CN103740796B - Method for extracting polypeptides from skimmed protein milk supernatant - Google Patents
Method for extracting polypeptides from skimmed protein milk supernatant Download PDFInfo
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- CN103740796B CN103740796B CN201410015528.6A CN201410015528A CN103740796B CN 103740796 B CN103740796 B CN 103740796B CN 201410015528 A CN201410015528 A CN 201410015528A CN 103740796 B CN103740796 B CN 103740796B
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Abstract
The invention discloses a method for extracting polypeptides from skimmed protein milk supernatant. The method comprises the following steps: (1) thermally grinding, pulping and centrifuging lipid-containing plants so as to obtain light phase liquid serving as vegetable fat, and obtain heavy phase liquid serving as skimmed protein milk; (2) regulating the pH to be 4.0-5.0, and precipitating under the isoelectric point of the skimmed protein milk so as to obtain skimmed milk supernatant and protein concentrated pulp; (3) collecting the skimmed milk supernatant, regulating the pH of the skimmed milk supernatant to be 8.2-8.9, adding trypsin for performing enzymolysis for 3.5-6 hours so as to obtain an enzymolysis solution; (4) performing enzyme deactivation on the enzymolysis solution to obtain an enzyme-free solution; (5) adding activated carbon into the enzyme-free solution, removing the activated carbon by utilizing a centrifugal machine so as to obtain the supernatant; and (6) concentrating the supernatant, performing spray drying so as to obtain the polypeptide powder. According to the method, a step of extracting the proteins is reduced, and the process flow is short, the operation is simple, and the processing cost is low and industrial production is facilitated.
Description
Technical field
The present invention relates to the preparation method of a peptide species, particularly from containing the method extracting polypeptide fat plant apolipoprotein breast supernatant liquor.
Background technology
Polypeptide is that a-amino acid links together with peptide chain and the compound formed, and it is also the intermediate product of proteolysis.Two amino acid molecular dehydrating condensations can form dipeptides, and multiple amino acid molecular dehydrating condensation can form tripeptides, tetrapeptide, pentapeptide etc.Usually the compound formed by 10 ~ 100 amino acid molecular dehydrating condensations is polypeptide, and its molecular weight is lower than 10, and 000 dalton, can through semi-permeable membranes, not by trichoroacetic acid(TCA) and ammonium sulfate precipitate.
Human body, to the absorption of protein, is mainly carried out the degradation of proteins into the form of low peptide by the effect of proteolytic enzyme.Experiment shows that peptide is compared amino acid and more easily, faster can be absorbed by mucous membrane of small intestine.Protein after enzymic hydrolysis, can obtain the mixture of polypeptide, and these mixtures solvability in acid condition improves greatly, and has the low good characteristic of concentration high viscosity.Although reach certain level to the research of vegetable-protein bioactive peptide both at home and abroad, and related products is applied to health care, but be mainly soybean active peptide product, the bioactive peptide research for other plant is just at the early-stage, related products is very few, cannot meet foodstuffs industry demand.
The extraction of domestic polypeptide mainly isolates protein from feed grouts, then polypeptide is extracted through enzymolysis, no matter be traditional milling process or lixiviation process, in the obtained dregs of rice all can there is sex change to a certain degree in protein, this brings a very large difficult problem to the deep processing of protein, cause the extraction yield of polypeptide low, protein utilization is not high.There is a lot of defect in the dregs of rice that Conventional processing methods obtains, as in milling process, the residual oil content in the dregs of rice is high, causes very large difficulty to the separation and purification of protein, and milling process obtains the dregs of rice through high temperature, and protein denaturation is serious, is more unfavorable for the extraction of protein; The dregs of rice residual organic solvent that lixiviation process is obtained, directly affect the quality of protein separation, the dregs of rice become waste liquid after proteins extraction, cannot carry out processing and utilization again, cause prepared using insufficient, waste resource.
