CN1032868C - Method for compound zymohydrolysis of silkworm chrysalis protein - Google Patents

Abstract

The present invention relates to a method for the composite zymohydrolysis of protein in silkworm pupas. In the method, silkworm pupas are used as raw materials, neutral protease and papain are used to compositely react with the protein liquid of the pupas for 60 to 90 minutes at 55 to 65 DEG C under neutral ordinary pressure, and a hydrolysate of the protein in the silkworm pupas is obtained after deodorization. The hydrolysate contains complete kinds and high content of amino acids necessary for human bodies, and can be used for preparing high-grade and middle-grade nutritive health-care products. The method has the advantages of simple technology, no need of corrosion resistance on devices, and no pollution of waste water, waste gas and waste slag in production. Compared with a single enzymolysis method, the method improves the yield by about 25% and lowers cost by over 25%.

Description

Method for compound zymohydrolysis of silkworm chrysalis protein

The present invention relates to a kind of biochemical method of enzymolysis pupa albumen.

It adopts papoid enzymolysis under the following conditions to disclose a kind of (method that is prepared nitrogen base acid complex liquid by cocoon chrysalis) (patent No. 86106166) on the 3rd 44 phases of volume at Gazette of Patent for Invention: (a) enzyme amount: bright pupa amount=1: 200～400 (weight ratios), (b) bright pupa amount: the water yield=1: 3.0～4.5 (weight ratio) be hydrolysis temperature (c): 60～70 ℃ of (d) enzymolysis times: reacting liquid pH value=7～8 during 60～120 minutes (e) enzymolysis.Its weak point is to adopt single papain enzymolysis pupa albumen, because the effect point of contact of enzyme is limited, therefore proteic utilization ratio is not very high, if prolong action time, will certainly spin out the production cycle in indefinitely.

In view of above-mentioned weak point of the prior art, the object of the present invention is to provide the method for the compound zymohydrolysis of silkworm chrysalis protein that can improve the pupa albumen utilization ratio that a kind of production technique is simple, cost is low.

Purpose of the present invention can realize by following measure: a kind of method of compound zymohydrolysis of silkworm chrysalis protein is characterized in that adopting neutral protease and papoid to carry out enzymolysis under the following conditions:

A, enzyme amount: bright pupa amount=1: 500～600 (weight ratios) (papoid vigor 600,000 units/g)

1: 200～300 (weight ratios) (neutral protease vigor 200,000 units/g)

B, bright pupa amount: the water yield=1: 3.0～5.0 (weight ratio)

C, hydrolysis temperature: 55～65 ℃

D, enzymolysis time: 60～90 minutes

E, enzyme digestion reaction liquid pH value: 6.0～8.0.

The top condition of its enzymolysis is:

A, enzyme amount: bright pupa amount=1: 540 (weight ratio) (papoid vigor 600,000 units/g)

=1: 250 (weight ratios) (neutral protease vigor 200,000 units/g)

B, bright pupa amount: the water yield=1: 4.0 (weight ratio)

C, hydrolysis temperature: 60 ℃

D, enzymolysis time: two kinds of enzymes added 60 minutes simultaneously

Two kinds of enzymes successively added 80 minutes

E, enzyme digestion reaction pH value: 7.5

Fig. 2 is the preparation technology's flow process with papoid and neutral protease compound zymohydrolysis of silkworm chrysalis protein:

In this preparation process, be the principal reaction part with neutral protease and papoid compound zymohydrolysis of silkworm chrysalis protein, therefore must be noted that the condition of control enzyme digestion reaction.

1. the consumption of enzyme.

