CN101676403A - Screening method of microbial strain for xylitol production - Google Patents

Abstract

The invention discloses a screening method of a microbial strain for xylitol production and relates to the method for obtaining the high-yield microbial strain of xylitol by natural screening. The screening method comprises the technological steps of (1) screening of a culture medium formula; (2) prepration of the liquid microbial strain; (3) screening of the microbial strain; (4) fermentation ofthe xylitol and (5) separation and purification of fermentation liquid. The high-yield microbial strain of the xylitol obtained by adopting the screening method belongs to candida tropicalis, therebyhaving the advantages of fast growth speed, high conversion rate of the xylitol and low residual sugar, and being the strain with important promotional value.

Description

The screening method of microbial strain for xylitol production

Technical field

The invention belongs to biological technical field, relate to a kind of by screening the method for the Xylitol superior strain that obtains naturally.

Technical background

The research that microbe transformation method is produced Xylitol starts from the seventies in 20th century, it is the focus of domestic and international expert's research always, at present, the screening Xylitol is produced the Zhejiang University that mainly contains of bacterial classification, Sichuan University etc., they utilize the corn cob hydrolyzed solution through simple detoxification (as ammonia treatment) to come xylitol zymolysis production, though found the variation bacterial classification that to produce Xylitol through domestication mutagenesis, but the poor stability of bacterial classification, restorative sudden change takes place in actual applications easily, be difficult for being applicable to secular Xylitol production, therefore screen the Xylitol high-yield strains development Xylitol industry is had important economic implications.

Summary of the invention

At the deficiencies in the prior art, the purpose of this invention is to provide a kind of screening method of microbial strain for xylitol production, to reduce the production cost of Xylitol, better realize safety in production promoting the application of Xylitol in people's daily life.

The technical solution adopted for the present invention to solve the technical problems is made up of following processing step:

(1) screening and culturing based formulas: in 1L water, add wood sugar 1-20g, yeast powder 0.1-1.0g, MgSO ₄7H ₂O 0.1-0.5g, CaCl ₂2H ₂O 0.05-0.3g, KH ₂PO ₄1-5g, (NH ₄) ₂SO ₄2-10g forms screening culture medium;

(2) preparation of liquid spawn: in 1L water, add MgSO ₄7H ₂O 0.1-1.0g, CaCl ₂2H ₂O0.05-0.5g, KH ₂PO ₄0.5-4.0g, (NH ₄) ₂SO ₄1.0-8.0g, in every liter of solution, add wood sugar 10-40g, glucose 10-50g, yeast extract paste 5-20g in addition, form seed culture medium; Under aseptic condition, in the 250ml triangular flask, add the above-mentioned seed culture medium of 50-100ml, and insert a ring bacterial classification, at 20-40 ℃, shaking culture 10-30h under the 100-400r/min condition gets liquid spawn;

(3) screening step: each 1g of sample that gets xylose mother liquid, the middle product of wood sugar, three bacterial classifications sources of soil respectively, add in the numbered 250ml triangular flask, add the 50ml pure water in each triangular flask again, in 100-400r/min, shake a bottle enrichment 20-50h under 20-40 ℃, shake a bottle pregnant solution to three kinds respectively then and do 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Gradient dilution, and spread plate is cultivated the bacterium colony that detects in the flat board behind the 30-60h and is distributed and quantity in 20-40 ℃ biochemical incubator, find that extent of dilution is that the effect of 10-6 is best; Picking list bacterium colony is drawn flat board in the flat board of coating, after the separation and purification of several times plate streaking, picking 30-45 bacterial strain, and in 2-6 ℃ malt extract medium, preserve; Adopt the liquid screening substratum in the step (1) that the bacterial strain of selecting is done fermentation test, detect once every 24h, wood sugar approach exhaustion in substratum, by shake flask test sieve a strain Xylitol high yield bacterium, can make that the content of Xylitol reaches 80-100% in the fermented liquid;

