A compound microecological preparation and its application in improving intestinal short-chain fatty acids

A technology of composite micro-ecology and micro-ecological preparations, which is applied in microorganism-based methods, bacteria used in food preparation, applications, etc., can solve the problem that the synchronous growth of xylo-oligosaccharides and the relative activity of xylanase cannot be realized. and other problems, to achieve the effect of improving intestinal short-chain fatty acid content, increasing short-chain fatty acid content, and increasing acetic acid content

Active Publication Date: 2022-04-12
QILU UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the prior art, it is mainly concentrated in Escherichia coli, and it is not secreted to the outside of Escherichia coli, and it is impossible to realize the synchronous growth of bacteria and the production of xylooligosaccharides by fermentation.
[0007] At present, in the research of heterologous expression of xylanase in Escherichia coli, the intracellular expression is mainly used. The intracellular xylanase is obtained by ultrasonication and purification, but the relative activity of xylanase will be reduced.

Method used

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  • A compound microecological preparation and its application in improving intestinal short-chain fatty acids
  • A compound microecological preparation and its application in improving intestinal short-chain fatty acids
  • A compound microecological preparation and its application in improving intestinal short-chain fatty acids

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Embodiment 1

[0053] The novel xylanase gene sequence (GenBank: AGN35000.1) derived from Bacillus was optimized by Escherichia coli codon preference, and the optimized xylanase gene sequence was obtained as shown in SEQ ID NO.2. The amino acid sequence of the glycanase is shown in SEQ ID NO.1.

[0054] The specific method of constructing the recombinant expression strain BL21(DE3) / pET22b(+)-xyl is as follows:

[0055] After amplifying the xylanase xyl gene sequence SEQ ID NO.2 with primer 0-Xyl-F and primer 0-Xyl-R, the product was purified using the kit. The homologous recombination method was used to connect to the vector pET22b(+), the ligated product was transformed into Escherichia coli XL1-Blue, and the plasmid was extracted with a kit to obtain a recombinant expression plasmid. Finally, it was transformed into Escherichia coli DE3 competent to obtain the recombinant expression strain BL21(DE3) / pET22b(+)-xyl.

[0056] The specific method of constructing the recombinant expression st...

Embodiment 2

[0076] The recombinant expression strain BL21(DE3) / pET22b(+)-xyl and the recombinant expression strain BL21(DE3) / pET22b(+)-Fae20-xyl constructed in Example 1 were cultured at 37°C until the OD600 was 0.5-0.6, Add IPTG to continue induction at 37°C for 24h. The bacterial solution was centrifuged at 10,000 rpm at 4°C for 5 min, and the supernatant was taken to prepare extracellular protein samples. The cells were centrifuged and resuspended in phosphate buffer for ultrasonic disruption (400w, 4s, pause 4s) for 20min, and the cell disruption solution was centrifuged at 12000rpm for 5min, and the cell disruption solution and centrifugation supernatant were taken to prepare whole cell protein samples and Intracellular protein samples. For 10% SDS-PAGE analysis. Analysis results such as figure 1 As shown, lanes 1-3 are xylanase xyl whole-cell protein samples, intracellular protein samples, and extracellular protein samples; lanes 4-6 are xylanase Fae20-xyl whole-cell protein samp...

Embodiment 3

[0079] Xylanase enzyme activity assay method:

[0080] After reacting 1% xylan substrate with appropriately diluted enzyme solution for 10 min, the reducing sugar (calculated as xylose) was determined by 3,5-dinitrosalicylic acid (DNS) method. The amount of reducing sugar and the corresponding enzyme activity were calculated by the standard curve. The specific measurement steps are as follows:

[0081] (1) Preparation of xylose standard solution

[0082] Prepare xylose into standard solution according to the concentration of 0.1, 0.25, 0.5, 1, 2.5, 5g / L

[0083] (2) Determination of xylose standard curve:

[0084] Take clean and dry EP tubes, label them and set them in parallel, add 150 μL of pH7 phosphate buffer to each tube, then add 50 μL of xylose solutions of various concentrations and 150 μL of DNS reagent, put them in a boiling water bath for 5 minutes and let them stand at room temperature.

[0085] Take 200 μL to a 96-well plate, and use a microplate reader to mea...

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Abstract

The invention relates to a composite microecological preparation and its application in improving intestinal short-chain fatty acids. The components of the composite microecological preparation include: xylooligosaccharides and lactic acid bacteria; the lactic acid bacteria are: Lactobacillus pumilus, intestinal One or more of coccus, Pediococcus lactis or Weissella species; the composite microecological preparation of the present invention can significantly increase the content of short-chain fatty acids in the cecum, and increase the content of acetic acid, propionic acid, and butyric acid , the most significant increase in acetic acid content, better play a probiotic role, and improve intestinal health.

Description

technical field [0001] The invention belongs to the technical field of microorganisms utilizing prebiotics, and in particular relates to a compound microecological preparation and its application in improving intestinal short-chain fatty acids. Background technique [0002] Short chain fatty acids (SCFA) are organic fatty acids composed of 1 to 6 carbon atoms. In the human body, it can be produced by the fermentation of non-digestible carbohydrates by colonic microorganisms, mainly including acetic acid, propionic acid, butyric acid, lactic acid, etc. Studies at home and abroad have shown that oligosaccharides have the effects of preventing constipation, colon cancer, tumors, diabetes, coronary heart disease, etc., especially the role of regulating intestinal microecological balance. Intestinal probiotics can regulate intestinal health by fermenting oligosaccharides to produce SCFAs. Fermenting oligosaccharides can produce higher levels of SCFAs than other carbon source sub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P39/00C12P7/54C12P7/52C12N9/42C12N15/56C12N15/70C12N1/21C12P19/14A23K10/18C12R1/24C12R1/01
CPCC12N1/20C12P39/00C12P7/54C12P7/52C12N9/2482C12Y302/01008C12N15/70C12P19/14A23K10/18C12N2800/22A23V2400/121A23V2400/413
Inventor 徐振上王婷王家朋
Owner QILU UNIV OF TECH
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