CN1294144C - Process for producing soy-bean whey separator - Google Patents
Process for producing soy-bean whey separator Download PDFInfo
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- CN1294144C CN1294144C CNB031500722A CN03150072A CN1294144C CN 1294144 C CN1294144 C CN 1294144C CN B031500722 A CNB031500722 A CN B031500722A CN 03150072 A CN03150072 A CN 03150072A CN 1294144 C CN1294144 C CN 1294144C
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Abstract
To fractionate soybean whey to soybean trypsin inhibitor concentrates (preferably, fractions mainly comprising BBI type and Kunitz type), respectively, and a soybean [beta]-amylase concentrate and a soybean oligosaccharide concentrate without adding salt. The method for producing soybean whey-fractionated product comprises (a) a process for concentrating the soybean whey and collect aggregated precipitation precipitated under an acidic condition and (b) a process for fractionating the supernatant to a higher molecular fraction and a lower molecular fraction. By the method, the soybean whey can be fractionated into 3 fractions, the soybean trypsin inhibitor concentrate, the soybean [beta]-amylase concentrate and/or the soybean oligosaccharide concentrate. (C)2005,JPO&NCIPI.
Description
Technical field
The simple manufacture method of trypsin inhibitor that the present invention relates to contain in the soybean and amylase-useful isolates such as beta-amylase enriched material with physiologically active.
Background technology
One of physiologically active substance that contains in the soybean is a trypsin inhibitor, and it is that 4.5 Kunitz type and molecular weight about 8,000 and iso-electric point are 4.2 BBI type that known its has molecular weight about 20,000 and iso-electric point.And, relevant its physiological action has the report as cancer chest abdomen ponding inhibitor (special fair 6-23113 communique), the hyperfunction inhibitor of struvite edema (spy opens flat 7-10772 communique) and use in the disposable absorbent article with the composite skin care product that contains enzyme inhibitors (special table 2002-505916 communique) etc.
Soya-beta amylase is that hydrolyzed starch becomes the enzyme of maltose form from non-reduced terminal cutting amylose starch, and its molecular weight about 57,000 and iso-electric point are 5.5.This enzyme is used for the manufacturing of molasses sugar honey and maltose, and the oxidation inhibitor that can be used as starch is directly used in bread and dessert.In addition, α-Dian Fenmei is active few in the soybean, and the thermotolerance of soya-beta amylase is stronger than the thermotolerance of the beta-amylase of other plant, therefore thinks that it has superiority on highly purified maltose manufacturing or food antioxidant purposes.
The a variety of oligose that contain in the soybean are the carbohydrates with physiologically active, and these carbohydrates comprise stachyose, raffinose, verbascose, pinite, chiro-inositol, inositol and sucrose etc.The carbohydrate that wherein has the effect of promotion bifidobacterium growth has stachyose, raffinose, verbascose, and the spy opens flat 3-22971 communique and the clear 60-066978 communique of Te Kai has been reported its application as bifidus factor.
About the separation method of Trypsin inhibitor SBTI, on laboratory level, proposed the combination isoelectric precipitation, saltoutd, the various method for refining of ion-exchange chromatography, gel-filtration etc.As commercial run, the spy opens the method that flat 6-145198 communique has been put down in writing combination ultra-filtration membrane and ion-exchange chromatography, and the spy opens the method that flat 10-066572 communique has been put down in writing combination trypsin inhibitor extraction method and ion-exchange chromatography.
About the separation method of soya-beta amylase, special public clear 57-11636 communique and the flat 7-107974 communique of Te Kai have been reported the method for utilizing ultra-filtration membrane, and special public clear 57-52386 communique has been reported the method for synthetic aluminium silicate as sorbent material of utilizing.
About the separation method of soybean oligosaccharide, the spy opens clear 60-66978 communique and has reported in the presence of calcium chloride, uses in the calcium hydroxide and soybean whey, reduces proteic method simultaneously to produce precipitation; Specially permit No. 2635210 communique and reported, adjust pH below 3.0 or 3.0, reduce proteic method simultaneously to produce precipitation with phosphoric acid after the soybean whey heating.
As mentioned above, Trypsin inhibitor SBTI, soya-beta amylase and soybean oligosaccharide are very useful materials, if can obtain by industrialized simple separation technology, can make a significant contribution to society.
On the other hand, though prior art can list the separation method of Trypsin inhibitor SBTI, the separation method of soya-beta amylase and the separation method of soybean oligosaccharide, but, these methods all are the special treatment method to target substance, and its technology can not fully utilize in the soybean whey that contains various useful component.
Use the proteic method of recovery of laboratory level, need in soybean whey, add a large amount of salt, therefore be unpractical method, and in order to reclaim the appointment composition, it also is unpractical directly supplying with the soybean whey that mixes a plurality of compositions in ion-exchange chromatography.
Method with the ultra-filtration membrane protein isolate is effective, but, be the precipitation of main component owing in soybean whey, produced gradually, and block the ultrafiltration fenestra easily with the trypsin inhibitor, reduce transit dose, therefore be difficult to obtain the soybean whey enriched material of high density.
In addition, the difference of the molecular weight of Trypsin inhibitor SBTI and soya-beta amylase is very little, and therefore, it is inappropriate directly utilizing the ultrafiltration membrane treatment soybean whey to separate each composition.Certainly, it almost is impossible separating BBI type trypsin inhibitor composition, Kunitz type trypsin inhibitor composition and beta-amylase composition respectively with ultra-filtration membrane from the soybean whey of lower concentration.
