Producing a lactobacillus strain

Abstract

With the present invention is described a process for the production of a Lactobacillus strain with low viability, and Lactobacillus strains obtained by the process.

Description

[0001] PRODUCING A LACTOBACILLUS STRAIN

[0002] Reference to a deposit of biological material

[0003] This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see last paragraph of the description.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a process for the production of a Lactobacillus strain with low viability and Lactobacillus strains obtained by the process.

[0006] BACKGROUND OF THE INVENTION

[0007] Helicobacter pylori (H. pylori) is a type of bacteria that infects the stomach lining, causing inflammation and damage to the gastric mucosa and is associated with a number of health problems, including e.g. peptic ulcers, gastritis and stomach cancer.

[0008] Lactobacillus strains such as Lactobacillus reuteri are commonly used in the production of various foods, supplements, dietary supplements and pharmaceutical products due to their favorable properties such as acid tolerance, bile tolerance, and adhesion to the human intestinal epithelium. In some cases, it may be desirable to use a Lactobacillus strain with reduced viability as a post- biotic such as e.g. against Helicobacter pylori infection, which is a non-viable microbial cell or a microbial cell component that can confer health benefits to the host.

[0009] However, obtaining a Lactobacillus strain with a sufficiently low viability can be challenging, as many of the common methods for reducing the viability of bacterial cells can also damage or denature the beneficial effects of the cells such as the effect on bacteria such as e.g. H. pylori. For example, heat treatment is a commonly used method for reducing the viability of bacterial cells, but it can also denature proteins and enzymes that are important for the beneficial effects of the postbiotic. Similarly, chemical treatments such as acids or oxidizing agents can disrupt the bacterial cell membrane and cause the release of cellular components that may be harmful to the host.

[0010] Other methods such as radiation treatment or pressure treatment may be effective in reducing the viability of Lactobacillus cells without damaging the beneficial features, but these methods can also be expensive, difficult to scale up, or may require specialized equipment.

[0011] WO 2007 / 073709 describes co-aggregates of Lactobacilli with Helicobacter pylori, which can be utilized for reducing or preventing Helicobacter pylori infections. Patent application ON 114480231 A describes a lactobacillus reuteri strain deposited at the Chinese Type Culture Collection under deposit number CCTCC no: M2020597, where the strain is capable of resisting helicobacter pylori infection. There is a need for improved methods for reducing the viability of Lactobacillus strains such as Lactobacillus reuteri in a way that retains their beneficial features while ensuring a low CFU count.

[0012] SUMMARY OF THE INVENTION

[0013] The invention provides a process for the production of a Lactobacillus strain. In an aspect, the process comprises the steps of: a) Fermenting a Lactobacillus strain at a pH below pH 6.0 b) Optionally increasing the pH to between pH 6.0 and pH 7.8 c) Setting the temperature to between 45 to 69 degrees Celsius and keeping the temperature constant for at least 3 hours, and d) Drying the resulting strain wherein an optional separation step is added before step b) or after step c), and / or an optional step of adding a carrier is added before step b) or after step c), and wherein step d optionally is carried out before step c).

[0014] The Lactobacillus strain is in an aspect Lactobacillus reuteri such as e.g. Lactobacillus reuteri deposited as DSM 17648.

[0015] In a further aspect, the process may further comprise adding the resulting strain to a composition to form a product which comprises a low amount of living Lactobacillus cells. In another or yet further aspect, the resulting strain may be added to a food product, e.g. a milk based food product such as yoghurt.

[0016] Also comprised herein are Lactobacillus strains obtained by the process.

[0017] BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 shows CFU / g of Lactobacillus reuteri cells deposited as DSM 17648 and co-aggregation of Lactobacillus reuteri deposited as DSM 17648 with Helicobacter pylori cells at different timepoints before and after thermal and pH treatment as described in Example 1 .

[0019] Figure 2 shows CFU / g of Lactobacillus reuteri cells deposited as DSM 17648 and co-aggregation of Lactobacillus reuteri deposited as DSM 17648 with Helicobacter pylori cells at different timepoints before and after thermal and pH treatment as described in Example 2.

[0020] Figure 3 shows CFU / g of Lactobacillus reuteri cells deposited as DSM 17648 and co-aggregation of Lactobacillus reuteri deposited as DSM 17648 with Helicobacter pylori cells before and after thermal and pH treatment, with and without additions of Nutriose as described in Example 3.

[0021] Figure 4 shows CFU / g of Lactobacillus reuteri cells deposited as DSM 17648 and co-aggregation of Lactobacillus reuteri deposited as DSM 17648 with Helicobacter pylori cells before and after thermal treatment as described in Example 4. Figure 5 shows the pH profile used in Example 6 during fermentation and inactivation of Lactobacillus reuteri DSM 17648 produced in large-scale (450 L) fermentation .

[0022] Figure 6 shows CFU / g of Lactobacillus reuteri cells deposited as DSM 17648 cells before and after the thermal treatment described in Example 6.

[0023] Figure 7 shows co-aggregation of Lactobacillus reuteri cells deposited as DSM 17648 with Helicobacter pylori cells before and after the heat treatment as described in Example 6.

[0024] DEFINITIONS

[0025] Co-aggregates: Alternatively termed “coaggregates”, are aggregates formed by co-aggregation as defined herein. In one aspect, co-aggregates have a size of at least 1 pm to 1 ,000 pm, in another aspect co-aggregates have a size of at least 50 pm or 100 pm. In one aspect, the at least two types of cells forming the co-aggregates are Lactobacillus cells and Helicobacter pylori cells. Non-limiting examples of co-aggregates are described in US 2007 / 0148149.

