CN101381694B - Bacteria cellulose producing bacteria and method for preparing bacteria cellulose using above bacterial strain - Google Patents
Bacteria cellulose producing bacteria and method for preparing bacteria cellulose using above bacterial strain Download PDFInfo
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Abstract
The invention provides bacterial cellulose (BC) producing bacteria and a method for preparing the bacterial cellulose by using the bacterial strain. The bacterial strain is a selected gluconic acid acetobacter xylinum GD-BC-1 with an accession number of CCTCC M 208149. The method is to use the bacterial strain as a fermentation bacterial strain and use glucose as a main fermentation material for fermentation so as to obtain the BC. The preparation method comprises the following steps of: activating a slant bacterial strain, liquid-activating the slant bacterial strain, inoculating the liquid-activated slant bacterial strain in a fermentation culture medium to perform dynamic or static fermentation and extracting products of BC from the fermentation solution. The preparation method uses the bacterial strain of gluconic acid acetobacter xylinum GD-BC-1 with the accession number of CCTCC M 208149 for fermentation so as to produce the BC. The BC can be produced through dynamic or static fermentation in a conical flask with glucose as the main fermentation material, and the yield is high and different methods can be used in different production environment to produce BC.
Description
Technical field
The present invention relates to a kind of method of producing the microorganism strains of bacteria cellulose and utilizing this strain fermentation production bacteria cellulose.
Background technology
Mierocrystalline cellulose is a biological polymer the abundantest on the earth, is the chief component as phytomass, and the annual Mierocrystalline cellulose that is produced by plant reaches hundreds of millions tons on the earth.Except that plant, some lower animal can produce tunicin (Tunlcin), and some bacterium can more efficient than plant in the heterotrophism mode, more high purity produce the outer bacteria cellulose of born of the same parents, and (Bacterial cellulose is hereinafter to be referred as BC.A.J.Brown in 1886 with regard to first report by acetobacter xylinum (Acetobacter xylinum) can synthesize gelatinous material outside a kind of born of the same parents (A.J.Brown.On an aceticferment forms cellulose[J] .J.Chem.Soc, 1886,49:432-439.), but because the output of no suitable laboratory facilities and fiber rope is lower, therefore be not subjected to enough attention for many years always, bacteria cellulose causes that people more note also in 20 th century later, use A.xylinum as a kind of model bacterium, further investigation BC is synthetic from Hestrin etc., he proved leave standstill with freeze dried bacterium vinegar cell can synthetic cellulose (Hestrin when glucose and oxygen are arranged, S.and Schramm, M., Synthesis of cellulose by Acetobacterxylinum:preparation of freeze dried cells capable of polymerizing glucose to cellulose, Biochem.J., 1954,58:345.); Secondly, Colvin[
4] in containing A.xylinum cell extract, glucose and ATP sample, observed cellulosic synthetic (Colvin JR.Formation of cellulose microfibrils in a homogenate of Acetobacter xylinum[J] .Arch.Biochem Biophys, 1957,70: 294-295.).BC belongs to elementary metabolic special outcome, and (PC) is the same with plant cellulose, and BC mainly also is the effect of playing a kind of protective layer.The bacterium that can produce BC mainly contains Acetobacter, Rhizobium, Agrobacterium, with (JonasR such as Sarcina, Farah LF.Production and application of microbial cellulose[J] .Polymer Degradation and Stability, 1998,59:101-106.).BC has the character of many uniquenesses.As: (1) vegetable fibre mainly is made up of Mierocrystalline cellulose, but mixes other many carbohydrates, as hemicellulose or xylogen.And BC is a kind of ＂ pure cellulose ＂, and high chemical purity and high-crystallinity are arranged; (2) Young's modulus of BC is more than the several times to ten times of general vegetable fibre, and the tensile strength height.(3) BC has very strong water-holding power; (4) BC has higher biological fitness and good biodegradability.(5) Modulatory character during the BC biosynthesizing.In addition, bacteria cellulose also has a lot of advantages with respect to traditional cellulose resource, as adds man-hour without the reject xylogen; The paper that can synthesize special purpose; Be processed into the nonwoven fabric of Any shape; Can be used as good food fibre etc., therefore, bacteria cellulose can be widely used in also can be used for industries such as makeup, medicine as biomaterial in papermaking, textiles and the food, has become one of focus of current new microorganism synthetic materials research about the research of BC.
