CN103695515B - Preparation Chinese chestnut method of protein - Google Patents
Preparation Chinese chestnut method of protein Download PDFInfo
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- CN103695515B CN103695515B CN201310589684.9A CN201310589684A CN103695515B CN 103695515 B CN103695515 B CN 103695515B CN 201310589684 A CN201310589684 A CN 201310589684A CN 103695515 B CN103695515 B CN 103695515B
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Abstract
The invention discloses the method preparing Chinese chestnut albumen, the method comprises the following steps: the rice-chestnut powder aqueous solution is carried out ultrasonic disruption process; Cellulase is utilized to carry out the first enzymolysis processing to the described rice-chestnut powder aqueous solution through ultrasonic disruption process; Utilize the first α-amylase that first enzyme hydrolysis products is carried out the second enzymolysis processing; Second enzymolysis processing product is carried out alkali and proposes process; Alkali being carried product carries out first centrifugal, and collects supernatant liquor, to obtain protein alkali extract; Utilize the second α-amylase that this protein alkali extract is carried out the 3rd enzymolysis processing; Utilize isoelectric point precipitation the 3rd enzymolysis product to be carried out acid to sink; The product that acid sunk carries out standing centrifugal with second successively, obtains precipitation to collect; And this precipitation is carried out being washed to neutrality, to obtain described Chinese chestnut protein.Utilize the method can effectively improve Chinese chestnut protein extracting ratio, and due to extraction conditions gentleness, so can not destroy the activity of albumen, obtained Chinese chestnut protein-active is good.
Description
Technical field
The present invention relates to preparation Chinese chestnut method of protein.
Background technology
Containing protein 5% ~ 11% in Chinese chestnut, content is higher than rice, and Methionin, Isoleucine, methionine(Met), halfcystine, Threonine, the amino acid whose content such as α-amino-isovaleric acid, phenylalanine, tyrosine are more than the standard of FAO/WHO, and Methionin is paddy rice, wheat, corn, and the first limiting amino acids of large beans, Threonine is paddy rice, second limiting amino acid of wheat, tryptophane and methionine(Met) are second limiting amino acid of corn and beans respectively.As can be seen here, edible Chinese chestnut or directly edible Chinese chestnut protein can supplement the deficiency of limiting amino acid in Cereal and beans, and the nutritive ingredient of meals can be made more perfect.
But present stage extracts preparation Chinese chestnut method of protein and still haves much room for improvement.
Summary of the invention
The present invention completes based on the following discovery of contriver:
Mainly contain two kinds to the extracting method of Chinese chestnut albumen both at home and abroad, a kind of is traditional buck method, and the shortcoming of this method is that extraction yield is low.Another kind is Subcritical Water Extraction method, and the shortcoming of this method is the sex change that the process extracting albumen easily causes albumen.
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind of method preparing Chinese chestnut albumen that extraction yield is high, protein-active is good.
According to an aspect of the present invention, the present invention proposes a kind of method preparing Chinese chestnut albumen.According to embodiments of the invention, the method comprises the following steps: the rice-chestnut powder aqueous solution is carried out ultrasonic disruption process, to obtain the rice-chestnut powder aqueous solution through ultrasonic disruption process; Cellulase is utilized to carry out the first enzymolysis processing, to obtain first enzyme hydrolysis products to the described rice-chestnut powder aqueous solution through ultrasonic disruption process; Utilize the first α-amylase that described first enzyme hydrolysis products is carried out the second enzymolysis processing, to obtain second enzyme hydrolysis products; Described second enzymolysis processing product is carried out alkali and proposes process, to obtain alkali carrying product; Described alkali being carried product carries out first centrifugal, and collects supernatant liquor, to obtain protein alkali extract; Utilize the second α-amylase that described protein alkali extract is carried out the 3rd enzymolysis processing, to obtain the 3rd enzymolysis product; Utilize isoelectric point precipitation described 3rd enzymolysis product to be carried out acid to sink, to obtain the heavy product of acid; The product that described acid sunk carries out standing centrifugal with second successively, obtains precipitation to collect; And described precipitation is carried out being washed to neutrality, to obtain described Chinese chestnut protein.Contriver is surprised to find, and utilize method of the present invention effectively can extract preparation Chinese chestnut albumen, and Chinese chestnut protein extracting ratio is high, active good.Particularly, method of the present invention utilizes ultrasonic wave and cellulase to carry out the stripping of enzymolysis promotion Chinese chestnut albumen, and utilize enzymolyzing alpha-amylase to destroy the tetra-inclusion complex of protein (in this article sometimes also by " protein " referred to as " albumen ") and starch, thus effectively improve the extraction yield of Chinese chestnut albumen, protein extracting ratio can up to 66%, 20% is improve relative to traditional method, and protein content can up to 88% in the Chinese chestnut albumen obtained, purity is high, quality is good, and because extraction conditions is gentle, the activity of Chinese chestnut albumen can be kept well.
In addition, preparation Chinese chestnut method of protein according to the above embodiment of the present invention can also have following additional technical characteristic:
It should be noted that, be applicable to raw material of the present invention and comprise fresh Chinese chestnut, dried Chinese chestnut, rice-chestnut powder.The main extraction agent adopted in method of the present invention is deionized water.The content of the kind of raw material, quality and albumen, will produce certain impact to the quality of finished product and yield, but all high than adopting prior art to prepare the efficiency of Chinese chestnut protein, quality better.
According to embodiments of the invention, under 400w ultrasonic power, carry out ultrasonic cell-break process 6-14min, preferred 10min.Thereby, it is possible to make cellularstructure loosen, strengthen the action effect of subsequent fiber element enzyme.According to embodiments of the invention, the solid-liquid ratio of the described rice-chestnut powder aqueous solution is 1g:15ml.In other words, the described rice-chestnut powder aqueous solution is by rice-chestnut powder and water being obtained according to the ratio mixed preparing of 1g:15ml.Thus, stripping and the extraction of following protein is conducive to.In addition, it should be noted that, aforesaid rice-chestnut powder can prepare by the following method: selecting without the fresh Chinese chestnut damaged by worms that goes mouldy is raw material, pulverizes after peeling, section, lyophilize through pulverizer, crosses 60 mesh sieves and makes rice-chestnut powder.
According to embodiments of the invention, in 40-60 degree Celsius preferably 50 degrees Celsius, under pH4-6 preferred pH4.5 condition, carry out described first enzymolysis processing 5-15min, preferred 10min.Thereby, it is possible to effectively promote the stripping of Chinese chestnut albumen, and protein-active can be kept.According to other embodiments of the present invention, when carrying out described first enzymolysis processing, the ratio of described cellulase and the described rice-chestnut powder aqueous solution through ultrasonic disruption process is 50-150U/g, preferred 100U/g.Thereby, it is possible to effectively destroy cell walls to promote albumen stripping, be conducive to follow-up protein extraction.
According to embodiments of the invention, in 30-50 degree Celsius preferably 40 degrees Celsius, under pH5.5-6.5 preferred pH6.1 condition, carry out described second enzymolysis processing 25-35min, preferred 30min.Thereby, it is possible to make the effective enzymolysis of chestnut starch, and then efficient solution can remove starch to the envelope of protein, thus be conducive to albumen stripping.According to other embodiments of the present invention, when carrying out described second enzymolysis processing, the ratio of described first α-amylase and described first enzyme hydrolysis products is 80-160U/g, preferred 120U/g.Thereby, it is possible to effectively improve the enzymolysis efficiency of chestnut starch, remove starch to the envelope of protein, and the activity of protein can be kept.
According to preferred exemplary more of the present invention, under 40 degrees Celsius, the first α-amylase is utilized to carry out described second enzymolysis processing 30min according to the ratio of 120U/g.Thus, amylorrhexis efficiency is high, few to the envelope of protein, and can keep the structure and activity of protein.
According to embodiments of the invention, described alkali is proposed process and is comprised: described second enzymolysis processing product being carried out pH regulator to pH is 8.0-10.0 preferably 10.0, and under 30-50 degree Celsius preferably 40 degrees Celsius, carries out described alkali carry 25-35min, preferred 30min.Thereby, it is possible to improve the solubility rate of albumen, the alkali obtaining rich in proteins carries product.According to concrete examples more of the present invention, described second enzymolysis processing product is carried out pH regulator to pH10.0, and under 40 degrees Celsius, carry out described alkali carry 30min.Thereby, it is possible to make the efficient stripping of albumen.
According to embodiments of the invention, centrifugal again after the filter residue obtained after first is centrifugal can being added deionized water wash, ultrasonication, then collect supernatant liquor and integrate with described protein alkali extract, to improve the extraction yield of Chinese chestnut albumen.
It should be noted that, contriver finds, in protein alkali extract, starch content is still higher, and thus, method of the present invention adds amylorrhexis starch wherein in protein alkali extract, so that the separation of albumen.According to embodiments of the invention, in 30-50 degree Celsius preferably 40 degrees Celsius, under pH5.5-6.5 preferred pH6.1 condition, carry out described 3rd enzymolysis processing 25-35min, preferred 30min.Thereby, it is possible to effectively break the tetra-inclusion complex of starch to albumen, facilitate the carrying out that follow-up acid is heavy.According to embodiments of the invention, when carrying out described 3rd enzymolysis processing, the ratio of described second α-amylase and described protein alkali extract is 80-160U/g, preferred 120U/g.Thus, chestnut starch dissolved efficiency is high, good to the starch removal effect of envelope Chinese chestnut protein, and can not destroy the activity of protein.
According to embodiments of the invention, described acid is sunk and to be carried out under isoelectric pH 4.2 condition.Thereby, it is possible to make Chinese chestnut protein effectively precipitate.
According to embodiments of the invention, this is prepared Chinese chestnut method of protein and comprises further described Chinese chestnut albumen is carried out vacuum lyophilization.Thereby, it is possible to effectively obtain Chinese chestnut albumen crude extract.Particularly, according to some embodiments of the present invention, vacuum freeze drier is utilized to carry out described vacuum lyophilization.Thereby, it is possible to effectively realize vacuum lyophilization, obtain Chinese chestnut albumen crude extract.
According to concrete examples more of the present invention, preparation Chinese chestnut method of protein of the present invention can also comprise the following steps: by rice-chestnut powder and water in 1:15(g/mL) ratio dissolves and obtains the rice-chestnut powder aqueous solution, then it carried out ultrasonic disruption process 10min under ultrasonic power 400w.Then, in the rice-chestnut powder aqueous solution through ultrasonic disruption process, add cellulase in the ratio of 100U/g, adjust pH to 4.5, put into 50 DEG C of waters bath with thermostatic control and stir enzymolysis 10min.Then pH=6.1 is adjusted, add low temperature alpha-amylase, enzyme addition is 120U/g, and at 40 DEG C, continue insulated and stirred enzymolysis 30min, then, after adjusting pH to 10.0 with 1mol/mLNaOH, put into 40 DEG C of water-bath lixiviate 30min, then, centrifuging and taking supernatant liquor, after adjusting the pH to 6.1 of supernatant liquor, low temperature alpha-amylase is added by the enzyme addition of 120U/g, and continue insulated and stirred enzymolysis 30min at 40 DEG C after, under isoelectric pH 4.2 condition, carry out acid sink, centrifuging and taking precipitates, namely Chinese chestnut protein is obtained after water washing and precipitating to neutrality, then its vacuum lyophilization is preserved.