(4) fermentation test of wood sugar: the 0.1-0.5% by its dry matter content adds the somatomedin of corn steep liquor as Xylitol high yield bacterium in the product in the middle of wood sugar, the 1-5% that presses the middle product total amount of wood sugar adds ammonium sulfate as nitrogenous source, 2-6% by product total amount in the middle of the wood sugar adds the liquid spawn that is obtained by step (2) cultivation, the control mixing speed is 100-400r/min, leavening temperature 28-31 ℃, preceding 20h air flow is 3.5-3.9L/min, air flow is 1.1-1.4L/min later on, and fermentation total time is 68-72 hour;

(5) separation and purification of fermented liquid: fermented liquid stopped to stir static 3 hours, and the whole flocculating settlings of thalline are emitted supernatant liquor, and thalline reclaims and continues fermentation; Supernatant liquor is heated to 85-90 ℃, removes by filter throw outs such as protein,, make Xylitol concentration reach 70-85%, adopt the way of gradient cooling, obtain high-quality crystalline xyhose alcohol product through evaporation concentration.

Positively effect of the present invention is: adopt Xylitol high yield Pseudomonas that this screening method obtains in candida tropicalis, and fast growth, Xylitol transformation efficiency height, residual sugar is low, is the bacterial classification with important promotional value.It is the Xylitol high yield bacterium of substrate with the wood sugar from a strain of row filter that the contriver adopts the laboratory, xylitol products purity height by fermentative production, almost colourless element, wood sugar purifying and high temperature, highly compressed chemistry hydrogenation step have been saved, reduced production cost, realized safety in production, using value is very big.

Embodiment

The invention will be further described below in conjunction with specific embodiment:

(1) preparation screening culture medium: in 1L water, add 15g wood sugar, 0.5g yeast powder, 0.3g MgSO ₄7H ₂O, 0.1g CaCl ₂2H ₂O, 2g KH ₂PO ₄, 5g (NH ₄) ₂SO ₄, form screening culture medium;

(2) preparation liquid spawn: in 1L water, add 0.5g MgSO ₄7H ₂O, 0.1g CaCl ₂2H ₂O, 2.0g KH ₂PO ₄, 5.0g (NH ₄) ₂SO ₄, 20g wood sugar, 30g glucose, 10g yeast extract paste, form seed culture medium, then under aseptic condition, get the above-mentioned seed culture medium of 80ml and pour in the 250ml triangular flask, and insert a ring bacterial classification, at 30 ℃, shaking culture 20h under the 300r/min condition gets liquid spawn;

(3) screening of bacterial classification: each 1g of sample that gets xylose mother liquid, the middle product of wood sugar, three bacterial classifications sources of soil respectively, add in the numbered 250ml triangular flask, add the 50ml pure water in each triangular flask again, in 200r/min, shake a bottle enrichment 35h under 30 ℃, shake a bottle pregnant solution to three kinds respectively then and do 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Gradient dilution, and spread plate is cultivated the bacterium colony that detects in the flat board behind the 50h and is distributed and quantity in 30 ℃ biochemical incubator, find that extent of dilution is 10 ^-6Effect best; Picking list bacterium colony is drawn flat board in the flat board of coating, after 6 plate streaking separation and purification, picking 39 bacterial strains, and in 4 ℃ malt extract medium, preserve; Adopt the liquid screening substratum in (1) that 39 bacterial strains selecting are done fermentation test, detect once every 24h, wood sugar approach exhaustion in substratum, by shake flask test sieve a strain Xylitol high yield bacterium, can make that the content of Xylitol reaches 90% in the fermented liquid;

(4) fermentation of wood sugar: product 300g pours in the 500ml triangular flask in the middle of getting mass percent concentration and be 17% wood sugar, add the 0.16g corn steep liquor, 1.5g ammonium sulfate, liquid spawn among the 2g (2), the control mixing speed is 200r/min, 30 ℃ of leavening temperatures, and preceding 20h air flow is 3.6L/min, air flow is 1.2L/min later on, and fermentation total time is 70 hours;

