CN103865844B - Bacteroides uniformis L8 and the application in degraded agar-agar or agaropectin oligose - Google Patents
Bacteroides uniformis L8 and the application in degraded agar-agar or agaropectin oligose Download PDFInfo
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Abstract
The invention discloses a strain new strains--bacteroides uniformis (Bacteroides? uniformis) L8 and the application in degraded agar-agar or agaropectin oligose, molecular weight can be that the agar-agar of 100 ~ 300kDa is degraded to an agaropectin oligose step of going forward side by side and is degraded to D-semi-lactosi by this bacterial strain, or the agaropectin oligose of molecular weight 0.5 ~ 10kDa is finally degraded to D-semi-lactosi, does agaropectin oligose has specificity simultaneously promote Bacteroides? the effect of uniformis growth, thus reach the related indication effect regulating obesity to cause.
Description
(1) technical field
The present invention relates to the degradation bacteria strains of a kind of agar-agar or agaropectin oligose, the particularly application of bacteroides uniformis (Bacteroidesuniformis) L8 in degraded agar-agar or agaropectin oligose.
(2) background technology
Agar-agar is the polysaccharide in a kind of marine red alga source, primarily of (l → 3)-β-D-semi-lactosi (calling G in the following text) and (1 → 4)-3,6-inner ether-α-L-semi-lactosi (calling A in the following text) is alternately formed by connecting, and agar-agar is widely used in food service industry because of its special gel property as foodstuff additive.Agar-agar can obtain the relatively low agaropectin oligose of molecular weight (calling AO in the following text) through enzyme liberating or acid degradation, recent studies have found that agaropectin oligose has the prebiotic effect promoting bifidobacterium growth in enteron aisle.
To live away from home in the gi tract of Healthy People miscellaneous intestinal microflora, be approximately 10
14left and right.Intestinal microflora and being in all the time in dynamic balance state between host and environment, forms one and depends on each other for existence, and the system of restriction mutually, thus when body defenses function is normal, it is useful and harmless to human body.Normal the gut flora has many important physiological functions, as the regulating effect to host's diabetes, hypertension, hyperlipidemia, immunization, toxin expelling effect and antitumor action etc., but once there is alteration of intestinal flora, these normal physiological functions will certainly be upset.
Edible probiotic bacterium can reach the object maintaining gastrointestinal health, and the probiotic bacterium that current China ratifies to use in food has more than 20 to plant, mainly Bacterium lacticum and bifidus bacillus.Research display probiotic bacterium has multiple physiological hygiene function, comprises cellular immunization promoter action, process lactose intolerance, reduces serum cholesterol, regulate human intestinal microflora balance, antianaphylaxis, suppress carcinogenic substance and other harmful microorganisms.
Prebiotics is a kind of dietary supplements, by optionally stimulating the growth of one or more bacteriums with active and produce wholesome effect to host, thus improves host health.Successful prebiotics should be when by upper digestive tract, major part not digested and can ferment by intestinal microflora.The most important thing is that it just stimulates the growth of profitable strain, instead of have the unwanted bacteria of potential pathogenic or corrupt activity.It is generally acknowledged, prebiotics provides " food " to probiotic bacterium, can be decomposed and absorb, promote beneficial bacteria growth and breeding by beneficial bacteria in enteron aisle.
Document (Gauffinetal, PLoSOne, 2012,7 (7): e41079) is reported, BacteroidesuniformisCECT7771 has the effect of metabolism and the immunologic function disorder regulating obesity to cause, and has potential prebiotic effect.Our result of study finds, agaropectin oligose has the effect that specificity promotes Bacteroidesuniformis growth.Therefore, when agaropectin oligose and Bacteroidesuniformis use jointly, agaropectin oligose by promoting the growth of Bacteroidesuniformis, thus can play the related indication effect regulating obesity to cause.
(3) summary of the invention
The object of the invention is to provide a strain new strains--bacteroides uniformis (Bacteroidesuniformis) L8 and the application in degraded agaropectin oligose (AO) or agar-agar (AP).
The technical solution used in the present invention is:
The invention provides a strain new strains--bacteroides uniformis (Bacteroidesuniformis) L8, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNO.8711, preservation date is on January 10th, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
The present invention also provides the application of described bacteroides uniformis L8 in degraded agar-agar or agaropectin oligose, concrete described application method is: the seed liquor obtained after seed culture by bacteroides uniformis L8 is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volumetric concentration 2 ~ 10%, 25 ~ 42 DEG C of cultivations, thin layer chromatography (TLC) tracing detection to polymerizable raw material degree is reduced to 7(and molecular weight 1098Da) below, realize the degraded of agar-agar or agaropectin oligose;
Described agar medium final concentration consists of: agar-agar 0.5 ~ 1g/L, Tryptones 3g/L, peptone 3g/L, yeast extract 4.5g/L, Saliva Orthana 0.5g/L, No. 3 cholate 0.4g/L, L-cysteine hydrochloride 0.8g/L, protoheme 0.05g/L, 1ml/L tween 80, NaCl4.5g/L, KCl2.5g/L, MgCl
26H
2o4.5g/L, CaCl
26H
2o0.2g/L, KH
2pO
40.4g/L, micro-2ml/L, solvent is distilled water, and pH value is 6.4 ~ 6.5; Described micro-final concentration consists of: MgSO
47H
2o3.0g/L, CaCl
22H
2o0.1g/L, MnCl
24H
2o0.32g/L, FeSO
47H
2o0.1g/L, CoSO
47H
2o0.18g/L, ZnSO
47H
2o0.18g/L, CuSO
45H
2o0.01g/L, NiCl
26H
2o0.092g/L;
Described agaropectin oligose substratum final concentration consists of: replaced by the 1 ~ 50g/L of the agar-agar in agar medium agaropectin oligose, other form same agar medium.
Further, in described agar medium, the concentration of agar-agar is 0.5 ~ 1g/L, and the molecular weight of described agar-agar is 100 ~ 300kDa, and in preferred agar medium, the concentration of agar-agar is 0.8 ~ 1g/L, and the molecular weight of described agar-agar is 100 ~ 300kDa.
Further, in described agaropectin oligose substratum, the concentration of agaropectin oligose is 1 ~ 50g/L, the molecular weight of described agaropectin oligose is 0.5 ~ 10kDa, and in preferred agaropectin oligose substratum, the concentration of agaropectin oligose is 10 ~ 30g/L, and the molecular weight of described agaropectin oligose is 0.5 ~ 10kDa.
Further, the seed liquor that described bacteroides uniformis L8 obtains after seed culture is inoculated with the inoculum size of volume ratio 4% ~ 6%.
Further, described bacteroides uniformis L8 being applied as in degraded agar-agar or agaropectin oligose: (1) slant culture: bacteroides uniformis L8 is seeded to slant medium, cultivates 2 ~ 4 days for 25 ~ 42 DEG C, obtains inclined-plane thalline; Described slant medium is the agar powder adding quality final concentration 1.5% in agaropectin oligose substratum;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking colony, cultivates 2 ~ 4 days for 25 ~ 42 DEG C, obtains seed liquor; Described seed culture medium is with agaropectin oligose substratum;
(3) fermentation culture: seed liquor is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volume ratio 4% ~ 6%, 25 ~ 42 DEG C of cultivations, thin layer chromatography (TLC) tracing detection to polymerizable raw material degree is reduced to 7(and molecular weight 1098Da) below, realize the degraded of agar-agar or agaropectin oligose.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides a strain new strains--bacteroides uniformis (Bacteroidesuniformis) L8, molecular weight can be that the agar-agar of 100 ~ 300kDa is degraded to D-semi-lactosi by this bacterial strain, or the agaropectin oligose of molecular weight 0.5 ~ 10kDa is degraded to D-semi-lactosi, agaropectin oligose (molecular weight is 0.5 ~ 10kDa) has the effect that specificity promotes Bacteroidesuniformis growth simultaneously, thus reaches the related indication effect regulating obesity to cause.
(4) accompanying drawing explanation
Fig. 1 is that TLC detects B.uniformisL8 to the thin-layer chromatogram of the Degradation of AO, Gal:D-semi-lactosi.
Fig. 2 is that HPLC detects B.uniformisL8 degraded AO end product color atlas, and curve 1 is monose standard substance HPLC stratographic analysis figure, Man-seminose, Rha-rhamnosyl, GalA-galacturonic acid, Glc-glucose, Gal-D-semi-lactosi, Xyl-rhamnosyl; Curve 2 is bacteroides uniformis L8 degraded agaropectin oligose 192h fermented liquid HPLC stratographic analysis figure.
Fig. 3 is that TLC detects B.uniformisL8 to the thin-layer chromatogram of the Degradation of AP.
Fig. 4 is that HPLC detects B.uniformisL8 degraded AP end product color atlas, and curve 1 is monose standard substance HPLC stratographic analysis figure, Man-seminose, Rha-rhamnosyl, GalA-galacturonic acid, Glc-glucose, Gal-semi-lactosi, Xyl-rhamnosyl; Curve 2 is bacteroides uniformis L8 degraded agar-agar 192h fermented liquid HPLC stratographic analysis figure.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The isolation identification of embodiment 1 bacterial strain L8
(1) preparation of agaropectin oligose and molecular weight determination
10g agar-agar to be added in 1L distilled water after (10mg/mL) heating for dissolving, adding the 1mol/LHCl aqueous solution to HCl final concentration is 0.1mol/L, 60 DEG C of heating in water bath for reaction 1h, after reaction terminates, in reaction solution, add 1mol/LNaOH aqueous solution neutralization reaction liquid pH value to 7, then adopt molecular weight cut-off to be after the nanofiltration membrane desalination of 200 ~ 500Da, trapped fluid 50 DEG C of concentrating under reduced pressure evaporates to dryness, obtain agaropectin oligose 8g, be designated as AO.
Get AO 0.1mol/LNa
2sO
4preparation 5mg/ml solution, with TSK-GELG3000PWXL gel chromatographic columns (30cm × 7.8mm), adopt the analysis of HP1260 type high performance liquid chromatograph to measure, moving phase is 0.1mol/LNa
2sO
4the aqueous solution, column temperature 25 DEG C, flow velocity 0.5ml/min, Composition distribution (G1362A, Agilent company of the U.S.) and multiple angle laser light scattering instrument (DOWNHeleos II, Wyatt company of the U.S.) is adopted to detect, molecular weight through measurement AO is the structure of 0.5 ~ 10kDa, AO is G-[A-G]
n(n is positive integer, 1≤n≤31, and G represents D-semi-lactosi, and A represents 3,6-inner ether semi-lactosi).
(2) preparation of substratum
Preparation VI-AO liquid nutrient medium, concrete composition is as follows: AO(molecular weight 0.5 ~ 10kDa) 5g/L, Tryptones 3g/L, peptone 3g/L, yeast extract 4.5g/L, Saliva Orthana 0.5g/L, 3# cholate 0.4g/L, L-cysteine hydrochloride 0.8g/L, protoheme 0.05g/L, tween 80 1ml, NaCl4.5g/L, KCl2.5g/L, MgCl
26H
2o4.5g/L, CaCl
26H
2o0.2g/L, KH
2pO
40.4g/L, micro-2ml/L, solvent is distilled water, and pH value is 6.4 ~ 6.5, and sterilizing is placed on anaerobism workstation.
Trace element final concentration consists of: MgSO
47H
2o3.0g/L, CaCl
22H
2o0.1g/L, MnCl
24H
2o0.32g/L, FeSO
47H
2o0.1g/L, CoSO
47H
2o0.18g/L, ZnSO
47H
2o0.18g/L, CuSO
45H
2o0.01g/L, NiCl
26H
2o0.092g/L.
(3) pre-treatment of ight soil
Get 1 volunteer's fresh excreta, with PBS(pH7.0) be made into 20%(wt/vol) suspension, fully rear the diameter of mixing is the metallic screen filtration of 2mm, removes large food particles, obtains ight soil PBS solution.
(4) inoculation culture
Gained ight soil PBS solution is seeded to VI-AO substratum (inoculation volume final concentration is 2%), and 37 DEG C of Anaerobic culturel 24h carry out preliminary enrichment culture.Nutrient solution is adopted 10 times of dilution method coated plates, dull and stereotyped add final concentration 2wt% agar for VI-AO liquid nutrient medium.After flat board is placed in anaerobism workstation 37 DEG C cultivation 72h, sample for detecting degraded situation after 37 DEG C of cultivation 144h in picking list bacterium colony to VI-AO liquid nutrient medium in anaerobism workstation.
(5) thin layer chromatography (TLC) detects degraded situation
Supernatant 0.2 μ L point sample on silica-gel plate is got after institute's sample thief is centrifugal, using D-semi-lactosi standard substance and reaction 0h sample as reference, be placed in formic acid: propyl carbinol: after water (volume ratio is 6:3:1) developping agent launches, dry up, dry up after infiltrating in orcinol developer (orcinol-sulphuric acid soln), 120 DEG C of heating 3min develop the color.Obtain the positive colony of degradable AO according to TLC result, be designated as bacterial strain L8, Fig. 1 is that TLC detects bacterial strain L8 to the degraded of AO, and after cultivating 48h as can be seen from Figure 1, agaropectin oligose is degraded, and degrades to end product D-semi-lactosi after cultivating 96h.