Adopt the synthetic bacteria cellulose of microbial fermentation processes, it is less that present bacteria cellulose produces the bacterial classification class, bacteria cellulose microorganism synthetic method has dynamic fermentation or static fermentation method, and most bacterial classification can only adopt a kind of the carrying out synthesizing of bacteria cellulose in dynamic fermentation or the static fermentation.Dynamically the fermentation method power consumption is higher relatively, technology is also complicated, but relatively is fit to scale operation, and bacteria cellulose output may be higher; The power consumption of static fermentation method is low, technology is simple, but output may be not high, is fit to small-scale production.
Summary of the invention
The purpose of this invention is to provide a kind of bacteria cellulose of making raw material with glucose etc. produces bacterium and utilizes this bacterial strain to prepare the method for bacteria cellulose, this bacteria cellulose produces bacterium and can adopt dynamic fermentation method can produce bacteria cellulose with the static fermentation method again, and the bacteria cellulose yield of two kinds of methods is all higher.
Microorganism strains provided by the present invention is bacteria cellulose generation bacterium--glyconic acid bacillus aceticus (Gluconacetobactersp.) GD-BC-1 through seed selection, this bacterial strain is preserved in Chinese typical culture collection center (CCTCC) on October 9th, 2008, is numbered: CCTCCM208149.This bacterial strain is hereinafter to be referred as glyconic acid bacillus aceticus GD-BC-1CCTCC M208149.
The contact data at China typical culture collection center is as follows:
Phone: 027-87682319 fax: (027) 87883833E-mail:[email protected]; Address: Wuhan Wuhan University postcode: 430072.
Glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 bacterial strains are at solid medium (glucose 100g, yeast extract paste 10g, lime carbonate 20g, agar 20g, distilled water is settled to 1L, adjust pH to 7.0～7.2.) upward grew 48 hours, the bacterium colony that forms is tiny, and colony diameter is no more than 3mm, is mostly circular, projection, neat in edge is creamy white or little yellow, can form the hide-like mycoderm, it is unicellular, tyrothricin that microscope is observed thalli morphology for 16 * 100 times, and its major physiological biochemical character sees the following form.
Annotate :+the positive ,-feminine gender
The genetics feature of glyconic acid bacillus aceticus GD-BC-1CCTCC M208149 bacterial strain: adopt 16S rRNA order-checking to carry out the taxonomy Molecular Identification.The bacterial classification extracting genome DNA adopts acetone to destroy bacteria cell wall membrane lipid structure, and lysate is abolished bacterial cell membrane, discharges nucleic acid in the born of the same parents, removes protein and polysaccharide, dehydrated alcohol deposit D NA, TE damping fluid dissolving DNA through phenol/chloroform/primary isoamyl alcohol.Adopt the 16S rDNA of two kinds of synthetic universal primer PCR amplification bacteriums, primer is as follows: 27F:5 '-AGA GTT TGA TCC TGG CTC AG-3 '; 1541R:5 '-AAG GAA GTG ATG CAG CCG CA-3 '.Directly carry out sequencing behind the pcr amplification product purifying, obtain the 16S rDNA sequence (its sequence is shown in SEQ1) of this bacterial strain by sequencing analysis.
The 16S rDNA sequence total length of glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 bacterial strains is 1436bp.Send GenBank to be Blast relatively the sequence that order-checking obtains, wherein the bacterial strain that homology is the highest is that (the Genbank accession number is: AY741144.1), homology reaches 97% to Gluconacetobacter sp.4L.In conjunction with the physiological and biochemical property test-results, judge that this bacterial strain is glyconic acid bacillus aceticus (Gluconace tobacter sp.).
With glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 provided by the present invention, with glucose as the triangular flask of main fermentation raw material in vibration fermentation or standing for fermentation all can produce bacteria cellulose, thereby can produce bacteria cellulose as fermentation strain with it.
