CN107513548A - A kind of method that peptone is prepared using fish meal pressed liquor - Google Patents
A kind of method that peptone is prepared using fish meal pressed liquor Download PDFInfo
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- CN107513548A CN107513548A CN201711045294.XA CN201711045294A CN107513548A CN 107513548 A CN107513548 A CN 107513548A CN 201711045294 A CN201711045294 A CN 201711045294A CN 107513548 A CN107513548 A CN 107513548A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a kind of method that peptone is prepared using fish meal pressed liquor, and it passes through following steps：（I）Fish meal pressed liquor, which is stood, removes fish oil；（II）The fish meal pressed liquor negative pressure low temperature that step I is obtained concentrates；（III）Heated in the concentrate that step II is obtained and citric acid is added in concentrate；（IV）The Plus acidic protease hydrolyzed in the mixture that step III is obtained；（V）Disodium hydrogen phosphate is added in the mixture that step IV is obtained, bean dregs, adds trypsin digestion then to be inactivated；（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered；（VII）Filtered fluid is concentrated and peptone powder product is obtained by spray drying.The present invention carries out mixed enzymolysis and by optimizing enzymatic hydrolysis condition by adding bean dregs so that prepared peptone has especially outstanding bacteria culture media characteristic.The method skill of the present invention is reasonable, advanced technology, workable, can be widely applied to the manufacture of the culture medium of bacterium.
Description
Technical field
The present invention relates to the preparation of peptide, especially a kind of method that peptone is prepared using fish meal pressed liquor.
Background technology
It is known that it with one or more low value fish is raw material that fish meal, which is, through squeezing, deoiling, being dehydrated, drying after boiling
The high-protein feed raw material obtained after dry, crushing and processing.It forms the unprocessed directly discharge of fish meal wastewater after squeezing.Due to
It is easily corrupt containing the solid contents such as dissolving Fish protein and the composition such as fish oil and amino acid, trace element in fish meal wastewater, cause
The eutrophication of China coastal seas seawater, serious pollution is caused to ecological environment, have impact on China coast beach and seawater fishery.
Therefore, how to prepare peptone using fish meal processing waste liquid is a urgent problem to be solved.
The content of the invention
In order to overcome deficiency of the prior art, it is an object of the invention to provide a kind of rational technology, advanced technology, can
The method that peptone is prepared using fish meal pressed liquor of strong operability.
The technical solution adopted for the present invention to solve the technical problems is：It is a kind of to prepare peptone using fish meal pressed liquor
Method, it is characterised in that：It passes through following steps：
（I）Fresh, pollution-free, the normal fish meal pressed liquor of smell is chosen, is put into and stands in tank, fish oil is removed by standing；
（II）The fish meal pressed liquor negative pressure low temperature concentration that step I is obtained calculates content as 16-18% until solid content using weight in wet base,
The weight of concentration mixture is calculated as 10000 parts by weight；
（III）40-45 degrees Celsius is heated in the concentrate that step II is obtained, and citric acid 45-70 is added in concentrate
Parts by weight；
（IV）Plus acidic protease is to 2000-4500 U/mL in the mixture that step III is obtained, in 40-45 degrees Celsius of lower enzyme
Solve 2-4 hours；
（V）Disodium hydrogen phosphate 60-180 parts by weight are added in the mixture that step IV is obtained, bean dregs 600-1200 parts by weight, are added
Trypsase continues to digest 8-16 hours, and go out under 80-90 degrees Celsius to 3000-5000U/mL under 40-45 degrees Celsius
It is living；
（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered, goes the removal of impurity, obtains peptone filtered fluid；
Wherein, the filter cloth of described plate and frame filter press is 100~200 mesh, and sheet frame pressure is 0.3~0.5MPa；
（VII）The filtered fluid that step VI is obtained is sent into vacuum concentrator and concentrated, and peptone is obtained by spray drying
Powder product.
In currently preferred aspect, in step II, described being concentrated under 5-10 degrees Celsius is carried out.