(6) qualification of degradation bacteria
A, bacterial strain L8 physio-biochemical characteristics
Table 1 bacterial strain L8 biochemical characteristic (wherein-represent negative ,+represent positive)
B, 16SrDNA sequential analysis
The extraction of DNA: bacterial strain L8 step (5) obtained adopts QIAGEN ight soil test kit (Qiagen company, Germany) to extract DNA.
16SrDNA total length increases:
Primer sequence:
27f(5'-CAGAGTTTGATCCTGGCT-3')，
1492r(5'-AGGAGGTGATCCAGCCGCA-3')
Amplification system: reaction system 25 μ L, DNA profiling 100ng, 10 × PCRBuffer2.5 μ L, dNTPmix (10Mmeach) 0.5 μ L, 10 μMs of each 0.5 μ L of upstream and downstream primer, Taq enzyme (5U/ μ L) 0.2 μ L, adds deionized water and complements to 25 μ L.
Amplification condition: denaturation 94 DEG C of 5min, circulate 94 DEG C of 35S, 55 DEG C of 35S, 72 DEG C of 1min, 35 circulations, extends 8min.
Sheng Gong biotechnology company limited (Shanghai is delivered to after PCR primer purifying, China) carry out DNA sequencing (shown in SEQIDNO:1), sequencing result is submitted in ncbi database and carry out Blast comparison, comparison result shows this bacterial strain and B.uniformis homology is 99%, according to physio-biochemical characteristics and 16SrDNA sequence alignment, bacterial strain L8 is accredited as bacteroides uniformis (B.uniformis), called after bacteroides uniformis (B.uniformis) L8.
SEQIDNO:1 sequence is:
CAGCATGAACTTAGCTTGCTAAGTTTGATGGCGACCGGCGCACGGGTGAGTAACACGTATCCAACCTGCCGATGACTCGGGGATAGCCTTTCGAAAGAAAGATTAATACCCGATGGCATAATTCTTCCGCATGGTAGAACTATTAAAGAATTTCGGTCATCGATGGGGATGCGTTCCATTAGGTTGTTGGCGGGGTAACGGCCCACCAAGCCTTCGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGACGAGAGTCTGAACCAGCCAAGTAGCGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTTATACGGGAATAAAGTGAGGCACGTGTGCCTTTTTGTATGTACCGTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGACGCTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGGTGTCTTGAGTACAGTAGAGGCAGGCGGAATTCGTGGTGTAGCGGTGAAATGCTTAGATATCACGAAGAACTCCGATTGCGAAGGCAGCTTGCTGGACTGTAACTGACGCTGATGCTCGAAAGTGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACACAGTAAACGATGAATACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAATTGCAACTGAATGATGTGGAGACATGTCAGCCGCAAGGCAGTTGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCGATAGTTACCATCAGGTTATGCTGGGGACTCTGTCGAGACTGCCGTCGTAAGATGTGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGGGTACAGAAGGCAGCTACACGGCGACGTGATGCTAATCCCGAAAGCCTCTCTCAGTTCGGATTGGAGTCTGCAACCCGACTCCATGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGAAAGCCGGGGGTACCTGAAGTGCGTAACCGCAAGGAGCGCCCTAGGGTAAAACTGGTGACTGGGGCTAAG
Embodiment 2 bacteroides uniformis L8 is to the Degradation of agaropectin oligose
AO in VI-AO liquid nutrient medium in embodiment 1 is replaced with respectively the agaropectin oligose (molecular-weight average 0.5 ~ 10kDa) of final concentration 1g/L, 30g/L and 50g/L, obtain VI-AO-1, VI-AO-30 and VI-AO-50 substratum.Slant medium is the agar powder adding final concentration 1.5wt% in embodiment 1 in VI-AO liquid nutrient medium, and seed liquor substratum is VI-AO liquid nutrient medium in embodiment 1.
