CN101323849B - Preparation of liquid beta-diastase - Google Patents
Preparation of liquid beta-diastase Download PDFInfo
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- CN101323849B CN101323849B CN2008100231941A CN200810023194A CN101323849B CN 101323849 B CN101323849 B CN 101323849B CN 2008100231941 A CN2008100231941 A CN 2008100231941A CN 200810023194 A CN200810023194 A CN 200810023194A CN 101323849 B CN101323849 B CN 101323849B
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Abstract
The invention relates to a preparation method of a liquid Beta-diastase, which is characterized by taking wheat bran as raw material. The preparation method comprises the following technological steps: the extraction of the wheat bran, solid-liquid separation, the fine filtration of an extract, ultra-filtration concentration, prescription, secondary fine filtration and the adjustment of enzyme activity. The preparation method adopts a preservative with proper matching and proper temperature and Ph so as to effectively inhibit bacterial growth and avoid putrefaction and enzyme deactivation; the obtained products have low content of impurity, contains low concentrate of industrial salt compared with that of a solid enzyme preparation and meets the requirements of General Standard for Food Additives easily. The preparation method of the invention is a brand new preparation technology of the liquid Beta-diastase and has the advantages of simple technology, high production efficiency, low production cost and being suitable for industrialized production.
Description
Technical field
The present invention relates to the preparation method of zymin, be specifically related to utilize plant to prepare the preparation method of liquid beta-diastase.
Background technology
Beta-amylase (claim α-1 again, 4-D dextran maltose lytic enzyme E.C.3.2.1.2) is a kind of circumscribed-type amylolytic enzyme, and it can be from the non reducing end of starch, catalytically hydrolyzing alpha-1 successively, and the 4-D glucoside bond generates maltose.Meanwhile, Walden translocation reaction (Waldeninversion) takes place, make product become β-type by α-type, so claim beta-amylase.
Since beta-amylase can not the hydrolysis amylopectin α-1,6 keys, can not stride across tapping point α-1,6 keys and cut inner α-1,4 keys and α-1, near 2～3 α-1,4 keys 6 keys are so often hang with the β-limit dextrin of 2～3 glucosyl residues outside residual downside props up in hydrolysate.When even beta-amylase acts on amylose starch, also can only make 70%～90% of starch be hydrolyzed into maltose, other is trisaccharide maltose and oligosaccharides.Beta-amylase industrial be a kind of important enzyme, it can change into maltose to starchy material, the different maltose product of content can be used for as foodstuffs industry such as candy, beer fermentations, and the also alternative glucose infusion liquid of highly purified Fructus Hordei Germinatus liquid glucose is used for the diabetics.
In currently reported production technology, the commodity beta-amylase of useful Production by Microorganism Fermentation, this enzyme heat-resistant stability can be poor, except that producing maltose, also produce the trisaccharide maltose of a great deal of, and the plant beta-amylase heat-resistant stability is good, and the action pH scope is wide, can produce more maltose.But the existing beta-amylase that extracts from plant all is a solid phase prod, be raw product foreign matter content height, can not be as foodstuff additive (seeing the preparation technology of patent publication No. CN1225943A soya-beta amylase), and in preparation process, adopt the technology of saltouing, sodium sulfate reclaims difficulty, has the liquid waste disposal problem.Multigelation makes complicated operation, the cost height.And the patent method of beta-amylase " extract " (seeing patent publication No. CN1491279A) only provides the employing cellulase to extract beta-amylase from barley or wheat, but cellulase price height, production cost height.Up to the present, owing to lack effective preparation technology, the commercial scale production of still being unrealized of the liquid plant beta-amylase preparation of high enzymatic activity.
Summary of the invention
Have problems such as production cost height, product foreign matter content height and liquid waste disposal at above-mentioned existing beta-amylase preparation method, the applicant provides a kind of preparation method of liquid beta-diastase.