[0026] Co-aggregation: “Co-aggregation” is herein used interchangeably with “coaggregation” and is understood as the formation of cell aggregates, also called agglomerates, between at least two different types of cells. Co-aggregation may e.g. be formed by mixing at least two cells such as Lactobacillus cells and Helicobacter pylori cells in a solution to form a suspension. An example of co-aggregation is found in Example 3 of US 2014 / 0147427.

[0027] Freeze drying: The terms “freeze drying”, “lyophilization” and “cryodesiccation” are terms known to the person skilled in the art and are used interchangeably herein.

[0028] Nutriose: Nutriose is known to the person skilled in the art and is the name for a digestion-resistant maize dextrin that is a carbohydrate which is made from corn starch.

[0029] Lactobacillus reuteri: is herein the same as Limosilactobacillus reuteri. It will be appreciated by the person skilled in the art that that the Lactobacillus genus taxonomy was updated in 2020 and Lactobacillus reuteri was changed to Limosilactobacillus reuteri. The new taxonomy is disclosed in Zheng, Jinshui et al., (2020) Int J Syst Evol Microbiol, 70 (4): 2782-2858.

[0030] Specific activity: The term is herein used for activity of strains that is important for the function of the strain. The specific activity may e.g. be binding to a pathogen such as e.g. Helicobacter pylori, E. coli, Clostridium difficile, Salmonella, fecal coliform bacteria, Streptococcus gordonii or mutants thereof. In one aspect, the specific activity is co-aggregation of the Lactobacillus strain produced by the process of the invention to Helicobacter pylori.

[0031] Thermal treatment: Is herein used interchangeably with heat treatment and means the heating of a substance or mixture to above room temperature. DETAILED DESCRIPTION OF THE INVENTION

[0032] Herein described is a process for the production of Lactobacillus strains. With the present invention, the challenge of reducing viable cell counts in Lactobacillus strains is addressed without losing the specific activity of the strains. By developing a process that effectively reduces viable cell counts while maintaining coaggregation behavior and strain activity, regulatory requirements for reduced cell viability are met without compromising the effectiveness of the product.

[0033] Lactobacillus cells forming co-aggregates with bacterial cells such as Helicobacter pylori cells may be used to prevent bacterial infections. For example, co-aggregates between Lactobacillus cells and Helicobacter pylori cells prevent Helicobacter pylori from penetrating into the stomach lining. The Helicobacter pylori cells, notably the cell surfaces thereof, are masked by the Lactobacillus cells, so that the Helicobacter pylori cells are no longer able to bind to the gastric epithelial cells. Inflammatory reactions are avoided as a result of the prevented binding of Helicobacter pylori to gastric epithelial cells. The masked, and thus inactivated, Helicobacter pylori cells are channeled through the gastrointestinal tract in the form of co-aggregates and excreted. It is thus possible to reduce or eradicate the Helicobacter pylori cells in the stomach.

[0034] The inventors of the present invention have identified the optimal process conditions for producing a Lactobacillus strain having reduced viability while the specific activity such as the co-aggrega- tion activity is retained.

[0035] The process for the production of a Lactobacillus strain comprises the steps of: a) Fermenting a Lactobacillus strain at a pH below pH 6.0 b) Optionally increasing pH to between pH 6.0 and pH 7.8 c) Setting the temperature to between 45 to 69 degrees Celsius and keeping the temperature constant for at least 3 hours, and d) Drying the resulting strain wherein an optional separation step is added before step b) or after step c), and / or an optional step of adding a carrier is added before step b) or after step c), and wherein step d optionally is carried out before step c).

[0036] The Lactobacillus strains produced in the production process may be any Lactobacillus strains. In one aspect, the Lactobacillus strains are Lactobacillus strains that coaggregate with Helicobacter pylori. In one aspect, the Lactobacillus strains are Lactobacillus reuteri strains. In a further aspect, the Lactobacillus reuteri strains are Lactobacillus reuteri strains that coaggregate with Helicobacter pylori. In one aspect, the Lactobacillus strains is Lactobacillus reuteri deposited as DSM 17648.

[0037] The fermentation step a) may be carried out in any suitable growth medium containing suitable nutrients such as e.g. one or more carbon sources, one or more nitrogen sources, one or more amino acids, one or more vitamins and / or one or more minerals. In one aspect, the growth medium is selected from the group consisting of: de Man, Rogosa and Sharpe broth (MRS broth); Tryptic Soy Broth (TSB broth); Elliker broth; Lactobacillus acidophilus, Lactobacillus plantarum, Tween 80 and glucose broth (LAPTg broth); Reinforced Clostridial Medium (RCM broth); Sabouraud dextrose broth; and Selective Lactobacillus Medium (SLM broth).

[0038] The pH in step a) may be any pH below pH 6.0 that is suitable for ensuring growth of the Lactobacillus strain. In one aspect, pH in step a) is at least pH 5.0. In a further aspect, pH in step a) is between pH 5.0 and pH 6.0. In yet a further aspect, pH in step a) is below pH 5.8. In still a further aspect, pH in step a) is about pH 5.5.

[0039] The temperature in step a) may be any temperature suitable for ensuring growth of the Lactobacillus strain. In one aspect, the temperature in step a) is between 25 to 40 degrees Celsius. In a further aspect, the temperature in step a) is about 37 degrees Celsius.

[0040] The fermentation step a) may be carried out as long as needed to obtain a satisfactory growth of the Lactobacillus strain. In one aspect, step a) carried out for 24 to 20 hours such as 7 to 20 hours, such as for 7 to 15 hours. In a further aspect, step a) is carried out for about 10 hours.

[0041] The fermentation method may be any suitable method such as e.g. batch-fermentation, fed-batch fermentation, continuous fermentation, and two-stage fermentation.