In currently preferred aspect, in step III, concentrate is heated to 42 degrees Celsius, and in concentrate
Add the parts by weight of citric acid 55.
In currently preferred aspect, in step iv, Plus acidic protease is extremely in the mixture that step III is obtained
3000 U/mL, digested 3 hours under 42 degrees Celsius.
In currently preferred aspect, in step V, disodium hydrogen phosphate 125 is added in the mixture that step IV is obtained
Parts by weight, the parts by weight of bean dregs 800, trypsase is added to continue enzymolysis under 42 degrees Celsius 12 hours to 4000 U/mL.
In currently preferred aspect, in step VI, the fermenting mixture plate and frame filter press that step V is obtained was carried out
Filter, removes the removal of impurity, peptone filtered fluid；Wherein, the filter cloth of described plate and frame filter press is 160 mesh, and sheet frame pressure is 0.4
MPa。
The method of the present invention realizes the efficient production of peptone by will be added between two kinds of protease timesharing with step.
And mixed enzymolysis is carried out by adding bean dregs in being digested in second step, and by optimizing enzymatic hydrolysis condition so that prepared
The peptone arrived has especially outstanding bacteria culture media characteristic.The method skill of the present invention is reasonable, advanced technology, operability
By force, it can be widely applied to the manufacture of the culture medium of bacterium, especially bacillus brevis.
Embodiment
Unless additionally illustrating, " fish meal pressed liquor " refers to during fish meal is produced, and fish are being carried out to crush it
Afterwards, the liquid waste obtained in expressing process is carried out.After extraction, obtained solid matter is further dried to obtain fish
Powder, described fish meal are used as the purposes such as feed addictive.
Unless additionally illustrating, the acid protease and trypsase in each embodiment of the invention are purchased from eastern permanent China
Road bio tech ltd.Two kinds of enzymes are 800,000 U/ grams of dry powder.When adding, water is added to be made 100,000 in the dry powder first
U/mL enzyme solutions, then the enzyme solutions of certain volume are added in seafood mixture, reach the amount of expectation.
Unless additionally illustrating, comparative example 1-5 shares the step III of embodiment 1 product, therefore starts it in step IV
Before, the experiment of embodiment 1 can be considered as parallel experiment with the material composition of comparative example.
Embodiment 1
A kind of method that peptone is prepared using fish meal pressed liquor, it passes through following steps：
（I）Fresh, pollution-free, the normal fish meal pressed liquor of smell is chosen, is put into and stands in tank, fish oil is removed by standing；
（II）By concentration under the obtained fish meal pressed liquor negative pressure of step I and 5-10 degrees Celsius until solid content calculates content with weight in wet base
For 16-18%, the weight for concentrating mixture is calculated as 10000 parts by weight（Herein by concentrator is limited, can not disposably carry out
All concentrations, so the solid content weight of each concentration result is different.And due to complete solid content can not be carried out
Separation test, so the measurement result is sampling and measuring, has larger uncertainty, can only obtain a value range.Together
Sample, thickening temperature is controlled in 5-10 degree Celsius ranges, also is difficult to ensure the temperature of equipment all areas in large-scale production
Degree is consistent and does not fluctuate.Herewith illustrate in following examples）；
（III）42 degrees Celsius are heated in the concentrate that step II is obtained, and the parts by weight of citric acid 55 are added in concentrate；
（IV）Plus acidic protease digests 3 hours to 3000 U/mL under 42 degrees Celsius in the mixture that step III is obtained；
（V）The parts by weight of disodium hydrogen phosphate 125 are added in the mixture that step IV is obtained, the parts by weight of bean dregs 800, add trypsase
To 4000 U/mL, continue enzymolysis under 42 degrees Celsius 12 hours, and inactivated under 85 degrees Celsius；
（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered, goes the removal of impurity, obtains peptone filtered fluid；
Wherein, the filter cloth of described plate and frame filter press is 160 mesh, and sheet frame pressure is 0.4MPa；
（VII）The filtered fluid that step VI is obtained is sent into vacuum concentrator and concentrated, and peptone is obtained by spray drying
Powder product.6.26 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Embodiment 2
A kind of method that peptone is prepared using fish meal pressed liquor, it passes through following steps：
（I）Fresh, pollution-free, the normal fish meal pressed liquor of smell is chosen, is put into and stands in tank, fish oil is removed by standing；
（II）By concentration under the obtained fish meal pressed liquor negative pressure of step I and 5-10 degrees Celsius until solid content calculates content with weight in wet base
For 16-18%, the weight for concentrating mixture is calculated as 10000 parts by weight；
（III）40 degrees Celsius are heated in the concentrate that step II is obtained, and the parts by weight of citric acid 45 are added in concentrate；
（IV）Plus acidic protease digests 4 hours to 4500 U/mL under 40 degrees Celsius in the mixture that step III is obtained；
（V）The parts by weight of disodium hydrogen phosphate 60 are added in the mixture that step IV is obtained, the parts by weight of bean dregs 600, add trypsase
To 5000 U/mL, continue enzymolysis under 40 degrees Celsius 16 hours, and inactivate at 80 degrees celsius；
（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered, goes the removal of impurity, obtains peptone filtered fluid；
Wherein, the filter cloth of described plate and frame filter press is 100 mesh, and sheet frame pressure is 0.3 MPa；
（VII）The filtered fluid that step VI is obtained is sent into vacuum concentrator and concentrated, and peptone is obtained by spray drying
Powder product.5.88 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Embodiment 3
A kind of method that peptone is prepared using fish meal pressed liquor, it passes through following steps：
（I）Fresh, pollution-free, the normal fish meal pressed liquor of smell is chosen, is put into and stands in tank, fish oil is removed by standing；
（II）By concentration under the obtained fish meal pressed liquor negative pressure of step I and 5-10 degrees Celsius until solid content calculates content with weight in wet base
For 16-18%, the weight for concentrating mixture is calculated as 10000 parts by weight；
（III）45 degrees Celsius are heated in the concentrate that step II is obtained, and the parts by weight of citric acid 70 are added in concentrate；
（IV）Plus acidic protease digests 2 hours to 2000 U/mL under 45 degrees Celsius in the mixture that step III is obtained；
（V）The parts by weight of disodium hydrogen phosphate 180 are added in the mixture that step IV is obtained, the parts by weight of bean dregs 1200, add tryptose
Enzyme continues enzymolysis 8 hours, and inactivated under 90 degrees Celsius to 3000 U/mL under 45 degrees Celsius；
（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered, goes the removal of impurity, obtains peptone filtered fluid；
Wherein, the filter cloth of described plate and frame filter press is 200 mesh, and sheet frame pressure is 0.5 MPa；
（VII）The filtered fluid that step VI is obtained is sent into vacuum concentrator and concentrated, and peptone is obtained by spray drying
Powder product.5.76 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Comparative example 1
In this comparative example, the parts by weight of disodium hydrogen phosphate 125 and the parts by weight of bean dregs 800 are added without in step V, other and implementation
Example 1 is identical.4.51 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Comparative example 2
In this comparative example, the parts by weight of disodium hydrogen phosphate 125 and the parts by weight of bean dregs 800 are added without in step V；By in step V
Trypsase added together with acid protease in step iv, carry out altogether enzymolysis 14 hours, other are same as Example 1.
4.28 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Comparative example 3
In this comparative example, step V is not performed, and step VI enzymolysis time is extended to 14 hours, other and the phase of embodiment 1
Together.3.19 kilograms of peptone powder product has finally been made in the concentration mixture of double centner.
Comparative example 4
In this comparative example, step IV is not performed, directly performs step V, other are same as Example 1.The concentration of double centner mixes
2.96 kilograms of peptone powder product has finally been made in compound.
Comparative example 5
In this comparative example, the disodium hydrogen phosphate of 125 parts by weight in step V is substituted for the sodium carbonate of 78 parts by weight, other
It is same as Example 1.Through measurement, after the sodium carbonate of 78 parts by weight is added, the pH for digesting mixed liquor is 7.9.And embodiment 1
The pH of enzyme mixation is 7.8 after middle addition disodium hydrogen phosphate.Both have similar pH value.The concentration mixture of double centner
4.92 kilograms of peptone powder product finally has been made.
