CN103849590A - Acid-resistant bifidobacterium breve BB8dpH and application thereof - Google Patents
Acid-resistant bifidobacterium breve BB8dpH and application thereof Download PDFInfo
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Abstract
The invention provides an acid-resistant bifidobacterium breve BB8dpH and an application thereof. The nucleotide sequence of the 16SrRNA gene part of the bifidobacterium breve BB8dpH is shown in SEQ ID NO.1, and is stored with the preservation number of CGMCCNo.8370. The bifidobacterium breve BB8dpH is a strain obtained by separating from the dejecta of healthy young people and further screening under the pH 3.2 acid condition. According to the fermentation and culturing process, anaerobic culture at 37 DEG C is carried out in a BL liquid substrate (containing 0.05% cysteine hydrochloride) for 24 hours. The bifidobacterium breve BB8dpH has remarkably better acid resistance than the common strains, and the acid resistance has genetic stability; the bifidobacterium breve BB8dpH has different percent contents of cell membrane fatty acids from the common strains, the average carbon chain length of the bifidobacterium breve BB8dpH is remarkably longer than that of the common strains, and the cell membrane fluidity of the bifidobacterium breve BB8dpH is remarkably lower than that of the common strains; and the bifidobacterium breve BB8dpH is used in the production field of daily fermented food, health-care food and medicines.
Description
Technical field
The present invention relates to microorganism, be specifically related to the acidproof bifidobacterium breve BB8dpH of a strain and application thereof.
Background technology
Various bacteriums in human gastrointestinal tract field planting, can be divided into probiotics, harmful bacteria and neutral bacterium according to it to the impact of HUMAN HEALTH.Probiotics has the probiotic bacterium of prebiotic effect to human body, wherein bifidus bacillus and Bacterium lacticum are the most representative probiotic bacteriums that is colonizated in human gastrointestinal tract.In the time that accounting for human gastrointestinal tract advantage quantity, probiotic bacterium is conducive to HUMAN HEALTH.
Bifidus bacillus belongs to gram-positive microorganism, is a kind of obligatory anaerobic bacteria, without movement properties, does not produce gemma, without pod membrane and flagellum in growth cycle.
Bifidus bacillus is very responsive to the variation of environment pH.Its most suitable growth pH is 6.7～7.0, once higher than 8.0 or lower than 5.0, bifidus bacillus will stop growing.When under low pH condition, can cause a large amount of death of bacterial cell, particularly the non-constant of humanized bifidus bacillus acid tolerance.
Bifidus bacillus is also very sensitive to the variation of envrionment temperature.When temperature is during between 37～41 ℃, grow the rapidest, when temperature is higher than 43 ℃ or during lower than 28 ℃, its growth is extremely slowly or stop growing.In production application, the optimum temperuture of bifidus bacillus is 35～40 ℃.
Bifidus bacillus is as important probiotic bacterium, it has multiple potential physiological function to human body, as regulating intestinal canal colony balance, anticancer antitumor, suppress pathogenic bacterium, anti-inflammatory, the diarrhoea that anti-ageing, prevention is caused by microbiotic, reduce cholesterol, alleviate the functions such as lactose intolerance and enhancing body immunizing power.
At present, along with going deep into of bifidus bacillus functional study, its application in the new product developments such as daily leavened food, protective foods and medicine is more and more.And bifidus bacillus is as important probiotic bacterium, many national legislations require the viable count in probiotic foods within the quality guaranteed period, must reach 106～108CFU/ml (g).In order to make it in the effectively prebiotic function of performance of human gastrointestinal tract, bifidus bacillus must have good storage stability and to GI tolerance (as acid resistance etc.) performance.Therefore, to adverse environment (as gastrointestinal tract environment) adaptability, stronger humanized bifidus bacillus strain is applicable important potential bacterial strain.
Summary of the invention
Object of the present invention, exactly for strain bifidobacterium breve BB8dpH and an application thereof is provided, bifidobacterium breve BB8dpH has the acid resistance of advantage, and acid resistance has genetic stability, and there is different cell membrane fat acid content per-cent and low cell membrane fluidity, can be used as potential probiotic strain.
Bifidobacterium breve BB8dpH of the present invention has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18th, 2013, be called for short CGMCC, address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica; Preservation registration number is CGMCC No.8370.Its Classification And Nomenclature is: bifidobacterium breve, latin name: Bifidobacterium breve.
To achieve these goals, technical scheme of the present invention is: the acidproof bifidobacterium breve of a strain (Bifidobacterium breve) BB8dpH, its preservation registration number is CGMCC No.8370, and its 16S rRNA Gene Partial nucleotide sequence is as shown in SEQ ID NO.1.
Above-mentioned bifidobacterium breve BB8dpH has following characteristic:
(1) acid resistance of advantage
After pH3.2 processes 3h, the logarithmic value log CFU/ml=of every milliliter of bacterium liquid viable count is 8.38 ± 0.35, and its acid resistance is obviously better than common bacterial strain;
(2) acid resistance has genetic stability
The bacterial strain BB8dpH newly screening is after pH3.2 processes 3h, logarithmic value log CFU/ml=8.62 ± 0.51 of its every milliliter bacterium liquid viable count, continuous passage 20 times is also processed after 3h logarithmic value log CFU/ml=9.11 ± 0.04 of its every milliliter bacterium liquid viable count through pH3.2;
(3) different cell membrane fat acid content per-cent
The C16 of the bacterial strain BB8dpH newly screening: 0 and C16: 1 fatty acid content per-cent is respectively 13.190 ± 0.802 and 3.705 ± 0.215, significantly lower than common bacterial strain; And C18: 0, C18: 1 and cycC19: 0 fatty acid content per-cent is respectively 6.307 ± 0.893,61.160 ± 0.932 and 10.699 ± 0.582, is significantly higher than common bacterial strain; Average carbon number is 17.496 ± 0.091, is significantly longer than common bacterial strain;
(4) there is lower cell membrane fluidity
Microviscosity r=0.144 ± 0.001 of the bacterial strain BB8dpH cytolemma newly screening, apparently higher than common bacterial strain, cell membrane fluidity is starkly lower than common bacterial strain.
The acquisition methods of above-mentioned bifidobacterium breve BB8dpH comprises the following steps:
A, 5 grams of fresh Healthy Youth human faecal mass are vibrated into ight soil homogenate with 50ml ight soil diluent, by in pH7.4BL liquid nutrient medium fresh 1ml ight soil homogenate access 9ml, 37 ℃ of anaerobism are cultivated 24h, with transfering loop picking bacterium liquid at the flat lining out of the TPY of pH6.5 solid medium, 37 ℃ of anaerobism are cultivated after 24h, picking list colony inoculation is in the fresh pH7.4BL liquid nutrient medium of 50ml, and 37 ℃ of anaerobism incubated overnight are to plateau, and the centrifugal 10min of 7600g obtains thalline; Thalline is transferred in the fresh BL liquid nutrient medium of pH3.2, after 37 ℃ of anaerobism incubated overnight, the centrifugal 10min of 7600g obtains thalline again;
B, use again after the phosphoric acid buffer washed twice of pH7.4, be resuspended in the phosphoric acid buffer of 500 μ l pH7.4, then coat in the TPY solid medium of pH6.5,37 ℃ of anaerobism are cultivated 3～4 days to growing single bacterium colony, picking list bacterium colony carries out microscopy, confirm to reach bacterial strain Pure strain separation, and this bacterial strain of Molecular Identification is bifidobacterium breve, called after bifidobacterium breve BB8dpH;
C, bifidobacterium breve BB8dpH is inoculated in fresh pH7.4BL liquid nutrient medium with 1% inoculum size, 37 ℃ of anaerobism are cultivated 24h, obtain the fermented liquid of bifidobacterium breve BB8dpH bacterial strain.