[0042] In one aspect, the pH increase in step b) is mandatory. In a further aspect, pH in step b) is between pH 6.0 and pH 7.8, such as between pH 6.2 and pH 7.8, or between pH 6.5 and pH 7.7. In yet a further aspect, pH in step b) is about pH 7.5.

[0043] In one aspect the temperature in step c) is between 45 to 69 degrees Celsius for at least 3 hours, such as between 50 to 69 degrees Celsius, between 40 to 60 degrees Celsius, between 50 to 60 degrees Celsius, between 50 to 55 degrees Celsius or between 55 to 60 degrees Celsius for at least 3 hours. In a further aspect, the temperature in step c) is about 55 degrees Celsius.

[0044] The temperature set in step c) is kept for at least 3 hours. In one aspect, the temperature in step c) is kept for between 4 to 10 hours, such as between 4 to 9 hours, between 4 to 8 hours, between 4 to 7 hours, or between 4 to 6 hours. In a further aspect, the temperature in step c) is kept for about 5 hours. In a further or alternative aspect, the temperature in step c) is kept for about 4 hours.

[0045] When pH is increased in step b), the pH of step b) and the temperature of step c) are kept for at least 3 hours. In one aspect, the pH of step b) and the temperature in step c) are kept for between 4 to 10 hours, such as between 4 to 9 hours, between 4 to 8 hours, between 4 to 7 hours, or between 4 to 6 hours. In a further aspect, the pH of step b) and the temperature in step c) are kept for about 5 hours. In a further or alternative aspect, the pH of step b) and the temperature in step c) are kept for about 4 hours. Drying may be carried out by any method known to the person skilled in the art and may be carried out in any suitable apparatus. In one aspect, drying in step c) is selected from the group consisting of: Freeze drying, spray drying, and fluidized bed drying. In a further aspect, drying in step c) is freeze drying. In an alternative aspect, drying in step c) is spray drying.

[0046] The process for the production of a Lactobacillus strain provides Lactobacillus strains for use in a product comprising a low amount of living Lactobacillus cells. In one aspect, provided are Lactobacillus strains for use in a product comprising less than 1 E+8 CFU / g Lactobacillus cells such as less than 1 E+7 CFU / g, 1 E+6 CFU / g, 1 E+5 CFU / g, 1 E+4 CFU / g, 1 E+3 CFU / g, 1 E+2 CFU / g or 1 E+1 CFU / g Lactobacillus cells. In one aspect, the Lactobacillus strains are for use in a product comprising less than 1 E+6 CFU / g Lactobacillus cells. In one aspect, the Lactobacillus strains are for use in a product comprising less than 1 E+5 CFU / g Lactobacillus cells. In one aspect, the Lactobacillus strains are for use in a product comprising less than 1 E+4 CFU / g Lactobacillus cells. In one aspect, the Lactobacillus strains are for use in a product comprising less than 1 E+3 CFU / g Lactobacillus cells. In another or further aspect, the Lactobacillus strains are for use in a postbi- otic. In another or yet further aspect, the Lactobacillus strains are for use in a solid beverage.

[0047] Use of Lactobacillus cells produced by the production method

[0048] Lactobacillus cells produced by the production method of the invention can be used in a pharmaceutical, supplement, dietary supplement, nutraceutical, food and / or dietary composition comprising a physiologically effective dose of the obtained Lactobacillus cells and a physiologically compatible carrier. The pharmaceutical compositions are compositions which serve solely therapeutic or prophylactic purposes. Supplement and dietary supplement compositions are human health compositions that are intended to supplement the diet and promote overall health and well-being. Nutraceutical compositions have health benefits beyond basic nutrition and are typically marketed as foods or dietary supplements. Dietary compositions within the meaning of the present invention are composition which, in addition to the Lactobacillus cells produced according to the invention, comprise a food or foodstuff (see, for example, not exhaustively, EU Directive 2002 / 46 / EC of June 10, 2002) and / or dietary supplement, optionally comprising adjuvants and additives. The invention further relates to the use or application of Lactobacillus cells produced according to the invention for producing a pharmaceutical or dietary composition, or a pharmaceutical product, supplement, nutraceutical, food or a dietary supplement, comprising the Lactobacillus cells or a pharmaceutical, supplement, dietary supplement, nutraceutical, food or dietary composition, in particular for the prophylaxis and / or the treatment of diseases caused by Helicobacter pylori infections, for example gastrointestinal conditions. These include in particular gastritis, stomach ulcers and stomach cancer. Also covered are those diseases and symptoms such as abdominal discomfort, in particular discomfort of the upper abdomen, gastric heaviness, gastric spasms, stomach pain, pain or pressure in the upper abdomen, burning sensation in the upper abdomen, chronic- recurrent abdominal disorders, permanent feeling of fullness, lack of appetite, fasting pain, bloating, heartburn, diarrhea, irregular bowel movements, indisposition, nausea, sickness and vomiting, food intolerance, malabsorption, upset stomach (functional dyspepsia), gastritis, damage to the mucous membrane, or gastroduodenal ulcer.

[0049] Also described is the use or application of the Lactobacillus cells produced according to the invention, or a pharmaceutical product, supplement, dietary supplement, nutraceutical, food or a dietary supplement comprising Lactobacillus cells produced according to the invention or a pharmaceutical or dietary composition, for eradicating or for the eradication therapy of Helicobacter pylori, optionally in combination with further suitable active ingredients, such as antibiotics.

[0050] The reference quantity here is a unit of administration, for example a tablet. The composition is preferably prepared for oral administration.

