CN108179164A - The method that polypeptide is extracted from degreasing protein breast supernatant - Google Patents
The method that polypeptide is extracted from degreasing protein breast supernatant Download PDFInfo
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- CN108179164A CN108179164A CN201810129326.2A CN201810129326A CN108179164A CN 108179164 A CN108179164 A CN 108179164A CN 201810129326 A CN201810129326 A CN 201810129326A CN 108179164 A CN108179164 A CN 108179164A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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Abstract
The invention discloses from degreasing protein breast supernatant extract polypeptide method, the specific steps are：Into lipid containing plant plus water, defibrination centrifuge, and obtain light-phase liquid as vegetable fat, and heavy-phase liquid is degreasing protein breast；The pH of degreasing protein breast is adjusted, is precipitated under the isoelectric point of degreasing protein breast, skimmed milk supernatant and albumen underflow is obtained, skimmed milk supernatant sterilizes after the two separation；The pH of skimmed milk supernatant is adjusted, composite bacteria is inoculated with, adds arabinose, then fermented and cultured obtains zymotic fluid；The pH of zymotic fluid is adjusted, adds in chitosan oligosaccharide, is stirred, centrifugation, supernatant concentration, freeze-drying is polypeptide.It has the beneficial effect that：The extracting method simple possible of polypeptide of the present invention, extraction efficiency, yield and purity are high, and polypeptide bioactivity obtained is high, without bitter taste, and extraction cost is low.
Description
Technical field
The present invention relates to technical field of biological extraction, and the side of polypeptide is extracted more particularly to from degreasing protein breast supernatant
Method.
Background technology
Peptide of the molecular structure between amino acid and protein, be have the function of protein structure and segment, tool
What the protein for having physiological function of the number in terms of necessarily was realized by peptide.Peptide have the function of such numerous and complicated structure with by
Form the amino acid classes of peptide, different numbers is determined with putting in order.Dipeptides and tripeptides are taken off by 2 or 3 amino acid
Water condensation forms, tetrapeptide, pentapeptide can and so on.It is, in general, that oligopeptides is that have amino acid number within 10 on peptide chain,
Polypeptide is exactly 10-50 amino acid number, and protein is 50 or more amino acid numbers.Small peptide or oligopeptide i.e. few
In peptide two, tripeptides.Polypeptide is a-amino acid with the compound that peptide chain links together and is formed, it is also protein hydrolysis
Intermediate product.Two amino acid molecular dehydrating condensations can form dipeptides, multiple amino acid molecular dehydrating condensations can be formed tripeptides,
Tetrapeptide, pentapeptide etc..Usually by the compound that 10~50 amino acid molecular dehydrating condensations are formed polypeptide, semi-permeable membrane can be penetrated,
It is not precipitated by trichloroacetic acid and ammonium sulfate.
It can be divided into existing natural endogenous polypeptide in body according to different sources and be present in animals and plants, micro- life
Exogenous polypeptid in object and after protein degradation.The peptide hormone of some important secretion glandular secretions is mainly endogenous in vivo
Property polypeptide.Directly or indirectly derive from animal food protein is exogenous polypeptid, such as animal milk.Although both at home and abroad to planting
The research of object protein active peptide has reached certain level, and Related product has been applied to health care, but predominantly soybean lives
Property peptide product, for other plant active peptide research just start to walk, Related product is very few, can not meet food industry need
It asks.Raw material used in domestic enzyme process polypeptide maturation process is the soybean protein isolate that protein content is 90%.Soybean protein isolate
The problems such as serious environmental pollution can be generated in process of production, China's strategy of sustainable development is not met, will be gradually eliminated.
Remaining enzymolysis polypeptide by the use of forage protein as raw material, is sunk after extracting protein by alkali soluble acid mostly, recycles protease
Polypeptide powder is made in enzymolysis.This method has extraction process complicated, and waste is too many, the problems such as protein extraction difficulty.
