CN1966705A - Process for preparing soybean isoflavone aglycon by microorganism enzyme method - Google Patents
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Abstract
The invention discloses a method for producing soybean isoflavone aglycone through microbe's enzyme method, and is culturing Aspergillus oryzae with soybean isoflavone powder as raw materials for fermentation to produce beta-glucosidase, and converting soybean isoflavone to soybean isoflavone aglycone. The method is characterized in comprising the following preparation steps: the said raw materials are 2-90% soybean isoflavone powder, the said strain is Aspergillus oryzae 3042, preparing seed culture liquid with the strain, preparing solid fermentation medium, fermenting to obtain fermenting product containing beta-glucosidase, adding soybean isoflavone powder for preparing enzyme converting solution, centrifuging for separation to obtain deposit, drying at low temperature to obtain soybean isoflavone aglycone. The invention avoids the restriction of the raw materials purity, converting rata can reach to over 90%. The invention has the advantages of short procedure, low cost, high bioavailability.
Description
Technical field
The present invention relates to make the method for isoflavone genin, particularly relate to microbial enzyme method is converted into isoflavone genin by beta-glucosidase preparation method with soybean isoflavones.
Background technology
It is a compounds of parent nucleus that soybean isoflavones (sorbean isoflovone) is meant with 3-phenyl chromone, is to have multiple bioactive soybean nutritional composition.At present, the soybean isoflavones that scientist has been found that has 15 kinds of isomer, is divided into the aglycon of free type and the glucosides two big classes of mating type.Wherein glucosides accounts for the 95-98% (w/w) of soybean isoflavones total amount, and aglycon accounts for the 2-5% (w/w) of soybean isoflavones total amount.Rakult microbe research center, Tokyo current research shows: the aglycon of free type is less with respect to the mating type polyglycoside molecule, and hydrophobicity is stronger, so biological utilisation is better.Research also shows: only contain the soybean isoflavones of 0.5-2.5% in the soybean, and the bioavailability of soybean isoflavones also has only 10-40%, this mainly is because glucosides type or derivatives thereof is difficult to directly be absorbed by human body.Have only the aglycon of generation form, just easily be absorbed and used.This shows, soybean isoflavone glucoside type and glycosides derivatives are changed into the aglycon form, is the prerequisite that improves its bioavailability.In recent years foreign scholar's patent report prepare daidzein method have: utilize commercial enzyme (as: beta-glucosidase, esterase etc.) that isoflavones is hydrolyzed into aglycon, utilize chemical process to synthesize, extract daidzein, strong acid, highly basic method for hydrolysis prepare daidzein, these methods because exist complicated operation, yield is low, reaction conditions is violent, cost is high, organic solvent residual causes shortcomings such as environmental pollution, all fail to be widely used.Disclosed Shuanghe Songnen Soybean Bioengineering LLC, Helongjiang No. 200410058379.3 patent applications, disclosed the processing method of producing isoflavone genin with β-glucose and membrane technique, this method is to be basic raw material with the soybean isoflavones powder, make inductor with soybean protein and soybean isoflavones, with cultivating the extracellular enzyme beta-glucoside enzyme hydrolysis of soybean isoflavone that mould discharges, and utilize the membrane technique that is selected from ultrafiltration and dialysis to carry out the aftertreatment of enzyme hydrolyzate, obtain isoflavone genin.Also there is certain defective in this method in raw material suitability and process aspect: 1, owing to adopt the soybean isoflavones powder of content more than 20%, transformation efficiency reaches more than 90%, and experiment shows, is lower than 20% as the soybean isoflavones powder content, and transformation efficiency can obviously reduce; Particularly material content is lower than at 10% o'clock, if adopt above-mentioned technology, can increase greatly and filter refining cost because impurity is many; 2, technical process is longer, and fermentative preparation beta-glucosidase raw material is more, requires harsh to filter plant and technical qualification.The present invention compared with prior art is not subjected to the restriction of material purity, and the same adaptation of the design of its processing condition is lower than 20% material purity, and transformation efficiency all reaches more than 90%, has bigger advantage aspect the adaptability of cost of manufacture and raw material.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, provide a kind of microbial enzyme method to prepare the method for isoflavone genin, this method is not limited by material purity, be lower than 20% at the raw material composition, even when lower, still have higher conversion, and technical process is shortened, the conversion cost is low, and bioavailability obviously improves, can be directly as medicine, healthy food material.