[0051] The galenic preparation of a pharmaceutical, supplement, nutraceutical, food or dietary composition, in particular of a supplement, dietary supplement or pharmaceutical product (drug), can be carried out in a way that is common practice in the art. Suitable solid or liquid galenic forms of preparation include, for example, granules, powders, sugar-coated tablets, tablets (micro)cap- sules, hard capsules, suppositories, syrups, juices, suspensions or emulsions, in the production of which conventional adjuvants such as excipients, disintegrants, binders, coating agents, swelling agents, glidants, lubricants, flavor additives, sweetening agents and solubilizers, are employed. Adjuvants that should be mentioned include magnesium stearate, sodium chloride, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum, milk protein, gelatin, starch, cellulose and the derivatives thereof, animal and vegetable oils such as cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycols and solvents, such as sterile water, and monohydric or polyhydric alcohols, such as glycerin. The pharmaceutical composition can be produced by mixing cells of at least one Lactobacillus strain in a defined dosage with a pharmaceutically suitable and physiologically compatible carrier, and optionally with further suitable active ingredients, additives or adjuvants having defined dosages, and preparing the desired form of administration. Possible carriers include in particular substances that are selected from the group consisting of dextrin, maltodextrin, microcrystalline cellulose, starch, in particular corn starch, levulose, lactose, dextrose, and mixtures of such substances. The composition may comprise or consist of 0.1 to 95% by weight carrier and 5 to 99.9% by weight spray-dried Lactobacillus cells, based on the total quantity of cells and carrier.

[0052] The reference quantity is a unit of administration, for example a packaging unit of a foodstuff for sale to an end consumer. The physiologically compatible carrier will generally be a food, which in particular is selected from the group consisting of dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparations. Suitable foods or foodstuffs, including water are those as defined, not exhaustively, for example, in (EC) Regulation No. 178 / 2002 of January 28, 2002. The invention further relates a method for producing a pharmaceutical and / or dietary composition according to the invention, in particular a supplement, dietary supplement or pharmaceutical product (drug), wherein the Lactobacillus cells produced by the production method are mixed with the physiologically compatible carrier and are preferably prepared for oral administration.

[0053] The Lactobacillus cells produced by the production method may also be used a method for the prophylaxis or the treatment of a human, in particular a patient or test subject suffering, or at risk of suffering, from a condition caused by a Helicobacter pylori infection, in particular gastritis or stomach ulcer, wherein this person is provided such as administered a physiologically effective dosage of a pharmaceutical, supplement and / or dietary composition comprising the Lactobacillus cells produced by the production method of the invention once to five times a day. The administration can take place over a limited period of time, for example 1 to 30 weeks, or without limitations in terms of time. In particular the latter is suited for permanent prophylaxis and for the prevention of relapses.

[0054] PREFERRED EMBODIMENTS

[0055] 1. A process for the production of a Lactobacillus strain comprising the steps of: a) Fermenting a Lactobacillus strain at a pH below pH 6.0 b) Optionally increasing the pH to between pH 6.0 and pH 7.8 c) Setting the temperature to between 45 to 69 degrees Celsius and keeping the temperature constant for at least 3 hours, and d) Drying the resulting strain wherein an optional separation step is added before step b) or after step c), and / or an optional step of adding a carrier is added before step b) or after step c), and wherein step d optionally is carried out before step c).

[0056] 2. The process according to embodiment 1 , wherein step c) is carried out before step d).

[0057] 3. The process according to embodiment 1 , wherein step d) is carried out before step c).

[0058] 4. The process according to any one of the preceding embodiments , wherein the temperature in step c) is kept constant for up to 20 days.

[0059] 5. The process according to any one of the preceding embodiments , wherein the temperature in step c) is kept constant for up to 15 days.

[0060] 6. The process according to any one of the preceding embodiments , wherein the temperature in step c) is kept constant for up to 9 days.

[0061] 7. The process according to embodiment 2, wherein the temperature in step c) is kept for between 3 to 10 hours. 8. The process according to embodiment 2, wherein the temperature in step c) is kept for between 4 hours and 10 hours.

[0062] 9. The process according to embodiment 2, wherein the temperature in step c) is kept for between 4 hours and 6 hours.

[0063] 10. The process according to embodiment 2, wherein the temperature of step c) is kept for about 5 hours.

[0064] 11. The process according to embodiment 2, wherein the temperature of step c) are kept for about 4 hours.

[0065] 12. The process according to embodiment 2, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for between 3 to 10 hours.

[0066] 13. The process according to embodiment 2, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for between 4 to 10 hours.

[0067] 14. The process according to embodiment 2, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for between 4 to 6 hours.

[0068] 15. The process according to embodiment 2, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for about 5 hours.

[0069] 16. The process according to embodiment 2, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for about 4 hours.

[0070] 17. The process according to embodiment 3, wherein the temperature in step c) is kept for between 24 hours and 20 days.

[0071] 18. The process according to embodiment 3, wherein the temperature in step c) is kept for between 24 hours and 15 days.

[0072] 19. The process according to embodiment 3, wherein the temperature in step c) is kept for between 24 hours and 9 days.

[0073] 20. The process according to embodiment 3, wherein the temperature in step c) is kept for between 24 hours and 7 days.

[0074] 21. The process according to embodiment 3, wherein the temperature in step c) is kept for between 24 hours and 96 hours.

[0075] 22. The process according to embodiment 3, wherein the temperature in step c) is kept for between 48 hours and 9 days. 23. The process according to embodiment 3, wherein the temperature in step c) is kept for between 48 hours and 7 days.

[0076] 24. The process according to embodiment 3, wherein the temperature in step c) is kept for between 72 hours and 9 days.

[0077] 25. The process according to embodiment 3, wherein the temperature in step c) is kept for between 72 hours and 7 days.

[0078] 26. The process according to embodiment 3, wherein the temperature in step c) is kept for between 72 hours and 96 hours.

