CN102250984B - Method for preparing functional extracellular polysaccharide of lactic acid bacteria - Google Patents
Method for preparing functional extracellular polysaccharide of lactic acid bacteria Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 115
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 115
- 150000004676 glycans Chemical class 0.000 title claims abstract description 102
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 39
- 241000894006 Bacteria Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000004310 lactic acid Substances 0.000 title claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 21
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 20
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 19
- 230000026731 phosphorylation Effects 0.000 claims abstract description 17
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 12
- 239000002244 precipitate Substances 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 9
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- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 238000005342 ion exchange Methods 0.000 claims abstract description 5
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- -1 phosphoric acid polysaccharide Chemical class 0.000 claims description 20
- 229910052711 selenium Inorganic materials 0.000 claims description 19
- 239000011669 selenium Substances 0.000 claims description 19
- 229940091258 selenium supplement Drugs 0.000 claims description 19
- 241000194035 Lactococcus lactis Species 0.000 claims description 14
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 14
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- DRWSDIDOJRQEKK-UHFFFAOYSA-N C(C)(=[O+][Se-])O Chemical compound C(C)(=[O+][Se-])O DRWSDIDOJRQEKK-UHFFFAOYSA-N 0.000 claims description 4
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- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 3
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- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 4
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for preparing functional extracellular polysaccharide of lactic acid bacteria, which is characterized by comprising the following steps of: inoculating a Lactococcus lactis subsp. lactis strain fermentation agent for culture, performing ultrafiltration concentration, precipitation and ethanol extraction on fermentation liquor, centrifuging a precipitate, and performing freeze drying to obtain powdery crude extracellular polysaccharide; performing separation and purification by using a diethylaminoethanol (DEAE) cellulose ion exchange column and SepharoseCL-6B gel column chromatography in turn, desalting, performing ultrafiltration concentration, precipitating by using an ethanol solution, and performing freeze drying on a precipitate to obtain pure extracellular polysaccharide; and performing phosphorylation and selenide formation on the pure extracellular polysaccharide. The method has the advantages that: the extracellular polysaccharide produced by the Lactococcus lactis subsp. lactis strain is subjected to selenide formation and phosphorylation modification by a dry heating method, and the functional extracellular polysaccharide of lactic acid bacteria with obvious antioxidant and immunity enhancing effects is obtained.
Description
Technical field
The present invention relates to a kind of Microbial exopolysaccharides, especially relate to a kind of preparation method of functional lactobacillus exocellular polysaccharide.
Background technology
In recent decades, owing to Microbial exopolysaccharides is researched and developed energetically at the peculiar advantage that has aspect product structure, performance and the production, the exploitation of Microbial exopolysaccharides has become one of focus of industrial microorganism research.Milk-acid bacteria exocellular polysaccharide (Exopolysaccharide, EPS) be that milk-acid bacteria is secreted into glue polysaccharide or the capsular polysaccharide outside the cell walls in the growth metabolism process, effects such as they have thickening, emulsification, preserve moisture and stablize, help to improve the rheological properties of cultured milk prod, reduce whey and separate out phenomenon, improve fermented-milk quality and aesthetic quality, some milk-acid bacteria exocellular polysaccharides also have immunity of organisms, the effect such as antitumor regulated.Because milk-acid bacteria is food grade industrial production bacterium, compare with other bacterium safe, so in recent years the research of milk-acid bacteria exocellular polysaccharide is increased gradually, particularly it in cultured milk prod effect be one of focus of Chinese scholars research and development always.But because its output and functionally active are lower, be the principal element one of its scale operation of restriction.
Discover that the activity of polysaccharide is subjected to the influence of its molecular structure directly or indirectly, take certain method that the polysaccharide molecule structure is modified, can improve or give the more activity of polysaccharide, reduce its toxic side effect.The common methods of at present polysaccharide being modified has sulfation, carboxymethylation, acetylize, alkylation, sulfonylation etc.; the biological activity of many natural polysaccharides behind sulphation modification strengthens; especially antiviral, antitumor, anti-AIDS, the body's immunological function aspect is significantly strengthened, thereby receive very big concern.