The prior art such as Authorization Notice No. is the Chinese invention patent of CN103740796B, is disclosed a kind of from degreasing protein
The method that polypeptide is extracted in newborn supernatant, includes the following steps：1）By lipid containing plant heat grinding slurry, centrifugation, obtaining light-phase liquid is
Vegetable fat, heavy-phase liquid are degreasing protein breast；2）Its pH=4.0~5.0 is adjusted, then under the isoelectric point of degreasing protein breast
It is precipitated, obtains skimmed milk supernatant and albumen underflow；3）Collect the skimmed milk supernatant, and adjust its pH=8.2~
8.9,3.5~6h of trypsin digestion is added in, obtains enzymolysis liquid；4）The enzymolysis liquid enzyme deactivation is obtained into no enzyme solution；5）To described
Activated carbon is added in no enzyme solution, centrifuge is recycled to remove activated carbon, obtains supernatant；6）By the supernatant concentration, spraying
Drying is to get the polypeptide powder.The method reduce the extraction step of protein, technological process is short, easy to operate, processing cost
It is low, suitable for industrialized production.But the preparation method carries out debitterizing and decoloring with activated carbon, can adsorb polypeptide, reduce polypeptide
Yield.
Invention content
The purpose of the present invention is to provide a kind of extracting method simple possibles, and extraction efficiency, yield and purity are high, obtained
Polypeptide bioactivity is high, without bitter taste, the low method that low molecular peptide is extracted from dairy products of extraction cost.
The problem of present invention in above-mentioned technology for mentioning, the technical solution taken is：
Microorganism used therefor of the present invention and its purchase enterprise are：Xuzhou Sai Fu bio tech ltd.
The method that polypeptide is extracted from degreasing protein breast supernatant, prepares, skimmed milk supernatant system including degreasing protein breast
Standby, fermentation, separation, the specific steps are：
It is prepared by degreasing protein breast：It is 1 by solid-liquid ratio:3-5（g/mL）Add water into lipid containing plant, slurry is ground at 45-55 DEG C extremely
Fineness is 0.2-1 μm, is subsequently placed in the centrifuge that rotating speed is 4000-6000r/min and centrifuges 25-35min, obtaining light-phase liquid is
Vegetable fat, heavy-phase liquid are degreasing protein breast, and spare, which can effectively remove the grease in lipid containing plant, avoid grease
Partially protein caused by oxidation is denaturalized, and improves the content of protein in albumin milk；
It is prepared by skimmed milk supernatant：The pH to 4.0-5.0 of degreasing protein breast is adjusted, is sunk under the isoelectric point of degreasing protein breast
It forms sediment, obtains skimmed milk supernatant and albumen underflow, skimmed milk supernatant sterilizes 20- at 115-125 DEG C after the two separation
30min, it is spare；
Fermentation：The pH to 6.5-8.0 of skimmed milk supernatant is adjusted, is inoculated with by inoculum concentration for 0.8-1.2% into skimmed milk supernatant
Composite bacteria adds the arabinose of composite bacteria total amount 0.02-0.5%, then the fermented and cultured under conditions of 25-35 DEG C
30-40h obtains zymotic fluid, spare, and above-mentioned composite bacteria is that weight ratio is 1:The bacillus subtilis of 0.5-0.8 and aspergillus niger, should
The proteolytic enzyme protolysate matter that step is generated using microorganism prepares polypeptide, and existing endopeptidase has end peptide again in generated enzyme
Enzyme can be cut in peptide middle-of-chain and both ends simultaneously, and the polypeptide yield height of preparation, performance are good, while fermentation process energy
Bitter polypeptide is enough sloughed, the polypeptide of preparation does not need to carry out de- suffering reason, so as to considerably reduce technological process, reduce life
Cost is produced, and the generation of fragranced substance is also had in fermentation process, the flavor and taste of polypeptide can be improved；
Separation：The pH to 3.5-4.5 of zymotic fluid is adjusted, chitosan oligosaccharide is added in for 0.1-0.2mg/mL by additive amount, at 30-40 DEG C
Under the conditions of stir 10-20min, be subsequently placed in rotating speed be 4000-6000r/min centrifuge in centrifuge 8-15min, take supernatant
8-15 times of concentration, freeze-drying are polypeptide, since the microorganism zymolyte comparison of ingredients of protein is complicated, existing inabundant water
The protein macromolecule of solution, also there is the polypeptide that molecular size range does not wait and the amino acid to dissociate, which can improve polypeptide
Separative efficiency, obtained polypeptide bioactivity are high, safe and non-toxic.
Preferably, arabinose contains the D-arabinose of 0.1-1.5%, the presence of the arabinose in fermentation step
Growth for microorganism provides carbon source, can promote the growth metabolism of microorganism, improves microorganism yield of enzyme and increases, makes to be laid eggs
White enzymatic activity is in higher level, shortens the extraction time of polypeptide, improves the yield of polypeptide, and the addition of D-arabinose can
The ratio of endopeptidase and peptide ending enzyme is adjusted, it can also hydrophobic group in modified amino acid molecule while the yield for improving polypeptide
Effect, improve the flavor of polypeptide.
Preferably, the preparation method of chitosan oligosaccharide is in separating step：It is 1 by solid-liquid ratio:12-16（g/L）By chitosan
Be added to the water, adjust its pH to 4.5-5.5, then add in complex enzyme for 5-7% by enzyme concentration, in complex enzyme papain and
The proportioning of cellulase is 1:0.4-0.6 digests 4-6h under conditions of being 40-50 DEG C in temperature, is aided with peroxide in enzymolysis process
Change hydrogen degradation, then adjust the pH value of enzymolysis liquid to neutrality, precipitate undegradable chitosan, supernatant concentration is dried in vacuo, so
Chitosan oligosaccharide is crosslinked to obtain with Fe3O4 magnetic particles afterwards, which combines hydrogen peroxide using chitosan as raw material, using double enzymolysis method
Method degradation chitosan prepares chitosan oligosaccharide, can cut off the glycosidic bond between GlcN and GlcNAC, preferential degradation compared with long chain and
The higher part of the degree of polymerization, relative molecular weight is low and the chitosan oligosaccharide of narrowly distributing for production, and the preparation method reaction condition is mild, easily
In control, free from environmental pollution, speed is fast.
Further preferably, hydrogenperoxide steam generator adds in the time and terminates preceding 50-70min for enzymolysis, hydrogenperoxide steam generator it is dense
It spends for 8-12%, the volume ratio of H2O2 and enzyme solution is 1:6-10.The effect of the assistant degradation of hydrogen peroxide is good, and adds in height too early
Concentration H2O2 solution can influence the vigor of enzyme, hinder the release of reduced sugar, and the condition can reach hydrolysis result and degradation effect
To best, while chitosan dissolves just in weak acid solution, and is not easy to form ammonium salt, and oxidation site is more, and degradation speed is fast so that
Molecular weight of product is low, and hydrogen peroxide degradation method is at low cost, easy to operate, while the subsequent processing of product is influenced small.
For optimisation technique method, the measure taken further includes：In hydrogenperoxide steam generator containing 0.2-0.3mM catechols and
0.01-0.1mM ellagic acids, catechol and ellagic acid can enrich the functional group of chitosan oligosaccharide by chemical modification, improve its object
Change property, while the stability of amino in chitosan, hydroxyl isoreactivity group amino can be protected, improve the adsorptivity of chitosan oligosaccharide
Can, and it is avoided that the generation of monosaccharide, the yield of chitosan oligosaccharide is improved, the final separative efficiency for improving polypeptide reduces extraction cost.
Compared with prior art, the advantage of the invention is that：1）The extracting method simple possible of polypeptide of the present invention, extraction effect
Rate, yield and purity are high, and the bioactivity of polypeptide obtained is high, without bitter taste, is easy to industrial-scale production, have it is good should
With value and market potential；2）The present invention prepares polypeptide using the proteolytic enzyme protolysate matter that microorganism generates, polypeptide yield is high,
Performance is good, does not need to carry out de- suffering reason, considerably reduces technological process, reduces production cost, and in fermentation process
There is the generation of fragranced substance, improve the flavor and taste of polypeptide；3）The separation method that extracting method of the present invention uses causes more
The separative efficiency of peptide is high, and obtained polypeptide bioactivity is high, safe and non-toxic, can be applied to food, medicine, field of health care products.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The method that polypeptide is extracted from degreasing protein breast supernatant is prepared including degreasing protein breast, prepared by skimmed milk supernatant, hair
Ferment, separation, the specific steps are：
1）It is prepared by degreasing protein breast：It is 1 by solid-liquid ratio:5（g/mL）Add water into lipid containing plant, slurry is ground at 45 DEG C to fineness
It is 1 μm, is subsequently placed in the centrifuge that rotating speed is 4000r/min and centrifuges 35min, obtains light-phase liquid as vegetable fat, heavy-phase liquid
For degreasing protein breast, spare, which can effectively remove the grease in lipid containing plant, part egg caused by avoiding the oxidation of grease
White matter is denaturalized, and improves the content of protein in albumin milk；
2）It is prepared by skimmed milk supernatant：The pH to 4.0 of degreasing protein breast is adjusted, is sunk under the isoelectric point of degreasing protein breast
It forms sediment, obtains skimmed milk supernatant and albumen underflow, skimmed milk supernatant sterilizes 20min at 125 DEG C after the two separation, it is standby
With；