Method of producing bacteria cellulose provided by the present invention with glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 bacterial strains, be characterized in doing fermentation strain with glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149, utilize glucose to ferment as main fermentation raw material, dynamically shake flask fermentation 4-10 days, can produce bacteria cellulose 2.0～8.0g/L; Standing for fermentation 4-10 days, can produce bacteria cellulose 1.5～4.4g/L.
Utilize the specific embodiment of glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 strain fermentations production bacteria cellulose as follows:
(1) slant strains activation: (glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149) lines on the culture medium slant of new preparation with cultured preservation inclined-plane kind, culture medium slant is composed as follows: glucose 80～120g, yeast extract paste 8～12g, lime carbonate 15～25g, agar 15～22g, tap water 1L, adjust pH to 6.5～7.2..Cultivated 24～48 hours for 28-32 ℃, refrigerate again in 4 ℃ 1～5 day.
(2) liquid of bacterial classification activation: with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is inoculated in the liquid activation medium, and the liquid activation medium is composed as follows: glucose 80～120g, yeast extract paste 8～12g, peptone 5～10g, tap water 1L, adjust pH to 6.0～7.0..The liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 28-32 ℃ of reciprocating type shaking table shaking culture 18～36 hours, shaking speed was 80-110r/m, and stroke is 65-75mm.
(3) fermentation: get cultured liquid activation and plant, press the 5-10% inoculum size, insert in the fermention medium and dynamically ferment or standing for fermentation.If adopt dynamic fermentation process, the bottled fermention medium of triangle of available 500ml (more volume also can), loading amount is 10% of a triangular flask volume, if use the 500ml triangular flask, then adorn fermention medium 50ml, in 28-32 ℃, stroke 65-80mm, the reciprocating type shaking table shaking culture of rotating speed 60-110r/m 4-10 days stops fermentation; If adopt the standing for fermentation method, its step is with dynamically fermentation process is basic identical, and difference is needn't shaking culture, but, stops to ferment after 4-10 days in 28-32 ℃ of static placement, gets final product.
(4) fermented liquid that finishes of fermentation promptly gets bacterial cellulose product through extraction process.
Because bacteria cellulose is the gel solid substance in the fermented liquid that fermentation finishes, therefore extracting bacterial cellulose product only needs to get final product according to the following steps: the fermented liquid that fermentation finishes carries out solid-liquid separation, solid is a bacterial cellulose gel, liquid is fermented waste fluid, take out bacterial cellulose gel, distilled water repeatedly washes, and removes surface impurity.Bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L, 80 ℃ of insulation 60min remove remaining thalline and substratum again.Wash repeatedly with deionized water then.This moment gel be creamy white translucent, again with bacterial cellulose gel in 60～80 ℃ of dryings, promptly obtain bacteria cellulose.
In step (3), suitable fermention medium composition following (calculating): 20-60g glucose, 5-10g peptone, 0.5g-1.5g yeast extract paste, 6-10gNa by every liter of contained grammes per square metre g/L of substratum
2HPO
4, 10-20g ethanol, 1-2g Trisodium Citrate, all the other compositions are water.To 4.5-7.0, available NaOH and HCl regulate based on the preceding adjust pH of sterilization in cultivation.121 ℃ of sterilizations in back 15 minutes, standby.
Maximum characteristics are to produce bacteria cellulose with a gluconobacter GD-BC-1 CCTCC M 208149 strain fermentations among the present invention, with glucose as the triangular flask of main fermentation raw material in vibration fermentation or standing for fermentation all can produce bacteria cellulose, and productive rate is higher, under the optimal dynamic technological condition for fermentation, produce bacteria cellulose and reach as high as 8.0g/L, under best standing for fermentation processing condition, produce bacteria cellulose and reach as high as 4.4g/L, thereby the present invention is fit to produce bacteria cellulose with different methods under the different production environments.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
The inclined-plane kind of using in the following examples adopts following method to cultivate: glyconic acid bacillus aceticus GD-BC-1 CCTCC M 208149 lines on the solid inclined-plane of new preparation, the solid inclined-plane is composed as follows: glucose 80～120g, yeast extract paste 8～12g, lime carbonate 15～25g, agar 15～22g, distilled water is settled to 1L, adjust pH to 6.5～7.2..Cultivated 24～48 hours, and refrigerated in 4 ℃ of refrigerators standby again for 28-32 ℃.