[0079] 27. The process according to embodiment 3, wherein the temperature in step c) is kept for between 96 hours and 9 days.

[0080] 28. The process according to embodiment 3, wherein the temperature in step c) is kept for between 96 hours and 7 days.

[0081] 29. The process according to embodiment 3, wherein the temperature in step c) is kept for between 7 and 9 days.

[0082] 30. The process according to any one of the preceding embodiments, wherein the pH is at least pH 5.0 in step a).

[0083] 31. The process according to any one of the preceding embodiments, wherein the pH is between pH 5.0 and pH 6.0 in step a).

[0084] 32. The process according to any one of the preceding embodiments, wherein the pH is below pH 5.8 in step a).

[0085] 33. The process according to any one of the preceding embodiments, wherein pH in step a) is about pH 5.5.

[0086] 34. The process according to any one of the preceding embodiments, wherein the temperature in step a) is between 35 to 40 degrees Celsius.

[0087] 35. The process according to any one of the preceding embodiments, wherein the temperature in step a) is about 37 degrees Celsius.

[0088] 36. The process according to any one of the previous embodiments, wherein step b) is mandatory.

[0089] 37. The process according to any one of the preceding embodiments, wherein the pH is between pH 6.2 and pH 7.8 in step b). 38. The process according to any one of the preceding embodiments, wherein the pH is between pH 6.5 and pH 7.7 in step b).

[0090] 39. The process according to any one of the preceding embodiments, wherein pH in step b) is about pH 7.5

[0091] 40. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 45 to 60 degrees Celsius for at least 3 hours.

[0092] 41. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 45 to 55 degrees Celsius for at least 3 hours.

[0093] 42. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 45 to 50 degrees Celsius for at least 3 hours.

[0094] 43. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 50 to 69 degrees Celsius for at least 3 hours.

[0095] 44. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 50 to 60 degrees Celsius for at least 3 hours.

[0096] 45. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 50 to 55 degrees Celsius for at least 3 hours.

[0097] 46. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 55 to 69 degrees Celsius for at least 3 hours.

[0098] 47. The process according to any one of the preceding embodiments, wherein the temperature in step c) is between 55 to 60 degrees Celsius for at least 3 hours.

[0099] 48. The process according to any one of the preceding embodiments, wherein the temperature in step c) is about 55 degrees Celsius for at least 3 hours.

[0100] 49. The process according to any one of the preceding embodiments, wherein the drying in step d) is performed by lyophilization.

[0101] 50. The process according to any one of the preceding embodiments, wherein the drying in step d) is performed by spray-drying.

[0102] 51. The process according to any one of the preceding embodiments, wherein a carrier is added in step d).

[0103] 52. The process according to embodiment 51 , wherein the carrier is selected from the group consisting of dextrin, maltodextrin, maize dextrin, potassium chloride, starch, lactose, Nutri- ose, sucrose, glucose, whey, gelatin, casein and / or albumin. 53. The process according to any one of the preceding embodiments, wherein a step of adding a carrier is mandatory and the carrier is dextrin, maize dextrin or Nutriose.

[0104] 54. The process according to embodiment 51 , wherein a step of adding a carrier is mandatory and the carrier is dextrin.

[0105] 55. process according to embodiment 51 , wherein a step of adding a carrier is mandatory and the carrier is maize dextrin.

[0106] 56. Process according to embodiment 51 , wherein a step of adding a carrier is mandatory and the carrier is Nutriose.

[0107] 57. The process according to any one of the preceding embodiments, wherein the process comprises a separation step before step b) or after step c) such as a centrifugation, microfiltration or ultrafiltration step.

[0108] 58. The process according to any one of the preceding embodiments, wherein the Lactobacillus strain is Lactobacillus reuteri.

[0109] 59. The process according to any one of the preceding embodiments, wherein the Lactobacillus strain is Lactobacillus reuteri deposited as DSM 17648.

[0110] 60. The process according to any one of the preceding embodiments, further comprising adding the resulting strain to a composition to form a product which comprises a low amount of living Lactobacillus cells.

[0111] 61 . The process according to any one of the preceding embodiments, further comprising adding the resulting strain to a composition to form a product which comprises less than 1 E+8 CFU / g Lactobacillus cells.

[0112] 62. The process according to embodiment 61 , wherein the product comprises less than 1 E+7 CFU / g, 1 E+6 CFU / g, 1 E+5 CFU / g, 1 E+4 CFU / g, 1 E+3 CFU / g, 1 E+2 CFU / g or 1 E+1 CFU / g Lactobacillus cells.

[0113] 63. The process according to embodiment 61 , wherein the product comprises less than 1 E+6 CFU / g Lactobacillus cells.

[0114] 64. The process according to embodiment 61 , wherein the product comprises less than 1 E+4 CFU / g Lactobacillus cells.

[0115] 65. The process according to embodiment 61 , wherein the product comprises no measurable live Lactobacillus cells. 66. The process according to any one of embodiments 61 to 65, wherein the formed product is a postbiotic.

[0116] 67. The process according to any one of embodiments 61 to 65, wherein the formed product is a solid beverage.

[0117] 68. The process according to any one of the preceding embodiments, further comprising adding the resulting strain to a food product.

[0118] 69. The process according to any one of the preceding embodiments, further comprising adding the resulting strain to a milk based food product such as yoghurt.

[0119] 70. The process according to any one of the preceding embodiments, wherein the process is for production of a product comprising a low amount of living Lactobacillus cells.

[0120] 71. The process according to any one of the preceding embodiments, wherein the process is for production of a product comprising less than E+8 CFU / g Lactobacillus cells.

[0121] 72. The process according to any one of the preceding embodiments, wherein the process is for production of a product comprising less than 1E+7 CFU / g, 1 E+6 CFU / g, 1 E+5 CFU / g, 1 E+4 CFU / g, 1E+3 CFU / g, 1E+2 CFU / g or 1 E+1 CFU / g Lactobacillus cells.

