CN105440153A - Water-insoluble exopolysaccharide of leuconostoc mesenteroides and preparation method thereof - Google Patents
Water-insoluble exopolysaccharide of leuconostoc mesenteroides and preparation method thereof Download PDFInfo
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Abstract
The invention discloses water-insoluble exopolysaccharide of leuconostoc mesenteroides and a preparation method thereof. The water-insoluble exopolysaccharide of leuconostoc mesenteroides is glucan of which the main chain is formed through connection of alpha-(1,6) glycosidic bonds and the branched chain is formed through connection of alpha-(1,3) and alpha-(1,4) glycosidic bonds, wherein the molar ratio of the glycosidic bonds alpha-(1,6), alpha-(1,3) and alpha-(1,4) is 1:0.2-0.3:0.3-0.4. Compared with other water-insoluble polysaccharide generated by bacterial strains coming from other sources, the water-insoluble exopolysaccharide possesses a special structure, and is a water-insoluble glucan simultaneously possessing alpha-(1,3) and alpha-(1,4) glycosidic bonds. The source of the water-insoluble exopolysaccharide is safe and reliable, the preparation method is relatively simple, is relatively suitable for industrialized production and application, and possesses extremely extensive application prospect.
Description
Technical field
The invention belongs to biological technical field, water-insoluble exocellular polysaccharide being specifically related to a kind of Leuconostoc mesenteroides and preparation method thereof.
Background technology
Water-insoluble glucan (Insolubleglucan, IG) be the water insoluble solution of a class, take glucose as a kind of carbohydrate polymer that one-component is polymerized, the carbohydrate not only wide material sources that this kind of physico-chemical property is special, all report is had in the meta-bolites of plant, fungi and bacterium, and of a great variety.The more IG of current report has dextran, curdlan and cellulose tri-kinds, from constructional feature, the branch of these IG reported is only by α-(1,2), α-(1,3) or α-(1,4) side chain and main chain couple together by the one in glycosidic link, seldom relate to the branched structure of 2 kinds or more glycosidic links.Therefore, the screening of new configuration IG is not only IG family with discovery and adds new member, and has greatly promoted the Disciplinary Frontiers of IG structure about research.
Summary of the invention
Technical problem to be solved by this invention is that current water-insoluble polysaccharide source is narrow, the syndeton glycosidic link type of side chain and main chain is single, preparation method is loaded down with trivial details, for the preparation of bacterial classification be the problems such as harmful bacteria, water-insoluble exocellular polysaccharide that the invention provides a kind of Leuconostoc mesenteroides and preparation method thereof.
The present inventor finds that preserving number is the water-insoluble exocellular polysaccharide that the fermented liquid preparation obtained after Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749 of CGMCCNo.10064 cultivates in synthetic medium can have special construction, and contriver completes the present invention thus.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides, described water-insoluble exocellular polysaccharide is that a kind of main chain is by α-(1,6) glycosidic link is formed by connecting, side chain is by α-(1,3) and α-(1,4) dextran that is formed by connecting of glycosidic link, wherein, glycosidic link α-(1,6): the molar ratio of α-(1,3): α-(Isosorbide-5-Nitrae) is 1: 0.2 ~ 0.3: 0.3 ~ 0.4.
Described water-insoluble exocellular polysaccharide is the water-insoluble glucan that one has α-(1,3) and α-(Isosorbide-5-Nitrae) glycosidic link simultaneously, preferably in white powder.
Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749 that described water-insoluble exocellular polysaccharide is preferably CGMCCNo.10064 by deposit number produces.
The water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides can be prepared by following preparation method and obtain as previously mentioned.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of preparation method of water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides, and described preparation method comprises the following steps:
1) be inoculated in substratum by Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749, fermentation culture obtains fermented liquid;
2) by step 1) after the lyophilize of gained fermented liquid, get the lixiviate of lyophilized powder alkali lye, get supernatant, regulate pH to 7, get precipitation, lyophilize and get final product.
Step 1 of the present invention) be: be inoculated in substratum by Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749, fermentation culture obtains fermented liquid.
