CN102703408A - Method for extracting glutamic acid decarboxylase from banana peel and method for producing r-reanal - Google Patents

Method for extracting glutamic acid decarboxylase from banana peel and method for producing r-reanal Download PDF

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CN102703408A
CN102703408A CN2012102152353A CN201210215235A CN102703408A CN 102703408 A CN102703408 A CN 102703408A CN 2012102152353 A CN2012102152353 A CN 2012102152353A CN 201210215235 A CN201210215235 A CN 201210215235A CN 102703408 A CN102703408 A CN 102703408A
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gad
gaba
glutamic acid
concentration
pericarpium musae
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缪冶炼
王琲
陈玲
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Nanjing Tech University
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Nanjing Tech University
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Abstract

The invention relates to a method for extracting glutamic acid decarboxylase from a banana peel, which comprises the steps of smashing the banana peel to paste to separate fiber and protein, wherein the length of fiber does not exceed 2mm; adding a phosphate buffer which is 5.0-7.0 in pH and contains 0.01-0.2g/L vitamin B6, shaking uniformly and standing and extracting for 1-5h, wherein each kilogram of banana peel corresponds to 2-7 liters of phosphate buffer; centrifugalizing at 6000-10000rpm for 10-50min to obtain a GAD (Glutamic Acid Decarboxylase) enzyme solution. The invention further provides a method for producing r-reanal by extracting glutamic acid decarboxylase from the banana peel. According to the invention, the waste in banana processing industry can be effectively used, the GAD and GABA (r-reanal) production costs are reduced, and the method can be used to produce GABA in a large scale.

Description

The method of from Pericarpium Musae, extracting L-Glutamic decarboxylase and producing γ-An Jidingsuan
Technical field
The invention belongs to food function composition production technique field, relate to a kind of method of from Pericarpium Musae, extracting L-Glutamic decarboxylase and producing γ-An Jidingsuan.
Background technology
(GABA has another name called 4-propalanine, aminobutyric acid and croak pyridine acid to γ-An Jidingsuan, and molecular formula is HOOCCH 2CH 2CH 2NH 2Molecular weight is 103.12); It is a kind of functional amino of nonprotein; Extensively be present in nature; Be the important inhibitory nerve mediator in the mammalian central nervous system, have the important physical function, blood pressure regulation and heart rate, blood pressure regulation, trophic nerve cell are arranged, treat epilepsy, impel ataraxy, promote the brain blood flow, promote the brain vigor, promote growth hormone secretion, assisting therapy asthma, activation hepatic and renal function, prevention of obesity, promotion alcohol metabolism, improve multiple efficacies such as climacteric syndrome.
The content of GABA is few in the natural food materials, and the content of GABA is generally 0.13～32.15 μ molg in the higher plant tissue -1, the GABA content in the 100 g brown rice is merely 3.8 mg, for needs improve healthy crowd, is nowhere near.For example, GABA for climacterium obstacle and first old phase mental disorder improve effect, competence exertion comes out in the time of need reaching 26 mg/ days intake.Therefore, let people can absorb enough GABA and come preventing disease, improve health, must work out can be simply, fast and low cost method produce more GABA.
At present, the working method of GABA has chemical synthesis, microbe fermentation method and concentration method.In the chemical synthesis, GABA is made through calcium hydroxide and bicarbonate of ammonia hydrolysis by pyrrolidone.The reaction of chemical synthesis is very fast, yield is high, but reaction conditions is comparatively harsh, and energy consumption is bigger, and objectionable constituent are easy residual, and application difficulty is bigger on food.Microbe fermentation method promptly utilizes the L-Glutamic decarboxylase (GAD) of microorganisms producing that substrate glutamic acid or Sodium Glutamate are changed into GABA.Microbe fermentation method content is higher, can be used safely in food, but it is long also to have a production cycle, and transformation efficiency is lower, and the downstream separation difficulty is bigger, the more high shortcoming of full-scale plant cost.Enrichment ratio juris and microbe fermentation method are similar, are about to L-glutamic acid or Sodium Glutamate and generate GABA through L-Glutamic decarboxylase catalysis, comprise single-material nature concentration method, add substrate single-material concentration method and many materials concentration method.