Summary of the invention
In view of described situation, the objective of the invention is not need to add salt, and soybean whey is separated into various soybean trypsin inhibitor concentrations (being main component with BBI type and Kunitz type preferably) respectively, soya-beta amylase enriched material and soybean oligosaccharide enriched material.
Present inventors are by concentrating the soybean whey that produces as by product in the manufacture method of soy protein isolate, adjust its pH then in stated limit, find that the trypsin inhibitor that exists in the soybean whey can be used as the amount recovery 80% or 80% or more of throw out with its activity value.
Then, add polar solvent in the above-mentioned steps separated liquid supernatant, test respectively finds that the beta-amylase that exists in the soybean whey can be used as the amount recovery 85% or 85% or more of throw out with its activity value.
On the other hand, find in specifying the pH scope, in the throw out of the trypsin inhibitor that reclaims, to add water, it is partly dissolved, separable composition based on BBI type and Kunitz type.
Then, isolating supernatant liquor in the above-mentioned steps is carried out concentrating of beta-amylase with ultra-filtration membrane, find, because in advance producing sedimentary component separating as trypsin inhibitor, so transit dose does not almost reduce, beta-amylase can be used as the amount recovery 85% or 85% or more of concentrated solution with its activity value in the soybean whey.
The present inventor has finished the present invention based on above-mentioned discovery.
That is, the present invention relates to the manufacture method of soy-bean whey separator, it is characterized in that, comprising: (a) concentrate soybean whey, the recycling step of the agglutination precipitate that recovery pH separates out in 1.7～5.2 scopes; And the step that (b) supernatant liquor that obtains is separated into macromolecule component and low molecular composition.
And the invention still further relates to following every, that is:
The step of separating out agglutination precipitate of described manufacture method is carried out in pH is 2.7～4.8 scopes.
In the described step of manufacturing (a), soybean whey concentrated is under pH is 3.5～8.5 condition, soybean whey is concentrated into its solids component carries out in the scope of 30 weight %～50 weight %.
In the described manufacture method, soybean whey concentrated be 4.5～7.0 at pH, temperature is to carry out under 20 ℃～65 ℃ the condition.
In the described manufacture method, the separating step (b) that described supernatant liquor is separated into macromolecule component and low molecular composition is to add polar solvent in supernatant liquor, and separates the throw out separate out and the separating step (b-1) of supernatant liquor; Or use ultrafiltration membrane treatment, be separated into concentrated solution and see through the separating step (b-2) of liquid; Or the step of its combination.
In the described manufacture method, described polar solvent is an ethanol, and step (b-1) is that alcohol concn in adjusting described supernatant liquor is 30 weight %～85 weight %, and implement under the temperature condition in-5 ℃～15 ℃ scopes.
Described step of manufacturing (b-1) is that the alcohol concn in adjusting described supernatant liquor is 50 weight %～80 weight %, and implement 0 ℃～10 ℃ temperature range.
Be that the ultra-filtration membrane of 5000～250000 scopes is separated into described supernatant liquor concentrated solution and sees through liquid with the isolated molecule amount in the described step of manufacturing (b-2).
Described step of manufacturing (b-2) be in temperature be below 65 ℃ or 65 ℃, pH 4.0～7.0, the solids component of supernatant liquor implements at 50 weight % or the scope below the 50 weight %.
In the described manufacture method, supernatant liquor that step (b-1) is obtained or step (b-2) obtain sees through liquid to be concentrated into solids component is 50 weight %～80 weight %.
In the described manufacture method, the agglutination precipitate that step (a) reclaims is a soybean trypsin inhibitor concentration, the concentrated solution that throw out that step (b-1) obtains or step (b-2) obtain is the soya-beta amylase enriched material, and what supernatant liquor that step (b-1) obtains or step (b-2) obtained is soybean oligosaccharide through liquid.
In the described manufacture method, add water in the throw out that step (a) obtains, throw out is partly dissolved in pH is 2.0～4.8 scope, the step of going forward side by side is carried out solid-liquid separation.
In the described manufacture method, make throw out part dissolving again in pH is 3.0～4.4 scope.
In the above-mentioned manufacture method, the liquid portion that described solid-liquid separation obtains is a BBI type trypsin inhibitor concentration, and solid part is a Kunitz type trypsin inhibitor concentration.
Description of drawings
Fig. 1 represents that the throw out of the supernatant liquor of step (a), step (a), the throw out of step (a) dissolve the throw out that obtains again, the throw out of step (a) dissolves the proteic SDS electrophoretic patten of the supernatant liquor that obtains again.
Symbol among Fig. 1 is as follows
1. telltale
2. the supernatant liquor of step (a)
3. the throw out of step (a)
4. the throw out of step (a) dissolves the throw out that obtains again
5. the throw out of step (a) dissolves the supernatant liquor that obtains again
Fig. 2 represents the proteic SDS electrophoretic patten of supernatant liquor
Symbol among Fig. 2 is as follows
1. telltale
2. precipitate entirely
3. pH is 4.0
4. pH is 4.4
5. pH is 4.8
6. pH is 5.2
7. pH is 5.6
8. pH is 6.0
Concrete form of implementation
Soybean whey among the present invention can be isolating as the so-called soybean whey behind the glycinin of soybean protein isolate or soybean protein concentrate etc. from soybean, preferably utilizes and do not experience the soybean whey that is subjected to thermal process that causes soybean trypsin or soya-beta amylase inactivation in this step.