In described BL liquid nutrient medium, TPY solid medium and phosphoric acid buffer, all contain weight percentage and be 0.05% cysteine hydrochloride.
Above-mentioned bifidobacterium breve BB8dpH bacterial strain is for the production field of daily leavened food, protective foods and medicine.
The acid tolerance performance of bifidobacterium breve BB8dpH of the present invention is significantly better than common bacterial strain, and acid resistance has genetic stability, has different cell membrane fat acid content per-cent and lower cell membrane fluidity.
Accompanying drawing explanation
Fig. 1 is the genetic stability analysis diagram of the acid resistance of bifidobacterium breve BB8dpH advantage.
Embodiment
The acquisition of bifidobacterium breve BB8dpH of the present invention: separate and further screen the bacterial classification obtaining from healthy young people fresh excreta under pH3.5 acidic conditions.Gramstaining is positive, and obligate anaerobic, without movement properties, does not produce gemma, without pod membrane and flagellum in growth cycle.
This bifidobacterium breve on October 18th, 2013 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC (address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica) preservation, preservation registration number is CGMCC No.8370.Its Classification And Nomenclature is: bifidobacterium breve, latin name: Bifidobacteriumlongum.
Further illustrate the present invention below in conjunction with embodiment and accompanying drawing 1:
Embodiment 1: the screening of bacterial strain and acquisition
5 grams of fresh Healthy Youth human faecal mass are vibrated into homogenate with 50ml ight soil diluent, by in pH7.4BL liquid nutrient medium (containing 0.05% cysteine hydrochloride) test tube fresh 1ml ight soil homogenate access 9ml, 37 ℃ of anaerobism are cultivated 24h, with transfering loop picking bacterium liquid at the TPY of pH6.5 (containing 0.05% cysteine hydrochloride) the flat lining out of solid medium, 37 ℃ of anaerobism are cultivated after 24h, picking list colony inoculation is in the fresh pH7.4BL liquid nutrient medium of 50ml (containing 0.05% cysteine hydrochloride), 37 ℃ of anaerobism incubated overnight are to plateau, the centrifugal 10min of 7600g obtains thalline, again thalline is transferred to pH value and is 3.2 fresh in 0.05% cysteine hydrochloride BL liquid nutrient medium, after 37 ℃ of anaerobism incubated overnight, the centrifugal 10min of 7600g obtains thalline, again with containing after phosphoric acid buffer (pH7.4) washed twice of 0.05% cysteine hydrochloride, be resuspended in 500 μ l containing in the phosphoric acid buffer (pH7.4) of 0.05% cysteine hydrochloride, then coat in TPY (containing the 0.05% cysteine hydrochloride) solid medium of pH6.5, 37 ℃ of anaerobism are cultivated 3～4 days to growing single bacterium colony, picking list bacterium colony carries out microscopy, confirm to reach bacterial strain Pure strain separation, and this bacterial strain of Molecular Identification is bifidobacterium breve, called after bifidobacterium breve BB8dpH.
Embodiment 2: the microscopy of bacterial strain
Under aseptic condition, transfering loop picking bifidobacterium breve BB8dpH, is applied on slide glass, and film-making gramstaining, examine under a microscope thalli morphology.
Embodiment 3: strain fermentation
Bifidobacterium breve BB8dpH is inoculated in fresh pH7.4BL liquid nutrient medium (containing 0.05% cysteine hydrochloride) with 1% inoculum size, and 37 ℃ of anaerobism are cultivated 24h, obtain the fermented liquid of bifidobacterium breve BB8dpH bacterial strain.