Single-material nature concentration method is exactly through changing some culture condition, the GAD endogenous enzyme in the active material, enrichment GABA.Chinese patent CN1939146A discloses a kind of method of enriching gamma-aminobutyric aid from rice germ.Being processed into the rice germ that is produced in the rice process with rice is raw material; Raw material carries out skimming treatment earlier; Place it in again and soaked 4-5 hour in 35-40 ℃ the water and control pH value, obtain the product of alpha-aminobutyric acid content through sterilization, dry, expanded, pulverizing again at 300-400mg/100g at 5.0-5.5.Chinese patent CN1939144A discloses and a kind of paddy has been processed brown rice earlier, carries out germination treatment again, carries out the enrichment method for processing again, makes alpha-aminobutyric acid content reach 500-900mg/100g even higher.It is raw material with long-grained nonglutinous rice or japonica rice brown rice that Chinese patent CN101352216B discloses a kind of; Through the method that is rich in γ-An Jidingsuan, functional Nutritive Rice sheet or ground rice that cleaning and sterilizing, immersion, germination, cleaning making beating, insulation conversion, roller drying or spraying drying etc. are processed, alpha-aminobutyric acid content is 1000-1400mg/100g.Chinese patent CN101273765A discloses a kind of preparation method who is rich in the γ-An Jidingsuan nutrient rice; With rice bran, rice or to crack rice be raw material; Wherein rice bran separates the batching powder that obtains being rich in γ-An Jidingsuan through enzymolysis; The rice or the process of cracking rice are pulverized and are obtained ground rice, and both are mixed according to a certain percentage, obtain the nutrient rice that alpha-aminobutyric acid content reaches 5%-8% through extruding again.Chinese patent CN1259854C discloses a kind of preparation technology of rice containing gamma amino-butyric acid, with the paddy paddy of hulling, removes husk and obtains brown rice; Add entry again, use ultrasonic cleaning; Activation under suitable temperature, sterilization destroys the kind skin of brown rice again through the method for enzymolysis, be drying to obtain finished product at last, and the alpha-aminobutyric acid content in the rice that makes is 30-300mg/100g.Japanese Patent Laid is opened 2004-159617 and is disclosed manufacture method that is rich in the GABA sprouted unpolished rice and the food that utilizes sprouted unpolished rice; Brown rice soaked 0.5～2 h in 35～45 ℃ of water after; Taking out the back keeps 1～8 h to make its germination under 35～45 ℃ of conditions; The GABA content of brown rice germ is 25～50 mg/100 g before the microbial reproduction problem that 8 h can avoid 12 h to germinate and occur with interior germinating time, enrichment, and GABA approximately brings up to 300～400 mg/100 g after the enrichment.Chinese patent CN100577031C discloses a kind of through the method for soaking, freezing and course of defrosting increases alpha-aminobutyric acid content in the soybean, and alpha-aminobutyric acid content is up to 1.9144mg/g-dry soybean.Japanese Patent Laid is opened 2006-345708 and is disclosed germinated soybean powder and manufacture method thereof; In 27～42 ℃ of water, soak 24 h; Make soybean germination; GABA content is increased to 142 mg/100 g-soybean from 0 in the soybean in this germination process, and total isoflavone content is increased to 305.2 mg/100 g from 69.5 mg/100 g, and soyasaponins content is increased to 470 mg/100 g from 310 mg/100 g.Single-material nature concentration method adopts natural single-material to carry out enrichment, and technical process is simple relatively, but substrate need rely on the L-glutamic acid of material itself, can cause reaction not exclusively, can't make full use of L-Glutamic decarboxylase.
