CN110093387A - A method of chitosan oligosaccharide is prepared using chitin in bacillus and Paecilomyces varioti Synergistic degradation shrimp and crab shells - Google Patents
A method of chitosan oligosaccharide is prepared using chitin in bacillus and Paecilomyces varioti Synergistic degradation shrimp and crab shells Download PDFInfo
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Abstract
The present invention provides a kind of method for preparing chitosan oligosaccharide using chitin in bacillus and Paecilomyces varioti Synergistic degradation shrimp and crab shells, makes fermentation medium by raw material of the shrimp and crab shells after decalcification, is inoculated with bacillus and Paecilomyces varioti, and through everfermentation, degradation obtains chitosan oligosaccharide.The present invention belongs to a kind of completely new technique using the technique of chitin fermenting and producing chitosan oligosaccharide in shrimp and crab shells.Chitinase is produced by bacillus and Paecilomyces varioti produces deacetylase; under the synergistic effect of the two; realize that shrimp shell takes off the bioprocess of albumen, chitin and/or Enzymatic Hydrolysis of Chitosan, chitin and/or chitin oligo saccharide deacetylation; obtain water soluble chitosan, chitosan oligosaccharide and Glucosamine; the biotransformation efficiency that chitin in shrimp and crab shells is converted into chitosan oligosaccharide is effectively increased, the effective technical way of development and utilization ocean chitin resource will be become；The preparation method does not need highly basic or hydrochloric acid, and free from environmental pollution, working condition is mild, and low energy consumption, meets energy-saving and emission-reduction.
Description
Technical field
It is the present invention relates to the technical field that microorganism processing prepares chitosan oligosaccharide, in particular to a kind of using bacillus and quasi-
The method that chitin prepares chitosan oligosaccharide in mould Synergistic degradation shrimp and crab shells.
Background technique
China is the maximum sea-farming big country in the whole world, and mariculture industry is after decades of development, it has also become littoral
One of the important industry of area's marine economy development.Counted and shown according to national fishery economic statistical communique, the annual cultivation in the whole nation and
The shell-fish aquatic products total amount of fishing occupies significant proportion in aquaculture and fishing total output, and the shell of shell-fish aquatic products contains abundant
Calcareous, protein and the substances such as chitin, can be processed to extract and obtain many kinds of substance.
Chitosan oligosaccharide be origin derived from shrimp and crab shells degradation of chitosan at the small molecule oligosaccharides with amino, have complete molten
Yu Shui, it is easy to many unique properties such as absorption, high, nontoxic, the good biocompatibility of bioactivity, application field is wide, easy
The outstanding features such as be absorbed by the body, and is known as the sixth-largest vital principle of human body, the good reputation of soft gold in foreign countries.Chitosan oligosaccharide is as new generation
It records perspective biological technology products, has broad application prospect." catalogue of feed additive varieties " (2013 editions) of the Ministry of Agriculture
Approval is using chitosan oligosaccharide and chitosan oligomer as feed addictive ingredient.National health State Family Planning Commission No. 6 bulletin in 2014 is also criticized
Quasi- chitosan oligosaccharide is as new raw-food material.Known chitosan can be made by chitin by deacetylation, therefore using chitin
Matter prepares chitosan oligosaccharide.
Chitin prepares the traditional handicraft of chitosan oligosaccharide, is to first pass through chemical method chitin deacetylation is obtained chitosan
Afterwards, it then is degraded to obtain low molecular chitosan oligosaccharide with chemical method or enzymatic isolation method, therefore, the deacetylation of chitin and degradation are production
Key.But this conventional method has the shortcomings that obvious: (1) consuming a large amount of concentrated base and hydrochloric acid, processing cost is high；(2) sewage
It is high containing organic matter and inorganic salt concentration, it is difficult to handle, causes serious environmental pollution；(3) chitosan through papain (or
Papain and cellulase) enzymatic hydrolysis prepares chitosan oligosaccharide, expensive, the high production cost of enzyme.