Technical scheme of the present invention is as follows:
The preparation method of liquid beta-diastase, its processing step comprises:
With the wheat bran is raw material, and its processing step comprises lixiviate, solid-liquid separation, the essence filter of vat liquor, ultrafiltration and concentration, prescription, the smart filter of secondary, the enzyme activity adjustment of wheat bran, and division is as follows:
Lixiviate:
(1) preparation lixiviate water: in water, add sanitas ethyl p-hydroxybenzoate 0.01～0.03%, potassium sorbate 0.1～0.25%, Sodium Benzoate 0.2～0.5%, reductive agent Sodium Pyrosulfite 0.5～1.5%, Sodium Hydrosulphite 0.5～1.5% by (weight/volume);
(2) lixiviate: the lixiviate water that wheat bran is added step (1) preparation by (weight/volume) 1: 4～7; Under the normal pressure, extraction temperature is controlled at 40～55 ℃, and pH is controlled at 4.5～6.5, lixiviate 6～8 hours;
Solid-liquid separation: carry out solid-liquid separation after the lixiviate.
Smart filter: isolated vat liquor is as cold as rapidly below 20 ℃, and isolated vat liquor adds the diatomite of 1～2% (weight/volume), crosses smart filter plate frame and carries out the essence filter;
Ultrafiltration and concentration: the smart filtrate of gained is carried out ultrafiltration and concentration, and the temperature of ultrafiltration and concentration is controlled at below 20 ℃;
Prescription: ultrafiltration and concentration is after the enzyme activity requirement of required specification, gained ultrafiltration and concentration liquid is put into the prescription jar, add the sodium-chlor, 8～10% sodium-acetate, 5～8% maltodextrins of stablizer 18～22%, 0.1～0.25% potassium sorbate, 0.2～0.5% Sodium Benzoate by (weight/volume), stirring and dissolving mixes;
The smart filter of secondary: add the diatomite of 1～2% (weight/volume) in the gained prescription liquid, cross smart filter plate frame and carry out the essence filter.
Enzyme activity is adjusted: collect the filtrate of the smart filter of secondary, sampling makes the product check, with diluent the enzyme activity of the smart filter of secondary filtrate is adjusted to 300000U/g or 600000U/g.
It is the sodium-chlor, 8～10% sodium-acetate, 5～8% maltodextrins, 0.1～0.25% potassium sorbate, the aqueous solution of 0.2～0.5% Sodium Benzoate that adds stablizer 18～22% by (weight/volume) that described enzyme activity is adjusted used diluent.
The liquid plant beta-amylase preparation of 300000U/g, the 600000U/g that produces according to above-mentioned preparation technology, product is brown liquid, free from extraneous odour, can dissolves each other with water, product Heat stability is good, catalytic activity are strong, and the most suitable operative temperature is between 55 ℃～60 ℃.When temperature surpassed 65 ℃, speed of response was accelerated, but inactivation is also accelerated.The most suitable action pH value scope of this product is between 5.0～6.8.Do not contain α-Dian Fenmei in this product.
Gained finished product of the present invention can reach following index: enzyme activity 300000U/g, 600000U/g, unit weight: 1.15～1.25g/ml, pH5.0～6.0, total number of bacterial colony≤10000cfu/ml.The vigor storage stability is high, below 25 ℃, preserves after 6 months enzyme activity still greater than 90%.
The total enzyme of the above technology rate of recovery 〉=74% of living, above-mentioned technology and index all are better than the zymin goods that obtain with other technology.The present invention is brand-new liquid beta-diastase preparation technology, and technology is simple, production efficiency is high, production cost is low, is suitable for suitability for industrialized production.