Li Xionghui, cross new victory to wait and in " research of the soybean polypeptide extraction process " literary composition delivered in 09 phase in 1999 " Food science ", soybean is first carried out low temperature desolventizing and obtain dregs of beans, tentatively protein is obtained again through water extraction process, use the heavy way of acid to carry out protein purification afterwards, finally use two enzyme to be hydrolyzed to protein and obtain polypeptide.This method raw material is single, extracts protein processes complexity, high cost from soybean.Xu Huaide, Chen Jinhai etc. disclose a kind of making method of walnut polypeptide powder in 200710017281.1, the method not only remains the multiple nutritional components of walnut, and the content of walnut polypeptide powder polypeptide reaches 60-80%, first this invention process is pulverized by walnut dregs, then adopts supersonic method extracted protein.Use papoid to carry out enzymolysis to the protein after vacuum-drying, through the enzyme that goes out, centrifugal, get supernatant liquor dialysis, through concentrated, after vacuum-drying, can polypeptide be obtained.It is large that this method extracts protein difficulty from the dregs of rice, and need further to purify, and energy consumption is large.
The raw material that domestic enzyme process polypeptide maturation process uses to be protein content be 90% soybean protein isolate.Soybean protein isolate can produce the problems such as serious environmental pollution in process of production, does not meet China's strategy of sustainable development, will progressively be eliminated.All the other enzymolysis polypeptides utilize feedstuff protein as raw material mostly, and after extracting protein by alkali extraction and acid precipitation, recycling proteolytic enzyme carrys out enzymolysis and makes polypeptide powder.The problems such as this method has leaching process complicated, and waste is too many, protein extraction difficulty.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of method extracting polypeptide from apolipoprotein breast supernatant liquor, the method reduce the extraction step of protein, technical process is short, simple to operate, is suitable for suitability for industrialized production.
For this reason, technical scheme of the present invention is as follows:
From apolipoprotein breast supernatant liquor, extract a method for polypeptide, comprise the steps:
1) will containing fat plant heat grinding slurry, through whizzer process, obtaining light-phase liquid is Vegetable oil lipoprotein, and heavy-phase liquid is apolipoprotein breast;
2) regulate described apolipoprotein breast pH=4.0 ~ 5.0, then precipitate under the iso-electric point of described apolipoprotein breast, obtain skimming milk supernatant liquor and albumen underflow;
3) collect described skimming milk supernatant liquor, and regulate its pH=8.2 ~ 8.9, then add the trypsinase of described skimming milk supernatant liquor quality 2 ~ 4% wherein, at 40 DEG C, enzymolysis 3.5 ~ 6h under agitation condition, obtain enzymolysis solution;
4) described enzymolysis solution is heated 4 ~ 8min under 85 ~ 95 DEG C of conditions, the enzyme that goes out obtains without enzyme liquid;
5) to described without adding the gac accounting for its quality 4 ~ 6% in enzyme liquid, stir 15 ~ 40min debitterizing and decoloring; Then remove gac wherein, obtain supernatant liquor;
6) be carry out spraying dry after 30 ~ 40wt% by described supernatant concentration to solids content, obtain polypeptide powder.
Described is walnut kernel, Semen arachidis hypogaeae, corn, soybean, sunflower seeds, linseed oil, vegetable seed, sesame or almond containing fat plant.
Step 1) described in apolipoprotein Ruzhong protein content be 21 ~ 46wt%.
Step 2) in deposition rate when obtaining skimming milk supernatant liquor and albumen underflow be 73 ~ 88%.
In step 2) in by HCl adjust ph.
In step 3) in by NaOH adjust ph.
Step 5) removing gac use vibration discharging centrifugal machine, helical-conveyer centrifugal.
Step 6) in the concentration method of supernatant liquor for described supernatant liquor is utilized falling-film evaporator, at vacuum tightness 850 ~ 890mbar, at temperature 48 ~ 52 DEG C, carry out vacuum concentration.
