Protection of microbial cells from acidic degradation

Abstract

A simple cellulose sulphate based microencapsulation technology has been applied to encapsulate bacterial or other microbial cells, which produce and release digestive enzymes and thereby provides an acid resistant shelter for these microbial cells. Surprisingly, the resulting spheres were found to provide sufficient protection for encapsulated cells from treatment with aqueous acidic solutions. Thereby the cellulose sulphate microencapsulated cells, such as probiotics are now enabled to survive passage, for example, through the stomach after consumption by a human or animal with a higher survival rate than those not within a microcapsule. After passing the stomach these cells are delivering products produced by them, e.g. enzymes or other nutrition factors. This technology therefore proves to be very useful in providing digestive or otherwise beneficial enzymes and/or of living microbial cells, into the lower gastrointestinal tract, where they could confer their health benefit to the host.

1 . Encapsulated microbial cells, wherein the microbial cells are encapsulated in a microcapsule having a porous capsule wall, wherein the porous capsule wall comprises a complex formed from sodium cellulose sulphate and poly[dimethyldiallyl-ammonium chloride], and wherein the microcapsule protects the microbial cells from being degraded by acidic aqueous solution. 
     
     
         2 . The encapsulated microbial cells according to  claim 1  wherein the cells produce and excrete digestive enzymes, and wherein the porous capsule wall is permeable to said digestive enzymes. 
     
     
         3 . The encapsulated microbial cells according to  claim 1 , wherein the acidic aqueous solution is simulated gastric juice, gastric juice or gastric acid. 
     
     
         4 . The encapsulated microbial cells according to  claim 2 , wherein the microcapsules release the enzymes generated by the microbial cells upon treatment with intestinal fluid or duodenal fluid. 
     
     
         5 . The encapsulated microbial cells according to  claim 4 , wherein the intestinal fluid is simulated intestinal fluid (SIF) and the duodenal fluid is simulated duodenal fluid (SDF). 
     
     
         6 . The encapsulated microbial cells according to  claim 1 , wherein the majority of encapsulated microbial cells survive a treatment with acidic aqueous solution according to  claim 3  having a pH range between 1.0 and 3.0, preferably between 1.5 and 2.5, most preferably of 2.0 for at least 1.5 hours, preferably for at least 2.5, and more preferably for at least 4 hours. 
     
     
         7 . The encapsulated microbial cells according to  claim 6 , wherein the majority of microbial cells survive a passage through the stomach of an animal. 
     
     
         8 . The encapsulated microbial cells according to  claim 7 , wherein the majority is defined as a value selected from the group consisting of at least 51% of the cells, as 60% to 90% of the cells, as 60% to 80% of the cells and as 60% of the cells. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The encapsulated microbial cells according to  claim 1 , wherein the microbial cells are at least partially released in the gut of an animal. 
     
     
         13 . The encapsulated microbial cells according to  claim 1 , wherein the animal is a mammal or an avian. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The encapsulated microbial cells according to  claim 1  wherein the microcapsules have a diameter of between 0.01 and 5 mm, preferably between 0.05 and 3 mm, and most preferably between 0.1 and 1 mm. 
     
     
         17 . The encapsulated microbial cells according to  claim 1 , wherein the surface pores of the porous capsule wall have a molecular weight cut off between 50 and 200 kDa, preferably between 60-150 kDa and most preferably between 60 and 100 kDa. 
     
     
         18 . The encapsulated microbial cells according to  claim 1 , wherein the microbial cells are selected from the group comprising bacterial cells, yeast cells, fungal cells and probiotic cells. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . The encapsulated bacterial cells according to  claim 18 , wherein the bacterial cells are selected from the group comprising  Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, Geobacillus  and probacteria such as  Lactobacillus.    
     
     
         23 . The encapsulated probiotic cells according to  claim 18 , wherein the probiotic cells are selected from the group comprising  Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus bulgaricus, Lactobacillus casei  subsp.  casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbrueckii  subsp.  lactis, Lactobacillus fermentum, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lacti, Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus  ( Lactobacillus  GG),  Lactobacillus sake, Lactobacillus salivarius, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus, and    Staphylococcus xylosus.    
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . Encapsulated  Lactobacillus acidophilus  or  Bacillus subtilis  cells, wherein the encapsulated cells produce and excrete digestive enzymes and wherein the cells are encapsulated in a microcapsule having a porous capsule wall, wherein the porous capsule wall comprises a complex formed from sodium cellulose sulphate and poly[dimethyldiallyl-ammonium chloride], thereby providing that these encapsulated cells are resistant to a treatment with acidic aqueous solution of a pH value of 2 for a time period of 2 to 4 hours. 
     
     
         28 . The encapsulated cells according to  claim 2 , wherein the digestive enzymes are selected from the group comprising alpha amylases, glucoamylases, alpha galactosidases, proteases, bromelain proteases, subtilisin, cellulases, pectinases and lipases. 
     
     
         29 . (canceled) 
     
     
         30 . The encapsulated microbial cells according to  claim 1  formulated as a food supplement comprising an excipient acceptable for food use. 
     
     
         31 . The encapsulated microbial cells according to  claim 1  formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a suitable carrier. 
     
     
         32 . A method to protect microbial cells from being degraded by treatment with an acidic aqueous solution by microencapsulation comprising
 a) suspending the living microbial cells in an aqueous solution of a polyelectrolyte sodium cellulose sulphate   b) introducing the suspension in form of preformed microcapsules into a precipitation bath containing an aqueous solution of the counter-charged polyelectrolyte poly[dimethyldiallyl-ammonium chloride],   c) terminating the reaction in the bath after 1-10 minutes, preferably 3-5 minutes, and more preferably after 4 minutes,   d) harvesting the encapsulated cells from the bath.   
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled)