CN102766671B - Method for extracting proteins from vitamin C fermented mash - Google Patents
Method for extracting proteins from vitamin C fermented mash Download PDFInfo
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Abstract
The invention discloses a method for extracting proteins from vitamin C fermented mash, which comprises the following steps: ultrafiltering the vitamin C fermented mash via an ultrafiltering membrane, and collecting the intercepted components; adding water into the intercepted components so as to uniformly mix, quickly freezing the intercepted component solution via an ultralow temperature quick-freezing technology, and then crushing the quick-frozen intercepted component solution via a high-pressure homogenizer; adding a compound protease into the crushed suspension, extracting the protein components via enzymolysis, centrifugally separating enzymatically decomposed solution obtained by enzymolysis, and collecting liquid supernatant; adjusting the pH value of the liquid supernatant, and carrying out flocculating settling for the proteins; and drying the precipitate to obtain the albumen powder. According to the invention, the albumen powder is extracted by a vitamin C fermented by-product, a wide variety of amino acid in the extracted proteins is capable of being taken as feed proteins and raw materials for deeply processing the proteins, thereby realizing comprehensively and effectively utilizing the vitamin C fermented by-product, increasing a new channel of the resource of the feeding albumen powder, and saving the resources and protecting the environment.
Description
Technical field
The present invention relates to a kind of from vitamin c fermenting mash method for extracting proteins, particularly a kind of mycelium is collected production technology broken and protein separation extraction, belongs to biological technical field.
Background technology
Vitamins C is a kind of indispensable material that maintains health of human body.It is viable cell redox reaction catalyzer, participate in multiple metabolism in body, have and promote multiple synthetic physiological action rapidly in body.Easily there is vitamin C deficiency in the C that is deficient in vitamin in body, when serious, can cause death.Clinical application: prevent and treat vitamin C deficiency; Promote organism metabolism, strengthen anti-infection ability, stop and produce carcinogenic substance; The first aid of various diseases; Make tissue produce collagen and can promote wound healing; Can help enzyme that cholesterol is changed into the solid acid of courage and drain, to alleviate the fragility of vessel wall, prevention coronary heart disease; Intravenous High-Dose is for the treatment of Keshan disease.Food applications: for foodstuff additive.As the nutritional additive of the nitrite inhibitor of the properties-correcting agent of the antiager of the antioxidant of food, beer, flour, tinned pre-and cake, candy, beverage, milk-product.
VC is for cosmetic industry and fodder additives etc.
At present, vitamins C is mainly to take W-Gum as raw material, adopts two-step fermenting to produce.Two-step fermenting is that first the Tengerdy of the U.S. and Huang etc. find, its process be the approach of L-sorbose, the L-sorbose that L-sorbyl alcohol first transforms through bacterium, the latter generates vitamin C precursor 2-KGA through fermentation using bacteria again.Early, L-sorbose pathways metabolism and metabolic mechanism research are also more deep, but are not used to so far production practice due to its 2-KGA low conversion rate in external two-step fermenting research.China Vitamin C Two-step Fermentation Fa Shiyou Institute of Microorganism, Academia Sinica cooperates in early 1970s invention with BJ Pharmaceutical Co., Ltd., it is current unique industrial microbe transformation method of vitamins C that is successfully applied to, this way belongs to L-sorbose approach, and its feature is that second step fermentation is completed by concomitance bacterium mixed fermentations such as oxidizing glucose acidfast bacilli and bacillus megateriums.One group of strain excellent that can utilize L-sorbose to produce 2-KGA that China filters out is at first oxidizing glucose acidfast bacilli and concomitance bacterium striped pseudomonas thereof, with by constantly practice improvement, by pilot scale, identify also and promote the use of in China gradually in June, 1976.Vitamin C Two-step Fermentation working system can produce the by products such as a large amount of mycelium, protein, account for 4% of fermented liquid, through ultrafiltration, hold back component viscosity larger, contain certain sodium colombate composition, the smell is awful, cannot be directly as animal-feed, much production of vitamin C producer holds back into the barnyard stacking that is placed in, boiler burning or direct outer selling by this part.The processing not in time of vitamin c fermenting by product pollutes air, affects factory and holds, and is therefore necessary vitamin c fermenting by product to fully utilize, and there are to doulbe-sides' victory meaning in society and development of company.China is that feed consumption Guo， China aquaculture the biggest in the world increases day by day to the demand of feedstuff protein, and for example 70% of feeding soybean protein dependence import (referring to fourth national literature, expand non-grain feed raw material supplying [J]. Chinese feed, 2006,22:22-23).Therefore, from vitamin c fermenting by product, extract protein as feedstuff protein significant.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of from vitamin c fermenting by product---method for extracting proteins vitamin c fermenting mash, the method can extract protein easily and efficiently from mash, realized the comprehensive utilization of resource, solved the problem of fermentation liquid to environment, turn waste into wealth again and reduced the waste of resource, there is good economics and sociology meaning.