The method reduce the extraction step of protein, technical process is short, simple to operate, and tooling cost is low, is suitable for suitability for industrialized production.And obtained polypeptide can be widely used in protective foods, has very high nutritive value.
Embodiment
Below in conjunction with embodiment, preparation method of the present invention is described in detail.
Embodiment 1
The method of polypeptide is extracted from walnut kernel skimming milk supernatant liquor
1) after walnut kernel being soaked 2 hours in the aqueous sodium hydroxide solution of 2wt%, rinse well with clear water, then use the peracetic acid disinfectant of 0.35wt%, then use clear water spray irrigation clean;
2) to step 1) add the water of its 5 times of quality in walnut kernel after process, at about 50 DEG C use grinding-wheel grinder pulp grinder heat grinding slurries, isolate slag crust and discolor; Again gained slurries colloidal mill being milled to fineness is 2 ~ 4 μm; Then colloidal mill is ground rear slurry and insert disk centrifugal separator at about 45 DEG C, centrifugation under rotating speed 7310r/min condition, obtaining light-phase liquid is Vegetable oil lipoprotein; Heavy-phase liquid is apolipoprotein breast; Slag-drip opening obtains the slag crust with high added value;
3) the apolipoprotein breast obtained is inserted paste roller mill, carry out 10 defibrinations, utilizing Kjeldahl determination to record total nitrogen is 3.5%, and the content being converted into protein is 22%;
4) then to step 3) obtain adding HCl in solution, regulate its pH=4.9, precipitate under the iso-electric point of described apolipoprotein breast again, obtain skimming milk supernatant liquor and albumen underflow when deposition rate is 80%, after both are separated, described albumen underflow is concentrated, spraying dry obtains protein powder;
5) described skimming milk supernatant liquor is got, recording total nitrogen with Kjeldahl determination is 0.6%, being converted into protein content is 3.8%, add the NaOH of 1mol/L wherein, regulate its pH=8.5, add the trypsinase accounting for its quality 4%, at 40 DEG C, enzymolysis 3.5 hours under agitation condition, obtain enzymolysis solution;
6) described enzymolysis solution is heated 6 minutes in 90 DEG C of waters bath with thermostatic control, carry out going out enzyme, obtain without enzyme liquid;
7) be cooled to rapidly 30 DEG C by described without enzyme liquid again, add the gac accounting for its quality 4%, Keep agitation 25 minutes, debitterizing and decoloring; Inserted helical-conveyer centrifugal again, centrifugal treating under 4000r/min condition, the supernatant liquor of the gac that is removed;
8) described supernatant liquor is utilized falling-film evaporator, vacuum concentration is carried out under the condition of vacuum tightness 850mbar, temperature 50 C, when being about 35wt% to solids content, utilize drying machine with centrifugal spray, be 160 DEG C in inlet temperature, under pressure is the condition of 0.40MPa, carries out spraying dry and obtain polypeptide powder.
