CN105112333A - Bifidobacterium longum with good intestinal tract colonizing ability and screening method and application of bifidobacterium longum - Google Patents
Bifidobacterium longum with good intestinal tract colonizing ability and screening method and application of bifidobacterium longum Download PDFInfo
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Abstract
The invention relates to bifidobacterium longum with good intestinal tract colonizing ability. The bifidobacterium longum is named CCFM 729, the bifidobacterium longum is preserved in China General Microbiological Culture Collection Center, which is located at No.3, No.1 courtyard, Beichen West Road, Chaoyang District, Beijing, on May 28th, 2015, and the preservation number of the bifidobacterium longum is CGMCC No. 10932. The bifidobacterium longum has the advantages that the bifidobacterium longum CCFM 729 is initially derived from the excrement of one-week-old infants, in vitro experiments show that the bifidobacterium longum has acid resistance and bile salt resistance, cell experiments show that the bifidobacterium longum can well adhere to colon cancer cells HT-29, animal experiments show that the bifidobacterium longum can colonize in a mouse for 10 days even more than 10 days, and the bifidobacterium longum is good in intestinal tract colonizing ability, huge in application and commercial value, capable of being used to produce bifidobacterium powder, capable of being jointly fermented with streptococcus thermophilus and lactobacillus bulgaricus to produce yogurt, capable of being made into probiotics chocolate and the like, and promising in application prospect.
Description
Technical field
The invention belongs to microbial technology field, especially a kind of bifidus longum bb and purposes with good enteron aisle colonization ability.
Background technology
Bifidus bacillus is as important beneficial bacteria of intestinal tract, it plays an important role in the infection suppressing the growth and breeding of harmful intestinal tract bacteria, opposing cause of disease mattress, bifidus bacillus can produce the organic acids such as acetic acid, propionic acid, butyric acid and lactic acid stimulates intestines peristalsis, promotes defecation, prevents constipation.Bifidus bacillus can also stimulate human immune system, thus improve resistance against diseases, delay senility.But, if bifidus bacillus can play above-mentioned effect, must can in human body long-term existence, namely surely grow.Research finds, part bifidus bacillus has the ability that enteron aisle is grown surely.Such as, S.Fujiwara etc. find that the bifidus longum bb SBT2929 with Streptomycin sulphate and rifampicin resistance surely can grow for a long time and affect composition and the metabolism of enteric microorganism in human body; The discoveries such as NathanP.McNulty, Bifidobacteriumanimalissubsp.lactis can at the aseptic mouse Colonization inside plants more than 28 days taking in high-carbohydrate diet.
Can detection bifidus bacillus mainly contain three kinds: one in enteron aisle method of growing decided at the higher level but not officially announced is utilize the Selective agar medium of bifidus bacillus that enumeration is carried out in the coating of ight soil gradient dilution rear plate; Two is utilize the test kit of API50CHL to transcribe in conjunction with end the qualification that PCR carries out bifidus bacillus in ight soil; Three is the methods utilizing DGGE (denaturing gradient gel electrophoresis), extracts the grand genome of ight soil and then to increase the 16SV3 district of bacterium, carries out band comparison determine that can bifidus bacillus grow enteron aisle is decided at the higher level but not officially announced with target bifidus bacillus.The method of plate count, there is obvious defect: because part bifidus bacillus exists harsher life condition, so can not cultivate on substratum, even therefore also false-negative result can may be there is in this method qualification of the enteron aisle bifidus bacillus grown decided at the higher level but not officially announced; Further, bifidus bacillus not of the same race can not make a distinction by Selective agar medium well.The method of test kit is comparatively novel, compensate for the deficiency that Selective agar medium exists, can carry out considering of bifidus bacillus colonization ability preferably.Meanwhile, the method for PCR-DGGE is relatively ripe, and easy and simple to handle due to technology, can distinguish bifidus bacillus not of the same race well, and generally can find out that from the brightness of band the colonization ability of bifidus bacillus in enteron aisle is strong and weak.
In human intestinal bifidus bacillus quantity with the age growth ratio minimizing, the unbalance meeting of enteric microorganism kind and ratio brings many health problems, such as constipation, diarrhoea, hypoimmunity etc., therefore need to take measures the bifidus bacillus in enteron aisle is bred.The way that in enteron aisle, bifidus bacillus increases is made to have two kinds: one to be the oral drink containing bifidus bacillus or biotechnological formulation; Two is breedings that supplementary bifidus factor (as oligose etc.) promotes the intrinsic bifidus bacillus of human body.In view of WHO is to oligose, as the recommended intake of xylo-oligosaccharide, oligofructose etc., experiment shows that it has some limitations the propagation of bifidus bacillus and effect is not too obvious, and the method effect therefore supplementing bifidus factor is little.The oral drink containing bifidus bacillus or biotechnological formulation, as long as bifidus bacillus can overcome the adverse environment of enteron aisle, just directly can play a role in enteron aisle.So, filter out and can make related products the enteron aisle bifidus bacillus that grows decided at the higher level but not officially announced, there is huge application and commercial value.
By retrieval, not yet find the patent publication us relevant to patent application of the present invention.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, one is provided to have acidproof, bile tolerance characteristic, cell experiment shows that it can adhere to colon cancer cell HT-29 well, experimentation on animals shows that it can in mouse Colonization inside plants 10 days even longer for some time, its good characteristic in enteron aisle colonization ability, have the bifidus longum bb with good enteron aisle colonization ability and the purposes of huge application and commercial value, this bifidus longum bb can be applied to be produced in Bifidobacteria powder, Yoghourt and bifidus bacillus probiotic bacterium chocolate.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of bifidus longum bb with good enteron aisle colonization ability, name is called CCFM729, specific name is Bifidobacteriumlongum, deposit number is: CGMCCNo.10932, preservation date: on 05 28th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
And described bifidus longum bb has following character:
(1) have acid resistance, well-grown under pH3.0-9.0 envrionment conditions, under pH2.5 environment, survival is good;
(2) can bile tolerance, under 0.3% cholate existent condition, survival is good;
(3) there is adhesivity, can stick to preferably on colon cancer cell HT-29;
(4) can in the mouse Colonization inside plants time of more than 10 days.
