CN112760265A - Bacterial cellulose strain suitable for dynamic culture and application thereof - Google Patents
Bacterial cellulose strain suitable for dynamic culture and application thereof Download PDFInfo
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Abstract
The invention discloses a strain suitable for dynamic culture and bacterial cellulose production, which is obtained by separating and screening from persimmons, is identified to be Gluconacetobacter xylinum, is named as Gluconacetobacter xylinum 171027-PER1(Gluconacetobacter xylinum), is preserved in the general microorganism strain preservation center of China Committee for culture and management of microorganisms, has the preservation number of CGMCC No.15234 and the preservation date of 2018, 1 month and 17 days. The invention also discloses a method for producing bacterial cellulose by the dynamic fermentation of the xyloglucan acetobacter 171027-PER1, wherein the yield of the dynamic fermentation cellulose can reach 6g/L, the fiber diameter is low, and the water holding capacity is stronger.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and relates to separation, identification and culture of a bacterial cellulose-producing strain suitable for dynamic culture, and a method for producing bacterial cellulose by using the bacterial strain.
Background
Bacterial Cellulose (BC) is a Cellulose synthesized by bacteria of the genus foal (Komagataeibacter), Rhizobium (Rhizobium), Sarcina (Sarcina) and the like and secreted to the outside of cells. BC is a linear long chain polymerized from D-glucopyranose molecules with beta-1, 4 glycosidic bonds. Generally consisting of fine fibers (nanofibers) having a width of about 20nm to 80nm, have many excellent characteristics such as high purity, high crystallinity, high tensile strength and elastic modulus.
At present, two culture methods, namely static culture and dynamic culture, are adopted at home and abroad to culture bacteria cellulose producing bacteria, and the produced bacteria cellulose is compact and filmy in the static culture mode. The bacterial cellulose produced by static culture has been applied to products such as food, sound vibration films, high-strength paper, novel wound binding materials and the like, has been developed completely, and has huge market prospect; however, the method for producing bacterial cellulose by dynamic fermentation is blank in the domestic market and needs to be developed, the dynamic fermentation often generates various forms such as flakes, floccules and spheres, the yield is limited, and the large-scale production and industrialization cannot be realized. For example, the highest BC yield is 2.0g/L in a dynamic fermentation optimized acetobacter xylinum 1.1812 culture medium (research on bacterial cellulose produced by acetobacter xylinum 1.1812 fermentation, Nature science board 2005 (05)) of grand Dongping, Nanjing university of science and technology; the pilot-scale research of xuyunhua and the like is carried out, and the mechanical stirring fermentation yield of the bacterial cellulose is improved from 2.2g/L to 2.8g/L (pilot-scale experimental research of bacterial cellulose produced by airlift fermentation, xuyunhua, food research and development 2016, 37 (19)).
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a bacterial cellulose strain suitable for dynamic culture and application thereof in bacterial cellulose production.
The invention provides a bacterial cellulose production strain, which is gluconacetobacter xylinus 171027-PER1 (also called xylose colatopsis 171027-PER1) which is deposited in China general microbiological culture Collection center (CGMCC) in 2018, 1 and 17 days, and the preservation number is CGMCC No. 15234. The preservation address is Beijing in China.
The strain is acetobacter xylinum (xylose colatole), and the bacterium is blue short rod-shaped, has no flagellum, spore and other structures and is gram-negative bacterium under an optical microscope through gram staining; after 3d of culture in a solid culture medium, the colony is in a white round shape, the edge is smooth and has no bulge, and no hydrolysis ring is arranged around the colony;
the gene sequence of the strain is shown in SEQ ID NO. 1.
The invention also provides a screening method of bacterial cellulose-producing strains, which comprises the following steps:
(1) primary enrichment culture: inoculating rotten fruits into a seed culture medium with the pH of 4.0-6.0 (preferably 4.0) and containing nystatin and absolute ethyl alcohol, shaking a test tube, and culturing in a constant-temperature incubator at 28-34 ℃ (preferably 30 ℃);
(2) secondary enrichment culture: washing the mycoderm which is enriched and grown for the first time, putting the mycoderm into a seed culture medium, adding nystatin, and culturing in a constant-temperature incubator at 28-34 ℃ (preferably 30 ℃);
(3) diluting and coating a plate: shearing the mycoderm subjected to secondary enrichment growth, diluting, coating the mycoderm on a solid seed culture medium plate containing a fluorescent whitening agent 28, and culturing in a constant-temperature incubator at 28-34 (preferably 30 ℃);
(4) preservation of the inclined plane: observing a flat plate with a single colony under an ultraviolet lamp, selecting a fluorescent colony, inoculating the fluorescent colony to a seed culture medium for slant amplification culture, culturing in a constant temperature incubator at 28-34 ℃ (preferably 30 ℃), and storing the slant in a refrigerator at 2-4 ℃ (preferably 4 ℃) after microscopic examination to obtain a strain culture.