With about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, and is inoculated in the liquid activation medium, and the liquid activation medium is composed as follows: glucose 80～120g, yeast extract paste 8～12g, peptone 5～10g, tap water 1L, adjust pH to 6.0～7.0..The liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 28-32 ℃ of reciprocating type shaking table shaking culture 18～36 hours, shaking speed was 80-110r/m, and stroke is 65-75mm.
Embodiment one:
Get 5 days inclined-plane kind of preservation in 4 ℃ of refrigerators [composition of slant medium (g/L): glucose 80, yeast extract paste 8, lime carbonate 15, agar 15, all the other compositions are water, pH6.5.Cultivated 24 hours for 28 ℃], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be transferred in the liquid activation medium of new configuration [composition of liquid activation medium (g/L): glucose 80, yeast extract paste 8, peptone 5, all the other compositions are water, adjust pH to 6.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 28 ℃ of reciprocating type shaking table shaking culture 18 hours, shaking speed was 80r/m, and stroke is 65mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 5% inoculum size (2.5ml), (g/L) is as follows for the composition of fermention medium: 20g glucose, 5g peptone, 0.5g yeast extract paste, 6gNa
2HPO
4, 10g ethanol, 1g Trisodium Citrate, all the other compositions are water, NaOH adjust pH to 7.0.Fermention medium is loaded in the 500ml triangular flask, and the triangular flask loading amount is 50ml, 32 ℃ of static placements 4 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, takes out bacterial cellulose gel, distilled water flushing 2 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 80 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 1.5g/L.
Embodiment two:
Get 5 days inclined-plane kind of preservation in 4 ℃ of refrigerators [composition of slant medium (g/L): glucose 80, yeast extract paste 8, lime carbonate 15, agar 15, all the other compositions are water, pH6.5.Cultivated 24 hours for 28 ℃], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be transferred in the liquid activation medium of new configuration [composition of liquid activation medium (g/L): glucose 80, yeast extract paste 8, peptone 5, all the other compositions are water, adjust pH to 6.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 28 ℃ of reciprocating type shaking table shaking culture 18 hours, shaking speed was 100r/m, and stroke is 75mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 5% inoculum size (2.5ml), (g/L) is as follows for the composition of fermention medium: 20g glucose, 5g peptone, 0.5g yeast extract paste, 6gNa
2HPO
4, 10g ethanol, 1g Trisodium Citrate, all the other compositions are water, NaOH adjust pH to 7.0.Fermention medium is loaded in the 500ml triangular flask, the triangular flask loading amount is 50ml, in 28 ℃, the reciprocating type shaking table shaking culture of stroke 65mm, rotating speed 60r/m 4 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, take out bacterial cellulose gel, distilled water flushing 2 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 80 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 2.0g/L.
Embodiment three:
Get 3 days inclined-plane kind of preservation in 4 ℃ of refrigerators, [composition of slant medium (g/L): glucose 100, yeast extract paste 10, lime carbonate 25, agar 18, all the other compositions are water, pH7.2.Cultivated 36 hours for 32 ℃], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be transferred in the liquid activation medium of new configuration [composition of liquid activation medium (g/L): glucose 100, yeast extract paste 10, peptone 7.5, all the other compositions are water, adjust pH to 7.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 32 ℃ of reciprocating type shaking table shaking culture 28 hours, shaking speed was 90r/m, and stroke is 75mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 7% inoculum size (3.5ml), (g/L) is as follows for the composition of fermention medium: 40g glucose, 7.5g peptone, 1g yeast extract paste, 8g Na
2HPO
4, 15g ethanol, 1.5g Trisodium Citrate, all the other compositions are water, HCl adjust pH to 4.5.Fermention medium is loaded in the 500ml triangular flask, and the triangular flask loading amount is 50ml, 28 ℃ of static placements 7 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, takes out bacterial cellulose gel, distilled water flushing 3 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 70 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 3.1g/L.