First, peptone molecular weight measurement
Obtained embodiment 1-3 and comparative example 1-5 peptone powder product powder presents faint yellow, has preferable thing
Rationality shape.The molecular weight point of embodiment 1-3 and comparative example 1-5 peptone powder product is measured using SDS-PAGE methods
Cloth.As a result show, the peptone of above each group is respectively provided with 600-3000Da molecular weight distribution.In the peptone powder by more than
When the aqueous solution instills saturation sodium sulphate, precipitation is occurred without.This shows that the peptone products of the above are to meet general albumen in itself
What peptone required.
2nd, peptone quality test
Bacillus brevis used in this experiment can be bacillus brevis QSYL, and bacillus brevis QSYL actual is short bud
Spore bacillus 7316, QSYL represent 7316 Chinese prefix.The strain grinds bio tech ltd purchased from Shanghai one.Use with
Under method evaluate the quality of above-described each group peptone products:（1）Cultivate by recovery, passed through with slant culture
The physiological saline bacteria suspension containing 1,000,000,000 bacillus brevis QSYL is made in grinding；（2）It is 0.1% chlorine to prepare containing mass fraction
Change sodium, about 0.015% sodium dihydrogen phosphate, the aqueous solution of 1% peptone, and be distributed into 5ml/ pipes（Embodiment 1-3, the nothing of comparative example 4
Additional sodium dihydrogen phosphate is needed, because just containing the sodium dihydrogen phosphate close to this content in its peptone）；（3）Take 1ml bacteria suspensions
Add in 1 tubulin peptone water, be well mixed, three Duplicate Samples are done simultaneously labeled as dilution factor 1；Taken from the pipe of dilution factor 1
1ml is added in 1 tubulin peptone water, is well mixed, and three Duplicate Samples are done simultaneously labeled as dilution factor 2；By that analogy until inciting somebody to action
1ml bacteria suspensions, which are diluted to dilution factor 9, to be terminated；5 to 9 continuous 5 dilution factors are taken, every kind of peptone amounts to 15 by all means, is put into training
Support 36 ± 1 DEG C of case to cultivate 96 hours, observe once and record the appearance positive within every 12 hours（There is hypha body）Pipe number, the note
Record the good and bad judgement that result is used for peptone quality.
The result of the test in the peptone obtained by embodiment 1 and comparative example 1-5 is have recorded in table 1-2.Wherein, 3/3 table
Show occur the positive in three groups of parallel samples, the positive findings for occurring two groups in 2/3 three groups of parallel samples of expression, by that analogy.
All positive findingses are added and obtain the integration of the group.Integration is higher, and it is more early to illustrate that positive findings occurs.
Table 1：The bacillus brevis cultivation results of embodiment 1 and comparative example 1-2 peptones
Table 2：The bacillus brevis cultivation results of comparative example 3-5 peptones
From can be seen that embodiment 1 with upper table 1-2 result of the test there is optimal Bacteria Culture to show, being secondly comparative example
2.The performance of comparative example 1 and comparative example 5 is also relatively good, and is closer to.Worst is comparative example 3 and 4., will in comparative example 5
Disodium hydrogen phosphate replaces with sodium carbonate, it is found that the culture performance of peptone has declined.In view of containing in each group culture medium
The sodium dihydrogen phosphate of similar content, eliminates sodium dihydrogen phosphate in incubation, this shows group of the disodium hydrogen phosphate to peptone
Influence beyond into the work pH for bringing pH conciliation enzymes, makes it have more preferably Bacteria Culture performance.From embodiment 1 and contrast
Example 1-3 contrast can be seen that addition disodium hydrogen phosphate and bean dregs so that peptone has more preferable cultural character.From embodiment
1 and the contrast of comparative example 4 can be seen that and simply digested using trypsase and bean dregs, it is impossible to so that peptone has
Good culture performance.