In one embodiment, the method for screening bacterial cellulose-producing strains comprises the following steps:
(1) primary enrichment culture:
selecting various rotten fruits, respectively inoculating into seed culture medium with pH adjusted to 4.0 with acetic acid, wherein the seed culture medium contains 150ul nystatin and 300ul absolute ethanol, slightly shearing the fruits, shaking the small test tubes, and culturing in a constant temperature incubator at 30 ℃;
(2) secondary enrichment culture:
washing the primary enriched and grown mycoderm with normal saline for three times, putting into seed culture medium, shearing, adding 150ul nystatin, shaking small test tube, and culturing in 30 deg.C constant temperature incubator;
(3) diluting and coating a plate:
cutting the secondary enrichment-grown mycoderm, diluting with three gradient of 10-1，10-2，10-3Respectively coating the seeds on solid seed culture medium plates containing fluorescent whitening agent 28, and culturing the coated seed culture medium plates in a constant temperature incubator at 30 ℃;
(4) preservation of the inclined plane:
observing the plate with single colony under 254nm ultraviolet lamp, selecting fluorescent colony, inoculating to seed culture medium, culturing at 30 deg.C, examining to obtain strain culture, and storing at 4 deg.C.
In some embodiments, the spoiled fruit of step (1) is selected from at least one of cherry, nectarine, apple, mango, orange, persimmon, and the like.
In some embodiments, the seed medium of step (1) and/or step (2) comprises: 1-3% (preferably 2%) of glucose, 0.5-1% (preferably 0.5%) of yeast powder, 0.5-1% (preferably 0.5%) of peptone, 0.1-0.5% (preferably 0.27%) of disodium hydrogen phosphate, 0.1-0.2% (preferably 0.115%) of citric acid, and the pH value is 5.0-7.0 (preferably 6.0); preferably, the glucose content is 2%, the yeast powder content is 0.5%, the peptone content is 0.5%, the disodium hydrogen phosphate content is 0.27%, the citric acid content is 0.115%, and the pH value is 6.0.
In some embodiments, the solid seed medium component of step (3) is agar added to the seed medium at a concentration of 1.5% to 2.0% (preferably 2.0%).
The invention also provides a method for identifying strains through molecular biology, which comprises the following steps:
inoculating the strain to 30mL of seed culture medium, culturing at 30 ℃ and 160r/min for 20h, streaking onto an HS culture medium plate, and culturing at 30 ℃ until a bacterial colony grows. Activating a single colony, inoculating the single colony into a seed culture medium added with cellulase (6.7%,. gtoreq.45 units/mg), culturing for 24h, extracting DNA for detecting the purity concentration of the DNA, and amplifying by using bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGC TACCTTGTTACGACTT-3'). The PCR reaction program comprises pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 40s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles, final extension at 72 ℃ for 5min, and storage of the product at 4 ℃. And verifying the amplification product by agarose gel electrophoresis, then sequencing by Shanghai Meiji biological medicine science and technology limited, and correcting and splicing the obtained 16S rDNA sequence in a forward and reverse direction by using software CExpress. The sequences after splicing were subjected to BLAST comparison analysis of National Center for Biotechnology Information (NCBI) database, the top 10 model strain sequences with the highest homology were downloaded as reference strains, sequence characteristics were analyzed using the software MEGA7.0 and a Neighbor-Joining phylogenetic tree was constructed to obtain strain identification results.
The invention also provides a method for producing bacterial cellulose by utilizing the dynamic fermentation of the acetobacter xylinum (Xylactinatus) 171027-PER1, which takes a culture medium containing cane sugar as a fermentation raw material, and dynamically ferments the acetobacter xylinum (Xylactinatus) 171027-PER1 to obtain the bacterial cellulose, and comprises the following steps:
the method comprises the following steps: inoculating acetobacter xylinum (Xylobacillus colans) 171027-PER1 into a seed culture medium, and performing static culture in a constant-temperature incubator at 28-34 ℃ (preferably 30 ℃) for 24-30h (preferably 24h) to obtain a 171027-PER1 culture solution;
step two: filtering the 171027-PER1 culture solution obtained in the first step by using absorbent cotton to obtain 171027-PER1 seed solution without containing a bacterial cellulose membrane;
step three: inoculating 171027-PER1 seed liquid into a triangular flask filled with 30-70 mL (preferably 50mL) of fermentation culture medium according to the inoculation amount of 8-10% (preferably 8%), and performing shake culture at the constant temperature of 28-34 ℃ (preferably 30 ℃), 100-200 rpm (preferably 120rpm) for 4-7 d (preferably 4d) to obtain bacterial cellulose fermentation liquid;
step four: and (4) centrifuging, washing, alkali boiling, neutralizing and drying the bacterial cellulose fermentation liquor obtained in the step three to obtain bacterial cellulose powder.