Bacteroides uniformis L8 is seeded to slant medium, cultivates 3 days for 37 DEG C, obtain inclined-plane thalline; Mono-clonal is chosen to seed liquor substratum from inclined-plane thalline, cultivate 3 days for 37 DEG C, obtain seed liquor, seed liquor is seeded to VI-AO-1 respectively with 6% volume ratio, VI-AO-30 and VI-AO-50 liquid nutrient medium, at 37 DEG C of Anaerobic culturel, be cultured to 48h, 96h, 144h, 192h sampling respectively, analyze degraded situation, degraded product structure and degradation capability.
(1) degraded situation analysis
Adopt thin layer chromatography method (method is with embodiment 1 step (5)) to detect bacteroides uniformis L8 to the degraded of 1g/L, 30g/L and 50g/LAO, concrete degraded situation is in table 2, and as shown in Table 2, bacteroides uniformis L8 can degrade the AO within the scope of 1 ~ 50g/L.
Table 2B.uniformisL8 is to the degraded situation of different concns AO
| AO concentration (g/L) | Whether degrade |
| 1 | Degraded |
| 30 | Degraded |
| 50 | Degraded |
(2) HPLC analyzes the structure of degraded end product
Get the 100 μ L bacteroides uniformis L8 fermented liquid that fermentation culture 192h obtains in the VI-AO liquid nutrient medium of AO final concentration 30g/L, adopt (the concrete grammar reference: pay Haining etc. of 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) pre-column derivatization-high performance liquid chromatography monose composition, four kinds of chromatographic processes of fucoidin monosaccharide composition analysis compare, Chinese Sea medicine, 2008,27 (4): 30).By with the contrasting of monose standard substance (seminose, rhamnosyl, galacturonic acid, glucose, semi-lactosi, wood sugar) retention time, determine that final degraded product is D-semi-lactosi (see Fig. 2), molecular weight is 180Da.
(3) residue AO content after Phenol-sulphate acid method analysis degraded
The standard D-semi-lactosi aqueous solution of accurate preparation 0.5mg/mL, accurate absorption 10,20,30,40,50 μ L, add water and mend to 100 μ L, add 6%(W/V) phenol 200 μ L, add the vitriol oil (mass concentration 98%) 1.5mL fast, vibration shakes up, 100 DEG C of heating in water bath 10min, surveys absorbancy after cooling at 490nm.Operate as blank using 100 μ L distilled water by same, using D-semi-lactosi quality as X-coordinate, absorbancy draws D-semi-lactosi typical curve as ordinate zou, and regression equation is y=0.0157x+0.0115(R
2=0.9991), x is D-semi-lactosi quality, and y is light absorption value.
Get the operation of the 100 μ L bacteroides uniformis L8 fermented liquid centrifuging and taking 40 μ L supernatant liquor that fermentation culture 192h obtains in the VI-AO liquid nutrient medium of AO final concentration 30g/L by D-semi-lactosi typical curve, 490nm detects absorbancy, with D-semi-lactosi typical curve regression equation calculation fermented liquid total sugar content.As calculated, remaining AO content in fermented liquid is 3.25g/L, and prove that bacteroides uniformis L8 can degrade AO, degradation rate is 89%.
Embodiment 3 bacteroides uniformis L8 is to the Degradation of agar-agar (AP)
It is 100 ~ 300kDa that the AO of VI-AO liquid nutrient medium in embodiment 1 is replaced to AP(molecular weight), in substratum, the final concentration of AP is respectively 0.5g/L and 1g/L, preparation VI-AP-0.5 and VI-AP-1 liquid nutrient medium.Slant medium is the agar powder adding final concentration 1.5wt% in embodiment 1 in VI-AO liquid nutrient medium, and seed liquor substratum is VI-AO liquid nutrient medium in embodiment 1.
Bacteroides uniformis L8 is seeded to slant medium, cultivate 3 days for 37 DEG C, choose mono-clonal from inclined-plane thalline and be seeded to seed culture medium, cultivate 3 days for 37 DEG C, after obtaining seed liquor, seed liquor is seeded to VI-AP-0.5 liquid nutrient medium and VI-AP-1 liquid nutrient medium respectively with the inoculum size of volumetric concentration 6%, 37 DEG C of Anaerobic culturel, respectively at 0h, 48h, 96h, 144h and 192h sampling, analyze degraded situation, degraded product structure and degradation capability.
(1) degraded situation
Thin layer chromatography method is adopted to detect degraded situation, when in TLC display substratum, AP final concentration is 0.5g/L and 1g/L, B.uniformisL8 can degrade AP(table 3), Fig. 3 is the degraded situation of TLC when to analyze AP final concentration be 1g/L, after Fig. 3 shows reaction 48h, agar-agar is degraded to agaropectin oligose, and the laggard step-down solution of reaction 96h is to end product.
