CN106834379A - A kind of preparation method and applications of new fine jade tetrose - Google Patents
A kind of preparation method and applications of new fine jade tetrose Download PDFInfo
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- CN106834379A CN106834379A CN201610850690.9A CN201610850690A CN106834379A CN 106834379 A CN106834379 A CN 106834379A CN 201610850690 A CN201610850690 A CN 201610850690A CN 106834379 A CN106834379 A CN 106834379A
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- 150000003538 tetroses Chemical class 0.000 title claims abstract description 69
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- 229920000936 Agarose Polymers 0.000 claims abstract description 30
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 21
- 101710110830 Beta-agarase Proteins 0.000 claims abstract description 14
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- 230000000694 effects Effects 0.000 abstract description 11
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- 230000002255 enzymatic effect Effects 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
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- 241000193830 Bacillus <bacterium> Species 0.000 description 2
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- 241000204294 Bacteroides stercoris Species 0.000 description 1
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000206572 Rhodophyta Species 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
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- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 description 1
- 229960000479 ceftriaxone sodium Drugs 0.000 description 1
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- 230000000593 degrading effect Effects 0.000 description 1
- FDRNWTJTHBSPMW-GNXCPKRQSA-L disodium;(6r,7r)-7-[[(2e)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(2-methyl-6-oxido-5-oxo-1,2,4-triazin-3-yl)sulfanylmethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound [Na+].[Na+].S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)/C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C([O-])=NN1C FDRNWTJTHBSPMW-GNXCPKRQSA-L 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
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Abstract
The invention discloses the preparation method and applications of a kind of new fine jade tetrose for belonging to agaropectin oligose preparing technical field.The method obtains new fine jade tetrose sterling using β agarase AgWH50B enzyme liquids and β agarase AgOGA118 enzyme liquids joint enzymolysis.The present invention is hydrolyzed agarose and is prepared new fine jade tetrose using the coordinated enzymatic hydrolysis effect of β agarase AgWH50B and β agarases AgOGA118, and enzymolysis efficiency is good, product purity is high.The efficient preparation of the present invention new fine jade tetrose of achievable high-purity, and the new fine jade tetrose for preparing has good gut flora regulating power, supported to provide theory and technology as development of raw materials functional food and health products with new fine jade tetrose, with good application potential and prospect.
Description
Technical field
The invention belongs to agaropectin oligose preparing technical field, and in particular to a kind of enzymolysis agarose prepares the side of new fine jade tetrose
Method and its application in terms of intestinal microecology regulation.
Background technology
New fine jade oligosaccharides is that (a kind of linear polysaccharide is mainly derived from the thin of the red algaes such as fragrant plant mentioned in ancient texts, agar, seaweed by agarose
Cell wall) degrade and obtain, typically it is made up of 2-10 monose, with D- galactolipins as reducing end under neutral, with various physiologically actives.Newly
The preparation method of fine jade oligosaccharides mainly has two kinds of acid hydrolyzation and enzymatic isolation method, and the application of wherein acid hydrolyzation is more, but acid hydrolyzation has dirt
Dye environment, product are complicated and react violent shortcoming, constrain the large-scale application of this method.Enzymatic isolation method has enzymolysis process
Gently, the advantages of environment-friendly and product purity higher, be that the preparation of new fine jade oligosaccharides opens new direction.
Although enzymatic isolation method has many advantages, such as compared with acid hydrolyzation, used as the substrate for being digested, agarose molecules amount is big,
Carbon chain lengths are long, solution viscosity is high, the exposure rate of enzyme-to-substrate limited to a certain extent, and then influence the effect of enzymolysis process
Rate.Therefore, the enzymolysis efficiency for how improving agarose is the key technology that enzymatic isolation method prepares new fine jade oligosaccharides.Meanwhile, new fine jade oligosaccharides
Physiologically active and its degree of polymerization have very big correlation, existing report all concentrates on the preparation of new fine jade oligosaccharides muscovado and physiology is lived
Journal of Sex Research, lacks the preparation and functional activity research of the single new fine jade oligosaccharides of the degree of polymerization.How the single degree of polymerization of high-purity is prepared
New fine jade oligosaccharides, and its physiologically active is studied, there is important theory value and promote for the development and application of new fine jade oligosaccharides and make
With.、
Patent CN201410148086 discloses β-agarase that a kind of agarose of degrading generates new fine jade tetrose, the β-agar-agar
Enzyme is AgWH50B, and the β-agarase independent transformation time is more long, and conversion ratio is low, and the product purity of preparation is not high.
