in slimming medicine.
8. apply as claimed in claim 7, it is characterized in that described slimming medicine comprises the supernatant or the dried mycopowder of supernatant concentration that pharmaceutically acceptable auxiliaries and described enterococcus faecalis KQ 2.6 obtain through fermentation culture.
9. apply as claimed in claim 8, it is characterized in that described pharmaceutically acceptable auxiliaries comprises sodium carboxymethyl cellulose or carboxymethyl starch sodium.
10. apply as claimed in claim 8, it is characterized in that in described slimming medicine, enterococcus faecalis KQ2.6 content is 5 × 10
7~ 5 × 10
12cFU/g.
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|---|---|---|---|---|
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| US11123378B2 (en) | 2017-05-22 | 2021-09-21 | 4D Pharma Research Limited | Compositions comprising bacterial strains |
| US11123379B2 (en) | 2017-06-14 | 2021-09-21 | 4D Pharma Research Limited | Compositions comprising bacterial strains |
| US11224620B2 (en) | 2016-07-13 | 2022-01-18 | 4D Pharma Plc | Compositions comprising bacterial strains |
| US11266698B2 (en) | 2011-10-07 | 2022-03-08 | 4D Pharma Research Limited | Bacterium for use as a probiotic for nutritional and medical applications |
| CN114901296A (en) * | 2019-12-27 | 2022-08-12 | 国立大学法人神户大学 | Capillary disease inhibitor, capillary disease improver, and capillary neogenesis promoter |
| US11723933B2 (en) | 2014-12-23 | 2023-08-15 | Cj Bioscience, Inc. | Composition of bacteroides thetaiotaomicron for immune modulation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373172B (en) * | 2011-11-03 | 2013-03-20 | 北京龙科方舟生物工程技术有限公司 | Enterococcus faecium and application thereof |
-
2014
- 2014-12-30 CN CN201410850022.7A patent/CN104560820B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373172B (en) * | 2011-11-03 | 2013-03-20 | 北京龙科方舟生物工程技术有限公司 | Enterococcus faecium and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| K SIVIERI等: "Probiotic Enterococcus faecium CRL 183 inhibit chemically induced colon cancer in male Wistar rats", 《EUROPEAN FOOD RESEARCH AND TECHNOLOGY》 * |
| PENG SUN等: "屎肠球菌（SF68）对小鼠免疫功能的影响", 《中国畜牧兽医》 * |
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