(5) separation and purification of fermented liquid: fermentation stops and static 3 hours, and the whole flocculating settlings of thalline are emitted supernatant liquor, and thalline reclaims and continues fermentation; Supernatant liquor is heated to 88 ℃, remove by filter throw outs such as protein, detecting its Xylitol content is 91.2%, through evaporation concentration, making its mass percent concentration is 81%, and the speed by 1 ℃/h is cooled to 35 ℃ then, carries out centrifugal, obtain crystalline xyhose alcohol product 30g, Xylitol content is 99.8%.

Claims (1)

1, a kind of screening method of microbial strain for xylitol production is characterized in that this screening method is made up of following processing step:

(1) in 1L water, adding wood sugar 1-20g, yeast powder 0.1-1.0g, MgSO ₄7H ₂O 0.1-0.5g, CaCl ₂2H ₂O 0.05-0.3g, KH ₂PO ₄1-5g, (NH ₄) ₂SO ₄2-10g forms screening culture medium;

(2) preparation of liquid spawn: in 1L water, add MgSO ₄7H ₂O 0.1-1.0g, CaCl ₂2H ₂O0.05-0.5g, KH ₂PO ₄0.5-4.0g, (NH ₄) ₂SO ₄1.0-8.0g,, in every liter of solution, add wood sugar 10-40g, glucose 10-50g, yeast extract paste 5-20g in addition, form seed culture medium; Under aseptic condition, in the 250ml triangular flask, add the above-mentioned seed culture medium of 50-100ml, and insert a ring bacterial classification, at 20-40 ℃, shaking culture 10-30h under the 100-400r/min condition gets liquid spawn;

(3) bacterial strain screening: each 1g of sample that gets xylose mother liquid, the middle product of wood sugar, three bacterial classifications sources of soil respectively, add in the numbered 250ml triangular flask, add the 50ml pure water in each triangular flask again, in 100-400r/min, shake a bottle enrichment 20-50h under 20-40 ℃, shake a bottle pregnant solution to three kinds respectively then and do 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Gradient dilution, and spread plate, the bacterium colony that detects in the flat board behind the training 30-60h in 20-40 ℃ biochemical incubator distributes and quantity, finds that extent of dilution is 10 ^-6Effect best; Picking list bacterium colony is drawn flat board in the flat board of coating, after the separation and purification of several times plate streaking, picking 30-45 bacterial strain, and in 2-6 ℃ malt extract medium, preserve; Adopt the liquid screening substratum in the step (1) that the bacterial strain of selecting is done fermentation test, detect once every 24h, wood sugar approach exhaustion in substratum, by shake flask test sieve a strain Xylitol high yield bacterium, can make that the content of Xylitol reaches 80-100% in the fermented liquid;

(4) fermentation of wood sugar: the 0.1-0.5% by its dry matter content adds the somatomedin of corn steep liquor as Xylitol high yield bacterium in the product in the middle of wood sugar, the 1-5% that presses the middle product total amount of wood sugar adds ammonium sulfate as nitrogenous source, 2-6% by product total amount in the middle of the wood sugar adds the liquid spawn that is obtained by step (2) cultivation, the control mixing speed is 100-400r/min, leavening temperature 28-31 ℃, preceding 20h air flow is 3.5-3.9L/min, air flow is 1.1-1.4L/min later on, and fermentation total time is 68-72 hour;

(5) separation and purification of fermented liquid: fermented liquid stopped to stir static 3 hours, and the whole flocculating settlings of thalline are emitted supernatant liquor, and thalline reclaims and continues fermentation; Supernatant liquor is heated to 85-90 ℃, removes by filter throw outs such as protein,, make Xylitol concentration reach 70-85%, adopt the way of gradient cooling, obtain high-quality crystalline xyhose alcohol product through evaporation concentration.