CN103865844A - Bacteroides uniformis L8 and application thereof in degrading agar or agar oligosaccharide - Google Patents
Bacteroides uniformis L8 and application thereof in degrading agar or agar oligosaccharide Download PDFInfo
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- CN103865844A CN103865844A CN201410054976.7A CN201410054976A CN103865844A CN 103865844 A CN103865844 A CN 103865844A CN 201410054976 A CN201410054976 A CN 201410054976A CN 103865844 A CN103865844 A CN 103865844A
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- 241000606219 Bacteroides uniformis Species 0.000 title claims abstract description 58
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Abstract
The invention discloses a new bacteria strain-bacteroides uniformis L8 and an application thereof in degrading agar or agar oligosaccharide. The bacteria strain can degrade agar with molecular weight of 100-300kDa into agar oligosaccharide and further degrade the agar oligosaccharide into D-galactose, or degrade the agar oligosaccharide with molecular weight of 0.5-10kDa into D-galactose finally, in addition, the agar oligosaccharide has the specificity to promote growth of the bacteroides uniformis, so as to play a role in regulating associated symptoms caused by obesity.
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 (Bacteroides uniformis) L8 in degraded agar-agar or agaropectin oligose.
(2) background technology
Agar-agar is the polysaccharide in a kind of marine red alga source, mainly by (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 as foodstuff additive because of its special gel property.Agar-agar is through enzyme liberating or acid degradation can obtain the relatively low agaropectin oligose of molecular weight (calling AO in the following text), recent studies have found that agaropectin oligose has the prebiotic effect that promotes bifidobacterium growth in enteron aisle.
Miscellaneous intestinal microflora of living away from home in the gi tract of Healthy People, is approximately 10
14left and right.Between intestinal microflora and host and environment, all the time in dynamic balance state, form one and depend on each other for existence, the system of restriction mutually, thereby it is useful and harmless to human body in the time that body defense function is normal.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., once but there is alteration of intestinal flora, will certainly upset these normal physiological functions.
Edible probiotic bacterium can reach the object that maintains gi tract health, and the probiotic bacterium that China ratifies to use in food at present has more than 20 to plant, and is mainly Bacterium lacticum and bifidus bacillus.Studies show that probiotic bacterium has multiple physiological hygiene function, comprise cellular immunization promoter action, process lactose intolerance, reduce serum cholesterol, regulate human intestinal microflora balance, antianaphylaxis, suppresses carcinogenic substance and other harmful microorganisms.
Prebiotics is a kind of dietary supplements, with activity, host is produced to wholesome effect, thereby improve host's health by the growth that optionally stimulates one or more bacteriums.Successfully prebiotics should be when by upper digestive tract, and major part is digested and can be by intestinal microflora fermented.The most important thing is that it just stimulates the growth of profitable strain, rather than 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 by beneficial bacteria in enteron aisle, promotes beneficial bacteria growth and breeding.
Document (Gauffin et al, PLoS One, 2012,7 (7): e41079) report, Bacteroides uniformis CECT7771 has the fat metabolism causing of adjusting and the effect of immunologic function disorder, has potential prebiotic effect.Our result of study discovery, agaropectin oligose has the effect that specificity promotes that Bacteroides uniformis grows.Therefore, when agaropectin oligose and Bacteroides uniformis use jointly, agaropectin oligose can pass through to promote the growth of Bacteroides uniformis, thereby plays the fat related indication effect causing that regulates.
(3) summary of the invention
The object of the invention is to provide the new bacterial strain of a strain--bacteroides uniformis (Bacteroides uniformis) 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 the new bacterial strain of a strain--bacteroides uniformis (Bacteroides uniformis) L8, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.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 that bacteroides uniformis L8 is obtained after seed culture is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volumetric concentration 2～10%, 25～42 ℃ of cultivations, thin layer chromatography (TLC) tracking detects and is reduced to 7(to polymerizable raw material degree is 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, Cys 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, 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: the 1～50g/L of the agar-agar in agar medium agaropectin oligose is replaced, and 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 preferably in 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 preferably in 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 with the inoculum size inoculation 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 ℃, obtain inclined-plane thalline; Described slant medium is the agar powder that adds quality final concentration 1.5% in agaropectin oligose substratum;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking colony, cultivate 2～4 days for 25～42 ℃, obtain 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 ℃ of cultivations, thin layer chromatography (TLC) tracking detects and is reduced to 7(to polymerizable raw material degree is 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 the new bacterial strain of a strain--bacteroides uniformis (Bacteroides uniformis) L8, the agar-agar that this bacterial strain can be 100～300kDa by molecular weight is degraded to D-semi-lactosi, 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 that Bacteroides uniformis grows simultaneously, thereby reaches the fat related indication effect causing that regulates.
