Method of double-coating lactic acid bacteria

Abstract

The present disclosure relates to production of double-coated lactic acid bacteria using peptides and polysaccharide. Double-coated lactic acid bacteria show improved heat-resistance, acid-resistance, bile-resistance, storage stability and excellent survival rate when reaching intestine.

1 . A method of preparing double-coated lactic acid bacteria, the method comprising:
 providing a protein aqueous solution comprising either or both of skim milk and isolated soybean protein as a protein component;   adding a proteolytic enzyme to the protein aqueous solution, which causes partial hydrolysis of the protein component in the protein aqueous solution to provide a hydrolyzed product comprising water-soluble peptides and water semi-soluble peptides;   cultivating lactic acid bacteria in a fermentation solution comprising the hydrolyzed product, wherein at least part of the water-soluble peptides from the hydrolyzed product are consumed by the lactic acid bacteria, wherein cultivating provides a liquid mixture comprising the lactic acid bacteria, the water-soluble peptides and the water-semisoluble peptides;   centrifuging the liquid mixture to deposit the water-semisoluble peptides along with the lactic acid bacteria such that deposited water-semisoluble peptides enclose lactic acid bacteria to provide peptide-coated lactic acid bacteria,   collecting, from the centrifugation result, the peptide-coated lactic acid bacteria,   mixing, in an aqueous solution, the collected peptide-coated lactic acid bacteria with a polysaccharide and a cryoprotectant to provide a homogenized aqueous mixture comprising the peptide-coated lactic acid bacteria, the polysaccharide and the cryoprotectant,   freezing the homogenized aqueous mixture to provide a frozen homogenized mixture in which the peptide-coated lactic acid bacteria are enclosed in a polysaccharide coating, and   lyophilizing the frozen homogenized mixture, thereby forming double-coated lactic acid bacteria coated with the water-semisoluble peptides and further with the polysaccharide.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein lyophilizing is performed at a temperature ranging from 0° C. to 40° C. 
     
     
         4 . The method of  claim 1 , wherein the polysaccharide is selected from the group consisting of xanthan gum, cellulose, levan and mixtures of the foregoing. 
     
     
         5 . The method of  claim 1 , wherein the lactic acid bacteria are selected from the group consisting of  Lactobacillus acidophilus, Streptococcus thermophilus , and  Bifidobacterium longum.    
     
     
         6 . The method of  claim 1 , further comprising:
 lyophilizing the peptide-coated lactic acid bacteria prior to mixing with the polysaccharide.   
     
     
         7 . The method of  claim 1 , wherein the cryoprotectant is selected from the group consisting of trehalose, malto-dextrin, mannitol, skim milk and mixtures of the foregoing. 
     
     
         8 . The method of  claim 1 , wherein in the step of freezing, the homogenized aqueous mixture solution is chilled to a temperature up to −55° C. 
     
     
         9 . The method of  claim 1 , wherein mixing with the polysaccharide comprises stirring the aqueous solution comprising peptide-coated lactic acid bacteria with the polysaccharide and the cryoprotectant at 5,000 RPM. 
     
     
         10 . The method of  claim 1 , wherein centrifuging for depositing the water-semisoluble peptides along with the lactic acid bacteria is conducted using a high speed centrifuge at 15,000 RPM or higher. 
     
     
         11 . The method of  claim 1 , wherein the double-coated lactic acid bacteria has a survival rate of 85.3% or higher after subjecting to an acidic juice at pH 2.1 for 120 minutes. 
     
     
         12 . The method of  claim 1 , wherein the double-coated lactic acid bacteria has a survival rate of 81.3% or higher after subjecting to an Oxgall solution 0.5% for 120 minutes. 
     
     
         13 . The method of  claim 1 , wherein in the step of mixing, the polysaccharide and the cryoprotectant are added to the collected peptided-coated lactic acid in the form of a polysaccharide aqueous solution and a cryoprotectant aqueous solution, respectively. 
     
     
         14 . (canceled)