Have a screening method for the bifidus longum bb of good enteron aisle colonization ability as above, step is as follows:
(1) collect the infant faeces in one week age of some parts of births, by faecal samples enrichment 12h in MRS+0.5 ‰-1 ‰ (mass percent) the halfcystine substratum containing oligofructose;
(2) gradient dilution is coated on the solid plate of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of interpolation 0.02% (mass percent) purpurum bromocresolis, cultivates 24-48h;
(3) the obvious single bacterium colony of picking variable color circle, line obtains single bacterium colony of purifying repeatedly;
(4), by after single bacterium colony liquid culture 24h of purifying, carry out the mensuration of fructose-6-phosphate salt phosphoketolase, Rapid identification its whether be bifidus bacillus;
(5) choose the bacterium of the F6PPK positive, extract its 16SrDNA and check order;
(6) choose milk-acid bacteria that sequencing result is accredited as bifidus bacillus and carry out acidproof, bile tolerance and adherence test;
(7) choose acidproof, bile tolerance and the good bifidus bacillus of adhesion property carries out the test of enteron aisle colonization ability, wherein, acidproof: pH=2 ~ 9, bile tolerance: cholate 0.3%, massfraction, adherence test: there is adhesive capacity to colon cancer cell HT-29;
(8) amplify the 16SV3 district of stool in mice flora, denaturing gradient gel electrophoresis detects the object bacteria in stool in mice, filters out the bifidus bacillus that enteron aisle colonization ability is excellent, must have the bifidus longum bb of good enteron aisle colonization ability.
And concrete steps are as follows:
(1) separation screening of milk-acid bacteria
(1) collect the infant faeces in one week age of some parts of births, by faecal samples enrichment 12h in MRS+0.5 ‰-1 ‰ (mass percent) the halfcystine substratum containing oligofructose;
(2) coat after faecal samples being carried out gradient dilution and with the addition of on the solid plate of MRS+0.5 ‰-1 ‰ halfcystine that mass percent is 0.02% purpurum bromocresolis, cultivate 24-48h;
(3) choose variable color circle obvious, and the single bacterium colony meeting milk-acid bacteria grown form carries out plate streaking purifying, screening and separating goes out milk-acid bacteria;
(4) carry out gramstaining after above-mentioned single bacterium colony being cultivated 24h in liquid MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, choose gram-positive microorganism and carry out follow-up test;
(2) preliminary evaluation of bifidus bacillus: fructose-6-phosphate salt phosphoketolase assay method
By step () screen the milk-acid bacteria obtained cultivate 24h in liquid MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, then get the centrifugal 2min of 1mL culture 8000rpm;
(2) with the 0.05MKH of the pH=6.5 containing 0.05% (mass percent) halfcystine
2pO
4solution washing twice;
(3) be resuspended in the above-mentioned phosphate buffered saline buffer that 200uL with the addition of 0.25% (mass percent) TritonX-100;
(4) add mixed solution that 50uL concentration is 6mg/mL Sodium Fluoride and 10mg/mL sodium iodoacetate and 50uL concentration is the fructose-6-phosphate of 80mg/mL, hatch 1h for 37 DEG C;
(5) to add 300uL concentration be 0.139g/mL, pH be 6.5 oxammonium hydrochloride, and place 10min in room temperature;
(6) add trichoroacetic acid(TCA) and the 4MHCl of 200uL15% (mass percent) respectively;
(7) add the 0.1MHCl that 200uL contains 5% (mass percent) iron trichloride, if system becomes redness rapidly, be the F6PPK positive, tentatively can conclude that it is bifidus bacillus;
(3) molecular biology identification of bifidus bacillus
(1) the genome extracting of single bacterium
A. by step (two) screen the milk-acid bacteria overnight incubation obtained, get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
B., after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
C. 200uLSDS lysate is added, 80 DEG C of water-bath 30min;
D. add phenol-chloroform solution 200uL in cellular lysate liquid, wherein the moiety of phenol-chloroform solution and volume ratio are: the saturated phenol of Tris: chloroform: primary isoamyl alcohol=25:24:1, and after putting upside down mixing, the centrifugal 5-10min of 12000rpm, gets supernatant 200uL;
E. add 400uL ice ethanol or ice Virahol in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing 1h, 12000rpm, abandon supernatant;
F. add the resuspended precipitation of 500uL70% (percent by volume) ice ethanol, the centrifugal 1-3min of 12000rpm, abandons supernatant;
G.60 DEG C oven for drying, or naturally dry;
H.50uLddH
2the heavy molten precipitation of O is in order to PCR;
⑵16SrDNAPCR
A. bacterial 16 S rDNA50uLPCR reaction system:
10 × Taqbuffer, 5uL; DNTP, 5uL; 27F, 0.5uL; 1492R, 0.5uL; Taq enzyme, 0.5uL;
Template, 0.5uL; ddH
2o, 38uL.
