CN103070293A - Method for extracting biological protein from aurantiochytrium sp residue - Google Patents
Method for extracting biological protein from aurantiochytrium sp residue Download PDFInfo
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Abstract
The invention provides a method for extracting a biological protein from an aurantiochytrium sp residue. The method comprises the following steps: performing fermentation culture on an aurantiochytrium sp strain, separating obtained fermentation broth, collecting fronds, washing the collected fronds, and performing oil extraction to obtain crude oil; and alkali-refining, decolorizing and deodorizing the prepared crude oil to obtain aurantiochytrium sp oil and an aurantiochytrium sp residue, wherein the protein content of the aurantiochytrium sp residue is up to more than 50% through secondary enzymolysis, most of the protein exists in the form of small peptide capable of being easily digested, and the content of DHA (Docosahexaenoic Acid) in a polyunsaturated fatty acid is 0.02%-2%, so that the aurantiochytrium sp residue is a good fish meal protein feed additive. According to the method for extracting the biological protein from the aurantiochytrium sp residue, the waste aurantiochytrium sp residue obtained after extracting the aurantiochytrium sp oil is used fully so as to recycle the waste and improve the economic benefit; the comprehensive utilization of microalgae cells is realized; and the cleaner production of an unsaturated fatty acid is realized.
Description
Technical field
The present invention relates to a kind of method of extracting bioprotein, especially a kind of method of from split kettle algae algae-residue, extracting bioprotein.
Background technology
Fish meal is as one of important feedstuff, have the protein content height, balanced in nutrition, be beneficial to absorbing of letting animals feed; The polyunsaturated fatty acid that contains larger proportion in the fatty,fiss such as DHA (DHA), eicosapentaenoic acid (EPA), is grown the normal growth of the letting animals feed young and to be had good facilitation; Fish meal also is the source of the mineral matters such as good calcium, phosphorus, iodine, selenium, and in addition, fish meal also contains multivitamin, so fish meal is the good feedstuff that is widely used and is difficult to substitute.Yet along with the excessive predation of socioeconomic development and resource, limited fish meal resource is exhausted gradually, has brought more serious ecological problem.To level and the worry of vertical transmission and the lasting deterioration of environmental pollution of disease, aquatic products become more and more by pollutions such as toxic compounds such as heavy metal, PCB, agricultural chemicals, in the urgent need to development can Peru Fish Dietary the novel protein feed resource.Although carried out the research that much utilizes the plant protein resource Peru Fish Dietary, yet be difficult to reach the effect of perfect Peru Fish Dietary, mainly contain following reason: the harmony (fish meal contains high-caliber essential amino acid and sulfur-containing amino acid) that amino acid forms, the bioavilability of protein (amino acid mainly exists with the little peptide form that is very easy to digest and assimilate in the fish meal), essential content of polyunsaturated fatty acid (containing more polyunsaturated fatty acid in the fish meal, is 0.03%～0.9% such as the content of DHA DHA) etc.
Containing abundant aliphatic acid, protein and vitamins and other nutritious components in the marine oil-producing microalgae, is a kind of good renewable resource, also is the primary source of the polyunsaturated fatty acid in the Fish.To split kettle algae (Aurantiochytrium sp) as example, its fat content can reach more than 50% of dry cell weight, and wherein the content of DHA (DHA) can account for about 50% of content of fatty acid.Extracting that protein content significantly improves in the algae-residue of aliphatic acid, up to about 50～60%, also have simultaneously remaining polyunsaturated fatty acid, is a kind of well comprehensive protein feed resource of Peru Fish Dietary.Split the kettle algae prepare polyunsaturated fatty acid and frond as the security of aquatic feed oneself through extensively being approved, the single cell protein accessory substance that how to utilize to greatest extent oil-producing microalgae to extract the polyunsaturated fatty acid production process is the problem that needs solve.
This shows that prior art awaits further developing.
Summary of the invention
The present invention has proposed a kind of method of extracting bioprotein from split kettle algae algae-residue for solving defective of the prior art, this bioprotein can be used as the additive of fish meal protein matter feed, the raw material that the method adopts is the discarded kettle algae algae-residue that splits, not only energy savings but also can turn waste into wealth.
For solving the problems of the technologies described above, the present invention program comprises:
A kind of method of extracting bioprotein from split kettle algae algae-residue said method comprising the steps of:
Add 0.1～1.5% pectase in the kettle algae algae-residue to splitting, 40～60 ℃ of temperature, hydrolysis 2～5h; Then add 0.1～1.5% protease, 40～60 ℃ of temperature, hydrolysis 2～5h; Spray-drying, 140～190 ℃ of EATs, 60～90 ℃ of leaving air temps make the dry powder that contains bioprotein at last.
The above-mentioned kettle algae algae-residue that splits prepares by the following method:
1) step of fermented and cultured is carried out in the strain of counterincision kettle phycomycete;
2) zymotic fluid that obtains is separated, collects frond, with the frond of collecting wash, grease after extracting crude oil;
3) with step 2) crude oil that makes obtains splitting kettle algae grease after alkali refining, decolouring and deodorization, and remaining being split kettle algae algae-residue.
The above-mentioned kettle phycomycete strain fermented and cultured of splitting prepares by the following method:
The bacterial strain access that 1) will be kept at the glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in 20～30 ℃ shaking table, with the rotating speed of 150～200rpm, cultivates 24～48h, obtains primary seed solution;
2) above-mentioned primary seed solution access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in 20～30 ℃ shaking table, rotating speed 150～200rpm cultivates 24～48h, obtains secondary seed solution;
3) the secondary seed solution access is equipped with in the fermentation tank of fermentation medium, inoculum concentration 2～10%v/v, throughput 0.2～2vvm, rotating speed 200～800rpm, 20～30 ℃ of tank temperature, pH6～7 obtain splitting kettle algae grease.
Carbon source content is 30～60g/L in the above-mentioned seed culture medium, and nitrogenous source content is 10～20g/L, and solvent is the mixture of seawater and distilled water, and described seawater and distilled water are that 1:1 mixes according to mass ratio; Described carbon source is glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulosic saccharified liquid; Described nitrogenous source is organic nitrogen source or inorganic nitrogen-sourced, described organic nitrogen source is yeast extract, peptone, tryptone, corn steep liquor, beef extract, soybean protein, sodium glutamate or urea, described inorganic nitrogen-sourced be ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, ammoniacal liquor or potassium nitrate.
Comprise following component in the above-mentioned fermentation medium: glucose 20～60g/L, yeast extract 5～30g/L, peptone 5～20g/L, potassium dihydrogen phosphate 0.5～8g/L, magnesium sulfate 0.5～5g/L, natrium citricum 0.5～5g/L, sea crystal 5～30g/L, VB11 0～100mg/L, vitamin B6 10～100mg/L, cobalamin 3～50mg/L, biotin 2～50mg/L.
Above-mentioned steps 3) fermentation mode in is batch fermentation, feed supplement-batch fermentation, continuously ferments or semicontinuous fermentation.
Above-mentioned steps 2) is separated into centrifugation, plate-frame filtering or membrane filtration in; Described grease is extracted as machine solvent extraction, high-pressure homogeneous broken wall or protease hydrolytic broken wall.
Above-mentioned organic solvent is cyclohexane, n-hexane, ethyl acetate, acetone, ethanol, benzinum, ether, toluene, chloroform or several mixtures.
Above-mentioned bioprotein can be used as fish meal protein matter feed addictive.
The invention provides a kind of utilize autonomous screening split that kettle algae superior strain obtains by fermentation split kettle algae algae-residue, and utilize this to split the method that kettle algae algae-residue extracts bioprotein, its algae-residue is through secondary enzymolysis, protein content reaches more than 50%, most of protein all exists with the little peptide form that is very easy to digestion, polyunsaturated fatty acid DHA content is 0.02% ~ 2%, is a kind of good fish meal protein matter feed addictive; The present invention takes full advantage of and extracts the algae-residue of discarding behind the aliphatic acid, and turning waste into wealth has increased economic benefit, and has realized the comprehensive utilization of microalgae cell, realizes the cleaner production of polyunsaturated fatty acid.
The specific embodiment
Below by specific embodiment method of the present invention is further described in detail, but the present invention is not limited thereto.
Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and raw material if no special instructions, all can obtain from commercial channels.
The kettle algae algae-residue that splits used among the embodiment is to split kettle phycomycete strain (this splits the strain of kettle phycomycete and can obtain) through fermented and cultured from existing channel, after fermented and cultured is finished, the zymotic fluid that obtains is separated, collects frond, with the frond of collecting wash, grease after extracting crude oil; The crude oil that gets is obtained splitting kettle algae grease and splits kettle algae algae-residue after alkali refining, decolouring and deodorization.Concrete preparation method is as follows: the preparation of primary seed solution: the kettle phycomycete strain access of splitting that will be kept at the glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in 20～30 ℃ shaking table, rotating speed with 150～200rpm, cultivate 24～48h, obtain primary seed solution, above-mentioned seed culture medium contains the carbon source of 30～60g/L, the nitrogenous source of 10～20g/L, solvent is the mixture of seawater and distilled water, and seawater and distilled water are that 1:1 mixes according to mass ratio; Carbon source can adopt glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulosic saccharified liquid; Nitrogenous source can adopt organic nitrogen source or inorganic nitrogen-sourced, organic nitrogen source is yeast extract, peptone, tryptone, corn steep liquor, beef extract, soybean protein, sodium glutamate or urea, and inorganic nitrogen-sourced is ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, ammoniacal liquor or potassium nitrate.
The preparation of secondary seed solution: above-mentioned primary seed solution access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in 20～30 ℃ shaking table, rotating speed 150～200rpm cultivates 24～48h, obtain secondary seed solution, above-mentioned seed culture medium is identical with the culture medium that primary seed solution adopts.
The secondary seed solution access is equipped with in the fermentation tank of fermentation medium, inoculum concentration 2～10%v/v, throughput 0.2～2vvm, rotating speed 200～800rpm, 20～30 ℃ of tank temperature, pH6～7, obtain splitting kettle algae grease, comprise following component in the above-mentioned fermentation medium: glucose 20～60g/L, yeast extract 5～30g/L, peptone 5～20g/L, potassium dihydrogen phosphate 0.5～8g/L, magnesium sulfate 0.5～5g/L, natrium citricum 0.5～5g/L, sea crystal 5～30g/L, VB11 0～100mg/L, vitamin B6 10～100mg/L, cobalamin 3～50mg/L, biotin 2～50mg/L, above-mentioned fermentation mode can be batch fermentation, feed supplement-batch fermentation, continuously ferment or semicontinuous fermentation.In above-mentioned sweat, can also increase and mend the sugar operation, guarantee that the concentration of glucose is 20～60g/L, described fermentation time is 80～110h, concentration of glucose is not higher than 10g/L during fermentation ends.
Split the refining of kettle algae grease and split being collected in the following specific embodiment of kettle algae algae-residue and describe in detail.
Embodiment 1:
Above-mentioned zymotic fluid is carried out centrifugation, and wash insoluble matter 2 times, collect the insoluble matter frond, add the suitable quantity of water adjusting viscosity, carry out high-pressure homogeneous broken wall, pressure 40Mpa circulates 2 times;
Add n-hexane and ethanol extraction 3 times, the organic phase solvent evaporated gets crude oil, obtains polyunsaturated fatty acid refined oil 562 grams after alkali refining, decolouring and deodorization, contains DHA38.5%;
The residue algae-residue adds 1% pectase, 50 ℃ are hydrolyzed 3 hours, add 1% protease, temperature 50 C again, 3 hours action time, spray-drying, 160 ℃ of EATs, 80 ℃ of leaving air temps, get about 350 grams of dry powder, protein content is about 56.5%, and DHA content 0.02% can be used as the protein feeds additive.
Embodiment 2:
Zymotic fluid is carried out centrifugation, and wash insoluble matter 2 times, collect the insoluble matter frond, add the suitable quantity of water adjusting viscosity, carry out enzymatic shell-broken;
Add n-hexane and ethanol extraction 3 times, the organic phase solvent evaporated gets crude oil, obtains polyunsaturated fatty acid refined oil 551 grams after alkali refining, decolouring and deodorization, contains DHA38.3%;
The residue algae-residue adds 0.1% pectase, and 60 ℃ are hydrolyzed 5 hours, add 0.1% protease again, and 60 ℃ are hydrolyzed 5 hours, spray-drying, 190 ℃ of EATs, 90 ℃ of leaving air temps get about 354 grams of dry powder, protein content is about 56.9%, and DHA content 0.05% can be used as the protein feeds additive.
Embodiment 3:
Zymotic fluid is carried out centrifugation, and wash insoluble matter 2 times, collect the insoluble matter frond, add the suitable quantity of water adjusting viscosity, carry out enzymatic shell-broken, carry out high-pressure homogeneous broken wall again, pressure 40Mpa circulates 1 time;
Centrifugation free oil and emulsion layer get crude oil behind the freeze thawing breakdown of emulsion, obtain polyunsaturated fatty acid refined oil 502 grams after alkali refining, decolouring and deodorization, contain DHA40.1%;
The residue algae-residue adds 1.5% pectase, and 40 ℃ are hydrolyzed 2 hours, add 1.5% protease again, and 40 ℃ are hydrolyzed 2 hours, spray-drying, 140 ℃ of EATs, 60 ℃ of leaving air temps get about 392 grams of dry powder, protein content is about 52.6%, and DHA content 2% can be used as the protein feeds additive.
Table 1 is analyzed data for the protein feeds amino acid and the total protein that make, and wherein, 1-16 adopts GB/T5009.124-2003 to survey data, and 17 adopt GB/T6432-1994 to survey data.
Table 1 protein biology feedstuff amino acid and total protein composition analysis
Should be understood that; above-mentioned description for preferred embodiment is comparatively detailed; can not therefore think the restriction to scope of patent protection of the present invention; those of ordinary skill in the art is under enlightenment of the present invention; do not breaking away from the scope situation that claim of the present invention protects; can also make the various deformation such as replacement, simple combination, the scope of asking for protection of the present invention should be as the criterion with claims.
Claims (9)
1. method of extracting bioprotein from split kettle algae algae-residue is characterized in that: said method comprising the steps of:
Add 0.1～1.5% pectase in the kettle algae algae-residue to splitting, 40～60 ℃ of temperature, hydrolysis 2～5h; Then add 0.1～1.5% protease, 40～60 ℃ of temperature, hydrolysis 2～5h; Spray-drying, 140～190 ℃ of EATs, 60～90 ℃ of leaving air temps make the dry powder that contains bioprotein.
2. a kind of method of from split kettle algae algae-residue, extracting bioprotein according to claim 1, it is characterized in that: the described kettle algae algae-residue that splits prepares by the following method:
1) step of fermented and cultured is carried out in the strain of counterincision kettle phycomycete;
2) zymotic fluid that obtains is separated, collects frond, with the frond of collecting wash, grease after extracting crude oil;
3) with step 2) crude oil that makes obtains splitting kettle algae grease after alkali refining, decolouring and deodorization, and remaining being split kettle algae algae-residue.
3. a kind of method of from split kettle algae algae-residue, extracting bioprotein according to claim 2, it is characterized in that: the described kettle phycomycete strain fermented and cultured of splitting prepares by the following method:
The bacterial strain access that 1) will be kept at the glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in 20～30 ℃ shaking table, with the rotating speed of 150～200rpm, cultivates 24～48h, obtains primary seed solution;
2) above-mentioned primary seed solution access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in 20～30 ℃ shaking table, rotating speed 150～200rpm cultivates 24～48h, obtains secondary seed solution;
3) the secondary seed solution access is equipped with in the fermentation tank of fermentation medium, inoculum concentration 2～10%v/v, throughput 0.2～2vvm, rotating speed 200～800rpm, 20～30 ℃ of tank temperature, pH6～7 obtain splitting kettle algae grease.
4. a kind of method of from split kettle algae algae-residue, extracting bioprotein according to claim 3, it is characterized in that: carbon source content is 30～60g/L in the described seed culture medium, nitrogenous source content is 10～20g/L, solvent is the mixture of seawater and distilled water, and described seawater and distilled water are that 1:1 mixes according to mass ratio; Described carbon source is glucose, glycerine, fructose, wood sugar, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulosic saccharified liquid; Described nitrogenous source is organic nitrogen source or inorganic nitrogen-sourced, described organic nitrogen source is yeast extract, peptone, tryptone, corn steep liquor, beef extract, soybean protein, sodium glutamate or urea, described inorganic nitrogen-sourced be ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, ammoniacal liquor or potassium nitrate.
5. a kind of method of from split kettle algae algae-residue, extracting bioprotein according to claim 3, it is characterized in that: comprise following component in the fermentation medium of described step 3): glucose 20～60g/L, yeast extract 5～30g/L, peptone 5～20g/L, potassium dihydrogen phosphate 0.5～8g/L, magnesium sulfate 0.5～5g/L, natrium citricum 0.5～5g/L, sea crystal 5～30g/L, VB11 0～100mg/L, vitamin B6 10～100mg/L, cobalamin 3～50mg/L, biotin 2～50mg/L.
6. a kind of method of extracting bioprotein from split kettle algae algae-residue according to claim 3, it is characterized in that: the fermentation mode in the described step 3) is batch fermentation, feed supplement-batch fermentation, continuously ferments or semicontinuous fermentation.
7. a kind of method of extracting bioprotein from split kettle algae algae-residue according to claim 2 is characterized in that: be separated into centrifugation, plate-frame filtering or membrane filtration described step 2); Described grease is extracted as machine solvent extraction, high-pressure homogeneous broken wall or protease hydrolytic broken wall.
8. a kind of method of extracting bioprotein from split kettle algae algae-residue according to claim 7, it is characterized in that: described organic solvent is cyclohexane, n-hexane, ethyl acetate, acetone, ethanol, benzinum, ether, toluene, chloroform or several mixtures.
9. each described bioprotein according to claim 1～8 is characterized in that: described bioprotein can be used as fish meal protein matter feed addictive.
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| CN106811285B (en) * | 2017-02-23 | 2020-04-17 | 湖北福星生物科技有限公司 | Method for physically extracting DHA grease from DHA fermentation liquor |
| CN106811285A (en) * | 2017-02-23 | 2017-06-09 | 湖北福星生物科技有限公司 | A kind of method that physics extracts DHA grease in zymotic fluid from DHA |
| CN109371084A (en) * | 2017-11-16 | 2019-02-22 | 中国水产科学研究院南海水产研究所 | It is a kind of to split the chelated calcium preparation method of pot algae peptide with antioxidant activity |
| CN108977470B (en) * | 2018-08-17 | 2022-04-15 | 中国科学院青岛生物能源与过程研究所 | Method for producing polyunsaturated fatty acid-rich oil by adopting lignocellulose |
| CN108977470A (en) * | 2018-08-17 | 2018-12-11 | 中国科学院青岛生物能源与过程研究所 | The method that polyunsaturated fatty acid grease is rich in using lignocellulosic production |
| CN117568176A (en) * | 2023-10-08 | 2024-02-20 | 山东悦翔生物有限公司 | DHA extraction process based on microalgae extraction |
| CN117247849A (en) * | 2023-11-20 | 2023-12-19 | 山东悦翔生物有限公司 | Microalgae extraction method |
| CN117247849B (en) * | 2023-11-20 | 2024-02-06 | 山东悦翔生物有限公司 | Microalgae extraction method |
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