CN101880699B

CN101880699B - Method for producing chitooligosaccharides by using microbial fermentation

Info

Publication number: CN101880699B
Application number: CN201010212749.4A
Authority: CN
Inventors: 尤越; 林克明; 林明煌
Original assignee: Fujian Sanju Biological Science & Technology Co Ltd
Current assignee: Fujian Sanju Biological Scientech Co., Ltd.
Priority date: 2010-06-29
Filing date: 2010-06-29
Publication date: 2015-07-01
Anticipated expiration: 2030-06-29
Also published as: CN101880699A

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

A kind of fermentable produces the method for chitin oligosaccharide

Technical field:

The present invention relates to biological technical field, particularly a kind of fermentable produces the method for chitin oligosaccharide.

Background technology:

Present Domestic is to the utilization of chitin, and mainly based on chitosan, and chitin oligosaccharide is as a kind of functional oligose, and be with a wide range of applications in fields such as medicine, food, its market value is higher than chitosan about 10 times.Chitin oligosaccharide is existing at home to be produced, and be adopt acid and alkali hydrolysis method mostly, also have employing enzymolysis process, but be all adopt combined-enzyme method, its cost intensive, scale are less.External also only have a few countries such as Japan to have scale production.In recent years to the function of chitin oligosaccharide, the going deep into of character research, it is applied in is paid close attention to both at home and abroad widely, wide market.

The raw material that chitin oligosaccharide is produced, derives from the waste material such as the shrimp of marine products processing factory, crab shell.China's oceanic area is extremely vast, and sea-food turnout is very large, and these resources are very sufficient very well, but are still not operatively developed so far and utilize, and thus cause potential threat to environment.So this resource of rational exploitation and utilization, not only there is economics meaning, also have very important ecological significance.

Traditional method preparing chitin oligosaccharide is deacetylation chemistry method, and because production process expends, the energy is many, required equipment is complicated, adopt a large amount of soda acid consumptions, simultaneously also contaminate environment.Therefore; people start to find biological method deacetylation; mostly adopt enzymolysis process now; owing to there is no single, efficient, narrow spectrum enzyme so far; need the enzymolysis adopting compound; cost costly, 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:

For traditional preparation method be deacetylation chemistry method; its production process expends that the energy is many, required equipment is complicated, adopt a large amount of soda acid consumptions; the simultaneously also problem such as contaminate environment; and the biologic enzymolysis method adopted now; this working system lacks cheap, efficient, specificity enzyme and rational reactive system, makes production lack the present situation of capability of industrialization.The invention provides a kind of method of microbial bacteria fermentative production chitin oligosaccharide, the method utilizes a kind of microbial bacteria and Paecilomyces lilacinus Paecilomyces lilacinus oxygen consumption submerged fermentation degraded shrimp, crab shell, and pass through feeding method again in the fermentation middle and later periods, produce higher concentration chitin oligosaccharide, effectively prevent traditional chitin oligosaccharide production process and adopt a large amount of soda acid consumptions, effectively improve the utilization ratio of the energy, cheapness can be realized again simultaneously, low-cost industrial produces chitin oligosaccharide.

The object of the present invention is achieved like this:

A kind of fermentable produces the method for chitin oligosaccharide, its with fresh shrimp shell or crab shell for raw material, through the preparation of raw material, fermenation raw liquid preparation, fermenation raw liquid sterilizing, inoculation fermentation, flow feeding, centrifugally remove thalline, vacuum concentration, spraying dry, product purification, obtain product chitin oligosaccharide.

Fresh shrimp shell or crab shell are dried, is ground into 120 object dry powder, then mix with water with the shrimp of weight ratio 2.5%, crab shell dry powder, then add 0.02% peptone, make fermenation raw liquid.The fermenation raw liquid prepared is pumped into fermentor tank, airtight tank body, add steam and carry out sterilizing, make material temperature rise to 121 DEG C, be incubated 30 minutes, be then cooled to 28 DEG C gradually; Inoculate in the fermented liquid that sterilizing is good into the good zymophyte of seed tank culture, air blow and agitation ferments, firm inoculation rear venting amount is 1:0.05v/v.min, in the process of fermentation, again according to the generation of carbonic acid gas, progressively increase ventilation, and progressively improve leavening temperature, at fermenting process results of regular determination reducing sugar content, and the growing amount of chitin oligosaccharide.Wherein the present invention screening microbial bacteria and Paecilomyces lilacinus, the outer chitinase of a certain amount of born of the same parents can be secreted, under artificial fermentation control condition, make it have the ability of sustaining degradation chitin, chitin is degraded and forms the chitin oligosaccharide of certain molecular weight (3000 ~ 4000) and concentration.Shrimp, crab shell (chitin) are on the one hand for the breeding of this bacterium provides C/N nutrition during the fermentation, are converted into fermentating metabolism product (chitin oligosaccharide) on the other hand.In the production process of fermentation, we pass through the way of manual control fermentation manufacturing technique and feeding method again, flow feeding is 10% shrimp, crab shell powder mixing suspension, be placed in stream and add tank, pass into steam and be warming up to 121 DEG C, be incubated sterilizing in 30 minutes, for subsequent use after cooling, when the middle and later periods of fermenting, points three times progressively stream add fermentor tank; The content of tunning is made to obtain enrichment to a certain extent.Survey containing chitin oligosaccharide content during fermentation ends, directly pump into whizzer, in whizzer 5000rpm, centrifugal 30 minutes, obtain supernatant liquor, supernatant liquor is pumped in storage tank for subsequent use; Fermentation liquor bactofugation, the supernatant liquor obtained is certain density chitin oligosaccharide product.Concentrate through concentration tank and then, supernatant liquor is pumped in double-effect evaporation concentration tank, carry out vacuum concentration, be concentrated into concentrated solution chitin oligosaccharide content 10 ~ 15% time, pump into storage tank for subsequent use, the concentration of chitin oligosaccharide can be made to improve further, last again by the method for low temperature spray drying, by chitin oligosaccharide concentrated solution, adopt spray-drier, by regulating inlet temperature to be 150 DEG C, discharge port temperature 70 ~ 75 DEG C, carries out spraying dry, obtained chitin oligosaccharide xeraphium product, chitin oligosaccharide yield can reach more than 96%, good water solubility.In spray-dired powder, also containing a certain amount of inorganic salts ingredients, therefore the content purity of chitin oligosaccharide 3000 ~ 4000 molecular weight is 93.6%.This product eats as agricultural, herding, and purity is feasible, as needed to improve content product purification further, carrying out purifying, can obtain 99% content chitin oligosaccharide product by the edible ethanols of 2 times, completely water-soluble.

The present invention has this project integrated use fermentable law technology and produces chitin oligosaccharide, effectively prevent traditional chitin oligosaccharide production process and adopt a large amount of soda acid consumptions, effectively improve the utilization ratio of the energy, and avoid with the higher production cost of enzyme process.Can obtain the chitin oligosaccharide product that purity is higher more at an easy rate, this product can be applicable to the fields such as medicine, agricultural, food, and technology is reached advanced world standards.

Accompanying drawing illustrates:

Fig. 1 is the method process flow sheet that a kind of fermentable of the present invention produces chitin oligosaccharide.

Embodiment:

Embodiment 1:

Inoculate in the fermented liquid that sterilizing is good into the good zymophyte of seed tank culture, air blow and agitation ferments, firm inoculation rear venting amount is 1:0.05v/v.min, in the process of fermentation, again according to the generation of carbonic acid gas, progressively increase ventilation, when carbon dioxide content every cube gas volume often increases by 5 gram equivalents, ventilation just increases 0.05v/min, and progressively improve leavening temperature, divide and improve temperature four times, initial temperature is 28 DEG C, first time temperature raising in 10 hours is 29 DEG C, 16 hours second time temperature raisings 30 DEG C, 20 hours third time temperature raisings 31 DEG C, 28 to 30 hours the 4th temperature raising 33.5 DEG C.

Embodiment 2:

The preparation of bacterial classification: the preservation inclined-plane taking out Paecilomyces lilacinus bacterial classification from refrigerator, is transferred on production inclined-plane, and transferring 5 production inclined-planes in a general preservation inclined-plane, carries out actication of culture, then the incubator that the production inclined-plane of having transferred puts into 28 DEG C is cultivated 3 days; Cultivate 3 days when 28 DEG C can be inoculated in one-level triangular flask after covering with inclined-plane, the substratum in triangular flask is 2.5% shrimp, crab shrimp shell powder suspension and 0.02% peptone make substratum; Through microscopy as in mycelia stalwartness i.e. accessible secondary triangular flask 28 DEG C cultivate 2 days, secondary triangular flask substratum is identical with one-level; Through microscopy as in mycelia stalwartness i.e. accessible seeding tank 28 DEG C cultivate 36 hours, inoculum size is 5%V/V.

Embodiment 3:

For improving the content of chitin oligosaccharide further, the edible ethanol of dry complete powder chitin oligosaccharide solid volume 1.5 times amount of using spray dissolves, the inorganic salt dissolving in ethanol are dissolved in ethanol because chitin oligosaccharide does not allow ethanol with other soluble impurities, stir 20min, Plate Filtration, obtain dry-matter, low temperature vacuumizes drying, just can obtain 99% content chitin oligosaccharide.