Summary of the invention
To solve the problems, such as to mention in background technique, the present invention, which provides, a kind of utilizes bacillus and Paecilomyces varioti Synergistic degradation
The method that chitin prepares chitosan oligosaccharide in shrimp and crab shells makes fermentation medium by raw material of the shrimp and crab shells after decalcification, is inoculated with gemma
Bacillus and Paecilomyces varioti, through everfermentation, degradation obtains chitosan oligosaccharide.
Further, comprising the following steps:
Step 1: shrimp and crab shells are ground, precipitating is collected by centrifugation after decalcification is handled, adds nitrogen source and microelement, is made
Fermentation medium, it is spare；
Step 2: by bacillus streak inoculation on the double-layer plate culture medium using chitin as sole carbon source, culture
It 25-45 DEG C of temperature, after cultivating 24-48h, refrigerates spare；By Paecilomyces varioti using chitin as the double-layer plate culture of sole carbon source
Streak inoculation on base, after cultivating 36-96h, refrigerates spare by 20-37 DEG C of cultivation temperature；
Step 3: picking step 2 Bacillus, it is inoculated into using chitin as in the fluid nutrient medium of sole carbon source,
It is 25-45 DEG C of cultivation temperature, shake culture 12-24h, spare；Picking step 2 Paecilomyces varioti strain, it is unique for being inoculated into chitin
It is 20-37 DEG C of cultivation temperature, shake culture 24-72h, spare in the fluid nutrient medium of carbon source；
Step 4: the liquid spawn of step 3 bacillus and Paecilomyces varioti is inoculated into fermentation medium made of step 1
In, 23-37 DEG C of cultivation temperature, shake culture 24-72h, obtain the product containing chitosan oligosaccharide.
Further, the Bacillus strain is dirty white or yellowish, bacterium colony height in the colony characteristics of LB culture medium
Protrusion, bacterium colony rough surface is opaque, soft；
The Bacillus strain is being to have significantly using chitin as the colony characteristics of the plating medium of sole carbon source
Transparent circle.
Further, the Paecilomyces varioti bacterial strain is canescence in the colony characteristics of peptone dextrose agar medium, is put down
Exhibition, villiform is fine and close, later period powdery, and the back side is colourless；
The Paecilomyces varioti bacterial strain is being bacterium solution in pale purple by the colony characteristics of the fluid nutrient medium of sole carbon source of chitin
Color；
The Paecilomyces varioti bacterial strain is using chitin as sole carbon source and the plating medium that adds 4- nitracetanilide
Colony characteristics are to have apparent transparent circle, and the culture medium within and around bacterium colony becomes yellow；
The Paecilomyces varioti bacterial strain is being no transparent circle by the colony characteristics of the plating medium of sole carbon source of chitosan.
Further, the composition of the fermentation medium are as follows: 40-200g shrimp and crab shells powder, 2-200g yeast, 0.4-1.2g
KH2PO4, 0.1-0.4g MgSO4, 0.005-0.02gFeSO4, 0.25-0.4mg ZnSO4, 1000mL water, PH=5.0-6.0.
Further, the bacillus is bacillus licheniformis, bacillus alcalophilus, bacillus amyloliquefaciens, withered grass
One or more of bacillus, bacillus thuringiensis and Paenibacillus polymyxa.
Further, the Paecilomyces varioti is Paecilomyces lilacinus, in Paecilomyces hepiali chen, Paecilomyces cicadae, paecilomyces farinograph
One or more.
Compared with traditional chitin degrading production, the present invention is had a characteristic that
(1) it the present invention provides a kind of new technique using chitin fermenting and producing chitosan oligosaccharide in shrimp and crab shells, utilizes
The synergistic effect of bacillus and Paecilomyces varioti realizes that shrimp shell takes off albumen, chitin and/or Enzymatic Hydrolysis of Chitosan, chitin and/or several
The bioprocess of fourth oligosaccharides deacetylation obtains the chitosan oligosaccharide and Glucosamine of water soluble chitosan, different molecular weight, effectively
Improve the biotransformation efficiency that chitin in shrimp and crab shells is converted into chitosan oligosaccharide.
(2) present invention is given birth to using chitin in the synergistic effect efficient degradation shrimp and crab shells of bacillus and Paecilomyces varioti
It produces, does not need highly basic or hydrochloric acid, free from environmental pollution, working condition is mild, and low energy consumption, meets energy-saving and emission-reduction.