Embodiment 4: the acid resistance of bifidobacterium breve BB8dpH advantage
The bacterial strain of pending acid resistance evaluation is inoculated in fresh pH7.4BL liquid nutrient medium (containing 0.05% cysteine hydrochloride) with 1% inoculum size, and go down to posterity 2 times, the 3rd generation fermented liquid cultivate 18h to plateau 37 ℃ of anaerobism, getting 1ml bacterium liquid is transferred in 1.5ml centrifuge tube, the centrifugal 10min of 7600g obtains thalline, by in the thalline fresh BL liquid nutrient medium that to be transferred to 10ml pH value be 3.2 (containing 0.05% cysteine hydrochloride), respectively at 0h and 3h, after 37 ℃ of anaerobism are cultivated, get 100 μ l bacterium liquid with coating containing the dilution bacterium liquid of getting 100 μ l after phosphoric acid buffer (pH7.4) gradient dilution of 0.05% cysteine hydrochloride in TPY (containing 0.05% cysteine hydrochloride) solid medium, 37 ℃ of anaerobism are cultivated enumeration after 3～4 days, and statistics is cultivated the cell concentration after 0h and 3h in the BL of pH3.2 liquid nutrient medium (containing 0.05% cysteine hydrochloride).The present embodiment adopts the separation of laboratory preservation from the bifidobacterium breve BBM of commercially produced product bacterial strain in contrast.The acid resistance of the BB8dpH advantage of bifidobacterium breve is as shown in table 1.
The acid resistance (pH=3.5 processes 4h) of table 1 bifidobacterium breve BB8dpH advantage
From table 1, bifidobacterium breve BB8dpH of the present invention is compared with commercially produced product bifidobacterium breve BBM, and its acid resistance has a clear superiority in.
Embodiment 5: the acid resistance of bifidobacterium breve BB8dpH advantage has genetic stability
To bifidobacterium breve BB8dpH bacterial strain 20 generations of continuous passage, and from the 3rd generation in 20 generations of continuous passage to the acid resistance of every generation evaluate.Bacterial strain to be evaluated is inoculated in the pH7.4BL liquid nutrient medium that 10ml is fresh (containing 0.05% cysteine hydrochloride) with 1% inoculum size, 37 ℃ of anaerobism are cultivated, and go down to posterity 2 times, the 3rd generation fermented liquid cultivate 18h to plateau 37 ℃ of anaerobism, getting 1ml bacterium liquid is transferred in 1.5ml centrifuge tube, the centrifugal 10min of 7600g obtains thalline, thalline is transferred in the fresh BL liquid nutrient medium (containing 0.05% cysteine hydrochloride) of 10ml pH value 3.2, respectively at 0h and 3h, after 37 ℃ of anaerobism are cultivated, get the bacterium liquid of 100 μ l with coating containing the dilution bacterium liquid of getting 100 μ l after phosphoric acid buffer (pH7.4) gradient dilution of 0.05% cysteine hydrochloride in TPY (containing 0.05% cysteine hydrochloride) solid medium, 37 ℃ of anaerobism are cultivated enumeration after 3-4 days, and calculate in the BL of pH3.2 liquid nutrient medium (containing 0.05% cysteine hydrochloride) and cultivate the cell concentration after 0h and 3h.
As seen from Figure 1, the advantage acid resistance of bifidobacterium breve BB8dpH bacterial strain of the present invention has genetic stability.
Embodiment 6: bifidobacterium breve BB8dpH cell membrane fat acid compositional analysis
The bacterial strain of pending fatty acid analysis 1% inoculum size is inoculated in fresh pH7.4BL liquid nutrient medium (containing 0.05% cysteine hydrochloride), and go down to posterity 2 times, the 3rd generation fermented liquid cultivate 18h to plateau 37 ℃ of anaerobism, get 200ml bacterium liquid in 7600g, 4 ℃ of centrifugal 20min, again with sterile distilled water washing 2 times, carry out lyophilize after thalline being refrigerated to-20 ℃ and obtain bacterium powder.Pour in test tube broken bacterium grinding again, after adding the 2ml volume ratio benzene/sherwood oil of 1: 1 in test tube, mix, adding 2ml mass volume ratio is after potassium hydroxide/methyl alcohol of 2.25% mixes again, test tube is placed in after 40 ℃ of water-bath 30min, after adding again 5ml distilled water, leave standstill 10min after layering obviously, get upper organic phase in brown sample bottle, when fatty acid analysis, sample size is 1 μ l.Fatty acid analysis carries out in HP6890 gas chromatograph, capillary column be Agilent company produce J & WHP-88 (30 × 0.25mm × 0.25 μ m).Furnace temperature program is: starting temperature is 80 ℃, maintains 5min, then maintains 2min after rising to 100 ℃ with 2 ℃/min, maintains 5min after then rising to 160 ℃ with 1 ℃/min.Carrier gas is nitrogen, and injector temperature and detector temperature are respectively 250 ℃ and 280 ℃.Fatty acid component by with standard substance go out peak retention time relatively come to determine.Lipid acid average carbon number calculates: average carbon number=∑ FAP × C/100%.The degree that wherein FAP is lipid acid, C is carbon atom number.The present embodiment adopts the separation of laboratory preservation from the bifidobacterium breve BBM of commercially produced product bacterial strain in contrast.The BB8dpH cell membrane fat acid content per-cent of bifidobacterium breve is as shown in table 2.