[0122] 73. The process according to embodiment 71 , wherein the product comprises less than 1 E+6 CFU / g Lactobacillus cells.

[0123] 74. The process according to embodiment 71 , wherein the product comprises less than 1 E+4 CFU / g Lactobacillus cells.

[0124] 75. The process according to embodiment 71 , wherein the product comprises no measurable live Lactobacillus cells.

[0125] 76. The process according to any one of embodiments 70 to 75, wherein the product is a postbiotic strain.

[0126] 77. The process according to any one of embodiments 70 to 76, wherein the product is a solid beverage.

[0127] 78. The process according to any one of embodiments 70 to 76, wherein the product is a food product.

[0128] 79. The process according to any one of embodiments 70 to 75, wherein the product is a milk based food product such as yoghurt.

[0129] 80. The process according to any one of the preceding embodiments, wherein the steps are: a) Fermenting a Lactobacillus strain at about pH 5.5 b) Increasing the pH to about pH 7.5 c) separating the solid phase from the liquid phase step using centrifugation d) optionally adding Nutriose as a carrier e) Increasing the temperature to about 55 degrees Celsius for about 5 hours f) if a carrier is not added in step d), adding Nutriose as a carrier g) Freeze or spray-drying the resulting strain.

[0130] 81. The process according to embodiment 80, wherein step g) is freeze-drying the resulting strain.

[0131] 82. The process according to embodiment 80, wherein step fgis spray-drying the resulting strain.

[0132] 83. The process according to any one of the preceding embodiments, wherein the steps are: a) Fermenting a Lactobacillus strain at about pH 5.5 b) separating the solid phase from the liquid phase step using centrifugation c) Increasing the temperature to about 55 degrees Celsius for about 7 to 9 hours d) Freeze or spray-drying the resulting strain.

[0133] 84. The process according to any one of the preceding embodiments, wherein the steps are: a) Fermenting a Lactobacillus strain at about pH 5.5 b) separating the solid phase from the liquid phase step using centrifugation c) Increasing the temperature to about 55 degrees Celsius for about 7 days d) Freeze or spray-drying the resulting strain.

[0134] 85. The process according to embodiment 83 or 84, wherein step d) is freeze-drying the resulting strain.

[0135] 86. The process according to embodiment 83 or 84, wherein step d) is spray-drying the resulting strain.

[0136] 87. A Lactobacillus strain obtained by the process of any one of the preceding embodiments.

[0137] EXAMPLES

[0138] Materials and Methods

[0139] Cell Storage:

[0140] Lactobacillus reuteri deposited as DSM 17648 were stored in a frozen state at -80°C. For storage, one ml of culture grown to the stationary phase in MRS (55 g / l, pH 6.5; Difco, USA) medium was mixed with 500 pl of a 50% (v / v) sterile glycerol solution.

[0141] Helicobacter pylori stored similarly, one ml of culture in Brucella broth supplemented with 10% fetal calf serum was mixed with 500 pl of 50% (v / v) sterile glycerol and stored at -80°C.

[0142] Cultivation: Lactobacillus reuteri deposited as DSM 17648: Cultured in MRS medium at 37°C under anaerobic conditions until the stationary phase was reached, as indicated by the OD600 measurements.

[0143] Helicobacter pylori'. Cultured in Brucella broth supplemented with 10% fetal calf serum at 37°C under microaerophilic conditions until reaching stationary phase.

[0144] Fermentation of Lactobacillus:

[0145] Conducted in MRS medium at 37°C. The fermentation process was monitored for pH, temperature, and OD600. Post-fermentation, the pH was adjusted as per experimental requirements.

[0146] Centrifugation:

[0147] Following fermentation process, the cultures were centrifuged at 5000g for 30 minutes to concentrate the bacterial cells.

[0148] Carrier Addition and Preparation for Spray Drying:

[0149] Nutriose Addition: Post-centrifugation, Nutriose was used as carrier and added to the concentrated bacterial cells at a 1 :9 ratio based on dry cell weight.

[0150] Spray Drying: The treated bacterial suspensions were then subjected to spray drying using standard parameters (inlet temperature of 140-180 °C, outlet temperature of 60-90°C).(Buchi Labor- technik AG, Flawil, Switzerland)

[0151] CFU Counting:

[0152] Samples were prepared for CFU counting by dissolved and serial diluted in sterile saline solution. The diluted samples were plated using a Spiral Plater (Eddy Jet, IUL, SA, Spain) to ensure uniform distribution and accuracy. The agar plates were then incubated for a period of 48 to 72 hours at a consistent temperature of 37 °C.

[0153] The CFU per gram of the original sample was calculated based on the number of colonies, taking into account the dilution factor and the volume of the sample that was plated.

[0154] Co-Aggregation Assay and Quantitative Analysis

[0155] A co-aggregation assay was utilized to evaluate the interaction between Lactobacillus strains and Helicobacter pylori. Equal volumes of Lactobacillus and H. pylori suspensions were mixed and incubated at room temperature to mimic natural interactions.

[0156] Cell counts for Lactobacillus were determined using a Thoma counting chamber. Based on these counts, suspensions with different cell concentrations were prepared. 2.5 ml of each suspension (H. pylori and Lactobacillus) was mixed and agitated for 5 minutes, followed by a resting period of 2 minutes to allow for visible flocculation.

[0157] The degree of co-aggregation was quantified by measuring the OD600 of the supernatant before and after co-aggregation.

