Controlled activation of the reuterin-production machinery of
Methods for the controlled activation of the reuterin-production machinery of Lactobacillus reuteri by adding glycerol and other substances during the manufacture of cell-cultures and keeping the produced reuterin in the bacterial cell during preservation and storage. In particular this invention relates to the manufacture of large amounts of L. reuteri that are loaded with reuterin, and the use of such loaded bacteria for applications such as prevention and treatment of diseases, for food applications and the like.
1. A method for producing freeze-dried Lactobacillus reuteri cells having reuterin retained inside the cells, comprising:
growing cells of a Lactobacillus reuteri in a fermentation step;
adding a priming substance selected from the group consisting of glycerol and 1,2-propanediol to the cells of the Lactobacillus reuteri at the start of the fermentation step so that the Lactobacillus reuteri cells form microcompartments and produce reuterin therein;
adding glycerol at the end of the fermentation step to the Lactobacillus reuteri cells to further produce reuterin by the Lactobacillus reuteri cells;
freeze-drying the cells of the Lactobacillus reuteri containing microcompartments and reuterin in the cells; and
storing the freeze-dried culture of Lactobacillus reuteri cells;
wherein the microcompartments enable the reuterin to be retained inside the freeze-dried cells of Lactobacillus reuteri during storage in an amount greater than in cells lacking microcompartments.
2. The method of claim 1 further comprising washing the cells and freeze-drying the cells after fermenting.
3. The method of claim 2 wherein the step of washing the cells after the fermenting step further comprises adding at least one cryo-protectant together with the glycerol after the fermenting step and the washing process, and before the freeze-drying.
4. The method of claim 2 wherein the freeze-drying takes place about 30-45 minutes after reuterin production has begun.
5. The method of claim 1 further comprising adding at least one cryo-protectant to the cells.
6. The method of claim 1 , wherein the fermenting step comprises adding at least one of cobalt or vitamin B-12 to the cells at the beginning of the fermenting step.
7. The method of claim 1 , wherein the priming substance is glycerol.
8. The method of claim 1 , wherein the priming substance is 1,2-propanediol.
9. The method of claim 1 , further comprising adding a survival improving substance selected from the group consisting of vitamin B12, cobalt and vitamin C.
10. The method of claim 7 , further comprising adding a survival improving substance selected from the group consisting of vitamin B12, cobalt and vitamin C.
11. The method of claim 8 , further comprising adding a survival improving substance selected from the group consisting of vitamin B12, cobalt and vitamin C.
12. The method of claim 11 , wherein the survival improving substance are vitamin B12 and cobalt.