The content of the invention
The application of the method and new fine jade tetrose of new fine jade tetrose is efficiently prepared it is an object of the invention to provide a kind of enzymatic isolation method,
So as to make up the deficiencies in the prior art.
A kind of preparation method of new fine jade tetrose, is carried out in accordance with the following steps：
(1) configuration quality fraction is the agarose solution of 0.3-0.7%；
(2) by weight (0.2-1):100 are separately added into β-agarase AgWH50B enzyme liquids and β-agarase AgOGA118 enzymes
Liquid, 35-45 DEG C of incubation 6-10h, obtains enzymolysis liquid；
(3) it is enzymolysis liquid is concentrated under reduced pressure in 55-65 DEG C, 2-5 times of volume absolute ethyl alcohol is added, in 8000-10000rpm, 0-
8 DEG C of low-temperature centrifugation 10-20min, collect supernatant；
(4) in 55-65 DEG C of supernatant concentrated under reduced pressure, after 0.35-0.55 μm of membrane filtration, over-molecular sieve purifying is freezed supernatant
Sample after purification is dried, new fine jade tetrose sterling is obtained.
Using the buffer solution configuration agarose solution of pH 7.0 in step (1).
The molecular sieve is Bio-Gel P2 posts.
Step (4) over-molecular sieve after purification, detects by TLC (thin-layer chromatography), and purity reaches 90%, and to carry out freezing again dry
Dry purifying.
The new fine jade tetrose of above-mentioned preparation answering in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
With.
Further, the new fine jade tetrose is in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
Promote Bifidobacterium in enteron aisle using, its described new fine jade tetrose, lactobacillus, the propagation of heavy wall bacterium and bacteroid.
Further, the new fine jade tetrose is in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
Using its described new fine jade tetrose can improve mouse intestinal flora structure.
Beneficial effects of the present invention：The present invention utilizes the coordinated enzyme of β-agarase AgWH50B and β-agarase AgOGA118
Solution is acted on, and hydrolysis agarose prepares new fine jade tetrose, and enzymolysis efficiency is good, product purity is high.Meanwhile, adjusted by mouse intestinal Tiny ecosystem
Section Effect study finds, compared with new fine jade muscovado, the enteric microorganism regulation of new fine jade tetrose and recovery capability are most strong.The present invention can
Realize the efficient preparation of the new fine jade tetrose of high-purity, and the new fine jade tetrose for preparing has good gut flora regulating power, be with
New fine jade tetrose is that development of raw materials functional food and health products provide theory and technology support, with good application potential with
Prospect.
Brief description of the drawings
The TLC analyses of the new fine jade tetrose of Fig. 1 enzymolysis products.
Each experimental mice excrement total bacteria count spirograms of Fig. 2.
Each experimental mice faecal bifidobacteria quantity figures of Fig. 3.
Each experimental mice excrement lactobacillus quantity figures of Fig. 4.
Each experimental mice excrement heavy wall bacterium number spirograms of Fig. 5.
Each experimental mice Bacteroides stercoris quantity figures of Fig. 6.
Fig. 7 mouse intestinal flora DGGE electrophoretograms.
Fig. 8 DGGE electrophoresis pattern virtual graphs.
Specific embodiment
The present invention will be further described with specific embodiment below in conjunction with the accompanying drawings.
Following examples prepare new fine jade tetrose using two kinds of β-agarase concerted catalysis agaroses, and to new fine jade tetrose sterling
Gut flora adjustment effect evaluated.The β used in embodiment-agarase AgWH50B and β-agarase AgOGA118
Gene can bite clone in agar-agar bacterium WH0801 (NRRL B-59247 (T) or CGMCC 1.10131 (T)) and obtain from faint yellow,
Can also be obtained by gene chemical synthesis according to two kinds of nucleotide sequences of enzyme gene of coding.
Embodiment 1：The preparation of AgWH50B enzyme liquids
According to AgWH50B sequences using the-CCGGAATTCATGACATTTACTAAAAGC-3 ' and 5 ' of primer 5 '-
CGAGCTCTTTTTTTTGTAACGCAG-3 ', with the faint yellow agar-agar bacterium WH0801 that bites as template enters performing PCR amplification, will expand
To AgWH50B genes be connected to pET21a carriers, build AgWH50B expression vectors pET50B.Expression vector is transferred to large intestine
In bacillus BL21 (DE3), recombination bacillus coli PET50B/BL21 is obtained.By recombination bacillus coli in 5052 self-induction culture mediums
In 20 DEG C of culture 36h, culture finishes thalline is collected by centrifugation.Using the resuspended thalline of buffer solution of pH 7.0, ultrasonication, in centrifuging and taking
Clearly, AgWH50B enzyme liquids are obtained.
Embodiment 2：The preparation of AgOGA118 enzyme liquids
According to AgOGA118 sequences using the-CGGAATTCATGTTAAAGCGCCACC-3 ' and 5 ' of primer 5 '-
CCCTCGAGTTGGCAAGTATAACCTGAC-3 ', with the faint yellow agar-agar bacterium WH0801 that bites as template enters performing PCR amplification, will expand
The AgOGA118 genes for obtaining are connected to pET21a carriers, build AgOGA118 expression vectors pET118.Expression vector is transferred to
In e. coli bl21 (DE3), recombination bacillus coli PET118/BL21 is obtained.Recombination bacillus coli is trained in 5052 self-inductions
20 DEG C of culture 36h in base are supported, culture finishes and thalline is collected by centrifugation.Using the resuspended thalline of buffer solution of pH 7.0, ultrasonication, centrifugation
Supernatant is taken, AgOGA118 enzyme liquids are obtained.
Embodiment 3：AgWH50B digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgWH50B enzyme liquids,
It is well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 8h.Enzymolysis is concentrated under reduced pressure in 60 DEG C by enzymolysis liquid after terminating, and concentrate adds
Enter 3 times of volume absolute ethyl alcohols, in 9000rpm, 4 DEG C of low-temperature centrifugation 15min collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant
After 0.45 μm of membrane filtration, the purifying of Bio-Gel P2 posts is crossed, TLC detections, freeze-drying sample after purification obtains new fine jade tetrose pure
Product.It is 96% to be computed the new fine jade tetrose purity of gained, and substrate conversion efficiency is 52%.
Embodiment 4：AgWH50B digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgWH50B enzyme liquids,
By weight 1:1000 add obsidian powder, well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 8h.Enzymolysis will after terminating
Enzymolysis liquid is concentrated under reduced pressure in 60 DEG C, and concentrate adds 3 times of volume absolute ethyl alcohols, and in 9000rpm, 4 DEG C of low-temperature centrifugation 15min are received
Collection supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant crosses the purifying of Bio-Gel P2 posts after 0.45 μm of membrane filtration, and TLC detections are cold
Dry sample after purification is freezed, new fine jade tetrose sterling is obtained.It is 97% to be computed the new fine jade tetrose purity of gained, and substrate conversion efficiency is
88%.
Embodiment 5：AgWH50B digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgWH50B enzyme liquids,
It is well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 24h.Enzymolysis is concentrated under reduced pressure in 60 DEG C by enzymolysis liquid after terminating, and concentrate adds
Enter 3 times of volume absolute ethyl alcohols, in 9000rpm, 4 DEG C of low-temperature centrifugation 15min collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant
After 0.45 μm of membrane filtration, the purifying of Bio-Gel P2 posts is crossed, TLC detections, freeze-drying sample after purification obtains new fine jade tetrose pure
Product.It is 96% to be computed the new fine jade tetrose purity of gained, and substrate conversion efficiency is 71%.
