CN105176967A - Preparation method of magnetic immobilized enzyme microspheres for producing low-lactose drinking milk - Google Patents

Abstract

The invention discloses a preparation method of magnetic immobilized enzyme microspheres for producing low-lactose drinking milk, belonging to the field of lactase. The preparation method comprises the steps of synthesizing iron oxide microparticles by taking iron dichloride, iron chloride and ammonia water as main raw materials and using a chemical co-precipitation method; then, preparing magnetic chitosan microspheres from the iron oxide microparticles and chitosan; adding a lactase solution into the magnetic chitosan microspheres to carry out cross-linking reaction; and immobilizing a lactase by using the chitosan microspheres by virtue of magnetism to produce the magnetic immobilized enzyme microspheres for the low-lactose drinking milk. According to the preparation method, the magnetic chitosan microspheres are used as carriers to immobilize the lactase, so that the lactase keeps the natural activity of an enzyme and is conveniently separated from a reaction solution and reused, and the storage stability is good.

Description

A kind of preparation method of the immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk

Technical field

The invention discloses a kind of preparation method of the immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk, belong to Sumylact L field.

Background technology

After lactose in Low lactose milk city milk enters small intestine, due to the shortage of Sumylact L, lactose can not be broken down into glucose and semi-lactosi is absorbed, and is called the bad and lactose malabsorption of lactose digestion.After lactose enters colon, by fermentation using bacteria generate short chain organic acid as acetic acid, propionic acid, butyric acid etc. with gas as methane, H2, CO2 etc., lactose fermentation process can cause borborygmus, stomachache, rectum gas and osmotic diarrhea, be called lactose intolerance when there are these symptoms, serious lactose digestion is bad or malabsorption is many occurs in 30 minutes to a few hours after a certain amount of lactose of absorption.Have the people of about 20% to there is lactose intolerance symptom in lactase deficiencies person, and lactose intolerance symptom individual difference is very large.The lactose yield of the number of intolerance shape and severity and lactase activity, absorption in small intestine and whether to take in other based food relevant simultaneously.

The lactose of general hydrolysis 70% ~ 80% can solve the problem of lactose intolerance.At present, the method reducing lactose has three kinds: ultrafiltration process, chemical acid-hydrolysis method and enzymolysis process, the wherein the most applicable production low-lactose milk of enzymolysis process.With the lactose in beta galactose former times enzymic hydrolysis cow's milk or milk-product, effectively can eliminate the intolerance shape of human body to lactose, for development China dairy industry, the health level tool improving the people is of great significance.But directly added in cow's milk by general for beta galactose enzyme, the activity of enzyme is lower, the transformation period is short, enzyme can only use once, utilization ratio is low, causes production cost higher, limits the application of beta galactose former times enzyme in dairy products industry and can not adapt to the growing market requirement.For overcoming these problems, by physics or the method for chemistry, Sumylact L molecule is strapped on carrier, makes it both keep the natural radioactivity of enzyme, be convenient to again be separated with reaction solution, and can reuse, be i.e. immobilized lactase.As a part for immobilized enzyme, the various performances of structures and characteristics to immobilized enzyme of solid support material have tremendous influence.

Summary of the invention

The technical problem that the present invention mainly solves: at present general for beta galactose enzyme directly being added in cow's milk, the activity of enzyme is lower, transformation period is short, enzyme can only use once, utilization ratio is low, cause production cost higher, limit the problem of the application of beta galactose former times enzyme in dairy products industry, provide a kind of preparation method of the immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk, the present invention is with iron protochloride, iron(ic) chloride, ammoniacal liquor is main raw material, adopt coprecipitation synthesis ferric oxide particles, chitosan magnetic micro-sphere is made again together with chitosan, add lactase soln in magnetotropism chitosan microball and carry out crosslinking reaction, by magnetic, Sumylact L is fixed by chitosan microball, produce the immobilized enzyme magnetic microsphere of Low lactose milk city milk, the present invention's chitosan magnetic micro-sphere conduct

