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Strain for efficiently converting stevioside into rubusoside and culture method and application thereof

A cultivation method and rubusoside technology, applied in the field of microorganisms, can solve problems such as low conversion rate, low catalytic efficiency, and poor specificity, and achieve the effects of easy operation, simple cultivation method, good pH stability and temperature stability

Active Publication Date: 2020-11-17
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems of low catalytic efficiency, poor specificity and low conversion rate of enzymes in the enzymatic production of rubusoside, the present invention develops a new strain Chryseobacterium sp. 1433, which can efficiently and specifically convert various sources of The stevioside or pure stevioside in the crude stevioside extract is converted into rubusoside with a conversion rate of 100%, while rebaudioside A (RA) and rubusoside are not hydrolyzed

Method used

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  • Strain for efficiently converting stevioside into rubusoside and culture method and application thereof
  • Strain for efficiently converting stevioside into rubusoside and culture method and application thereof
  • Strain for efficiently converting stevioside into rubusoside and culture method and application thereof

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Effect test

Embodiment 1

[0039] Screening and isolation of embodiment 1 bacterial strain

[0040] The screening process of the above-mentioned Chryseobacterium sp. 1433 is as follows:

[0041] (1) Colony screening

[0042] Take 1000mL of offshore seawater outside the east gate of Qingdao Campus of Shandong University and filter it with a 0.22μm filter membrane, and paste the filtered filter membrane on the stevioside sole carbon source agar medium and culture it at 28-35°C for 3 days; Shandong University 5g each of the coastal sludge outside the east gate of the Qingdao campus and the humus of the seaside forest were diluted with 50mL of sterile water, and 200μL of each was inoculated on stevioside-only carbon source agar plates, and cultured at 28-35°C for 3 days; A total of 198 colonies were grown in the culture, and the growth of the strains was observed. 48 colonies with different shapes and appearances were selected and inoculated into the improved M9 liquid medium for re-screening. The liquid m...

Embodiment 2

[0048] The identification of embodiment 2 bacterial strains

[0049] (1) Characteristics of strains

[0050] Biological characteristics of the strain: Gram-negative bacteria, aerobic, non-motile, non-flagellate, short rod-shaped, 0.40-0.46×1.0-1.27 μm in size, the colony is yellow, protruding, round and paste-like, YPD solid Grow on the culture medium at 30°C for 1 to 2 days, and form round colonies with a diameter of 0.5 to 2mm, such as figure 1 shown.

[0051] (2) Identification of strains

[0052] Obtain the 16SrDNA gene sequence of Chryseobacterium sp. 1433 through DNA extraction, PCR amplification and sequencing, as shown in SEQ ID NO.1, length is 1481bp, carry out BLAST alignment on GenBank and draw phylogenetic tree, phylogenetic tree is as follows figure 2 shown.

[0053] According to the results of 16S rDNA sequence alignment and the biological characteristics of the strain, the strain was identified as Chryseobacterium sp. and named Chryseobacterium sp. 1433.

Embodiment 3

[0054] The cultivation of embodiment 3 bacterial strains

[0055] The culture method of Chryseobacterium sp. (Chryseobacterium sp.) 1433 described in embodiment 1, comprises steps as follows:

[0056] Streak inoculation of Chryseobacterium sp. 1433 on the solid medium, culture at 30°C for 30h, and activate the strain; inoculate the activated strain into the liquid medium, and cultivate at 30°C and 300rpm 30h, prepare the seed liquid; inoculate the seed liquid into the liquid medium according to the volume ratio of 2%, and cultivate for 30h under the conditions of 30°C and 300rpm to obtain the bacterial liquid.

[0057] The solid medium is YPD solid medium; the composition of the YPD solid medium is: 1% yeast extract, 2% peptone, 2% glucose, 2% agarose, all in mass percentage, pH 7.0.

[0058] The liquid medium is YPD liquid medium or LB liquid medium; the composition of the YPD liquid medium is: 1% yeast extract, 2% peptone, 2% glucose, all in mass percentage, pH 7.0; LB liqu...

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Abstract

The invention relates to a strain for efficiently converting stevioside into rubusoside and a culture method and application thereof. A strain of (Chryseobacterium sp.) 1433 is preserved in the ChinaGeneral Microbiological Culture Collection Center on July 6, 2020, the preservation number is CGMCC No.20188 and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Yard, West Beichen road, Chaoyang District, Beijing. The invention also relates to the culture method of the strain and the application of the strain in preparing the rubusoside. The (Chryseobacterium sp.1433) screened and separated by the invention can hydrolyze sophorose of the stevioside into glucosyl to generate the rubusoside and cannot further hydrolyze the rubusoside, so that the conversion rate of the biological strain to the stevioside reaches 100 percent, the yield of the rubusoside reaches 100 percent and the strain is completely superior to the reported related strains.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacterial strain for efficiently transforming stevioside into rubusoside, a cultivation method and application thereof. Background technique [0002] Rubusoside is the extract of sweet tea from Rubus genus Rosaceae which grows in Guangxi Zhuang Autonomous Region, China. It is a natural and high-efficiency sweetener. Its sweetness is 300 times that of sucrose, and its taste is close to that of sucrose. It is safe and non-toxic. It has good effects of lowering blood sugar, blood fat and caries (Kim et al., 2019). It is a good sugar substitute natural sweetener for people with diabetes, hyperglycemia and obesity. It is widely used in food, health care products and other natural non-toxic Calorie Sweeteners Market. In addition, rubusoside is also a good natural co-solvent, which can help insoluble drugs dissolve to improve drug efficacy, such as anticancer drugs...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/56C12R1/01
CPCC12N1/20C12P19/56C12R2001/01C12N1/205
Inventor 肖敏阎振鑫徐莉
Owner SHANDONG UNIV
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