In recent years, the selenizing of polysaccharide is modified and is also begun to cause people's attention, the investigator carries out selenium-enriching cultivation or cultivation to plant or microorganism, by their growth metabolism, selenium is carried out enrichment and bio-transformation, or modify by the polysaccharide selenizing, selenium or the functional group that contains selenium are grafted on the polysaccharide molecule, to obtain active stronger selenium polysaccharide.Studies have shown that the biological activity of selenium polysaccharide generally is higher than polysaccharide and selenium, be easier to be absorbed by body and utilize, as cultivating the glossy ganoderma selenium polysaccharide that obtains has more significant inhibition tumor growth than ganoderan activity by rich selenium.But the rich selenium of milk-acid bacteria exocellular polysaccharide carries training or cultivates the gained selenium polysaccharide, have the not high deficiency of selenium content, and the selenizing modification technique of existing polysaccharide often uses a large amount of organic solvents, product is dangerous but also contaminate environment easily not only, and has shortcomings such as the selenium structure is single relatively.
Phosphorylation modification is a kind of covalent modification, is the process that the hydroxyl on the side chain is replaced by phosphate radical.The polysaccharide that phosphorylated polysaccharide can make some not have activity has originally shown the antitumor physiologically active of Denging.But mainly concentrate on starch, chitosan and Mierocrystalline cellulose etc. about phosphorylation modification at present, the research of milk-acid bacteria exocellular polysaccharide phosphorylation modification aspect does not see that so far report is arranged.
Summary of the invention
Technical problem to be solved by this invention provides a kind of exocellular polysaccharide that can produce Lactococcus lactis breast subspecies bacterial strain simultaneously and carries out selenizing and phosphorylation modification, make the preparation method of the functional lactobacillus exocellular polysaccharide of selenium content abundance and safety and environmental protection, this functional lactobacillus exocellular polysaccharide has significantly anti-oxidant and immune-enhancing effect.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of preparation method of functional lactobacillus exocellular polysaccharide specifically comprises the steps:
(1) preparation of milk-acid bacteria crude extracellular polysaccharide
With the Lactococcus lactis breast subspecies strain fermentation agent inoculum size of per-cent 3.0-5.0% by volume, be seeded on the improved BLX substratum, cultivate 20 h down at 37 ℃ earlier, cultivate 10-15h down at 28-32 ℃ again, the centrifugal 15-20min of 6000-7000rpm removes the fermented liquid that somatic cells must contain exocellular polysaccharide, then fermented liquid is carried out ultrafiltration and concentration, press 10% the adding Tricholroacetic Acid precipitation of concentrated solution quality again, 0-4
oPlaced 8-12 hour under the C, the centrifugal 15-20min of 6000-7000rpm then, get supernatant liquor and carry out ultrafiltration and concentration, get the Crude polysaccharides concentrated solution, adding volume again is the ethanolic soln of the 90-95% of 3 times of Crude polysaccharides concentrated solutions, leaves standstill 10-12 hour at 4 ℃, then the centrifugal 20-25min of 6000-7000rpm, the taking precipitate lyophilize gets pulverous milk-acid bacteria crude extracellular polysaccharide;
(2) separation and purification of milk-acid bacteria crude extracellular polysaccharide
The milk-acid bacteria crude extracellular polysaccharide that step (1) is obtained is dissolved in a small amount of distilled water, earlier through the separation and purification of DEAE-cellulose ion exchange column, collection contains the elutriant of first elution peak of milk-acid bacteria crude extracellular polysaccharide, freeze concentration is to 5-6mL, again through the separation and purification of Sepharose CL-6B gel filtration chromatography, collection contains the elutriant of second elution peak of milk-acid bacteria crude extracellular polysaccharide, be the daltonian nanofiltration membrane nanofiltration of 280-320 desalination with elutriant through molecular weight cut-off, again through ultrafiltration and concentration, the ethanolic soln precipitation of 90-95%, the taking precipitate lyophilize obtains the outer holosaccharide of lactic acid mycetocyte;
(3) phosphorylation of the outer holosaccharide of lactic acid mycetocyte