(4) accompanying drawing explanation
Fig. 1 is the thin-layer chromatogram that TLC detects the Degradation of B.uniformis L8 to AO, Gal:D-semi-lactosi.
Fig. 2 is that HPLC detects B.uniformis L8 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 the thin-layer chromatogram that TLC detects the Degradation of B.uniformis L8 to AP.
Fig. 4 is that HPLC detects B.uniformis L8 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 is 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 ℃ of heating in water bath for reaction 1h, after reaction finishes, in reaction solution, add 1mol/L NaOH aqueous solution neutralization reaction liquid pH value to 7, after then adopting molecular weight cut-off to be the nanofiltration membrane filtration desalination of 200～500Da, 50 ℃ of concentrating under reduced pressure evaporates to dryness of trapped fluid, obtain agaropectin oligose 8g, be designated as AO.
Get AO 0.1mol/L Na
2sO
4preparation 5mg/ml solution, with TSK-GEL G3000PWXL gel chromatographic columns (30cm × 7.8mm), adopts the analysis of HP1260 type high performance liquid chromatograph to measure, and moving phase is 0.1mol/L Na
2sO
4the aqueous solution, 25 ℃ of column temperatures, flow velocity 0.5ml/min, adopt differential detector (G1362A, Agilent company of the U.S.) and multiple angle laser light scattering instrument (DOWN Heleos II, Wyatt company of the U.S.) to detect, be 0.5～10kDa through the molecular weight of measuring AO, the structure of AO is G-[A-G]
n(n is positive integer, 1≤n≤31, and G represents D-semi-lactosi, 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, Cys 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 mix the rear metallic screen that is 2mm with diameter and filter, remove large food particles, obtain 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 ℃ of anaerobism are cultivated 24h and carried out preliminary enrichment culture.Nutrient solution is adopted to 10 times of dilution method coated plates, dull and stereotyped add final concentration 2wt% agar for VI-AO liquid nutrient medium.Flat board is placed in 37 ℃ of anaerobism workstations and cultivates after 72h, in anaerobism workstation picking list bacterium colony in VI-AO liquid nutrient medium 37 ℃ cultivate 144h after sampling for detection of degraded situation.
(5) thin layer chromatography (TLC) detects degraded situation
After institute's sample thief is centrifugal, get supernatant 0.2 μ L point sample on silica-gel plate, 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, after infiltrating in orcinol developer (orcinol-sulphuric acid soln), dry up, 120 ℃ 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 the degraded of bacterial strain L8 to AO, cultivates as can be seen from Figure 1 the later agaropectin oligose of 48h and is degraded, and after cultivation 96h, degrades to end product D-semi-lactosi.
(6) evaluation of degradation bacteria
A, bacterial strain L8 physio-biochemical characteristics
Table 1 bacterial strain L8 biochemical characteristic (wherein-represent feminine gender ,+expression is positive)
B, 16S rDNA sequential analysis
The extraction of DNA: the bacterial strain L8 that step (5) is obtained adopts QIAGEN ight soil test kit (Qiagen company, Germany) to extract DNA.
The amplification of 16S rDNA total length:
Primer sequence:
27f(5'-CAGAGTTTGATCCTGGCT-3')，
1492r(5'-AGGAGGTGATCCAGCCGCA-3')
Amplification system: reaction system 25 μ L, DNA profiling 100ng, 10 × PCR Buffer2.5 μ L, dNTP mix (10Mm each) 0.5 μ L, the each 0.5 μ L of 10 μ M upstream and downstream primer, Taq enzyme (5U/ μ L) 0.2 μ L, adds deionized water and complements to 25 μ L.
