CN101445796A - Method for manufacturing liquid xylanase through fermentation - Google Patents

Abstract

The invention relates to a method for manufacturing liquid xylanase through fermentation. The method adopts aspergillus niger mutagenic fungi as the manufacture bacterial strain which is subjected to liquid deep level ventilation fermentation on a low sugar culture medium to manufacture xylanase preparations. The method can enable enzymic synthesis to grow rapidly without interruption. Xylanase manufactured by using the method is nontoxic and harmless, and can be applied to the fields of light industry paper making, food, energy, fodder, environment protection and the like, in particular to the mass production of plant fiber ethanol, thereby pioneering in the field of using agricultural waste to generate energy. Moreover, the method has the advantages of high ratio of enzyme activity to cost, straightforward technology, short fermentation period, and stable product quality, and is nontoxic and causes no pollution. Meanwhile, solid residues separated by fermentation from enzyme preparations contain a certain amount of nutrient substances such as xylanase, mycoprotein and the like, and therefore can achieve remarkable benefits when being used as feed additives.

Description

A kind of fermentation method for producing of liquid xylanase

Technical field

The invention belongs to the preparation technique field of enzyme bacterium, is a kind of fermentation method for producing of liquid xylanase.

Background technology

The research and development of biomass alcoholic acid are emphasis of current various countries new resources strategy.Countries in the world, especially developed countries such as the U.S., Canada, Germany, Sweden, Japan, all, still also there is not at present a tame technical scale to utilize biomass material to produce the enterprise of alcohol fuel in the world in the biomass energy utilization technologies of being devoted to develop clean and effective.Its major obstacle is: the enzyme cost is too high, and Mierocrystalline cellulose and hemicellulose can utilize the transformation efficiency of monose on the low side to yeast;

Hemicellulose is the abundant polysaccharide of content second in the plant cell wall, accounts for 30 one 40% of biomass material dry weight.Xylan is a kind of poly five-carbon sugar, and main component is the D-wood sugar, is the important component part of plant hemicellulose.Zytase be a class with β-1 in inscribe and the circumscribed mode degradation of xylan molecule, the enzyme of 4-wood sugar glycosidic bond system is the general name of one group of enzyme of degradation of hemicellulose xylan, this enzyme system can be widely used in fields such as papermaking, food, fodder additives.Utilizing cellulose raw material to produce in the field of alcohol, then require zytase that hemicellulose is decomposed into wood sugar, rather than xylo-oligosaccharide, so that the yeast of assimilation wood sugar is converted into alcohol to wood sugar, increase the ethanol output capacity of cellulose raw material.In recent years, attach great importance to research in the world to this enzyme, and the zytase that prior art is produced, the product that forms when decomposing hemicellulose mainly is an xylo-oligosaccharide, the content of wood sugar is lower, being unsuitable for the cellulose raw material Alcohol Production uses, this mainly is generally to lack xylosidase in the zytase enzyme component of cultivating, can only be converted into wood oligose to hemicellulose, can not further be hydrolyzed to wood oligose can be for the monose of yeast utilization, these have all seriously hindered its industrial applications aspect biomass fuel ethanol, how to improve the xylosidase vigor in Xylanase activity and the enzyme component, and high enzyme is lived, the zytase production technology also is not resolved at present cheaply.Thereby the suitability for industrialized production of alcohol is a raw material with grain still at present, and consumed resource is big, and the production cost height is unfavorable for promoting the use of of fuel alcohol.On the other hand, a large amount of plant straws that contains hemicellulose is usually discarded or burn, and causes great waste, also brings in various degree pollution to environment.

Summary of the invention

It is high to the purpose of this invention is to provide a kind of enzyme work, and cost is low, and easy to operate, fermentation period is lacked, constant product quality, the fermentation method for producing of the liquid xylanase of nontoxic pollution-free.

Realize that the technical scheme that purpose of the present invention is taked is: the fermentation method for producing of liquid xylanase may further comprise the steps:

(1) bacterial classification is chosen: in zytase is produced bacterial strain, pick out the aspergillus niger mutagenic fungi as produce bacterial strain be placed on preserve on the PDA inclined-plane lawn standby;

(2) spawn culture: the vigorous slant strains of picking lawn is put into culturing bottle, under 28～32 ℃ temperature condition, cultivated 1～2 day, in seeding tank, add substratum then, the weight percent of again the culturing bottle bacterial classification being pressed substratum 2%～10% inserts in the seeding tank, under 28～34 ℃ of temperature, ventilating ratio 0.2～0.6v/v condition, cultivated 12～40 hours, wherein substratum is the bran water mixed solution of 2～5% weight percent concentration, makes fermented bacterium;