Leuconostoc mesenteroides BD3749 described in preparation method of the present invention is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on November 26th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101.The Classification And Nomenclature of the suggestion of this bacterial strain is: Leuconostoc mesenteroides Leuconostocmesenteroides, and the deposit number of this bacterial strain is: CGMCCNo.10064, and the culture name of institute's preservation is called: BD3749.
The preparation method of Leuconostoc mesenteroides BD3749 of the present invention is conventional, preferably also comprises this Leuconostoc mesenteroides BD3749 and activates the step obtaining Leuconostoc mesenteroides seed.Described Leuconostoc mesenteroides seed is that ordinary method obtains, method preferably for comprising the following steps: the lyophilized powder of Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749 is dissolved with a small amount of sterile distilled water, getting with transfering loop the M17 solid medium that a ring lines containing 5% sucrose (buys from OXOID, Britain), 30 DEG C of aerobic cultivation 48h take out, putting into 10mL with transfering loop picking list bacterium colony (buys from OXOID containing the M17 liquid nutrient medium of 5% sucrose, Britain), turbula shaker is used to be dispersed in liquid nutrient medium by bacterium colony, 30 DEG C, 180rpm shaking culture 24h takes out, (buy from OXOID with the M17 liquid nutrient medium that 2% (v/v) inoculum size is inoculated in containing 5% sucrose, Britain), 30 DEG C, after 180rpm shaking culture 24h, by culture 15, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed (also known as Leuconostoc mesenteroides seed liquor) fermented.The lyophilized powder of described Leuconostoc mesenteroides BD3749 is prepared by following methods: picking 2 bacterium colonies, and cultivate 4h for 30 DEG C in the skimming milk solution being inoculated in 10% (w/w) of 300mL ,-80 DEG C of pre-freezes are spent the night ,-20 DEG C of lyophilizes.
Wherein, described substratum is the substratum of this area routine, as long as can cultivate Leuconostoc mesenteroides, be preferably synthetic medium, described synthetic medium comprises following component: peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034% and sucrose 5 ~ 30%, the content of sucrose is preferably 10 ~ 20%, and be more preferably 10%, surplus is water, and described per-cent is mass percent.The preparation method of described synthetic medium is the preparation method of this area routine.The pH of described substratum is preferably 5 ~ 8, is more preferably 7.
Wherein, described fermentation culture is this area conventional culture methods, and the inoculum size of described Leuconostoc mesenteroides is preferably 1 ~ 5%, is more preferably 2 ~ 4%, and be 3% best, described per-cent is volume percent.Described fermentation culture is preferably shaking culture, and described shaking culture is conventional, and hunting speed is preferably 100 ~ 300rpm, is more preferably 150 ~ 250rpm, is 200rpm best.The leavening temperature of described fermentation culture is preferably 15 ~ 37 DEG C, is more preferably 25 DEG C ~ 33 DEG C, is 30 DEG C best, and fermentation time is preferably 24 ~ 96 hours, is more preferably 48 ~ 84 hours, is 72 hours best.
Step 2 of the present invention) be: by step 1) after the lyophilize of gained fermented liquid, get the lixiviate of lyophilized powder alkali lye, get supernatant, regulate pH to 7, get precipitation, lyophilize and get final product.Wherein, described lyophilize is freeze-drying (i.e. lyophilize) technology of this area routine, as long as reach removal moisture, obtains the object of solid substance.Described alkali lye is conventional, and be preferably NaOH solution, the concentration of described NaOH solution is 1 ~ 5M, is preferably 2 ~ 4M, is more preferably 3M.Described lixiviate is conventional, and during described lixiviate, the ratio of lyophilized powder and alkali lye is 1g: 1mL ~ 1g: 5mL, is preferably 1g: 2mL ~ 1g: 4mL, is more preferably 1g: 3mL.Described supernatant is that ordinary method obtains, and is preferably centrifugal, described centrifugal be conventional, centrifugal speed is preferably 8,000 ~ 15,000g, is more preferably 10,000 ~ 12,000g, and the centrifugal time is preferably 5 ~ 15 minutes, is more preferably 8 ~ 12 minutes.Described pH uses ordinary method to regulate, and is preferably to regulate by acid solution, and described acid solution is conventional, and be preferably HCl solution, described HCl strength of solution is 1 ~ 3M, is preferably 1.5 ~ 2.5M, is more preferably 2M.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
1, the invention solves current water-insoluble polysaccharide (especially water-insoluble glucan) to originate narrow present situation, the source for water-insoluble polysaccharide provides alternative selection.