(3) present invention can also produce high activity chitinase, chitin deacetylase and chitosan enzyme.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of creative work.
It should be noted that in the examples where no specific technique or condition is specified, it is described according to the literature in the art
Technology or conditions or carried out according to product description, reagents or instruments used without specified manufacturer, be that can lead to
Cross the conventional products of commercially available acquisition.
The preparation of culture medium:
A liquid (1% colloid chitosan solution) is prepared: being weighed 1g Chitosan powder, 1% acetic acid solution of 80mL is added, in water
50 DEG C of heating water bath dissolutions in bath.Natural cooling, it is fixed after the homogenate of high-speed homogenization machine with the NaOH tune pH to 6.0 of 2mol/L
Hold to 100mL, adds 2g agar.
B liquid (1% tobacco brown spot pathogen solution) is prepared: being claimed 1g chitin that 10ml concentrated hydrochloric acid is added, (to be walked in 40 DEG C of water-baths
Corridor operation) stirring 3 minutes, it is allowed to dissolve.4 DEG C of 100ml of cold water is added in solution, chitin is just heavy as soliquid
Get off in shallow lake.Colloidal precipitation, after being washed with distilled water 2 times, the NaOH tune pH to 6.0 of 2mol/L, with high-speed homogenization machine are collected in centrifugation
After homogenate, it is settled to 100ml, adds 2g agar.
C liquid is prepared: 0.24g KH2PO4, 0.004g FeSO4·2H2O, 0.2g MgSO4·7H2O, 0.0004g ZnSO4,
0.8g yeast extract, 200ml water adjust pH 7.0.
Chitosan solid culture medium: the first liquid of falling A, the rear liquid of falling C；
Chitin solid medium: the first liquid of falling B, the rear liquid of falling C；
Above-mentioned culture medium passes through conventional sterilant processing.
The Bacillus that the present invention uses belongs to gram-positive bacteria, produces brood cell, and on LB culture medium, bacterium colony is high
Protrusion, bacterium colony rough surface is opaque, dirty white or yellowish, soft；The optimum growth temperature 28-37 of the bacillus
DEG C, the most suitable growth pH7.2-7.4.
The bacillus that the present invention uses is cultivated for 24 hours on the plating medium using chitin as sole carbon source, degrades several
Fourth matter generates significant transparent circle, indicates that this plant of bacterium has the biological nature for producing high activity chitinase.
The bacillus that the present invention uses (chitin content in the fluid nutrient medium using chitin as sole carbon source
1.0%) cultivate 36-48h, can efficient degradation chitin, chitin, which is completed, is converted into flocculent substance.
The Paecilomyces varioti strain that the present invention uses, on peptone dextrose agar medium, diameter reaches the left side 50mm after 7 days
The right side, open and flat, canescence, villiform is fine and close, later period powdery, and the back side is colourless；Using chitin as the fluid nutrient medium of sole carbon source
On, after shake culture 36h, bacterium solution is in lavender；It as sole carbon source and is adding the flat of 4- nitracetanilide using chitin
Cultivate 48h on plate culture medium, degradation chitin generates significant transparent circle, the diameter ratio of transparent circle and bacterium colony 2 or more, and
Culture medium within and around bacterium colony becomes yellow, indicates that this plant of bacterium has the biology for producing high activity chitin deacetylase special
Property；But 48h is cultivated on the plating medium using chitosan as sole carbon source, does not generate significant transparent circle, instruction is poly- without shell
Anase activity.The optimum growth temperature of this plant of bacterium is 23~30 DEG C, and the most suitable growth pH is 5.5~6.0.
Embodiment 1
A, feedstock processing:
Shrimp and crab shells are placed in pulverizer and grind, and crushed material decalcification is handled, and precipitating is collected by centrifugation, heavy containing chitin with this
Carbon source and nitrogen source of the starch as fermenting and producing add microelement, fermentation medium are made.
The composition of fermentation medium: 40g shrimp and crab shells powder, 2g yeast, 0.4g KH2PO4, 0.1g MgSO4,
0.005gFeSO4, 0.25mg ZnSO4, 1000mL water, PH=5.