Add substrate single-material concentration method and in material, add substrate glutamic acid (or Sodium Glutamate, Stimulina) exactly, carry out the GABA enrichment.Chinese patent CN100415119C discloses working method of a kind of GABA of being rich in coated mill rice and products thereof, brown rice is immersed add CaCl 2, Sodium Glutamate (or Stimulina), xitix (or lactic acid) and Y factor form in the water nutrient solution and cultivate 12～72h, the long 0.5～4.0mm of rice bud.Endogenous L-Glutamic decarboxylase is activated, and the L-glutamic acid of catalysis self and the Sodium Glutamate of interpolation or Stimulina make its enrichment GABA.After drying, pearling, obtain being rich in the coated mill rice of GABA.The high-content of GABA reaches 15mg/100g in the finished product.Chinese patent CN1248596C discloses a kind of preparation method and application of the GABA of being rich in germ meal: (1) vibrates the rice embryo under pH5.6,40 ℃ of conditions; Activate endogenous enzyme; Add calcium chloride 500～700 μ mol/L, the GABA content in the rice embryo rises to 615 mg/100 g from 28 mg/100 g of raw material; (2) adopt trypsin hydrolyzing rice embryo, the GABA in the dry rice embryo powder can reach 2.3 g/100 g; (3) in the mixed solution of rice embryo and pH5.6 phosphoric acid buffer, add Sodium Glutamate, after reaction 6 h under 40 ℃ of conditions, the transformation efficiency of Sodium Glutamate reaches 100%, and GABA output reaches 20.7 g/100 g rice embryos.It is the method for feedstock production high density γ-An Jidingsuan powder with the rice bran that Chinese patent CN100478447C discloses a kind of; With the rice bran is raw material, adds L-glutamic acid, calcium chloride and Vitazechs, 40 ℃ of temperature of reaction; PH5.6; Obtain being rich in the reaction solution of γ-An Jidingsuan under the condition of 6～8 h, then through spinning, ultrafiltration, concentrate and spraying drying after, obtain content and be 50% GABA powder.Chinese patent CN101273769B discloses a kind of preparation method of germinated brown rice with high-content gamma-aminobutyric acid, to be raw material when the fresh paddy of producing per year, through rice huller paddy, the rough separation of paddy and the selected brown rice that obtains; Brown rice obtains finished product through washing, sterilization, immersion, cultivation, sterilization and drying again.Adopt during cultivation and include phosphate buffered saline buffer 2-5mmol/L Sodium Glutamate, 0.05-0.1mmol/L calcium chloride and 0.01-0.05mmol/L Vitazechs, pH 5.6-5.8 as nutrient solution; Improve the activity of endogenous L-Glutamic decarboxylase; The contained L-glutamic acid and the Sodium Glutamate of interpolation are prepared the sprout brown rice that alpha-aminobutyric acid content is 200mg/100g in the catalytically decomposed brown rice.Chinese patent CN101294155A discloses the activation method for enzyme of L-Glutamic decarboxylase in a kind of rice bran; Through adding L-glutamic acid (Glu), Sodium Glutamate (Glu-Na), calcium chloride, Vitazechs (PLP), VB6; Utilize phosphoric acid buffer or xitix or lactic acid to regulate pH; Activate L-Glutamic decarboxylase at a certain temperature, obtain being rich in the reaction solution of GABA.Chinese patent CN1840672A discloses and a kind of L-glutamic acid is joined in the pumpkin and is the method for γ-An Jidingsuan through inherent enzymatic conversion in the pumpkin; The pumpkin that employing is handled without the salt marsh preservation; Preferred results back produces processed squash product at 7 ~ 14 days pumpkin of 8 ~ 12 ℃ of following subzero treatment, can obtain containing the processed squash product of 10% ~ 20% γ-An Jidingsuan.Japanese Patent Laid is opened 2008-245527 and is disclosed a kind of method of utilizing banana and L-glutamic acid or L-sodium glutamate to produce the GABA food materials; Wherein the addition of L-glutamic acid or L-sodium glutamate is 0.5%～10% of a banana edible part weight; Best pH is 4.5～6.5; Optimum temperature is 20～50 ℃, reaction time 30～120 min.Above-mentioned concentration method is to improve concentration of substrate through interpolation L-glutamic acid or Sodium Glutamate in food materials, increases GABA output.But, in food materials, have L-Glutamic decarboxylase (GAD) and GABA transaminase (GABA-T) simultaneously.On the one hand, L-glutamic acid or Sodium Glutamate generate GABA through decarboxylic reaction under the effect of GAD, and on the other hand, ammonia takes place to change under the effect of GABA-T for GABA and α-Tong Wuersuan.Therefore, the GABA production efficiency of interpolation substrate single-material concentration method is still not high.
In addition, multiple food materials concentration method adopts two kinds of differing materials to carry out the GABA enrichment.Chinese patent CN101810322A is disclosed a kind ofly will to be rich in the L-Glutamic decarboxylase food materials and to be rich in the L-glutamic acid food materials and to mix and carry out decarboxylic reaction, obtain being rich in the method for GABA food materials.This method can make full use of the activity of L-Glutamic decarboxylase, has also guaranteed the abundance of substrate glutamic acid, has also proposed the new approaches of GABA enriching method simultaneously.