The inventive method is the method that microbial enzyme method prepares isoflavone genin, be to be raw material with the soybean isoflavones powder, with cultivating aspergillus oryzae fermentative preparation beta-glucosidase, the soybean isoflavones glycosides is converted into isoflavone genin, it is characterized in that this preparation method may further comprise the steps: a) described raw material is 2～90% soybean isoflavones powder; B) Shai Xuan bacterial classification is an aspergillus oryzae 3042, with this strain preparation seed culture fluid; C) prepare the solid fermentation substratum, obtain containing the fermented product of beta-glucosidase; D) the soybean isoflavones powder is joined the conversion fluid of producing enzyme in the beta-glucoside enzyme solution of fermented extracted, carry out centrifugal solid-liquid again and separate, the throw out cryodrying obtains isoflavone genin.
The method that the present invention prepares isoflavone genin in more detail may further comprise the steps:
One, raw material
Basic raw material is 2～90% soybean isoflavones powder, and it is 5～20% soybean isoflavones meal that technology of the present invention is suitable for content too.
Two, bacterial classification
Screening is an aspergillus oryzae (Aspergillus oryzae) 3042 with bacterial classification
The strain inclined plane medium preparation method that goes down to posterity: PDA medium preparation method
Three, solid fermentation method prepares beta-glucosidase and purifying, enzyme activity determination
1, the preparation of seed culture fluid and cultural method:
Zulkovsky starch 2%, glucose 1%, potassium primary phosphate 0.1%, sal epsom 0.1%, yeast extract paste 0.5%, SODIUMNITRATE 0.2%, the PH nature is in 121 ℃ of sterilization 15min; Aseptic technique, the spore on the picking proper amount of fresh inclined-plane is transferred in the 80ml seed culture medium, in 20～28 ℃, 160～180r/min concussion cultivation, 38～48hr.
2, solid fermentation substratum and cultural method
With wheat bran: water cooperates according to 1: 1.5～3.0 weight ratio, the PH nature, 121 ℃ of sterilization 30min, inoculum size with 1～7% inserts fermentation culture in the solid medium with the seed for preparing, cultivate 20～28 ℃, humidity 60～70%, incubation time 60～84hr obtains containing the solid fermentation thing of beta-glucosidase;
3, the extraction of beta-glucosidase crude enzyme liquid
Above-mentioned solid fermentation thing is added the distilled water of 10 times of amounts, stir and soak 2～6hr, in 4～10 ℃, centrifugally under 5000～10000r/min condition carry out liquid-solid separation, the gained supernatant liquor is the beta-glucosidase crude enzyme liquid.
4, beta-glucosidase preliminary purification
For ease of preserving, need crude enzyme liquid is carried out purification process, with crude enzyme liquid with 75% ethanol, carrying out alcohol precipitation under 4～10 ℃ spends the night, under this temperature with 5000～10000r/min centrifugation, throw out obtains the beta-glucosidase liquid of preliminary purification with dissolved in distilled water, or obtains the solid beta-glucosidase-40～-50 times lyophilizes.Behind the preliminary purification, use the DEAE-sephadexA-50 ion-exchange resin purification, the molecular weight of measuring beta-glucosidase with SDS is 45kb.
5, the vigour-testing method of beta-glucosidase
(1) making of glucose typical curve
Adopt spectrophotometry.Get 8 and have graduated colorimetric cylinder, the accurate glucose standard of drawing is used liquid (1mg/ml) 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4ml; Replenish 2ml with distilled water; Add the 1.5mlDNS test solution respectively, boiling water bath 5min, fixed molten with distilled water to 10ml, in the 540nm colorimetric, the drawing standard curve.
(2) mensuration of beta-glucoside enzyme activity in the sample
Get 4 and have graduated colorimetric cylinder, add 0.5% saligenin 0.5ml respectively, control tube adds DNS test solution 1ml, and sample hose and control tube are placed on water-bath 5min under 50 ℃ of environment simultaneously; Each pipe adds enzyme liquid 0.5ml respectively, and behind 50 ℃ of water-bath 30min, sample hose adds DNS test solution 1ml, and boiling water bath 5 minutes is settled to 10ml with distilled water, measures absorbance in the 540nm wavelength.Enzyme is lived, and per hour to be defined as the needed enzyme amount of glucose that generates 1umol by substrate be the enzyme unit (U) that lives in unit.