Amplification condition: 94 ℃ of 5min of denaturation, the 94 ℃ of 35S that circulate, 55 ℃ of 35S, 72 ℃ of 1min, 35 circulations, extend 8min.
After PCR product purification, deliver to Sheng Gong biotechnology company limited (Shanghai, China) carry out DNA sequencing (shown in SEQ ID NO:1), sequencing result is submitted to and in ncbi database, carried out Blast comparison, this bacterial strain of comparison result shows and B.uniformis homology are 99%, according to physio-biochemical characteristics and 16S rDNA sequence alignment, bacterial strain L8 is accredited as to bacteroides uniformis (B.uniformis), called after bacteroides uniformis (B.uniformis) L8.
SEQ ID NO:1 sequence is:
CAGCATGAACTTAGCTTGCTAAGTTTGATGGCGACCGGCGCACGGGTGAGTAACACGTATCCAACCTGC?CGATGACTCGGGGATAGCCTTTCGAAAGAAAGATTAATACCCGATGGCATAATTCTTCCGCATGGTAGAACTA?TTAAAGAATTTCGGTCATCGATGGGGATGCGTTCCATTAGGTTGTTGGCGGGGTAACGGCCCACCAAGCCTTC?GATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGAGG?CAGCAGTGAGGAATATTGGTCAATGGACGAGAGTCTGAACCAGCCAAGTAGCGTGAAGGATGACTGCCCTAT?GGGTTGTAAACTTCTTTTATACGGGAATAAAGTGAGGCACGTGTGCCTTTTTGTATGTACCGTATGAATAAGGA?TCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAG?GGAGCGTAGGCGGACGCTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGG?TGTCTTGAGTACAGTAGAGGCAGGCGGAATTCGTGGTGTAGCGGTGAAATGCTTAGATATCACGAAGAACTC?CGATTGCGAAGGCAGCTTGCTGGACTGTAACTGACGCTGATGCTCGAAAGTGTGGGTATCAAACAGGATTAG?ATACCCTGGTAGTCCACACAGTAAACGATGAATACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAA?GCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACA?AGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTGAATTGCAACTGAATGA?TGTGGAGACATGTCAGCCGCAAGGCAGTTGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGT?GTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCGATAGTTACCATCAGGTTATGCTGGGGACTCTGTCGA?GACTGCCGTCGTAAGATGTGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTAC?ACACGTGTTACAATGGGGGGTACAGAAGGCAGCTACACGGCGACGTGATGCTAATCCCGAAAGCCTCTCTCA?GTTCGGATTGGAGTCTGCAACCCGACTCCATGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCG?GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGAAAGCCGGGGGTACCTGAAGTGCGTAA?CCGCAAGGAGCGCCCTAGGGTAAAACTGGTGACTGGGGCTAAG
The Degradation of embodiment 2 bacteroides uniformis L8 to agaropectin oligose
The agaropectin oligose (molecular-weight average 0.5～10kDa) that AO in VI-AO liquid nutrient medium in embodiment 1 is replaced with respectively to final concentration 1g/L, 30g/L and 50g/L, obtains VI-AO-1, VI-AO-30 and VI-AO-50 substratum.Slant medium is the agar powder that adds 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 ℃, obtain inclined-plane thalline; Choose mono-clonal to seed liquor substratum from inclined-plane thalline, cultivate 3 days for 37 ℃, obtain seed liquor, seed liquor is seeded to respectively to VI-AO-1 with 6% volume ratio, VI-AO-30 and VI-AO-50 liquid nutrient medium, cultivate 37 ℃ of anaerobism, be cultured to respectively 48h, 96h, 144h, 192h sampling, 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 the bacteroides uniformis L8 degraded to 1g/L, 30g/L and 50g/L AO, specifically degraded situation is in table 2, as shown in Table 2, and the bacteroides uniformis L8 AO within the scope of 1～50g/L that can degrade.
The degraded situation of table 2B.uniformis L8 to 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 process comparisons of fucoidin monose compositional analysis, 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 (seeing Fig. 2), molecular weight is 180Da.