The outer holosaccharide of the lactic acid mycetocyte that step (2) is obtained and phosphate solution are in the ratio mixing of mass ratio 6:1～3, and adjusting pH to 4.0-6.0, lyophilize is to Powdered, move in the loft drier then, at 70-90 ℃ of following reacting by heating 4-6h, remove unnecessary salt with the ethanolic soln flushing of 70-75% then, place rotatory evaporator distillation 2-3 hour again, remove residual ethanol, vacuum lyophilization, obtain the solid phosphoric acid polysaccharide, wherein phosphate radical grafting amount reaches 1.64-1.70 mg/g;
(4) selenic acidization
The solid phosphoric acid polysaccharide that step (3) is obtained is soluble in water, and to obtain mass percent be 15% phosphorylated polysaccharide solution, with phosphorylated polysaccharide solution and the selenizing agent ratio mixing in mass ratio 9:1, and adjusting pH to 1.5-2.5, lyophilize is to Powdered, move in the loft drier then, at 45-55 ℃ of following reacting by heating 12-15h, remove unnecessary selenizing agent with the ethanolic soln flushing of 70-75% then, place rotatory evaporator to distill again, remove residual ethanol, put into dialysis tubing, place distilled water dialysis to remove residual selenizing agent, after dialysis finishes, with the polysaccharide soln in the dialysis tubing to going into beaker, the 90-95% ethanol sedimentation that adds 3 times of polysaccharide soln volumes, 4 ℃ left standstill 10-12 hour, the centrifugal 20-25min of 6000-7000rpm, taking precipitate lyophilize, namely get phosphorylation and selenic acid polysaccharide product is the functional lactobacillus exocellular polysaccharide, wherein the content of selenium is 0.17-0.18 mg/g.
The viable count content of described Lactococcus lactis breast subspecies strain fermentation agent reaches 10
5-7Individual/mL.
Described improvement BLX substratum, it is composed as follows: 1.5-2.0% glucose, 1.0-1.2% fructose, the 1.3-1.5% soy peptone, the 1.3-1.5% Tryptones, initial pH is 6.0-6.5.
Ultrafiltration and concentration process described in step (1), the step (2) is: adopting the aperture is housed is 50 kilodalton inner pressed hollow-fibre membranes, be 0.25-0.40MPa at working pressure, service temperature is under 20-25 ℃ the condition, and exocellular polysaccharide in the Lactococcus lactis breast subspecies fermented liquid is carried out ultrafiltration and concentration.
To be phosphoric acid salt and water form by the mixed of mass ratio 20:1 phosphate solution described in the step (3).
Described phosphoric acid salt refers to Sodium hexametaphosphate 99 (Na
6P
6O
18), tripoly phosphate sodium STPP (Na
5P
3O
10) and trisodium phosphate (Na
4P
2O
7) in any; Described selenizing agent refers to acetic acid selenide or Sodium Selenite (Na
2SeO
3) or the two mixture.
Compared with prior art, the invention has the advantages that: the preparation method of a kind of functional lactobacillus exocellular polysaccharide of the present invention, produce crude extracellular polysaccharide by the preparation lactobacillus-fermented, the separation and purification of DEAE-cellulose ion exchange column and gel filtration chromatography post, ultrafiltration and concentration, the nanofiltration desalination, ultrafiltration and concentration, heated phosphoricization and selenic acidization, lyophilize obtains having anti-oxidant and milk-acid bacteria exocellular polysaccharide immunoloregulation function again, this method discloses dry heat legal system the get everything ready phosphorylation of significantly anti-oxidant and immunoloregulation function and the method for selenic acid polysaccharide utilized first, compare with other selenic acid method, this method has does not use poisonous organic solvent, have succinct, environmental protection, safety, characteristics such as efficient, under the same concentrations condition, compare the Total antioxidant capacity of gained phosphorylation and selenic acid polysaccharide with the polysaccharide of phosphorylation and selenic acidization not, anti-superoxide anion (O
2-) free radical, suppress the hydroxy radical qiao ability and improved 30%, 35%, 39% respectively, the phagocytic index that endoxan is caused immunocompromised mouse monokaryon scavenger cell has been improved 16.32%, leukocyte count has increased by 15.68%, and spleen index and thymus index have improved 11.52% and 15.46% respectively.
In sum, the present invention has set up a kind of namely when selenizing agent and phosphoric acid salt exist, method with dry heat, the exocellular polysaccharide that Lactococcus lactis breast subspecies bacterial strain is produced carries out selenizing and phosphorylation modification, acquisition has the functional Lactococcus lactis exocellular polysaccharide of significantly anti-oxidant and immune-enhancing effect, provides theoretical foundation for developing new healthy ingredient, functional foodstuff or medicine.
