CN101880699A - Method for producing chitooligosaccharides by using microbial fermentation - Google Patents
Method for producing chitooligosaccharides by using microbial fermentation Download PDFInfo
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- CN101880699A CN101880699A CN2010102127494A CN201010212749A CN101880699A CN 101880699 A CN101880699 A CN 101880699A CN 2010102127494 A CN2010102127494 A CN 2010102127494A CN 201010212749 A CN201010212749 A CN 201010212749A CN 101880699 A CN101880699 A CN 101880699A
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Abstract
The invention discloses a process for producing chitooligosaccharides through microbial fermentation by using microbial bacteria, namely paecilomyces lilacinus as zymophyte and using shrimp shell meal as a raw material. The process comprises the following steps of: degrading shrimp and crab shells by using aerobic submerged fermentation of the microbial bacteria, namely the paecilomyces lilacinus, enriching a fermentation product to a certain degree at the middle late stage of the fermentation by a re-flowing process, centrifuging fermentation solution and removing the bacteria to obtain supernate, namely a chitooligosaccharides product with certain concentration, and concentrating the supernate to further produce the high-concentration chitooligosaccharides. The obtained chitooligosaccharides can be used for the fields of medicament, agriculture and food; and the whole production process is simple, convenient and efficient, has wide industrialized development prospect, and develops a green application path for the shrimp and crab shells.
Description
Technical field:
The present invention relates to biological technical field, particularly a kind of method of producing chitin oligosaccharide with microbial fermentation.
Background technology:
Current domestic utilization to chitin, mainly based on chitosan, and chitin oligosaccharide is with a wide range of applications in fields such as medicine, food as a kind of functional oligose, and its market value is higher about 10 times than chitosan.Chitin oligosaccharide is existing at home to be produced, and is to adopt the acid and alkali hydrolysis method mostly, and the employing enzymolysis process is also arranged, but all is to adopt combined-enzyme method, and its cost costliness, scale are less.Also have only a few countries such as Japan that scale production is arranged abroad.In recent years to the going deep into of the function of chitin oligosaccharide, property research, it is applied in and is subjected to both at home and abroad paying close attention to widely, and market outlook are wide.
The raw material that chitin oligosaccharide is produced derives from the waste materials such as shrimp, crab shell of sea-food source mill.China's oceanic area is extremely vast, and the sea-food turnout is very big, and these resources are fine very sufficient, but are not developed effectively yet so far and utilize, thereby causes potential to threaten to environment.So this resource of rational exploitation and utilization not only has economics meaning, also has very important ecological significance.
Traditional method for preparing chitin oligosaccharide is the deacetylation chemical method, and the energy is many, required equipment is complicated because production process expends, adopt a large amount of soda acid consumptions, and the while is contaminate environment also.Therefore; people begin to seek the biological method deacetylation; mostly adopt enzymolysis process now; owing to there is not single, efficient, narrow spectrum enzyme so far; need to adopt the compound enzymolysis; cost is expensive, and does not have science, standardization, suitable enzymatic reaction system at enzymolysis process, still has with a certain distance from large-scale industrial production.
Summary of the invention:
At traditional preparation method is the deacetylation chemical method; its production process expends the soda acid consumption that the energy is many, required equipment is complicated, employing is a large amount of; also problem such as contaminate environment simultaneously; and the biologic enzymolysis method that adopts now; that this working system lacks is cheap, efficient, specificity enzyme and rational reactive system, makes and produces the present situation that lacks capability of industrialization.The invention provides a kind of method with microbial bacteria fermentative production chitin oligosaccharide, it is Paecilomyces lilacinus bacterium Paecilomyces lilacinus oxygen consumption submerged fermentation degraded shrimp, crab shell with microbial bacteria that this method is utilized a kind of, and add technology by stream again in the fermentation middle and later periods, produce the higher concentration chitin oligosaccharide, effectively avoided traditional chitin oligosaccharide production process to adopt a large amount of soda acid consumptions, improve rate of energy effectively, can realize cheapness, low-cost suitability for industrialized production chitin oligosaccharide simultaneously again.
The object of the present invention is achieved like this:
A kind of method of producing chitin oligosaccharide with microbial fermentation, it is a raw material with fresh shrimp shell or crab shell, through preparation, fermenation raw liquid preparation, fermenation raw liquid sterilization, inoculation fermentation, flow feeding, centrifugal thalline, vacuum concentration, spraying drying, the product purification of going of raw material, obtain the product chitin oligosaccharide.
Fresh shrimp shell or crab shell are dried, be ground into 120 purpose dry powder, shrimp, crab shell dry powder and the water with weight ratio 2.5% mixes then, adds 0.02% peptone again, makes fermenation raw liquid.The fermenation raw liquid for preparing is pumped into fermentor tank, and airtight tank body adds steam and sterilizes, and makes material temperature rise to 121 ℃, is incubated 30 minutes, is cooled to 28 ℃ then gradually; In the good fermented liquid of sterilization, inoculate the good zymophyte of seed tank culture, stir ventilating fermentation, rigidly connecting kind of a rear venting amount is 1:0.05v/v.min, process in fermentation, according to the generation of carbonic acid gas, progressively increase ventilation again, and progressively improve leavening temperature, growing amount at fermenting process results of regular determination reducing sugar content and chitin oligosaccharide.Wherein the microbial bacteria of the present invention's screening is the Paecilomyces lilacinus bacterium, can secrete the outer chitin enzyme of a certain amount of born of the same parents, under artificial fermentation control condition, make it have the ability that continues the degraded chitin, chitin is degraded forms the chitin oligosaccharide of certain molecular weight (3000～4000) and concentration.Shrimp, crab shell (chitin) for this bacterium breeding provides C/N nutrition, are converted into fermentating metabolism product (chitin oligosaccharide) on the one hand on the other hand during the fermentation.In the production process of fermentation, we add the way of technology again by artificial control fermentation manufacturing technique and stream, flow feeding is 10% shrimp, crab shell powder mixing suspension, place stream to add jar, feed steam and be warming up to 121 ℃, be incubated sterilization in 30 minutes, the cooling back is standby, when the middle and later periods of fermentation, divide progressively stream adding fermentor tank three times; Make the content of tunning obtain to a certain degree enrichment.Survey contains chitin oligosaccharide content during fermentation ends, directly pumps into whizzer, in whizzer 5000rpm, and centrifugal 30 minutes, obtain supernatant liquor, pump into supernatant liquor in the storage tank standby; Fermented liquid is through bactofugation, and the supernatant liquor that obtains is certain density chitin oligosaccharide product.Concentrate through concentration tank and then, supernatant liquor is pumped in the double-effect evaporation concentration tank, carry out vacuum concentration, be concentrated into concentrated solution chitin oligosaccharide content at 10～15% o'clock, it is standby to pump into storage tank, the concentration of chitin oligosaccharide is further improved, last again by the method for low temperature spray drying, with the chitin oligosaccharide concentrated solution, adopt spray-drier, by regulating the opening for feed temperature is 150 ℃, 70～75 ℃ of discharge port temperature are carried out spraying drying, make chitin oligosaccharide xeraphium product, the chitin oligosaccharide yield can reach more than 96%, good water solubility.In the spray-dired powder, also contain a certain amount of inorganic salt composition, so the content purity of chitin oligosaccharide 3000～4000 molecular weight is 93.6%.This product eats as agricultural, herding, and purity is feasible fully, further improves the content product purification as need, can carry out purifying by 2 times edible ethanol, can obtain 99% content chitin oligosaccharide product, and is water-soluble fully.
The present invention has this project integrated use microbial fermentation law technology and produces chitin oligosaccharide, effectively avoided traditional chitin oligosaccharide production process to adopt a large amount of soda acid consumptions, improved rate of energy effectively, and avoided with the higher production cost of enzyme process.Can obtain the higher chitin oligosaccharide product of purity more at an easy rate, this product can be applicable to fields such as medicine, agricultural, food, and technology is reached advanced world standards.
Description of drawings:
Fig. 1 is a kind of method process flow sheet of producing chitin oligosaccharide with microbial fermentation of the present invention.
Embodiment:
Embodiment 1:
In the good fermented liquid of sterilization, inoculate the good zymophyte of seed tank culture, stir ventilating fermentation, rigidly connecting kind of a rear venting amount is 1:0.05v/v.min, process in fermentation, again according to the generation of carbonic acid gas, progressively increase ventilation, when 5 gram equivalents of every increases of every cube of gas volume of carbon dioxide content, ventilation just increases 0.05v/min, and progressively improves leavening temperature, divide and improve temperature four times, initial temperature is 28 ℃, and temperature raising for the first time in 10 hours is 29 ℃, and temperature raising for the second time is 30 ℃ about 16 hours, temperature raising for the third time is 31 ℃ about 20 hours, and the 4th temperature raising is 33.5 ℃ about 28 to 30 hours.