Four, the beta-glucosidase bio-transformation of soybean isoflavones
This step can be crude enzyme liquid or the purified enzyme liquid of producing in the above-mentioned steps in order to the enzyme liquid that transforms.
1, the preparation of isoflavone genin
The ratio of raw material and beta-glucosidase is 1000～4000U/g, 40～50 ℃ of invert points, PH4.5～7.0, stirring velocity is 160～180r/min, transformation time 6～10hr obtains the conversion fluid of enzyme, again with this conversion fluid in 4～10 ℃, centrifugally under the condition of 5000～10000r/min carry out liquid-solid separation, the throw out cryodrying obtains isoflavone genin.
2, the processing of parting liquid
Conversion fluid carries out supernatant liquor after the liquid-solid separation and carries out alcohol precipitation with 75% edible ethanol and spend the night, at 4～10 ℃, centrifugal liquid-solid separation, the sedimentary beta-glucosidase of carrying out under 5000～10000r/min condition, use dissolved in distilled water, utilize again behind the mensuration enzyme activity; Reclaim ethanol in the supernatant liquor, obtain medicinal extract and get isoflavone genin, merge with for the first time liquid-solid isoflavone genin that obtains that separates in conversion back in 50～80 ℃ of drying under reduced pressure.
The present invention compared with prior art has following advantage:
1, not limited by material purity, at material content 2～20% o'clock, transformation efficiency still can reach more than 90%, and technical process is obviously shortened, and it is low to transform cost, and bioavailability improves several 10 times, can be directly as medicine, healthy food material;
2, it is residual to utilize the Bata-glucuroide of bent 3042 fermentative production of rice to carry out the conversion organic solvent-free of soybean isoflavones, and technology is simple, strong operability, be produced on a large scale;
3, the substratum composition is simple, and culture condition is easy to control, transforms used enzyme and ethanol and all can repeat to recycle non-environmental-pollution;
4, improve the bioavailability and the biological activity of soybean isoflavones, filled up high activity soy bean isoflavones preparation technology's blank.
With specific embodiment method of the present invention is described below, the embodiment in the specific embodiments of the invention can not constitute any restriction to protection domain of the present invention.
Accompanying drawing is a process flow diagram of the present invention.
Embodiment 1:
Raw material: the soybean ketone flavones meal (Tianjin spike natural product company limited product) that adopts content 10%
Bacterial classification: institute is brewageed in aspergillus oryzae (Aspergillus oryzae) 3042 Shanghai
The strain inclined plane medium preparation method that goes down to posterity: PDA medium preparation
1, the preparation of solid fermentation method beta-glucosidase and enzyme activity determination
(1) preparation of seed culture medium and spawn culture thereof:
Precision takes by weighing Zulkovsky starch 10.0g, glucose 5.0g, potassium primary phosphate 0.5g, sal epsom 0.5g, yeast extract paste 2.5g, saltpetre 1.0g is with dissolved in distilled water and be diluted to 500ml, PH6.5, divide to install in the 500ml triangular flask every bottle of 80ml, 121 ℃ of sterilization 15min.Aseptic technique, the spore of rice bent 3042 is transferred in the seed culture medium on an amount of fresh inclined-plane of picking, cultivates 42h in 27 ℃, 160r/min concussion.Promptly obtain the needed liquid spawn of solid fermentation.(note: above needed chemical reagent is analytical pure)
(2) preparation of solid fermentation substratum and culture of strains thereof
Take by weighing wheat bran 1000g, according to wheat bran: the ratio of water 1: 2 (w/v) is configured the solid fermentation substratum, PH nature, 121 ℃ of sterilization 30min.Aseptic technique inserts the liquid spawn for preparing in the solid medium with 5% inoculum size, and in the culture tank of packing into behind the mixing, the thickness of substratum is about 3cm, fermentation culture in aseptic proving room.Culture temperature is 27 ℃, and humidity remains on 65-70%, and incubation time is 84 hours.
