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CN-122029271-A - Novel method for producing lactobacillus rhamnosus

CN122029271ACN 122029271 ACN122029271 ACN 122029271ACN-122029271-A

Abstract

The present invention relates to a method for producing lactobacillus rhamnosus, wherein the method comprises the steps of a) fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermenter and removing a fermentation broth from the fermenter, wherein the fermentation is performed in the presence of acetate or acetate salt, b) subjecting the fermentation broth to shear and optionally concentrating the fermentation broth. The compositions thus obtained and the use of such compositions are also described.

Inventors

  • Hendricus Bernadus Albertus Weigekamp
  • Paul Zu'er

Assignees

  • 帝斯曼知识产权资产管理有限公司

Dates

Publication Date
20260512
Application Date
20240610
Priority Date
20230608

Claims (19)

  1. 1. A method of producing lactobacillus rhamnosus, wherein the method comprises the steps of: (a) Fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermentor and removing fermentation broth from the fermentor, wherein the fermentation is performed in the presence of acetate or acetate; (b) Applying shear to the fermentation broth and optionally concentrating the fermentation broth.
  2. 2. A method of producing a composition comprising lactobacillus rhamnosus, wherein the method comprises the steps of: (a) Fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermentor and removing fermentation broth from the fermentor, wherein the fermentation is performed in the presence of acetate or acetate; (b) Applying shear to the fermentation broth and optionally concentrating the fermentation broth.
  3. 3. The method of claim 1 or 2, wherein the acetate salt is ammonium acetate.
  4. 4. The method according to any of the preceding claims, Wherein the fermentation is carried out in the presence of ammonium acetate, and Wherein the fermentation is performed at a pH point or the fermentation is performed towards a final pH point, the pH point being equal to or greater than pH 3.0, wherein the pH is controlled by adding a titrant, wherein the titrant is selected from the group consisting of sodium hydroxide and potassium hydroxide.
  5. 5. The method according to any of the preceding claims, wherein the fermentation medium is a solution, suspension or dispersion, preferably an aqueous solution, suspension or dispersion, wherein preferably acetate is present as dissociated or non-dissociated ammonium acetate salt.
  6. 6. The method of any one of the preceding claims, wherein the fermentation medium has a volume equal to or greater than 50 liters.
  7. 7. The method of any one of the preceding claims, wherein step (b) comprises homogenizing the unconcentrated fermentation broth, wherein the homogenizing is performed at a speed in the range of greater than 10000 revolutions per minute (rpm) to equal to or less than 50000 revolutions per minute (rpm) for a time in the range of equal to or greater than 1 minute to less than 5 minutes.
  8. 8. The method according to any of the preceding claims, wherein the shear rate applied is in the range of equal to or greater than 0.5 s -1 , preferably equal to or greater than 1.0 s -1 , more preferably equal to or greater than 10 s -1 , yet more preferably equal to or greater than 100 s -1 , yet more preferably equal to or greater than 500 s -1 , to equal to or less than 500000 s -1 , preferably equal to or less than 100000 s -1 , more preferably equal to or less than 50000 s -1 , yet more preferably equal to or less than 10000 s -1 , yet more preferably equal to or less than 5000 s -1 .
  9. 9. The method according to any of the preceding claims, wherein the applied shear stress is in the range of equal to or greater than 1.10 -9 pascals, preferably equal to or greater than 1.10 -8 pascals, more preferably equal to or greater than 1.10 -7 pascals, still more preferably equal to or greater than 1.10 -6 pascals, still more preferably equal to or greater than 1.10 -5 pascals, to equal to or less than 1.10 -1 pascals, more preferably equal to or less than 1.10 -2 pascals, still more preferably equal to or less than 1.10 -3 pascals, still more preferably equal to or less than 1.10 -4 pascals.