Embodiment
Describe in further detail below in conjunction with the present invention of embodiment.
One, measuring method
1, measurement of the polysaccharide content
(1) reducing sugar content that total sugar content-the DNS method records that records of the content ≈ phenolsulfuric acid method of polysaccharide;
(2) the phenolsulfuric acid method is surveyed total sugar content in the fermented liquid: the mensuration of total sugar content phenolsulfuric acid method in the sample.Sample thief liquid 1.0mL adds 1.0mL distilled water, adds 6.0% phenol solution 1.0mL again, adds the 5.0mL vitriol oil at last.Each sample do three times parallel, do a contrast with distilled water again.Reagent add neat after, static 10min shakes up again, room temperature is placed behind the 20min in its OD value of 490nm place survey.After the mensuration, get the OD mean value of measured sample, the regression equation y=26.133x+0.628 according to typical curve calculates corresponding total sugar content, and wherein x is OD(490nm) value, y is glucose concn (mg/L);
(3) mensuration of reducing sugar content in the sample: sample thief liquid 1.0mL, add 1.0mL distilled water again, add 1.5mL3 ' at last, 5 '-dinitrosalicylic acid (DNS).Each sample is made parallel sample three times, does a contrast with distilled water again.With each pipe solution mixing, in boiling water bath, heat 5min, add 21.5mL distilled water to every pipe again with the cold water cool to room temperature immediately after the taking-up, shake up, survey the OD value in 520nm wavelength place.After the mensuration, get the OD mean value of measured sample, the regression equation y=54.617x+1.4934 according to the DNS typical curve calculates corresponding reducing sugar content, and wherein x is OD(490nm) value, y is glucose concn (mg/L).
2, the mensuration of phosphate radical (molybdenum blue colorimetric method)
Be standard substance with the potassium primary phosphate, place the colorimetric cylinder of 25mL respectively, add the ammonium molybdate solution of 5% 2.0mL successively, shake up, leave standstill several seconds after, the Resorcinol solution that adds 2% 1.0mL sodium sulfite solution and 0.5% 1.0mL respectively, shake up, add water to scale, leave standstill 30min after, with the reference zeroing, measure absorbancy at the 660nm place
[16], be that X-coordinate, corresponding absorbancy are that ordinate zou is made typical curve (y=1.0100x-0.1472, X are absorbance) with the phosphate concentration.
Get an amount of sample in beaker, add the vitriol oil and the concentrated nitric acid of 1mL respectively, be heated at electric furnace and smolder, 30% the hydrogen peroxide that adds 1mL then after cooling, slowly heating again repeats above step and no longer smolders in bottle, and solution is water white transparency or faint yellow.Cooling back adds the hydrochloric acid of 1mL 6mol/mL, in the electric furnace heating acid is thoroughly decomposed, and is transferred to constant volume in the volumetric flask of 50mL, gets 5ml and measures absorbancy by the typical curve working method, calculates the content of phosphorus according to regression curve.
3, Determination of Selenium Contents
Be standard substance with the Sodium Selenite, add 8mL 0.1% o-phenylenediamine solution, 80% formic acid adjust pH is 1.5-2.5, transfers in the 50 mL volumetric flasks constant volume.In the dark place 50 min.With the extraction of 10 mL toluene, leave standstill.The selenium standardized solution of extraction is transferred in the 100 mL volumetric flasks, is settled to scale, toluene is reference, at λ=334nm place maximum absorption is arranged, the production standard curve.Accurately take by weighing 20.0 mg selenium polysaccharides and place Erlenmeyer flask, add 2 mL nitration mixture HCIO
4+ H
2S0
4+ HN0
3(volume ratio 1:l:4) digests cooling when clarifying to solution, is transferred in the 10 mL volumetric flasks, and constant volume is measured absorbancy, calculates selenium content according to typical curve.
