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Efficient post-extraction method of curdlan

An extraction method and polysaccharide technology, which are applied in the field of high-efficiency post-extraction of available polysaccharides, can solve the problems of colloid loss, large usage of organic solvents, long drying time, etc., and achieve improved recovery efficiency, reduced production cost, and improved drying efficiency. Effect

Inactive Publication Date: 2019-08-30
TAIXING DONGSHENG FOOD TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of dissolving curdlan polysaccharide with alkaline solution and precipitating colloid with acid solution, the recovery efficiency of curdlan polysaccharide is low and the colloid is lost due to incomplete dissolution or precipitation of colloid; the use of ethanol in the dehydration and drying process has the use of organic solvents Large size, high cost, and technical defects of potential safety hazards; if the alcohol dehydration process is not used, the dehydration efficiency is low and the drying time is long
In addition, the gel strength of the prepared curdlan polysaccharide product is lower than that of the crude product of the fermentation broth

Method used

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  • Efficient post-extraction method of curdlan
  • Efficient post-extraction method of curdlan
  • Efficient post-extraction method of curdlan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning and sequencing analysis of protease DSNJ-rPE gene

[0039] Search several protease sequences from NCBI website, carry out bioinformatics analysis, use CODEHOP (Consensus-degenerate hybrid oligonucleotide primers) program to select two highly conserved amino acid sequences and design degenerate primers.

[0040] Using the soil genomic DNA extraction kit from MP bio ( Spin Kit for Soil) to extract the total genomes of 50 soil samples from different sources.

[0041] In the 50μl system, add upstream primer: 5′-gcaccggattccgacgacag-3′, and downstream primer: 5′-tcgccgcaggccgactctag-3′, the final concentration is 0.8μM, the final concentration of dNTPs is 0.2mM, and the total DNA of soil genome is 10ng, 2U Pfu DNA polymerase. The amplification program was 94°C for 5min; 35×(94°C for 30s, 55°C for 60s, 72°C for 60s); 72°C for 10min. The PCR products were sent to Shanghai Sangon Bioengineering Company for sequencing.

[0042] Analyzing the sequencing r...

Embodiment 2

[0043]Embodiment 2: the construction of prokaryotic expression vector of protease DSNJ-rPE gene (attached figure 1 )

[0044] According to the MTG gene sequence obtained by sequencing, SacI recognition sequence (GAGCTC) and XhoI recognition sequence (CTCGAG) were added to the 5' and 3' ends respectively, and Shanghai Sangon Bioengineering Company was commissioned to synthesize the gene. The synthesized gene and vector pET-23a were digested with appropriate amount of the same restriction endonuclease BamHI and ligated with T4 DNA ligase. The recombinant vector transformed Escherichia coli DH5α. Randomly pick a single colony from the transformation plate, insert it into LB liquid medium, shake culture, extract a small amount of plasmid, electrophoresis, use the plasmid with electrophoresis delay as a template for PCR verification, and send it to Shanghai Sangon Bioengineering Co., Ltd. after confirming that the connection is successful sequencing.

Embodiment 3

[0045] Embodiment 3: the expression of protease DSNJ-rPE gene in Escherichia coli

[0046] Transform the recombinant expression plasmid pET-23a-DSNJ-rPE containing DSNJ-rPE gene into Escherichia coli expression host strain BL21(DE3)pLysS, culture at 37°C for 10-11 hours, pick small colonies, and insert them into 50ml LB containing ampicillin Liquid medium, culture at 30°C at 70-90rpm overnight, take the seed solution according to the volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until OD600 is about 0.6, add IPTG (final concentration 100 μg / ml) induction. After 1.5 hours, the cells were collected by centrifugation. Break up the bacteria, centrifuge to collect the supernatant, and obtain the recombinant DSNJ-rPE protease crude enzyme solution.

[0047] Recombinant protease DSNJ-rPE activity assay method: Dissolve fluorescently labeled casein in 0.2mL phosphate buffer (pH 7.0), mix well, absorb 50μL of su...

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Abstract

The invention discloses an efficient post-extraction method of curdlan. The preparation method comprises the following steps: firstly, adding a recombinant microbial protease DSNJ-rPE into a fermentation broth producing curdlan agrobacterium spp, and carrying out reacting at 20-45 DEG C for 1-2 hours; carrying out filtering and collecting thallus, and dissolving the thallus by using an alkali solution to obtain curdlan; then, carrying out centrifuging or plate-frame filtration to remove the thallus; neutralizing the supernate by an acid solution to separate out curdlan reversible gel; washingthe gel with deionized water for 2-4 times, carrying out centrifuging or plate-frame filtration, collecting the colloid, and cutting the colloid into particles by a chopper mixer; carrying out quick-freezing treatment on the granular colloid by liquid nitrogen or dry ice; dehydrating and drying the granular colloid by adopting a boiling drying method; and finally, further crushing the dried colloidal particles to obtain the curdlan refined product. According to the method disclosed by the invention, production cost can be reduced, and the polysaccharide recovery rate and production efficiencycan be improved. Meanwhile, gel strength of the curdlan extracted by using the method is higher than gel strength of curdlan extracted by a conventional method.

Description

technical field [0001] The invention belongs to the field of food industry biotechnology, and relates to a high-efficiency post-extraction method of curdlan polysaccharide. Background technique [0002] Curdlan (Chinese name: Candlan polysaccharide, also known as Curdlan gum) is a high molecular polymer produced by fermentation of microorganisms such as Agrobacterium sp. with glucose, sucrose and other sugars as raw materials. It is a straight-chain polyglucose composed of glucose structural units connected by β-1,3-glucosidic bonds, and this type of polydextrose is water-insoluble. Curdlan has the property of rapidly forming a gel under heating conditions. It can form a thermally irreversible gel after being treated at a temperature above 85°C for more than 10 minutes, so it is also called a thermal gel. As early as 1996, the US FDA approved Curdlan as a food raw material for use in the food industry. Curdlan is the third microbial macromolecule glue approved by FDA after...

Claims

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Application Information

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IPC IPC(8): C08B37/02
CPCC08B37/0003C08B37/0024
Inventor 张晓健
Owner TAIXING DONGSHENG FOOD TECH
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