CN109762855A - A kind of new method for defatted rice bran sulphation of fermenting - Google Patents
A kind of new method for defatted rice bran sulphation of fermenting Download PDFInfo
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Abstract
The invention discloses a kind of new methods of defatted rice bran sulphation of fermenting, and first half dry fermentation of defatted rice bran, polysaccharide molecule are transformed by the process of biology, is then directly over sulphation, traditional first extraction, purified polysaccharide, then multiple steps of sulphation is omitted；And reduces polysaccharide molecular weight by biology and the double processes of chemistry, make polysaccharide functionalization, increase the utilization rate of polysaccharide.Present invention fermentation defatted rice bran sulphation new method, first passing through bioprocess reduces polysaccharide molecular weight, and it is easy to dissociate, direct sulphation is carried out after bioprocess, it reduces and the sulphation conventional methods again such as first extracts, purifies, process environmental protection saves human and material resources, high income, sulphation process uses tasteless, low boiling point, easy to handle reagent instead from environmental angle.
Description
Technical field
The invention belongs to biology, chemical modification natural goods molecular applications in the field of antiviral bioactivity, and in particular to
To biology, chemical modification defatted rice bran, obtains fermentation polysaccharides sulfuric ester and show the fermentation polysaccharides for being better than non-sulphation in vitro
Antiviral activity, a kind of new method for defatted rice bran sulphation of fermenting.
Background technique
Rice bran non-starch polysaccharide includes cellulose, hemicellulose, pectin, is the main component for constituting cell wall, wherein fine
Dimension element is played a supporting role in cell wall, and referred to as structural polysaccharide, hemicellulose and pectin class are played in cell wall and filled out
Use use and connection function, referred to as matrix polysaccharide as.Hemicellulose includes that alpha-glucans, araboxylan, glucose sweet dew are poly-
Sugar etc., Pectic polysaccharides include araban, arabogalactan, poly- rhamno-galacturonic acid, same to polygalacturonic acid.
Rice bran about contains 50% dietary fiber, 20% protein and 10% phytic acid calcium after extracting grease, can be used to
High value-added product is developed, such as rice bran non-starch polysaccharide, rice bran protein, phytic acid.Under different extraction processes, due to different knots
The cell wall properties of structure layer are different, to prepare the dissolubility of rice bran polysaccharide, monosaccharide composition, molecular weight, sugar chain conformation, space knot
Structure, bioactivity difference.For polysaccharide, functional group is less on monosaccharide residue, degree of branching is not high, molecular weight phase
It is relatively low to higher polysaccharide bioactivity, and its lower water-soluble hair for even more further limiting its bioactivity
It waves, therefore the chemical research persons method that has studied various molecular modifications prepares the derivative of polysaccharide, improves its bioactivity.Often
The Polysaccharide Modification seen has physical modification, chemical modification (sulphation, phosphorylation, acetylation etc.) and bioengineering modification.It is withered
Careless bacillus can produce a large amount of protease, phytase, cellulase, amylase, lipase, fruit during growth metabolism
Glue enzyme and other beneficial agents.First half dry fermentation of defatted rice bran, wherein the distribution of polysaccharide molecular weight and monosaccharide can be varied.
Sulfation is that a kind of modified relatively conventional method of structural modification is carried out to polysaccharide.Through Sulfation moditied processing
Later, the molecular weight of polysaccharide reduce, it is water-soluble increase, charge changes, bioactivity obviously increases, and in certain molecular weight and
Within the scope of sulfuric acid degree of substitution, the functional activity and sulfuric acid degree of substitution of polysaccharide sulfate, which are presented, to be positively correlated, therefore polysaccharide is carried out sulphur
The method of Esterification modification just can be widely used and study.