Embodiment four:
Get 3 days inclined-plane kind of preservation in 4 ℃ of refrigerators, [composition of slant medium (g/L): glucose 100, yeast extract paste 10, lime carbonate 25, agar 18, all the other compositions are water, pH7.2.Cultivated 36 hours for 32 ℃], scrape with the bacterial classification of inoculating needle about 1cm2 area on the above-mentioned inclined-plane, be transferred to [the composition of liquid activation medium (g/L): glucose 100 in the liquid activation medium of new configuration, yeast extract paste 10, peptone 7.5, all the other compositions are water, adjust pH to 7.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 32 ℃ of reciprocating type shaking table shaking culture 28 hours, shaking speed was 90r/m, and stroke is 75mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 7% inoculum size (3.5ml), (g/L) is as follows for the composition of fermention medium: 40g glucose, 7.5g peptone, 1g yeast extract paste, 8g Na
2HPO
4, 15g ethanol, 1.5g Trisodium Citrate, all the other compositions are water, HCl adjust pH to 4.5.Fermention medium is loaded in the 500ml triangular flask, the triangular flask loading amount is 50ml, in 28 ℃, the reciprocating type shaking table shaking culture of stroke 75mm, rotating speed 90r/m 7 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, take out bacterial cellulose gel, distilled water flushing 3 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 70 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 5.6g/L.
Embodiment five:
Get 1 day inclined-plane kind of preservation in 4 ℃ of refrigerators, [composition of slant medium (g/L): glucose 120, yeast extract paste 12, lime carbonate 20, agar 22, all the other compositions are water, pH7.0.Cultivated 48 hours for 30 ℃], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be transferred in the liquid activation medium of new configuration [composition of liquid activation medium (g/L): glucose 120, yeast extract paste 12, peptone 10, all the other compositions are water, adjust pH to 6.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 30 ℃ of reciprocating type shaking table shaking culture 36 hours, shaking speed was 110r/m, and stroke is 70mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 10% inoculum size (5ml), (g/L) is as follows for the composition of fermention medium: 60g glucose, 10g peptone, 1.5g yeast extract paste, 10g Na
2HPO
4, 20g ethanol, 2g Trisodium Citrate, all the other compositions are water, adjust pH to 5.5.Fermention medium is loaded in the 500ml triangular flask, and the triangular flask loading amount is 50ml, 30 ℃ of static placements 10 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, takes out bacterial cellulose gel, distilled water flushing 3 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 60 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 4.4g/L.
Embodiment six:
Get 1 day inclined-plane kind of preservation in 4 ℃ of refrigerators, [composition of slant medium (g/L): glucose 120, yeast extract paste 12, lime carbonate 20, agar 22, all the other compositions are water, pH7.0.Cultivated 48 hours for 30 ℃], with inoculating needle with about 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be transferred in the liquid activation medium of new configuration [composition of liquid activation medium (g/L): glucose 120, yeast extract paste 12, peptone 10, all the other compositions are water, adjust pH to 6.0.], the liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 30 ℃ of reciprocating type shaking table shaking culture 36 hours, shaking speed was 110r/m, and stroke is 70mm.Get above-mentioned cultured liquid activation medium and be inoculated into new configuration, 121 ℃ of sterilizations 15 minutes and be cooled on the fermention medium of room temperature by 10% inoculum size (5ml), (g/L) is as follows for the composition of fermention medium: 60g glucose, 10g peptone, 1.5g yeast extract paste, 10g Na
2HPO
4, 20g ethanol, 2g Trisodium Citrate, all the other compositions are water, adjust pH to 5.5.Fermention medium is loaded in the 500ml triangular flask, the triangular flask loading amount is 50ml, in 30 ℃, the reciprocating type shaking table shaking culture of stroke 70mm, rotating speed 110r/m 10 days, stop fermentation, the fermented liquid that fermentation is finished carries out solid-liquid separation, take out bacterial cellulose gel, distilled water flushing 3 times, bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L 80 ℃ of insulation 60min, after using deionized water rinsing 2 times then, again bacterial cellulose gel is dried to constant weight in 60 ℃, weighs in the balance heavily, the output that draws bacteria cellulose is 8.0g/L.
Sequence table
＜110〉Guangdong Microbes Inst
＜120〉bacteria cellulose produces bacterium and utilizes this bacterial strain to prepare the method for bacteria cellulose
<160>1
<210>1
<211>1436
<212>DNA
＜213〉glyconic acid bacillus aceticus (Gluconacetobacter sp.) GD-BC-1
<400>1
Claims (4)
1. a bacteria cellulose produces bacterium, it is characterized in that this bacterial strain is glyconic acid bacillus aceticus (Gluconacetobacter sp.) GD-BC-1CCTCC M 208149.
