Novel method for producing lactobacillus rhamnosus

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 fermenter in a lactose-free, preferably milk-free culture medium and removing the fermentation broth from the fermenter wherein the fermentation is carried out in the presence of acetate or acetate; b) applying shear to the fermentation broth and optionally concentrating the fermentation broth. The invention also relates to the compositions thus obtained and uses of such compositions.

Description

Technical Field

[0001] This invention relates to the production of Lactobacillus rhamnosus (Lactobacillus rhamnosus) Lactobacillus rhamnosus This invention relates to a new method for producing compositions containing *Lactobacillus rhamnosus*, and to the use of such compositions in food or beverages and for medical purposes. Background Technology

[0002] Combinations of bacteria with probiotic activity are becoming increasingly popular as part of the diets of humans and animals due to their beneficial health effects. In addition to supporting gut health and function, these health benefits include refilling the gut after antibiotic treatment, counteracting lactose intolerance, supporting the immune system, and lowering cholesterol. Lactic acid bacteria, primarily from the genera *Lactobacillus* and *Bifidobacterium*, which can help improve or maintain gut health and function, are generally referred to as probiotics (also known as probiotics in this article).

[0003] Lactobacillus rhamnosus, especially Lactobacillus rhamnosus GG, is one of the most popular probiotics.

[0004] In the article titled "Lebeer et al. published in Applied and Environmental Microbiology, Volume 78, Number 1, January 2012, pages 185-193",... Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells In their study, they analyzed the function of the fimbriae of *Lactobacillus rhamnosus* GG. They pointed out that *Lactobacillus rhamnosus* GG is crucial for effective adhesion to intestinal epithelial cell lines and biofilm formation.

[0005] Pili (also known as cilia) are small, hair-like fibrous proteins present on the surface areas of many bacteria. To improve the bioavailability of 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.

[0006] Furthermore, probiotics are preferably marketed based on their colony-forming units (CFU) count per gram. Consumers consider the CFU count per gram as a measure of the probiotic's viability. Therefore, probiotic manufacturers consider a high CFU / g count in their products an advantage.

[0007] Therefore, there remains a need in the art for a method to produce Lactobacillus rhamnosus with high bioavailability and / or high bacterial surface area and / or small bacterial particle size and / or high CFU / g count and / or high viable cell count. Summary of the Invention

[0008] Novel methods for producing Lactobacillus rhamnosus and for producing compositions containing Lactobacillus rhamnosus have been found. These methods can improve the bioavailability of Lactobacillus rhamnosus fimbriae and / or bacterial particle size and / or CFU / g count and / or viable cell count.

[0009] Therefore, in a first aspect, the present invention provides a method for producing Lactobacillus rhamnosus, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shear to the fermentation broth and optionally concentrate the fermentation broth.

[0010] Furthermore, in a second aspect, the present invention provides a method for producing a composition comprising Lactobacillus rhamnosus, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shear to the fermentation broth and optionally concentrate the fermentation broth.

[0011] In a third aspect, the present invention provides Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles, or compositions comprising Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles, which are obtained or can be obtained by any of the methods described above.

[0012] In a fourth aspect, the present invention provides *Lactobacillus rhamnosus* cells or *Lactobacillus rhamnosus* bacterial particles, wherein the bacterial particles having a particle size or diameter 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, even 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, even more preferably equal to or less than 6.5 micrometers, and most preferably equal to or less than 6.0 micrometers, preferably equal to or less than 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 micrometers, wherein the particle size is suitably determined by laser diffraction particle size analysis, and the cells preferably have a particle size equal to or greater than 2.00 × 10⁻⁶. 10 CFU / g cell viability, more preferably equal to or greater than 2.10·10 10 CFU / g cell viability, or more preferably equal to or greater than 1.50 × 10⁻⁶. 11 The optimal cell viability is 2.00 CFU / g or greater. 11 CFU / gram of cell viability.

[0013] In a fifth aspect, the present invention provides a composition comprising *Lactobacillus rhamnosus* cells or *Lactobacillus rhamnosus* bacterial particles, wherein the bacterial particles have a particle size or diameter of 90% w / w or greater, preferably 95% w / w or greater, more preferably 99% w / w or greater, even more preferably 99.5% w / w or greater, still more preferably 99.9% w / w or greater, and most preferably 100.0% w / w or greater, having a particle size or diameter of 8.0 μm or less, preferably 7.5 μm or less, more preferably 7.0 μm or less, even more preferably 6.5 μm or less, and most preferably 6.0 μm or less, preferably 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 μm or less, wherein the particle size is suitably determined by laser diffraction particle size analysis, and the composition preferably contains 2.00 × 10⁻⁶ μm. 10 CFU / g cell viability, more preferably equal to or greater than 2.10·10 10 CFU / g cell viability, or more preferably equal to or greater than 1.50 × 10⁻⁶. 11 The optimal cell viability is 2.00 CFU / g or greater. 11 CFU / gram of cell viability.