In summary, this experiment is demonstrated by combining bean dregs addition fermentation, and enzymolysis auxiliary agent is used as using disodium hydrogen phosphate
So that peptone has good Bacteria Culture performance.
3rd, peptone dilution factor is tested
In order to describe the problem and save test material, the experiment that dilution factor is 7-9 has also been carried out to embodiment 2-3.Its result
The dilution factor of performance and embodiment 1 is that 7-9 experiment is essentially identical.Embodiment 2-3 is equal after 12 hours when dilution factor is 7
It is positive for three pipes.24 it was as a child the three pipes positive when dilution factor is 8, embodiment 2 is when 12 hours and dilution factor are 8
2 pipes are positive, and embodiment 3 is that 1 pipe is positive.Embodiment 2 is positive for 1 pipe at 12 hours under dilution factor 9, and 24 hours are 2 pipes sun
Property, it is positive for 3 pipes after 36 hours.Performance of the embodiment 3 with embodiment 1 under dilution factor 9 is identical.As can be seen here, embodiment 2-3
There is similar culture performance compared with Example 1.
Claims (6)
- A kind of 1. method that peptone is prepared using fish meal pressed liquor, it is characterised in that：It passes through following steps：（I）Fresh, pollution-free, the normal fish meal pressed liquor of smell is chosen, is put into and stands in tank, fish oil is removed by standing；（II）The fish meal pressed liquor negative pressure low temperature concentration that step I is obtained calculates content as 16-18% until solid content using weight in wet base, The weight of concentration mixture is calculated as 10000 parts by weight；（III）40-45 degrees Celsius is heated in the concentrate that step II is obtained, and citric acid 45-70 is added in concentrate Parts by weight；（IV）Plus acidic protease is to 2000-4500 U/mL in the mixture that step III is obtained, in 40-45 degrees Celsius of lower enzyme Solve 2-4 hours；（V）Disodium hydrogen phosphate 60-180 parts by weight are added in the mixture that step IV is obtained, bean dregs 600-1200 parts by weight, are added Trypsase continues to digest 8-16 hours, and go out under 80-90 degrees Celsius to 3000-5000U/mL under 40-45 degrees Celsius It is living；（VI）The fermenting mixture plate and frame filter press that step V is obtained is filtered, goes the removal of impurity, obtains peptone filtered fluid； Wherein, the filter cloth of described plate and frame filter press is 100~200 mesh, and sheet frame pressure is 0.3~0.5MPa；（VII）The filtered fluid that step VI is obtained is sent into vacuum concentrator and concentrated, and peptone is obtained by spray drying Powder product.
- A kind of 2. method that peptone is prepared using fish meal pressed liquor according to claim 1, it is characterised in that：In step In II, described being concentrated under 5-10 degrees Celsius is carried out.
- A kind of 3. method that peptone is prepared using fish meal pressed liquor according to claim 1, it is characterised in that：In step In III, concentrate is heated to 42 degrees Celsius, and the parts by weight of citric acid 55 are added in concentrate.
- A kind of 4. method that peptone is prepared using fish meal pressed liquor according to claim 1, it is characterised in that：In step In IV, Plus acidic protease digests 3 hours to 3000 U/mL under 42 degrees Celsius in the mixture that step III is obtained.
- A kind of 5. method that peptone is prepared using fish meal pressed liquor according to claim 1, it is characterised in that：In step In V, the parts by weight of disodium hydrogen phosphate 125 are added in the mixture that step IV is obtained, the parts by weight of bean dregs 800, add trypsase extremely 4000 U/mL, continue enzymolysis 12 hours under 42 degrees Celsius.
- A kind of 6. method that peptone is prepared using fish meal pressed liquor according to claim 1, it is characterised in that：In step In VI, the fermenting mixture plate and frame filter press that step V is obtained is filtered, removes the removal of impurity, peptone filtered fluid；Wherein, institute The filter cloth for the plate and frame filter press stated is 160 mesh, and sheet frame pressure is 0.4 MPa.
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