Enzyme amount and the ratio of bright pupa amount directly influence the effect of enzymolysis, in bright pupa amount one regularly, along with amino acid whose content in the increase product of enzyme amount also increases thereupon, but not proportional relation, even it is also very little to increase the amplitude that amino acid whose content increases in the enzyme volume production thing after the enzyme amount reaches certain numerical value, consider to select suitable enzyme amount from economic angle, that is:

Enzyme amount: bright pupa amount=1: 500～600 (weight ratios) (papoid vigor 600,000 units/gram)

1: 200～300 (weight ratios) (neutral protease vigor 200,000 units/gram)

Comparatively suitable, wherein again with the enzyme amount: bright pupa amount=1: 540 (weight ratio) (papoid vigor 600,000 units/gram)

1: 250 (weight ratio) (neutral protease vigor 200,000 units/gram) are good

As shown in table 1, be both the bright pupa of 60 grams, aminoacids content situation contrast in the different enzyme volume production things when other condition is identical:

Table 1

Enzyme amount: aminoacids content (grams per liter) in pupa amount (weight ratio) product

1: 270 (wood) 1: 540 (in) 10.00

1: 270 (wood) 1: 250 (in) 11.97

1: 540 (wood) 1: 250 (in) 12.21

1: 540 (wood) 1: 125 (in) 10.92

1: 540 (wood) 1: 500 (in) 7.93

Annotate: (wood) be papoid, (in) be neutral protease

Because every batch of the vigor of enzyme is different, both made with of the prolongation of a collection of enzyme along with storage period, the vigor of enzyme also descends thereupon, must measure the vigor of enzyme before therefore using, and (method is attached) converses the consumption of actual required enzyme by following formula according to the vigor of the enzyme that records:

2. reactant concn

When the low water yield that promptly adds of reactant concn more for a long time, not only total amino acid content is lower in the product, and has reduced usage ratio of equipment; Though and plant factor is higher when concentration of reactants is higher, but because system thickness enzyme can not fully act on the pupa albumen, make that amino acid whose total amount is not high yet in the product, bright pupa amount is 60 grams in the table 2, the contrast of total amino acid content situation in products therefrom when other condition is all identical except that the concentration of reactants difference:

Table 2 water: aminoacids content (grams per liter) in bright pupa (weight ratio) product

2.5 9.69

3.0 12.5

3.5 13.09

4.0 13.15

4.5 12.90

5.0 10.9

5.5 8.78

Work as the water yield as can be seen from the table: bright pupa amount=3.0～5.0: 1 effect is better, and works as the water yield: bright pupa amount=4.0: 1 best results.

3. time of enzymolysis

The proteic reaction needed regular hour of protease hydrolysis pupa, regularly amino acid whose content also increases thereupon in the product along with the reaction times increases when other condition one, and as can be seen from Figure 1 aminoacids content is not proportional in enzymolysis time and the product.When reaction times during at 60～90 minutes, aminoacids content is higher in the product, when surpassing 90 minutes, though increase the reaction times but aminoacids content increases in the product amplitude is also little, from shortening reaction time, the angle of enhancing productivity is considered, get 60～90 minutes comparatively suitable, because the present invention has adopted two kinds of proteolytic enzyme, thereby the mode that drops into also can be two kinds, a kind of mode is that two kinds of proteolytic enzyme add simultaneously, another kind of mode is that two kinds of proteolytic enzyme divide adding successively, each reacted 40 minutes, the result shows that result that dual mode carries out makes in the product total amino acid content difference little, the actual consideration of the production of the metallization processes that conforms to the principle of simplicity, and the mode that adopts two kinds of enzymes to add is simultaneously reacted 60 minutes for best.

4. the temperature of enzymolysis

The optimum temperature of papoid is 65 ℃, and the optimum temperature of neutral protease is 55 ℃, because carrying out two kinds of enzymes of complex enzyme hydrolysis adds reaction simultaneously, need to seek the optimum temperature of an optimum temps near these two kinds of enzymes, in table 3, be both the bright pupa of 60 grams when other condition is identical, aminoacids content situation contrast in the product under differing temps:

Aminoacids content (grams per liter) in the table 3 hydrolysis temperature ℃ product

50 7.22

55 11.53

60 12.20

65 10.57

70 8.29

From above-mentioned test-results show temperature of reaction 55～65 ℃ comparatively suitable, and 60 ℃ near the optimum temperuture of two kinds of enzymes.