B.PCR condition:
95℃5min；95℃10s；55℃30s；72℃30s；step2-430×；72℃5min；12℃2min；
(3) prepare 1% sepharose, PCR primer mixed with 10000 × loadingbuffer afterwards, applied sample amount 5uL, 120V run 30min, then carry out gel imaging;
(4) the PCR primer of 16SrDNA is carried out sequencing analysis, use BLAST in GeneBank, carry out search and similarity comparison the sequence results obtained, choose the milk-acid bacteria that sequencing result is accredited as bifidus bacillus ,-80 DEG C of preservations are for subsequent use;
(4) acidproof, the bile tolerance of bifidus bacillus detect
(1) get the bifidus bacillus of-80 DEG C of preservations in step (three), rule once at the solid plate of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, anaerobism workstation cultivate be transferred to MRS+0.5 ‰-1 ‰ (mass percent) halfcystine after 24-48h liquid tube in the middle of, go down to posterity twice;
(2) be inoculated into the liquid tube of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=7.0 ± 0.2 respectively with the inoculum size of 4%, it is blank group; The liquid tube of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=2, pH=2.5, pH=3, pH=4, pH=5, pH=6, pH=8, pH=9, these several groups is acidproof experimental group; MRS+0.5 ‰-1 ‰ (mass percent) halfcystine+0.1%, 0.2%, 0.3%, in the liquid tube of 0.4% bovine bile, these several groups is bile tolerance experimental group;
By be inoculated in the middle of aforesaid liquid test tube bacteria suspension vibration mixing after, rapidly it is transferred on 96 orifice plates with the amount in 200uL/ hole, often group do three parallel, measured its OD every two hours by microplate reader
600value;
(4) at each time point, experimental group data and control group data are compared, its tolerance performance of the less explanation of data difference is better, and the tolerance comparing bacterium is strong and weak;
(5) because the pH of hydrochloric acid in gastric juice in human intestinal environment is about 2.5, the cholate content of secretion is 0.1% ~ 0.5%, the viable bacteria taken in is by time of enteron aisle at 2 hours, so to be chosen in 2 hours, survival under pH=2.5 environment is good, the good bifidus bacillus that survives under 0.3% cholate existent condition carries out follow-up test;
(5) bifidus bacillus measures the adhesive capacity of colon cancer cell HT-29
(1) the cryopreservation tube of the HT-29 cell be positioned in liquid nitrogen container being placed in rapidly 37 DEG C of water-baths makes it melt;
(2) after it being mixed with PRMI-1640 perfect medium, be placed in culture dish, in 5% CO2gas incubator, 37 DEG C of Secondary Culture, every other day change a nutrient solution;
Wherein, described PRMI-1640 perfect medium is PRMI-1640 basic medium+10% (mass percent) foetal calf serum+1% (mass percent) mycillin;
(3) after passage cell 3-4 time, after again covering with 80% Tissue Culture Plate, trysinization, after PBS cleaning, centrifugal, be 10 with PRMI-1640 basic medium adjustment cell concn
5cells/mL, and 6h is cultivated in the Tissue Culture Plate of inserting slide glass in advance;
By step (four) screen the bifidus bacillus incubated overnight obtained, be 10 by the concentration of bacteria suspension of PRMI-1640 basic medium adjustment incubated overnight
7cfu/mL, with above-mentioned HT-29 co-culture of cells 4h;
(5) PBS cleans 4 times, then fixes 5-10min with methyl alcohol, and the addition of methyl alcohol is advisable to be paved with Tissue Culture Plate, dyes afterwards with Ji's nurse Sa dye liquor;
(6) rinse dye liquor to colourless by PBS washing lotion, be placed to seasoning in 37 DEG C of incubator dried overnight or normal temperature;
(7) take out slide glass, observe under the oily mirror of 100 times, choose the milk-acid bacteria belonging to and there is strong adhesive capacity; Wherein, bacterial count total in the middle of the visual field of 20 random selecting is greater than 100, namely belongs to the bacterium that adhesive capacity is strong;
(6) bifidus bacillus determining in mouse intestinal situation of growing measures
Test mice is 6-8 SPF level female mice in age in week, every day gavage be resuspended in step (five) in physiological saline screen the milk-acid bacteria obtained, bacteria concentration is 10
8cfu/mL, the gavage cycle is 14 days, stool in mice is gathered once before gavage, stool in mice is gathered every other day after terminating with gavage in gavage, extract the ight soil genome of mouse afterwards with FastDNAkitforsoil, the 16SV3 district of amplification gene group, denaturing gradient gel electrophoresis detects the object bacteria in stool in mice, filter out the bifidus bacillus that enteron aisle colonization ability is excellent, the bifidus longum bb of good enteron aisle colonization ability must be had.
The bifidus longum bb as above with good enteron aisle colonization ability is producing the application in Bifidobacteria powder.
And the preparation process of described Bifidobacteria powder is as follows:
(1) the preparation of substratum: the fermention medium of bifidus longum bb is add the MRS substratum that mass percent is the Cys of 0.5 ‰-1 ‰, and pH is 6.8 ± 0.2;
(2) protectant preparation: use water to mix with lyophilized vaccine raw material, preparing containing 100g/L ~ 150g/L skim-milk, 100g/L ~ 200g/L trehalose, 100g/L ~ 150g/L sucrose, surplus is the lyophilized vaccine of water;
(3) step bifidus longum bb CCFM729 bacterial classification being inoculated into sterilizing 20min at 115 DEG C according to the inoculum size of 2-4% (1) in substratum, then under the condition of temperature 37 DEG C, in anaerobism workstation, cultivate 24h, the centrifugal 15min of 6000 × g; Clean 2-3 time with the phosphate buffered saline buffer of pH=7.2; By step, (2) middle lyophilized vaccine is resuspended afterwards, makes the concentration of bacterium reach 10
10cfu/mL; Then this bacteria suspension is cultivated 1h under the condition of 37 DEG C of anaerobism, then at-20 DEG C of pre-freeze 12h; Finally carry out vacuum lyophilization and obtain described Bifidobacteria powder.
There is the bifidus longum bb of good enteron aisle colonization ability as above in the application of producing in Yoghourt or the application in production bifidus bacillus probiotic bacterium chocolate.
And, the composite use of described bifidus longum bb fermentation strain.
And described fermentation strain is thermophilus streptococcus and lactobacillus bulgaricus.
And the preparation process of described Yoghourt is as follows:
(1) first make bifidobacterium yoghurt starter, described bifidobacterium yoghurt starter is Bifidobacteria powder according to claim 6;
(2) the preparation of substratum: the ratio of skimmed milk powder according to 12-15:100 (g/mL) mixed with water, then 105 DEG C of sterilizing 20min, obtain skimming milk;
By step (1) in bifidobacterium yoghurt starter and thermophilus streptococcus, lactobacillus bulgaricus add in step skimming milk (2) according to the ratio that mass ratio is 1:1:1, stir;
(4) ferment 8-12h, and leavening temperature is: 37 DEG C, at 4 DEG C of refrigeration about 8h after having fermented, obtains Yoghourt;
Or the preparation process of described bifidus bacillus probiotic bacterium chocolate is as follows:
(1) first make Bifidobacteria powder, described Bifidobacteria powder is Bifidobacteria powder according to claim 6;
(2) commercially available cocoa powder, theobroma oil, granulated sugar are mixed in proportion and are constantly stirred to paste in hot water;
By step (1) in Bifidobacteria powder join step (2) in gained paste, and to stir rapidly, pour mould into, naturally cooling, obtain probiotic bacterium chocolate.