The invention discloses a kind of from vitamin c fermenting mash method for extracting proteins, its step is for collecting vitamin c fermenting mash ultrafiltration filter residue, pass through super low temperature quick frozen, high pressure homogenizer carries out fragmentation to materials such as mycelium, enzymatic hydrolysis extracts albumen, centrifugal, flocculating settling, the dry protein powder that obtains, concrete technical scheme is as follows:
A method for extracting proteins from vitamin c fermenting mash, is characterized in that specifically comprising the following steps:
(1) vitamin c fermenting mash is carried out to uf processing with ultra-filtration membrane, the macromolecular substance such as mycelium wherein and protein are held back out from mash, collect and hold back composition;
(2) add water to stir the composition of holding back of step (1) ultrafiltration gained, then adopt super low temperature quick frozen technology will hold back the quick-frozen of composition solution, obtain suspension liquid, and then with high pressure homogenizer, gained suspension liquid is carried out to break process;
(3) albumen prozyme is added in the suspension liquid after step (2) is processed to enzymolysis and extraction protein component wherein;
(4) enzymolysis solution of enzymolysis gained is carried out to centrifugation, collect supernatant liquor;
(5) pH of the described supernatant liquor of regulating step (4), makes protein flocculating settling, crosses leaching precipitation;
(6) precipitation in step (5) is dried, obtains protein powder.
In above-mentioned steps (1), the molecular weight cut-off of described ultra-filtration membrane is 1000～2000 dalton; The condition that fermentation liquid carries out ultrafiltration is: intake pressure 0.2～0.5Mpa, 29～39 ℃ of ultrafiltration temperature, fermentation liquid pH value 5.0～7.5.
In above-mentioned steps (2), water to add volume and the volume ratio of holding back composition be 1:0.5～1:5; Super low temperature quick frozen temperature is-35～-50 ℃, and the quick-frozen time is 5～10min; Suspension liquid is 100～150Mpa by the pressure of high pressure homogenizer, and flow velocity is 150-260m/s.
In above-mentioned steps (3), described albumen prozyme is comprised of carboxypeptidase and enzyme A, and enzyme A is selected from one or more in following enzyme: trypsinase, papoid, Sumizyme MP; Wherein the mass ratio of carboxypeptidase and enzyme A is 1:1.0-2.0, and preferred described albumen prozyme is comprised of carboxypeptidase, Sumizyme MP and trypsinase, and their mass ratio is carboxypeptidase: Sumizyme MP: trypsinase=1:1.2:0.5.
In above-mentioned steps (3), the consumption of albumen prozyme is 0.35%～1.2% of suspension liquid quality, and hydrolysis temperature is 40～60 ℃, and during enzymolysis, suspension liquid pH is 5.5～8.5, and enzymolysis time is 2～3.5h.
In above-mentioned steps (4), carry out centrifugation at 30～40 ℃, rotating speed is 800～1500rpm, and the time is 30～90min.
In above-mentioned steps (5), by supernatant liquor pH regulator to 2.0～4.5, make protein flocculating settling, the protein settling time is 6-14h.
In above-mentioned steps (6), the spray-dired method of dry employing, the dry inlet temperature of spraying is 200～300 ℃, air outlet temperature is 50～100 ℃.
The advantage that the present invention has and positively effect:
1. the present invention utilizes vitamin c fermenting by product to extract protein powder; extract rich amino acids in albumen; can be used as feedstuff protein; also can be used as the raw material of protein deep processing; having realized the comprehensive of vitamin c fermenting by product effectively utilizes; increase a new channel in albumen powder for feed source, saved resource, protected environment.