Common Sulfation method mainly has sulphate method, chlorosulfonic acid-pyridine method, the organic alkaline process of sulfur trioxide-.Three kinds
Method is all first by sulfur acidizing reagent (concentrated sulfuric acid, chlorosulfonic acid, sulfur trioxide-pyridine or sulfur trioxide-triethylamine) in cooling
Under the conditions of be dissolved in solvent, be prepared into esterifying reagent, then the solution reaction with polysaccharide or polysaccharide that heats up.Sulphate method can be certain
There is low yield in Polysaccharides in degree, obtained product.Chlorosulfonic acid-pyridine method yield is preferable, is domestic common
Sulfation method, but chlorosulfonic acid has certain risk, is state control raw material.Sulfur trioxide-organic base rule is more to pacify
Complete, reaction condition is mild but reagent costly, be not easy to react.
Natural and artificial synthesized polysaccharide sulfate is also proved in anticoagulation by numerous tests, AntiHIV1 RT activity, antiviral, anti-swells
Tumor, enhancing immunity of organism activity etc. have remarkable efficacy, and after Sulfation is modified, activity has significantly many polysaccharide
Raising.
Summary of the invention
The present invention has made improvement for the shortcomings of the prior art, provides a kind of fermentation defatted rice bran sulphur
The new method of acidification, what the present invention was realized particular by following technical solution:
The invention discloses a kind of new methods of defatted rice bran sulphation of fermenting to pass through first half dry fermentation of defatted rice bran
Polysaccharide molecule is transformed in the process of biology, is then directly over sulphation, traditional first extraction, purified polysaccharide, then sulfuric acid is omitted
The multiple steps changed；And reduces polysaccharide molecular weight by biology and the double processes of chemistry, make polysaccharide functionalization, increase the benefit of polysaccharide
With rate.
As a further improvement, specific steps of the invention are as follows:
1) defatted rice bran, is weighed, certain pure water is added to ferment with bacillus subtilis semidry method；
2), take organic solvent in dry bottle, it is cooling, lower dropwise addition oleum is stirred, it is spare to obtain Sulfation reagent；
3) dry ferment rice bran, is weighed, organic solvent is added and under agitation, is slowly added dropwise in step 2) after cooling
Sulfation reagent, be added dropwise after being fully cooled, then at stirred in water bath react, cool down after completion of the reaction, be neutralized to pH
=7, it filters, rotary evaporation, is freeze-dried up to crude product；
4), the crude product for obtaining step 3) is further dialysed, post separation is handled, up to sterling after freeze-drying.
As a further improvement, defatted rice bran semidry method of the present invention carry out fermentation be plus 15% pure water with
0.1% bacillus subtilis is fermented 72 hours.
As a further improvement, in step 3) of the present invention, the mass ratio of ferment rice bran and organic solvent is
1:10.
As a further improvement, in step 3) of the present invention, the mass ratio of ferment rice bran and oleum
For 1:10~18.
As a further improvement, the time of ferment rice bran sulphation was at 1~4 hour in step 3) of the present invention.
As a further improvement, organic solvent of the present invention is acetonitrile, the Sulfation reagent is smoke
The acetonitrile solvent of sulfuric acid, the mass ratio that the oleum accounts for acetonitrile solution is 50~70%.
As a further improvement, rice bran sulfated polysaccharides degree of substitution of the present invention is 1.0~1.4.
The present invention uses oleum acetonitrile solution, acetonitrile boiling point, low easy to handle, logical first half dry fermentation of defatted rice bran
The process transformation polysaccharide molecule for crossing biology, is then directly over sulphation, be omitted first extract, purified polysaccharide, then sulphation
More steps, and reduce polysaccharide molecular weight by biology and the double processes of chemistry, make polysaccharide functionalization, and increase mentioning for polysaccharide
Take rate.
The present invention first passes through bioprocess, followed by the double processes of chemical modification are directly changed rice bran, make the polysaccharide of rice bran
Molecular weight distribution changes；The accounting of the monosaccharide of the polysaccharide of rice bran changes, and the ferment rice bran sulphation being prepared is more
Sugar has inhibiting effect to virus.The polysaccharide CY1 of the sulphation of acquisition, by purifying, it is real to carry out extracorporeal antivirus effect cell toxicant for identification
It tests, activity is better than the rice bran polysaccharide of non-sulphation.
The advantage of patent:
1, fermentation defatted rice bran sulphation new method, first passing through bioprocess reduces polysaccharide molecular weight,
And it is easy to dissociate.
2, direct sulphation is carried out after bioprocess, is reduced and sulphation conventional methods again, the process ring such as is first extracted, purifies
It protects, save human and material resources, high income.
3, sulphation process uses tasteless, low boiling point, easy to handle reagent instead from environmental angle.
Detailed description of the invention
Fig. 1 is absorbance to sulfate concentration canonical plotting (potassium sulfate)；
Fig. 2 is the canonical plotting of different molecular weight glucan；
Fig. 3 is the graph of molecular weight distribution of sample 1 (4 hours products of ferment rice bran sulphation)；
Fig. 4 is the graph of molecular weight distribution of sample 2 (4 hours products of non-ferment rice bran sulphation)；
Fig. 5 is 90 μ g/mg monosaccharide standard chromatograms；
Fig. 6 is the monosaccharide distribution map of sample 1 (4 hours products of ferment rice bran sulphation)；
Fig. 7 is the monosaccharide distribution map of sample 2 (4 hours products of non-ferment rice bran sulphation).
Specific embodiment
Further illustrate the present invention with reference to embodiments.
Embodiment 1
500g defatted rice bran semidry method is claimed to ferment.Add 15% pure water and 0.1% bacillus subtilis, ferments
72 hours.It is dry.
Take anhydrous acetonitrile 1000g in dry bottle, it is 0 DEG C cooling, stir lower dropwise addition oleum 2000g, as sulfuric acid
Esterifying reagent, it is spare.
50g is dry, and ferment rice bran adds 500g acetonitrile, after being cooled to 0 DEG C, under agitation, Sulfation examination is slowly added dropwise
Agent 1350g after being added dropwise, after 0 DEG C is fully cooled, reacts 4 hours then at 35 DEG C of stirred in water bath.After completion of the reaction will
0 DEG C of cooling of flask, 20% sodium hydroxide solution of agitation and dropping neutralizes, until pH=7, filters, rotary evaporation, is freeze-dried up to thick
Product 46g.
It uses MD55 bag filter deionized water dialysis 2 days respectively, after concentration, crosses gel chromatographic columns (ultra-fine Sephadex LH-
20, deionized water), mobile phase is deionized water, and flow velocity 0.4mL/min, TLC detect ingredient (solvent acetone: n-butanol: water
=1:1:1, color developing agent: 10% sulfuric acid-methanol solution), target fraction is collected, is freeze-dried up to sterling 30g, obtains sample 1
(CY1)。
Comparative example 1
50g drying defatted rice bran adds 500g acetonitrile, after being cooled to 0 DEG C, under agitation, Sulfation examination is slowly added dropwise
Agent 1350g after being added dropwise, after 0 DEG C is fully cooled, reacts 4 hours then at 35 DEG C of stirred in water bath.After completion of the reaction will
0 DEG C of cooling of flask, 20% sodium hydroxide solution of agitation and dropping neutralizes, until pH=7, filters, rotary evaporation, is freeze-dried up to thick
Product 42g.
It uses MD55 bag filter deionized water dialysis 2 days respectively, after concentration, crosses gel chromatographic columns (ultra-fine Sephadex LH-
20, deionized water), mobile phase is deionized water, and flow velocity 0.4mL/min, TLC detect ingredient (solvent acetone: n-butanol: water
=1:1:1, color developing agent: 10% sulfuric acid-methanol solution), target fraction is collected, is freeze-dried up to sterling 27g, obtains sample 2.
Embodiment 2
500g defatted rice bran semidry method is claimed to ferment.Add 15% pure water and 0.1% bacillus subtilis, ferments
72 hours.It is dry.