The seed culture medium mainly comprises the following components: 1-3% (preferably 2%) of glucose, 0.5-1% (preferably 0.5%) of yeast powder, 0.5-1% (preferably 0.5%) of peptone, 0.1-0.5% (preferably 0.27%) of disodium hydrogen phosphate, 0.1-0.2% (preferably 0.115%) of citric acid, and the pH value is 5.0-7.0 (preferably 6.0); preferably, the glucose content is 2%, the yeast powder content is 0.5%, the peptone content is 0.5%, the disodium hydrogen phosphate content is 0.27%, the citric acid content is 0.115%, and the pH value is 6.0.
The fermentation medium mainly comprises the following components: 4-5% of sucrose, (preferably 5%), 0.5-1.2% of yeast powder, (preferably 0.9%), 0.3-0.7% of peptone, (preferably 0.5%), 0.1-0.5% of dipotassium phosphate, (preferably 0.3%), 0.2-1.0% of acetic acid, (preferably 0.6%), 1-2% of magnesium sulfate, (preferably 2%), and a pH value of 4.0-6.0, (preferably 5.0); preferably, the composition comprises 5% of sucrose, 0.9% of yeast powder, 0.5% of peptone, 0.3% of dipotassium hydrogen phosphate, 0.6% of acetic acid, 2% of magnesium sulfate and 5.0 of pH value.
And step two, the absorbent cotton is filtered, namely absorbent cotton is placed in a needle cylinder, 171027-PER1 culture solution is poured into the needle cylinder, and the uniform 171027-PER1 seed solution without the bacterial cellulose membrane is obtained through natural filtration.
In the fourth step, the centrifugation is to put the bacterial cellulose fermentation liquor into a centrifuge bottle and centrifuge for 5min at 9000 rpm; the step of washing is to add the centrifuged bacterial cellulose fermentation liquor into ultrapure water, pour the ultrapure water into a centrifugal bottle, centrifuge the fermentation liquor at 9000rpm for 5min and remove supernatant; the alkali boiling is to add the bacterial cellulose fermentation liquor after water washing into 1 percent sodium hydroxide solution, and then carry out boiling water bath for 1h at the temperature of 80-100 ℃; the neutralization is to add the bacterial cellulose after the alkali cooking into 100mL of 1% acetic acid to neutralize the pH; and the drying is to bake the neutralized bacterial cellulose fermentation liquor in an oven at 80 ℃ for 12 h.
The invention also provides bacterial cellulose prepared by the method.
Triangular flasks and fermentation tanks are important for obtaining bacterial cellulose with the thickness of only 27nm and high uniformity through fermentation.
In the triangular flask system, the bacterial cellulose produced by the acetobacter xylinum (coltsia xylosus) 171027-PER1 is uniformly dispersed, and is different from the bacterial cellulose producing strains which produce flaky or nodular bacterial cellulose.
In a fermentation tank system, the bacterial cellulose produced by the gluconacetobacter xylinus (Xylaria colauba) 171027-PER1 is uniformly dispersed, and is different from the bacterial cellulose producing strains which produce the bacterial cellulose in a sheet or a block shape.
The average diameter of the bacterial cellulose produced by the gluconacetobacter xylinus 171027-PER1 is only 27 nm.
In the above steps of the invention, the triangular flask and the fermentation tank are important: the bacterial cellulose is secreted from the outside of cells while consuming carbon sources in the bacterial growth process, the generated bacterial cellulose can wrap bacteria, and most of thallus fermentation processes can gather in a lump or block shape due to continuous generation of the bacterial cellulose. In addition, most of the bacteria producing bacterial cellulose are strict aerobic bacteria, and when the bacteria are wrapped by too much cellulose, the oxygen and energy supply of the bacteria can be influenced, so that the bacteria are in worse and worse states, and the yield is reduced.
In the invention, in the triangular flask dynamic fermentation, although the shaking table is continuously oscillated, the fermentation liquid is static relative to the thalli in the whole process, so that the mass or block cellulose is easily formed.
In the fermentation tank, cellulose is easy to adhere and wrap the stirring paddle, and large blocks of bacterial cellulose are formed and wound on the stirring paddle, so that less bacterial cellulose is in the fermentation tank.
The invention also provides application of the gluconacetobacter xylinus (Xylobacillus colatyi) 171027-PER1 in fermentation production of bacterial cellulose.