No matter know from above-mentioned, in the production of GABA, be microbe fermentation method or plants enriched method, and L-Glutamic decarboxylase (GAD) has played critical effect.The GAD that is applied to GABA production at present mainly extracts from mikrobe.Chinese patent CN102120995A discloses a kind of immobilization L-Glutamic decarboxylase and preparation method thereof.In milk-acid bacteria, add acetic acid-acetate buffer, being mixed with every milliliter, to contain the bacterium number be the bacteria suspension of 300-500cfu, and ultrasonication under the ice-water bath condition obtains the crude enzyme liquid of L-Glutamic decarboxylase; Use napped cotton fabric to be carrier, carry out pre-treatment with sodium hydroxide, pyridine, Tosyl chloride successively; Place the L-Glutamic decarboxylase crude enzyme liquid to soak, in solution, add LUTARALDEHYDE then, realize the immobilization of L-Glutamic decarboxylase.But the objectionable constituent of should technology using are more, difficult separation the, difficult enforcement on food applications.Chinese patent CN100562581C discloses a kind of method of microorganisms producing γ-An Jidingsuan.The bacterial cell disruption that will contain L-Glutamic decarboxylase after the sodium-alginate immobilization, carries out enzymic catalytic reaction with immobilization L-Glutamic decarboxylase and L-glutamic acid or glutaminate in reaction column, obtain γ-An Jidingsuan.It is long that extraction GAD and Production by Microorganism Fermentation GABA equally also have the production cycle from microorganism cells, and the downstream separation difficulty is big, shortcomings such as production cost height.GAD compares with mikrobe, and food materials GAD more can guarantee food safety, and easy to operate, production cost is low.But, also from food materials, do not extract food grade GAD at present, and carry out the technology of GABA safety in production with it.
Summary of the invention
The purpose of this invention is to provide a kind of method of from Pericarpium Musae, extracting L-Glutamic decarboxylase, solve the problem that present Pericarpium Musae only abandons, is not utilized effectively as rubbish.
The present invention also provides a kind of use from Pericarpium Musae, to extract the method that L-Glutamic decarboxylase is produced γ-An Jidingsuan, and Pericarpium Musae is applied to GABA production, reduces the production cost of GABA, and guarantees the edible safety of GABA product.
The present invention realizes through following technical scheme: a kind of method of from Pericarpium Musae, extracting L-Glutamic decarboxylase, and this method may further comprise the steps:
(1) Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is no more than 2mm;
(2) to add pH be 5.0～7.0, contain the vitamins B of 0.01 ~ 0.2g/L 6Phosphoric acid buffer, shake up, leave standstill and extract 1～5h, wherein, the corresponding 2～7L phosphoric acid buffer of every 1kg Pericarpium Musae;
Centrifugal 10～the 50min of (3) 6000～10000rpm obtains GAD enzyme liquid.
Further preferably, a kind of method of from Pericarpium Musae, extracting L-Glutamic decarboxylase, this method may further comprise the steps:
(1) Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is no more than 2mm;
(2) to add pH be 5.5, contain the vitamins B of 0.1g/L 6Phosphoric acid buffer, shake up, leave standstill and extract 4h, wherein, the corresponding 5L phosphoric acid buffer of every 1kg Pericarpium Musae;
(3) the centrifugal 30min of 9000rpm obtains GAD enzyme liquid.
A kind of use extracted the method that L-Glutamic decarboxylase is produced γ-An Jidingsuan from Pericarpium Musae, also comprise step (4), is catalyzer with GAD enzyme liquid; With concentration is that the L-glutamic acid of 50～250mg/100mL is substrate; Produce GABA, wherein, the consumption of GAD is 1.5～15U/100mg-L-glutamic acid; Temperature of reaction is 37 ℃, and the reaction times is 5～60min.
Adopt the positively effect of technique scheme: starting material and reagent that (1) is used are food grade, can guarantee food safety; (2) effectively utilize the waste of banana secondary industry, reduce GAD and GABA production cost, help protecting environment simultaneously; (3) production technique is simple, and is easy to operate, and cost is lower, can be used for mass production GABA, satisfies people's various demands; (4) effectively utilize a large amount of GAD that contain in the Pericarpium Musae, solved the enrichment problem of GAD, can improve the output of GABA greatly.
Description of drawings
Fig. 1 is the relation of light absorption value A and GABA concentration C a.