The present invention is a raw material with the wheat bran, and production cost is low; In the production process of liquid beta-diastase, because activity time is long, corruption easily causes the inactivation of enzyme, and the present invention adopts properly mixed sanitas and suitable temperature and pH, and effective bacteria growing inhibiting prevents the inactivation of corruption and enzyme; The present invention is a kind of beta-amylase preparation of liquid state, and Industrial Salt concentration contained for the solid zymin is low, easily reaches the food grade standard-required; In process of production, temperature only need be controlled at below 20 ℃, has reduced production energy consumption; The present invention adopts the step of smart filter-ultrafiltration and concentration-smart filter of secondary, and the product foreign matter content of gained is low, reaches the requirement of foodstuff additive standard; Wheat bran after the separation is capable of using as feed after drying, does not have useless solid thing and handles problems.
Description of drawings
Fig. 1 is a process flow sheet of the present invention;
Fig. 2 is the DNS semi-quantitative method canonical plotting that is used for enzyme activity determination.
Embodiment
The present invention will be further described by the following examples:
Embodiment 1:
1, lixiviate:
(1) preparation lixiviate water: add sanitas ethyl p-hydroxybenzoate 0.025Kg, potassium sorbate 0.625Kg, Sodium Benzoate 0.875Kg, reductive agent Sodium Pyrosulfite 1.25Kg, Sodium Hydrosulphite 3.75Kg in the 250L water, stirring and dissolving;
(2) lixiviate: take by weighing the 50Kg wheat bran and add in the lixiviate water of step (1) preparation; Under the normal pressure, warming-in-water to 45 ℃ insulation, transferring pH is 4.5, per hour stirs once lixiviate 6 hours;
2, solid-liquid separation: carry out solid-liquid separation with sheet frame or screw press after the lixiviate;
3, smart filter: isolated vat liquor is as cold as rapidly below 20 ℃, and vat liquor adds the diatomite of 3.75Kg, crosses smart filter plate frame and carries out the essence filter;
4, ultrafiltration and concentration: the smart filtrate of step 3 gained enters the ultrafiltration and concentration device and carries out ultrafiltration and concentration, and the temperature of ultrafiltration and concentration is controlled at below 20 ℃, and what ultrafiltration and concentration adopted is 4 inches rolled films, and the molecular weight that dams is 10K dalton;
5, prescription: ultrafiltration and concentration is emitted when the 390000U/g to enzyme activity, obtain about 6.14L ultrafiltration and concentration liquid, place the prescription jar, add sodium-chlor, the sodium-acetate of 491g, 307g maltodextrin, sanitas 6.14g potassium sorbate, the dissolving of 12.28g Sodium Benzoate of 1105g, stir.
6, the smart filter of secondary: add 79.8g diatomite in prescription liquid, filtration stirs.Get finished product 7.25Kg, enzyme activity 320000U/g, yield 77%.
7, enzyme activity adjustment: collect the filtrate of the smart filter of secondary, sampling makes the product check, records unit weight: 1.18g/ml, pH5.5, total number of bacterial colony≤10000cfu/ml.Carry out the enzyme activity adjustment with diluent, used diluent is the sodium-chlor, 8～10% sodium-acetate, 5～8% maltodextrins, 0.1～0.25% potassium sorbate, the aqueous solution of 0.2～0.5% Sodium Benzoate that adds stablizer 18～22% by (weight/volume), and enzyme activity is adjusted to 300000U/g.
Embodiment 2:
1, lixiviate:
(1) preparation lixiviate water: add sanitas ethyl p-hydroxybenzoate 1.4Kg, potassium sorbate 14Kg, Sodium Benzoate 14Kg, reductive agent Sodium Pyrosulfite 70Kg, Sodium Hydrosulphite 70Kg in the 7000L water, stirring and dissolving;
(2) lixiviate: take by weighing the 1000Kg wheat bran and add in the lixiviate water of step (1) preparation; Under the normal pressure, warming-in-water to 55 ℃ insulation, transferring pH is 5.0, per hour stirs once lixiviate 7 hours;
2, solid-liquid separation: carry out solid-liquid separation with sheet frame or screw press after the lixiviate;
3, smart filter: isolated vat liquor is as cold as rapidly below 20 ℃, and vat liquor adds the diatomite of 84Kg, crosses smart filter plate frame and carries out the essence filter;
4, ultrafiltration and concentration: the smart filtrate of step 3 gained is carried out ultrafiltration and concentration, and the temperature of ultrafiltration and concentration is controlled at below 20 ℃, and what ultrafiltration and concentration adopted is 8 inches rolled films, and the molecular weight that dams is 20K dalton;
5, prescription: ultrafiltration and concentration is emitted when the 780000U/g to enzyme activity, obtain 59.6L ultrafiltration and concentration liquid, place sodium-chlor, the sodium-acetate of 5.62kg, 4.1kg maltodextrin, the sanitas of prescription jar adding 12.49kg: 125g potassium sorbate, the dissolving of 219g Sodium Benzoate stir.