Embodiment 2:
The preparation of bacterial classification: take out the preservation inclined-plane of Paecilomyces lilacinus bacterial classification from refrigerator, be transferred on the production inclined-plane, 5 production inclined-planes of switching, a general preservation inclined-plane carry out actication of culture, again 28 ℃ incubator are put on the good production inclined-plane of switching and are cultivated 3 days; Can inoculate in the one-level triangular flask 28 ℃ and cultivate 3 days after covering with the inclined-plane, the substratum in the triangular flask is that 2.5% shrimp, crab shrimp shell powder suspension and 0.02% peptone are made substratum; Can insert in the secondary triangular flask 28 ℃ through microscopy such as mycelia stalwartness and cultivate 2 days, secondary triangular flask substratum is identical with one-level; Can insert in the seeding tank 28 ℃ through microscopy such as mycelia stalwartness and cultivate 36 hours, inoculum size is 5%V/V.
Embodiment 3:
For further improving the content of chitin oligosaccharide, the edible ethanol of 1.5 times of amounts of powder chitin oligosaccharide solid volume that available spraying drying is intact dissolves, make and dissolve in alcoholic acid inorganic salt and other soluble impurities and be dissolved in the ethanol because chitin oligosaccharide is not allowed ethanol, stir 20min, Plate Filtration, obtain dry-matter, low temperature vacuumizes drying, just can obtain 99% content chitin oligosaccharide.
Embodiment 4:
Bacterial classification separates the multiple Paecilomyces lilacinus bacterial strain of acquisition from multiple soil, with the good bacterial strain of Analysis of Plate crust degradation capability, this bacterial strain is source Nan Jing subtropics virgin forest soil, separate the Paecilomyces lilacinus bacterial strain that obtains, again by cobalt-60 and alternately mutagenesis of ultraviolet ray, obtain the shrimp of directly to degrade, crab shell powder, form the new Paecilomyces lilacinus bacterial strain of chitin oligosaccharide (3000～4000 molecular weight) product, by inoculation triangular flask shrimp repeatedly, fermentation of crab shell powder and the fermentation of inoculation seeding tank, adopt under meter, the carbonic acid gas detector, thermometer, acidometer, the mensuration of instruments such as liquid chromatograph, obtain one group of fermentation parameter, according to the fermentation engineering principle, determine suitable leavening temperature and fermentation ventilation.Fermentation condition is promptly: 28 ℃, one time temperature raising to 29 of initial temperature ℃, secondary temperature raising to 30 ℃, three temperature raisings to 31 ℃, four temperature raisings to 33 ℃; Fermentation period is 60 hours; The fermentation ventilation is: initial air quantity 1:0.05, secondary air flow 1:0.1, tertiary air quantity 1:0.15, four air quantity 1:0.2, five air quantity 1:0.25.The final product that obtains certain content satisfies the needed concentration of suitability for industrialized production substantially, and production cost is lower, and product purity is higher, good water solubility.
Claims (3)
1. method of producing chitin oligosaccharide with microbial fermentation, it is characterized in that with fresh shrimp shell or crab shell be raw material, through preparation, fermenation raw liquid preparation, fermenation raw liquid sterilization, inoculation fermentation, flow feeding, centrifugal thalline, vacuum concentration, spraying drying, the product purification of going of raw material, obtain the product chitin oligosaccharide;
(1) preparation of raw material: fresh shrimp shell or crab shell dry, and it is standby to be ground into 120 purpose dry powder;
(2) fermenation raw liquid preparation: shrimp, crab shell powder and water with weight ratio 2.5% mix, and add 0.02% peptone again, make fermenation raw liquid;
(3) fermenation raw liquid sterilization: the fermenation raw liquid for preparing is pumped into fermentor tank, and airtight tank body adds steam and sterilizes, and makes to expect temperature rise to 121 ℃ to be incubated 30 minutes, is cooled to 28 ℃ then gradually;
(4) inoculation fermentation: in the good fermented liquid of sterilization, inoculate the good zymophyte of 5% seed tank culture, stir ventilating fermentation, rigidly connecting kind of a rear venting amount is 1:0.05v/v.min, process in fermentation, according to the generation of carbonic acid gas, progressively increase ventilation again, and progressively improve leavening temperature, growing amount at fermenting process results of regular determination reducing sugar content and chitin oligosaccharide;
(5) flow feeding: stream is reinforced to be 10% shrimp, crab shell powder mixing suspension, places stream to add jar, and feeding steam is warming up to 121 ℃, is incubated sterilization in 30 minutes, and the cooling back is standby, when the middle and later periods of fermentation, divides progressively stream adding fermentor tank three times;
(6) the centrifugal thalline that goes: need survey during fermentation ends and contain chitin oligosaccharide content, directly pump into whizzer, in whizzer 5000rpm, centrifugal 30 minutes, obtain supernatant liquor, supernatant liquor is pumped in the storage tank standby, sampling Detection contains the chitin oligosaccharide amount;
(7) vacuum concentration: supernatant liquor is pumped in the double-effect evaporation concentration tank, carry out vacuum concentration, be concentrated into concentrated solution chitin oligosaccharide content at 10～15% o'clock, it is standby to pump into storage tank;
(8) spraying drying: with the chitin oligosaccharide concentrated solution, adopting spray-drier, is 150 ℃ by regulating the opening for feed temperature, 70～75 ℃ of discharge port temperature are carried out spraying drying, make chitin oligosaccharide xeraphium product, the chitin oligosaccharide yield can reach more than 96%, good water solubility.