[0158] The formula for calculating co-aggregation percentage is: r,£.n / (ODH. pylori OD|_actobacillus) " OD / - / . py / or / .+Lactobacillus

[0159] Degree of co-aggregation % = - x 100

[0160] ODH. pylori + OD|_actobacillus In this formula, 0% co-aggregation represents no change in OD600 after mixing, indicating no co-aggregation. A value above 50% indicates significant co-aggregation activity, suggesting effective interaction between the bacterial strains.

[0161] Example 1 - Impact of pH on Heat Treatment Efficacy for Lactobacillus Strains (1)

[0162] Fermentation and Pre-Heat Treatment pH Adjustment:

[0163] Fermentation of Lactobacillus reuteri deposited as DSM 17648 was conducted at 37 °C. As the cultures reached the stationary phase, indicated by a plateau in OD600 values, the fermentation medium was divided into two batches. One batch was left at the native pH of 5.2, while the pH of the second batch was adjusted to 7.0.

[0164] Heat Treatment Implementation:

[0165] Subsequently, both batches of the fermentation medium were subjected to a heat treatment at 56 °C ± 1 °C for 6 hours. Hourly samples were collected for analysis.

[0166] Sample Processing:

[0167] Each sample was centrifuged at 5000g for 30 minutes to pellet the bacterial cells preparing them for the subsequent addition of carriers and further analyses.

[0168] Carrier Addition Before Spray Drying:

[0169] Prior to spray drying, Nutriose was added as a carrier to the pelleted bacterial cells in a 1 :9 ratio based on dry cell weight.

[0170] Pre-Heat Treatment Analysis:

[0171] Before the heat treatment, samples from both pH conditions (5.2 and 7.0) were analyzed to establish baseline data for CFU / g (Colony Forming Units per gram) and co-aggregation capabilities.

[0172] Effects of Heat Treatment on Viability (Fig 1):

[0173] Untreated Lactobacillus samples showed an initial CFU / g of approximately 1 E+9. Post heat treatment, a significant reduction in CFU / g was observed in samples from both pH conditions. At pH 5.2 (native), CFU / g levels decreased to just below 1 E+4, while at pH 7, CFU / g levels dropped below the detection limit.

[0174] Co-Aggregation Dynamics (Fig 1):

[0175] Prior to heat treatment, co-aggregation was assessed for both pH-treated and untreated samples.

[0176] During the heat treatment, the native pH 5.2 samples displayed a decrease in co-aggregation. In contrast, samples adjusted to pH 7 maintained stable co-aggregation throughout the 6-hour heating period.

[0177] Example 2 - Impact of pH on Heat Treatment Efficacy for Lactobacillus Strain (2)

[0178] Fermentation and Pre-Heat Treatment pH Adjustment:

[0179] Fermentation of Lactobacillus reuteri deposited as DSM 17648 was conducted at 37 °C. As the cultures reached the stationary phase, indicated by a plateau in OD600 values, the fermentation medium was divided into three batches. The pH of the broth was adjusted to different pH levels, A (pH 7.2), B (pH 7.5), and C (pH 7.8). Subsequently, the fermentation vessels were heated to a temperature of 56 °C ± 1 °C for 6 hours.

[0180] Analysis of CFU / g and Co-Aggregation Activity (Fig 2):

[0181] Figure 2 provides an analysis of the CFU / g and the co-aggregation activity against Helicobacter pylori for all treated and untreated samples at the initial time point (T=0).

[0182] Co-Aggregation Observations:

[0183] Across all treated and untreated samples, a stable co-aggregation was observed. The average sedimentation power was noted to be around 60%.

[0184] CFU / g Reduction in Treated Samples:

[0185] For samples treated at pH 7.2 and 7.5, the CFU / g was reduced to approximately 1 E+5 after 6 hours of heat treatment. In contrast, samples treated at pH 7.8 showed a more significant reduction in CFU / g, falling far below 1 E+4 over the 6-hour heat treatment period.

[0186] Example 3 - Impact of Carrier on Heat Treatment Efficacy for Lactobacillus Strain

[0187] The Lactobacillus reuteri deposited as DSM 17648 was first fermented at 37 °C and then subjected to a concentration step. Subsequent to this concentration, the bacterial suspension was divided into two sets for treatment comparison: one with the addition of Nutriose before the pH and heat treatment and the other after pH and heat treatment.

[0188] The pH of concentrated samples was then adjusted to 7.7 to standardize conditions for the subsequent heat treatment. A controlled heat treatment at 56 °C was applied for 3 hours. Following this thermal stress, both the Nutriose- treated and untreated samples were prepared for spray dried.

[0189] Assessment of Co-Aggregation and Viability (Figure 3):

[0190] Figure 3 presents the initial CFU / g and co-aggregation activity against Helicobacter pylori for both Nutriose-treated and untreated samples, measured at T=0 (the start of the experiment). Co-Aggregation Analysis:

[0191] A consistent co-aggregation was observed across all samples, with no significant difference between treated and untreated groups, maintaining an average sedimentation power of approximately 60%.

[0192] CFU / g Outcomes:

[0193] For the Nutriose-treated samples, after 4 hours, the CFU / g was effectively reduced to below detection point, demonstrating the efficacy of the treatment process. In contrast, the untreated samples maintained a CFU / g of approximately 1 E+08.

[0194] Example 4 - Impact of Post-Spray Drying Heat Treatment of Lactobacillus Powders (1)

[0195] Heat Treatment Post-Spray Drying:

[0196] Spray-dried powders comprising Lactobacillus reuteri deposited as DSM 17648 were prepared as described in Example 2 of WO 2012 / 168468. The spray-dried powders (A, B, C, D with different starting CFU / g) were subjected to a continuous heat treatment at set temperatures of 50°C and 55°C for a duration of 96 hours without prior pH adjustment.