Table 2 bifidobacterium breve BB8dpH cell membrane fat acid content per-cent
* represent p < 0.05, the C16 of 2 strain bacterial strains is described: 0, C16: 1, C18: 0, C18: 1, cycC19: 0 fatty acid content per-cent and average carbon number exist significant difference.
From table 2, its C16 of bifidobacterium breve BB8dpH bacterial strain: 0 and C16: 1 fatty acid content per-cent is respectively 13.190 ± 0.802 and 3.705 ± 0.215, significantly lower than control strain; And C18: 0, C18: 1 and CycC19: 0 fatty acid content per-cent is respectively 6.307 ± 0.893,61.160 ± 0.932 and 10.699 ± 0.582, is significantly higher than control strain; Average carbon number is 17.496 ± 0.091, is significantly longer than control strain.
Embodiment 7: bifidobacterium breve BB8dpH has lower cell membrane fluidity
The bacterial strain of pending fatty acid analysis 1% inoculum size is inoculated in fresh pH7.4BL liquid nutrient medium (containing 0.05% cysteine hydrochloride), and go down to posterity 2 times, the 3rd generation fermented liquid cultivate 18h to plateau 37 ℃ of anaerobism, get bacterium liquid and regulate concentration to OD
450=0.25, with after 37 ℃ of fixing 1h of 0.25% formaldehyde, phosphoric acid buffer (pH7.4) washed twice containing 0.25% formaldehyde for thalline, bacterial cell is with 5 × 10
-6m1.6-phenylbenzene-1,3,5-hexatriene (DPH) is bathed mark 1h in 37C temperature, uses fluorescent spectrophotometer assay fluorescence intensity, adds polarising means.Excitation wavelength 360nm, emission wavelength 430nm, slit is 6nm.The calculating of microviscosity r value: r=(I
vv-G I
vh)/(I
vv+ 2G I
vh), wherein I
vvand I
vhrepresent respectively: the polarized light that exciting light is vertical direction, and the fluorescence intensity that utilizing emitted light records while being respectively the polarized light of vertical and horizontal direction; G is grating improvement factor (G=I
hv/ I
hh), I
hvand I
hhrepresent that respectively exciting light is the polarized light of horizontal direction, and the fluorescence intensity that utilizing emitted light records while being respectively the polarized light of vertical and horizontal direction.The larger explanation cell membrane fluidity of microviscosity r value is lower.The present embodiment adopts the separation of laboratory preservation from the bifidobacterium breve BBM of commercially produced product bacterial strain in contrast.The cell membrane fluidity that the BB8dpH of bifidobacterium breve is lower is as shown in table 3.
Table 3 bifidobacterium breve BB8dpH has lower cell membrane fluidity
* p < 0.05 illustrates the acid resistance significant difference of 2 strain bacterial strains.
From table 3, bifidobacterium breve BB8dpH of the present invention, compared with commercially produced product bifidobacterium breve BBM, has lower cell membrane fluidity.
