Isolation, Identification and Application of a Bacterial Cellulose-Producing Strain

A technology of bacterial cellulose and bacterial strains, applied in the field of microorganisms, can solve the problems of difficult-to-adjacent species and strains, and achieve the effects of excellent biological affinity, rapid film production and film production, and simple operation

Active Publication Date: 2020-07-10
HEC PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the current identification of BC membrane-producing strains is mainly through the combination of 16S rDNA and physiological and biochemical methods. This method may have the limitation that it is difficult to distinguish adjacent species. Therefore, in some studies on BC-producing bacteria In the patents of various species, the strains are only identified to the genus, such as Chinese invention patent CN200810218500.7, bacterial cellulose producing bacteria and a method for preparing bacterial cellulose using the strain; Chinese invention patent CN201010515946.3, a bacterial cellulose strain

Method used

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  • Isolation, Identification and Application of a Bacterial Cellulose-Producing Strain
  • Isolation, Identification and Application of a Bacterial Cellulose-Producing Strain
  • Isolation, Identification and Application of a Bacterial Cellulose-Producing Strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Screening of film-producing strains

[0040] (1) Pick about 1 cm of pulp with decayed tissue from rotten apples, pears, citrus, melons, and mangoes 3 Small pieces, rinsed in 10 mL sterile water.

[0041] (2) Pipette 100 μL of rinse liquid into the PA bottle containing 10 mL of primary screening liquid medium. After static culture at 30°C for 4 days, a thin film appeared; transfer the film to sterile water with sterilized tweezers and rinse once to wash off the bacteria on the surface, then transfer the film to a 5mL centrifuge tube and rinse with sterile water. Mash the tip of the bacterial pipette as much as possible; add 4 mL of sterilized water and mix well; absorb 100 μL of the liquid and perform 5-fold gradient dilution to finally obtain 1-fold, 5-fold, 25-fold, 125-fold, and 625-fold diluted bacterial suspensions. Take 100 μL of each serial dilution and spread it on the primary screening solid medium. Among them, the composition of the primary scr...

Embodiment 2

[0044] Embodiment 2: Morphological observation of bacterial strain

[0045] Colony morphology: cultured at 30°C, cultivated on the first-screened solid plate medium for 72 hours, the colony is round, off-white, 2-3 mm in diameter, raised, dry, uneven, with irregular edges, easy to pick. The colony increases with time, and after 10 days, the diameter can reach more than 8mm, forming cellulose film blocks on the surface of the culture medium.

[0046] Cell morphology: The bacterial cells were observed by scanning electron microscope. The HEC-004 strain was rod-shaped, with a width of about 0.4-0.8 μm and a length of about 4 μm (see figure 1 ); the Gram reaction of this strain was negative.

Embodiment 3

[0047] Embodiment 3: the identification of strain

[0048] The main steps of amplifying the 16S rDNA, dnaK, groEL and rpoB genes of the HEC-004 strain are as follows:

[0049] A single colony was picked from the HS solid plate and inoculated into the primary screening liquid medium containing 1% cellulase, and cultured at 30°C and 150rpm for 48h.

[0050] Use a 1.5mL centrifuge tube to collect 1mL of the culture solution, and centrifuge at 12000rpm to collect the bacteria and remove the supernatant. The cell pellet was resuspended and washed with sterilized water, and the supernatant was removed after centrifugation. Repeat the cell wash once, and finally suspend the cells in 100 μL of water.

[0051] 16S rDNA, dnaK, groEL, rpoB were amplified by PCR method. The PCR system is: 1 μL of bacterial solution, PCR Master Mix (Takara, Japan) 25 μL, forward primer 2 μL, reverse primer 2 μL, add deionized water to 50 μL. PCR program: 95°C for 10 minutes; 30 cycles of 95°C for 30...

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Abstract

The invention belongs to the technical field of microorganisms, and in particular relates to the isolation, identification and application of a bacteria strain producing bacterial cellulose. The strain is isolated from spoiled fruit and has the characteristics of rapid film production and high film production. The present invention comprehensively uses 16S rDNA, and three housekeeping genes dnaK, groEL, and rpoB to carry out molecular analysis on the isolated HEC‑004 strain. Through horizontal identification, it was determined that the isolated strain was Gluconacetobacter nataicola, which has been preserved in the China General Microorganism Culture Collection Management Center with the preservation number: CGMCC No.11588.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to the isolation and identification of a bacteria strain producing bacterial cellulose, and the static fermentation culture of bacterial cellulose membranes. Background technique [0002] Bacterial cellulose (BC) refers to cellulose synthesized by bacteria. It is a polymer compound formed by the polymerization of glucose through β-1,4-glycosidic bonds. Its structure is very close to that of natural cellulose. Compared with cellulose produced by plants, BC has the characteristics of high purity, high crystallinity, high Young's modulus, high water holding capacity, excellent biodegradability and good biocompatibility, so it is expected to be used as a new type of biological material. Degradable materials are used in fields such as food, chemical industry and medicine. [0003] According to existing reports, microorganisms with BC synthesis function include Gluconacetoba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/02C12Q1/04C12Q1/689C12P19/04C12R1/02
CPCC12N1/02C12P19/04C12Q1/689C12N1/205C12R2001/02
Inventor 谢文平张鸿鲍素敏吴冬青毛兴艳姚红涛李利佳
Owner HEC PHARMA CO LTD
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