The advantage that the present invention obtains and beneficial effect are:
1, bifidus longum bb of the present invention (Bifidobacteriumlongum) CCFM729 derives from the infant faeces in one week age of birth at first, experiment in vitro shows that it has acidproof, bile tolerance characteristic, cell experiment shows that it can adhere to colon cancer cell HT-29 well, experimentation on animals shows that it can in mouse Colonization inside plants 10 days even longer for some time, its good characteristic in enteron aisle colonization ability, has huge application and commercial value.
2, bifidus longum bb of the present invention can be applied to and makes Bifidobacteria powder and produce Yoghourt etc. with thermophilus streptococcus, lactobacillus bulgaricus combined ferment, and the Yoghourt made has preferably mouthfeel and quality, has broad application prospects.
3, bifidus longum bb of the present invention can make bacterium powder through fermentation final vacuum lyophilize, and combines with cocoa powder, theobroma oil, granulated sugar etc. and be made into probiotic bacterium chocolate, and the viable count of the bifidus bacillus probiotic bacterium chocolate made reaches 10
9cfu/g, has mouthfeel silk cunning, delicious food concurrently and improves the prebiotic function of intestinal microflora, will have wide market outlook.
Accompanying drawing explanation
Fig. 1 is the colonial morphology figure of bifidus longum bb CCFM729 of the present invention;
Fig. 2 is thalli morphology (1000 ×) figure of bifidus longum bb CCFM729 of the present invention;
Fig. 3 is the growth curve chart of bifidus longum bb CCFM729 of the present invention;
Fig. 4 is the adhesion figure of bifidus longum bb CCFM729 of the present invention to colon cancer cell HT-29;
Fig. 5 is that bifidus longum bb CCFM729 of the present invention determining in mouse intestinal grows situation DGGE schematic diagram, before wherein Pre, gavage, postday2, postday10 and M represent gavage bifidus bacillus respectively, gavage time, gavage terminate 2 days, gavage terminates 10 days and Marker.
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The raw material used in the present invention, if no special instructions, is conventional commercially available prod; The method used in the present invention, if no special instructions, is the ordinary method of this area.
A kind of bifidus longum bb with good enteron aisle colonization ability, name is called CCFM729, specific name is Bifidobacteriumlongum, deposit number is: CGMCCNo.10932, preservation date: on 05 28th, 2015, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center.
Utilize the Microbiological Characteristics such as morphological specificity, cultural colony and physiological and biochemical property and 16SrDNA equimolecular biological means to be accredited as bifidus longum bb (Bifidobacteriumlongum) CCFM729 to this milk-acid bacteria to detect, the biological characteristics of this bifidus longum bb CCFM729 is as follows:
Colony characteristics: well-grown on the substratum of MRS+0.5 ‰ (mass percent) halfcystine, form oyster white, translucent, convex bacterium colony, diameter is about 3mm, bacterium colony smooth surface, moistening, neat in edge, as shown in Figure 1.Cellular form is long and bend, club-like, and part, in dumb-bell shape, without mobility, does not produce gemma, as shown in Figure 2.
Growth characteristics: the minimum growth temperature of this bacterial strain is 20 DEG C, and maximum growth temperature is 45 DEG C, and this bacterial strain is with the growth temperature of 37 DEG C for the best, and the highest and minimum initial growth pH is 9.0 and 3.0, and the initial pH of the most suitable growth is 6.8; The lag period of bifidus longum bb CCFM729 bacterial strain of the present invention is relatively short, and about 4h starts to enter logarithmic phase 12h and just reaches stationary phase.Growth curve as shown in Figure 3.
The above-mentioned bifidus longum bb with good enteron aisle colonization ability is screened from some parts of infant faeces by great many of experiments, and its concrete screening method is as follows:
(1) separation screening of milk-acid bacteria
(1) collect the infant faeces in one week age of some parts of births, by faecal samples enrichment 12h in MRS+0.5 ‰-1 ‰ (mass percent) the halfcystine substratum containing oligofructose;
(2) coat after faecal samples being carried out gradient dilution and with the addition of on the solid plate of MRS+0.5 ‰-1 ‰ halfcystine that mass percent is 0.02% purpurum bromocresolis, cultivate 24-48h;
(3) choose variable color circle obvious, and the single bacterium colony meeting milk-acid bacteria grown form (oyster white, translucent, circular, bacterium colony surface wettability, neat in edge etc.) carries out plate streaking purifying, screening and separating goes out milk-acid bacteria;
(4) carry out gramstaining after above-mentioned single bacterium colony being cultivated 24h in liquid MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, choose gram-positive microorganism and carry out follow-up test.
(2) preliminary evaluation (F6PPK mensuration) of bifidus bacillus
By in step () screen the milk-acid bacteria obtained cultivate 24h in liquid MRS+0.5 ‰-1 ‰ halfcystine, then get the centrifugal 2min of 1mL culture 8000rpm;
(2) with the 0.05MKH of the pH=6.5 containing 0.05% halfcystine
2pO
4wash twice;
(3) be resuspended in the above-mentioned phosphate buffered saline buffer that 200uL with the addition of 0.25%TritonX-100;
(4) add mixed solution that 50uL concentration is 6mg/mL Sodium Fluoride and 10mg/mL sodium iodoacetate and 50uL concentration is the fructose-6-phosphate of 80mg/mL, hatch 1h for 37 DEG C;
(5) to add 300uL concentration be 0.139g/mL, pH be 6.5 oxammonium hydrochloride, and place 10min in room temperature;
(6) add trichoroacetic acid(TCA) and the 4MHCl of 200uL15% respectively;
(7) add the 0.1MHCl that 200uL contains 5% iron trichloride, if system becomes redness rapidly, be the F6PPK positive, tentatively can conclude that it is bifidus bacillus.
(3) molecular biology identification of bifidus bacillus
(1) the genome extracting of single bacterium
A. get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
B., after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
C. 200uLSDS lysate is added, 80 DEG C of water-bath 30min;
D. add phenol-chloroform (the saturated phenol of Tris: chloroform: the volume ratio of primary isoamyl alcohol is 25:24:1) 200uL in cellular lysate liquid, put upside down the rear centrifugal 5-10min of 12000rpm of mixing, get supernatant 200uL;
E. add 400uL ice ethanol (or ice Virahol) in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing about 1h, 12000rpm, abandon supernatant;
F. add the resuspended precipitation of 500uL70% (percent by volume) ice ethanol, the centrifugal 1-3min of 12000rpm, abandons supernatant;
G.60 DEG C oven for drying, or naturally dry;
H.50uLddH
2the heavy molten precipitation of O is in order to PCR.
⑵16SrDNAPCR
A. bacterial 16 S rDNA50uLPCR reaction system:
10 × Taqbuffer, 5uL; DNTP, 5uL; 27F, 0.5uL; 1492R, 0.5uL; Taq enzyme, 0.5uL;
Template, 0.5uL; ddH
2o, 38uL.
B.PCR condition:
95℃5min；95℃10s；55℃30s；72℃30s；step2-430×；72℃5min；12℃2min；
(3) prepare 1% sepharose, PCR primer mixed with 10000 × loadingbuffer afterwards, applied sample amount 5uL, 120V run 30min, then carry out gel imaging.
(4) the PCR primer of 16SrDNA is carried out sequencing analysis, use BLAST in GeneBank, carry out search and similarity comparison the sequence results obtained, choose the milk-acid bacteria that sequencing result is accredited as bifidus bacillus ,-80 DEG C of preservations are for subsequent use.
(4) drafting of bifidus longum bb CCFM729 growth curve and acidproof, bile tolerance detect
Get the bifidus longum bb CCFM729 of-80 DEG C of preservations, it is streak culture on the solid plate of MRS+0.5 ‰ halfcystine.After 37 DEG C of anaerobism workstations cultivate 48 hours, from well-grown bifidus longum bb list bacterium colony, picking bifidobacterium cells to be inoculated in the liquid nutrient medium of MRS+0.5 ‰ halfcystine in anaerobism workstation quiescent culture 36 hours, and culture temperature is 37 DEG C.Then by 2% inoculum size this is activated after bifidus longum bb CCFM729 nutrient solution to be inoculated in the liquid nutrient medium of MRS+0.5 ‰ halfcystine in anaerobism workstation quiescent culture 36 hours, culture temperature is 37 DEG C, OD value under getting 5mL nutrient solution use ultraviolet spectrophotometer survey 600nm every 3 hours also carries out gradient dilution rear plate coating counting, draw the growth curve of bifidus longum bb CCFM729 according to result, its result as shown in Figure 3.As can be seen from Figure 3: bifidus longum bb CCFM729 grows rapidly in the substratum of MRS+0.5 ‰ halfcystine, and lag phase is shorter, enters logarithmic phase greatly after 3h, and about 21h enters stationary phase.Along with the prolongation of incubation time, strain growth produces acid, and pH constantly reduces, and after entering stationary phase, pH downtrending is gradually delayed.At the end of 36h cultivates, the pH value of nutrient solution is 4.12, and in nutrient solution, viable bacteria concentration can reach 5.6 × 10
9cfu/mL.
Acidproof, bile tolerance detects: (1) gets the bifidus bacillus of-80 DEG C of preservations, rule once at the solid plate of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, anaerobism workstation cultivate be transferred to MRS+0.5 ‰-1 ‰ (mass percent) halfcystine after 24-48h liquid tube in the middle of, go down to posterity twice;
(2) liquid tube (blank group) of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=7.0 ± 0.2 is inoculated into respectively with the inoculum size of 4%; The liquid tube (acidproof experimental group) of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=2, pH=2.5, pH=3, pH=4, pH=5, pH=6, pH=8, pH=9; MRS+0.5 ‰-1 ‰ (mass percent) halfcystine+0.1%, 0.2%, 0.3%, in the liquid tube of 0.4% bovine bile (bile tolerance experimental group);
(3) by be inoculated in the middle of aforesaid liquid test tube bacteria suspension vibration mixing after, rapidly it is transferred on 96 orifice plates with the amount in 200uL/ hole, often group do three parallel, measured its OD every two hours by microplate reader
600value;
(4) at each time point, experimental group data and control group data are compared, its tolerance performance of the less explanation of data difference is better, and the tolerance comparing bacterium is strong and weak;
(5) because the pH of hydrochloric acid in gastric juice in human intestinal environment is about 2.5, the cholate content of secretion is about 0.3%, the viable bacteria taken in is by time of enteron aisle at 2 hours, so to be chosen in 2 hours, survival under pH=2.5 environment is good, the good bifidus bacillus that survives under 0.3% cholate existent condition carries out follow-up test.
After testing, this bacterium has acid resistance, well-grown under pH3.0-9.0 envrionment conditions, and under pH2.5 environment, survival is good;
And it can bile tolerance, under 0.3% cholate existent condition, survival is good.
(5) bifidus longum bb CCFM729 measures the adhesive capacity of colon cancer cell HT-29
(1) the cryopreservation tube of the HT-29 cell be positioned in liquid nitrogen container being placed in rapidly 37 DEG C of water-baths makes it melt;
(2), after it being mixed with PRMI-1640 perfect medium (PRMI-1640 basic medium+10% foetal calf serum+1% mycillin), be placed in culture dish, in 5% CO2gas incubator, 37 DEG C of Secondary Culture, every other day change a nutrient solution;
(3) after passage cell 3-4 time, after again covering with (80%) Tissue Culture Plate, trysinization, after PBS cleaning, centrifugal, be 10 with PRMI-1640 basic medium adjustment cell concn
5cells/mL, and 6h is cultivated in the Tissue Culture Plate of inserting slide glass in advance;
By step (four) screen the bifidus bacillus incubated overnight obtained, the concentration of bacteria suspension adjusting incubated overnight with PRMI-1640 is 10
7cfu/mL, with above-mentioned HT-29 co-culture of cells 4h;
(5) PBS cleans 4 times, then fixes 5-10min with methyl alcohol, and the amount of methyl alcohol is advisable to be paved with Tissue Culture Plate, dyes afterwards with Ji's nurse Sa dye liquor;
(6) rinse dye liquor to colourless, in 37 DEG C of incubator dried overnight (or normal temperature is placed to seasoning) by PBS washing lotion;
(7) observe with the oily mirror of 100 times, as shown in Figure 4.As can be seen from Fig. 4 result, bifidus longum bb CCFM729 can adhere to the surface of colon cancer cell HT-29 well, and according to the saying of C.N.JACOBSEN, bifidus longum bb CCFM729 belongs to the milk-acid bacteria with strong adhesive capacity.
(6) bifidus longum bb CCFM729 determining in mouse intestinal situation of growing measures
Test mice is 6-8 SPF level female mice in age in week, and every day, gavage was resuspended in the bifidus longum bb CCFM729 in physiological saline, and bacteria concentration is 10
8cfu/mL.The gavage cycle is 14 days, gathers stool in mice once before gavage, every other day gathers stool in mice after terminating in gavage with gavage.Extract the ight soil genome of mouse afterwards with FastDNAkitforsoil, the 16SV3 district of amplification gene group, then runs DGGE, and result as shown in Figure 5.As can be seen from the result of Fig. 5, bifidus longum bb CCFM729 can't detect before gavage in the ight soil of mouse, can detect in gavage, within 2,4,6,8,10 days after gavage terminates, all can detect, result can be inferred thus, bifidus longum bb CCFM729 can mouse Colonization inside plants at least 10 days, or even longer for some time.
The bifidus longum bb that the present invention has good enteron aisle colonization ability has following character:
(1) have acid resistance, well-grown under pH3.0-9.0 envrionment conditions, under pH2.5 environment, survival is good;
(2) can bile tolerance, under 0.3% cholate existent condition, survival is good;
(3) there is adhesivity, can stick to preferably on colon cancer cell HT-29;
(4) can mouse Colonization inside plants 10 days, the even longer time.
The present invention has the related application of the bifidus longum bb of good enteron aisle colonization ability:
Application examples 1: be applied in the making of Bifidobacteria powder
(1) preparation of substratum: the fermention medium of described bifidus longum bb is the MRS substratum adding mass percent 0.5 ‰ Cys, and the pH of described fermention medium is 6.8 ± 0.2;
(2) protectant preparation: use water and lyophilized vaccine mixed raw material for the lyophilized vaccine that to obtain containing 100g/L skim-milk, 100g/L trehalose, 150g/L sucrose, surplus be water;
(3) step bifidus longum bb CCFM729 bacterial classification being inoculated into sterilizing 20min at 115 DEG C according to the inoculum size of 2-4% (1) in substratum, then under the condition of temperature 37 DEG C, in anaerobism workstation, cultivate 24h, the centrifugal 15min of 6000 × g; Clean 2-3 time with the phosphate buffered saline buffer of pH=7.2; By step, (2) middle lyophilized vaccine is resuspended afterwards, makes the concentration of bacterium reach 10
10cfu/mL; Then this bacteria suspension is cultivated 1h under the condition of 37 DEG C of anaerobism, then at-20 DEG C of pre-freeze 12h; Finally carry out vacuum lyophilization and obtain described Bifidobacteria powder.
(4) through flat board coating counting, the concentration of the Bifidobacteria powder bacterium of gained reaches 10
10cfu/g, vacuum packaging after refrigerating 1 month, viable count still remains on 10
10cfu/g.
Application examples 2: the composite production Yoghourt of the compound strain such as bifidus bacillus and thermophilus streptococcus
(1) first make bifidobacterium yoghurt starter, described bifidobacterium yoghurt starter is Bifidobacteria powder obtained in application examples 1;
(2) the preparation of substratum: the ratio of skimmed milk powder according to 12-15:100 (g/mL) mixed with water, then 105 DEG C of sterilizing 20min, obtain skimming milk;
(3) by step, (1) middle bifidobacterium yoghurt starter and thermophilus streptococcus, lactobacillus bulgaricus add in step skimming milk (2) according to the ratio of 1:1:1;
(4) ferment 8-12h, and leavening temperature is: 37 DEG C, at 4 DEG C of refrigeration about 8h after having fermented, obtains Yoghourt.
(5), through flat board coating counting, the Yoghourt viable count of gained reaches 10
9cfu/g, 2-6 DEG C of refrigeration 7 days, still have preferably mouthfeel, viable count still can reach 10
8cfu/g.
Application examples 3: bifidus bacillus is chocolate for making bifidus bacillus probiotic bacterium
(1) first make Bifidobacteria powder, described Bifidobacteria powder is Bifidobacteria powder according to claim 6;
(2) commercially available cocoa powder, theobroma oil, granulated sugar are constantly stirred to paste by mixed being incorporated in hot water of suitable proportion;
By step (1) in Bifidobacteria powder join in step (2) gained paste, and to stir rapidly, pour mould into, naturally cooling, obtain probiotic bacterium chocolate.
(5) chocolate is crushed also dull and stereotyped coating counting, the chocolate viable count of probiotic bacterium of gained reaches 10
9cfu/g, at 2-6 DEG C of refrigeration two weeks, viable count still can reach 10
8cfu/g.
Claims (10)
1. one kind has the bifidus longum bb of good enteron aisle colonization ability, it is characterized in that: name is called CCFM729, specific name is Bifidobacteriumlongum, deposit number is: CGMCCNo.10932, preservation date: on 05 28th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2. the bifidus longum bb with good enteron aisle colonization ability according to claim 1, is characterized in that: described bifidus longum bb has following character:
(1) have acid resistance, well-grown under pH3.0-9.0 envrionment conditions, under pH2.5 environment, survival is good;
(2) can bile tolerance, under 0.3% cholate existent condition, survival is good;
(3) there is adhesivity, can stick to preferably on colon cancer cell HT-29;
(4) can in the mouse Colonization inside plants time of more than 10 days.
3. there is a screening method for the bifidus longum bb of good enteron aisle colonization ability as claimed in claim 1 or 2, it is characterized in that: step is as follows:
(1) collect the infant faeces in one week age of some parts of births, by faecal samples enrichment 12h in MRS+0.5 ‰-1 ‰ (mass percent) the halfcystine substratum containing oligofructose;
(2) gradient dilution is coated on the solid plate of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of interpolation 0.02% (mass percent) purpurum bromocresolis, cultivates 24-48h;
(3) the obvious single bacterium colony of picking variable color circle, line obtains single bacterium colony of purifying repeatedly;
(4), by after single bacterium colony liquid culture 24h of purifying, carry out the mensuration of fructose-6-phosphate salt phosphoketolase, Rapid identification its whether be bifidus bacillus;
(5) choose the bacterium of the F6PPK positive, extract its 16SrDNA and check order;
(6) choose milk-acid bacteria that sequencing result is accredited as bifidus bacillus and carry out acidproof, bile tolerance and adherence test;
(7) choose acidproof, bile tolerance and the good bifidus bacillus of adhesion property carries out the test of enteron aisle colonization ability, wherein, acidproof: pH=2 ~ 9, bile tolerance: cholate 0.3%, massfraction, adherence test: there is adhesive capacity to colon cancer cell HT-29;
(8) amplify the 16SV3 district of stool in mice flora, denaturing gradient gel electrophoresis detects the object bacteria in stool in mice, filters out the bifidus bacillus that enteron aisle colonization ability is excellent, must have the bifidus longum bb of good enteron aisle colonization ability.