2. utilize clarifixator to carry out after first quick-frozen compositions such as vitamin c fermenting mycelium broken, the protein component in fermentation byproduct is extracted, by albumen prozyme, extract the protein in fermentation process for vitamin production by product, protein extracting ratio is high.
3. raw materials cost of the present invention is low, and equipment is simple, easy to operate, and energy consumption is little, and byproduct comprehensive utility value is high, is easy to accomplish scale production, and has reduced the pollution of production of vitamin C to environment, has improved industry added value.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete technology condition of embodiment, material proportion and result thereof be only for the present invention is described, and should also can not limit the present invention described in claims.
In following embodiment, adopt the hydrochloric acid soln of 15-30wt%, the sulphuric acid soln of 15-40wt%, sodium hydroxide or its aqueous solution, potassium hydroxide or its aqueous solution regulate pH.
embodiment 1
(1) fermentation liquid uf processing
Get vitamin c fermenting mash 100g, by the daltonian ultrafiltration membrane system of molecular weight cut-off 2000, process, intake pressure is controlled at 0.4～0.5Mpa, regulate fermentation liquid pH value 5.0,35～39 ℃ of service temperatures, after uf processing, the by product such as mycelium and protein is held back, and rejection reaches 98.0%, collects and holds back composition.
(2) break process
Composition is held back in collection, add distilled water and stir, water is 1:0.5 with the volume ratio of holding back composition, then mixed solution is added to super low temperature quick frozen equipment, at-35 ℃ of quick-frozen 5min, and then at pressure 120Mpa, under flow velocity 200m/s condition, by high pressure homogenizer, suspension liquid is carried out to break process, bacterial cell disruption rate is 94%.
(3) enzymatic hydrolysis
Albumen prozyme (carboxypeptidase, Sumizyme MP, papoid mass ratio are 1:1:1) toward adding slurries quality 0.35% in the slurries after fragmentation, carries out enzymolysis to protein, and temperature of reaction is 50～55 ℃, and pH is 7～8, and the reaction times is 3.5h.
(4) centrifugation
Utilize whizzer to carry out centrifugation to enzymolysis solution, centrifugal treating rotating speed is 800rpm, and centrifuging temperature is 30 ℃, and centrifugation time is 60min, collects centrifuged supernatant, and protein yield is 95%.
(5) flocculating settling
Add hydrochloric acid soln and regulate supernatant liquor pH value to 3.5-4.5, natural subsidence 6h, abandoning supernatant, protein yield is 86%.
(6) dry
Utilize spray-dryer to carry out drying treatment to sedimentation component, inlet temperature is 200 ℃, in spray-drying process, protein slurry is stirred, and air outlet temperature is 80 ℃, can obtain faint yellow protein powder 49.62g.
embodiment 2
(1) fermentation liquid uf processing
Get vitamin c fermenting mash 100g, by the daltonian ultrafiltration membrane system of interception 1500, process, intake pressure is controlled at 0.2-0.3Mpa, regulate fermentation liquid pH value 7.5, service temperature 30-34 ℃, the by product such as mycelium and protein is held back, and rejection reaches 97.5%, collects and holds back composition.
(2) break process
Composition is held back in collection, add distilled water and stir, water is 1:1 with the volume ratio of holding back composition, then mixed solution is added to super low temperature quick frozen equipment, at-50 ℃ of quick-frozen 8min, and then at pressure 100Mpa, under flow velocity 260m/s condition, by high pressure homogenizer, suspension liquid is carried out to break process, bacterial cell disruption rate is 95.5%.
(3) enzymatic hydrolysis
In the slurries after fragmentation, add the albumen prozyme (carboxypeptidase, papoid mass ratio are 1:1) of slurries quality 1.2%, protein is carried out to enzymolysis, temperature of reaction is 55-60 ℃, and pH is 5.5-6.0, and the reaction times is 3h.
(4) centrifugation
Utilize whizzer to carry out centrifugation to enzymolysis solution, centrifugal treating rotating speed is 1500rpm, and centrifuging temperature is 35 ℃, and centrifugation time is 30min, collects centrifuged supernatant, and protein yield is 93%.