Claims (5)
1. the acidproof bifidobacterium breve of a strain (Bifidobacterium breve) BB8dpH, its preservation registration number is CGMCC No.8370, its 16S rRNA Gene Partial nucleotide sequence is as shown in SEQ ID NO.1.
2. bifidobacterium breve BB8dpH as claimed in claim 1, is characterized in that, described bifidobacterium breve BB8dpH has following characteristic:
(1) acid resistance of advantage
After pH3.2 processes 3h, the logarithmic value log CFU/ml=of every milliliter of bacterium liquid viable count is 8.38 ± 0.35, and its acid resistance is obviously better than common bacterial strain;
(2) acid resistance has genetic stability
The bacterial strain BB8dpH newly screening is after pH3.2 processes 3h, logarithmic value log CFU/ml=8.62 ± 0.51 of its every milliliter bacterium liquid viable count, continuous passage 20 times is also processed after 3h logarithmic value log CFU/ml=9.11 ± 0.04 of its every milliliter bacterium liquid viable count through pH3.2;
(3) different cell membrane fat acid content per-cent
The C16 of the bacterial strain BB8dpH newly screening: 0 and C16: 1 fatty acid content per-cent is respectively 13.190 ± 0.802 and 3.705 ± 0.215, significantly lower than common bacterial strain; And C18: 0, C18: 1 and cycC19: 0 fatty acid content per-cent is respectively 6.307 ± 0.893,61.160 ± 0.932 and 10.699 ± 0.582, is significantly higher than common bacterial strain; Average carbon number is 17.496 ± 0.091, is significantly longer than common bacterial strain;
(4) there is lower cell membrane fluidity
Microviscosity r=0.144 ± 0.001 of the bacterial strain BB8dpH cytolemma newly screening, apparently higher than common bacterial strain, cell membrane fluidity is starkly lower than common bacterial strain.
3. the acquisition methods of bifidobacterium breve BB8dpH described in claim 1, is characterized in that, comprises the following steps:
A, 5 grams of fresh Healthy Youth human faecal mass are vibrated into ight soil homogenate with 50ml ight soil diluent, by in pH7.4BL liquid nutrient medium fresh 1ml ight soil homogenate access 9ml, 37 ℃ of anaerobism are cultivated 24h, with transfering loop picking bacterium liquid at the flat lining out of the TPY of pH6.5 solid medium, 37 ℃ of anaerobism are cultivated after 24h, picking list colony inoculation is in the fresh pH7.4BL liquid nutrient medium of 50ml, and 37 ℃ of anaerobism incubated overnight are to plateau, and the centrifugal 10min of 7600g obtains thalline; Thalline is transferred in the fresh BL liquid nutrient medium of pH3.2, after 37 ℃ of anaerobism incubated overnight, the centrifugal 10min of 7600g obtains thalline again;
B, use again after the phosphoric acid buffer washed twice of pH7.4, be resuspended in the phosphoric acid buffer of 500 μ l pH7.4, then coat in the TPY solid medium of pH6.5,37 ℃ of anaerobism are cultivated 3～4 days to growing single bacterium colony, picking list bacterium colony carries out microscopy, confirm to reach bacterial strain Pure strain separation, and this bacterial strain of Molecular Identification is bifidobacterium breve, called after bifidobacterium breve BB8dpH;
C, bifidobacterium breve BB8dpH is inoculated in fresh pH7.4BL liquid nutrient medium with 1% inoculum size, 37 ℃ of anaerobism are cultivated 24h, obtain the fermented liquid of bifidobacterium breve BB8dpH bacterial strain.
4. the acquisition methods of bifidobacterium breve BB8dpH as claimed in claim 3, is characterized in that, all contains weight percentage and be 0.05% cysteine hydrochloride in described BL liquid nutrient medium, TPY solid medium and phosphoric acid buffer.
5. the application of bifidobacterium breve BB8dpH as claimed in claim 1, is characterized in that: described bifidobacterium breve BB8dpH bacterial strain is for the production field of daily leavened food, protective foods and medicine.
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