In other words, be exactly that Trypsin inhibitor SBTI and soya-beta amylase in the soybean whey has activity.
Therefore, the soybean whey that uses among the present invention is, for example low sex change skimmed soy beans water after extracting under about neutrallty condition, remove the residue from beans after making composition, make skimmed soy milk near iso-electric point (for example, pH be 4.5 near) thus the coagulation sedimentation that soybean protein takes place is removed the solution that soybean protein obtains.
Usually, use the soybean whey that produces as by product in the isolating soybean protein step of industrial manufacturing, can obtain a large amount of and stay-in-grade raw material like this, therefore most suitable.
Usually, the solids component of soybean whey is about 3%, and its solids component is made up of about 20% crude protein, about 20% ash content and about 60% carbohydrate.Even change the water extraction number of times of skimmed soy beans, the solids component of soybean whey is formed also almost not variation.
Among the present invention, soybean whey preferably has the activity of Trypsin inhibitor SBTI and soya-beta amylase, if solids component is about 3%, wherein, the activity of preferred Trypsin inhibitor SBTI is about 5 units/ml, and the activity of soya-beta amylase is about 100 units/ml.
In addition, the measuring method of soybean trypsin inhibitor activity can adopt the BAPA method in the method for formulating according to A.O.C.S. among the present invention, promptly measure the method for reacting the reducing sugar that generates as the yam starch and the whey (amylase solution) of matrix with the Somogi-Nelson method, wherein, 1 unit enzyme amount be meant in 40 ℃, 10 minutes generate be equivalent to 1mg glucose sugared the time required enzyme amount.
Trypsin inhibitor SBTI is stable below 8.0 at pH.Its thermostability is stable below 50 ℃, can keep 60% activity at 70 ℃.
In addition, beta-amylase stable below 65 ℃, is 4.0 o'clock at pH in pH is 4.5～7.0 scopes, stable below 45 ℃, is 3.5 o'clock at pH, and stable below 20 ℃, so pH is 3.0 o'clock, is necessary to maintain the temperature at the low temperature below 20 ℃.
Therefore, when handling soybean whey, suitably select pH and temperature, to keep the activity of two kinds of enzymes.Usually, select pH in 3.5～8.0 scope.
Reclaiming proteic method by soybean whey has salting-out process, still, when for example Trypsin inhibitor SBTI being precipitated with salt, is 3% soybean whey with respect to solids component, need to add a large amount of salt of 14%, so this is unpractical.
In addition, the iso-electric point of Kunitz type trypsin inhibitor is that pH is 4.5, and the iso-electric point of BBI type trypsin inhibitor is that pH is 4.2, and the iso-electric point of beta-amylase is that pH is 5.5.But the pH that attempts directly to adjust soybean whey carries out isoelectric precipitation on iso-electric point separately, do not obtain the agglutination precipitate of target substance yet or the amount that obtains few.
Description of step (a) at first.
In the throw out of step (a), add water, in pH is 2.0～4.8 scopes, throw out is partly dissolved, can further carry out solid-liquid separation.The liquid portion that obtains from this solid-liquid separation is a BBI type trypsin inhibitor concentration, and solid part is a Kunitz type trypsin inhibitor concentration.
That is, emphasis of the present invention is by concentrating soybean whey and by adjusting pH in 1.7～5.2 scopes, preferably in 2.7～4.8 scope, separates the agglutination precipitate and the supernatant liquor of separating out.This agglutination precipitate can be used as soybean trypsin inhibitor concentration.
In addition, the pH that is concentrated in of soybean whey is in 3.5～8.5 the scope, preferably carries out in pH is 4.5～7.0 scope; Solution temperature preferably carries out 20 ℃～65 ℃ scopes 20 ℃～80 ℃ scopes; Being concentrated into solids component is 30 weight %～50 weight %, is preferably 35 weight %～45 weight %.
Concentration method can adopt known method, if decompression concentrates down, can prevent that temperature from rising, and concentrated effect is better.
In addition,, for example, during pH less than 3.5, cause the beta-amylase instability easily, and pH surpasses at 8.5 o'clock, causes the trypsin inhibitor instability easily if pH exceeds above-mentioned scope.
In addition, during 20 ℃ of solution temperature less thaies, the mechanical property of reliever is restricted, and temperature is when surpassing 65 ℃, and the activity of beta-amylase has the trend of reduction.
Its reason is, solids component is being adjusted into 30 weight %～50 weight %, and during preferred 35 weight %～45 weight % scopes, the ash content concentration in the whey increases relatively, can obtain and the identical effect of saltouing separable especially trypsin inhibitor composition.If described pH, temperature and concentration exceed above-mentioned scope, the rate of recovery of the Trypsin inhibitor SBTI in the soybean whey is with the trend of activity value in respect of reduction, and the beta-amylase activity that reclaims in the step (b-2) also has the trend of reduction.
In the time of particularly in solids component being adjusted to the scope that can from whey, be settled out the Trypsin inhibitor SBTI composition, pH can be adjusted in described scope (pH is 1.7～5.2), but, even exceed this scope, if give one's full attention to the inactivation that low pH produces beta-amylase, controlled temperature effectively, for example, during pH less than 4.0, carry out below 20 ℃, also can obtain good effect about.
Concentrate soybean whey, being recovered in pH is the agglutination precipitate of separating out in 1.7～5.2 o'clock, and residual supernatant liquor is separable to be macromolecule component and low molecular composition (b step).
The separating step (to call the b step in the following text) that this supernatant liquor is separated into macromolecule component and low molecular composition comprises and adds polar solvent as the 1st step in supernatant liquor, separate the throw out separate out and supernatant liquor separating step (b-1) and as the 2nd step the supernatant liquor ultrafiltration membrane treatment, be separated into concentrated solution and through the separating step (b-2) of liquid.
Description of step (b-1) at first.
In the supernatant liquor that step (a) obtains, add polar solvent, separate the throw out and the supernatant liquor of separating out.This throw out is the soya-beta amylase enriched material.
In addition, concentrate this supernatant liquor, as the raw material use of oligose.Preferably this supernatant concentration being become solids component is 50 weight % or more than the 50 weight %.
In addition, the used polar solvent of the present invention can be the polar solvent that allow to add in food, is preferably ethanol, and adjusts preferably that alcohol concn is 30 weight %～85 weight % in the supernatant liquor, and places-5 ℃～15 ℃ temperature range to implement.
Usually, alcohol concn is 20 weight % of supernatant liquor or 20 weight % when above, produces agglutination precipitate, and still, beta-amylase dissolves in low-concentration ethanol solution, and carbohydrate is separated out when alcohol concn is high.Therefore, the alcohol concn of step (b-1) suitably in the scope of 30 weight %～85 weight %, preferred 50 weight %～80 weight %.And, soybean whey is nutritious and the solution of easy propagate microorganisms, but can not heat-sterilization when separating effective ingredient, so long more possible more propagate microorganisms of raw material whey elapsed time, therefore, if consider sterilization effect when reclaiming beta-amylase from step (b-1), concentration of ethanol is 65 weight %～75 weight % when more preferably precipitating.
Ethanol makes beta-amylase composition inactivation, and its reason not only is concentration of ethanol, and relevant with high temperature, so ethanol sedimentation should carry out at-5 ℃～15 ℃, preferably at 0 ℃～10 ℃.
In addition, 30 weight %～50 weight % at high proportion because solids component is about for the supernatant liquor that obtains from step (a), and the oligose in the mother liquor distributes more in the throw out that step (b-1) obtains, therefore when implementing ethanol sedimentation, the supernatant liquor of dilution step (a) suitably, oligose is dissolved in the supernatant liquor, can improves the rate of recovery.
Preferably supernatant concentration to the oligose composition that step (b-1) is obtained is 50 weight % or more than the 50 weight %, can reduce volume like this so that keeping or transportation, and can prevent to rot.
For example, during as the raw material of oligose, preferably have flowability and corrupt few concentration, normally contain the solids component of 50 weight %～85 weight %, preferably contain the solids component of 55 weight %～80 weight % at this supernatant liquor.
This supernatant concentration thing is carried out refinement treatment such as desalination, can obtain the purified oligose.
By above-mentioned series of steps, can from soybean whey, separate Trypsin inhibitor SBTI, soya-beta amylase and soybean oligosaccharide effectively.
Then being partly dissolved of the agglutination precipitate that reclaims from step (a) of explanation below.
The soybean trypsin inhibitor concentration that at pH is 1.7～5.2 o'clock coagulation sedimentations is 2.0～4.8 at pH, preferably in being 3.0～4.4 scope, pH is dissolved in water (being partly dissolved), the salt concn of the condensed whey that produces agglutination precipitate is reduced, therefore BBI type trypsin inhibitor dissolves again, Kunitz type trypsin inhibitor does not dissolve, separable thus both be supernatant liquor and throw out.
Described separation (solid-liquid separation) method can be utilized known equipment for separating liquid from solid, for example centrifugal separating device, membrane separation unit etc.
Described agglutination precipitate is a soybean trypsin inhibitor concentration, and the liquid portion that obtains from solid-liquid separation is a BBI type trypsin inhibitor concentration, and solid part is a Kunitz type trypsin inhibitor concentration.
The following describes step of the present invention (b-2).
The supernatant liquor that step (a) is obtained is separated into concentrated solution and sees through liquid with ultra-filtration membrane.This concentrated solution can be used as the soya-beta amylase enriched material.
See through the raw material that liquid can be used as soybean oligosaccharide.
Used here ultra-filtration membrane preferably uses isolated molecule amount 5000～250000, more preferably the ultra-filtration membrane of isolated molecule amount 10000～100000.
The supernatant liquor that step (a) obtains need not dilute and can directly use, but when concentrating, along with increasing of the solids component of concentrated solution, transit dose (seeing through flow) reduces, therefore need to keep the solids component of concentrated solution below 50 weight %, below the preferred 45 weight %, can take suitably to add the method that water carries out ultrafiltration for this reason.
The temperature of solution that sees through ultra-filtration membrane is high more, and transit dose is high more.But, in order not cause the inactivation of beta-amylase, be chosen in 65 ℃, preferably carrying out ultrafiltration step below 55 ℃.This moment, preferred pH was 4.0～7.0 for the stability of beta-amylase, and more preferably pH is 4.5～6.0.Beta-amylase is 6.0 or 6.0 also stable when above at pH, but is 6.0 or 6.0 when above at pH, and the dissolved phytic acid can not dissolve under acidic conditions, and produces precipitation, so is unfavorable.
The liquid that sees through that obtains from step (b-2) contains 50 weight % or the oligose more than the 50 weight %, and contains 20 weight % or the above ash content of 20 weight %, after the desalination, can be used as foodstuffs material.
In addition, the oligose composition that sees through liquid that preferably step (b-2) is obtained is concentrated into 50 weight % or more than the 50 weight %, can reduces volume like this so that take care of or transportation, and can prevent corruption.
For example, should see through the raw material of liquid, preferably have flowability and corrupt few concentration, be generally the solids component of 50 weight %～85 weight %, the solids component of preferred 55 weight %～80 weight % as oligose.
Should see through the liquid enriched material and carry out refinement treatment such as desalination, can obtain refining oligose.
Embodiment
Below by embodiment form of implementation of the present invention is described.
Embodiment 1
Step (a)
The soybean whey 70kg that will from the soy protein isolate manufacturing step, obtain (solids component 2.9 weight %, pH is 4.6, total trypsin inhibitor activity value 357000 units, total beta-amylase activity value 9120000 units, crude protein content 20.1 dry weights (dry) %, ash content 19.7 dry weight %, carbohydrate 60.2 dry weight %) with made vaporizer (CEP-L type) concentrating under reduced pressure under 50 ℃ of (80 ℃ of Heating temperatures) conditions of boiling temperature of soybean whey of the former making in great river, obtain solids component 41.3 weight %, total trypsin inhibitor activity value 333000 units, the condensed whey 4600g (because of reasons such as absorption lose about 300g) of total beta-amylase activity value 8530000 units.
Condensed whey is cooled to 15 ℃, and adjusting pH with phosphoric acid is 4.0, leave standstill 3 hours after, carry out centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out.
Separating the supernatant liquor (supernatant liquor of (a) step) that obtains is 4297g, and its solids component is 40.0 weight %, total trypsin inhibitor activity value 40000 units, total beta-amylase activity value 8200000 units; The throw out that obtains (throw out of (a) step) is 324g, and its solids component is 55.0 weight %, total trypsin inhibitor activity value 305000 units, total beta-amylase activity value 700000 units.
Therefore, the partition ratio of two kinds of enzymic activitys is that trypsin inhibitor is 11.6% in supernatant liquor, is 88.4% in precipitation; Beta-amylase is 92.1% in supernatant liquor, is 7.9% in precipitation.
Step (b-1)
The supernatant liquor 4200g of step (a) is cooled to 8 ℃, one-level ethanol (purity 99.5%) 4000g (alcohol concn is 61.3 weight %) helical stir limit, limit is slowly added, form softish cake class agglutinator, can directly separate, but also can centrifugation after 15 minutes (1500G * 10 minute), be separated into supernatant liquor and throw out.
Separating the throw out (throw out of (b-1) step) that obtains is 2028g, and its solids component is 55.2 weight %, total beta-amylase activity value 7970000 units, total trypsin inhibitor activity value 9800 units.
Supernatant liquor distills (65 ℃ of Heating temperatures) with rotatory evaporator, removes ethanol, obtains solution (supernatant liquor of (b-1) step) 2530g of solids component 29.8 weight %.
The water that adds 3 times of amounts in the throw out of step (b-1), with homogenizer (3000rpm) stirring and dissolving, almost therefore all dissolvings, can implement the highly purified of beta-amylase with this throw out as raw material.
The supernatant liquor 2500g of step (b-1) further uses film type flasher (the MF-10A type of Tokyo natural sciences system) in 60 ℃ of boiling temperatures, 90 ℃ of Heating temperatures, carries out concentrating under reduced pressure, and obtaining solids component is the enriched material 1120g of 60.1 weight %.
In this enriched material, with respect to solids component, contain the carbohydrate of 76.3 weight %, wherein 32.0% is the above oligose of three sugar.
Embodiment 2
Use the soybean whey identical with embodiment 1, concentrating under reduced pressure uses the same method, the condensed whey 300g that solids component is 51.5 weight % (total trypsin inhibitor activity value 26000 units will be concentrated into, total beta-amylase activity value 698000 units) be cooled to 15 ℃, adjusting pH is 4.0, after leaving standstill 3 hours, carry out centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out.
Separating the supernatant liquor that obtains is 271g, and its solids component is 50.9 weight %, total trypsin inhibitor activity value 15000 units, total beta-amylase activity value 611000 units; The throw out that obtains is 34g, and its solids component is 48.8 weight %, total trypsin inhibitor activity value 13000 units, total beta-amylase activity value 101000 units.
When being concentrated into solids component and being 51.5 weight %, the selective precipitation degree of trypsin inhibitor is poorer than embodiment 1.
Embodiment 3
Use the soybean whey identical with embodiment 1, concentrating under reduced pressure uses the same method, being concentrated into solids component is condensed whey 300g (total trypsin inhibitor activity value 15000 units of 28.7 weight %, total beta-amylase activity value 361000 units), enriched material is cooled to 15 ℃, and adjusting pH is 4.0, leave standstill 3 hours after, carry out centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out.
Separating the supernatant liquor that obtains is 286g, and its solids component is 27.9 weight %, total trypsin inhibitor activity value 7300 units, total beta-amylase activity value 320000 units; The throw out that obtains is 18g, and its solids component is 35.2 weight %, total trypsin inhibitor activity value 8100 units, total beta-amylase activity value 54000 units.
When solids component is the concentration of 28.7 weight %, compare with embodiment 1, the precipitation of trypsin inhibitor is insufficient.
Embodiment 4
The identical implementation step of method (a) with embodiment 1, at solids component is among the supernatant liquor 300g (total beta-amylase activity value 625000 units) of the step (a) of 42.3 weight %, under room temperature (25 ℃), after slowly adding 270g ethanol (alcohol concn is 60.6%) while stirring, by centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out.The throw out that obtains is 145g, and its solids component is 56.4 weight %, total beta-amylase activity value 313000 units.
Among the embodiment 1, from the supernatant liquor of step (a) precipitation through step (b-1), the solids component yield that obtains is 66.6%, and in contrast to this, the solids component yield of embodiment 4 is 64.4%, and is almost equal.
But, be that 97% almost full dose reclaims and compares with the beta-amylase actives rate of recovery of embodiment 1, embodiment 4 only is 50%, its reason may be to have produced inactivation by temperature.
Application examples 1
Adding 500g water in the throw out 100g (solids component 56.1 weight %, total trypsin inhibitor activity value 95000 units) of the step (a) of the method preparation identical with embodiment 1, after homogenizer (3000rpm * 15 minute) stirring and dissolving, with centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out, obtain supernatant liquor 580g (solids component 7.9 weight %, total trypsin inhibitor activity value 93000 units) and throw out 20g (solids component 51.5 weight %, total trypsin inhibitor activity value 3500 units).
According to this result, the trypsin inhibitor of coagulation sedimentation can dissolve by adding water again in step (a), therefore, and can be from the refining trypsin inhibitor of the throw out camber of step (a).
Embodiment 5
Step (a)
With soybean whey 200kg (exsiccant solids component 3.0 weight %, pH is 4.6, total trypsin inhibitor activity value 960000 units, total beta-amylase activity value 22400000 units, crude protein content 20.3 dry weight %, ash content 20.1 dry weight %, carbohydrate 59.6 dry weight %) with made vaporizer (CEP-L type) concentrating under reduced pressure under 50 ℃ of (80 ℃ of Heating temperatures) conditions of boiling temperature of soybean whey of the former making in great river, obtain exsiccant solids component 42.2 weight %, total trypsin inhibitor activity value 930000 units (66 units/g), (the condensed whey 14000g (because of reasons such as absorption lose about 200g) of 1572 units/g) of total beta-amylase activity value 22010000 units.
Condensed whey is cooled to 15 ℃, with phosphoric acid adjust pH be leave standstill 3 hours under 4.0,10 ℃ after, carry out centrifugation (1500G * 10 minute), be separated into supernatant liquor and throw out.
Separating the supernatant liquor (supernatant liquor of (a) step) obtain be 12850g, and its exsiccant solids component is 41.3 weight %, total trypsin inhibitor activity value 110500 units (8.6 units/g), total (1595 units/g) of beta-amylase activity value 20500000 units; The throw out that obtains (throw out of (a) step) is 1150g, and its exsiccant solids component is 52.3 weight %, total trypsin inhibitor activity value 820000 units ((1330 units/g) of 713 units/g), total beta-amylase activity value 1530000 units.
Therefore, the partition ratio of two kinds of enzymic activitys is that trypsin inhibitor is 11.9% in supernatant liquor, is 88.1% in precipitation; Beta-amylase is 93.1% in supernatant liquor, is 6.9% in precipitation.
The sedimentary dissolving again of step (a)
Add 2000g water in the throw out of 1000g step (a), keeping pH is 4.0, after homogenizer (4000rpm * 15 minute) stirring and dissolving, with centrifugation (5000G * 10 minute), is separated into supernatant liquor and throw out.
Obtain supernatant liquor 2767g, its solids component is 14.2 weight %, total trypsin inhibitor activity value 595000 units (215 units/g); Obtain throw out 233g, its solids component is 55.8 weight %, total trypsin inhibitor activity value 221000 units (948 units/g).
Supernatant liquor and sedimentary albumen are analyzed with the SDS electrophoresis.
The albumen of the supernatant liquor that the throw out that obtains after the throw out of the precipitation of the supernatant liquor of step (a), step (a), Sugar receptors, step (a) dissolves again, the throw out of step (a) obtain after dissolving again, the figure of usefulness SDS electrophoretic analysis as shown in Figure 1.
Fig. 1 represents, the following proteic situation of molecular weight 42kDa, and 2. there is trypsin inhibitor (only residual BBI types) in line in 1. hardly, all distribute online.
2. line adds water and dissolves and obtain line throw out 3. and have Kunitz type trypsin inhibitor, and line supernatant liquor 4. has BBI type trypsin inhibitor.
As mentioned above, from the sedimentary different solvabilities of step (a), but initial gross separation Kunitz type trypsin inhibitor and BBI type trypsin inhibitor.
Step (b-2)
The concentration of step (a) supernatant liquor is added water to be adjusted into 40 Brixs (Brix) (solids component is about 38%, beta-amylase activity value 1470 units/g), ultra-filtration membrane (isolated molecule amount 30000, the membrane area 1.4m of the Diasen film (strain) of use system
2, the tubular wire pattern).For the concentration that keeps concentrating part at 40Brix, when suitably adding water, at flow 1m
3/ h, back-pressure 3kg/cm
2, 45 ℃ of liquid temperature, pH be under 4.5 the condition, the beta-amylase activity value to be concentrated to about 10 times, its result is as shown in table 1.
Table 1
| Time (minute) | Accumulative total sees through liquid measure (kg) | Film is handled liquid measure (kg) | Amount of water (kg) | Transit dose (kg/m 2/h) | Concentrated solution concentration (Brix) |
| 0 | 0 | 12.8 | 40.0 | ||
| 10 | 1.6 | 11.2 | 6.9 | 40.0 | |
| 20 | 2.9 | 9.9 | 5.6 | 40.1 | |
| 30 | 4.1 | 8.6 | 5.3 | 40.5 | |
| 40 | 5.2 | 7.5 | 4.7 | 41.0 | |
| 50 | 6.1 | 6.6 | 3.8 | 42.0 | |
| 60 | 6.8 | 6.7 | 0.43 | 2.9 | 39.5 |
| 70 | 7.7 | 5.8 | 3.9 | 41.0 | |
| 80 | 8.5 | 5.0 | 3.4 | 43.0 | |
| 90 | 9.2 | 4.9 | 0.53 | 2.9 | 39.7 |
| 100 | 10.2 | 3.9 | 4.4 | 43.5 | |
| 110 | 11.0 | 3.5 | 0.50 | 3.6 | 39.5 |
| 120 | 12.0 | 3.4 | 0.87 | 4.3 | 33.5 |
| 130 | 13.3 | 2.2 | 5.3 | 40.1 |
The supernatant liquor concentration of step (a) is adjusted into 40Brix, handles 12.8kg.
For the concentration of UF membrane concentration liquid is remained on 40Brix, in the time of 60 minutes, 90 minutes, 110 minutes, 120 minutes, add water respectively.
When concentrating, along with increasing of concentration, transit dose reduces, but because adjust concentration by adding water, can recover transit dose and not produce stifled film phenomenon.Move after 130 minutes and finish, transit dose does not significantly reduce, and can further concentrate.
Average transit dose is 4.1kg/m
2/ h, the drying solid composition of the concentrated solution that obtains is about 38.0%, and (dry thing is scaled 35500 units/g) in beta-amylase activity value 13500 units/g.
In addition, the UF film is as shown in table 2 through the composition of liquid.
Table 2
| The exsiccant solids component | Crude protein | Ash content | Carbohydrate | The beta-amylase activity value |
| 28.5％ | 9.5 dry weight % | 22.5 dry weight % | 68.0 dry weight % | 0 unit |
In addition, with the high performance liquid chromatography (detector/system L-6200 of Hitachi, pump/L3350, post/Shodex Sugar SC-1011), with water as mobile phase, analyze carbohydrate, its result, the drying solid composition is the soybean oligosaccharide enriched material that contains oligose (raffinose+stachyose) 27.8%, the upright sugar 7.3% of pinane, chiro-inositol 1.0%, inositol 2.6% etc.
Embodiment 6
Add water at the UF film through in the liquid, adjusting the drying solid composition is 20% (ash content 4.5%), carry out desalination with electrodialysis unit (the micro-ア シ ラ イ ザ of Asahi Chemical Industry's system--S3, cartridge/AC-220-550), its result, specific conductivity is reduced to 1.0mS/cm (ash content 0.2%, the drying solid composition is 16.5%) time, sapid soybean oligosaccharide enriched material can be obtained.
Embodiment 7 (combination UF film (ultra-filtration membrane) concentrates the embodiment with ethanol sedimentation)
The concentration of the supernatant liquor of step (a) is added water to be adjusted into 40Brix (solids component is about 38%, and beta-amylase activity value 1470 units/g) are with diasen film (strain) system ultra-filtration membrane (isolated molecule amount 30000, membrane area 1.4m
2, the tubular wire pattern), for the concentration that keeps concentrating part at 40Brix, when suitably adding water, at flow 1m
3/ h, back-pressure 3kg/cm
2, 45 ℃ of liquid temperature, pH be under 4.5 the condition, the beta-amylase activity value to be concentrated to about 10 times, obtains that the drying solid composition is about 38.0%, (dry thing is scaled the concentrated solution of 35500 units/g) in beta-amylase activity value 13500 units/g.The liquid temperature of this concentrated solution 100g is adjusted to 8 ℃, and the ethanol temperature is also adjusted to 8 ℃, and 150g ethanol is joined in the concentrated solution, leave standstill 10 minutes after, obtain throw out with centrifugation.
The throw out that obtains is that solids component is about 59.2%, and the beta-amylase activity value is that (dry thing is scaled 39500 units/g), finish further concentrating of beta-amylase thus to 23400 units/g.
In addition, there is not the beta-amylase activity in the supernatant liquor with ethanol sedimentation.
Embodiment 8
With the method identical with embodiment 1 soybean whey being evaporated to solids component is 40.6 weight %, get spissated condensed whey 450g (total trypsin inhibitor activity value is 30500 units), divide and install in the beaker of 9 100ml, each beaker 50g (total trypsin inhibitor activity value be 3300 units) that packs into, adjusting pH respectively with hydrochloric acid or sodium hydroxide after being cooled to 15 ℃ is 1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5, left standstill 8 hours, centrifugation then (1500G * 10 minute), be separated into supernatant liquor and throw out, measure the activity of total trypsin inhibitor of supernatant liquor.
The activity value of total trypsin inhibitor of supernatant liquor becomes pH1.5/2500 unit, pH2.0/1650 unit, pH2.5/1330 unit, pH3.0/820 unit, pH3.5/540 unit, pH4.0/350 unit, pH4.5/980 unit, pH5.0/1480 unit, pH5.5/2120 unit respectively, has the activity value of total trypsin inhibitor of 7 one-tenth to transfer in the throw out in pH is 3.0～4.5 scope.