(3) fermentative processing: 2～10% production bacterial classification by weight percentage, the corn cob that 2～8% grinding particle size 0.5mm are following, 0.5～6% corn steep liquor, 0.5～1% nutritive salt, 75～95% water is mixed into the fermentation slurry, the slurry that will ferment is put into fermentor tank by ventilation 0.2～0.6v/v, mixing speed 150～350rpm ventilates and stirs fermentation, and behind fermentation 24h, added 0.1～3% corn steep liquor and 0.01～0.2% corn cob by initial fermentation slurry weight stream every 2～6 hours, temperature is controlled at 25～35 ℃, fermented 3～5 days, the residue in Plate Filtration elimination fermentation slurries promptly can be made into liquid xylanase.

(4) residue treatment: will ferment and filter the solid residue that obtains, and sell as fodder additives.

The liquid xylanase of Sheng Chaning according to the method described above; adopt high yield enzyme aspergillus niger mutagenic fungi alive as producing bacterial strain; with corn cob; corn steep liquor is as the fermentation base-material; can guarantee that the mould-growth breeding is fast; the zytase of producing is nontoxic; harmless; contain inscribe β ,-1,4 zytases and circumscribed β-xylosidase; and the auxiliary enzymes of degraded substitution in side chain base; can be wood oligose and wood sugar with xylan hydrolysis, it is simple that this method also has production technique, and operation is implemented convenient; raw material sources are extensive; production cost is low, and is pollution-free, is suitable for large-scale industrial production etc. advantage.

The liquid xylanase that present method is produced both can be applicable to fields such as light industry papermaking, food, the energy, feed and environmental protection, more can be used for bio-ethanol, improve biomass material can utilize transformation efficiency from monose to yeast, thereby improve the efficient that biomass material obtains alcohol fuel, fill up the blank that lacks xylosidase in the cellulose ethanol industry xylan, effectively improved the level of comprehensive utilization of stalk resource.And utilizing corn cob, corn steep liquor as low sugar culture-medium, isolated solid residue contained nutritive substances such as a certain amount of zytase and tropina after fermentation obtained zymin, and is obvious as the fodder additives benefit.

Embodiment

The present invention produces the method for liquid xylanase, adopt the aspergillus niger mutagenic fungi as producing bacterial strain, on low sugar culture-medium, carry out the deep liquid ventilating fermentation and produce xylanase preparation, can make enzymic synthesis do not checked and grow rapidly, enzyme is lived and cost ratio height, technology is easily grasped, fermentation period short, constant product quality, nontoxic pollution-free.The zytase of being produced, its composition mainly comprises inscribe β,-1,4 zytases and circumscribed β-xylosidase, and the auxiliary enzymes of degraded substitution in side chain base, can be wood oligose and wood sugar with xylan hydrolysis, improve biomass material can utilize transformation efficiency from monose to yeast, thereby improve the efficient that biomass material obtains alcohol fuel, can be applied in light industry papermaking, food, the energy, field such as feed and environmental protection, filled up the blank that lacks xylosidase in the vegetable fibre ethanol industry xylan simultaneously, isolated solid residue contained nutritive substances such as a certain amount of zytase and tropina after fermentation obtained zymin, can be used as fodder additives, can improve the level of comprehensive utilization of stalk resource effectively, further specify below in conjunction with embodiment.

Embodiment 1

Be that example is done with detailed explanation the present invention with the 50L fermentor tank below:

At first in zytase is produced bacterial strain, pick out the aspergillus niger mutagenic fungi as produce bacterial strain be placed on preserve on the PDA inclined-plane lawn standby; The vigorous slant strains of picking lawn is put into culturing bottle again, under 28 ℃ temperature condition, cultivated 2 days, in seeding tank, add substratum then, the weight percent of again the culturing bottle bacterial classification being pressed substratum 2% inserts in the seeding tank, under 28 temperature, ventilating ratio 0.2～0.6v/v condition, cultivated 40 hours, wherein substratum is the bran water mixed solution of 2% weight percent concentration, makes the production bacterial classification; The following corn cob of 10% production bacterial classification, 8% grinding particle size 0.5mm, 6% corn steep liquor, 1% nutritive salt, 75% water are mixed into the fermentation slurry by weight percentage, and the slurry that will ferment is put into 5m ³Ventilate by ventilation 0.2～0.6v/v, mixing speed 150～350rpm in the fermentor tank and stir fermentation, and behind fermentation 24h, added 0.1% corn steep liquor and 0.01% corn cob by initial fermentation slurry weight stream every 2 hours, temperature is controlled at 25 ℃, fermented 5 days, the manual residue that filters in the elimination fermentation slurries of 100 order filter clothes promptly can be made into the zytase of liquid.