B, prepared by strain:
(1) plate Spawn incubation: bacillus is crossed on the double-layer plate culture medium using chitin as sole carbon source
Inoculation, incubator are 28 DEG C, and culture for 24 hours, it is spare to have cultivated postposition refrigerator；By Paecilomyces varioti using chitin as the double of sole carbon source
Streak inoculation on layer plating medium, cultivates 72h, it is spare to have cultivated postposition refrigerator by 23 DEG C of incubator temperature.
(2) strain cultivation: with cultured Bacillus in oese picking (1), 50mL is inoculated into several
It in the fluid nutrient medium of Ding Zhiwei sole carbon source, is cultivated on shaking table, 28 DEG C of temperature, revolving speed 150rpm, cultivates 16h, cultivate
It is spare；50mL is inoculated into Paecilomyces varioti strain cultured in oese picking (1) to train by the liquid of sole carbon source of chitin
It supports in base, is cultivated on shaking table, 23 DEG C of temperature, revolving speed 150rpm, for 24 hours, culture is good spare for culture.
C, fermenting and producing
By fermentation medium made of (2) cultured bacillus and Paecilomyces varioti liquid spawn access 200mL step a
In, inoculum concentration is respectively 0.8ml and 0.2ml, is cultivated on shaking table, 25 DEG C of temperature, revolving speed 150rpm, and 36h is cultivated, and chitin is complete
Fermentation is terminated after degradable, supernatant is collected in centrifugation, obtains the tunning containing chitosan oligosaccharide.
D, the purification of chitosan oligosaccharide
The distilled water of 1/5 column volume is first added in glass chromatography column, opens bottom of the pillar cock, bubble is discharged, then
The polyacrylamide gel resin of distilled water immersion is slowly poured into chromatographic column while stirring, about 340mL resin is added altogether；To
After pillar installs, constant flow pump is opened, with the 0.1molL of 2-3 times of volume-1NH4HCO3Resin is rinsed, until resin is compacted；It takes
Chitosan oligosaccharide product obtained above is dissolved in 2mL 0.1molL-1NH4HCO3Solution, until completely dissolved, with 0.22 μm of filter membrane mistake
Tunning solution, is then slowly added by filter along wall.After sample is added completely into column, cock is opened, makes solution is past to flow down
Out, when sample solution is equal with cylinder, constant flow pump is opened, 0.1molL is used-1NH4HCO3Solution is eluted.It opens automatic
Fraction collector is collected 1 pipe by certain every 10min of flow velocity, is detected after concentrated by rotary evaporation using TLC method, and by identical group
Division is simultaneously collected.
Embodiment 2
A, feedstock processing:
Shrimp and crab shells are placed in pulverizer and grind, and crushed material decalcification is handled, and precipitating is collected by centrifugation, heavy containing chitin with this
Carbon source and nitrogen source of the starch as fermenting and producing add microelement, fermentation medium are made.
The composition of fermentation medium: 80g shrimp and crab shells powder, 4g yeast, 0.9g KH2PO4, 0.2g MgSO4, 0.01gFeSO4,
0.25mg ZnSO4, 1000mL water, PH=5.
B, prepared by strain:
(1) plate Spawn incubation: bacillus is crossed on the double-layer plate culture medium using chitin as sole carbon source
Inoculation, incubator are 25 DEG C, and culture for 24 hours, it is spare to have cultivated postposition refrigerator；By Paecilomyces varioti using chitin as the double of sole carbon source
Streak inoculation on layer plating medium, cultivates 72h, it is spare to have cultivated postposition refrigerator by 20 DEG C of incubator temperature.
(2) strain cultivation: with cultured Bacillus in oese picking (1), 50mL is inoculated into several
It in the fluid nutrient medium of Ding Zhiwei sole carbon source, is cultivated on shaking table, 25 DEG C of temperature, revolving speed 150rpm, cultivates 12h, cultivate
It is spare；50mL is inoculated into Paecilomyces varioti strain cultured in oese picking (1) to train by the liquid of sole carbon source of chitin
It supports in base, is cultivated on shaking table, 20 DEG C of temperature, revolving speed 150rpm, for 24 hours, culture is good spare for culture.