50g is dry, and ferment rice bran adds 500g acetonitrile, after being cooled to 0 DEG C, under agitation, Sulfation examination is slowly added dropwise
Agent 1200g after being added dropwise, after 0 DEG C is fully cooled, reacts 4 hours then at 35 DEG C of stirred in water bath.After completion of the reaction will
0 DEG C of cooling of flask, 20% sodium hydroxide solution of agitation and dropping neutralizes, until pH=7, filters, rotary evaporation, is freeze-dried up to thick
Product 42g.
It uses MD55 bag filter deionized water dialysis 2 days respectively, after concentration, crosses gel chromatographic columns (ultra-fine Sephadex LH-
20, deionized water), mobile phase is deionized water, and flow velocity 0.4mL/min, TLC detect ingredient (solvent acetone: n-butanol: water
=1:1:1, color developing agent: 10% sulfuric acid-methanol solution), target fraction is collected, is freeze-dried up to sterling 25g.
Embodiment 3
The dry wheat bran semidry method of 500g is claimed to ferment.Add 15% pure water and 0.1% bacillus subtilis, ferments
72 hours.It is dry.
50g is dry, and fermenting wheat bran adds 500g acetonitrile, after being cooled to 0 DEG C, under agitation, Sulfation examination is slowly added dropwise
Agent 1350g after being added dropwise, after 0 DEG C is fully cooled, reacts 4 hours then at 35 DEG C of stirred in water bath.After completion of the reaction will
0 DEG C of cooling of flask, 20% sodium hydroxide solution of agitation and dropping neutralizes, until pH=7, filters, rotary evaporation, is freeze-dried up to thick
Product 40g.
It uses MD55 bag filter deionized water dialysis 2 days respectively, after concentration, crosses gel chromatographic columns (ultra-fine Sephadex LH-
20, deionized water), mobile phase is deionized water, and flow velocity 0.4mL/min, TLC detect ingredient (solvent acetone: n-butanol: water
=1:1:1, color developing agent: 10% sulfuric acid-methanol solution), target fraction is collected, is freeze-dried up to sterling 23g.
Embodiment 4
The characterization of two class polysaccharide sulfates compares:
Embodiment 1 is shown in the preparation of two class polysaccharide sulfates.It obtains sample 1 (ferment rice bran polysaccharide sulfate 4h)；Sample 2 is (not
Ferment rice bran polysaccharide sulfate 4h).
1. the degree of polysaccharide sulfate measures
Based on barium sulfate turbidimetry, the sulphur of barium sulfate turbidimetry measurement polysaccharide sulfate is used with reference to GB13580.6-1992
Acidizing degree.
Configuration: sulfate radical standard solution: 108.7mg potassium sulfate standard substance is weighed, is dissolved with water, is settled to 100mL.
(sulfate radical content 0.60mg/mL).- 0.5% polyethylene glycol of 1% barium chloride (MW=2000Da) solution: 1.1775g BaCl2·
2H2O, addition polymerization ethylene glycol (MW=2000Da) 0.5000g, is dissolved in water, is settled to 100mL.
Specification Curve of Increasing: take respectively sulfate radical standard solution 0.04,0.08,0.12,0.16,0.20,0.24,0.28,
0.32mL is diluted with water to 3.5mL or so, and -0.5% polyethylene glycol of 1.00mL1% barium chloride (MW=2000Da) solution is added,
It is settled to 5.00mL, is placed 20 minutes.Sulfate radical standard solution is replaced as above operate as blank with distilled water 0.20mL,
Standard curve is measured at λ=420nm.Y=353.71x is obtained, R2=0.9989, Fig. 1 are absorbances to sulfate concentration standard
Curve graph (potassium sulfate).
Ferment rice bran polysaccharide sulfate 11.3mg is weighed respectively in tool plug test tube, be added 1mol/L hydrochloric acid 5.00mL, 105
DEG C heating hydrolysis 8 hours.0.20mL sample solution is taken respectively, is diluted to about 3.5mL, and 1.00mL1% barium chloride -0.5% is added
Polyethylene glycol (MW=2000Da) solution is settled to 5.00mL, places 20 minutes.Sulfate radical standard is replaced with distilled water 0.20mL
Solution carries out as above operation as blank, and absorbance is measured at λ=420nm.Parallel laboratory test is three times.