(2) HPLC analyzes the structure of degraded product
Get the VI-AP-1 liquid nutrient medium 192h fermented liquid that 100 μ l bacteroides uniformis L8 are 1g/L at AP final concentration, PMP-derivatization method in embodiment 2 is adopted to measure the structure of end product, by with the contrasting of monose standard substance (seminose, rhamnosyl, galacturonic acid, glucose, D-semi-lactosi, wood sugar) retention time, determine that final degraded product is D-semi-lactosi (Fig. 4).
(3) Phenol-sulphate acid method remains AP content after measuring L8 degraded
The making of D-semi-lactosi typical curve is with embodiment 2.
Get the 100 μ L bacteroides uniformis L8 fermented liquid that fermentation culture 192h obtains in the VI-AP-1 liquid nutrient medium of AP final concentration 1g/L by embodiment 2 method, 490nm detects absorbancy, with typical curve regression equation calculation total sugar content.As calculated, remaining AP content in fermented liquid is 0.3g/L, and prove that bacteroides uniformis L8 can degrade AP, degradation rate is 70%.
Table 3B.uniformisL8 is to the degraded situation of different concns AP
| AP concentration (g/L) | Whether degrade |
| 0.5 | Degraded |
| 1.0 | Degraded |
Embodiment 4 agaropectin oligose promotes that B.uniformisL8 breeds in bottle fermentation
1. the preparation method of agaropectin oligose and molecular weight determination are with embodiment 1.
2. the preparation of substratum is with the preparation of VI-AO in embodiment 1.
3. the pre-treatment of ight soil
Get 3 volunteer's fresh excretas, treatment process, with embodiment 1, obtains ight soil PBS solution.
4. inoculation culture
Gained ight soil PBS solution is seeded to VI-AO liquid nutrient medium (inoculation volume final concentration is 2%) respectively, 37 DEG C of Anaerobic culturel, and respectively at 0h and 48h sampling, is numbered No.1, No.2 and No.3 respectively.
5. adopt QIAGEN ight soil test kit (Qiagen company, Germany) to extract the DNA of 0h and 48h fermented liquid and bacteroides uniformis L8.
6.RT-TimePCR compares the quantity of B.uniformisL8 in 0h and 48h fermented liquid
(1) Primer selection:
Adopt bibliographical information Bacteroidesuniformis special primer (EijiIshikawa etc., JBiosciBioeng.2013,116 (2): 265-70)
Buni188-F（TTCTTCCGCATGGTAGAAC），
Buni590-R（CTTTCACAACTGACTTAAGCG）
(2) amplification system: reaction system 25 μ L, DNA profiling 100ng(and BacteroidesuniformisL8DNA), 10 × PCRBuffer2.5 μ L, dNTPmix0.5 μ L, 10 μMs of each 0.5 μ L of upstream and downstream primer, Taq enzyme (5U/ μ L) 0.2 μ L, adds deionized water and complements to 25 μ L.
(3) amplification condition: denaturation 94 DEG C of 5mim, circulate 94 DEG C of 35S, 55 DEG C of 35S, 72 DEG C of 1min, 35 circulations, extends 8min.
(4) preparation of outer standard substance: the fragment that application specific primer Buni188-F and Buni590-R increases is carried out glue and reclaimed purifying after adopting electrophoresis detection, purified fragments is connected with pMD18-T carrier, be transformed in competent escherichia coli cell DH-5 α after connection, select positive colony order-checking, authenticated positive colony is extracted plasmid, adopt trace dna determinator NanoDrop2000(Thermo, USA), after measuring plasmid concentration, the concentration that substitution formula 1 calculates linearized plasmid solution is 2.94 × 10
8copy number/ng plasmid DNA (formula 1 namely: copy number=9.1 × 10 of every 1 μ g plasmid DNA
11the size (kb) of/plasmid DNA), plasmid deionized water extraction obtained, by 10 times of gradient dilutions, uses dilution rear 2.94 × 10
8-2.94 × 10
1copy number/ng plasmid DNA as the template of typical curve, for the quantitative assay of this Pseudomonas in subsequent sample.