(3) beta-glucoside enzyme extraction and preliminary purification
With the ratio adding distilled water of above-mentioned solid fermentation thing according to 1: 10, stir, under 4 ℃ of condition conditions, soaked 4 hours, centrifugal under the 5000r/min condition at 4 ℃, collect supernatant liquor and be the beta-glucosidase crude enzyme liquid.
Get crude enzyme liquid 500ml, slowly add edible ethanol, make ethanol concn reach that alcohol precipitation spends the night under 75%, 4 ℃ of condition, centrifugal under 10000r/min, the 4 ℃ of conditions, collecting precipitation, with the 500ml dissolved in distilled water or under-40 ℃ condition lyophilize preserve.Enzyme liquid with preliminary purification passes through the absorption of DEAE-sephadexA-50 ion exchange resin, sodium chloride solution gradient elution, dialysis, concentrates the bent 3042 beta-glucoside enzyme molecular weights of back SDS cataphoretic determination rice with 12% at 45kb.
(4) beta-glucosidase vigour-testing method
A) making of glucose typical curve
Adopt spectrophotometry.Get 8 and have graduated colorimetric cylinder, the accurate glucose standard of drawing is used liquid (1mg/ml) 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4ml; Supply 2ml with distilled water; Add the 1.5mlDNS test solution respectively, boiling water bath 5min is settled to 10ml with distilled water, in the 540nm colorimetric, and the drawing standard curve.
B) mensuration of beta-glucoside enzyme activity in the sample
Get 4 and have graduated colorimetric cylinder, add 0.5% saligenin 0.5ml respectively, control tube adds DNS test solution 1ml, and sample hose and control tube are placed on water-bath 5min under 50 ℃ of environment simultaneously; Each pipe adds enzyme liquid 0.5ml respectively, and behind 50 ℃ of water-bath 30min, sample hose adds DNS test solution 1ml, and boiling water bath 5 minutes is settled to 10ml with distilled water, measures absorbance in the 540nm wavelength.Enzyme is lived, and per hour to be defined as the needed enzyme amount of glucose that generates 1umol by substrate be the enzyme unit (U) that lives in unit.
The average enzyme activity for preparing 5 batches beta-glucosidase crude enzyme liquid according to the method in the case study on implementation is 1201U/ml, and the average enzyme activity behind the preliminary purification is 4750U/ml.
2, the preparation of isoflavone genin
The aforesaid crude enzyme liquid of enzyme liquid that hydrolysis is used or the enzyme of preliminary purification raw materials usedly are that daidzin content was 6.42% before 10% soybean isoflavones (Huabei Pharmaceutic Co., Ltd) transformed, Genistoside content is 2.02%, daidzin 2.24%, daidzein 0.46%, lignin 0.03%, glycitein 0.10%.
(1) takes by weighing 10% soybean isoflavones 100g, ratio according to substrate and enzyme 2250U/g adds crude enzyme liquid 187.3ml, adds distilled water to 1000ml (concentration of substrate is 0.1g/ml), the PH nature, under 180 rev/mins of conditions of 45 ℃ of stirring velocitys of temperature, transform 8 hours, termination reaction, with enzymatic conversion liquid at 10000r/min, centrifugal under 4 ℃ of conditions, collecting precipitation is in 70 ℃, drying under reduced pressure under the-0.1Mpa condition, the soybean isoflavones powder 80.1g after can obtaining after the pulverizing transforming.
(2) in " (1) " step, slowly add edible ethanol to 75% in the supernatant liquor of centrifugal collection, alcohol precipitation spends the night under 4 ℃ of conditions, centrifugal under 10000rmin, the 4 ℃ of conditions, collect supernatant liquor and reclaim ethanol, the lotion that obtains is with above-mentioned condition drying under reduced pressure, the soybean isoflavones powder 10.4g after obtaining after the pulverizing transforming.
(3) product that obtains for twice is merged soybean isoflavones powder 90.5g after obtaining altogether transforming, measure main active ingredient daidzin 0.347%, Genistoside 0.094%, daidzein 4.667%, genistein 1.227% with the HPLC method; The daidzin transformation efficiency is 95.1%, Genistoside transformation efficiency 95.8%.