6, the smart filter of secondary: add 0.9kg diatomite in prescription liquid, filtration stirs.Get finished product 70.25kg, enzyme activity 632000U/g, yield 74%.
7, enzyme activity adjustment: collect the filtrate of the smart filter of secondary, sampling makes the product check, records unit weight: 1.17g/ml, pH5.6, total number of bacterial colony≤10000cfu/ml.Enzyme activity is adjusted to 600000U/g, and used diluent is identical with embodiment 1.
Embodiment 3:
1, lixiviate:
(1) preparation lixiviate water: add sanitas ethyl p-hydroxybenzoate 9Kg, potassium sorbate 30Kg, Sodium Benzoate 150Kg, reductive agent Sodium Pyrosulfite 450Kg, Sodium Hydrosulphite 150Kg in the 30000L water, stirring and dissolving;
(2) lixiviate: take by weighing the 5000Kg wheat bran and add in the lixiviate water of step (1) preparation; Under the normal pressure, warming-in-water to 50 ℃ insulation, transferring pH is 6.5, per hour stirs once lixiviate 8 hours;
2, solid-liquid separation: carry out solid-liquid separation with sheet frame or screw press after the lixiviate;
3, smart filter: isolated vat liquor is as cold as rapidly below 20 ℃, and vat liquor adds the diatomite of 84Kg, crosses smart filter plate frame and carries out the essence filter;
4, ultrafiltration and concentration: the smart filtrate of step 3 gained is carried out ultrafiltration and concentration, and the temperature of ultrafiltration and concentration is controlled at below 20 ℃, and what ultrafiltration and concentration adopted is 8 inches rolled films, and the molecular weight that dams is 60K dalton;
5, prescription: ultrafiltration and concentration is emitted when the 780000U/g to enzyme activity, obtain 309.03L ultrafiltration and concentration liquid, place the prescription jar, add sodium-chlor, the sodium-acetate of 30.9kg, the 24.72kg maltodextrin of 67.98kg, sanitas 618g potassium sorbate, the dissolving of 1.54kg Sodium Benzoate stir.
6, the smart filter of secondary: add 3.71kg diatomite in prescription liquid, filtration stirs.Get finished product 370.8kg, enzyme activity 615000U/g, yield 75.7%.
7, enzyme activity adjustment: collect the filtrate of the smart filter of secondary, sampling makes the product check, records unit weight: 1.20g/ml, pH5.4, total number of bacterial colony≤10000cfu/ml.Enzyme activity is adjusted to 600000U/g, and used diluent is identical with embodiment 1.
The product of embodiment 1-3 gained, its enzyme activity determination method is as follows:
The mensuration of beta-amylase enzyme activity according to the habitual measuring method of industry, takes the DNS semi-quantitative method to measure.
The mensuration of the total enzyme activity of beta-amylase in the wheat bran takes by weighing the 50g wheat bran and places beaker.Get 200ml water and add ethyl p-hydroxybenzoate 0.02g, potassium sorbate 0.2g, Sodium Benzoate 0.4g, reductive agent Sodium Pyrosulfite 1g, Sodium Hydrosulphite 1g stirring and dissolving are transferred pH4.5.Pour in the beaker of putting wheat bran and mix.A lixiviate 8 hours is per hour stirred in warming-in-water to 40 ℃ insulation.Filter paper filtering, filtrate is measured enzyme activity with the method that describes below.Enzyme activity is measured by this method and is obtained in this specification sheets.