(5) RT-TimePCR measures the quantity of B.uniformisL8 in ight soil and fermented liquid
Reaction conditions: the reaction system of 20 μ L, wherein: 2 × SYBRGreenRealtimePCRMasterMix (Toyobo, Japan) 10 μ L, each 0.5 μM of upstream and downstream primer, DNA profiling (being respectively extraction 0 and 48h fermented liquid DNA in the plasmid DNA of (4) gained typical curve concentration used in step 6, step 5) adds 20ng.
Response procedures: 95 DEG C of denaturation 1min; 95 DEG C of sex change 15s, 55 DEG C of 15s, 72 DEG C extend 15s, carry out 40 circulations altogether.
The typical curve of fluorescent quantitation is with the concentration of PCR reaction template (getting log value) for X-coordinate, the curve about template concentrations and CP value relation done for ordinate zou with CrossingPoint value (CP value).The equation Y=-4.079X+47.258(R of this curve
2=0.991), Y is CP value, and X is template concentrations.
Calculate in 0h and 48h fermented liquid according to typical curve that B.uniformisL8 quantity is in table 3, as can be seen from the table, in fermented liquid, B.uniformisL8 quantity is apparently higher than ight soil, illustrates that AO can promote the propagation of B.uniformisL8.
B.uniformisL8 quantity in table 3 ight soil and fermented liquid
Claims (7)
1. bacteroides uniformis (
bacteroidesuniformis) L8, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNO.8711, preservation date is on January 10th, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
2. the application of bacteroides uniformis L8 described in claim 1 in degraded agar-agar or agaropectin oligose.
3. apply as claimed in claim 2, it is characterized in that described application method is: the seed liquor obtained after seed culture by bacteroides uniformis L8 is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volume ratio 2 ~ 10%, 25 ~ 42 DEG C of cultivations, thin layer chromatography tracing detection is reduced to less than 7 to polymerizable raw material degree, realizes the degraded of agar-agar or agaropectin oligose;
Described agar medium final concentration consists of: agar-agar 0.5 ~ 1g/L, Tryptones 3g/L, peptone 3g/L, yeast extract 4.5g/L, Saliva Orthana 0.5g/L, No. 3 cholate 0.4g/L, L-cysteine hydrochloride 0.8g/L, protoheme 0.05g/L, 1ml/L tween 80, NaCl4.5g/L, KCl2.5g/L, MgCl
26H
2o4.5g/L, CaCl
26H
2o0.2g/L, KH
2pO
40.4g/L, micro-2ml/L, solvent is distilled water, and pH value is 6.4 ~ 6.5; Described micro-final concentration consists of: MgSO
47H
2o3.0g/L, CaCl
22H
2o0.1g/L, MnCl
24H
2o0.32g/L, FeSO
47H
2o0.1g/L, CoSO
47H
2o0.18g/L, ZnSO
47H
2o0.18g/L, CuSO
45H
2o0.01g/L, NiCl
26H
2o0.092g/L;
Described agaropectin oligose substratum final concentration consists of: replaced by the 1 ~ 50g/L of the agar-agar in agar medium agaropectin oligose, other form same agar medium.
4. apply as claimed in claim 3, it is characterized in that described agar-agar molecular weight is 100 ~ 300kDa.
5. apply as claimed in claim 3, it is characterized in that described agaropectin oligose molecular weight is 0.5 ~ 10kDa, structure is G-[A-G]
n, n is positive integer, 1≤n≤31, and G represents D-semi-lactosi, and A represents 3,6-inner ether semi-lactosi.
6. apply as claimed in claim 3, it is characterized in that the seed liquor that described bacteroides uniformis L8 obtains after seed culture is inoculated with the inoculum size of volume ratio 4% ~ 6%.
7. apply as claimed in claim 3, it is characterized in that described being applied as:
(1) slant culture: bacteroides uniformis L8 is seeded to slant medium, cultivates 2 ~ 4 days for 25 ~ 42 DEG C, obtains inclined-plane thalline; Described slant medium is: in agaropectin oligose substratum, add the agar powder that quality final concentration is 1.5%;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking colony, cultivates 2 ~ 4 days for 25 ~ 42 DEG C, obtains seed liquor; Described seed culture medium is with agaropectin oligose substratum;
(3) fermentation culture: seed liquor is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volume ratio 4% ~ 6%, 25 ~ 42 DEG C of cultivations, thin layer chromatography tracing detection is reduced to less than 7 to polymerizable raw material degree, realizes the degraded of agar-agar or agaropectin oligose.
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