5. the pH value of enzymolysis

Neutral protease and papoid need to carry out enzymolysis under suitable pH value, reaction solution is the acid or alkaline activity that all can influence enzyme when other reaction conditions is identical, make that amino acid whose content reduces in the product, table 5 is contrasts of aminoacids content situation in the product under the condition of the bright pupa of 60 grams different pH values when other condition is identical:

Table 5

PH value aminoacids content (grams per liter)

5.5 7.76

6 9.78

6.5 10.02

7.0 11.85

7.5 12.20

8.0 10.91

8.5 5.14 The above results show that the pH value is amino in 6.0～8.0 o'clock products

The content of acid is more satisfactory, and the pH value is 7.5 o'clock best results, but is hydrolyzed gradually owing to protein in actual production, and the pH value of reaction system is lowered gradually, therefore needs to adopt yellow soda ash (30%) aqueous solution that the pH value of reaction solution is adjusted at any time.

Embodiment 1

Getting 60 kilograms of bright pupas rinses well, adding less water rubs through mincer, and then add water to 240 kilogram (bright pupa: water=1: 4), on colloidal mill, wear into homogenate (5～20 microns of particle diameters) and squeeze into reactor by beverage pump, chuck to reactor feeds steam, start agitator simultaneously, make slurries be warmed up to 60 ℃, and the pH value of slurries is transferred to 7.5 with the yellow soda ash of 30% (weight), add neutral enzymatic 220 grams (vigor 200,000 units/gram), papain 110 grams (vigor 600,000 units/gram) this moment.Keep reaction 60 minutes (intermittently stirring) with this understanding, be cooled to room temperature then, filter twice (terylene filter cloth) with whizzer (2000 rev/mins), add removal agent of raw meat smell veltol plus 100 gram again, alcohol mixes the back high-pressure filteration for 4 kilograms, at this moment add potassium sorbate sanitas 600 grams again, filtrate promptly obtains 200 kilograms of amino acid composite liquid finished products by the high-temperature short-time sterilization device, and aminoacids content is 12.3 grams per liters.

Embodiment 2

Repeat the operational condition of example 1, just when enzyme-added enzymolysis, add neutral enzymatic 220 gram (vigor 200,000 units/gram) reactions earlier after 40 minutes, add papain 110 gram (vigor 600,000 units/gram) reactions 40 minutes again, it can obtain 205 kilograms of amino acid composite liquid finished products, and aminoacids content is 12.0 grams per liters.

Embodiment 3

Repeat the operational condition of example 1, wherein add 200 kilograms in water (bright pupa amount: water=1: 3.3) neutral enzymatic 200 grams (vigor 200,000 units/gram), papain 120 grams (vigor 600,000 units/gram), 65 ℃ temperature maintenance reactions 90 minutes, can obtain amino acid composite liquid finished product 105 kg, aminoacids content is 10.88 grams per liters.

Embodiment 4

Repeat the operational condition of example 1, wherein add 300 kilograms in water (bright pupa amount: water=1: 5.0) neutral enzymatic 300 grams (vigor 200,000 units/gram), papain 100 grams (vigor 600,000 units/gram), 55 ℃ temperature maintenance reactions 60 minutes, can obtain 310 kilograms of amino acid composite liquid finished products, aminoacids content is 10.9 grams per liters.

Embodiment 5

Repeat the operational condition of example 1, wherein recording the papain vigor is 500,000 units/gram, and neutral protease vigor is 140,000 units/gram, and the amount that then adds enzyme is respectively:

Papain: 110 * 60/50=132 gram

Neutral enzymatic: 220 * 20/14=315 gram

Obtain 200 kilograms of amino acid composite liquid finished products, aminoacids content is 12.1 grams per liters.

The present invention has following advantage compared to existing technology:

1. improved amino acid whose transformation efficiency.