The beneficial effects of the invention include: compared with the currently reported strains, the bacterial cellulose strain 171027-PER1 obtained by the invention is suitable for dynamic fermentation, has the characteristics of high yield and finer cellulose, and is identified as acetobacter xylinum (Komagataeibacter xylinum) by 171027-PER1 in combination with morphological, physiological, biochemical and phylogenetic analyses. The strain of the invention is dynamically fermented to produce more dispersed filamentous bacterial cellulose, which is more suitable for dynamic fermentation; the yield is high, the fermentation time 4d reaches 6g/L, and the fiber diameter is lower and is only 27 nm. The fiber diameter reported in the literature at present is mainly 50-100nm, and the thinner fiber diameter has stronger water holding capacity and can absorb 60-700 times of water of the dry weight of the fiber.
Drawings
FIG. 1 is a phylogenetic analysis diagram of the bacterium Xylofoeniculi 171027-PER1 according to the present invention.
FIG. 2 is an infrared spectrum analysis chart of bacterial cellulose produced by the bacterium colatole 171027-PER 1.
FIG. 3 is a thermogravimetric analysis diagram of bacterial cellulose produced by Xylella colatonii 171027-PER 1.
FIG. 4 is a scanning electron microscope image of bacterial cellulose produced by Xylella xylofoenum 171027-PER 1.
FIG. 5 is a diameter distribution diagram of bacterial cellulose produced by the bacterium Xylobacter colatoides 171027-PER 1.
FIG. 6 is a graph showing the comparison of the dynamic fermentation yields of 171027-PER1 of Xylella coll of the present invention and a standard strain.
FIG. 7 is a graph comparing the morphology of bacterial cellulose produced in flasks of the bacterium Xylobacter colatoides 171027-PER1 of the present invention with that of the standard strain ATCC 53263. (FIG. A shows bacterial cellulose produced in Xylobacteroides xylocolae 171027-PER1 triangular flask, and FIG. B shows bacterial cellulose produced in standard strain ATCC 53263 triangular flask)
FIG. 8 is a graph comparing the morphology of bacterial cellulose produced by fermentors of the bacterium Xylobacter colatole 171027-PER1 of the present invention with that of the standard strain ATCC 53263. (FIG. A shows bacterial cellulose produced by the fermentation tank of Xylobacteroides xylocolae 171027-PER1, and FIG. B shows bacterial cellulose produced by the fermentation tank of Standard Strain ATCC 53263)
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1: and (3) separating bacterial cellulose-producing strains:
(1) primary enrichment culture:
selecting various rotten fruits, respectively inoculating into seed culture medium with pH adjusted to 4.0 with acetic acid, wherein the seed culture medium contains 150ul nystatin and 300ul absolute ethyl alcohol, slightly shearing the fruits, shaking the small test tubes, and culturing in a 30-DEG C constant-temperature incubator;
(2) secondary enrichment culture:
washing the primary enriched and grown mycoderm with normal saline for three times, putting into seed culture medium, shearing, adding 150ul nystatin, shaking small test tube, and culturing in 30 deg.C constant temperature incubator;
(3) dilution coating:
cutting the secondary enrichment-grown mycoderm, diluting with three gradient of 10-1，10-2，10-3Respectively coating the seeds on solid seed culture medium plates containing fluorescent whitening agent 28, and culturing the coated seed culture medium plates in a constant temperature incubator at 30 ℃;
(4) preservation of the inclined plane:
observing the flat plate with single bacterial colony under 254nm ultraviolet lamp, selecting fluorescent bacterial colony, inoculating to seed culture medium, culturing at 30 deg.C in constant temperature incubator, and storing at 4 deg.C after microscopic examination to obtain strain culture;
the putrefactive fruit is at least one selected from cherry, nectarine, apple, mango, orange, persimmon, etc.
The seed culture medium comprises the following main components: 2% of glucose, 0.5% of yeast powder, 0.5% of peptone, 0.27% of disodium hydrogen phosphate and 0.115% of citric acid, and the pH value is 6.0.
Example 2: identification of bacteria
(1) The 16S rDNA gene of 171027-PER1 strain is amplified through the following steps:
inoculating the strain to 30mL of seed culture medium, culturing at 30 ℃ and 160r/min for 20h, streaking onto an HS culture medium plate, and culturing at 30 ℃ until a bacterial colony grows. Activating a single colony, inoculating the single colony into a seed culture medium added with cellulase (6.7%,. gtoreq.45 units/mg), culturing for 24h, extracting DNA for detecting the purity concentration of the DNA, and amplifying by using bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCT TGTTACGACTT-3'). The PCR reaction program comprises pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 40s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles, final extension at 72 ℃ for 5min, and storage of the product at 4 ℃. And verifying the amplification product by agarose gel electrophoresis, then sequencing by Shanghai Meiji biological medicine science and technology Limited, and correcting and splicing the obtained 16S r DNA sequence in forward and reverse directions by using software CExpress.
Through sequence alignment, an evolutionary tree is made by using MEGA software, and the result shows that 171027-PER1 strain has the highest homology with Komagataeibacxylinus ATCC 53524 (the original name is Gluconacerobacterxylinus).