4, resistance of oxidation index determining
(1) mensuration of Total antioxidant capacity: add the exocellular polysaccharide of preliminary purification in the oxidizing reaction system, utilize the Fenton reaction system to produce hydroxy radical qiao, as positive control, reaction finishes the back and measures light absorption value in the 510nm place with xitix; Total antioxidant capacity is calculated by following formula:
(2) mensuration of ultra-oxygen anion free radical: in reactive system, the changing value that the ultra-oxygen anion free radical that every liter of sample suppresses at 37 ℃ of reaction 40min is equivalent to the ultra-oxygen anion free radical that the vitamins C of 1mg suppresses is a unit of activity
OD
1: the absorbancy of control tube; OD
2: the absorbancy of measuring pipe; OD
3: the absorbancy of standard pipe
(3) mensuration of hydroxy radical qiao: Fenton reaction is the chemical reaction of modal generation hydroxy radical qiao, H
2O
2Amount and Fenton reaction produce hydroxy radical qiao and be directly proportional, after giving electron acceptor(EA), use the gress reagent colour development, form red material, its colour generation and hydroxy radical qiao how much proportional
Standard pipe concentration is 8.824mmol/L; Sampling amount is 1mL; OD
1: the absorbancy of control tube; OD
2: the absorbancy of measuring pipe; OD
3: the absorbancy of standard pipe; OD
4: the absorbancy of blank pipe.
5, immune indexes is measured
(1) laboratory animal grouping and filling stomach
60 mouse are divided into 4 groups at random, 15 every group.Five groups are respectively normal control group, endoxan (CY) control group, CY+ exocellular polysaccharide group, CY+ phosphorylation exocellular polysaccharide group.After mouse adapts to a week, begin to irritate stomach, normal control group and CY control group are irritated stomach physiological saline 0.10ml/10g body weight every day, and common polysaccharide or phosphorylated polysaccharide group are irritated stomach 10 mg/ml exocellular polysaccharide solution 0.10ml/10g body weight, continuous 30 days respectively every day.Irritating preceding 5 days of stomach, except the normal control group, other the three groups of isometric endoxan 100mg/kg of abdominal injection every day body weight
(2) organ index calculation formula
Each is organized mouse and weighs behind last administration 24h, and the tail vein is got blood, after taking off cervical vertebra and putting to death mouse, cuts open and gets liver, spleen and thymus gland.Blot to weigh at electronic balance with filter paper and calculate spleen index and thymus index
Thymus gland (spleen) index=
(3) phagocytic index is measured
Clean up index
K=
, phagocytic index α=
K: the not calibrated index of engulfing; OD
1: blood specimen OD value in the time of 2 minutes; OD
2: blood specimen OD value in the time of 20 minutes.
Two, specific embodiment
Embodiment 1
A kind of preparation method of functional lactobacillus exocellular polysaccharide specifically may further comprise the steps:
(1) preparation of milk-acid bacteria crude extracellular polysaccharide:
Be 10 with viable count
73.0%(v/v is pressed in the Lactococcus lactis of individual/mL breast subspecies strain fermentation agent (purchasing in Chinese industrial microbial strains preservation administrative center)) inoculum size, be seeded on the improved BLX substratum, cultivate 20 h down at 37 ℃ earlier, cultivate 15h down at 28 ℃ again, the centrifugal 15-20min of 6000-7000rpm removes the fermented liquid that somatic cells must contain exocellular polysaccharide, with ultra-filtration equipment exocellular polysaccharide in the Lactococcus lactis breast subspecies fermented liquid is carried out ultrafiltration and concentration then, in concentrated solution, add 10% Tricholroacetic Acid precipitation TCA precipitation again, 0-4
oPlaced 8-12 hour under the C, the centrifugal 20min of 6000-7000rpm then, get supernatant liquor, through the hollow-fibre membrane ultrafiltration and concentration, get the Crude polysaccharides concentrated solution again, adding volume is the ethanol of the 90-95% of 3 times of Crude polysaccharides concentrated solutions, left standstill 10-12 hour at 4 ℃, the centrifugal 25min of 6000rpm, the taking precipitate lyophilize gets pulverous milk-acid bacteria crude extracellular polysaccharide;
(2) separation and purification of Crude polysaccharides:
The milk-acid bacteria crude extracellular polysaccharide that step (1) is obtained is dissolved in a small amount of distilled water, earlier through the separation and purification of DEAE-cellulose ion exchange column, collection contains the elutriant (milk-acid bacteria crude extracellular polysaccharide Determination on content method in measurement of the polysaccharide content method) by experiment of first elution peak of milk-acid bacteria crude extracellular polysaccharide, freeze concentration 5-6mL, again through the separation and purification of Sepharose CL-6B gel filtration chromatography, collection contains the elutriant of second elution peak of milk-acid bacteria crude extracellular polysaccharide, be the daltonian nanofiltration membrane nanofiltration of 280-320 desalination with elutriant through molecular weight cut-off, again through ultrafiltration and concentration, the ethanolic soln precipitation of 90-95%, the taking precipitate lyophilize obtains the outer holosaccharide of lactic acid mycetocyte;
(3) phosphorylation of polysaccharide:
The outer holosaccharide of the lactic acid mycetocyte that step (2) is obtained and Sodium hexametaphosphate 99 (Na
6P
6O
18) solution is in the ratio mixing of mass ratio 6:1, and adjusting pH to 5.0, lyophilize is to Powdered, it is interior at 70 ℃ of following reacting by heating 6h to move to loft drier then, removes unnecessary salt with the ethanolic soln flushing of 70-75% then, places rotatory evaporator distillation 2-3 hour again, remove residual ethanol, vacuum lyophilization obtains the solid phosphoric acid polysaccharide, and wherein phosphate radical grafting amount reaches 1.64-1.70 mg/g;
(4) selenic acidization:
The solid phosphoric acid polysaccharide that step (3) is obtained is soluble in water, and to obtain mass percent be 15% phosphorylated polysaccharide solution, with phosphorylated polysaccharide solution and the selenizing agent-Sodium Selenite ratio mixing in mass ratio 9:1, and adjusting pH to 1.5-2.5, lyophilize is to Powdered, move in the loft drier then, at 50 ℃ of following reacting by heating 13.5h, remove unnecessary selenizing agent with the ethanolic soln flushing of 70-75% then, place rotatory evaporator to distill again, remove residual ethanol, put into dialysis tubing, place distilled water dialysis to remove residual selenizing agent, after dialysis finishes, with the polysaccharide soln in the dialysis tubing to going into beaker, the 90-95% ethanol sedimentation that adds 3 times of polysaccharide soln volumes, 4 ℃ left standstill 10-12 hour, the centrifugal 20-25min of 6000-7000rpm, taking precipitate lyophilize, namely get phosphorylation and selenic acid polysaccharide product is the functional lactobacillus exocellular polysaccharide, wherein the content of selenium is 0.17-0.18 mg/g.
Specifically be in the example since then, improve the BLX substratum, it is composed as follows: 1.5-2.0% glucose, and 1.0-1.2% fructose, the 1.3-1.5% soy peptone, the 1.3-1.5% Tryptones, initial pH is 6.0-6.5.
Ultrafiltration and concentration process described in step (1) and the step (2) is: adopting the aperture is housed is 50 kilodalton inner pressed hollow-fibre membranes, be 0.25-0.40MPa at working pressure, service temperature is under 20-25 ℃ the condition, and exocellular polysaccharide in the Lactococcus lactis breast subspecies fermented liquid is carried out ultrafiltration and concentration.
To be phosphoric acid salt and water form by the mixed of mass ratio 20:1 phosphate solution.
Embodiment 2
With embodiment 1, its difference is: Lactococcus lactis breast subspecies strain fermentation agent viable count and inoculum size are respectively 10 in the preparation process of milk-acid bacteria crude extracellular polysaccharide
6Individual/mL and 4.0%(v/v); Cultivate 20 h down at 37 ℃ earlier, cultivate 12h down at 30 ℃ again; During phosphorylation reaction, used phosphoric acid salt is tripoly phosphate sodium STPP, and the quality that Powdered Crude polysaccharides and tripoly phosphate sodium STPP (being dissolved in a spot of water earlier) are pressed 6:2 is than mixing, and adjusting pH to 6.0, and lyophilize is at 80 ℃ of following reacting by heating 5h; The used selenizing agent of selenic acid reaction is the acetic acid selenide, at 45 ℃ of following reacting by heating 15h.
Embodiment 3
With embodiment 1, its difference is: Lactococcus lactis breast subspecies strain fermentation agent viable count and inoculum size are respectively 10 in the preparation process of milk-acid bacteria crude extracellular polysaccharide
5Individual/mL and 5.0%(v/v); Cultivate 20 h down at 37 ℃ earlier, cultivate 10h down at 32 ℃ again; During phosphorylation reaction, used phosphoric acid salt is trisodium phosphate, and the quality that Powdered Crude polysaccharides and tripoly phosphate sodium STPP (being dissolved in a spot of water earlier) are pressed 6:3 is than mixing, and adjusting pH to 4.0, and lyophilize is at 90 ℃ of following reacting by heating 4h; The used selenizing agent of selenic acid reaction is the mixture of acetic acid selenide and Sodium Selenite, at 55 ℃ of following reacting by heating 12h.