It should be noted that; relative to traditional method; preparation Chinese chestnut method of protein of the present invention considers that emphatically the structure of protection Chinese chestnut albumen while improving Chinese chestnut protein extracting ratio is not destroyed; by the effect of α-amylase, cellulase and ultrasonic cell disruption instrument; promote the stripping of Chinese chestnut albumen; decrease the residual of chestnut starch; and because extraction conditions is gentle; effectively can keep the activity of protein, thus be conducive to follow-up study and the suitability for industrialized production of biological activity and functional protein.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the schematic flow sheet preparing Chinese chestnut method of protein according to an embodiment of the invention.
Embodiment
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that below by the embodiment be described with reference to the drawings be exemplary, only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
General method:
With reference to Fig. 1, according to embodiments of the invention, preparation Chinese chestnut method of protein of the present invention generally comprises following steps:
First, the rice-chestnut powder aqueous solution is carried out ultrasonic disruption process, to obtain the rice-chestnut powder aqueous solution through ultrasonic disruption process.
Secondly, cellulase is utilized to carry out the first enzymolysis processing, to obtain first enzyme hydrolysis products to the described rice-chestnut powder aqueous solution through ultrasonic disruption process.
Again, utilize the first α-amylase that described first enzyme hydrolysis products is carried out the second enzymolysis processing, to obtain second enzyme hydrolysis products.
Then, described second enzymolysis processing product is carried out alkali and proposes process, to obtain alkali carrying product.
Next, described alkali is carried product and carry out first centrifugal, and collect supernatant liquor, to obtain protein alkali extract.
Then, utilize the second α-amylase that described protein alkali extract is carried out the 3rd enzymolysis processing, to obtain the 3rd enzymolysis product.
Then, utilize isoelectric point precipitation described 3rd enzymolysis product to be carried out acid and sink, to obtain the heavy product of acid.
Next, the product that described acid sunk carries out standing centrifugal with second successively, obtains precipitation to collect.
Then, described precipitation is carried out being washed to neutrality, to obtain described Chinese chestnut protein.
Embodiment 1
According to foregoing general method, with reference to Fig. 1, prepare Chinese chestnut protein according to following steps:
Selecting without the fresh Chinese chestnut damaged by worms that goes mouldy is raw material, pulverizes after peeling, section, lyophilize through pulverizer, crosses 60 mesh sieves and makes rice-chestnut powder.Then, by rice-chestnut powder and deionized water according to 1:15(g/mL) ratio dissolve and obtain the rice-chestnut powder aqueous solution and be placed in Ultrasonic Cell Disruptor (JAV-IV type Ultrasonic cell smash, Ao Bo ultrasonic wave Electrical Appliances Co., Ltd of Jining City) in, under 400w ultrasonic power, process 10min.Then in the rice-chestnut powder aqueous solution through ultrasonic disruption process, cellulase (Sigma-Aldrich is added according to the ratio of 100U/g, article No. 22178), adjust pH to 4.5 again, proceed to 50 DEG C of thermostat water baths (W201B type thermostat water bath, Shen Shun bio tech ltd, Shanghai) and stir enzymolysis 10min.Then, adjust pH=6.1, add α-amylase (Sigma-Aldrich, article No. A1031), enzyme addition is 120U/g, insulated and stirred enzymolysis 30min at 40 DEG C.Then, adjust pH to 10.0 with 1mol/mLNaOH, and put into 40 DEG C of water-baths (W201B type thermostat water bath, Shen Shun bio tech ltd, Shanghai) in after lixiviate 30min, centrifuging and taking supernatant liquor, adjust supernatant liquor pH to 6.1 again, then α-amylase (Sigma-Aldrich is added according to the enzyme addition of 120U/g, article No. A1031), and continue insulated and stirred enzymolysis 30min at 40 DEG C after, under isoelectric pH 4.2 condition, carry out acid sink, then centrifuging and taking precipitation, namely water washing and precipitating obtains Chinese chestnut protein to neutrality, again by its vacuum lyophilization (ThermoFisherLL1500 Freeze Drying Equipment, Thermo Fisher Scientific Inc. of the U.S.) preserve.
As calculated, Chinese chestnut extraction rate of protein is 66%, and because extraction conditions is gentle, obtained Chinese chestnut protein-active is very good.In addition, the protein content recording Chinese chestnut crude protein by Kjeldahl determination (KDY-9830 kjeldahl apparatus, Tong Run source, Beijing electromechanical technology co., Ltd) is 88.43%.
Comparative example 1
Selecting without the fresh Chinese chestnut damaged by worms that goes mouldy is raw material, pulverizes after peeling, section, lyophilize through pulverizer, crosses 60 mesh sieves and makes rice-chestnut powder.Then, utilize traditional buck method, extract preparation Chinese chestnut protein according to the step shown in following flow process:
Degreasing Chinese chestnut powder → by material-water ratio 1:15 (g/mL) add distilled water adjust pH to 9.0 → under 45 DEG C of water-baths lixiviate 140min → centrifugal (4000r/min, 10min) → get supernatant liquor → acid heavy (with 1mol/LHCl tune pH to 4.2) → collecting precipitation.
Described precipitation is Chinese chestnut albumen, and as calculated, Chinese chestnut protein extracting ratio is 39.69%.
Comparative example 2
Selecting without the fresh Chinese chestnut damaged by worms that goes mouldy is raw material, pulverizes after peeling, section, lyophilize through pulverizer, crosses 60 mesh sieves and makes rice-chestnut powder.Then, utilize Subcritical water chromotagraphy method, extract preparation Chinese chestnut protein according to the step shown in following flow process:
Degreasing Chinese chestnut powder → add distilled water by material-water ratio 1:15 (g/mL) to adjust pH to 9.0 → Subcritical Water Extraction (Extracting temperature 180 DEG C, extraction time 25min and under extracting pressure 4.0MPa condition) → cooling → centrifugal (4000r/min, 10min) → get supernatant liquor → acid heavy (with 1mol/LHCl tune pH to 4.2) → collecting precipitation.
Described precipitation is Chinese chestnut albumen, and as calculated, Chinese chestnut protein extracting ratio is 45.28%.
Comparative example 1, comparative example 1 and comparative example 2 are known, and the Chinese chestnut extraction rate of protein of preparation Chinese chestnut method of protein of the present invention, improves 26% relative to traditional buck method, improve 21% relative to Subcritical water chromotagraphy method.Show relative to traditional method, the Chinese chestnut extraction rate of protein of preparation Chinese chestnut method of protein of the present invention significantly improves.In addition, from embodiment 1, the Chinese chestnut purity of protein that preparation Chinese chestnut method of protein of the present invention obtains is high, and active good, protein content can up to 88%.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (17)
1. prepare a Chinese chestnut method of protein, it is characterized in that, comprise the following steps:
The rice-chestnut powder aqueous solution is carried out ultrasonic disruption process, under 400W ultrasonic power, carries out described ultrasonic disruption process 6-14min, to obtain the rice-chestnut powder aqueous solution through ultrasonic disruption process;
Utilize cellulase to carry out the first enzymolysis processing to the described rice-chestnut powder aqueous solution through ultrasonic disruption process, under 40-60 degree Celsius, pH4-6 condition, carry out described first enzymolysis processing 5-15min, to obtain first enzyme hydrolysis products;
Utilize the first α-amylase that described first enzyme hydrolysis products is carried out the second enzymolysis processing, under 30-50 degree Celsius, pH5.5-6.5 condition, carry out described second enzymolysis processing 25-35min, to obtain second enzyme hydrolysis products;
Described second enzymolysis processing product is carried out alkali and proposes process, to obtain alkali carrying product;
Described alkali being carried product carries out first centrifugal, and collects supernatant liquor, to obtain protein alkali extract;
Utilize the second α-amylase that described protein alkali extract is carried out the 3rd enzymolysis processing, under 30-50 degree Celsius, pH5.5-6.5 condition, carry out described 3rd enzymolysis processing 25-35min, to obtain the 3rd enzymolysis product;
Utilize isoelectric point precipitation described 3rd enzymolysis product to be carried out acid to sink, to obtain the heavy product of acid;
The product that described acid sunk carries out standing centrifugal with second successively, obtains precipitation to collect; And
Described precipitation is carried out being washed to neutrality, to obtain described Chinese chestnut protein.
2. method according to claim 1, is characterized in that, under 400W ultrasonic power, carries out described ultrasonic disruption process 10min.
3. method according to claim 1, is characterized in that, in 50 degrees Celsius, carries out described first enzymolysis processing 10min under pH4.5 condition.
4. method according to claim 1, is characterized in that, when carrying out described first enzymolysis processing, the ratio of described cellulase and the described rice-chestnut powder aqueous solution through ultrasonic disruption process is 50-150U/g.
5. method according to claim 4, is characterized in that, when carrying out described first enzymolysis processing, the ratio of described cellulase and the described rice-chestnut powder aqueous solution through ultrasonic disruption process is 100U/g.
6. method according to claim 1, is characterized in that, in 40 degrees Celsius, carries out described second enzymolysis processing 30min under pH6.1 condition.
7. method according to claim 1, is characterized in that, when carrying out described second enzymolysis processing, the ratio of described first α-amylase and described first enzyme hydrolysis products is 80-160U/g.
8. method according to claim 7, is characterized in that, when carrying out described second enzymolysis processing, the ratio of described first α-amylase and described first enzyme hydrolysis products is 120U/g.
9. method according to claim 1, is characterized in that, described alkali is proposed process and comprised:
It is 8.0-10.0 that described second enzymolysis processing product is carried out pH regulator to pH, and under 30-50 degree Celsius, carries out described alkali carry 25-35min.
10. method according to claim 9, is characterized in that, described alkali is proposed process and comprised:
It is 10.0 that described second enzymolysis processing product is carried out pH regulator to pH, and under 40 degrees Celsius, carries out described alkali carry 30min.
11. methods according to claim 1, is characterized in that, in 40 degrees Celsius, carry out described 3rd enzymolysis processing 30min under pH6.1 condition.
12. methods according to claim 1, is characterized in that, when carrying out described 3rd enzymolysis processing, the ratio of described second α-amylase and described protein alkali extract is 80-160U/g.
13. methods according to claim 12, is characterized in that, when carrying out described 3rd enzymolysis processing, the ratio of described second α-amylase and described protein alkali extract is 120U/g.
14. methods according to claim 1, is characterized in that, described acid is sunk and to be carried out under isoelectric pH 4.2 condition.
15. methods according to claim 1, is characterized in that, comprise further and described Chinese chestnut albumen is carried out vacuum lyophilization.
16. methods according to claim 15, is characterized in that, utilize vacuum freeze drier to carry out described vacuum lyophilization.
17. methods according to claim 1, is characterized in that, the solid-liquid ratio of the described rice-chestnut powder aqueous solution is 1g:15ml.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5738887A (en) * | 1993-08-17 | 1998-04-14 | Wu; Wencai | Process of preparing fruit/vegetable juice and protein emulsion with multi-enzyme system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5738887A (en) * | 1993-08-17 | 1998-04-14 | Wu; Wencai | Process of preparing fruit/vegetable juice and protein emulsion with multi-enzyme system |
Non-Patent Citations (3)
| Title |
|---|
| 亚临界水萃取板栗蛋白工艺研究;张海晖等;《食品工业》;20130420;第34卷(第4期);第67-68页第1.2.2节 * |
| 板栗水溶性蛋白质提取工艺的优化;常学东等;《食品科学》;20041115;第25卷(第11期);第141-143页 * |
| 板栗蛋白质的提取及多肽的制备;张帅等;《山西农业大学学报》;20110130;第31卷(第1期);摘要，第74页第1.2.1-1.2.2节，第75页第2.1节，图1-2 * |
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