(2) The xylose foal bacillus 171027-PER1 strain is subjected to physiological and biochemical tests according to the operation instructions of a physiological and biochemical kit, and is identified according to Bojie's Manual of identification of bacteria, and the identification results are shown in the following tables 1 and 2:
TABLE 1 physiological and biochemical characteristics of Strain 171027-PER1
TABLE 2 carbon Source utilization test of Strain 171027-PER1
| Reagent | Results | Reagent | Results |
| Glucose | + | Xylose | - |
| Sucrose | + | Arabinose | - |
| Rhamnose | _ | Galactose | + |
| Maltose | + | Sorbitol | - |
| Mannitol | _ | Inositol | - |
| Fructose | + |
Note: "+" indicates utilized, and "-" indicates not utilized
By comprehensive analysis, the strain separated by the invention is a bacterium of Xylobacter xylinum Komagataeibacter xylinum (originally named as gluconacetobacter xylinum Gluconoacetobacter xylinus).
Example 3: production of triangular flask bacterial cellulose
Inoculating bacillus coltsianthi 171027-PER1 preserved at 4 ℃ into a seed culture medium, carrying out static culture in a constant-temperature incubator at 30 ℃ for 28h to obtain 171027-PER1 culture solution, filtering by using absorbent cotton to obtain seed solution without a bacterial cellulose membrane, transferring 171027-PER1 seed solution into a 250mL triangular flask filled with 50mL of fermentation culture medium according to 9% of inoculum size, carrying out shake cultivation at 30 ℃ and 120rpm for 4d, after fermentation is finished, centrifuging for 5min at 9000rmp, removing the culture medium, and continuously washing and centrifuging the precipitated bacterial cellulose twice. Removing thallus and other impurities with 1% sodium hydroxide solution in boiling water bath for 1 h. And (3) carrying out centrifugal neutralization by using 1% acetic acid, washing the obtained product to be neutral, and then putting the purified bacterial cellulose into an oven at 80 ℃ for drying for 12 hours to calculate the yield.
The seed culture medium mainly comprises the following components: 2% of glucose, 0.5% of yeast powder, 0.5% of peptone, 0.27% of disodium hydrogen phosphate and 0.115% of citric acid, and the pH value is 6.0.
The fermentation medium mainly comprises the following components: 5 percent of sucrose, 0.9 percent of yeast powder, 0.5 percent of peptone, 0.3 percent of dipotassium phosphate, 0.6 percent of acetic acid, 2 percent of magnesium sulfate and 5.0 of pH value.
Example 4: production of bacterial cellulose in 10L fermentation tank
Inoculating bacillus colauda 171027-PER1 preserved at 4 ℃ into a seed culture medium, performing static culture in a constant-temperature incubator at 30 ℃ for 28h to obtain 171027-PER1 culture solution, filtering by using absorbent cotton to obtain a first-stage seed solution without a bacterial cellulose membrane, inoculating 171027-PER1 seed solution into a 250mL common triangular flask filled with 50mL of the seed culture medium according to 9% of inoculation amount, performing constant-temperature shaking culture at 30 ℃ and 120rpm for 24h to obtain a second-stage seed solution, inoculating into a 10L fermentation tank filled with 5.5L of fermentation culture medium according to 9% of inoculation amount, and fermenting at 30 ℃ and 200rpm for 4 d. After the fermentation is finished, sampling, centrifuging for 5min at 9000rmp, removing the culture medium, and continuously washing and centrifuging the precipitated bacterial cellulose twice. Removing thallus and other impurities with 1% sodium hydroxide solution in boiling water bath for 1 h. And (3) carrying out centrifugal neutralization by using 1% acetic acid, washing the obtained product to be neutral, and then putting the purified bacterial cellulose into an oven at 80 ℃ for drying for 12 hours to calculate the yield.
The seed culture medium mainly comprises the following components: 2% of glucose, 0.5% of yeast powder, 0.5% of peptone, 0.27% of disodium hydrogen phosphate and 0.115% of citric acid, and the pH value is 6.0.
The fermentation medium mainly comprises the following components: 5 percent of sucrose, 0.9 percent of yeast powder, 0.5 percent of peptone, 0.3 percent of dipotassium phosphate, 0.6 percent of acetic acid, 2 percent of magnesium sulfate and 5.0 of pH value.
Example 5: bacterial cellulose sample preparation
Bacterial cellulose was produced according to example 4 of the present invention. After the fermentation is finished, sampling, centrifuging for 5min at 9000rmp, removing the culture medium, and continuously washing and centrifuging the precipitated bacterial cellulose twice. Removing thallus and other impurities with 1% sodium hydroxide solution in boiling water bath for 1 h. The solution is neutralized by centrifugation with 1% acetic acid and washed to neutrality with water. And (3) drying the neutral bacterial cellulose in a freeze dryer, and crushing the neutral bacterial cellulose by a crusher to obtain bacterial cellulose powder after the drying is finished.