[0014] In a sixth aspect, the present invention provides a composition for consumption by animals and / or humans, preferably a pharmaceutical or food product or beverage product, comprising: - Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above; or - The composition containing Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above.

[0015] In a seventh aspect, the present invention provides the use of the Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above, or the composition comprising Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above, in the production of food or beverage products.

[0016] In an eighth aspect, the present invention provides the use of Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above, or compositions containing Lactobacillus rhamnosus cells or Lactobacillus rhamnosus bacterial particles as described above, in probiotic compositions and / or for medical purposes and / or in medicaments or as medicaments, preferably as medicaments for treating or preventing diseases or conditions in or related to the gastrointestinal tract of animals or humans.

[0017] Using the methods according to the invention and in the compositions and uses according to the invention, the bacterial surface area per gram of product can be increased, and / or the bacterial particle size can be reduced, and / or the CFU / g count can be increased, and / or the viable cell count can be increased. As illustrated in the examples, the methods according to the invention result in a synergistic effect, which leads to a high CFU / g count and small particle size. The high CFU / g count and small particle size together result in a high total bacterial particle surface area per gram of product. Furthermore, without wishing to be bound by any kind of theory, it is believed that, as a result of the applied methods, the pili on the cells can become less entangled and have higher bioavailability. Without wishing to be bound by any kind of theory, it is believed that this high total cell surface area and associated high bioavailability of pili on the cell surface can be beneficial when applied to the gastrointestinal tract of humans or animals. Without wishing to be bound by any kind of theory, it is believed that, for example, adhesion to and / or interaction with human or animal intestinal epithelial cells can be increased, for example, through pili on the cell surface of Lactobacillus rhamnosus cells. In other words, without being bound by any kind of theory, it is believed that the methods, compositions, and uses according to the invention that result in and / or have the aforementioned high cell surface area, small particle size, high CFU / g count, and / or high live cell count can benefit the probiotic activity of Lactobacillus rhamnosus cells.

[0018] Brief description of the attached figures The invention is illustrated by the following figures.

[0019] Figure 1 The bacterial particle size distribution of *Lactobacillus rhamnosus* GG is shown for samples derived from fermenters B1 and C1 as illustrated in the examples. As shown, compared to the case without shearing (“light gray line”), applying shear (“dark gray line”) allows a significant reduction in the percentage volume of particles larger than 5 micrometers (μm), and the second peak of this line for fermenter B1 (according to the invention) (just above 2%) is significantly lower than the second peak of this line for fermenter C1 (comparative example, just above 2.5%). Invention Details definition Unless otherwise defined or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0021] Throughout the specification and appended claims, the words “comprising” and “including”, as well as variations such as “containing”, should be interpreted as inclusive. That is, where the context permits, these words are intended to express the possibility of including other elements or integers not specifically stated.

[0022] When used in this text, the articles “a” and “an”, or when no quantifier is used, refer to one or more grammatical objects (i.e., one or at least one). For example, “an element” can mean one element or more elements. When a noun is mentioned in the singular (e.g., a compound, an additive, etc.), it implies the inclusion of the plural. Therefore, when referring to a specific part (e.g., “a strain”), it means “at least one” of that strain, e.g., “at least one strain”, unless otherwise specified.

[0023] When referring to a compound that has several isomers (e.g., D and L enantiomers), the compound in principle includes all enantiomers, diastereomers, and cis / trans isomers of the compound that can be used in specific aspects of the present invention; in particular, when referring to the compound, it includes natural isomers.

[0024] Unless otherwise expressly stated, the various embodiments of the invention described herein can be combined in various ways.

[0025] The term "milk" is intended to encompass milk derived from mammalian and plant sources or mixtures thereof. Mammal sources of milk include, but are not limited to, dairy cows, sheep, goats, buffalo, camels, llamas, horses, or reindeer. Plant sources of milk include, but are not limited to, milk derived from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, sesame seeds, and sunflower seeds. Furthermore, the term "milk" refers not only to whole milk but also to skim milk or any liquid component or reconstituted milk derived from it.

[0026] Lactobacillus rhamnosus The term lactic acid bacteria (LAB) is a general term for a group of Gram-positive bacteria that do not form spores, and whose main fermentation product is lactic acid. Examples of probiotics include LAB bacteria of the genera *Lactobacillus*, *Bifidobacterium*, and *Saccharomyces*. *Lactobacillus rhamnosus* is one of the most popular probiotics.

[0027] Preferably, Lactobacillus rhamnosus is one or more of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus BD0016 and / or Lactobacillus rhamnosus KF 7.

[0028] More preferably, in all aspects of the invention, *Lactobacillus rhamnosus* is *Lactobacillus rhamnosus* GG. 。 Lactobacillus rhamnosus GG is also formally known as Lactobacillus rhamnosus GG. These terms are used interchangeably in this article.