4. the screening method with the bifidus longum bb of good enteron aisle colonization ability according to right 3, is characterized in that: concrete steps are as follows:
(1) separation screening of milk-acid bacteria
(1) collect the infant faeces in one week age of some parts of births, by faecal samples enrichment 12h in MRS+0.5 ‰-1 ‰ (mass percent) the halfcystine substratum containing oligofructose;
(2) coat after faecal samples being carried out gradient dilution and with the addition of on the solid plate of MRS+0.5 ‰-1 ‰ halfcystine that mass percent is 0.02% purpurum bromocresolis, cultivate 24-48h;
(3) choose variable color circle obvious, and the single bacterium colony meeting milk-acid bacteria grown form carries out plate streaking purifying, screening and separating goes out milk-acid bacteria;
(4) carry out gramstaining after above-mentioned single bacterium colony being cultivated 24h in liquid MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, choose gram-positive microorganism and carry out follow-up test;
(2) preliminary evaluation of bifidus bacillus: fructose-6-phosphate salt phosphoketolase assay method
By step () screen the milk-acid bacteria obtained cultivate 24h in liquid MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, then get the centrifugal 2min of 1mL culture 8000rpm;
(2) with the 0.05MKH of the pH=6.5 containing 0.05% (mass percent) halfcystine
2pO
4solution washing twice;
(3) be resuspended in the above-mentioned phosphate buffered saline buffer that 200uL with the addition of 0.25% (mass percent) TritonX-100;
(4) add mixed solution that 50uL concentration is 6mg/mL Sodium Fluoride and 10mg/mL sodium iodoacetate and 50uL concentration is the fructose-6-phosphate of 80mg/mL, hatch 1h for 37 DEG C;
(5) to add 300uL concentration be 0.139g/mL, pH be 6.5 oxammonium hydrochloride, and place 10min in room temperature;
(6) add trichoroacetic acid(TCA) and the 4MHCl of 200uL15% (mass percent) respectively;
(7) add the 0.1MHCl that 200uL contains 5% (mass percent) iron trichloride, if system becomes redness rapidly, be the F6PPK positive, tentatively can conclude that it is bifidus bacillus;
(3) molecular biology identification of bifidus bacillus
(1) the genome extracting of single bacterium
A. by step (two) screen the milk-acid bacteria overnight incubation obtained, get the bacteria suspension 1mL of overnight incubation in 1.5mL centrifuge tube, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
B., after washing thalline with the piping and druming of 1mL sterilized water, the centrifugal 2min of 10000rpm, abandons supernatant and obtains thalline;
C. 200uLSDS lysate is added, 80 DEG C of water-bath 30min;
D. add phenol-chloroform solution 200uL in cellular lysate liquid, wherein the moiety of phenol-chloroform solution and volume ratio are: the saturated phenol of Tris: chloroform: primary isoamyl alcohol=25:24:1, and after putting upside down mixing, the centrifugal 5-10min of 12000rpm, gets supernatant 200uL;
E. add 400uL ice ethanol or ice Virahol in 200uL supernatant ,-20 DEG C of centrifugal 5-10min of standing 1h, 12000rpm, abandon supernatant;
F. add the resuspended precipitation of 500uL70% (percent by volume) ice ethanol, the centrifugal 1-3min of 12000rpm, abandons supernatant;
G.60 DEG C oven for drying, or naturally dry;
H.50uLddH
2the heavy molten precipitation of O is in order to PCR;
⑵16SrDNAPCR
A. bacterial 16 S rDNA50uLPCR reaction system:
10 × Taqbuffer, 5uL; DNTP, 5uL; 27F, 0.5uL; 1492R, 0.5uL; Taq enzyme, 0.5uL; Template, 0.5uL; ddH
2o, 38uL.
B.PCR condition:
95℃5min；95℃10s；55℃30s；72℃30s；step2-430×；72℃5min；12℃2min；
(3) prepare 1% sepharose, PCR primer mixed with 10000 × loadingbuffer afterwards, applied sample amount 5uL, 120V run 30min, then carry out gel imaging;
(4) the PCR primer of 16SrDNA is carried out sequencing analysis, use BLAST in GeneBank, carry out search and similarity comparison the sequence results obtained, choose the milk-acid bacteria that sequencing result is accredited as bifidus bacillus ,-80 DEG C of preservations are for subsequent use;
(4) acidproof, the bile tolerance of bifidus bacillus detect
(1) get the bifidus bacillus of-80 DEG C of preservations in step (three), rule once at the solid plate of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine, anaerobism workstation cultivate be transferred to MRS+0.5 ‰-1 ‰ (mass percent) halfcystine after 24-48h liquid tube in the middle of, go down to posterity twice;
(2) be inoculated into the liquid tube of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=7.0 ± 0.2 respectively with the inoculum size of 4%, it is blank group; The liquid tube of MRS+0.5 ‰-1 ‰ (mass percent) halfcystine of pH=2, pH=2.5, pH=3, pH=4, pH=5, pH=6, pH=8, pH=9, these several groups is acidproof experimental group; MRS+0.5 ‰-1 ‰ (mass percent) halfcystine+0.1%, 0.2%, 0.3%, in the liquid tube of 0.4% bovine bile, these several groups is bile tolerance experimental group;
By be inoculated in the middle of aforesaid liquid test tube bacteria suspension vibration mixing after, rapidly it is transferred on 96 orifice plates with the amount in 200uL/ hole, often group do three parallel, measured its OD every two hours by microplate reader
600value;
(4) at each time point, experimental group data and control group data are compared, its tolerance performance of the less explanation of data difference is better, and the tolerance comparing bacterium is strong and weak;
(5) because the pH of hydrochloric acid in gastric juice in human intestinal environment is about 2.5, the cholate content of secretion is 0.1% ~ 0.5%, the viable bacteria taken in is by time of enteron aisle at 2 hours, so to be chosen in 2 hours, survival under pH=2.5 environment is good, the good bifidus bacillus that survives under 0.3% cholate existent condition carries out follow-up test;
(5) bifidus bacillus measures the adhesive capacity of colon cancer cell HT-29
(1) the cryopreservation tube of the HT-29 cell be positioned in liquid nitrogen container being placed in rapidly 37 DEG C of water-baths makes it melt;