2, the water-insoluble exocellular polysaccharide adopting the present invention to prepare has two kinds of rare branched glucosidic keys and the form of depositing, and is a kind of polysaccharide of novel structure, and the theory also relatively many according to the polysaccharide function that structure is more complicated, therefore, has certain development significance.
The step (as SAVAGE method Deproteinization) of Deproteinization is often relate in the preparation process of 3, conventional polysaccharide, but fermented liquid does not regulate the direct lyophilize of pH in preparation method of the present invention, when acid fermentation liquid is by progressively freeze-drying dehydration, pH constantly declines, cause local overacidification, cause the rapid sex change of the floating preteins in fermented liquid, this part albumen is centrifuged removing when alkali lye lixiviate, thus reaches the object of Deproteinization.
4, Leuconostoc mesenteroides belongs to milk-acid bacteria category, so compared with the insoluble polysaccharide produced with other bacterial strains of originating (as sporeformer, aerogen, mould etc.), the exocellular polysaccharide from Leuconostoc mesenteroides has higher food safety.The present invention prepare gained extracellular polysaccharide extractive can as functional additive substitute other source water-insoluble polysaccharides be applied in food, pharmacy and association area, its application prospect is very wide.
biomaterial preservation information
Leuconostoc mesenteroides BD3749 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on November 26th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is: CGMCCNo.10064, culture title is BD3749, and Classification And Nomenclature is Leuconostoc mesenteroides Leuconostocmesenteroides.
Accompanying drawing explanation
Fig. 1 shows the monose composition measuring result of Leuconostoc mesenteroides BD3749 water-insoluble exocellular polysaccharide.
Fig. 2 shows the infrared measurement result of Leuconostoc mesenteroides BD3749 water-insoluble exocellular polysaccharide.
Fig. 3 shows the nuclear magnetic resonance measuring result of Leuconostoc mesenteroides BD3749 water-insoluble exocellular polysaccharide.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
" room temperature " described in the present invention refers to the temperature of carrying out the operation room tested, and is generally 15 ~ 25 DEG C, if the reagent used in embodiment does not add explanation, is analytical reagent, buys from traditional Chinese medicines group.
The preparation of the water-insoluble exocellular polysaccharide of embodiment 1 Leuconostoc mesenteroides
1, materials and methods
The preparation of seed (fermented bacterium): the lyophilized powder of Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749 is dissolved with a small amount of sterile distilled water, getting with transfering loop the M17 solid medium that a ring lines containing 5% sucrose (buys from OXOID, Britain), 30 DEG C of aerobic cultivation 48h take out, putting into 10mL with transfering loop picking list bacterium colony (buys from OXOID containing the M17 liquid nutrient medium of 5% sucrose, Britain), turbula shaker is used to be dispersed in liquid nutrient medium by bacterium colony, 30 DEG C, 180rpm shaking culture 24h takes out, (buy from OXOID with the M17 liquid nutrient medium that 2% (v/v) inoculum size is inoculated in containing 5% sucrose, Britain), 30 DEG C, after 180rpm shaking culture 24h, by culture 15, centrifugal 10 minutes of 000rpm, supernatant discarded, after thalline sterile distilled water washs 2 times, suspend with the sterile distilled water of former volume of culture, obtain the seed (also known as Leuconostoc mesenteroides seed liquor) fermented.The lyophilized powder of described Leuconostoc mesenteroides BD3749 is prepared by following methods: picking 2 bacterium colonies, and cultivate 4h for 30 DEG C in the skimming milk solution being inoculated in 10% (w/w) of 300ml ,-80 DEG C of pre-freezes are spent the night ,-20 DEG C of lyophilizes.
Synthetic medium (peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034%, sucrose 10%, described per-cent is mass percent) preparation: by peptone 10g, yeast extract 5g, sucrose 100g, K
2hPO
420g and CaCl
20.034g and 1L distilled water mixes, after fully dissolving, with Na
2cO
3regulate pH to 7, namely sterilizing obtains the synthetic medium of required sucrose concentration.