Sulfate radical degree of substitution calculation formula:
Wherein S% is sulfate radical SO4 in sample2-Mass fraction.
The absorbance of 1 counter sample of table, sulfate radical degree of substitution
4 hours degree of substitution of ferment rice bran sulphation 0.124.
2. the molecular weight determination of controlling sulfate polyose sample
Its molecular weight is measured using the purity of GPC-MALLS analysis polysaccharide sample simultaneously, gel permeation chromatography system uses
Chromatographic column is Shodex OHpak-SB 803, chromatographic column: Ohpak SB-804HQ (300 × 8mm), mobile phase: 0.1mol/L
NaNO3, flow velocity: 0.8mL/min, column temperature: 45 DEG C, sample amount: 100 μ L.Detection wavelength: 663.1nm.
Polysaccharide standard items (glucan) Fig. 2 is the canonical plotting of different molecular weight glucan；Sample 1: ferment rice bran, sulphur
Acidification 4 hours.There are 3 peaks, the 3rd peak accounting is most.1st peak molecular weight is 20,000.See Fig. 3 about ferment rice bran sulphation 4
The graph of molecular weight distribution of hour product；Specific data are shown in Table 2.
2 sample of table, 1 molecular weight distribution
Sample 2: non-ferment rice bran, sulphation 4 hours.There are 3 peaks, the 3rd amount accounting is most.1st peak molecular weight 34
Ten thousand.See graph of molecular weight distribution of the Fig. 4 about non-4 hours products of ferment rice bran sulphation；Specific data are shown in Table 3.
3 sample of table, 2 molecular weight distribution
Two samples are compared, the rice bran through everfermentation reduces by the 3rd peak accounting, and 2 accounting of the 1st, 2 peak relative sample increases；
Compared with sample 2, decrease in molecular weight after the fermentation of the 1st peak of sample 1, by 340,000 the 20,000, the 2nd peaks of drop by 9447 drops 5370.It can from data
See, rice bran fermentation process significant effect.
Note: Mn- number-average molecular weight；Mp-peak molecular weight；Mw- weight average molecular weight.
3. the monosaccharide component of controlling sulfate polyose is analyzed
Instrument ICS-5000+ ion chromatograph, electrochemical detector, four potential waveforms.Chromatographic condition Carbo PACTM
PA10 (2mm × 50mm) guard column, Carbo PACTM PA10 (2.0mm × 250mm) anion-exchange column.Leacheate condition: 0
~22min, 2.5%200mmol/L NaOH, 5%200mmol/L CH3COONa；22.1~30min, 4%200mmol/L
NaOH, 30%200mmol/L CH3COONa；30~40min, 100%200mmol/L NaOH；40~50min, 9%
200mmol/L NaOH.25 μ L of sampling volume.Ultrapure water.
The hydrolysis of sample: weighing the polysaccharide sample of certain mass, and 2mol/L TFA is added and seals immediately, in 110 DEG C of baking ovens
2h is hydrolyzed, is transferred in water-bath and is evaporated after cooling, adds water 1mL to wash, is evaporated, 3 times is washed repeatedly and is evaporated completely to TFA, add water
It shifts in 25mL measuring bottle, constant volume shakes up, after 0.25 μm of miillpore filter filters, for ion chromatography.
Ion chromatography operation: booting.1. opening nitrogen main valve, partial pressure is adjusted to 0.2Mpa, then adjust on ion chromatograph
Decompression list index be 5psi or so；(nitrogen partial pressure device being such as not configured, please ignore this step) 2. opens ICS5000 power supply；
3. opening AS-AP power supply；(AS-AP being such as not configured, please ignore this step) 4. opens computer, starts instrumentation controller；5. clicking
7 icon of Chromeleon on desktop enters software；6. confirming whether instrument on―line state is normal；7. bubble in blowdown pump；8.