FIG. 2 is an infrared spectrum of a sample of bacterial cellulose obtained in example 5 of the present invention, from which FIG. 2 a typical absorption peak of cellulose can be observed. 3343cm-1Is located at 2917cm and is a stretching vibration peak of intramolecular hydrogen bonds and hydroxyl groups-1Is C-H stretching vibration absorption peak, 1110cm-1And 1055cm-1Is generated by C-O-C stretching vibration, is consistent with the report of the literature, and indicates that the bacterial cellulose belongs to I type alpha cellulose.
FIG. 3 is a thermogravimetric analysis of a bacterial cellulose sample obtained in example 5 of the present invention, and it can be seen from FIG. 3 that the loss of weight of bacterial cellulose mainly has three stages, the mass loss in the first stage occurs near 100 ℃, the loss amount is 4.46%, and the loss is mainly some bound water on the surface of bacterial cellulose; the mass loss of the second stage is about 250 ℃, the loss amount is 34.38%, mainly due to the fact that bacterial cellulose contains a large amount of hydroxyl, and the hydroxyl is dehydrated, and the mass loss is consistent with the maximum weight loss of the second stage which is reported in the literature and mainly occurs at 238-329 ℃; the mass loss in the third stage is around 430 ℃, the loss amount is 15.16%, and the bacterial cellulose continues to be decomposed. Thermogravimetric analysis shows that the maximum weight loss temperature of the bacterial cellulose produced by the strain is 250 ℃, and the bacterial cellulose has the property of high temperature resistance.
FIG. 4 is an SEM image of a bacterial cellulose sample obtained in example 5 of the present invention, and it can be seen from FIG. 4 that the filaments produced by the dynamic fermentation method are closely interlaced and have significantly uneven thickness, and a large number of pore structures exist, so that the sample has a large specific surface area, and can absorb hundreds of times of water on its own dry weight.
FIG. 5 is a distribution diagram of fiber diameters of the bacterial cellulose samples obtained in example 5 of the present invention, and it can be seen from FIG. 5 that the average diameter is only 27nm as long as the fiber diameters are distributed between 20nm and 40nm, which is much smaller than the average diameter of 50 nm to 100nm commonly found in the literature
Example 6: comparison of bacterial cellulose produced by dynamic culture of xylofoal bacillus 171027-PER1 and model strain
Respectively inoculating bacillus colauda 171027-PER1 preserved at 4 ℃ and model strains ATCC 53263, DSM 2004, DSM 46603 and DSM11804 for producing bacterial cellulose into a seed culture medium, standing and culturing for 28h in a constant-temperature incubator at 30 ℃ to obtain a strain culture solution, filtering by using absorbent cotton to obtain a seed solution without a bacterial cellulose membrane, transferring the seed solution into a 250mL triangular flask filled with 50mL of fermentation culture medium according to 9 percent of inoculum size, culturing for 4d in a constant-temperature shaking table at 30 ℃ and 120rpm, centrifuging for 5min at 9000rmp after fermentation is finished, removing the culture medium, and continuously washing and centrifuging the precipitated bacterial cellulose twice. Removing thallus and other impurities with 1% sodium hydroxide solution in boiling water bath for 1 h. And (3) carrying out centrifugal neutralization by using 1% acetic acid, washing the obtained product to be neutral, and then putting the purified bacterial cellulose into an oven at 80 ℃ for drying for 12 hours to calculate the yield.
FIG. 6 is a graph comparing the yields of bacterial cellulose produced by dynamic culture of Corynebacterium colae 171027-PER1 and model strain in example 6 of the present invention, and it can be seen from FIG. 6 that the yield of bacterial cellulose dynamically cultured by the invention is 6g/L for Corynebacterium colae 171027-PER1, the yield of bacterial cellulose dynamically cultured by other model strains such as ATCC 53263 is 1.03g/L, the yield of bacterial cellulose dynamically cultured by DSM 2004 is 0.99g/L, the yield of bacterial cellulose dynamically cultured by DSM 46603 is 0.71g/L, and the yield of bacterial cellulose dynamically cultured by DSM11804 is 1.8 g/L. Therefore, the dynamic culture of the xylose foal bacillus 171027-PER1 is obviously higher than that of other model strains, and the yield is high.