2, the preparation of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
It is 10% that Leuconostoc mesenteroides seed liquor is inoculated in sucrose mass percent by the inoculum size of 3% (v/v), pH is in the synthetic medium of 7,30 DEG C, 200rpm oscillation and fermentation cultivation obtains fermented liquid in 72 hours, after fermented liquid freeze-drying (i.e. lyophilize), by the NaOH solution of lyophilized powder and 3M with 1g: 3mL ratio mixed room temperature lixiviate 2h, 15, the centrifugal 5min of 000rpm, get supernatant, regulate pH to 7,15 with the HCl solution of 2M, 000rpm is centrifugal, and 5min gets precipitation, and namely lyophilize obtains water-insoluble exocellular polysaccharide A.
3, the output of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
Through weighing and calculating, the output obtaining BD3749 water-insoluble exocellular polysaccharide A is 28.75g/L.
The preparation of the water-insoluble exocellular polysaccharide of embodiment 2 Leuconostoc mesenteroides
1, materials and methods
The preparation of seed (fermented bacterium): with embodiment 1.
Synthetic medium (peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034%, sucrose 30%, described per-cent is mass percent) preparation: by peptone 10g, yeast extract 5g, sucrose 300g, K
2hPO
420g and CaCl
20.034g and 1L distilled water mixes, after fully dissolving, with NaHCO
3regulate pH to 8, namely sterilizing obtains the synthetic medium of required sucrose concentration.
2, the preparation of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
It is 30% that Leuconostoc mesenteroides seed liquor is inoculated in sucrose mass percent by the inoculum size of 5% (v/v), pH is in the synthetic medium of 8,15 DEG C, 300rpm oscillation and fermentation cultivation obtains fermented liquid in 24 hours, after fermented liquid freeze-drying, by the NaOH solution of lyophilized powder and 1M with 1g: 5mL ratio mixed room temperature lixiviate 2h, 8, the centrifugal 15min of 000rpm, get supernatant, regulate pH to 7,8 with the HCl solution of 3M, 000rpm is centrifugal, and 15min gets precipitation, and namely lyophilize obtains water-insoluble exocellular polysaccharide B.
3, the output of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
Through weighing and calculating, the output obtaining BD3749 water-insoluble exocellular polysaccharide B is 12.48g/L.
The preparation of the water-insoluble exocellular polysaccharide of embodiment 3 Leuconostoc mesenteroides
1, materials and methods
The preparation of seed (fermented bacterium): with embodiment 1.
Synthetic medium (peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034%, sucrose 5%, described per-cent is mass percent) preparation: by peptone 10g, yeast extract 5g, sucrose 50g, K
2hPO
420g and CaCl
20.034g and 1L distilled water mixes, and after fully dissolving, regulate pH to 5 with NaOH, namely sterilizing obtains the synthetic medium of required sucrose concentration.
2, the preparation of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
It is 5% that Leuconostoc mesenteroides seed liquor is inoculated in sucrose mass percent by the inoculum size of 1% (v/v), pH is in the synthetic medium of 5,37 DEG C, 100rpm oscillation and fermentation cultivation obtains fermented liquid in 96 hours, after fermented liquid freeze-drying, by the NaOH solution of lyophilized powder and 5M with 1g: 1mL ratio mixed room temperature lixiviate 2h, 10, the centrifugal 12min of 000rpm, get supernatant, regulate pH to 7,10 with the HCl solution of 1M, 000rpm is centrifugal, and 12min gets precipitation, and namely lyophilize obtains water-insoluble exocellular polysaccharide C.
3, the output of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
Through weighing and calculating, the output obtaining BD3749 water-insoluble exocellular polysaccharide C is 6.94g/L.
The preparation of the water-insoluble exocellular polysaccharide of embodiment 4 Leuconostoc mesenteroides
1, materials and methods
The preparation of seed (fermented bacterium): with embodiment 1.
Synthetic medium (peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034%, sucrose 20%, described per-cent is mass percent) preparation: by peptone 10g, yeast extract 5g, sucrose 200g, K
2hPO
420g and CaCl
20.034g and 1L distilled water mixes, after fully dissolving, with Na
2cO
3and NaHCO
3(regulate pH to 6, namely sterilizing obtains the synthetic medium of required sucrose concentration.