Flow velocity is arranged in turn on pump；9. column oven temperature is arranged, column temperature heating mode is opened；10. as used ED detector, setting detection mould
Formula selects reference electrode, opens electrochemical voltage, selects waveform.
Fig. 5 is 90 μ g/mg monosaccharide standard chromatograms (specific data see the table below 4)
4 standard items testing result of table
1 monosaccharide component of sample analyzes (ferment rice bran sulfated polysaccharides (4 hours))
Fig. 6 is the monosaccharide distribution of sample 1 (4 hours products of ferment rice bran sulphation) (specific data see the table below 5)
The analysis of 5 sample of table, 1 monosaccharide component
Fucose, rhamnose, arabinose, galactolipin, glucose, xylose are with respect to accounting are as follows: 1.89,1.36,1,1.63,
4.77、1.68。
2 monosaccharide component of sample analyzes (non-ferment rice bran sulfated polysaccharides (4 hours))
Fig. 7 is the monosaccharide distribution of sample 2 (4 hours products of non-ferment rice bran sulphation) (specific data see the table below 6)
The analysis of 6 sample of table, 2 monosaccharide component
Do not ferment and ferment rice bran in glucose all accountings it is big, but after ferment rice bran sulphation, glucose accounting is reduced, phase
To glucose, fucose, rhamnose, Arab, galactolipin, xylose accounting is improved.The glucose in sulfated polysaccharides that do not ferment is more,
Other opposite monosaccharide accountings are few.
4. the Comparison study of liang class polysaccharide sulfate is tested:
Antiviral activity evaluation
Sample 1: ferment rice bran sulfated polysaccharides (4 hours)
Sample 2: non-ferment rice bran sulfated polysaccharides (4 hours)
HSV-1 herpes simplex virus；H1N1 influenza A virus；Mdck cell dog kidney passage cell
1,2 pairs of mdck cells of sample do not have the concentration range of obvious cytotoxicity are as follows: (0~100 μm of ol/L).
Virus activity: the TCID50 of H1N1 is 1 × 10- 4The TCID50 of/100 μ L, HSV-1 is 1 × 10- 3/100μL。
According to cytotoxicity experiment as a result, sample 1 or 2 to be diluted to respective concentration ladder in acellular poison concentration range
Degree, measures its Anti-viral activity in vitro respectively in following method.
Mdck cell uses the DMEM culture medium containing 10% fetal calf serum to press every milliliter 5 × 104The concentration of a cell is inoculated into
In 96 orifice plates, every 100 μ L of hole sets 37 DEG C, overnight incubation forms cell monolayer in 5%CO2 incubator.Discard supernatant liquid, cell
It is washed 2 times with PBS, addition influenza virus H1N1 or HSV-1 infection cell, after every hole 100 μ L, 37 DEG C of incubation 2h, discards virus
Liquid.It is separately added into cell dimension liquid (the DMEM culture medium containing 1% serum) of the drug containing various concentration, every 100 μ L of hole, while being set just
Normal cell controls group, virus control group and positive control drug Oseltamivir carboxylate (10 μm of ol/L) group, every group setting 4 multiple
Hole.It is placed in 37 DEG C, cultivates 72h in 5%CO2 incubator, observe cytopathy daily.And cell viability, experiment are measured with mtt assay
It is repeated 3 times altogether, calculates inhibiting rate, the medium effective concentration (EC50) of samples for viral.Viral suppression (%)=(dosing group is inhaled
Receipts degree-virus control group trap)/(normal cell controls group trap-virus control group trap) × 100.
As a result, the EC50 of sample 1 and 2 infected by influenza H1N1 of sample be respectively (14.45 ± 4.90), (34.54 ±
3.82)μmol/L；Positive drug Oseltamivir carboxylate is to the inhibiting rate of H1N1 virus at experimental concentration (10 μm of ol/L)
(56.74 ± 7.48) %.Illustrate that the inhibition virus capable of infected by influenza H1N1 sample 1 is strong.