Embodiment 2
The method of polypeptide is extracted from Semen arachidis hypogaeae skimming milk supernatant liquor
1) after Semen arachidis hypogaeae being soaked 1.5 hours in the aqueous sodium hydroxide solution of 1wt%, rinse well with clear water, then use the peracetic acid disinfectant of 0.35wt%, then use clear water spray irrigation clean;
2) to step 1) add the water of its 4 times of quality in Semen arachidis hypogaeae after process, at about 50 DEG C use grinding-wheel grinder pulp grinder heat grinding slurries, isolate slag crust and discolor; Again gained slurries colloidal mill being milled to fineness is 2 ~ 4 μm; Then colloidal mill is ground rear slurry and insert disk centrifugal separator at about 45 DEG C, centrifugation under rotating speed 7310r/min condition, obtaining light-phase liquid is Vegetable oil lipoprotein; Heavy-phase liquid is apolipoprotein breast; Slag-drip opening obtains the slag crust with high added value;
3) the apolipoprotein breast obtained is inserted paste roller mill, carry out 8 defibrinations, utilizing Kjeldahl determination to record total nitrogen is 5.68%, and the content being converted into protein is 31%;
4) then to step 3) obtain adding HCl in solution, regulate its pH=4.5, precipitate under the iso-electric point of described apolipoprotein breast again, when deposition rate is 73%, obtain skimming milk supernatant liquor and albumen underflow, after both are separated, described albumen underflow is concentrated, spraying dry obtains protein powder;
5) described skimming milk supernatant liquor is got, recording total nitrogen with Kjeldahl determination is 0.75%, being converted into protein content is 4.1%, add the NaOH of 1mol/L wherein, regulate its pH=8.5, add the trypsinase accounting for its quality 3%, at 40 DEG C, enzymolysis 4 hours under agitation condition, obtain enzymolysis solution;
6) described enzymolysis solution is heated 7 minutes under 90 DEG C of conditions, carry out going out enzyme, obtain without enzyme liquid;
7) be cooled to rapidly 30 DEG C by described without enzyme liquid again, add the gac accounting for its quality 5%, Keep agitation 30 minutes, debitterizing and decoloring; Inserted helical-conveyer centrifugal again, centrifugal treating under 4200r/min condition, the supernatant liquor of the gac that is removed;
8) described supernatant liquor is utilized falling-film evaporator, vacuum concentration is carried out under the condition of vacuum tightness 880mbar, temperature 49 DEG C, when being about 35% to solids content, utilize drying machine with centrifugal spray, be 160 DEG C in inlet temperature, under pressure is the condition of 0.40MPa, carries out spraying dry and obtain polypeptide powder.
Embodiment 3
The method of polypeptide is extracted from soybean skimming milk supernatant liquor
1) after soybean being soaked 2 hours in the aqueous sodium hydroxide solution of 2wt%, rinse well with clear water, then use the peracetic acid disinfectant of 0.35wt%, then use clear water spray irrigation clean;
2) to step 1) add the water of its 3 times of quality in soybean after process, at about 50 DEG C use grinding-wheel grinder pulp grinder heat grinding slurries, isolate slag crust and discolor; Again gained slurries colloidal mill being milled to fineness is 2 ~ 4 μm; Then colloidal mill is ground rear slurry and insert whizzer at about 45 DEG C, centrifugation under rotating speed 7310r/min condition, obtaining light-phase liquid is Vegetable oil lipoprotein; Heavy-phase liquid is apolipoprotein breast; Slag-drip opening obtains the slag crust with high added value;
3) the apolipoprotein breast obtained is inserted paste roller mill, carry out 6 defibrinations, utilizing Kjeldahl determination to record total nitrogen is 7.2%, and the content being converted into protein is 41%;
4) then to step 3) obtain adding HCl in solution, regulate its pH=4.5, precipitate under the iso-electric point of described apolipoprotein breast again, when deposition rate is 78%, obtain skimming milk supernatant liquor and albumen underflow, after both are separated, described albumen underflow is concentrated, spraying dry obtains protein powder;
5) described skimming milk supernatant liquor is got, recording total nitrogen with Kjeldahl determination is 0.79%, being converted into protein content is 4.5%, add the NaOH of 1mol/L wherein, regulate its pH=8.5, add the trypsinase accounting for its quality 3%, at 40 DEG C, enzymolysis 5 hours under agitation condition, obtain enzymolysis solution;
6) described enzymolysis solution is heated 4 minutes under 90 DEG C of conditions, carry out going out enzyme, obtain without enzyme liquid;
7) be cooled to rapidly 30 DEG C by described without enzyme liquid again, add the gac accounting for its quality 5%, Keep agitation 30 minutes, debitterizing and decoloring; Inserted vibration discharging centrifugal machine again, centrifugal treating under 4200r/min condition, the supernatant liquor of the gac that is removed;
8) described supernatant liquor is utilized falling-film evaporator, at vacuum tightness 890mbar, vacuum concentration is carried out when temperature 48 DEG C, when being about 35% to solids content, utilize drying machine with centrifugal spray, be 160 DEG C in inlet temperature, under pressure is the condition of 0.40MPa, carries out spraying dry and obtain polypeptide powder.