Carrier immobilized Sumylact L, makes Sumylact L keep the natural radioactivity of enzyme, is convenient to again be separated with reaction solution, conveniently reuses, and its excellent storage stability.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

(1) in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 10 ~ 12, move into water-bath and be warming up to 40 ~ 50 DEG C, be placed on magnetic stirrer and stir 20 ~ 30min with the rotating speed of 600 ~ 700r/min, continue to be warming up to 70 ~ 80 DEG C, insulation ageing 1 ~ 2h;

(2) mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 10 ~ 15min of 8000 ~ 9000r/min, be separated and be precipitated thing, by throw out deionized water rinsing repeatedly until pH is 7 ~ 8, obtain ferric oxide particles;

(3) taking 5 ~ 10g chitosan, to be dissolved in 200 ~ 300mL mass concentration be in the acetic acid solution of 10%, stirring with glass stick makes chitosan dissolve completely, add the above-mentioned obtained ferric oxide particles of 10 ~ 20g again, put into sonic oscillation instrument sonic oscillation mixing 10 ~ 20min;

(4) in the there-necked flask with agitator and heating unit, above-mentioned ultrasonic mixed solution is added, add above-mentioned ultrasonic mixed solution more wherein, add 80 ~ 90mL paraffin oil wherein again, 7 ~ 8mL Tween-80 and 0.3 ~ 0.5g Magnesium Stearate, be warming up to 40 ~ 50 DEG C, start agitator to stir with the rotating speed of 200 ~ 300r/min, and 3 ~ 5mL polyoxyethylene glycol is dripped in whipping process, keep rotating speed stirring reaction 1 ~ 2h, pH to 10 ~ 12 are regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 60 ~ 70 DEG C, continue reaction 2 ~ 3h, thing is precipitated after filtration, with deionized water rinsing 3 ~ 5 times, obtain chitosan magnetic micro-sphere,

(5) getting the above-mentioned obtained chitosan magnetic micro-sphere of 500 ~ 600mg joins in the triangular flask of 250mL, pour the fully swelling 12 ~ 18h of 50 ~ 100mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 1 ~ 2h is adsorbed at 4 ~ 6 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 30 ~ 40 DEG C, there is crosslinking reaction in insulation 40 ~ 60min;

(6) magnet is placed at the triangular flask bottom even of above-mentioned generation crosslinking reaction, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 3 ~ 5 times with phosphoric acid buffer, at 50 ~ 60 DEG C, dry 2 ~ 3h, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.

Application method of the present invention: the harden monitoring that the present invention obtains can add lactose to and drink in dairy products and carry out degraded lactose, immobilized enzyme and liquid enzymes are placed in 4 DEG C of Refrigerator stores, take out respectively at certain hour and survey its enzyme activity, after Sumylact L is immobilized, its package stability increases, place after 30 days, the enzyme activity of immobilized enzyme still can reach 60 ~ 65%, and the enzyme activity of liquid enzymes then drops to 28 ~ 30%.

The invention has the beneficial effects as follows:

(1) the present invention's chitosan magnetic micro-sphere is as carrier immobilized Sumylact L, makes Sumylact L keep the natural radioactivity of enzyme, is convenient to again be separated with reaction solution, conveniently reuses, and its excellent storage stability;

(2) the present invention utilizes external magnetic field can control mode of motion and the direction of magneticsubstance immobilized enzyme, substitutes traditional mechanical stirring mode, improves the catalytic efficiency of immobilized enzyme.