Claims (2)
1. the preparation method of a functional lactobacillus exocellular polysaccharide is characterized in that specifically comprising the steps:
(1) preparation of milk-acid bacteria crude extracellular polysaccharide
With the Lactococcus lactis breast subspecies strain fermentation agent inoculum size of per-cent 3.0-5.0% by volume, be seeded on the improved BLX substratum, cultivate 20 h down at 37 ℃ earlier, cultivate 10-15h down at 28-32 ℃ again, the centrifugal 15-20min of 6000-7000rpm removes the fermented liquid that somatic cells must contain exocellular polysaccharide, then fermented liquid is carried out ultrafiltration and concentration, press 10% the adding Tricholroacetic Acid precipitation of concentrated solution quality again, 0-4
oPlaced 8-12 hour under the C, the centrifugal 15-20min of 6000-7000rpm then, get supernatant liquor and carry out ultrafiltration and concentration, get the Crude polysaccharides concentrated solution, adding volume again is the ethanolic soln of the 90-95% of 3 times of Crude polysaccharides concentrated solutions, leaves standstill 10-12 hour at 4 ℃, then the centrifugal 20-25min of 6000-7000rpm, the taking precipitate lyophilize gets pulverous milk-acid bacteria crude extracellular polysaccharide; The viable count content of described Lactococcus lactis breast subspecies strain fermentation agent reaches 10
5-7Individual/mL, described improvement BLX substratum, it is composed as follows: 1.5-2.0% glucose, 1.0-1.2% fructose, the 1.3-1.5% soy peptone, the 1.3-1.5% Tryptones, initial pH is 6.0-6.5;
(2) separation and purification of milk-acid bacteria crude extracellular polysaccharide
The milk-acid bacteria crude extracellular polysaccharide that step (1) is obtained is dissolved in a small amount of distilled water, earlier through the separation and purification of DEAE-cellulose ion exchange column, collection contains the elutriant of first elution peak of milk-acid bacteria crude extracellular polysaccharide, freeze concentration is to 5-6mL, again through the separation and purification of Sepharose CL-6B gel filtration chromatography, collection contains the elutriant of second elution peak of milk-acid bacteria crude extracellular polysaccharide, be the daltonian nanofiltration membrane nanofiltration of 280-320 desalination with elutriant through molecular weight cut-off, again through ultrafiltration and concentration, the ethanolic soln precipitation of 90-95%, the taking precipitate lyophilize obtains the outer holosaccharide of lactic acid mycetocyte;
(3) phosphorylation of the outer holosaccharide of lactic acid mycetocyte
The outer holosaccharide of the lactic acid mycetocyte that step (2) is obtained and phosphate solution are in the ratio mixing of mass ratio 6:1～3, and adjusting pH to 4.0-6.0, lyophilize, to Powdered, move in the loft drier then, at 70-90 ℃ of following reacting by heating 4-6h, remove unnecessary salt with the ethanolic soln flushing of 70-75% then, place rotatory evaporator distillation 2-3 hour again, remove residual ethanol, vacuum lyophilization obtains the solid phosphoric acid polysaccharide, and wherein phosphate radical grafting amount reaches 1.64-1.70 mg/g; To be phosphoric acid salt and water form by the mixed of mass ratio 20:1 described phosphate solution, and phosphoric acid salt is Sodium hexametaphosphate 99 (Na
6P
6O
18), tripoly phosphate sodium STPP (Na
5P
3O
10) and trisodium phosphate (Na
4P
2O
7) in any;
(4) selenic acidization
The solid phosphoric acid polysaccharide that step (3) is obtained is soluble in water, and to obtain mass percent be 15% phosphorylated polysaccharide solution, with phosphorylated polysaccharide solution and the selenizing agent ratio mixing in mass ratio 9:1, and adjusting pH to 1.5-2.5, lyophilize is to Powdered, move in the loft drier then, at 45-55 ℃ of following reacting by heating 12-15h, remove unnecessary selenizing agent with the ethanolic soln flushing of 70-75% then, place rotatory evaporator to distill again, remove residual ethanol, put into dialysis tubing, place the distilled water dialysis to remove residual selenizing agent, after dialysis finishes, with the polysaccharide soln in the dialysis tubing to going into beaker, add the 90-95% ethanol sedimentation of 3 times of polysaccharide soln volumes, 4 ℃ left standstill 10-12 hour, the centrifugal 20-25min of 6000-7000rpm, the taking precipitate lyophilize, namely get phosphorylation and selenic acid polysaccharide product is the functional lactobacillus exocellular polysaccharide, wherein the content of selenium is 0.17-0.18 mg/g, and described selenizing agent refers to acetic acid selenide or Sodium Selenite or the two mixture.
