Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB) and a Fraction of its LPS-Elimination Protein.

Lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. Furthermore, inflammation of human intestines can be caused by a lipopolysaccharide (LPS) component in the cell walls of gram-negative bacteria. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30 °C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Cloning and Expression of Lipopolysaccharide Elimination Protein (LEP) in Lactic Acid Bacteria.

Lipopolysaccharide (LPS) is related to human inflammation. Therefore, in the probiotics research field, controlling the mechanisms of LPS neutralization and elimination of inflammation of human intestines are important. This chapter presents a description of the identification of LPS elimination protein (LEP) in lactic acid bacteria (LAB), cloning of its protein, and its expression. First, LEP is extracted from the LAB cell wall digestion fraction using Blue Native PAGE. Then LEP is identified by the elimination activity of LPS on gel pieces. Results show that the LEP is an approx. 200 kDa protein part of heat shock protein in lactic acid bacteria. After sequencing amino acids of LEP, LEP cloning is done using a Brevibacillus sp. expression system without a general transformation system but with Gram-negative Escherichia coli having LPS. Results presented in this chapter demonstrate the elimination activity of recombinant LEP.

Neutralization of Lipopolysaccharide by Heat Shock Protein in Pediococcus pentosaceus AK-23.

About 1000 species of bacteria are present in the human intestine. Some Gram-negative bacteria such as Escherichia coli or Salmonella spp. among intestinal bacteria have lipopolysaccharide (LPS), which might induce inflammation of human intestines. Actually, LPS, especially its lipid A constituent, is toxic. Small amounts of LPS in bacteria cause inflammation of mucosa and other tissues in humans. Such bacteria may be regulated by beneficial lactic acid bacteria to maintain human health. Many lactic acid bacteria show cancer prevention activity and anti-inflammatory activity in intestines. Recently, Pediococcus pentosaceus AK-23 was isolated from fermentative vegetable pickles for neutralization of LPS. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23, by ultrafiltration using a 300 kDa membrane and a 100 kDa membrane after cell wall digestion by lysozyme. Gel running blue native electrophoresis revealed the existence of a 217 kDa protein. The band of the protein having the ability to bind LPS on the gel was analyzed for amino acid homology. As the result, it is revealed as part of a subunit of heat shock protein (HSP). Furthermore, it displayed LPS binding or hydrophobic motifs. The protein neutralized LPS to release fatty acid as myristic acid and glucose from polysaccharide. These findings suggest that HSP in P. pentosaceus AK-23 neutralizes LPS to decompose it compising fatty acid and polysaccharide.

Isolation of Endotoxin Eliminating Lactic Acid Bacteria and a Property of Endotoxin Eliminating Protein.

Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein.