Process for removing endotoxin in bacteria polysaccharide by using macroporous resin

A technology of macroporous resin and bacterial polysaccharide, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc.

Active Publication Date: 2007-05-30
YUXI WALVAX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, macroporous resins are used to remove endotoxins from Gram-negative bacterial polysaccharide products. At present, there is no literature report at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Haemophilus influenzae type b was fermented and cultured in a bioreactor; after sterilizing with formaldehyde in the late logarithmic growth period, the supernatant was collected by centrifugation; cetyltrimethylammonium bromide was added to the supernatant to reach the final concentration 0.1%, stand at room temperature for 12 hours, centrifuge to collect the precipitate; add 1mol / L CaCl to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 50%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The polysaccharide after washing is subjected to vacuum freeze-drying to obtain the crude polysaccharide of Haemophilus influenzae type b.

[0029] Dissolve 50 g of raw sugar at 5 mg / ml in 1 / 10 (V / V) saturated neutral sodium ac...

Embodiment 2

[0041] Use a bioreactor to ferment and cultivate group A meningococci; after sterilizing with formaldehyde in the late logarithmic growth period, centrifuge to collect the supernatant; add cetyltrimethylammonium bromide to the supernatant to a final concentration of 0.1 %, stand at room temperature for 12 hours, centrifuge to collect the precipitate; add 1mol / LCaCl to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 90%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The polysaccharide after washing is subjected to vacuum freeze-drying to obtain the crude polysaccharide of group A meningococcus.

[0042] Dissolve 500 g of raw sugar at 50 mg / ml in 1 / 10 (V / V) saturated neutral sodium acetate solution, extract 10 times with...

Embodiment 3

[0054] Use a bioreactor to ferment and cultivate group C meningococcus; after sterilizing with formaldehyde in the late logarithmic growth period, centrifuge to collect the supernatant; add cetyltrimethylammonium bromide to the supernatant to a final concentration of 0.1 %, stand at room temperature for 12 hours, centrifuge to collect the precipitate; add 1mol / LCaCl to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 80%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The polysaccharide after washing is subjected to vacuum freeze-drying to obtain the crude polysaccharide of group C meningococcus.

[0055] 500g of raw sugar was dissolved in 1 / 10 (V / V) saturated neutral sodium acetate solution at 30mg / ml, extracted 6 times...

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Abstract

The invention discloses a removing method of endotoxin in the bacterial polysaccharide through large-hole resin in the biological technical domain, which comprises the following steps: fermenting bacteria through biological reactor; making rough sugar; extracting rough sugar through cooled phenol to remove protein; hyperfiltering through composite buffer of calcium ionic chelant-surface activator; removing endotoxin adsorbed by large-hole resin; obtaining bacterial polysaccharide with low endotoxin content.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for removing endotoxin in bacterial polysaccharides. Background technique: [0002] Bacterial capsular polysaccharides are important protective antigens of bacteria. Vaccination with these polysaccharides can protect the population over 2 years old. However, during the fermentation and cultivation of Gram-negative bacteria, in addition to producing capsular polysaccharides, a large amount of proteins, nucleic acids, endotoxins and other components are also synthesized. When the endotoxin content in the vaccine exceeds a certain standard, it may cause severe fever and allergic purpura and other side effects after vaccination. Therefore, the bacterial polysaccharide vaccine regulations issued by the World Health Organization (WHO) and the polysaccharide vaccine in the three parts of the "Chinese Pharmacopoeia" 2005 edition The national standards all have stri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C08B37/00B01D61/14B01D71/28C12R1/42C12R1/21C12R1/01
Inventor 黄镇向左云
Owner YUXI WALVAX BIOTECH CO LTD
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