Enzyme activity definition: at pH5.50, under the condition that temperature is 60 ℃, per hour to produce the required enzyme amount of 1mg maltose be an enzyme activity unit (u/g or u/ml) to hydrolysis 1.10% starch fluid.
Enzyme activity determination (DNS semi-quantitative method)
1 reagent
1.10.2M, the pH5.50 phosphoric acid buffer
First liquid: take by weighing Sodium phosphate dibasic (Na
2HPO
412H
2O) 53.65g is settled to 1000ml with dissolved in distilled water.
Second liquid: take by weighing SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
412H
2O) 27.8g is settled to 1000ml with dissolved in distilled water.
Get first liquid 6.5ml, second liquid 93.5ml mixes the back and proofreaies and correct pH5.50 with acidometer.
The 1.210%pH5.50 phosphoric acid buffer
Get above-mentioned 0.2M, pH5.50 phosphoric acid buffer 10ml adds 90ml distilled water, mix.
1.3DNS solution
Accurately take by weighing 3.5-dinitrosalicylic acid 1.0000g, phenol 0.2g, S-WAT 0.05g, sodium hydroxide 1g, Seignette salt 20g uses the distilled water heating for dissolving, cooling is settled to 100ml, stores in brown bottle, and placing can drawing standard curve (each preparation needs to draw) after a week.
1.41.1% starch damping fluid
Boil about 50ml distilled water, join in a small amount of dissolved in distilled water liquid of 1.1g starch, boil again to transparent, cooling,
Add 10ml, 0.2M, the pH5.50 phosphoric acid buffer is settled to 100ml.
2 instrument and equipments
2.1 0～100 ℃ of water bath with thermostatic control (precision ± 0.2 ℃)
2.2 stopwatch
2.325ml color-comparison tube
2.4 transfer pipet
2.5 volumetric flask
The line drawing of 3 typical curves (DNS semi-quantitative method)
Accurately take by weighing in (105～110 ℃) dried glucose 50mg, be settled to 50ml, be made into the 1mg/ml Glucose Liquid with dissolved in distilled water, boil 15min by table one operation sequential and boiling water, cooling adds 10.5ml distilled water, 722 type spectrophotometer 550nm colorimetrics.
Table one
Seeing Fig. 2, is ordinate zou with the OD value, and glucose mg number is an X-coordinate, and the drawing standard graphic representation (is calculated constant K (K=glucose mg/OD) with regression equation.Sample is found it and is equivalent to glucose mg number according to the OD value of being measured on typical curve, multiply by 1.9 times again, is maltose mg number.
4 determination steps
4.1 specimen preparation
Accurately take by weighing a certain amount of enzyme sample, dissolve constant volume, shake up with 10% phosphoric acid buffer.(about 20 units of every mg enzyme liquid are advisable, and the OD value is controlled between 0.2～0.4).
4.2 measure
A. accurately draw 1.4 solution 9.0ml,, add 1ml enzyme sample, the 30min of accurate timing reaction immediately in 60 ℃ of water bath with thermostatic control preheating 5min.
B. draw the 0.5ml reaction solution rapidly in the 25ml color-comparison tube that sucks DNS reagent 1.50ml, boil 15min, cooling.Add 10.50ml distilled water again, shake up.Use the 550nm colorimetric.Contrast: substitute the enzyme sample with distilled water, other operation together.
4.3 calculate
Enzyme activity unit u/mg=OD * 2 * 20 * 1.9 * K * n=OD * K * 76 * n
In the formula: K---the typical curve constant
N---diluted sample multiple
2---reaction 30min is converted into 60min
20---will draw the 0.5ml reaction solution and be converted into 10ml
1.9---the ratio of maltose glucose molecule amount
Record extracting solution enzyme activity 15064U/g, convert content 60256U/g wheat bran in the wheat bran.