(9) product purification: in the spray-dired powder, also contain a certain amount of inorganic salt composition, so the content purity of chitin oligosaccharide 3000～4000 molecular weight is 93.6%.This product eats as agricultural, herding, and purity is feasible fully, further improves content as need, can carry out purifying by 2 times edible ethanol, can obtain 99% content chitin oligosaccharide product, and is water-soluble fully.
2. a kind of method of producing chitin oligosaccharide with microbial fermentation according to claim 1, it is characterized in that 2.5% shrimp, crab shell powder mixing suspension are that 1.5% shrimp shell meal and 1% crab shell powder mix with water with 0.02 peptone and make fermented liquid, the stream liquid feeding is that 10% shrimp, crab shell powder mixing suspension are that 6% shrimp shell meal and 4% crab shell powder and water are mixed into suspension.
3. a kind of method of producing chitin oligosaccharide with microbial fermentation according to claim 1, it is characterized in that planting the good zymophyte of seed tank culture, its zymophyte is for cultivating the preparation of sophisticated Paecilomyces lilacinus bacterium bacterial classification: and slant preservation bacterial classification → activation production inclined-plane → one-level triangular flask spreads cultivation → and the secondary triangular flask spreads cultivation → and seed culture is a mature strains, inoculum size is 5% fermentation liquid measure.
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| CN103695315A (en) * | 2013-10-12 | 2014-04-02 | 福建三炬生物科技股份有限公司 | Method for producing chitooligosaccharide by microbial fermentation |
| CN104293700A (en) * | 2014-09-19 | 2015-01-21 | 中农颖泰林州生物科园有限公司 | Production process of bacillus licheniformis for feed |
| CN105234167A (en) * | 2015-11-16 | 2016-01-13 | 福建三炬生物科技股份有限公司 | Heavy metal bioremediation agent containing alga oligosaccharides and preparing method of agent |
| CN106747934A (en) * | 2016-11-21 | 2017-05-31 | 黄河三角洲京博化工研究院有限公司 | A kind of production method rich in chitosan oligosaccharide organic fertilizer |
| CN107087641A (en) * | 2017-02-08 | 2017-08-25 | 福建三炬生物科技股份有限公司 | A kind of marine oligosaccharide biological agent for improving crop salt-resistance and preparation method thereof |
| CN109053237A (en) * | 2018-09-18 | 2018-12-21 | 中国海洋大学 | A kind of preparation method of the foliar fertilizer of natural plants containing chitinous oligomers |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695315A (en) * | 2013-10-12 | 2014-04-02 | 福建三炬生物科技股份有限公司 | Method for producing chitooligosaccharide by microbial fermentation |
| CN103695315B (en) * | 2013-10-12 | 2016-01-20 | 福建三炬生物科技股份有限公司 | A kind of fermentable produces the method for chitin oligosaccharide |
| CN104293700A (en) * | 2014-09-19 | 2015-01-21 | 中农颖泰林州生物科园有限公司 | Production process of bacillus licheniformis for feed |
| CN105234167A (en) * | 2015-11-16 | 2016-01-13 | 福建三炬生物科技股份有限公司 | Heavy metal bioremediation agent containing alga oligosaccharides and preparing method of agent |
| CN105234167B (en) * | 2015-11-16 | 2017-12-08 | 福建三炬生物科技股份有限公司 | A kind of renovation agent of heavy metal biological containing alga oligosaccharides and preparation method thereof |
| CN106747934A (en) * | 2016-11-21 | 2017-05-31 | 黄河三角洲京博化工研究院有限公司 | A kind of production method rich in chitosan oligosaccharide organic fertilizer |
| CN107087641A (en) * | 2017-02-08 | 2017-08-25 | 福建三炬生物科技股份有限公司 | A kind of marine oligosaccharide biological agent for improving crop salt-resistance and preparation method thereof |
| CN107087641B (en) * | 2017-02-08 | 2021-04-13 | 福建三炬生物科技股份有限公司 | Marine oligosaccharide biological preparation for improving salt resistance of crops and preparation method thereof |
| CN109053237A (en) * | 2018-09-18 | 2018-12-21 | 中国海洋大学 | A kind of preparation method of the foliar fertilizer of natural plants containing chitinous oligomers |
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