Fig. 2 is influence (extraction time: 4h, the centrifugation time: 10min) of liquid material comparison enzyme liquid GAD concentration.
Fig. 3 is influence (extraction time: 4h, the centrifugation time: 10min) that the GAD vigor is extracted in the comparison of liquid material.
Fig. 4 is influence (the liquid material ratio: 5:1, centrifugation time: 10min) of extraction time to enzyme liquid GAD concentration.
Fig. 5 is influence (the liquid material ratio: 5:1, centrifugation time: 10min) of extraction time to extracting the GAD vigor.
Fig. 6 is influence (extraction time: 4h, the liquid material ratio: 5:1) of centrifugation time to enzyme liquid GAD concentration.
Fig. 7 is influence (extraction time: 4h, the liquid material ratio: 5:1) of centrifugation time to extracting the GAD vigor.
Fig. 8 is that GABA concentration is over time in the enzyme reaction.
Fig. 9 is the influence to final GABA concentration of GAD consumption and aminoglutaric acid concentration.
Figure 10 is the influence to L-glutamic acid conversion rate of GAD consumption and aminoglutaric acid concentration.
Embodiment
Through embodiment and Test Example technical scheme of the present invention is further specified below, but should not understand limitation of the present invention:
Experiment material among the present invention is that banana is produced in Hainan.This banana is bought banana wholesale department, north gate, Pukou District Zhujiang River town from Nanjing.
Embodiment 1
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is 2mm, is loaded in the triangular flask, adds 2L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 2
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is 2mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 3
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 2mm, is loaded in the triangular flask, adds 7L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 4
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 2mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 1h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 5
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.5mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 3h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 6
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.5mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 5h after, centrifugal 10min under the 9000rpm condition, crude enzyme liquid.
Embodiment 7
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.0mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 9000rpm condition, crude enzyme liquid.
Embodiment 8
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.0mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 50min under the 9000rpm condition, crude enzyme liquid.
Embodiment 9
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 2mm, is loaded in the triangular flask, adds 5L pH5.0, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 9000rpm condition, crude enzyme liquid.
Embodiment 10
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 2mm, is loaded in the triangular flask, adds 5L pH7.0, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 9000rpm condition, crude enzyme liquid.
Embodiment 11
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.5mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.01g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 9000rpm condition, crude enzyme liquid.
Embodiment 12
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.5mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.2g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 9000rpm condition, crude enzyme liquid.
Embodiment 13
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.0mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 6000rpm condition, crude enzyme liquid.
Embodiment 14
With soybean milk maker the 1kg Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is about 1.0mm, is loaded in the triangular flask, adds 5L pH5.5, contains the vitamins B of 0.1g/L 6Phosphoric acid buffer (Na 2HPO 4/ NaH 2PO 4), shake up, leave standstill extract 4h after, centrifugal 30min under the 10000rpm condition, crude enzyme liquid.
Embodiment 15
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 10U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 5min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 16
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 10U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 40min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 17
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 10U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 18
The glutamic acid solution of getting concentration and be 50mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 10U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 19
The glutamic acid solution of getting concentration and be 150mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 10U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 20
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 1.5U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 21
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 5U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Embodiment 22
The glutamic acid solution of getting concentration and be 250mg/100mL is a benchmark with the amount of glutamic acid in triangular flask, adds the GAD enzyme liquid of 15U/100mg-L-glutamic acid.Place 37 ℃ water-bath to react 60min triangular flask, boil the enzyme that goes out, GABA concentration in the colorimetric method for determining reaction solution.
Test Example 1The influence that liquid material comparison GAD extracts
Embodiment 1-3 is used to measure the influence that liquid material comparison GAD extracts.The GAD vigor of crude enzyme liquid is according to colorimetric method for determining.The L-glutamic acid 5mL that gets crude enzyme liquid 5mL and 10mmol/L mixes in test tube, in 37 degrees centigrade water-bath, is incubated 30min.Enzymolysis solution adopts spectrophotometric determination at the light absorption value at 630nm place.Being defined as of 1 GAD unit of activity, under the condition of optimum temperuture and pH, PM catalysis L-glutamic acid decarboxylic reaction produces the required enzyme amount of GABA of 1 μ mol.GAD vigor U in the Pericarpium Musae d(U/g-DM) calculation formula is following:
Figure 2012102152353100002DEST_PATH_IMAGE001
In the formula, U d: GAD vigor in the Pericarpium Musae, U/g-DM; C a: the GABA concentration of enzymolysis solution, μ mol/mL; V: enzymolysis solution volume, V=10mL; N: the ratio of crude enzyme liquid consumption in crude enzyme liquid TV and the enzyme digestion reaction; T: reaction times, min; m b: the quality of Pericarpium Musae, g; M b: the Pericarpium Musae thing benchmark water ratio that wets, %.