2. the preparation method of a kind of functional lactobacillus exocellular polysaccharide according to claim 1, it is characterized in that the ultrafiltration and concentration process described in step (1) and the step (2) is: adopting the aperture is housed is 50 kilodalton inner pressed hollow-fibre membranes, be 0.25-0.40MPa at working pressure, service temperature is under 20-25 ℃ the condition, and exocellular polysaccharide in the Lactococcus lactis breast subspecies fermented liquid is carried out ultrafiltration and concentration.
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| CN111621532A (en) * | 2020-07-15 | 2020-09-04 | 辽宁大学 | Preparation method of lactobacillus extracellular polysaccharide and derivatives thereof |
| CN112029807A (en) * | 2020-08-24 | 2020-12-04 | 东北农业大学 | Preparation method of lactobacillus rhamnosus exopolysaccharide |
| CN112167341A (en) * | 2020-09-27 | 2021-01-05 | 吉林农业大学 | Preparation method of dairy product containing lactobacillus plantarum JLAU103 exopolysaccharide |
| CN112646049B (en) * | 2020-12-30 | 2022-07-29 | 南方海洋科学与工程广东省实验室(湛江) | Phosphorylated Platymonas extracellular polysaccharide and preparation method thereof |
| CN113308501A (en) * | 2021-07-09 | 2021-08-27 | 甘肃省科学院生物研究所 | Preparation method of lactobacillus extracellular polysaccharide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235098A (en) * | 2008-01-09 | 2008-08-06 | 浙江大学 | Method for modifying functional plants polysaccharide |
| CN101445563A (en) * | 2008-10-10 | 2009-06-03 | 汤洁 | Method for manufacturing selenium-rich fucoidin |
| CN101597340A (en) * | 2009-07-09 | 2009-12-09 | 云南大学 | A kind of new preparation process of selenic acid polysaccharide |
-
2011
- 2011-07-11 CN CN 201110191610 patent/CN102250984B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101235098A (en) * | 2008-01-09 | 2008-08-06 | 浙江大学 | Method for modifying functional plants polysaccharide |
| CN101445563A (en) * | 2008-10-10 | 2009-06-03 | 汤洁 | Method for manufacturing selenium-rich fucoidin |
| CN101597340A (en) * | 2009-07-09 | 2009-12-09 | 云南大学 | A kind of new preparation process of selenic acid polysaccharide |
Non-Patent Citations (6)
| Title |
|---|
| 乳酸乳球菌胞外多糖的分离及其体外抗氧化活性研究;梅秀明等,0;《食品科学》;20090101;第30卷(第01期);232-235 * |
| 乳酸乳球菌胞外多糖的制备及对环磷酰胺所致免疫损伤小鼠的保护作用;梅秀明等,0;《营养学报》;20091015;第31卷(第05期);505-508 * |
| 乳酸菌发酵液中胞外多糖的超滤浓缩技术研究;梅秀明等,0;《食品科学》;20081215;第29卷(第12期);413-416 * |
| 梅秀明等,0.乳酸乳球菌胞外多糖的分离及其体外抗氧化活性研究.《食品科学》.2009,第30卷(第01期),232-235. |
| 梅秀明等,0.乳酸乳球菌胞外多糖的制备及对环磷酰胺所致免疫损伤小鼠的保护作用.《营养学报》.2009,第31卷(第05期),505-508. |
| 梅秀明等,0.乳酸菌发酵液中胞外多糖的超滤浓缩技术研究.《食品科学》.2008,第29卷(第12期),413-416. |
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