Utilize above method, the data list of embodiment 1-3 is as follows:
Claims (3)
1. the preparation method of a liquid beta-diastase is characterized in that with the wheat bran being raw material, and its processing step comprises lixiviate, solid-liquid separation, the essence filter of vat liquor, ultrafiltration and concentration, prescription, the smart filter of secondary, the enzyme activity adjustment of wheat bran, and division is as follows:
Lixiviate:
(1) preparation lixiviate water: in water by weight/volume ratio adds sanitas ethyl p-hydroxybenzoate 0.01～0.03%, potassium sorbate 0.1～0.25%, Sodium Benzoate 0.2～0.5%, reductive agent Sodium Pyrosulfite 0.5～1.5%, Sodium Hydrosulphite 0.5～1.5%;
(2) lixiviate: with wheat bran by weight/volume ratio 1: 4～7 adds the lixiviate water of steps (1) preparation; Under the normal pressure, extraction temperature is controlled at 40～55 ℃, and pH is controlled at 4.5～6.5, lixiviate 6～8 hours;
Solid-liquid separation: carry out solid-liquid separation after the lixiviate;
Smart filter: isolated vat liquor is as cold as rapidly below 20 ℃, and isolated vat liquor adds the diatomite of 1～2% by weight/volume, crosses smart filter plate frame and carries out the essence filter;
Ultrafiltration and concentration: the smart filtrate of gained is carried out ultrafiltration and concentration, and the temperature of ultrafiltration and concentration is controlled at below 20 ℃;
Prescription: ultrafiltration and concentration is after the enzyme activity requirement of required specification, gained ultrafiltration and concentration liquid is put into the prescription jar, by weight/volume ratio adds the sodium-chlor, 8～10% sodium-acetate, 5～8% maltodextrins of stablizer 18～22%, 0.1～0.25% potassium sorbate, 0.2～0.5% Sodium Benzoate, stirring and dissolving mixes;
The smart filter of secondary: add the diatomite of 1～2% by weight/volume in the gained prescription liquid, carry out essence and filter crossing smart filter plate frame below 20 ℃;
Enzyme activity is adjusted: collect the filtrate of the smart filter of secondary, sampling makes the product check, with diluent the enzyme activity of the smart filter of secondary filtrate is adjusted to 300000U/g or 600000U/g.
2. method according to claim 1 is characterized in that described ultrafiltration and concentration adopts rolled film, and the molecular weight that dams is 10～60K dalton.
3. method according to claim 1, it is characterized in that described enzyme activity adjust used diluent be by weight/volume ratio adds the sodium-chlor, 8～10% sodium-acetate, 5～8% maltodextrins, 0.1～0.25% potassium sorbate, the aqueous solution of 0.2～0.5% Sodium Benzoate of stablizer 18～22%.
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| PL2617823T3 (en) | 2006-09-21 | 2015-12-31 | Basf Enzymes Llc | Phytases, nucleic acids encoding them and methods for making and using them |
| FR2943686B1 (en) * | 2009-03-30 | 2013-11-01 | Roquette Freres | PROCESS FOR OBTAINING BETA AMYLASE PREPARATION FROM SOLUBLE FRUITS OF STARCH PLANTS |
| CN101962632B (en) * | 2010-05-11 | 2012-06-27 | 湖南鸿鹰祥生物工程股份有限公司 | Method for producing plant extract food-grade beta-amylase preparation |
| CN101979529B (en) * | 2010-10-14 | 2012-07-18 | 武汉工业学院 | Method for extracting liquid beta-amylase from bran |
| CN102399763B (en) * | 2011-09-18 | 2012-12-26 | 淮北市三和诺生物工程有限责任公司 | Novel method for producing food grade beta-amylase with ultrahigh activity |
| CN103789286B (en) * | 2014-02-28 | 2019-11-12 | 多多药业有限公司 | A kind of preparation method of amylase |
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