3）Fermentation：The pH to 8.0 of skimmed milk supernatant is adjusted, into skimmed milk supernatant compound bacteria is inoculated with for 0.8% by inoculum concentration
Kind, the arabinose of composite bacteria total amount 0.5% is added, then fermented and cultured 40h obtains zymotic fluid under conditions of 25 DEG C, standby
With above-mentioned composite bacteria is that weight ratio is 1:0.5 bacillus subtilis and aspergillus niger, the step utilize the egg that microorganism generates
White enzymatic protein prepares polypeptide, and existing endopeptidase has peptide ending enzyme again in generated enzyme, can be simultaneously in peptide middle-of-chain
Cut with both ends, the polypeptide yield of preparation is high, performance is good, while fermentation process can slough bitter polypeptide, preparation it is more
Peptide does not need to carry out de- suffering reason, so as to considerably reduce technological process, reduce production cost, and in fermentation process also
The generation of fragranced substance is had, the flavor and taste of polypeptide can be improved；
4）Separation：The pH to 4.5 of zymotic fluid is adjusted, chitosan oligosaccharide is added in for 0.1mg/mL by additive amount, is stirred under conditions of 40 DEG C
10min is mixed, is subsequently placed in the centrifuge that rotating speed is 6000r/min and centrifuges 8min, take 15 times of supernatant concentration, freeze-drying is
For polypeptide, since the microorganism zymolyte comparison of ingredients of protein is complicated, the existing protein macromolecule not being fully hydrolyzed also has
The polypeptide and free amino acid, the step that molecular size range does not wait can improve the separative efficiency of polypeptide, obtained polypeptide life
Object activity is high, safe and non-toxic.
In above-mentioned fermentation step arabinose contains 0.1% D-arabinose, which exists for microorganism
Growth provides carbon source, can promote the growth metabolism of microorganism, improves microorganism yield of enzyme and increases, makes at institute's protease production
In higher level, shorten the extraction time of polypeptide, improve the yield of polypeptide, and the addition of D-arabinose can adjust endopeptidase
With the ratio of peptide ending enzyme, while the yield for improving polypeptide can also in modified amino acid molecule hydrophobic group effect, change
The flavor of kind polypeptide.
The preparation method of chitosan oligosaccharide is in above-mentioned separating step：It is 1 by solid-liquid ratio:16（g/L）Chitosan is added to the water,
Its pH to 4.5 is adjusted, then adds in complex enzyme for 7% by enzyme concentration, the proportioning of papain and cellulase is in complex enzyme
1:0.4,4h is digested under conditions of being 50 DEG C in temperature, is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts enzymolysis liquid
PH value precipitates undegradable chitosan, supernatant concentration, then vacuum drying is crosslinked to obtain shell widow with Fe3O4 magnetic particles to neutrality
Sugar, hydrogenperoxide steam generator add in the time and terminate preceding 70min for enzymolysis, a concentration of 8%, the H2O2 of hydrogenperoxide steam generator and enzyme solution
Volume ratio is 1:10.The effect of the assistant degradation of hydrogen peroxide is good, and the work of enzyme can be influenced by adding in high concentration H2O2 solution too early
Power hinders the release of reduced sugar, and the condition can make hydrolysis result and degradation effect reach best, while shell gathers in weak acid solution
Sugared dissolving, and be not easy to form ammonium salt just, oxidation site is more, and degradation speed is fast so that molecular weight of product is low, and hydrogen peroxide drops
Solution is at low cost, easy to operate, while the subsequent processing of product is influenced small；Contain 0.2mM catechols in hydrogenperoxide steam generator
With 0.1mM ellagic acids, catechol and ellagic acid can enrich the functional group of chitosan oligosaccharide by chemical modification, improve its materialization
Matter, while the stability of amino in chitosan, hydroxyl isoreactivity group amino can be protected, the absorption property of chitosan oligosaccharide is improved, and
It is avoided that the generation of monosaccharide, improves the yield of chitosan oligosaccharide, the final separative efficiency for improving polypeptide reduces extraction cost；The preparation
Method prepares chitosan oligosaccharide using chitosan as raw material, using double enzymolysis method joint hydrogen peroxide method degradation chitosan, can cut off GlcN
Glycosidic bond between GlcNAC, the preferential degradation part higher compared with long chain and the degree of polymerization, production relative molecular weight are low
And the chitosan oligosaccharide of narrowly distributing, and the preparation method reaction condition is mild, easily controllable, free from environmental pollution, speed is fast.
Embodiment 2：
The method that polypeptide is extracted from degreasing protein breast supernatant is prepared including degreasing protein breast, prepared by skimmed milk supernatant, hair
Ferment, separation, the specific steps are：
1）It is prepared by degreasing protein breast：It is 1 by solid-liquid ratio:3（g/mL）Add water into lipid containing plant, slurry is ground at 55 DEG C to fineness
It is 0.2 μm, is subsequently placed in the centrifuge that rotating speed is 6000r/min and centrifuges 25min, obtains light-phase liquid as vegetable fat, heavy phase
Liquid is degreasing protein breast, spare；
2）It is prepared by skimmed milk supernatant：The pH to 5.0 of degreasing protein breast is adjusted, is sunk under the isoelectric point of degreasing protein breast
It forms sediment, obtains skimmed milk supernatant and albumen underflow, skimmed milk supernatant sterilizes 30min at 115 DEG C after the two separation, it is standby
With；
3）Fermentation：The pH to 6.5 of skimmed milk supernatant is adjusted, into skimmed milk supernatant compound bacteria is inoculated with for 1.2% by inoculum concentration
Kind, the arabinose of composite bacteria total amount 0.02% is added, then fermented and cultured 30h obtains zymotic fluid under conditions of 35 DEG C, standby
With above-mentioned composite bacteria is that weight ratio is 1:0.8 bacillus subtilis and aspergillus niger；
4）Separation：The pH to 3.5 of zymotic fluid is adjusted, chitosan oligosaccharide is added in for 0.2mg/mL by additive amount, is stirred under conditions of 30 DEG C
20min is mixed, is subsequently placed in the centrifuge that rotating speed is 4000r/min and centrifuges 15min, take 8 times of supernatant concentration, freeze-drying is
For polypeptide.
Arabinose contains 1.5% D-arabinose in above-mentioned fermentation step.
The preparation method of chitosan oligosaccharide is in above-mentioned separating step：It is 1 by solid-liquid ratio:12（g/L）Chitosan is added to the water,
Its pH to 5.5 is adjusted, then adds in complex enzyme for 5% by enzyme concentration, the proportioning of papain and cellulase is in complex enzyme
1:0.6,6h is digested under conditions of being 40 DEG C in temperature, is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts enzymolysis liquid
PH value precipitates undegradable chitosan, supernatant concentration, then vacuum drying is crosslinked to obtain shell widow with Fe3O4 magnetic particles to neutrality
Sugar, hydrogenperoxide steam generator add in the time and terminate preceding 50min for enzymolysis, a concentration of 12%, the H2O2 of hydrogenperoxide steam generator and enzyme solution
Volume ratio is 1:6；Contain 0.3mM catechols and 0.01mM ellagic acids in hydrogenperoxide steam generator.
Embodiment 3：
The method that polypeptide is extracted from degreasing protein breast supernatant is prepared including degreasing protein breast, prepared by skimmed milk supernatant, hair
Ferment, separation, the specific steps are：
1）It is prepared by degreasing protein breast：It is 1 by solid-liquid ratio:4（g/mL）Add water into lipid containing plant, slurry is ground at 50 DEG C to fineness
It is 0.6 μm, is subsequently placed in the centrifuge that rotating speed is 5000r/min and centrifuges 30min, obtains light-phase liquid as vegetable fat, heavy phase
Liquid is degreasing protein breast, spare；
2）It is prepared by skimmed milk supernatant：The pH to 4.5 of degreasing protein breast is adjusted, is sunk under the isoelectric point of degreasing protein breast
It forms sediment, obtains skimmed milk supernatant and albumen underflow, skimmed milk supernatant sterilizes 25min at 120 DEG C after the two separation, it is standby
With；
3）Fermentation：The pH to 7.2 of skimmed milk supernatant is adjusted, into skimmed milk supernatant compound bacteria is inoculated with for 1.0% by inoculum concentration
Kind, the arabinose of composite bacteria total amount 0.3% is added, then fermented and cultured 36h obtains zymotic fluid under conditions of 30 DEG C, standby
With above-mentioned composite bacteria is that weight ratio is 1:0.6 bacillus subtilis and aspergillus niger；
4）Separation：The pH to 4.0 of zymotic fluid is adjusted, chitosan oligosaccharide is added in for 0.15mg/mL by additive amount, is stirred under conditions of 35 DEG C
15min is mixed, is subsequently placed in the centrifuge that rotating speed is 5000r/min and centrifuges 10min, take 12 times of supernatant concentration, be freeze-dried
As polypeptide.