C, fermenting and producing
By fermentation medium made of (2) cultured bacillus and Paecilomyces varioti liquid spawn access 200mL step a
In, inoculum concentration is respectively 0.4ml and 0.6ml, is cultivated on shaking table, 23 DEG C of temperature, revolving speed 150rpm, and for 24 hours, chitin is complete for culture
Fermentation is terminated after degradable, supernatant is collected in centrifugation, obtains the tunning containing chitosan oligosaccharide.
D, the purification of chitosan oligosaccharide
The distilled water of 1/5 column volume is first added in glass chromatography column, opens bottom of the pillar cock, bubble is discharged, then
The polyacrylamide gel resin of distilled water immersion is slowly poured into chromatographic column while stirring, about 340mL resin is added altogether；To
After pillar installs, constant flow pump is opened, with the 0.1molL of 2-3 times of volume-1NH4HCO3Resin is rinsed, until resin is compacted；It takes
Chitosan oligosaccharide product obtained above is dissolved in 2mL 0.1molL-1NH4HCO3Solution, until completely dissolved, with 0.22 μm of filter membrane mistake
Tunning solution, is then slowly added by filter along wall.After sample is added completely into column, cock is opened, makes solution is past to flow down
Out, when sample solution is equal with cylinder, constant flow pump is opened, 0.1molL is used-1NH4HCO3Solution is eluted.It opens automatic
Fraction collector is collected 1 pipe by certain every 10min of flow velocity, is detected after concentrated by rotary evaporation using TLC method, and by identical group
Division is simultaneously collected.
Embodiment 3
A, feedstock processing:
Shrimp and crab shells are placed in pulverizer and grind, and crushed material decalcification is handled, and precipitating is collected by centrifugation, heavy containing chitin with this
Carbon source and nitrogen source of the starch as fermenting and producing add microelement, fermentation medium are made.
The composition of fermentation medium: 50g shrimp and crab shells powder, 3g yeast, 0.6g KH2PO4, 0.1g MgSO4, 0.01gFeSO4,
0.25mg ZnSO4, 1000mL water, PH=5.
B, prepared by strain:
(1) plate Spawn incubation: bacillus is crossed on the double-layer plate culture medium using chitin as sole carbon source
Inoculation, incubator are 37 DEG C, cultivate 48h, it is spare to have cultivated postposition refrigerator；By Paecilomyces varioti using chitin as the double of sole carbon source
Streak inoculation on layer plating medium, cultivates 120h, it is spare to have cultivated postposition refrigerator by 30 DEG C of incubator temperature.
(2) strain cultivation: with cultured Bacillus in oese picking (1), 50mL is inoculated into several
It in the fluid nutrient medium of Ding Zhiwei sole carbon source, is cultivated on shaking table, 37 DEG C of temperature, revolving speed 150rpm, culture for 24 hours, is cultivated
It is spare；50mL is inoculated into Paecilomyces varioti strain cultured in oese picking (1) to train by the liquid of sole carbon source of chitin
It supports in base, is cultivated on shaking table, 30 DEG C of temperature, revolving speed 150rpm, cultivate 48h, culture is good spare.
C, fermenting and producing
By fermentation medium made of (2) cultured bacillus and Paecilomyces varioti liquid spawn access 200mL step a
In, inoculum concentration is all 0.5ml and 0.5ml, is cultivated on shaking table, 30 DEG C of temperature, revolving speed 150rpm, and 48h is cultivated, and chitin is complete
Fermentation is terminated after degradation, supernatant is collected in centrifugation, obtains the tunning containing chitosan oligosaccharide.