  10. 10. The method according to any of the preceding claims, wherein the shearing is applied by subjecting the fermentation broth to a homogenization and/or centrifugation step.
  11. 11. The method according to any of the preceding claims, wherein the method comprises the steps of: (a) Fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermentor and removing fermentation broth from the fermentor, wherein the fermentation is performed in the presence of acetate or acetate; (b) Applying shear to the fermentation broth and concentrating the fermentation broth, and (C) The concentrated fermentation broth is frozen and/or dried.
  12. 12. The method of any one of the preceding claims, wherein the method comprises spray drying or freeze drying the concentrated fermentation broth.
  13. 13. The method according to any of the preceding claims, wherein the method further comprises adding one or more additives, preferably cryoprotectants and/or stabilizers, to the concentrated fermentation broth.
  14. 14. Lactobacillus rhamnosus bacterial particles or a composition comprising lactobacillus rhamnosus bacterial particles, obtained or obtainable by the method according to any of the preceding claims.
  15. 15. Lactobacillus rhamnosus bacterial particles, wherein the bacterial particles equal to or greater than 90% w/w, preferably equal to or greater than 95% w/w, more preferably equal to or greater than 99% w/w, still more preferably equal to or greater than 99.5% w/w, still more preferably equal to or greater than 99.9% w/w and most preferably equal to or greater than 100.0% w/w have a viable cell count of equal to or less than 8.0 microns, preferably equal to or less than 7.5 microns, more preferably equal to or less than 7.0 microns, yet more preferably equal to or less than 6.5 microns and most preferably equal to or less than 6.0 microns, and the bacterial particles preferably have a viable cell count of equal to or greater than 2.00.10 CFU/g, more preferably equal to or greater than 2.10.10. 10 CFU/g, yet more preferably equal to or greater than 1.50.10 11 CFU/g and most preferably equal to or greater than 2.00. 11 CFU/g, as determined by laser diffraction particle size analysis.
  16. 16. A composition comprising lactobacillus rhamnosus bacterial particles, wherein the bacterial particles equal to or greater than 90% w/w, preferably equal to or greater than 95% w/w, more preferably equal to or greater than 99% w/w, still more preferably equal to or greater than 99.5% w/w, still more preferably equal to or greater than 99.9% w/w and most preferably equal to or greater than 100.0% w/w have a viable cell count of equal to or less than 8.0 microns, preferably equal to or less than 7.5 microns, more preferably equal to or less than 7.0 microns, yet more preferably equal to or less than 6.5 microns and most preferably equal to or less than 6.0 microns, as determined by laser diffraction particle size analysis, and the composition preferably comprises a viable cell count of equal to or greater than 2.00-10 10 CFU/g, more preferably equal to or greater than 2.10-10 10 CFU/g, yet more preferably equal to or greater than 1.50-10- 11 CFU/g and most preferably equal to or greater than 2.00- 11 CFU/g.
  17. 17. A composition for animal and/or human consumption, preferably a pharmaceutical or food product or beverage product, comprising: lactobacillus rhamnosus bacterial particles according to claim 14 or 15, or -A composition comprising lactobacillus rhamnosus bacterial particles according to claim 14 or claim 16.
  18. 18. Use of the lactobacillus rhamnosus bacterial particles according to claim 14 or 15 or the composition comprising lactobacillus rhamnosus bacterial particles according to claim 16 or claim 17 in the production of a food or beverage product.
  19. 19. Use of the lactobacillus rhamnosus bacterial particles according to claim 14 or 15 or the composition comprising lactobacillus rhamnosus bacterial particles according to claim 16 or 17 in a probiotic composition and/or for medical purposes and/or in a medicament or as a medicament, preferably as a medicament for the treatment or prophylaxis of a disease or condition in or associated with the gastrointestinal tract of an animal or human.