3, the recycling of beta-glucosidase
Partly use the crude enzyme liquid that obtains in (3) step in an amount of dissolved in distilled water and the 1st operation to merge use the precipitation that centrifugal back in " (the 2) " step in this preparation section obtains.
Embodiment 2:
1,5% soybean isoflavones powder hydrolysis prepares isoflavone genin
The used enzyme liquid of hydrolysis is with 10% soybean isoflavones powder hydrolysising experiment: daidzin content was 3.37% before raw materials used 5% soybean isoflavones (the defatted soy flour allotment that 10% the soybean isoflavones powder of producing with Huabei Pharmaceutic Co., Ltd adds the extraction soybean isoflavones after drying of 1 times of amount forms) transformed, Genistoside content is 1.12%, daidzin 1.21%, daidzein 0.25%, genistein 0.02%, glycitein 0.04%.
(1) takes by weighing 5% soybean isoflavones 100g, ratio according to substrate and enzyme 2300U/g adds crude enzyme liquid 192ml, add distilled water to 1000 (concentration of substrate is 0.1g/ml), the PH nature under 180 rev/mins of conditions of 45 ℃ of stirring velocitys of temperature, transforms 8 hours, termination reaction, at 10000r/min, centrifugal under 4 ℃ of conditions, collecting precipitation is in 70 ℃ with enzymatic conversion liquid.Drying under reduced pressure under the-0.1Mpa condition, the soybean isoflavones powder 80.6g after can obtaining after the pulverizing transforming.
(2) in " (1) " step, slowly add edible ethanol in the centrifugal collection supernatant liquor, make ethanol concn to 75%, alcohol precipitation spends the night under 4 ℃ of conditions, centrifugal under 10000rmin, the 4 ℃ of conditions, collect supernatant liquor and reclaim ethanol, the lotion that obtains is with above-mentioned condition drying under reduced pressure, the soybean isoflavones powder 9.7g after obtaining after the pulverizing transforming.
(3) product that obtains for twice is merged soybean isoflavones powder 91.3g after obtaining altogether transforming, measure main active ingredient daidzin 0.236%, Genistoside 0.071%, daidzein 2.112%, genistein 0.731% with the HPLC method; The daidzin transformation efficiency is 93.6%, Genistoside transformation efficiency 94.2%.
2, the recycling of beta-glucosidase
Identical with the recycling step in the 10% soybean isoflavones hydrolysising experiment among the embodiment 1.
Embodiment 3:
1,20% soybean isoflavones powder hydrolysis prepares isoflavone genin
The used enzyme liquid of hydrolysis is with 10% soybean isoflavones powder hydrolysising experiment: raw materials usedly be that daidzin content in the 20% soybean isoflavones powder (Tianjin spike natural product company limited) is 7.98%, Genistoside content is 9.51%, daidzin 2.62%, daidzein 0.72%, genistein 0.61%, glycitein 0.19%.
(1) takes by weighing 20% soybean isoflavones 100g, ratio according to substrate and enzyme 3000U/g adds crude enzyme liquid 250ml, add distilled water to 1000 (concentration of substrate is 0.1g/ml), the PH nature under 180 rev/mins of conditions of 45 ℃ of stirring velocitys of temperature, transforms 8 hours, termination reaction, at 10000r/min, centrifugal under 4 ℃ of conditions, collecting precipitation is in 70 ℃ with enzymatic conversion liquid.Drying under reduced pressure under the-0.1Mpa condition, the soybean isoflavones powder 82.3g after can obtaining after the pulverizing transforming.
(2) in " (1) " step, slowly add edible ethanol in the supernatant liquor of centrifugal collection, make ethanol concn to 75%, alcohol precipitation spends the night under 4 ℃ of conditions, centrifugal under 10000rmin, the 4 ℃ of conditions, collect supernatant liquor and reclaim ethanol, the lotion that obtains is with above-mentioned condition drying under reduced pressure, the soybean isoflavones powder 10.3g after obtaining after the pulverizing transforming.
(3) product that obtains for twice is merged soybean isoflavones powder 92.6g after obtaining altogether transforming, measure main active ingredient daidzin 0.370%, Genistoside 0.359%, daidzein 5.874%, genistein 5.709% with the HPLC method; The daidzin transformation efficiency is 95.7%, Genistoside transformation efficiency 96.5%.