D, the purification of chitosan oligosaccharide
The distilled water of 1/5 column volume is first added in glass chromatography column, opens bottom of the pillar cock, bubble is discharged, then
The polyacrylamide gel resin of distilled water immersion is slowly poured into chromatographic column while stirring, about 340mL resin is added altogether；To
After pillar installs, constant flow pump is opened, with the 0.1molL of 2-3 times of volume-1NH4HCO3Resin is rinsed, until resin is compacted；It takes
Chitosan oligosaccharide product obtained above is dissolved in 2mL 0.1molL-1NH4HCO3Solution, until completely dissolved, with 0.22 μm of filter membrane mistake
Tunning solution, is then slowly added by filter along wall.After sample is added completely into column, cock is opened, makes solution is past to flow down
Out, when sample solution is equal with cylinder, constant flow pump is opened, 0.1molL is used-1NH4HCO3Solution is eluted.It opens automatic
Fraction collector is collected 1 pipe by certain every 10min of flow velocity, is detected after concentrated by rotary evaporation using TLC method, and by identical group
Division is simultaneously collected.
Embodiment 4
A, feedstock processing:
Shrimp and crab shells are placed in pulverizer and grind, and crushed material decalcification is handled, and precipitating is collected by centrifugation, heavy containing chitin with this
Carbon source and nitrogen source of the starch as fermenting and producing add microelement, fermentation medium are made.
The composition of fermentation medium: 200g shrimp and crab shells powder, 20g yeast, 1.2g KH2PO4, 0.4g MgSO4,
0.02gFeSO4, 0.40mg ZnSO4, 1000mL water, PH=5.
B, prepared by strain:
(1) plate Spawn incubation: bacillus is crossed on the double-layer plate culture medium using chitin as sole carbon source
Inoculation, incubator are 32 DEG C, cultivate 48h, it is spare to have cultivated postposition refrigerator；By Paecilomyces varioti using chitin as the double of sole carbon source
Streak inoculation on layer plating medium, cultivates 96h, it is spare to have cultivated postposition refrigerator by 28 DEG C of incubator temperature.
(2) strain cultivation: with cultured Bacillus in oese picking (1), 50mL is inoculated into several
It in the fluid nutrient medium of Ding Zhiwei sole carbon source, is cultivated on shaking table, 32 DEG C of temperature, revolving speed 150rpm, culture for 24 hours, is cultivated
It is spare；50mL is inoculated into Paecilomyces varioti strain cultured in oese picking (1) to train by the liquid of sole carbon source of chitin
It supports in base, is cultivated on shaking table, 28 DEG C of temperature, revolving speed 150rpm, cultivate 48h, culture is good spare.
C, fermenting and producing
By fermentation medium made of (2) cultured bacillus and Paecilomyces varioti liquid spawn access 200mL step a
In, inoculum concentration is respectively 0.6ml and 0.4ml, is cultivated on shaking table, 28 DEG C of temperature, revolving speed 150rpm, and 72h is cultivated, and chitin is complete
Fermentation is terminated after degradable, supernatant is collected in centrifugation, obtains the tunning containing chitosan oligosaccharide.
D, the purification of chitosan oligosaccharide
The distilled water of 1/5 column volume is first added in glass chromatography column, opens bottom of the pillar cock, bubble is discharged, then
The polyacrylamide gel resin of distilled water immersion is slowly poured into chromatographic column while stirring, about 340mL resin is added altogether；To
After pillar installs, constant flow pump is opened, with the 0.1molL of 2-3 times of volume-1NH4HCO3Resin is rinsed, until resin is compacted；It takes
Chitosan oligosaccharide product obtained above is dissolved in 2mL 0.1molL-1NH4HCO3Solution, until completely dissolved, with 0.22 μm of filter membrane mistake
Tunning solution, is then slowly added by filter along wall.After sample is added completely into column, cock is opened, makes solution is past to flow down
Out, when sample solution is equal with cylinder, constant flow pump is opened, 0.1molL is used-1NH4HCO3Solution is eluted.It opens automatic
Fraction collector is collected 1 pipe by certain every 10min of flow velocity, is detected after concentrated by rotary evaporation using TLC method, and by identical group
Division is simultaneously collected.