In the present invention, the measuring method of protein content is: utilize Kjeldahl determination to record total nitrogen content, be then converted into protein content.
Determining content of peptides:
Polypeptide powder obtained in embodiment 1 ~ 3 is added the trichloroacetic acid solution that concentration is 15wt%, be stirred well to high molecular weight protein to precipitate completely, and micromolecule polypeptide retains in the solution, precipitation in removing solution obtains the supernatant liquor being dissolved with polypeptide, according to the total nitrogen content of the test determines supernatant liquor of GB/T5009.5-2010, then extrapolate the content of polypeptide.
That is: in the obtained polypeptide powder of embodiment 1,2,3, the content of polypeptide is respectively 74%, 75% and 72%.
Utilize the polypeptide preparation yolk pie that the inventive method is obtained:
The method of the polypeptide preparation polypeptide group that embodiment 1,2,3 is obtained, comprises the steps:
1) heart fillings is sent in preparation: by polypeptide, functional oligose, egg is put into basin egg-whisk and beaten thin out to yolk color or turn white, and oligose all dissolves; Add the aqueous solution of polypeptide powder, stir, add weak strength flour, the aqueous solution of polypeptide powder is again added after stirring, stir and obtain mixed pulp, the little fire of described mixed pulp is endured slowly, is stirred to mixed pulp with rubber squeegee simultaneously and becomes pasty state, from fire, add butter rapidly, stir, add appropriate vanilla, milk flavour and emulsifying agent, obtain sending heart fillings, send heart fillings to cool to be placed on freezer compartment of refrigerator to be preserved by described.
2) prepare cake to stick with paste: shell egg, functional oligose are put into basin egg-whisk beat to expand to egg liquid turn white, liquid is dense thick; Add weak strength flour, Mierocrystalline cellulose, pectinose, full grain flour, sorbyl alcohol, malt sugar, baking powder, citric acid, salt and shortening stir, then add weak strength flour and stir and obtain cake and stick with paste;
3) polypeptide group is prepared: stuck with paste described cake and pour in mould to 1/4 full, put into the roasting 3min of the baking box being preheated to 180 DEG C, the filling heart that put into suitable size again in mould, that freeze, secondly being stuck with paste by cake to pour in mould expires to 9 one-tenths, put into baking box, bake at 180 DEG C and can obtain polypeptide group.
Suggestion massfraction raw materials used in aforesaid method is as follows: functional oligose 16%, weak strength flour 26%, egg 4%, full grain flour 10%, polypeptide powder 15%, water 8%, pectinose 1%, Mierocrystalline cellulose 2.7%, emulsifying agent 4%, shortening 8%, sorbyl alcohol 1%, malt sugar 3%, baking powder 0.7%, salt 0.4%, vanilla 0.1%, milk flavour 0.07%, citric acid 0.03%.
Containing polypeptide in the group that the method is obtained, have polypeptide concurrently and be easy to fast, anti-oxidant, hypotensive by intestinal absorption, absorption rate, decreasing cholesterol, antifatigue several functions.And the pectinose that contains and Mierocrystalline cellulose can suppress human body to the absorption of sucrose, promote to be suitable for designed for old people by intestines peristalsis in group.