2, the preparation of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
It is 20% that Leuconostoc mesenteroides seed liquor is inoculated in sucrose mass percent by the inoculum size of 2% (v/v), pH is in the synthetic medium of 6,25 DEG C, 250rpm oscillation and fermentation cultivation obtains fermented liquid in 48 hours, after fermented liquid freeze-drying, by the NaOH solution of lyophilized powder and 4M with 1g: 2mL ratio mixed room temperature lixiviate 2h, 12, the centrifugal 8min of 000rpm, get supernatant, regulate pH to 7,12 with the HCl solution of 1.5M, 000rpm is centrifugal, and 8min gets precipitation, and namely lyophilize obtains water-insoluble exocellular polysaccharide D.
3, the output of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
Through weighing and calculating, the output obtaining BD3749 water-insoluble exocellular polysaccharide D is 16.34g/L.
The preparation of the water-insoluble exocellular polysaccharide of embodiment 5 Leuconostoc mesenteroides
1, materials and methods
The preparation of seed (fermented bacterium): with embodiment 1.
Synthetic medium (peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034%, sucrose 15%, described per-cent is mass percent) preparation: by peptone 10g, yeast extract 5g, sucrose 150g, K
2hPO
420g and CaCl
20.034g and 1L distilled water mixes, after fully dissolving, with Na
2cO
3, NaHCO
3regulate pH to 6.5 with NaOH, namely sterilizing obtains the synthetic medium of required sucrose concentration.
2, the preparation of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
It is 15% that Leuconostoc mesenteroides seed liquor is inoculated in sucrose mass percent by the inoculum size of 4% (v/v), pH is in the synthetic medium of 6.5,33 DEG C, 150rpm oscillation and fermentation cultivation obtains fermented liquid in 84 hours, after fermented liquid freeze-drying, by the NaOH solution of lyophilized powder and 2M with 1g: 4mL ratio mixed room temperature lixiviate 2h, 14, the centrifugal 7min of 000rpm, get supernatant, regulate pH to 7,14 with the HCl solution of 2.5M, 000rpm is centrifugal, and 7min gets precipitation, and namely lyophilize obtains water-insoluble exocellular polysaccharide E.
3, the output of the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides
Through weighing and calculating, the output obtaining BD3749 water-insoluble exocellular polysaccharide E is 26.41g/L.
The monose composition measuring of the water-insoluble exocellular polysaccharide of embodiment 6 Leuconostoc mesenteroides
Get exocellular polysaccharide A prepared by aforesaid method, form with the monose of high-efficiency anion chromatography determination polysaccharide.
(1) polysaccharide hydrolysis
Take 3mg exocellular polysaccharide A and 2mL4moL/LTFA (trifluoroacetic acid, purchased from Sigma-AldrichCo.LLC, the U.S.) fully to mix, fill N
2tube sealing, is hydrolyzed 20h in 110 DEG C of baking ovens; Open lid after cooling, after adding 200 μ L methyl alcohol, use N
2dry up, so repeat to add methyl alcohol and use N
2blow 3 times, remove TFA, its residue water dissolution is settled to 5mL, supply sample introduction analysis with after 0.45 μm of micro-pore-film filtration.
(2) chromatographic condition
Chromatographic column: CarboPacPA203mmi.d. × 150mm;
Moving phase: A, H
2o; B, 250mmol/LNaOH; C, 1mol/LCH
3cOONa;
Ternary gradient elution: flow velocity: 0.5mL/min; Integrated pulsed amperometric detection device;
Au working electrode: Ag/AgCl reference electrode,
Sampling volume: 20 μ L; Column temperature: 30 DEG C.
The stratographic analysis of the glycosyl composition of Leuconostoc mesenteroides BD3749 water-insoluble exocellular polysaccharide A the results are shown in Figure 1, and this polysaccharide has single absorption peak at 5.875min, and the retention time of this absorption peak is consistent with glucose mark product retention time.
Conclusion: Leuconostoc mesenteroides BD3749 water-insoluble exocellular polysaccharide is made up of the single glycosyl of glucose, is a kind of dextran.