The embodiment of the present invention is with reference to " the deacetylated crust of GB 29941-2013 national food safety standard food additives
Plain (chitosan) " method analyze the deacetylation degree of obtained chitosan oligosaccharide, the deacetylation degree detection of embodiment 1-4 is tied
Fruit is as shown in table 1:
Table 1
According to the result of table 1, it is known that the present invention can effectively be degraded shrimp using the synergistic effect of bacillus and Paecilomyces varioti
Crab shell prepares chitosan oligosaccharide, and the chitosan oligosaccharide deacetylation degree obtained is high；Wherein, embodiment 4 is in preparation method by shrimp and crab shells
The ratio of powder and water is improved to 1:5, and the sugared deacetylation degree of chitotriose, shell tetrose, shell pentasaccharides and shell six obtained, which has, obviously to be mentioned
It rises, and after incubation time 72h, observes that chitin is degradable.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (7)
1. a kind of method for preparing chitosan oligosaccharide using chitin in bacillus and Paecilomyces varioti Synergistic degradation shrimp and crab shells, feature exist
In: fermentation medium is made by raw material of the shrimp and crab shells after decalcification, bacillus is inoculated with and Paecilomyces varioti is degraded through everfermentation
To chitosan oligosaccharide.
2. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the following steps are included:
Step 1: shrimp and crab shells are ground, precipitating is collected by centrifugation after decalcification is handled, adds nitrogen source and microelement, fermentation is made
Culture medium, it is spare；
Step 2: by bacillus streak inoculation on the double-layer plate culture medium using chitin as sole carbon source, cultivation temperature
It 25-45 DEG C, after cultivating 24-48h, refrigerates spare；By Paecilomyces varioti on the double-layer plate culture medium using chitin as sole carbon source
Streak inoculation, after cultivating 36-96h, refrigerates spare by 20-37 DEG C of cultivation temperature；
Step 3: picking step 2 Bacillus, is inoculated into using chitin to cultivate in the fluid nutrient medium of sole carbon source
It is 25-45 DEG C of temperature, shake culture 12-24h, spare；Picking step 2 Paecilomyces varioti strain, is inoculated into using chitin as sole carbon source
Fluid nutrient medium in, it is 20-37 DEG C of cultivation temperature, shake culture 24-72h, spare；
Step 4: the liquid spawn of step 3 bacillus and Paecilomyces varioti is inoculated into fermentation medium made of step 1,
23-37 DEG C of cultivation temperature, shake culture 24-72h, obtain the product containing chitosan oligosaccharide.
3. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the Bacillus strain is dirty white or yellowish, bacterium in the colony characteristics of LB culture medium
Drop height protrusion, bacterium colony rough surface is opaque, soft；
The Bacillus strain using chitin as the colony characteristics of the plating medium of sole carbon source be have it is apparent transparent
Circle.
4. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the Paecilomyces varioti bacterial strain is greyish white in the colony characteristics of peptone dextrose agar medium
Color, open and flat, villiform is fine and close, later period powdery, and the back side is colourless；
The Paecilomyces varioti bacterial strain is being bacterium solution in lavender by the colony characteristics of the fluid nutrient medium of sole carbon source of chitin；
The Paecilomyces varioti bacterial strain is using chitin as sole carbon source and the bacterium colony of the plating medium that adds 4- nitracetanilide
Feature is to have apparent transparent circle, and the culture medium within and around bacterium colony becomes yellow；
The Paecilomyces varioti bacterial strain is being no transparent circle by the colony characteristics of the plating medium of sole carbon source of chitosan.
5. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the composition of the fermentation medium are as follows: 40-200g shrimp and crab shells powder, 2-20g yeast, 0.4-
1.2g KH2PO4, 0.1-0.4g MgSO4, 0.005-0.02gFeSO4, 0.25-0.4mg ZnSO4, 1000mL water, PH=5.0-
6.0。
6. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the bacillus is bacillus licheniformis, bacillus alcalophilus, bacillus subtilis, solution
One or more of bacillus amyloliquefaciens, bacillus thuringiensis and Paenibacillus polymyxa.
7. chitin prepares shell in a kind of utilization bacillus according to claim 1 and Paecilomyces varioti Synergistic degradation shrimp and crab shells
The method of oligosaccharides, it is characterised in that: the Paecilomyces varioti is Paecilomyces lilacinus, Paecilomyces hepiali chen, Paecilomyces cicadae, the quasi- blueness of silty
It is one or more of mould.
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| CN111642617A (en) * | 2020-06-19 | 2020-09-11 | 福建省大丰山禽业发展有限公司 | Special microecological preparation for laying hens and preparation method thereof |
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