2, the recycling of beta-glucosidase
Identical with the recycling step in the 10% soybean isoflavones hydrolysising experiment.
Claims (7)
1, microbial enzyme method prepares the method for isoflavone genin, be to be raw material with the soybean isoflavones powder, with cultivating aspergillus oryzae fermentative preparation beta-glucosidase, the soybean isoflavones glycosides is converted into isoflavone genin, it is characterized in that this preparation method may further comprise the steps:
A) described raw material is 2～90% soybean isoflavones powder;
B) Shai Xuan bacterial classification is an aspergillus oryzae (Aspergillus oryzae) 3042, with this strain preparation seed culture fluid;
C) prepare the solid fermentation substratum, obtain containing the fermented product of beta-glucosidase;
D) the soybean isoflavones powder is joined the conversion fluid of producing enzyme in the beta-glucoside enzyme solution of fermented extracted, carry out centrifugal solid-liquid again and separate, the throw out cryodrying obtains isoflavone genin.
2, in accordance with the method for claim 1, the ratio that it is characterized in that raw material and beta-glucosidase is 1000～4000U/g, 40～50 ℃ of invert points, PH4.5～7.0, stirring velocity are 160～180r/min, transformation time 6～10hr, obtain the conversion fluid of enzyme, again with this conversion fluid in 4～10 ℃, centrifugally under the condition of 5000～10000r/min carry out liquid-solid separation, the throw out cryodrying obtains isoflavone genin.
3, in accordance with the method for claim 2, it is characterized in that conversion fluid carries out supernatant liquor after the liquid-solid separation and carries out the alcohol precipitation with 75% edible ethanol and spend the night, at 4～10 ℃, centrifugally under 5000～10000r/min condition carry out liquid-solid separation, sedimentary beta-glucosidase, use dissolved in distilled water, utilize again behind the mensuration enzyme activity; Reclaim ethanol in the supernatant liquor, obtain medicinal extract and get isoflavone genin in 50～80 ℃ of drying under reduced pressure.
4, in accordance with the method for claim 1, it is as follows to it is characterized in that solid fermentation method prepares the step that contains beta-glucosidase:
A) preparation of seed culture fluid and cultural method: Zulkovsky starch 2%, glucose 1%, potassium primary phosphate 0.1%, sal epsom 0.1%, yeast extract paste 0.5%, SODIUMNITRATE 0.2%, the PH nature is in 121 ℃ of sterilization 15min; Spore on an amount of inclined-plane of picking is transferred in the seed culture medium, in 20～28 ℃, 160～180r/min concussion cultivation, 38～48hr;
B) preparation of solid fermentation substratum and cultural method: with wheat bran: water cooperates according to 1: 1.5～3.0 weight ratio, the PH nature, 121 ℃ of sterilization 30min, inoculum size with 1～7% inserts fermentation culture in the solid medium with the seed for preparing, cultivate 20～28 ℃, humidity 60～70%, incubation time 60～84hr obtains containing the solid fermentation thing of beta-glucosidase;
C) extraction of beta-glucosidase crude enzyme liquid: the distilled water that above-mentioned solid fermentation thing is added 10 times of amounts, stir and soak 2～6hr, in 4～10 ℃, centrifugally under 5000～10000r/min condition carry out liquid-solid separation, the gained supernatant liquor is the beta-glucosidase crude enzyme liquid.
5, in accordance with the method for claim 4, it is characterized in that described c) the preliminary purification method of crude enzyme liquid in the step is: with crude enzyme liquid with 75% ethanol, carrying out alcohol precipitation under 4～10 ℃ spends the night, under this temperature with 5000～10000r/min centrifugation, throw out obtains the beta-glucosidase liquid of preliminary purification with dissolved in distilled water, or at-40～-50 ℃ of following lyophilizes acquisition solid beta-glucosidases, behind the preliminary purification, use the DEAE-sephadexA-50 ion-exchange resin purification, use its molecular weight of SDS cataphoretic determination at 45kb.
6, in accordance with the method for claim 1, the purity that it is characterized in that raw soybeans isoflavones powder selects 5～20% for use.
7,, it is characterized in that the beta-glucoside enzyme solution in order to transform is crude enzyme liquid or purified enzyme liquid according to claim 1 or 4 described methods.
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