Embodiment 6：AgWH50B digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgWH50B enzyme liquids,
By weight 1:1000 add obsidian powder, well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 24h.Enzymolysis will after terminating
Enzymolysis liquid is concentrated under reduced pressure in 60 DEG C, and concentrate adds 3 times of volume absolute ethyl alcohols, and in 9000rpm, 4 DEG C of low-temperature centrifugation 15min are received
Collection supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant crosses the purifying of Bio-Gel P2 posts after 0.45 μm of membrane filtration, and TLC detections are cold
Dry sample after purification is freezed, new fine jade tetrose sterling is obtained.It is 96% to be computed the new fine jade tetrose purity of gained, and substrate conversion efficiency is
91%.
Embodiment 7：AgOGA118 digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgOGA118 enzymes
Liquid, it is well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 8h.In 60 DEG C be concentrated under reduced pressure enzymolysis liquid after terminating by enzymolysis, concentrate
3 times of volume absolute ethyl alcohols are added, in 9000rpm, 4 DEG C of low-temperature centrifugation 15min collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, on
Clear to cross the purifying of Bio-Gel P2 posts after 0.45 μm of membrane filtration, TLC detections, freeze-drying sample after purification obtains Neoagarooctaose
With Neoagarodecaose muscovado.
Embodiment 8：AgOGA118 digests agarose
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 1:100 add AgOGA118 enzymes
Liquid, by weight 1:1000 add obsidian powder, well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 8h.After enzymolysis terminates
Enzymolysis liquid is concentrated under reduced pressure in 60 DEG C, and concentrate adds 3 times of volume absolute ethyl alcohols, in 9000rpm, 4 DEG C of low-temperature centrifugation 15min,
Collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant crosses the purifying of Bio-Gel P2 posts after 0.45 μm of membrane filtration, and TLC is detected,
Freeze-drying sample after purification, obtains new fine jade tetrose sterling.It is 91% to be computed the new fine jade tetrose purity of gained, and substrate conversion efficiency is
80%.
Embodiment 9：AgWH50B and AgOGA118 coordinated enzymatic hydrolysis agaroses
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 0.5:100 are separately added into
AgWH50B and AgOGA118 enzyme liquids, it is well mixed to carry out enzyme digestion reaction after 40 DEG C of incubation 8h.Enzymolysis terminate after by enzymolysis liquid in
60 DEG C are concentrated under reduced pressure, and concentrate adds 3 times of volume absolute ethyl alcohols, and in 9000rpm, 4 DEG C of low-temperature centrifugation 15min collect supernatant.In
60 DEG C of supernatants concentrated under reduced pressure, supernatant crosses the purifying of Bio-Gel P2 posts, TLC detections, freeze-drying purifying after 0.45 μm of membrane filtration
Sample, obtains new fine jade tetrose sterling afterwards, as shown in figure 1, being computed gained, newly fine jade tetrose purity is the TLC testing results of sample
96%, substrate conversion efficiency is 93%.
Embodiment 10：AgWH50B and AgOGA118 coordinated enzymatic hydrolysis agaroses
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 0.5:100 are separately added into
AgWH50B and AgOGA118 enzyme liquids, by weight 1:1000 add obsidian powder, well mixed to be carried out after 40 DEG C of incubation 8h
Enzyme digestion reaction.Enzymolysis is concentrated under reduced pressure in 60 DEG C by enzymolysis liquid after terminating, and concentrate adds 3 times of volume absolute ethyl alcohols, in
9000rpm, 4 DEG C of low-temperature centrifugation 15min, collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant after 0.45 μm of membrane filtration, mistake
Bio-Gel P2 posts are purified, and TLC detections, freeze-drying sample after purification obtains new fine jade tetrose sterling, are computed the new fine jade four of gained
Sugared purity is 98%, and substrate conversion efficiency is 99%.