Embodiment 9
Agglutination precipitate (the total trypsin inhibitor component) 50g that obtains with the method identical with embodiment 1, keep with sodium hydroxide or hydrochloric acid and to add water 150g when its pH is respectively pH4.0, pH4.4, pH4.8, pH5.2, pH5.6, pH6.0, and stir with homogenizer (4000rpm20 minute 20 ℃), promote it to dissolve once more.
After stirring dissolving again, with the centrifugation in 5000G/20 minute of the worker of Hitachi mechanism supercentrifuge, the proteic SDS-electrophoretic patten of the supernatant liquor that obtains (trypsin inhibitor component) as shown in Figure 2.
As can be seen from Figure 2, line 4. (pH4.4) shows there is not KSTI in the supernatant liquor, and 5. line under (pH4.8) and the higher pH, sneaks into KSTI in the supernatant liquor, can not separate BBI and KSTI.
In addition, with the pH lower limit of pH4.0, pH3.0, pH2.0, pH1.0 research separation KSTI and BBI, not expression is 3.0 o'clock at pH among the figure, does not have KSTI in the supernatant liquor, is to sneak into KSTI in the 2.0 or 2.0 following supernatant liquors at pH.
According to the present invention, soybean whey can be divided into substantially three kinds of compositions such as soybean trypsin inhibitor concentration, soya-beta amylase enriched material and/or soybean oligosaccharide enriched material.
And soybean trypsin inhibitor concentration can be divided into BBI type trypsin inhibitor and Kunitz type trypsin inhibitor, and soya-beta amylase is filtered thoroughly with the UF film, can carry out highly concentrated.
These isolates can be used as the intermediate raw material of Trypsin inhibitor SBTI, soya-beta amylase and soybean oligosaccharide.
Utilize step of the present invention, can from soybean whey, separate Trypsin inhibitor SBTI, soya-beta amylase and soybean oligosaccharide effectively, therefore can fully utilize soybean whey by a series of step.
Claims (11)
1, separate the method for Trypsin inhibitor SBTI, soya-beta amylase and soybean oligosaccharide from soybean whey, it is characterized in that, described method comprises:
(a) pH be under 3.5～8.5 the condition in 20 ℃～65 ℃ concentrating under reduced pressure soybean wheys, adjust pH then and be recovered in the recycling step that pH is the agglutination precipitate of the soybean trypsin inhibitor enriched material of separating out under 1.7～5.2 the condition by centrifugation;
(b) adopt and in the supernatant liquor that obtains, to add ethanolic soln, and separate the throw out of separating out and the separating step (b-1) of supernatant liquor; Or be the supernatant liquor that 30000 ultrafiltration membrane treatment obtains with the isolated molecule amount, be separated into concentrated solution and see through the separating step (b-2) of liquid; Or separating step (b-1) and combination (b-2), the supernatant liquor that obtains is separated into the separating step of macromolecule component soya-beta amylase enriched material and low molecular composition soybean oligosaccharide class.
2, the method for claim 1 is characterized by, and described recycling step of separating out agglutination precipitate is to carry out in pH is 2.7～4.8 scope.
3, the method for claim 1 is characterized by, and in the step (a), soybean whey is concentrated into its solids component in the scope of 30 weight %～50 weight %.
4, manufacture method as claimed in claim 3 is characterized by, and described soybean whey concentrated is to carry out under pH is 4.5～7.0 condition.
5, manufacture method as claimed in claim 1 is characterized by, and the alcohol concn of described step (b-1) in adjusting described supernatant liquor is 30 weight %～85 weight %, and implements under the temperature condition of temperature in-5 ℃～15 ℃ scopes.
6, manufacture method as claimed in claim 5 is characterized by, and the alcohol concn of described step (b-1) in adjusting described supernatant liquor is 50 weight %～80 weight %, and temperature is implemented 0 ℃～10 ℃ scope.
7, manufacture method as claimed in claim 1 is characterized by, described step (b-2) in temperature is below 65 ℃ or 65 ℃, pH 4.0～7.0, the supernatant liquor solids component implements at 50 weight % or the scope below the 50 weight %.
8, manufacture method as claimed in claim 1 is characterized by, and supernatant liquor that step (b-1) is obtained or step (b-2) obtain sees through liquid to be concentrated into solids component be 50 weight %～85 weight %.
9, manufacture method as claimed in claim 1 is characterized by, and adds water in the throw out that step (a) obtains, and makes its part dissolving again in pH is 2.0～4.8 scope, further to carry out solid-liquid separation.
10, manufacture method as claimed in claim 9 is characterized by, and described throw out is that 3.0～4.4 range section dissolves again at pH.
11, as claim 9 or 10 described manufacture method, wherein the liquid portion that obtains from described solid-liquid separation is a BBI type trypsin inhibitor concentration, and solid part is a Kunitz type trypsin inhibitor concentration.
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| JP2002223228 | 2002-07-31 | ||
| JP223228/2002 | 2002-07-31 | ||
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| JP2003094128 | 2003-03-31 |
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| CN103756987B (en) * | 2014-02-18 | 2015-08-26 | 山东绿健生物技术有限公司 | Prepare the method for beta-amylase and soybean oligosaccharide simultaneously |
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| CN1364763A (en) * | 2001-01-19 | 2002-08-21 | 北京中联捷思科技发展有限公司 | Method for extracting soy bean oligosaccharide from soy bean whey water |
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