2. the preparation method of a bacteria cellulose is characterized in that doing fermentation strain with glyconic acid bacillus aceticus GD-BC-1CCTCC M 208149, utilizes glucose to ferment as main fermentation raw material.
3. the preparation method of bacteria cellulose according to claim 2 is characterized in that technological process is as follows:
(1) slant strains activation: cultured glyconic acid bacillus aceticus GD-BC-1CCTCC M 208149 preservation inclined-plane kinds are lined on the culture medium slant of new preparation, culture medium slant is composed as follows: glucose 80～120g, yeast extract paste 8～12g, lime carbonate 15～25g, agar 15～22g, tap water 1L, adjust pH to 6.5～7.2, cultivated 24～48 hours for 28-32 ℃, refrigerate again in 4 ℃ 1～5 day;
(2) liquid of bacterial classification activation: with 1cm on the above-mentioned inclined-plane
2The bacterial classification of area scrapes, be inoculated in the liquid activation medium, the liquid activation medium is composed as follows: glucose 80～120g, yeast extract paste 8～12g, peptone 5～10g, tap water 1L, adjust pH to 6.0～7.0, the liquid activation medium is loaded in the 250ml triangular flask, the triangular flask loading amount is 20ml, in 28-32 ℃ of reciprocating type shaking table shaking culture 18～36 hours, shaking speed was 80-110r/m, and stroke is 65-75mm;
(3) fermentation: get cultured liquid activation and plant, press the 5-10% inoculum size, insert in the fermention medium, carry out dynamically or standing for fermentation; When adopting the dynamic approach fermentation, with the bottled fermention medium of the above volumetrical triangle of 500ml, loading amount is 10% of a triangular flask volume, in 28-32 ℃, and stroke 65-80mm, the reciprocating type shaking table shaking culture of rotating speed 60-110r/m 4-10 days stops fermentation; When the method fermentation was left standstill in employing, with the bottled fermention medium of the above volumetrical triangle of 500ml, loading amount was 10% of a triangular flask volume, after 4-10 days, stopped fermentation in 28-32 ℃ of static placement;
(4) fermented liquid that finishes of fermentation extracts bacterial cellulose product by following technology:
The fermented liquid that fermentation is finished passes through solid-liquid separation, obtain bacterial cellulose gel, repeatedly wash with distilled water, remove its surface impurity, again bacterial cellulose gel is immersed in the NaOH solution of 0.1mol/L, 80 ℃ of insulation 60min, remove remaining thalline and substratum, wash repeatedly with deionized water then, obtain the translucent gel that is creamy white, again with this gel in 60～80 ℃ of dryings, promptly get the bacteria cellulose product.
4. according to the preparation method of claim 2 or 3 described bacteria celluloses, it is characterized in that described fermention medium composition is calculated as by every liter of contained grammes per square metre g/L of substratum: 20-60g glucose, 5-10g peptone, 0.5g-1.5g yeast extract paste, 6-10gNa
2HPO
4, 10-20g ethanol, 1-2g Trisodium Citrate, all the other compositions are water; Cultivation based on the sterilization before adjust pH to 4.5-7.0.
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| CN103451252B (en) * | 2013-10-08 | 2017-09-29 | 黑龙江大学 | Chrysanthemum shape bacteria cellulose and its fermentation preparation |
| CN103667148B (en) * | 2013-12-14 | 2016-01-27 | 江南大学 | One plant height produces the high temperature resistant middle gluconacetobacter of bacteria cellulose |
| CN103740784B (en) * | 2013-12-27 | 2015-10-28 | 深圳先进技术研究院 | A kind of method reducing bacteria cellulose degree of crystallinity during the fermentation |
| CN107488607B (en) * | 2016-06-13 | 2020-07-10 | 广东东阳光药业有限公司 | Separation identification and application of bacterial cellulose producing strain |
| CN110295133A (en) * | 2019-08-02 | 2019-10-01 | 长春理工大学 | A kind of screening technique of bacteria cellulose synthesis bacterium |
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