Embodiment 11：AgWH50B and AgOGA118 coordinated enzymatic hydrolysis agaroses
0.5% agarose solution is configured using the buffer solution of pH 7.0, by weight 0.5:100 are separately added into
AgWH50B and AgOGA118 enzyme liquids, by weight 1:1000 add lady's-grass extract, well mixed to be carried out after 40 DEG C of incubation 8h
Enzyme digestion reaction.Enzymolysis is concentrated under reduced pressure in 60 DEG C by enzymolysis liquid after terminating, and concentrate adds 3 times of volume absolute ethyl alcohols, in
9000rpm, 4 DEG C of low-temperature centrifugation 15min, collect supernatant.In 60 DEG C of supernatants concentrated under reduced pressure, supernatant after 0.45 μm of membrane filtration, mistake
Bio-Gel P2 posts are purified, and TLC detections, freeze-drying sample after purification obtains new fine jade tetrose sterling, are computed the new fine jade four of gained
Sugared purity is 98%, and substrate conversion efficiency is 99%.
The extracting method of above-mentioned lady's-grass extract is：The cauline leaf of lady's-grass is dried, is clayed into power, plus 3-5 times of parts by weight
70% ethanol solution refluxing extraction 3 times, merging filtrate is evaporated and is made.
Embodiment 12：Mouse intestinal microorganism adjusts experiment packet
The Balb/c experiment mices that will be bought temporarily are supported one week, to shake down.Keeping each group average mice body weight phase
On the premise of near, 40 mouse are divided into 5 groups at random：It is normal group (NC), natural recovering group (NR), positive controls (FOS), mixed
Sugared group (NAOS) and tetrose group (NAT).Wherein, after modeling is completed, positive controls give FOS gavage, and muscovado group is given
Neoagarooctaose and Neoagarodecaose mixture gavage, tetrose group is given to give new fine jade tetrose gavage, normal group gives with natural recovering group
Normal saline gavage.Mouse is placed in the small mouse cage of Animal House and raises, Animal House room temperature between 20 to 25 DEG C, daily
12h illumination is provided, using bar-shaped feedstuff feeding, drinking water is distilled water.
Embodiment 13：Mouse intestinal microbial state treatment model
After adaptability is fed one week, Ceftriaxone Sodium gavage operation is carried out to remaining four groups in addition to normal group (NC),
Ceftriaxone na concn is 125mg/mL, and every mouse stomach amount is 0.2mL/ each, each gavage operating interval at least 6h.Just
Normal group carries out normal saline gavage, and daily gavage 2 times continues 8 days, to build mouse intestinal flora imbalance model.
Embodiment 14：New fine jade tetrose regulating intestinal canal flora effect detection sample preparation
After modeling successfully, new fine jade tetrose, new fine jade muscovado, FOS etc. is begun to use to carry out gavage.Wherein, normal group
(NC) and natural recovering group (NR) gavage reagent be physiological saline, positive controls (FOS) gavage reagent be FOS (gavage
Dosage is 150mg/kg), muscovado group (NAOS) gives the mix reagent of Neoagarooctaose and Neoagarodecaose, and (given low is 150mg/
Kg), tetrose group gives new fine jade tetrose gavage (given low is 150mg/kg).Gavage is carried out daily to operate 1 time, continues 14 days.
It is accurate at 14 days to weigh 0.1g stool in mice, 1mL 0.01mol/L PBS are added, smash rear whirlpool to pieces with the aseptic pipette tips of pipettor
Concussion is mixed.Sample is centrifuged at room temperature after mixing, and condition is 1500rpm, 5min.Collect supernatant, carry out at a high speed from
The heart, condition is that room temperature 10000rpm is centrifuged 3min, and collects thalline is precipitated, as far as possible supernatant discarded.Take precipitation 1mL 0.01mol/L
PBS solution is washed 2 times, and the aseptic water washings of 1mL 1 time are centrifuged (10000rpm, 3min) again at room temperature, abandon supernatant, are obtained
Obtain final bacterial sediment.
Embodiment 15：The RT-PCR analyses of new fine jade tetrose regulating intestinal canal flora effect
Extract the DNA in bacterial sediment, feature flora Bifidobacterium, lactobacillus, heavy wall bacterium according to enteric microorganism and
Bacteroid DNA characteristicses primers, carry out RT-PCR amplifications.Meanwhile, analyze common beneficial bacteria of intestinal tract using DGGE methods
The change of species.The feature Flora dynamics and total bacteria count result of variations of wherein each experimental group enteric microorganism are as shown in Fig. 2-Fig. 6.