To the inhibiting effect that has of HSV-1, EC50 is respectively (34.45 ± 2.90), (56.54 ± 3.02) for sample 1 and sample 2
μmol/L；It is obvious not as good as the inhibiting effect to H1N1 virus, but sample 1 should expression effect.Positive drug Oseltamivir carboxylic acid
Salt is (45.77 ± 2.34) % to the inhibiting rate of HSV-1 at experimental concentration (10 μm of ol/L).
Funded projects: state natural sciences fund 30870553；National International Sci & Tech Cooperation project 2010DFA34370；Zhejiang
River province International Sci & Tech Cooperation special project 2013C14012；Zhejiang Province Natural Science Fund In The Light LQY18B060002；2018 year Zan Yu section
Grind fund.
The above is not limitation of the present invention, it should be pointed out that: those skilled in the art are come
It says, under the premise of not departing from essential scope of the present invention, several variations, modifications, additions or substitutions can also be made, these improvement
It also should be regarded as protection scope of the present invention with retouching.
Claims (9)
1. a kind of new method for defatted rice bran sulphation of fermenting, which is characterized in that first half dry fermentation of defatted rice bran, pass through biology
Polysaccharide molecule is transformed in process, is then directly over sulphation, is omitted traditional first extraction, purified polysaccharide, then sulphation is more
A step；And reduces polysaccharide molecular weight by biology and the double processes of chemistry, make polysaccharide functionalization, increase the utilization rate of polysaccharide.
2. the new method of fermentation defatted rice bran sulphation according to claim 1, which is characterized in that specific steps are as follows:
1) defatted rice bran, is weighed, certain pure water is added to ferment with bacillus subtilis semidry method；
2), take organic solvent in dry bottle, it is cooling, lower dropwise addition oleum is stirred, it is spare to obtain Sulfation reagent；
3) dry ferment rice bran, is weighed, organic solvent is added and under agitation, the sulphur in step 2) is slowly added dropwise after cooling
Esterification reagent, is added dropwise after being fully cooled, and reacts then at stirred in water bath, cools down after completion of the reaction, be neutralized to pH=7,
It filters, rotary evaporation, is freeze-dried up to crude product；
4), the crude product for obtaining step 3) is further dialysed, post separation is handled, up to sterling after freeze-drying.
3. the new method of fermentation defatted rice bran sulphation according to claim 2, which is characterized in that the defatted rice bran
It is to add 15% pure water and 0.1% bacillus subtilis that semidry method, which carries out fermentation, is fermented 72 hours.
4. the new method of fermentation defatted rice bran sulphation according to claim 2, which is characterized in that the step 3)
In, the mass ratio of ferment rice bran and organic solvent is 1:10.
5. the new method of fermentation defatted rice bran sulphation according to claim 2, which is characterized in that the step 3)
In, the mass ratio of ferment rice bran and oleum is 1:10~18.
6. the new method of fermentation defatted rice bran sulphation according to claim 2, which is characterized in that the step 3)
In, the time of ferment rice bran sulphation was at 1~4 hour.
7. the new method of fermentation defatted rice bran sulphation according to claim 5 or 6, which is characterized in that described is organic
Solvent is acetonitrile, and the Sulfation reagent is the acetonitrile solvent of oleum, and the oleum accounts for acetonitrile solution
Mass ratio is 50~70%.
8. the new method of degreasing ferment rice bran sulphation according to claim 1 or 2 or 3 or 4 or 5 or 6, feature exist
In rice bran sulfated polysaccharides degree of substitution is 1.0~1.4.
9. the new method of degreasing ferment rice bran sulphation according to claim 1 or 2 or 3 or 4 or 5 or 6, feature exist
In the new method is suitable for the transformation of the biomass of wheat bran, maize peel.
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| CN113999325B (en) * | 2021-11-18 | 2022-09-09 | 国家粮食和物资储备局科学研究院 | Rice bran fermented polysaccharide, preparation and application |
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