[0197] Viability Assessment:

[0198] CFU / g was measured at time intervals (0, 24, 48, 72, and 96 hours) to monitor the viability of Lactobacillus reuteri under heat stress. At both temperatures, the viability of all batches decreased significantly over time, with no detectable CFU / g observed in any batch at 48 hours and beyond (figure 4).

[0199] Co-Aggregation Activity:

[0200] Co-aggregation assessment with H. pylori was exclusively performed at the 96-hour mark, after the completion of heat treatment.

[0201] The co-aggregation activity percentages at this time point were above 50% indicating that despite the loss of viability, the functional activity related to co-aggregation was retained (figure 4).

[0202] Example 5 - Impact of Post-Spray Drying Heat Treatment of Lactobacillus Powders (2)

[0203] Spray-dried powders comprising Lactobacillus reuteri deposited as DSM 17648 were pre-pared as e.g. described in Example 2 of WO 2012 / 168468. The spray-dried powders were packed in 10 kg aluminum pouches which were packed individually in cardboard boxes and stacked on a pallet in 3 layers with a 2x6 scheme. A heat chamber (France Etuves XXL04.5) was used to heat up the powder to 55 °C. The samples were kept for 7 and 9 days in the heat chamber. CFU and coaggregation activity were measure before and after the treatment. The results are shown in table 1.

[0204] Table 1 : Summary - Results of heart treatment of packed powders

[0205] Co-aggregation for all samples were above specification and CFU were successfully reduced for all batches.

[0206] Example 6 - Large-Scale Fermentation, Inactivation, and Drying of Lactobacillus reuteri (DSM 17648) pH and heat treatment was conducted on Lactobacillus reuteri DSM 17648 produced under large- scale (450 L) fermentation.

[0207] Fermentation was conducted at 37 °C and pH was stabilized around pH 5.5 during fermentation followed by a gradual increase to pH 7.6. prior to thermal inactivation as indicated in figure 5. The pH-adjusted culture was then subjected to a controlled heat treatment at 56 °C for 4 hours to achieve the desired inactivation level. Samples were collected before (untreated sample) and after treatment (treated sample) to measure CFU / g, providing a quantitative assessment of the reduction in viable cells.

[0208] Results:

[0209] The untreated sample exhibited a CFU / g of 2.93E+08 while the treated sample showed a significant reduction to 1.59E+03 CFU / g (figure 6). This confirms the efficacy of the pH and heat treatment in achieving a substantial decrease in viability, meeting the specifications for low-viability products.

[0210] To evaluate the retention of functional co-aggregation with Helicobacter pylori, samples were assessed for their co-aggregation capability. Co-aggregation percentage was compared across untreated, treated and control samples (where the control sample was the commercial Pylopass product).

[0211] The control sample exhibited a co-aggregation activity of 66.9%, the untreated sample showed a slight decrease to 58.1%, while the treated sample retained substantial co-aggregation activity at 54.3% (figure 7). This demonstrates the ability of the pH and heat treatment process to maintain functional co-aggregation despite significant CFU reduction.

[0212] This example demonstrates the scalability of the process for large-scale (450 L) fermentation and subsequent inactivation of Lactobacillus reuteri DSM 17648, confirming the effectiveness of the process in reducing CFU / g while maintaining co-aggregation activity. This adjustment ensures optimal conditions for reducing viable cell counts while preserving co-aggregation properties.

[0213] Deposit of Biological Material

[0214] The following biological material has been deposited under the terms of the Budapest Treaty with the Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7 B, D-38124 Braunschweig, Germany, and given the following accession number:

[0215] Deposited strain Accession Number Date of Deposit Country of origin

[0216] Lactobacillus reuteri DSM 17648 12 October 2005 Germany

[0217] The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Claims

CLAIMS1 . A process for the production of a Lactobacillus strain comprising the steps of: a) Fermenting a Lactobacillus strain at a pH below pH 6.0, b) Optionally increasing the pH to between pH 6.0 and pH 7.8, c) Setting the temperature to between 45 to 69 degrees Celsius and keeping the temperature constant for at least 3 hours, and d) Drying the resulting strain; wherein an optional separation step is added before step b) or after step c), and / or an optional step of adding a carrier is added before step b) or after step c), and wherein step d) optionally is carried out before step c).

2. The process according to claim 1 , wherein step c) is carried out before step d).

3. The process according to claim 1 , wherein step d) is carried out before step c).

4. The process according to claim 2, wherein the temperature in step c) is kept for between 3-10 hours.

5. The process according to claim 3, wherein the temperature in step c) is kept for between 24 hours to 20 days.

6. The process according to any one of the preceding claims, wherein the pH is below pH 5.8 in step a).

7. The process according to any one of the preceding claims, wherein step b) is mandatory.

8. The process according to any one of the preceding claims, wherein step b) is mandatory and the pH of step b) and the temperature of step c) are kept for between 3 to 10 hours.

9. The process according to any one of the preceding claims, wherein a carrier is added in step d).

10. The process according to claim 9, wherein the carrier is selected from the group consisting of dextrin, maltodextrin, maize dextrin, potassium chloride, starch, lactose, Nutriose, sucrose, glucose, whey, gelatin, casein and / or albumin.11 . The process according to any one of the preceding claims, wherein the Lactobacillus strain is Lactobacillus reuteri.

12. The process according to any one of the preceding claims, wherein the Lactobacillus strain is Lactobacillus reuteri deposited as DSM 17648.

13. The process according to any one of the preceding claims, further comprising adding the resulting strain to a composition to form a product which comprises a low amount of living Lactobacillus cells.

14. The process according to any one of the preceding claims, wherein the process is for production of a product comprising a low amount of living Lactobacillus cells.

15. A Lactobacillus strain obtained by the process of any one of the preceding claims.

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