(5) flocculating settling
Add hydrochloric acid soln and regulate supernatant liquor pH value to 3.0-4.0, natural subsidence 14h, abandoning supernatant, protein yield is 93%.
(6) dry
Utilize spray-dryer to carry out drying treatment to sedimentation component, in spray-drying process, protein slurry is stirred, inlet temperature is 300 ℃, and air outlet temperature is 100 ℃, obtains faint yellow protein powder 52.28g.
embodiment 3
(1) fermentation liquid uf processing
Get vitamin c fermenting mash 100g, by the daltonian ultrafiltration membrane system of interception 1000, process, intake pressure is controlled at 0.2-0.25Mpa, regulates fermentation liquid pH value 6.0, service temperature 29-33 ℃.The by product such as mycelium and protein is held back, and rejection reaches 99.0%, collects and holds back composition.
(2) break process
Composition is held back in collection, add distilled water and stir, water is 1:1.5 with the volume ratio of holding back composition, then mixed solution is added to super low temperature quick frozen equipment, at-50 ℃ of quick-frozen 10min, and then at pressure 150Mpa, under the condition of flow velocity 150m/s, by high pressure homogenizer, suspension liquid is carried out to break process, bacterial cell disruption rate is 99.0%.
(3) enzymatic hydrolysis
In the slurries after fragmentation, add the mixed enzyme (carboxypeptidase, Sumizyme MP, tryptic mass ratio are 1:1.2:0.5) of slurries quality 0.75%, protein is carried out to enzymolysis, temperature of reaction is 40-45 ℃, and pH is 7.5-8.5, and the reaction times is 2h.
(4) centrifugation
Utilize whizzer to carry out centrifugation to enzymolysis solution, centrifugal treating rotating speed is 1200rpm, and centrifuging temperature is 40 ℃, and centrifugation time is 90min, collects centrifuged supernatant, and protein yield is 99.3%.
(5) flocculating settling
Add hydrochloric acid soln and regulate supernatant liquor pH value to albumen iso-electric point 3.5-4.5, natural subsidence 10h, abandoning supernatant, protein yield is 95%.
(6) dry
Utilize spray-dryer to carry out drying treatment to sedimentation component, in spray-drying process, protein slurry is stirred, inlet temperature is 280 ℃, and air outlet temperature is 50 ℃, can obtain faint yellow protein powder 59.44g.
embodiment 4
Adopt the step of embodiment 3 to extract protein powder, different: mixed enzyme used is that mass ratio is carboxypeptidase, Sumizyme MP and the trypsinase of 1:0.5:0.5, centrifugal collection supernatant liquor, recording protein yield is 98%.
Claims (7)
1. a method for extracting proteins from vitamin c fermenting mash, is characterized in that comprising the following steps:
(1) vitamin c fermenting mash is carried out to uf processing with ultra-filtration membrane, the macromolecular substance such as mycelium wherein and protein are held back out from mash, collect and hold back composition;
(2) add water to stir the composition of holding back of step (1) ultrafiltration gained, then adopt super low temperature quick frozen technology will hold back the quick-frozen of composition solution, obtain suspension liquid, and then with high pressure homogenizer, gained suspension liquid is carried out to break process;
(3) albumen prozyme is added in the suspension liquid after step (2) is processed to enzymolysis and extraction protein component wherein;
(4) enzymolysis solution of enzymolysis gained is carried out to centrifugation, collect supernatant liquor;
(5) pH of the described supernatant liquor of regulating step (4), makes protein flocculating settling, crosses leaching precipitation;
(6) precipitation in step (5) is dried, obtains protein powder;
In step (2), water to add volume and the volume ratio of holding back composition be 1:0.5～1:5; Super low temperature quick frozen temperature is-35～-50 ℃, and the quick-frozen time is 5～10min; Suspension liquid is 100～150Mpa by the pressure of high pressure homogenizer, and flow velocity is 150-260m/s;
In step (3), described albumen prozyme is comprised of carboxypeptidase and enzyme A, and enzyme A is selected from one or more in following enzyme: trypsinase, papoid, Sumizyme MP; Wherein the mass ratio of carboxypeptidase and enzyme A is 1:1.0-2.0;
In step (3), the consumption of albumen prozyme is 0.35%～1.2% of suspension liquid quality, and hydrolysis temperature is 40～60 ℃, and during enzymolysis, suspension liquid pH is 5.5～8.5, and enzymolysis time is 2～3.5h.
2. method according to claim 1, is characterized in that: in step (1), the molecular weight cut-off of described ultra-filtration membrane is 1000～2000 dalton; The condition that fermentation liquid carries out ultrafiltration is: intake pressure 0.2～0.5Mpa, 29～39 ℃ of ultrafiltration temperature, fermentation liquid pH value 5.0～7.5.
3. method according to claim 1, is characterized in that: in step (3), described albumen prozyme is comprised of carboxypeptidase, Sumizyme MP and trypsinase, and their mass ratio is carboxypeptidase: Sumizyme MP: trypsinase=1:1.2:0.5.
4. method according to claim 1, is characterized in that: in step (4), carry out centrifugation at 30～40 ℃, rotating speed is 800～1500rpm, and the time is 30～90min.
5. method according to claim 1, is characterized in that: in step (5), by supernatant liquor pH regulator to 2.0～4.5, make protein flocculating settling, the protein settling time is 6-14h.
6. method according to claim 1, is characterized in that: in step (6), be dried the spray-dired method that adopts, the dry inlet temperature of spraying is 200～300 ℃, and air outlet temperature is 50～100 ℃.
7. a method for extracting proteins from vitamin c fermenting mash, is characterized in that comprising the following steps:
(1) fermentation liquid uf processing
Get vitamin c fermenting mash 100g, by the daltonian ultrafiltration membrane system of interception 1000, process, intake pressure is controlled at 0.2-0.25Mpa, regulate fermentation liquid pH value 6.0, service temperature 29-33 ℃, the by product such as mycelium and protein is held back, and rejection reaches 99.0%, collects and holds back composition;
(2) break process
Composition is held back in collection, add distilled water and stir, water is 1:1.5 with the volume ratio of holding back composition, then mixed solution is added to super low temperature quick frozen equipment, at-50 ℃ of quick-frozen 10min, and then at pressure 150Mpa, under the condition of flow velocity 150m/s, by high pressure homogenizer, suspension liquid is carried out to break process, bacterial cell disruption rate is 99.0%;
(3) enzymatic hydrolysis
In the slurries after fragmentation, add the mixed enzyme of slurries quality 0.75%, protein is carried out to enzymolysis, temperature of reaction is 40-45 ℃, and pH is 7.5-8.5, and the reaction times is 2h; Described mixed enzyme is that mass ratio is carboxypeptidase, Sumizyme MP and the tryptic mixture of 1:1.2:0.5;
(4) centrifugation
Utilize whizzer to carry out centrifugation to enzymolysis solution, centrifugal treating rotating speed is 1200rpm, and centrifuging temperature is 40 ℃, and centrifugation time is 90min, collects centrifuged supernatant, and protein yield is 99.3%;
(5) flocculating settling
Add hydrochloric acid soln and regulate supernatant liquor pH value to albumen iso-electric point 3.5-4.5, natural subsidence 10h, abandoning supernatant, protein yield is 95%;
(6) dry
Utilize spray-dryer to carry out drying treatment to sedimentation component, in spray-drying process, protein slurry is stirred, inlet temperature is 280 ℃, and air outlet temperature is 50 ℃, can obtain faint yellow protein powder 59.44g.
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| CN107674897A (en) * | 2016-09-30 | 2018-02-09 | 青岛大学 | One kind is by method for extracting proteins in bata-carotene zymotic fluid |
| CN107674899A (en) * | 2016-09-30 | 2018-02-09 | 青岛大学 | One kind is by method for extracting proteins in vitamin B12 zymotic fluid |
| CN107674898A (en) * | 2016-09-30 | 2018-02-09 | 青岛大学 | One kind is by method for extracting proteins in farnoquinone zymotic fluid |
| CN107417765B (en) * | 2017-09-26 | 2020-12-04 | 珠海联邦制药股份有限公司 | Method for separating and purifying recombinant protein in escherichia coli autolysis expression system |
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| CN101143789A (en) * | 2006-09-15 | 2008-03-19 | 中国科学院金属研究所 | Nano laminated Ta2AlC ceramic powder and preparing method thereof |
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