The structural determination of the water-insoluble exocellular polysaccharide of embodiment 7 Leuconostoc mesenteroides
(1) FTIR spectrum (FTIR) measures
After the water-insoluble exocellular polysaccharide A prepared by aforesaid method fully mixes with potassium bromide powder, be pressed into sheet, application Fourier infrared spectrograph (Nicolet6700, ThermoFisher company, the U.S.) is carried out FTIR spectrum (FTIR) and is measured.
Result as shown in Figure 2,919cm
-1the absorption peak at place proves that the carbon skeleton of this polysaccharide is made up of α-glucosides, 1004cm
-1the absorption peak at place shows to there is a large amount of α-(1,6) glycosidic link in this polysaccharide, 1102cm
-1place is the absorption peak of C-O key chattering in glucosyl residue, 1147cm
-1the absorption peak at place is the result of the stretching vibration of C-O-C on glycosidic link bridge.3281cm
-1the strong broad peak at place is intermolecular and hydroxyl group absorption peak in molecule, 2922cm
-1for the absorption peak of c h bond stretching vibration, 1645cm
-1the absorption peak at place is caused by Bound moisture.
Conclusion: BD3749 water-insoluble exocellular polysaccharide is mainly formed with α-(1,6) glycosidic link.
(2) nucleus magnetic resonance (NMR) measures
The water-insoluble exocellular polysaccharide A prepared by aforesaid method is dissolved in the deuterated sodium hydroxide solution of 1M by the concentration of 5mg/mL completely, and using NMR spectrometer (AvanceIII400MHz, Bruker company, Germany) carry out nucleus magnetic resonance (NMR) mensuration.
In Fig. 3, A is NMR-
1h composes, the chemical shift of 7 protons of show sample is respectively 4.945 (H-1), 3.970 (H-6), 3.887 (H-5), 3.787 (H-6 '), 3.705 (H-3), 3.554 (H-2), 3.477 (H-4) pm, 5.223 and the absorption peak at 5.315ppm place be respectively the result of glycosidic link vibration; In Fig. 3, B is NMR-
13c composes, and show sample is at 101.252 (C-1), and there is resonance at 77.245 (C-3), 74.843 (C-2), 73.449 (C-5), 72.850 (C-4) and 68.554 (C-6) ppm place.
Conclusion: FTIR with
1h,
13the data binding analysis of C spectrum shows, BD3749 water-insoluble exocellular polysaccharide is that a kind of main chain is formed by connecting by α-(1,6) glycosidic link, the dextran that side chain is formed by connecting by α-(1,3) and α-(Isosorbide-5-Nitrae) glycosidic link.
The mensuration of the glycosidic link ratio of the water-insoluble exocellular polysaccharide of embodiment 8 Leuconostoc mesenteroides
The BD3749 water-insoluble exocellular polysaccharide of preparation in embodiment 1 ~ 5 is carried out
1the detection of H-NMR, by comparing upper H-1 (α-(1 of color atlas (as Fig. 3 A), 6)), α-(1,3) and α-(1,4) integral area at absorption peak place determines the mol ratio relation of each glycosidic link, and the measurement result of each sample is as shown in table 1.
Table 1 α-(1,6), the mol ratio relation of α-between (1,3) and α-(Isosorbide-5-Nitrae)
As can be seen from Table 1, the molar ratio of glycosidic link α in BD3749 water-insoluble exocellular polysaccharide-(1,6): α-(1,3): α-(Isosorbide-5-Nitrae) is 1: 0.2 ~ 0.3: 0.3 ~ 0.4.
Comparative example 1
By the inoculum size in embodiment 2, synthetic medium pH, culture temperature, fermentation time, the speed of fermentation vibration, sucrose concentration and lixiviate time concentration of lye adjust one by one, obtain with next water-insoluble exocellular polysaccharide of preparing of group different methods, each group output is as shown in table 2.