The present invention adopts two kinds of akin enzymes of action condition to the pupa albumen acting in conjunction, because the kind difference of enzyme, therefore the action site of enzyme is also different, enzyme point of contact on the protein peptide chain is increased, be that protein transduction changes into amino acid whose quantity and increases, table 6 is to be all the bright pupa of 60 grams, the contrast of aminoacids content situation in outer all the other the condition homogeneous phases while products of kind of dezymotizing and different amts:

Table 6 enzyme amount: aminoacids content (grams per liter) in pupa amount (weight ratio) product

1: 540 (wood) 5.82

1: 270 (wood) 9.08

1: 540 (wood) 12.2

1: 250 (in) 10.0

1: 500 (in) 8.27

Two kinds of enzyme complex enzyme hydrolysis can improve about 25% than single enzymolysis productive rate as can be seen from the above table.

2. reduced production cost

The price of neutral protease can make cost reduce more than 25% than the low price of papoid, benefits suitability for industrialized production.

Annex 1 1398 neutral proteinase vigour-testing method

One, reagent:

1. Folin reagent:

In 2000 milliliters ground reflux, add sodium wolframate (Na ₂WO ₄2H ₂O) 100g, Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 25g, distilled water 700ml, 85% phosphoric acid 50ml, concentrated hydrochloric acid 100ml, little fiery boiling reflux 10 hours behind the removal condenser, adds Lithium Sulphate (Li ₂SO ₄) 50g, distilled water 50ml and several dense (99%) bromine waters shake up, and carry out in stink cupboard, boil 15 minutes again, to remove unnecessary bromine, (cold back is boiled and is removed excessive bromine if still have green need add bromine water again), cooling back adding distil water is settled to 1000ml, mix filtration, it is golden yellow that reagent should be, be stored in the brown bottle, during use with 1 part of former folin solution and 2 parts of distilled water mixings.

2. 0.4N yellow soda ash

Take by weighing anhydrous sodium carbonate (Na ₂CO ₃) 42.4g, be settled to 1000 milliliters with dissolved in distilled water.

3. 0.4M Tricholroacetic Acid:

Take by weighing the 65.4g Tricholroacetic Acid, be settled to 1000ml with dissolved in distilled water.

4.0.02M pH7.5 phosphoric acid buffer:

Take by weighing Na ₂HPO ₄12H ₂O6.02g and NaH ₂PO ₄2H ₂O 0.5g is settled to 1000ml with dissolved in distilled water.

5. casein solution:

Accurately take by weighing casein 2.000g, add an amount of 0.02M pH7.5 phosphoric acid buffer with the moistening back of a small amount of 0.5N NaOH earlier, heating should often be stirred and be made fully dissolving in boiling water bath, and cooling (should stir) back moves into 100 milliliters of volumetric flasks, with 0.02M pH7.5 phosphoric acid buffer constant volume to 100ml.

Attached: as 1) during preparation casein constant volume,, to purchase and to add 1-2 and drip the alcohol froth breaking if foam is too much.

2) casein solution is preserved a week at 4 ℃ of refrigerators, as finds rotten should the preparation again.

6. 100 mcg/ml standard tyrosine solutions.

Accurately take by weighing pre-L-tyrosine 0.1g, progressively add 1NHCl 6ml dissolving, with 0.2NHCl constant volume 100ml prior to 105 ℃ of dry 2-3 hours, promptly get the tyrosine solution of 1mg/ml, get this solution 10ml, with 0.2NHCl constant volume 100ml, the tyrosine solution of 100ug/ml.

Two, measuring method:

1. enzyme liquid preparation to be measured:

Accurately take by weighing 2.0000g, with the dissolving of the damping fluid of a small amount of 0.02M pH7.5, and smash with glass stick and to grind, in the careful impouring volumetric flask of upper strata liquid, the sediment part adds a small amount of above-mentioned damping fluid again, smash so repeatedly and ground 3～4 times, in last all immigrations volumetric flask, shake up to scale with the damping fluid constant volume, with 4 layers of filtered through gauze, according to enzyme activity, be diluted to suitable multiple with above-mentioned damping fluid again, use for measuring.

2. measure:

1). earlier casein solution is put into 40 ℃ of water bath with thermostatic control preheatings 3～5 minutes.

2). press Fig. 3 sequential operation:

Blank:

Should add Tricholroacetic Acid earlier in the sample, and then add casein solution.