Embodiment

First in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 10 ~ 12, move into water-bath and be warming up to 40 ~ 50 DEG C, be placed on magnetic stirrer and stir 20 ~ 30min with the rotating speed of 600 ~ 700r/min, continue to be warming up to 70 ~ 80 DEG C, insulation ageing 1 ~ 2h, mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 10 ~ 15min of 8000 ~ 9000r/min, is separated and is precipitated thing, by throw out deionized water rinsing repeatedly until pH is 7 ~ 8, obtain ferric oxide particles, taking 5 ~ 10g chitosan, to be dissolved in 200 ~ 300mL mass concentration be in the acetic acid solution of 10%, stirring with glass stick makes chitosan dissolve completely, add the above-mentioned obtained ferric oxide particles of 10 ~ 20g again, put into sonic oscillation instrument sonic oscillation mixing 10 ~ 20min, above-mentioned ultrasonic mixed solution is added in the there-necked flask with agitator and heating unit, add above-mentioned ultrasonic mixed solution more wherein, add 80 ~ 90mL paraffin oil wherein again, 7 ~ 8mL Tween-80 and 0.3 ~ 0.5g Magnesium Stearate, be warming up to 40 ~ 50 DEG C, start agitator to stir with the rotating speed of 200 ~ 300r/min, and 3 ~ 5mL polyoxyethylene glycol is dripped in whipping process, keep rotating speed stirring reaction 1 ~ 2h, pH to 10 ~ 12 are regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 60 ~ 70 DEG C, continue reaction 2 ~ 3h, thing is precipitated after filtration, with deionized water rinsing 3 ~ 5 times, obtain chitosan magnetic micro-sphere, getting the above-mentioned obtained chitosan magnetic micro-sphere of 500 ~ 600mg joins in the triangular flask of 250mL, pour the fully swelling 12 ~ 18h of 50 ~ 100mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 1 ~ 2h is adsorbed at 4 ~ 6 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 30 ~ 40 DEG C, there is crosslinking reaction in insulation 40 ~ 60min, magnet is placed at the triangular flask bottom even that crosslinking reaction occurs, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 3 ~ 5 times with phosphoric acid buffer, at 50 ~ 60 DEG C, dry 2 ~ 3h, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.

Example 1

First in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 10, move into water-bath and be warming up to 40 DEG C, be placed on magnetic stirrer and stir 20min with the rotating speed of 600r/min, continue to be warming up to 70 DEG C, insulation ageing 1h, mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 10 ~ 15min of 8000r/min, is separated and is precipitated thing, by throw out deionized water rinsing repeatedly until pH is 7, obtain ferric oxide particles, taking 5g chitosan, to be dissolved in 200mL mass concentration be in the acetic acid solution of 10%, and stirring with glass stick makes chitosan dissolve completely, then adds the above-mentioned obtained ferric oxide particles of 10g, puts into sonic oscillation instrument sonic oscillation mixing 10min, above-mentioned ultrasonic mixed solution is added in the there-necked flask with agitator and heating unit, add above-mentioned ultrasonic mixed solution more wherein, add 80mL paraffin oil wherein again, 7mL Tween-80 and 0.3g Magnesium Stearate, be warming up to 40 DEG C, start agitator to stir with the rotating speed of 200r/min, and in whipping process, drip 3mL polyoxyethylene glycol, keep rotating speed stirring reaction 1h, pH to 10 is regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 60 DEG C, continue reaction 2h, thing is precipitated after filtration, with deionized water rinsing 3 times, obtain chitosan magnetic micro-sphere, getting the above-mentioned obtained chitosan magnetic micro-sphere of 500mg joins in the triangular flask of 250mL, pour the abundant swelling 12h of 50mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 1h is adsorbed at 4 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 30 DEG C, there is crosslinking reaction in insulation 40min, magnet is placed at the triangular flask bottom even that crosslinking reaction occurs, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 3 times with phosphoric acid buffer, dry 2h at 50 DEG C, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.

Application method of the present invention: the harden monitoring that the present invention obtains can add lactose to and drink in dairy products and carry out degraded lactose, immobilized enzyme and liquid enzymes are placed in 4 DEG C of Refrigerator stores, take out respectively at certain hour and survey its enzyme activity, after Sumylact L is immobilized, its package stability increases, place after 30 days, the enzyme activity of immobilized enzyme still can reach 60%, and the enzyme activity of liquid enzymes then drops to 28%.