Embodiment 2

Below again with 5m ³Fermentor tank is that example is done to explain the present invention:

At first in zytase is produced bacterial strain, pick out the aspergillus niger mutagenic fungi as produce bacterial strain be placed on preserve on the PDA inclined-plane lawn standby; The vigorous slant strains of picking lawn is put into culturing bottle again, under 30 ℃ temperature condition, cultivated 35 hours, in seeding tank, add substratum then, the weight percent of again the culturing bottle bacterial classification being pressed substratum 6% inserts in the seeding tank, under 32 ℃ of temperature, ventilating ratio 0.2～0.6v/v condition, cultivated 25 hours, wherein substratum is the bran water mixed solution of 4% weight percent concentration, makes the production bacterial classification; The following corn cob of 2% production bacterial classification, 2% grinding particle size 0.5mm, 0.5% corn steep liquor, 0.5% nutritive salt, 95% water are mixed into the fermentation slurry by weight percentage, and the slurry that will ferment is put into 50m ³Ventilate by ventilation 0.2～0.6v/v, mixing speed 150～350rpm in the fermentor tank and stir fermentation, and behind fermentation 24h, added 2% corn steep liquor and 0.1% corn cob by initial fermentation slurry weight stream every 4 hours, temperature is controlled at 30 ℃, fermented 4 days, and promptly can be made into the cellulase of liquid with the residue in the Plate Filtration elimination fermentation slurries.

Embodiment 3

Below with 50m ³Fermentor tank is that example is done to explain the present invention:

At first in zytase is produced bacterial strain, pick out the aspergillus niger mutagenic fungi as produce bacterial strain be placed on preserve on the PDA inclined-plane lawn standby; The vigorous slant strains of picking lawn is put into culturing bottle again, under 32 ℃ temperature condition, cultivated 1 day, in seeding tank, add substratum then, the weight percent of again the culturing bottle bacterial classification being pressed substratum 10% inserts in the seeding tank, under 34 ℃ of temperature, ventilating ratio 0.2～0.6v/v condition, cultivated 12 hours, wherein substratum is the bran water mixed solution of 5% weight percent concentration, makes the production bacterial classification; The following corn cob of 6% production bacterial classification, 6% grinding particle size 0.5mm, 3.3% corn steep liquor, 0.7% nutritive salt, 84% water are mixed into the fermentation slurry by weight percentage, the slurry that will ferment is put into the 50L fermentor tank and is ventilated by ventilation 0.2～0.6v/v, mixing speed 150～350rpm and stir fermentation, and behind fermentation 24h, added 3% corn steep liquor and 0.2% corn cob by initial fermentation slurry weight stream every 6 hours, temperature is controlled at 35 ℃, fermented 3 days, and promptly can be made into the cellulase of liquid with the residue in the Plate Filtration elimination fermentation slurries.

Claims (1)

1, a kind of fermentation method for producing of liquid xylanase, it is characterized in that: it may further comprise the steps:

(1) bacterial classification is chosen: in zytase is produced bacterial strain, pick out the aspergillus niger mutagenic fungi as produce bacterial strain be placed on preserve on the PDA inclined-plane lawn standby;

(2) spawn culture: the vigorous slant strains of picking lawn is put into culturing bottle, under 28～32 ℃ temperature condition, cultivated 1～2 day, in seeding tank, add substratum then, the weight percent of again the culturing bottle bacterial classification being pressed substratum 2%～10% inserts in the seeding tank, under 28～34 ℃ of temperature, ventilating ratio 0.2～0.6v/v condition, cultivated 12～40 hours, wherein substratum is the bran water mixed solution of 2～5% weight percent concentration, makes fermented bacterium;

(3) fermentative processing: 2～10% production bacterial classification by weight percentage, the corn cob that 2～8% grinding particle size 0.5mm are following, 0.5～6% corn steep liquor, 0.5～1% nutritive salt, 75～95% water is mixed into the fermentation slurry, the slurry that will ferment is put into fermentor tank by ventilation 0.2～0.6v/v, mixing speed 150～350rpm ventilates and stirs fermentation, and behind fermentation 24h, added 0.1～3% corn steep liquor and 0.01～0.2% corn cob by initial fermentation slurry weight stream every 2～6 hours, temperature is controlled at 25～35 ℃, fermented 3～5 days, the residue in Plate Filtration elimination fermentation slurries promptly can be made into liquid xylanase.

(4) residue treatment: will ferment and filter the solid residue that obtains, and sell as fodder additives.