Claims (8)
1. from apolipoprotein breast supernatant liquor, extract a method for polypeptide, it is characterized in that: comprise the steps:
1) will containing fat plant heat grinding slurry, through whizzer process, obtaining light-phase liquid is Vegetable oil lipoprotein, and heavy-phase liquid is apolipoprotein breast;
2) regulate described apolipoprotein breast pH=4.0 ~ 5.0, then precipitate under the iso-electric point of described apolipoprotein breast, obtain skimming milk supernatant liquor and albumen underflow;
3) collect described skimming milk supernatant liquor, and regulate its pH=8.2 ~ 8.9, then add the trypsinase of described skimming milk supernatant liquor quality 2 ~ 4% wherein, at 40 DEG C, enzymolysis 3.5 ~ 6h under agitation condition, obtain enzymolysis solution;
4) described enzymolysis solution is heated 4 ~ 8min under 85 ~ 95 DEG C of conditions, the enzyme that goes out obtains without enzyme liquid;
5) to described without adding the gac accounting for its quality 4 ~ 6% in enzyme liquid, stir 15 ~ 40min debitterizing and decoloring; Then remove gac wherein, obtain supernatant liquor;
6) be carry out spraying dry after 30 ~ 40wt% by described supernatant concentration to solids content, obtain polypeptide powder.
2. the method for claim 1, is characterized in that: described is walnut kernel, Semen arachidis hypogaeae, corn, soybean, sunflower seeds, linseed oil, vegetable seed, sesame or almond containing fat plant.
3. the method for claim 1, is characterized in that: the Ruzhong of apolipoprotein described in step 1) protein content is 21 ~ 46wt%.
4. the method for claim 1, is characterized in that: step 2) in deposition rate when obtaining skimming milk supernatant liquor and albumen underflow be 73 ~ 88%.
5. the method for claim 1, is characterized in that: in step 2) in by HCl adjust ph.
6. the method for claim 1, is characterized in that: by NaOH adjust ph in step 3).
7. the method for claim 1, is characterized in that: step 5) removing gac uses vibration discharging centrifugal machine, helical-conveyer centrifugal.
8. the method for claim 1, is characterized in that: in step 6), the concentration method of supernatant liquor is for utilize falling-film evaporator by described supernatant liquor, at vacuum tightness 850 ~ 890mbar, carries out vacuum concentration at temperature 48 ~ 52 DEG C.
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| CN106889298A (en) * | 2015-12-21 | 2017-06-27 | 北京华昱安然医药科技有限公司 | A kind of fructus cannabis polypeptide powder and preparation method thereof |
| CN105622739A (en) * | 2016-02-15 | 2016-06-01 | 天津替代医学科技股份有限公司 | Method for extracting ubiquitin and like ubiquitin from protein milk |
| CN108179164A (en) * | 2018-02-08 | 2018-06-19 | 金华市铁骑士生物科技有限公司 | The method that polypeptide is extracted from degreasing protein breast supernatant |
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| WO2004085664A2 (en) * | 2003-03-25 | 2004-10-07 | Council Of Scientific And Industrial Research | Process for the preparation of angiotensin converting enzyme (ace) inhibitors and its use |
| CN102505034A (en) * | 2011-12-02 | 2012-06-20 | 安徽燕之坊食品有限公司 | Method for preparing active polypeptide from corn protein powder |
| CN102533918A (en) * | 2012-01-18 | 2012-07-04 | 安徽燕之坊食品有限公司 | Method for preparing rapeseed active polypeptides |
| CN103392904A (en) * | 2013-08-02 | 2013-11-20 | 江苏丘陵地区镇江农业科学研究所 | Method used for extracting black soya bean polypeptide by combining wet ball milling with enzyme method |
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Patent Citations (4)
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| WO2004085664A2 (en) * | 2003-03-25 | 2004-10-07 | Council Of Scientific And Industrial Research | Process for the preparation of angiotensin converting enzyme (ace) inhibitors and its use |
| CN102505034A (en) * | 2011-12-02 | 2012-06-20 | 安徽燕之坊食品有限公司 | Method for preparing active polypeptide from corn protein powder |
| CN102533918A (en) * | 2012-01-18 | 2012-07-04 | 安徽燕之坊食品有限公司 | Method for preparing rapeseed active polypeptides |
| CN103392904A (en) * | 2013-08-02 | 2013-11-20 | 江苏丘陵地区镇江农业科学研究所 | Method used for extracting black soya bean polypeptide by combining wet ball milling with enzyme method |
Non-Patent Citations (1)
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