Example 2

First in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 11, move into water-bath and be warming up to 45 DEG C, be placed on magnetic stirrer and stir 25min with the rotating speed of 650r/min, continue to be warming up to 75 DEG C, insulation ageing 1.5h, mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 13min of 8500r/min, is separated and is precipitated thing, by throw out deionized water rinsing repeatedly until pH is 7.5, obtain ferric oxide particles, taking 8g chitosan, to be dissolved in 250mL mass concentration be in the acetic acid solution of 10%, and stirring with glass stick makes chitosan dissolve completely, then adds the above-mentioned obtained ferric oxide particles of 15g, puts into sonic oscillation instrument sonic oscillation mixing 15min, above-mentioned ultrasonic mixed solution is added in the there-necked flask with agitator and heating unit, add above-mentioned ultrasonic mixed solution more wherein, add 85mL paraffin oil wherein again, 7.5mL Tween-80 and 0.4g Magnesium Stearate, be warming up to 45 DEG C, start agitator to stir with the rotating speed of 250r/min, and in whipping process, drip 4mL polyoxyethylene glycol, keep rotating speed stirring reaction 1.5h, pH to 11 is regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 65 DEG C, continue reaction 2.5h, thing is precipitated after filtration, with deionized water rinsing 4 times, obtain chitosan magnetic micro-sphere, getting the above-mentioned obtained chitosan magnetic micro-sphere of 550mg joins in the triangular flask of 250mL, pour the abundant swelling 15h of 75mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 1.5h is adsorbed at 5 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 35 DEG C, there is crosslinking reaction in insulation 50min, magnet is placed at the triangular flask bottom even that crosslinking reaction occurs, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 4 times with phosphoric acid buffer, dry 2.5h at 55 DEG C, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.

Application method of the present invention: the harden monitoring that the present invention obtains can add lactose to and drink in dairy products and carry out degraded lactose, immobilized enzyme and liquid enzymes are placed in 4 DEG C of Refrigerator stores, take out respectively at certain hour and survey its enzyme activity, after Sumylact L is immobilized, its package stability increases, place after 30 days, the enzyme activity of immobilized enzyme still can reach 63%, and the enzyme activity of liquid enzymes then drops to 29%.

Example 3

First in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 12, move into water-bath and be warming up to 50 DEG C, be placed on magnetic stirrer and stir 30min with the rotating speed of 700r/min, continue to be warming up to 80 DEG C, insulation ageing 2h, mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 15min of 9000r/min, is separated and is precipitated thing, by throw out deionized water rinsing repeatedly until pH is 8, obtain ferric oxide particles, taking 10g chitosan, to be dissolved in 300mL mass concentration be in the acetic acid solution of 10%, and stirring with glass stick makes chitosan dissolve completely, then adds the above-mentioned obtained ferric oxide particles of 20g, puts into sonic oscillation instrument sonic oscillation mixing 20min, above-mentioned ultrasonic mixed solution is added in the there-necked flask with agitator and heating unit, add above-mentioned ultrasonic mixed solution more wherein, add 90mL paraffin oil wherein again, 8mL Tween-80 and 0.5g Magnesium Stearate, be warming up to 50 DEG C, start agitator to stir with the rotating speed of 300r/min, and in whipping process, drip 5mL polyoxyethylene glycol, keep rotating speed stirring reaction 2h, pH to 12 is regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 70 DEG C, continue reaction 3h, thing is precipitated after filtration, with deionized water rinsing 5 times, obtain chitosan magnetic micro-sphere, getting the above-mentioned obtained chitosan magnetic micro-sphere of 600mg joins in the triangular flask of 250mL, pour the abundant swelling 18h of 100mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 2h is adsorbed at 6 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 40 DEG C, there is crosslinking reaction in insulation 60min, magnet is placed at the triangular flask bottom even that crosslinking reaction occurs, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 5 times with phosphoric acid buffer, dry 3h at 60 DEG C, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.