Embodiment 4:

Bacterial classification is separated and obtains multiple Paecilomyces lilacinus bacterial strain from multiple soil, with the bacterial strain that Analysis of Plate crust degradation capability is good, this bacterial strain is source Nan Jing subtropics virgin forest soil, be separated the Paecilomyces lilacinus bacterial strain obtained, mutagenesis is replaced again by cobalt-60 and ultraviolet, acquisition can directly be degraded shrimp, crab shell powder, form the new Paecilomyces lilacinus bacterial strain of chitin oligosaccharide (3000 ~ 4000 molecular weight) product, by inoculation triangular flask shrimp repeatedly, the fermentation of crab shell powder and the fermentation of inoculation seeding tank, adopt under meter, carbon dioxide detector, thermometer, acidometer, the mensuration of the instruments such as liquid chromatograph, obtain one group of fermentation parameter, according to fermentation engineering principle, determine suitable leavening temperature and fermentation ventilation.Fermentation condition is namely: initial temperature 28 DEG C, once lift temperature to 29 DEG C, secondary lifts temperature to lift temperature to for 30 DEG C, three times and lifts temperature to 33 DEG C 31 DEG C, four times; Fermentation period is 60 hours; 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 product of the certain content of final acquisition, substantially meet the concentration required for suitability for industrialized production, production cost is lower, and product purity is higher, good water solubility.

Claims

1. produce the method for chitin oligosaccharide with fermentable for one kind, it is characterized in that with fresh shrimp shell or crab shell for raw material, through the preparation of raw material, fermenation raw liquid preparation, fermenation raw liquid sterilizing, inoculation fermentation, flow feeding, centrifugally remove thalline, vacuum concentration, spraying dry, product purification, obtain product chitin oligosaccharide;

(1) preparation of raw material: fresh shrimp shell or crab shell dry, is ground into 120 object dry powder for subsequent use;

(2) fermenation raw liquid preparation: mix with water with the shrimp of weight ratio 2.5%, crab shell powder, then add 0.02% peptone, make fermenation raw liquid, wherein the amount of shrimp shell meal is 1.5%, and the amount of crab shell powder is 1%;

(3) fermenation raw liquid sterilizing: the fermenation raw liquid prepared is pumped into fermentor tank, airtight tank body, adds steam and carries out sterilizing, makes material temperature rise to 121 DEG C, is incubated 30 minutes, is then cooled to 28 DEG C gradually;

(4) inoculation fermentation: inoculate in the fermenation raw liquid that sterilizing is good into the good zymophyte of 5% seed tank culture, described zymophyte is Paecilomyces lilacinus (Paecilomyces lilacinus); Air blow and agitation ferments, and just inoculation rear venting amount is 1: 0.05v/v.min, in the process of fermentation, again according to the generation of carbonic acid gas, progressively increase ventilation, and progressively improve leavening temperature, when carbon dioxide content every cube gas volume increases by 5 gram equivalents, ventilation just increases 0.05V/min; Divide and improve temperature four times, initial temperature is 28 DEG C, and first time temperature raising in 10 hours is 29 DEG C, the temperature raising of 16 hours second time is 30 DEG C, and third time temperature raising in 20 hours is 31 DEG C, the 4th temperature raising in 28 to 30 hours 33.5 DEG C, at fermenting process results of regular determination reducing sugar content, and the growing amount of chitin oligosaccharide;

(5) flow feeding: it is 10% shrimp, crab shell powder mixing suspension that stream feeds in raw material, and is placed in stream and adds tank, pass into steam and be warming up to 121 DEG C, be incubated sterilizing in 30 minutes, for subsequent use after cooling, when the middle and later periods of fermenting, points three times progressively stream add fermentor tank;

(6) centrifugally remove thalline: need survey containing chitin oligosaccharide content during fermentation ends, directly pump into whizzer, under 5000rpm condition, centrifugal 30 minutes, obtain supernatant liquor, supernatant liquor is pumped in storage tank for subsequent use, sampling Detection is containing chitin oligosaccharide amount;

(7) vacuum concentration: supernatant liquor is pumped in double-effect evaporation concentration tank, carries out vacuum concentration, is concentrated into concentrated solution chitin oligosaccharide content 10 ~ 15% time, pumps into storage tank for subsequent use;

(8) spraying dry: by chitin oligosaccharide concentrated solution, adopts spray-drier, by regulating inlet temperature to be 150 DEG C, discharge port temperature 70 ~ 75 DEG C, carries out spraying dry, obtained chitin oligosaccharide xeraphium product, chitin oligosaccharide yield can reach more than 96%, good water solubility;

(9) product purification: the edible ethanol of spray-dired powder solid volume 1.5 times amount dissolves, stir 20min, Plate Filtration, obtains dry-matter, and low temperature vacuumizes drying, obtains 99% content chitin oligosaccharide.

2. a kind of fermentable produces the method for chitin oligosaccharide as claimed in claim 1, it is characterized in that described 10% shrimp, crab shell powder mixing suspension is the shrimp shell meal of 6% and the crab shell powder of 4% and water are mixed into suspension.

3. a kind of fermentable produces the method for chitin oligosaccharide as claimed in claim 1, it is characterized in that planting the good zymophyte of seed tank culture is cultivate ripe Paecilomyces lilacinus, being prepared as of bacterial classification: slant preservation bacterial classification-activation production inclined-plane-one-level triangular flask spreads cultivation-and secondary triangular flask spreads cultivation-and seed culture is mature strains, and inoculum size is 5% fermentation liquid measure.