(3) residue AO content after phenol sulfuric acid method analysis degraded
The accurately standard D-semi-lactosi aqueous solution of preparation 0.5mg/mL, accurately draw 10,20,30,40,50 μ L, add water and mend to 100 μ L, add 6%(W/V) phenol 200 μ L, add fast the vitriol oil (mass concentration 98%) 1.5mL, vibration shakes up, 100 ℃ of heating in water bath 10min, cooling after 490nm survey absorbancy.Operate as blank by same using 100 μ L distilled water, using D-semi-lactosi quality as X-coordinate, absorbancy is drawn 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 100 μ L bacteroides uniformis L8 fermented liquid centrifuging and taking 40 μ L supernatant liquors that fermentation culture 192h obtains in the VI-AO liquid nutrient medium of AO final concentration 30g/L by the operation of D-semi-lactosi typical curve, 490nm detects absorbancy, with D-semi-lactosi typical curve regression equation calculation fermented liquid total sugar content.As calculated, in fermented liquid, remaining AO content is 3.25g/L, proves the bacteroides uniformis L8 AO that can degrade, and degradation rate is 89%.
The Degradation of embodiment 3 bacteroides uniformis L8 to 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 that adds 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 ℃, choose mono-clonal from inclined-plane thalline and be seeded to seed culture medium, cultivate 3 days for 37 ℃, obtain after seed liquor, seed liquor is seeded to respectively to VI-AP-0.5 liquid nutrient medium and VI-AP-1 liquid nutrient medium with the inoculum size of volumetric concentration 6%, 37 ℃ of anaerobism are cultivated, respectively at 0h, 48h, 96h, 144h and 192h sampling, analyze degraded situation, degraded product structure and degradation capability.
(1) degraded situation
Adopt thin layer chromatography method to detect degraded situation, TLC shows when in substratum, AP final concentration is 0.5g/L and 1g/L, the B.uniformis L8 AP(table 3 of can degrading), Fig. 3 is the degraded situation of TLC when analyzing AP final concentration and being 1g/L, after Fig. 3 shows to react 48h, agar-agar is degraded to agaropectin oligose, after reaction 96h, further degrades 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, adopt the structure of PMP-derivatization method mensuration end product in embodiment 2, 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 sulfuric 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 methods, 490nm detects absorbancy, with typical curve regression equation calculation total sugar content.As calculated, in fermented liquid, remaining AP content is 0.3g/L, proves the bacteroides uniformis L8 AP that can degrade, and degradation rate is 70%.
The degraded situation of table 3B.uniformis L8 to different concns AP
| AP concentration (g/L) | Whether degrade |
| 0.5 | Degraded |
| 1.0 | Degraded |
Embodiment 4 agaropectin oligoses promote B.uniformis L8 propagation 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 respectively VI-AO liquid nutrient medium (inoculation volume final concentration is 2%), and 37 ℃ of anaerobism are cultivated, and respectively at 0h and 48h sampling, are numbered respectively No.1, No.2 and No.3.
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-Time PCR compares the quantity of B.uniformis L8 in 0h and 48h fermented liquid
(1) Primer selection:
Adopt bibliographical information Bacteroides uniformis special primer (Eiji Ishikawa etc., J Biosci Bioeng.2013,116 (2): 265-70)
Buni188-F（TTCTTCCGCATGGTAGAAC），
Buni590-R（CTTTCACAACTGACTTAAGCG）
(2) amplification system: reaction system 25 μ L, DNA profiling 100ng(is Bacteroides uniformis L8DNA), 10 × PCR Buffer2.5 μ L, dNTP mix0.5 μ L, the each 0.5 μ L of 10 μ M 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: 94 ℃ of 5mim of denaturation, the 94 ℃ of 35S that circulate, 55 ℃ of 35S, 72 ℃ of 1min, 35 circulations, extend 8min.
(4) preparation of outer standard substance: the fragment of application specific primer Buni188-F and Buni590-R amplification is carried out glue recovery purifying after adopting electrophoresis detection, purifying fragment is connected with pMD18-T carrier, after connection, be transformed in competent escherichia coli cell DH-5 α, select positive colony order-checking, the positive colony of verifying is extracted to plasmid, adopt trace dna determinator Nano Drop2000(Thermo, USA) measure after plasmid concentration, the concentration that substitution formula 1 calculates linear plasmid solution is 2.94 × 10
8copy number/ng plasmid DNA (formula 1: copy number=9.1 × 10 of every 1 μ g plasmid DNA
11the size (kb) of/plasmid DNA), the plasmid that extraction is obtained by 10 times of gradient dilutions, uses dilution rear 2.94 × 10 with deionized water
8-2.94 × 10
1copy number/ng plasmid DNA is as the template of typical curve, for the quantitative assay of this Pseudomonas in subsequent sample.