FIGS. 7 and 8 are morphological diagrams of bacterial cellulose produced by dynamic culture of the bacterium Xylofoeni 171027-PER1 and the model strain ATCC 53263 in a flask and a fermenter according to the present invention. As is clear from FIG. 7, FIG. 7A shows that the bacterial cellulose produced by dynamic culture in Eremobacter xylinum 171027-PER1 flasks was uniformly dispersed, and FIG. 7B shows that the bacterial cellulose produced by dynamic culture in model strain ATCC 53263 flasks was lumpy and unevenly distributed. As is clear from FIG. 8, FIG. 8A shows that the bacterial cellulose produced by dynamic culture in the Xylaria foenum 171027-PER1 fermenter was uniformly dispersed, and FIG. 8B shows that the bacterial cellulose produced by dynamic culture in the model strain ATCC 53263 fermenter was lumpy and unevenly distributed.
The seed culture medium mainly comprises the following components: 2% of glucose, 0.5% of yeast powder, 0.5% of peptone, 0.27% of disodium hydrogen phosphate and 0.115% of citric acid, and the pH value is 6.0.
The fermentation medium mainly comprises the following components: 5 percent of sucrose, 0.9 percent of yeast powder, 0.5 percent of peptone, 0.3 percent of dipotassium phosphate, 0.6 percent of acetic acid, 2 percent of magnesium sulfate and 5.0 of pH value.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims.
SEQUENCE LISTING
<110> university of east China
<120> bacterial cellulose strain suitable for dynamic culture and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1380
<212> DNA
<213> Komagataeibacter xylinus Per-1
<400> 1
gggcatgggg cagcttacca tgcaagtcgc acgaaccttt cggggttagt ggcggacggg 60
tgagtaacgc gtagggatct gtccatgggt gggggataac tttgggaaac tgaagctaat 120
accgcatgac acctgagggt caaaggcgca agtcgcctgt ggaggaacct gcgttcgatt 180
agctagttgg tggggtaaag gcctaccaag gcgatgatcg atagctggtc tgagaggatg 240
atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300
attggacaat gggcgcaagc ctgatccagc aatgccgcgt gtgtgaagaa ggttttcgga 360
ttgtaaagca ctttcagcgg ggacgatgat gacggtaccc gcagaagaag ccccggctaa 420
cttcgtgcca gcagccgcgg taatacgaag ggggcaagcg ttgctcggaa tgactgggcg 480
taaagggcgc gtaggcggtt gacacagtca gatgtgaaat tcctgggctt aacctggggg 540
ctgcatttga tacgtggcga ctagagtgtg agagagggtt gtggaattcc cagtgtagag 600
gtgaaattcg tagatattgg gaagaacacc ggtggcgaag gcggcaacct ggctcatgac 660
tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 720
tgtaaacgat gtgtgctgga tgttgggtga ctttgtcatt cagtgtcgta gttaacgcga 780
taagcacacc gcctggggag tacggccgca aggttgaaac tcaaaggaat tgacgggggc 840
ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgcagaacc ttaccagggc 900
ttgacatgcg gaggccgtgt ccagagatgg gcatttctcg caagagacct ccagcacagg 960
tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020
caaccctcgc ctttagttgc cagcacgtct gggtgggcac tctaaaggaa ctgccggtga 1080
caagccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatgtc ctgggctaca 1140
cacgtgctac aatggcggtg acagtgggaa gccaggtggt gacaccgagc cgatctcaaa 1200
aagccgtctc agttcggatt gcactctgca actcgagtgc atgaaggtgg aatcgctagt 1260
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320
caccatggga gttggtttga ccttaagccg gtgagcgaac cgcaaggacg cagccgacca 1380
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
tacggctacc ttgttacgac tt 22
Claims (11)
1. A bacterial cellulose strain is characterized in that the bacterial cellulose strain is named as gluconacetobacter xylinus 171027-PER1, is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, has a preservation number of CGMCC No.15234 and a preservation date of 2018, 1 month and 17 days.
2. The bacterial cellulose strain of claim 1, wherein said bacterial strain is acetobacter gluconicum, and said bacterial strain exhibits blue short rod-like, flagellate and spore-like structures, and is a gram-negative bacterium, as seen by gram staining under an optical microscope; after 3d of culture in a solid culture medium, the colony is in a white round shape, the edge is smooth and has no bulge, and no hydrolysis ring is arranged around the colony; and/or the gene sequence of the strain is shown as SEQ ID NO. 1.
3. A method for producing bacterial cellulose by dynamic fermentation of gluconacetobacter xylinus 171027-PER1 is characterized in that a culture medium containing cane sugar is used as a fermentation raw material, and dynamic fermentation of gluconacetobacter xylinus 171027-PER1 is carried out to obtain the bacterial cellulose, and the method specifically comprises the following steps:
the method comprises the following steps: inoculating the strain 171027-PER1 as claimed in claim 1 or 2 into a seed culture medium, and performing static culture in a constant temperature incubator at 28-34 ℃ for 24-30h to obtain a 171027-PER1 culture solution;
step two: filtering the 171027-PER1 culture solution obtained in the first step by using absorbent cotton to obtain 171027-PER1 seed solution without containing a bacterial cellulose membrane;
step three: inoculating 171027-PER1 seed liquid into a triangular flask filled with 30-70 mL of fermentation medium according to the inoculation amount of 8-10%, and performing shake culture at the constant temperature of 28-34 ℃ and 100-200 rpm for 4-7 days to obtain bacterial cellulose fermentation liquid;
step four: and (4) centrifuging, washing, alkali boiling, neutralizing and drying the bacterial cellulose fermentation liquor obtained in the step three to obtain bacterial cellulose powder.