Application method of the present invention: the harden monitoring that the present invention obtains can add lactose to and drink in dairy products and carry out degraded lactose, immobilized enzyme and liquid enzymes are placed in 4 DEG C of Refrigerator stores, take out respectively at certain hour and survey its enzyme activity, after Sumylact L is immobilized, its package stability increases, place after 30 days, the enzyme activity of immobilized enzyme still can reach 65%, and the enzyme activity of liquid enzymes then drops to 30%.

Claims (1)

1., for the production of a preparation method for the immobilized enzyme magnetic microsphere of Low lactose milk city milk, it is characterized in that concrete preparation process is:

(1) in molar ratio for 9:5 takes in the there-necked flask that massfraction 15% iron(ic) chloride and massfraction 25% ferrous sulfate mixing solutions pour into agitator and thermometer, dropwise instill in there-necked flask with the ammoniacal liquor that mass concentration is 30% by syringe afterwards, regulate pH to 10 ~ 12, move into water-bath and be warming up to 40 ~ 50 DEG C, be placed on magnetic stirrer and stir 20 ~ 30min with the rotating speed of 600 ~ 700r/min, continue to be warming up to 70 ~ 80 DEG C, insulation ageing 1 ~ 2h;

(2) mixture after ageing is moved into horizontal centrifuge, with the rotating speed high speed centrifugation process 10 ~ 15min of 8000 ~ 9000r/min, be separated and be precipitated thing, by throw out deionized water rinsing repeatedly until pH is 7 ~ 8, obtain ferric oxide particles;

(3) taking 5 ~ 10g chitosan, to be dissolved in 200 ~ 300mL mass concentration be in the acetic acid solution of 10%, stirring with glass stick makes chitosan dissolve completely, add the above-mentioned obtained ferric oxide particles of 10 ~ 20g again, put into sonic oscillation instrument sonic oscillation mixing 10 ~ 20min;

(4) in the there-necked flask with agitator and heating unit, above-mentioned ultrasonic mixed solution is added, add 80 ~ 90mL paraffin oil wherein again, 7 ~ 8mL Tween-80 and 0.3 ~ 0.5g Magnesium Stearate, be warming up to 40 ~ 50 DEG C, start agitator to stir with the rotating speed of 200 ~ 300r/min, and 3 ~ 5mL polyoxyethylene glycol is dripped in whipping process, keep rotating speed stirring reaction 1 ~ 2h, pH to 10 ~ 12 are regulated again with the sodium hydroxide solution that mass concentration is 10%, be warming up to 60 ~ 70 DEG C, continue reaction 2 ~ 3h, thing is precipitated after filtration, with deionized water rinsing 3 ~ 5 times, obtain chitosan magnetic micro-sphere,

(5) getting the above-mentioned obtained chitosan magnetic micro-sphere of 500 ~ 600mg joins in the triangular flask of 250mL, pour the fully swelling 12 ~ 18h of 50 ~ 100mL phosphate buffer solution into, add the lactase soln that mass concentration is 80% afterwards, put into ice-water bath, fully 1 ~ 2h is adsorbed at 4 ~ 6 DEG C, adding the glutaraldehyde solution that mass concentration is 40% again, be warming up to 30 ~ 40 DEG C, there is crosslinking reaction in insulation 40 ~ 60min;

(6) magnet is placed at the triangular flask bottom even of above-mentioned generation crosslinking reaction, magnetic microsphere in bottle is precipitated, topple over removal supernatant liquor, collecting precipitation thing also moves into baking oven after rinsing 3 ~ 5 times with phosphoric acid buffer, at 50 ~ 60 DEG C, dry 2 ~ 3h, obtains a kind of immobilized enzyme magnetic microsphere for the production of Low lactose milk city milk.