Description

Novel method for producing lactobacillus rhamnosus Technical Field The present invention relates to a novel method for producing lactobacillus rhamnosus (Lactobacillus rhamnosus) and a novel method for producing a composition comprising lactobacillus rhamnosus. Furthermore, the present invention relates to such a composition comprising lactobacillus rhamnosus, the use of such a composition in food or beverage and the use of such a composition for medical purposes. Background Bacterial compositions having probiotic activity are becoming increasingly popular as part of the human and animal diet due to their beneficial health effects. In addition to supporting intestinal health and function, these health benefits include refilling the intestine after antibiotic treatment, counteracting lactose intolerance, supporting the immune system, and lowering cholesterol. Lactic acid bacteria mainly from the genera lactobacillus and bifidobacterium, which may help to improve or maintain intestinal health and function, are often referred to as probiotic bacteria (also referred to herein as probiotics). Lactobacillus rhamnosus, in particular lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG), is one of the most popular probiotics. In Lebeer et al, APPLIED AND Environmental Microbiology, volume 78,Number 1,January 2012,pages 185-193, titled "Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells", they analyzed the function of the pili of lactobacillus rhamnosus GG. They indicated that lactobacillus rhamnosus GG is critical for efficient adhesion of intestinal epithelial cell lines and biofilm formation. Pili (also known as cilia) are small hair-like fibrous proteins that are present on many bacterial surface areas. To increase the bioavailability of the pili, it would be advantageous to increase the surface area of lactobacillus rhamnosus GG bacteria per gram of product, for example by having more bacterial particles per gram and/or by having smaller bacterial particles. Furthermore, probiotics are preferably marketed based on their Colony Forming Units (CFU) count per gram. The count of Colony Forming Units (CFU) per gram is considered by the consumer as a measure of the viability of the probiotic. Thus, manufacturers of probiotics consider it an advantage to have a high CFU/g count in their products. Thus, there remains a need in the art for a method of producing lactobacillus rhamnosus with high bioavailability and/or high bacterial surface area and/or small bacterial particle size and/or high CFU/gram count and/or high viable cell count of the pili. Disclosure of Invention A new method of producing lactobacillus rhamnosus and a new method of producing a composition comprising lactobacillus rhamnosus have now been found. By these methods, the bioavailability and/or bacterial particle size and/or CFU/gram count and/or viable cell count of the pili of lactobacillus rhamnosus can be improved. Accordingly, in a first aspect, the present invention provides a method of producing lactobacillus rhamnosus, wherein the method comprises the steps of: (a) Fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermentor and removing fermentation broth from the fermentor, wherein the fermentation is performed in the presence of acetate or acetate; (b) Applying shear to the fermentation broth and optionally concentrating the fermentation broth. Further, in a second aspect, the present invention provides a method of producing a composition comprising lactobacillus rhamnosus, wherein the method comprises the steps of: (a) Fermenting the lactobacillus rhamnosus in a lactose-free, preferably milk-free, medium in a fermentor and removing fermentation broth from the fermentor, wherein the fermentation is performed in the presence of acetate or acetate; (b) Applying shear to the fermentation broth and optionally concentrating the fermentation broth. In a third aspect, the present invention provides lactobacillus rhamnosus cells or lactobacillus rhamnosus bacterial particles, or a composition comprising lactobacillus rhamnosus cells or lactobacillus rhamnosus bacterial particles, which is obtained or obtainable by any of the above-described methods. In a fourth aspect, the present invention provides lactobacillus rhamnosus cells or lactobacillus rhamnosus bacterial particles wherein the bacterial particles equal to or greater than 90% w/w, preferably equal to or greater than 95% w/w, more preferably equal to or greater than 99% w/w, still more preferably equal to or greater than 99.5% w/w, still more preferably equal to or greater than 99.9% w/w and most preferably equal to or greater than 100.0% w/w have a particle size or diameter equal to or less than 8.0 micrometers (μm), preferably equal to or less than 7.5 micrometers, more preferably equal to or less than 7.0 micrometers, still more preferably equal to or