The making method of typical curve is following, and the GABA solution with the phosphoric acid buffer of pH5.5 preparation 20mmol/L is diluted to concentration 0,2,4,6,8 and 10mmol/L respectively with it again, makes reference liquid.The reference liquid of getting the 1mL different concns adds borate buffer 1mL, 9% Youxiaolin (reactive chlorine of Youxiaolin reagent is 5.26%) 1mL and 6% the phenol 5mL of pH9.0 respectively in test tube in test tube, test tube is placed boiling water bath 10min.Ethanol 1mL, the mixing of ice bath 20min, adding 60% in test tube are surveyed light absorption value A at wavelength 630nm place.Fig. 1 representes light absorption value A and GABA concentration C aRelation.
Light absorption value A and GABA concentration C aThe available following formula of relation represent:
Figure 839337DEST_PATH_IMAGE002
（R 2=0.996）
In the formula, A: light absorption value; C a: GABA concentration, mmol/L.
Fig. 2 and Fig. 3 represent the influence of liquid material comparison enzyme liquid GAD concentration and Pericarpium Musae GAD vigor respectively.When liquid material ratio was 5, enzyme liquid GAD concentration and Pericarpium Musae GAD vigor reached peak, are respectively 0.21U/mL, 13.04U/g.When solid-liquid ratio continues to increase, enzyme liquid GAD density loss, Pericarpium Musae GAD vigor then remains unchanged basically.Its reason possibly be, when low, the sugared concentration in the enzyme liquid is higher at the liquid material, and the viscosity of liquid is too big, does not utilize the extraction of enzyme; When the liquid material than greater than 5 the time, zymoprotein concentration begins to descend, and causes that GAD reduces in the 1mL enzyme liquid, and total Pericarpium Musae GAD vigor is constant.Therefore, this experiment is set in 5 with liquid material ratio.The righttest solid-liquid ratio when extracting GAD in the banana pulp is 2.5, possibly be because Pericarpium Musae and banana pulp all exist than big-difference in each side such as the content of enzyme, physical behavior, chemical ingredients compositions, so the righttest solid-liquid ratio also there are differences.
Test Example 2The influence that extraction time extracts GAD
Embodiment 2,4 ~ 6 is used to measure the influence that extraction time extracts GAD.Can know that by Fig. 4 and Fig. 5 the GAD vigor improves along with the prolongation of time, reach the highest to 4h, be 0.17 U/mL, and the GAD vigor descends afterwards.Its reason maybe for, the time is too short, the enzyme stripping is incomplete, along with the increase of time, the enzyme stripping quantity also increases, enzyme live to improve; Lixiviate reaches after the certain hour; The complete stripping of enzyme; No longer increase; But impurity such as the protein of stripping, proteolytic enzyme, polysaccharide are also many more in the vat liquor, and microbiological contamination also possibly bring the hydrolysis of proteolytic enzyme to make the change that reduces the physico-chemical property that also has enzyme itself alive of total enzyme, finally causes enzyme to be lived and reduces.Also there is same phenomenon to take place in the catalatic extraction of pork liver.
Test Example 3The influence that centrifugation time extracts GAD
Embodiment 2,7,8 is used to measure the influence that centrifugation time extracts GAD.Fig. 6 and Fig. 7 represent that centrifugation time is to crude enzyme liquid GAD concentration and the influence of extracting the GAD vigor.Crude enzyme liquid GAD vigor is the back downward trend that rises earlier with centrifugation time, and when centrifugation time was 30min, it was 0.83U/mL that crude enzyme liquid GAD vigor reaches peak.When centrifugation time more in short-term, impurity such as Mierocrystalline cellulose can't be removed fully, influence the contact of enzyme-to-substrate, and when centrifugation time surpassed 30min, crude enzyme liquid GAD was precipitated in centrifugal process, caused crude enzyme liquid GAD vigor to reduce.Therefore, can think comparatively suitable when centrifugation time is 30min.
Test Example 4GABA concentration over time
Embodiment 15 ~ 17 is used to measure GABA concentration over time.Press the ratio of 10U/100mg-L-glutamic acid, when adding GAD reacted in glutamic acid solution, GABA concentration was as shown in Figure 8 with the variation in reaction times.In the reaction GABA concentration in time increase and constantly rise.GABA concentration in the glutamic acid solution is increased to 40.1mg/100mL when reaction 40min, keep certain behind the 40min.For guaranteeing sufficient reacting, this experiment will be set in 60min in the reaction times.
Test Example 5GAD consumption and aminoglutaric acid concentration are to the influence of final GABA concentration
Embodiment 17 ~ 22 is used to measure the influence to final GABA concentration of GAD consumption and aminoglutaric acid concentration.By 2.5,5.0, the ratio of 10U/100mg-L-glutamic acid adds GAD when reacting in glutamic acid solution, GAD consumption and concentration of substrate are as shown in Figure 9 to the influence of final GABA concentration.In this scope of experiment, final GABA concentration increases with the increase of concentration of substrate and enzyme dosage.When concentration of substrate is that 250mg/100mL, GAD consumption are respectively 2.5,5.0 and during 10U/100mg-L-glutamic acid, final GABA concentration is 97,123 and 150mg/100mL.
Test Example 6GAD consumption and aminoglutaric acid concentration are to the influence of L-glutamic acid conversion rate
Embodiment 17,20 ~ 22 is used to measure the influence to L-glutamic acid conversion rate of GAD consumption and aminoglutaric acid concentration.By the final GABA concentration among Fig. 9, can calculate the L-glutamic acid conversion rate when being raw material with the glutamic acid solution, shown in figure 10.L-glutamic acid conversion rate increases with the increase of GAD consumption, but reduces with the increase of concentration of substrate.When concentration of substrate is that 50mg/100mL, GAD consumption are respectively 2.5,5 and during 10U/100mg-L-glutamic acid, L-glutamic acid conversion rate reaches 93.5%, 99.3% and 99.3% respectively; And when concentration of substrate increased to 250mg/100mL, L-glutamic acid conversion rate was reduced to 55.3%, 70% and 87.1% respectively.
When the GAD consumption is a 2.5U/100mg-L-glutamic acid, when concentration of substrate increased to 250mg/100mL from 50mg/100mL, L-glutamic acid conversion rate reduced about 40%.And work as the GAD consumption is 10U/100mg-L-glutamic acid, and when concentration of substrate increased to 250mg/100mL from 50mg/100mL, L-glutamic acid conversion rate only reduced about 12%.

Claims (3)

1. method of from Pericarpium Musae, extracting L-Glutamic decarboxylase, it is characterized in that: this method may further comprise the steps:
(1) Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is no more than 2mm;
(2) to add pH be 5.0～7.0, contain the vitamins B of 0.01 ~ 0.2g/L 6Phosphoric acid buffer, shake up, leave standstill and extract 1～5h, wherein, the corresponding 2～7L phosphoric acid buffer of every 1kg Pericarpium Musae;
Centrifugal 10～the 50min of (3) 6000～10000rpm obtains GAD enzyme liquid.
2. a kind of method that L-Glutamic decarboxylase is produced γ-An Jidingsuan of from Pericarpium Musae, extracting according to claim 1, it is characterized in that: this method may further comprise the steps:
(1) Pericarpium Musae is smashed to being the mud shape, made fiber and albumen sepn, staple length is no more than 2mm;
(2) to add pH be 5.5, contain the vitamins B of 0.1g/L 6Phosphoric acid buffer, shake up, leave standstill and extract 4h, wherein, the corresponding 5L phosphoric acid buffer of every 1kg Pericarpium Musae;
(3) the centrifugal 30min of 9000rpm obtains GAD enzyme liquid.
3. the use GAD enzyme liquid according to claim 1 or claim 2 method of producing γ-An Jidingsuan; It is characterized in that also comprising step (4): with GAD enzyme liquid is catalyzer, is that the L-glutamic acid of 50～250mg/100mL is substrate with concentration, produces GABA; Wherein, The consumption of GAD is 1.5～15U/100mg-L-glutamic acid, and temperature of reaction is 37 ℃, and the reaction times is 5～60min.
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