4. The method according to claim 3, wherein the fermentation medium comprises 4 to 5% of sucrose, 0.5 to 1.2% of yeast powder, 0.3 to 0.7% of peptone, 0.1 to 0.5% of dipotassium hydrogen phosphate, 0.2 to 1.0% of acetic acid, 1 to 2% of magnesium sulfate, and a pH value of 4.0 to 6.0;
the seed culture medium comprises the following components: 1-3% of glucose, 0.5-1% of yeast powder, 0.5-1% of peptone, 0.1-0.5% of disodium hydrogen phosphate, 0.1-0.2% of citric acid and 5.0-7.0 of pH value.
5. The method of claim 3, wherein in the second step, the absorbent cotton is filtered by placing the absorbent cotton in a syringe, then pouring the 171027-PER1 culture solution into the syringe, and naturally filtering to obtain the 171027-PER1 seed solution which is uniform and does not contain the bacterial cellulose membrane.
6. The method of claim 3, wherein in step four, the centrifugation is performed by filling the bacterial cellulose fermentation broth into a centrifuge bottle, and centrifuging at 9000rpm for 5 min; the step of washing is to add the centrifuged bacterial cellulose fermentation liquor into ultrapure water, pour the ultrapure water into a centrifugal bottle, centrifuge the fermentation liquor at 9000rpm for 5min and remove supernatant; the alkali boiling is to add the bacterial cellulose fermentation liquor after water washing into 1 percent sodium hydroxide solution, and then carry out boiling water bath for 1h at the temperature of 80-100 ℃; the neutralization is to add the bacterial cellulose after the alkali cooking into 100mL of 1% acetic acid to neutralize the pH; and the drying is to bake the neutralized bacterial cellulose fermentation liquor in an oven at 80 ℃ for 12 h.
7. Bacterial cellulose obtainable by a process according to any one of claims 3 to 6.
8. The bacterial cellulose of claim 7, wherein said bacterial cellulose produced by acetobacter xylinum 171027-PER1 is uniformly dispersed; and/or the average diameter of the bacterial cellulose produced by the gluconacetobacter xylinus 171027-PER1 is only 27 nm.
9. Use of acetobacter xylinum 171027-PER1 as claimed in claim 1 or 2 for the fermentative production of bacterial cellulose.
10. A screening method of acetobacter gluconicum is characterized by comprising the following steps:
(1) primary enrichment culture: inoculating rotten fruits into a seed culture medium with the pH of 4.0-6.0 and containing nystatin and absolute ethyl alcohol, shaking a test tube, and culturing in a constant-temperature incubator at the temperature of 28-34 ℃;
(2) secondary enrichment culture: washing the mycoderm which is enriched and grown for the first time, putting the mycoderm into a seed culture medium, adding nystatin, and culturing in a constant-temperature incubator at 28-34 ℃;
(3) diluting and coating a plate: shearing the mycoderm subjected to secondary enrichment growth, diluting, coating the mycoderm on a solid seed culture medium plate containing a fluorescent whitening agent 28, and culturing in a constant-temperature incubator at 28-34 ℃;
(4) preservation of the inclined plane: observing the flat plate with the single bacterial colony under an ultraviolet lamp, selecting fluorescent bacterial colony, inoculating the fluorescent bacterial colony to a seed culture medium, performing slant amplification culture, culturing in a constant-temperature incubator at 28-34 ℃, and storing the slant in a refrigerator at 2-4 ℃ after microscopic examination of the bacterial colony.
11. The screening method according to claim 10, wherein in the step (1), the putrefactive fruit is at least one selected from the group consisting of cherry, nectarine, apple, mango, orange, persimmon; in the step (1) or the step (2), the seed culture medium comprises the following components: 1-3% of glucose, 0.5-1% of yeast powder, 0.5-1% of peptone, 0.1-0.5% of disodium hydrogen phosphate and 0.1-0.2% of citric acid, and the pH value is 5.0-7.0.
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| CN116925974A (en) * | 2023-08-18 | 2023-10-24 | 江苏省农业科学院 | Bacterial cellulose-producing strain and application thereof |
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| CN116925974A (en) * | 2023-08-18 | 2023-10-24 | 江苏省农业科学院 | Bacterial cellulose-producing strain and application thereof |
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