Three, calculate:

1 gram enzyme powder or 1ml enzyme liquid are under the condition of 40 ℃ of pH7.5, and the enzyme amount that the per minute caseinhydrolysate produces 1ug tyrosine is an enzyme activity unit.

Enzyme activity unit=4/10 * K * 0.D * n

In the formula: 4-reaction reagent cumulative volume 4ml.

The 10-assaying reaction time is 10 minutes.

The 0.D value is the colorimeter constant by 1 suitable tyrosine micrograms (g/ml) in the K-typical curve.

N-enzyme liquid dilution general times.

Annex 2 papoid vigour-testing methods

One, reagent:

1. enzyme liquid: enzyme 0.1g puts in the mortar, adds a small amount of frozen water and grinds frozen water constant volume 100ml.

2. enzyme diluent: (face and use stylish preparation)

Get the 0.4577g cysteine hydrochloride, get 0.223gEDTANa ₂Transfer pH to 5.0 with 4N sodium hydroxide or 4N hydrochloric acid before the constant volume 10ml, constant volume.

3. 4N sodium hydroxide

4. 4N hydrochloric acid

5. casein solution: get 1.79g Sodium phosphate dibasic constant volume 100ml, be 0.05M Sodium phosphate dibasic liquid, get the 0.6g casein and add above-mentioned disodium phosphate soln 80ml, transfer constant volume 100ml behind the pH=7.0 with 1/10N hydrochloric acid after the stirring and dissolving.

6. trichloroacetic acid solution:

Get 99% glacial acetic acid 1.896ml, the back that is dissolved in water adds the 1.805g sodium acetate, adds 1.797g Tricholroacetic Acid dissolving back constant volume 100ml behind the dissolving mixing.

7. standard amino acid: (50.0g tyrosine/ml)

Get 5mg tyrosine 1/10N hydrochloric acid constant volume 100ml.

Two, measuring method:

1. get enzyme liquid 5ml and put in the 10ml volumetric bottle, enzyme-added diluent constant volume 10ml, this is for supplying to survey enzyme liquid.

2. get for examination enzyme liquid 1ml and in test tube with ground stopper, put 37 ℃ of water bath heat preservations 10 minutes, add the casein solution 5ml of preheating, the reaction 10 minutes of clocking.

3. add Tricholroacetic Acid 5ml during by 10 minutes, shake up filtration.

4. get confession examination enzyme liquid 1ml and add Tricholroacetic Acid 5ml, add casein solution 5ml after 10 minutes, filter, be blank solution.

5.275nm measure absorption value A.

6. the 275nm absorption value of the accurate tyrosine of mark (is with water blank) As.

Three, calculate:

The vigor of papoid (unit/g)

Claims (2)

1. the method for a compound zymohydrolysis of silkworm chrysalis protein is characterized in that adopting neutral protease and papoid to carry out enzymolysis under the following conditions:

A, enzyme amount: bright pupa amount=1: 500～600 (weight ratios) (papoid vigor 600,000 units/g)

1: 200～300 (weight ratios) (neutral protease vigor 200,000 units/g)

B, bright pupa amount: the water yield=1: 3.0～5.0 (weight ratio)

C, hydrolysis temperature: 55～65 ℃

D, enzymolysis time: 60～90 minutes

E, enzyme digestion reaction liquid pH value: 6.0～8.0.

2. the method for compound zymohydrolysis of silkworm chrysalis protein according to claim 1 is characterized in that adopting the condition of neutral protease and papoid complex enzyme hydrolysis to be:

A, enzyme amount: bright pupa amount=1: 540 (weight ratio) (papoid vigor 600,000 units/g)

=1: 250 (weight ratios) (neutral protease vigor 200,000 units/g)

B, bright pupa amount: the water yield=1: 4.0 (weight ratio)

C, hydrolysis temperature: 60 ℃

D, enzymolysis time: two kinds of enzymes added 60 minutes simultaneously

Two kinds of enzymes successively added 80 minutes

E, enzyme digestion reaction liquid pH value: 7.5

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