Arabinose contains 0.8% D-arabinose in above-mentioned fermentation step.
The preparation method of chitosan oligosaccharide is in above-mentioned separating step：It is 1 by solid-liquid ratio:14（g/L）Chitosan is added to the water,
Its pH to 5.0 is adjusted, then adds in complex enzyme for 6% by enzyme concentration, the proportioning of papain and cellulase is in complex enzyme
1:0.5,5h is digested under conditions of being 45 DEG C in temperature, is aided with hydrogen peroxide degradation in enzymolysis process, then adjusts enzymolysis liquid
PH value precipitates undegradable chitosan, supernatant concentration, then vacuum drying is crosslinked to obtain shell widow with Fe3O4 magnetic particles to neutrality
Sugar, hydrogenperoxide steam generator add in the time and terminate preceding 60min for enzymolysis, a concentration of 10%, the H2O2 of hydrogenperoxide steam generator and enzyme solution
Volume ratio is 1:8；Contain 0.25mM catechols and 0.05mM ellagic acids in hydrogenperoxide steam generator.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. from degreasing protein breast supernatant extract polypeptide method, including degreasing protein breast prepare, skimmed milk supernatant prepare,
Fermentation, separation, it is characterised in that：The fermentation step is：The pH of skimmed milk supernatant is adjusted, composite bacteria is inoculated with, adds
Arabinose, fermented and cultured obtains zymotic fluid, spare.
2. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：It is described to connect
Kind amount is 0.8-1.2%, and the composite bacteria is that weight ratio is 1:The bacillus subtilis of 0.5-0.8 and aspergillus niger.
3. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：The hair
The pH of ferment culture is 6.5-8.0, temperature is 25-35 DEG C, time 30-40h.
4. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：Ah
The additive amount for drawing uncle's sugar is the 0.02-0.5% of composite bacteria total amount.
5. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：Ah
Draw the sugared D-arabinose containing 0.1-1.5% of uncle.
6. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：Described point
It is from step：The pH to 3.5-4.5 of zymotic fluid is adjusted, chitosan oligosaccharide is added in for 0.1-0.2mg/mL by additive amount, at 30-40 DEG C
Under the conditions of stir 10-20min, be then centrifuged for, supernatant concentration, freeze-drying be polypeptide.
7. the method for polypeptide is extracted in the breast supernatant according to claim 1 from degreasing protein, it is characterised in that：The shell
The preparation method of oligosaccharides is：It is 1 by solid-liquid ratio:12-16（g/L）Chitosan is added to the water, adjusts its pH to 4.5-5.5, so
Complex enzyme is added in for 5-7% by enzyme concentration afterwards, the proportioning of papain and cellulase is 1 in complex enzyme:0.4-0.6, in temperature
Spend be 40-50 DEG C under conditions of digest 4-6h, be aided with hydrogen peroxide degradation in enzymolysis process, then adjust enzymolysis liquid pH value to
Then neutrality, supernatant concentration, vacuum drying are crosslinked to obtain chitosan oligosaccharide with Fe3O4 magnetic particles, contain in the hydrogenperoxide steam generator
Catechol and ellagic acid.
8. the method for polypeptide is extracted in the breast supernatant according to claim 7 from degreasing protein, it is characterised in that：The mistake
Hydrogen peroxide solution adds in the time and terminates preceding 50-70min for enzymolysis, a concentration of 8-12% of hydrogenperoxide steam generator, H2O2 and enzyme solution
Volume ratio is 1:6-10.
9. the method for polypeptide is extracted in the breast supernatant according to claim 7 from degreasing protein, it is characterised in that：The mistake
Contain 0.2-0.3mM catechols and 0.01-0.1mM ellagic acids in hydrogen peroxide solution.
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2018
- 2018-02-08 CN CN201810129326.2A patent/CN108179164A/en not_active Withdrawn
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| WO2004082397A1 (en) * | 2003-03-21 | 2004-09-30 | Upfront Chromatography A/S | Method for high throughput volumes in the fractionation of bio-molecules by chromatographic systems |
| CN103740796A (en) * | 2014-01-14 | 2014-04-23 | 天津替代医学科技有限公司 | Method for extracting polypeptides from skimmed protein milk supernatant |
| CN106244658A (en) * | 2016-09-19 | 2016-12-21 | 陕西科技大学 | A kind of preparation method of sweet potato polypeptide |
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| 梁天姣: "壳聚糖的制备及其在酵母多肽分离纯化上的应用研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
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