Table 2 different methods prepares gained water-insoluble exopolysaccharides
Can draw from the result shown in table 2; by inoculum size in the preparation method of exocellular polysaccharide of the present invention; synthetic medium pH; culture temperature; fermentation time; the speed of fermentation vibration, sucrose concentration and lixiviate time concentration of lye when adjusting to outside scope, the output of gained exocellular polysaccharide significantly reduces.All raw materials and reagent are all commercially above.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the water-insoluble exocellular polysaccharide of a Leuconostoc mesenteroides, it is characterized in that, described water-insoluble exocellular polysaccharide is that a kind of main chain is formed by connecting by α-(1,6) glycosidic link, side chain is by α-(1,3) and the dextran that is formed by connecting of α-(Isosorbide-5-Nitrae) glycosidic link, wherein, glycosidic link α-(1,6): the molar ratio of α-(1,3): α-(Isosorbide-5-Nitrae) is 1: 0.2 ~ 0.3: 0.3 ~ 0.4.
2. water-insoluble exocellular polysaccharide as claimed in claim 1, it is characterized in that, Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749 that described water-insoluble exocellular polysaccharide is CGMCCNo.10064 by deposit number produces.
3. a preparation method for the water-insoluble exocellular polysaccharide of Leuconostoc mesenteroides as claimed in claim 1 or 2, it is characterized in that, it comprises the following steps:
1) be inoculated in substratum by Leuconostoc mesenteroides (Leuconostocmesenteroides) BD3749, fermentation culture obtains fermented liquid;
2) by step 1) after the lyophilize of gained fermented liquid, get the lixiviate of lyophilized powder alkali lye, get supernatant, regulate pH to 7, get precipitation, lyophilize and get final product.
4. preparation method as claimed in claim 1, is characterized in that, step 1) described in the preparation method of Leuconostoc mesenteroides also comprise Leuconostoc mesenteroides BD3749 and activate the step obtaining Leuconostoc mesenteroides seed.
5. preparation method as claimed in claim 1, is characterized in that, step 1) described in the inoculum size of Leuconostoc mesenteroides be 1 ~ 5%, being preferably 2 ~ 4%, is more preferably 3%, and described per-cent is volume percent.
6. preparation method as claimed in claim 1, is characterized in that, step 1) described in substratum be synthetic medium, described synthetic medium comprises following component: peptone 1%, yeast extract 0.5%, K
2hPO
40.5%, CaCl
20.034% and sucrose 5 ~ 30%, the content of sucrose is preferably 10 ~ 20%, and be more preferably 10%, surplus is water, and described per-cent is mass percent; Or the pH of described substratum is 5 ~ 8, is preferably 7.
7. preparation method as claimed in claim 1, is characterized in that, step 1) described fermentation culture is shaking culture, hunting speed is preferably 100 ~ 300rpm, is more preferably 150 ~ 250rpm, is 200rpm best.
8. preparation method as claimed in claim 1, is characterized in that, step 1) described in the leavening temperature of fermentation culture be 15 ~ 37 DEG C, be preferably 25 DEG C ~ 35 DEG C, be more preferably 30 DEG C; Or fermentation time is 24 ~ 96 hours, being preferably 48 ~ 84 hours, is more preferably 72 hours.
9. preparation method as claimed in claim 1, is characterized in that, step 2) described alkali lye is NaOH solution, the concentration of described NaOH solution is 1 ~ 5M, is preferably 2 ~ 4M, is more preferably 3M; And/or, step 2) described lixiviate time lyophilized powder and the ratio of alkali lye be 1g: 1mL ~ 1g: 5mL, being preferably 1g: 2mL ~ 1g: 4mL, is more preferably 1g: 3mL.
10. preparation method as claimed in claim 1, is characterized in that, step 2) described supernatant is that centrifuging and taking obtains, centrifugal speed is preferably 8,000 ~ 15,000g is more preferably 10,000 ~ 12,000g, the centrifugal time is preferably 5 ~ 15 minutes, is more preferably 8 ~ 10 minutes; And/or, step 2) described pH be with acid solution regulate, described acid solution is preferably HCl solution, and described HCl strength of solution is 1 ~ 3M, is preferably 1.5 ~ 2.5M, is more preferably 2M.
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| CN109295127B (en) * | 2018-10-31 | 2022-04-01 | 光明乳业股份有限公司 | Alpha-glucan with oxygen pressure exclusion effect and preparation method and application thereof |
| CN112326711A (en) * | 2020-11-12 | 2021-02-05 | 暨南大学 | Analysis method for rapidly acquiring nuclear magnetic resonance carbon spectrum of achyranthes bidentata polysaccharide |
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