It can be seen from testing result, in terms of total bacteria count, each experimental group is obviously improved compared with natural recovering group, total bacteria count, wherein low
Fructooligosaccharides and new fine jade tetrose effect are the most notable.Prove that new fine jade oligosaccharides has proliferation function to beneficial bacteria of intestinal tract, wherein new fine jade tetrose
Cultivation effect is better than new fine jade muscovado.In prebiotic bacterium amount context of detection, new fine jade tetrose is to Bifidobacterium, lactobacillus, heavy wall bacterium and plan
The all good facilitation of the propagation of bacillus, and the facilitation effect of new fine jade tetrose is better than new fine jade muscovado.
Embodiment 16：The DGGE analyses of new fine jade tetrose regulating intestinal canal flora effect
The DNA in bacterial sediment is extracted, entering performing PCR amplification using 16S rDNA universal primers obtains genes of interest fragment,
And carry out after electrophoresis determines to expand successfully for DGGE analyses, analysis result is as shown in Figure 7 and Figure 8.Compared with normal group, each group
There is certain displacement in flora band.And compared with model group, muscovado group and tetrose group bin number also have certain with composition
Difference, but still compare similar with model group, the trend that wherein the oriented normal group of tetrose group is recovered.Result above is pointed out, addition
Especially new fine jade tetrose can to a certain extent improve mouse intestinal flora structure for FOS, new fine jade oligosaccharides.
The present invention can efficiently prepare new fine jade tetrose by enzymatic isolation method, and the new fine jade tetrose for preparing has preferably regulation intestines
Road flora effect, these features make the art of this patent and product have good development and application in functional food and field of health care products
Prospect.
Claims (7)
1. a kind of preparation method of new fine jade tetrose, it is characterised in that carry out in accordance with the following steps：
(1) configuration quality fraction is the agarose solution of 0.3-0.7%；
(2) by weight (0.2-1):100 are separately added into β-agarase AgWH50B enzyme liquids and β-agarase AgOGA118 enzyme liquids,
35-45 DEG C of incubation 6-10h, obtains enzymolysis liquid；
(3) it is enzymolysis liquid is concentrated under reduced pressure in 55-65 DEG C, 2-5 times of volume absolute ethyl alcohol is added, in 8000-10000rpm, 0-8 DEG C
Low-temperature centrifugation 10-20min, collects supernatant；
(4) in 55-65 DEG C of supernatant concentrated under reduced pressure, after 0.35-0.55 μm of membrane filtration, over-molecular sieve is purified supernatant, freeze-drying
Sample, obtains new fine jade tetrose sterling after purification.
2. a kind of preparation method of new fine jade tetrose according to claim 1, it is characterised in that pH 7.0 is used in step (1)
Buffer solution configuration agarose solution.
3. a kind of preparation method of new fine jade tetrose according to claim 1, it is characterised in that the molecular sieve is Bio-Gel
P2 posts.
4. a kind of preparation method of new fine jade tetrose according to claim 1, it is characterised in that step (4) over-molecular sieve is purified
Afterwards, detect that purity reaches 90% carries out freeze-drying purifying again by TLC.
5. the new fine jade tetrose that prepared by claim 1 is in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
Using.
6. according to claim 5 new fine jade tetrose in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
Application, it is characterised in that the new fine jade tetrose promotes Bifidobacterium in enteron aisle, lactobacillus, the propagation of heavy wall bacterium and bacteroid.
7. according to claim 5 new fine jade tetrose in terms of intestinal flora of mammals structure and intestinal beneficial bacterium propagation is recovered
Application, it is characterised in that the new fine jade tetrose can improve mouse intestinal flora structure.
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| CN111629733A (en) * | 2017-11-16 | 2020-09-04 | 高丽大学校产学协力团 | Method for producing algal-derived agarotriose and use thereof as probiotic |
| CN113736837A (en) * | 2021-10-11 | 2021-12-03 | 成都大学 | Method for preparing new agaro-oligosaccharide from agar |
| CN115089596A (en) * | 2022-06-20 | 2022-09-23 | 自然资源部第三海洋研究所 | New application of new agaro-oligosaccharide in preparing product for treating depression |
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Application publication date: 20170613 |