(2) after it being mixed with PRMI-1640 perfect medium, be placed in culture dish, in 5% CO2gas incubator, 37 DEG C of Secondary Culture, every other day change a nutrient solution;
Wherein, described PRMI-1640 perfect medium is PRMI-1640 basic medium+10% (mass percent) foetal calf serum+1% (mass percent) mycillin;
(3) after passage cell 3-4 time, after again covering with 80% Tissue Culture Plate, trysinization, after PBS cleaning, centrifugal, be 10 with PRMI-1640 basic medium adjustment cell concn
5cells/mL, and 6h is cultivated in the Tissue Culture Plate of inserting slide glass in advance;
By step (four) screen the bifidus bacillus incubated overnight obtained, be 10 by the concentration of bacteria suspension of PRMI-1640 basic medium adjustment incubated overnight
7cfu/mL, with above-mentioned HT-29 co-culture of cells 4h;
(5) PBS cleans 4 times, then fixes 5-10min with methyl alcohol, and the addition of methyl alcohol is advisable to be paved with Tissue Culture Plate, dyes afterwards with Ji's nurse Sa dye liquor;
(6) rinse dye liquor to colourless by PBS washing lotion, be placed to seasoning in 37 DEG C of incubator dried overnight or normal temperature;
(7) take out slide glass, observe under the oily mirror of 100 times, choose the milk-acid bacteria belonging to and there is strong adhesive capacity; Wherein, bacterial count total in the middle of the visual field of 20 random selecting is greater than 100, namely belongs to the bacterium that adhesive capacity is strong;
(6) bifidus bacillus determining in mouse intestinal situation of growing measures
Test mice is 6-8 SPF level female mice in age in week, every day gavage be resuspended in step (five) in physiological saline screen the milk-acid bacteria obtained, bacteria concentration is 10
8cfu/mL, the gavage cycle is 14 days, stool in mice is gathered once before gavage, stool in mice is gathered every other day after terminating with gavage in gavage, extract the ight soil genome of mouse afterwards with FastDNAkitforsoil, the 16SV3 district of amplification gene group, denaturing gradient gel electrophoresis detects the object bacteria in stool in mice, filter out the bifidus bacillus that enteron aisle colonization ability is excellent, the bifidus longum bb of good enteron aisle colonization ability must be had.
5. the bifidus longum bb as claimed in claim 1 or 2 with good enteron aisle colonization ability is producing the application in Bifidobacteria powder.
6. the bifidus longum bb with good enteron aisle colonization ability according to claim 5 is producing the application in Bifidobacteria powder, it is characterized in that: the preparation process of described Bifidobacteria powder is as follows:
(1) the preparation of substratum: the fermention medium of bifidus longum bb is add the MRS substratum that mass percent is the Cys of 0.5 ‰-1 ‰, and pH is 6.8 ± 0.2;
(2) protectant preparation: use water to mix with lyophilized vaccine raw material, preparing containing 100g/L ~ 150g/L skim-milk, 100g/L ~ 200g/L trehalose, 100g/L ~ 150g/L sucrose, surplus is the lyophilized vaccine of water;
(3) step bifidus longum bb CCFM729 bacterial classification being inoculated into sterilizing 20min at 115 DEG C according to the inoculum size of 2-4% (1) in substratum, then under the condition of temperature 37 DEG C, in anaerobism workstation, cultivate 24h, the centrifugal 15min of 6000 × g; Clean 2-3 time with the phosphate buffered saline buffer of pH=7.2; By step, (2) middle lyophilized vaccine is resuspended afterwards, makes the concentration of bacterium reach 10
10cfu/mL; Then this bacteria suspension is cultivated 1h under the condition of 37 DEG C of anaerobism, then at-20 DEG C of pre-freeze 12h; Finally carry out vacuum lyophilization and obtain described Bifidobacteria powder.
7. there is the bifidus longum bb of good enteron aisle colonization ability as claimed in claim 1 or 2 in the application of producing in Yoghourt or the application in production bifidus bacillus probiotic bacterium chocolate.
8. the application of bifidus longum bb in production Yoghourt with good enteron aisle colonization ability according to claim 7, is characterized in that: the composite use of described bifidus longum bb fermentation strain.
9. the application of bifidus longum bb in production Yoghourt with good enteron aisle colonization ability according to claim 8, is characterized in that: described fermentation strain is thermophilus streptococcus and lactobacillus bulgaricus.
10. the bifidus longum bb with good enteron aisle colonization ability according to claim 7 is producing the application in Yoghourt, it is characterized in that: the preparation process of described Yoghourt is as follows:
(1) first make bifidobacterium yoghurt starter, described bifidobacterium yoghurt starter is Bifidobacteria powder according to claim 6;
(2) the preparation of substratum: the ratio of skimmed milk powder according to 12-15:100 (g/mL) mixed with water, then 105 DEG C of sterilizing 20min, obtain skimming milk;
By step (1) in bifidobacterium yoghurt starter and thermophilus streptococcus, lactobacillus bulgaricus add in step skimming milk (2) according to the ratio that mass ratio is 1:1:1, stir;
(4) ferment 8-12h, and leavening temperature is: 37 DEG C, at 4 DEG C of refrigeration about 8h after having fermented, obtains Yoghourt;
Or the preparation process of described bifidus bacillus probiotic bacterium chocolate is as follows:
(1) first make Bifidobacteria powder, described Bifidobacteria powder is Bifidobacteria powder according to claim 6;
(2) commercially available cocoa powder, theobroma oil, granulated sugar are mixed in proportion and are constantly stirred to paste in hot water;
By step (1) in Bifidobacteria powder join step (2) in gained paste, and to stir rapidly, pour mould into, naturally cooling, obtain probiotic bacterium chocolate.
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