(5) RT-Time PCR measures the quantity of B.uniformis L8 in ight soil and fermented liquid
Reaction conditions: the reaction system of 20 μ L, wherein: 2 × SYBR Green Realtime PCR Master Mix (Toyobo, Japan) 10 μ L, the each 0.5 μ M of upstream and downstream primer, DNA profiling (be respectively extract in the plasmid DNA, step 5 of (4) gained typical curve concentration used in step 60 and 48h fermented liquid DNA) adds 20ng.
Response procedures: 95 ℃ of denaturation 1min; 95 ℃ of sex change 15s, 55 ℃ of 15s, 72 ℃ are extended 15s, carry out altogether 40 circulations.
The typical curve of fluorescent quantitation is take the concentration of PCR reaction template (getting log value) as X-coordinate, the curve about template concentrations and CP value relation of being done for ordinate zou with Crossing Point 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 according to typical curve that in 0h and 48h fermented liquid, B.uniformis L8 quantity is in table 3, as can be seen from the table, in fermented liquid, B.uniformis L8 quantity, apparently higher than ight soil, illustrates that AO can promote the propagation of B.uniformis L8.
B.uniformis L8 quantity in table 3 ight soil and fermented liquid
Claims (7)
1. bacteroides uniformis (Bacteroides uniformis) L8, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.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 in degraded agar-agar or agaropectin oligose described in a claim 1.
3. application as claimed in claim 2, it is characterized in that described application method is: the seed liquor that bacteroides uniformis L8 is obtained after seed culture is seeded in agar-agar or agaropectin oligose substratum with the inoculum size of volume ratio 2～10%, 25～42 ℃ of cultivations, thin layer chromatography is followed the tracks of to detect to polymerizable raw material degree and is reduced to below 7, 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, Cys 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, 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: the 1～50g/L of the agar-agar in agar medium agaropectin oligose is replaced, and other form same agar medium.
4. application as claimed in claim 3, is characterized in that described agar-agar molecular weight is 100～300kDa.
5. application as claimed in claim 3, is characterized in that described agaropectin oligose molecular weight is 0.5～10kDa.
6. application as claimed in claim 3, is characterized in that the inoculum size inoculation with volume ratio 4%～6% of seed liquor that described bacteroides uniformis L8 obtains after seed culture.
7. application as claimed in claim 3, 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 ℃, obtain inclined-plane thalline; Described slant medium is: in agaropectin oligose substratum, add quality final concentration 1.5% agar powder;
(2) seed culture: be seeded to seed culture medium from inclined-plane thalline picking colony, cultivate 2～4 days for 25～42 ℃, obtain 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 ℃ of cultivations, thin layer chromatography is followed the tracks of to detect to polymerizable raw material degree and is reduced to below 7, realizes the degraded of agar-agar or agaropectin oligose.
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| CN116676207A (en) * | 2023-03-14 | 2023-09-01 | 中国海洋大学 | Bacteroides sally strain and application thereof in degradation preparation of chondroitin sulfate oligosaccharide and hyaluronic acid oligosaccharide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107002022A (en) * | 2014-09-30 | 2017-08-01 | 上海交通大学医学院附属瑞金医院 | Purposes of the bacteroid in obesity-related disease is treated or prevented |
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| CN116676207A (en) * | 2023-03-14 | 2023-09-01 | 中国海洋大学 | Bacteroides sally strain and application thereof in degradation preparation of chondroitin sulfate oligosaccharide and hyaluronic acid oligosaccharide |
| CN116676207B (en) * | 2023-03-14 | 2024-06-04 | 中国海洋大学 | Bacteroides sally strain and application thereof in degradation preparation of chondroitin sulfate oligosaccharide and hyaluronic acid oligosaccharide |
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