[0029] The term “Lactobacillus rhamnosus GG” should be understood in this document to refer to Lactobacillus rhamnosus strain ATCC 53103, or its mutants or variants, which is deposited by Sherwood Gorbach and Barry Goldin at the American Center for Type Culture Collection.

[0030] Most preferably, in all aspects of the invention, *Lactobacillus rhamnosus* is the *Lactobacillus rhamnosus* GG strain preserved as ATCC 53103. 。

[0031] Lactobacillus rhamnosus can exist as Lactobacillus rhamnosus bacterial particles, wherein each bacterial particle may contain one or more cells. Advantageously, the method according to the invention allows for a reduction in bacterial particle size and an increase in bacterial surface area. Therefore, preferably, the Lactobacillus rhamnosus present in the composition according to the invention comprises or is composed of Lactobacillus rhamnosus bacterial particles, said bacterial particles preferably comprising or being composed of: equal to or less than 100 cells / particle, more preferably equal to or less than 50 cells / particle, even more preferably equal to or less than 20 cells / particle, even more preferably equal to or less than 10 cells / particle, and still more preferably equal to or less than 5 cells / particle. Most preferably, the Lactobacillus rhamnosus present in the composition according to the invention comprises or is composed of Lactobacillus rhamnosus bacterial particles, said bacterial particles comprising or being composed of equal to or less than 3 cells / particle.

[0032] Step (a) In step (a) of the method according to the invention, Lactobacillus rhamnosus GG is fermented in a fermenter in a lactose-free, preferably milk-free, medium, and the fermentation broth is removed from the fermenter.

[0033] A fermenter may include or consist of a fermentation reactor, which is sometimes also called a fermentation tank or fermentation vessel. A fermenter may or may not include a bubbling system and is a bubbling reactor; may or may not include an agitator and is a stirred reactor; and / or may or may not include a loop and is a loop reactor. Combinations are also possible. When the fermenter includes an agitator or is a stirred reactor, the agitator preferably operates at a stirring rate equal to or greater than 1 rpm, more preferably equal to or greater than 2 rpm, even more preferably equal to or greater than 5 rpm, even more preferably equal to or greater than 10 rpm, up to equal to or less than 500 rpm, more preferably equal to or less than 300 rpm, even more preferably equal to or less than 200 rpm, even more preferably equal to or less than 100 rpm, and most preferably equal to or less than 50 rpm.

[0034] When the fermenter is a laboratory fermenter, it can have a volume in the range of 1 liter or more, more preferably 5 liters or more, to 50 liters or less. More preferably, the fermenter is an industrial fermenter. Therefore, more preferably, the fermenter is a fermenter with a volume of 50 liters or more, more preferably 100 liters or more, even more preferably 500 liters or more, and most preferably 1000 liters or more, preferably 700,000 liters or less, more preferably 500,000 liters or less, and even more preferably 250,000 liters or less, and even more preferably 100,000 liters or less.

[0035] Lactobacillus rhamnosus is fermented in a culture medium in a fermenter. This culture medium may also be referred to herein as a fermentation medium. The culture medium may suitably be a solution, suspension, or dispersion. Preferably, the fermentation medium is an aqueous fermentation medium. Where the fermenter is a laboratory fermenter, the fermentation medium may be contained in a volume equal to or greater than 1 liter, more preferably equal to or greater than 5 liters, to equal to or less than 50 liters. More preferably, the fermenter is an industrial fermenter. Therefore, more preferably, the volume of the fermentation medium is equal to or greater than 50 liters, more preferably equal to or greater than 100 liters, even more preferably equal to or greater than 500 liters, and most preferably equal to or greater than 1000 liters and preferably equal to or less than 700,000 liters, more preferably equal to or less than 500,000 liters, and even more preferably equal to or less than 250,000 liters, and even more preferably equal to or less than 100,000 liters.

[0036] For use as a probiotic (and also by lactose-intolerant consumers), the culture medium must be lactose-free. "Lactose-free" is understood herein to mean that the fermentation medium preferably contains equal to or less than 1000 ppmw (parts per million by weight) of lactose, more preferably equal to or less than 100 ppmw of lactose, even more preferably equal to or less than 10 ppmw of lactose, even more preferably equal to or less than 1 ppmw of lactose, and still more preferably equal to or less than 0.1 ppmw of lactose. Most preferably, the culture medium contains no measurable amount of lactose and is completely lactose-free.

[0037] More preferably, the culture medium is milk-deficient or milk-free. "Milk-free" is understood herein to mean that the fermentation medium preferably contains equal to or less than 1000 ppmv (parts per million by volume), more preferably equal to or less than 100 ppmv, even more preferably equal to or less than 10 ppmv, still more preferably equal to or less than 1 ppmv, and even more preferably equal to or less than 0.1 ppmv. Most preferably, the culture medium does not contain any measurable milk and is completely milk-free.

[0038] As stated above, the term milk includes milk from mammalian sources and / or plant-based sources. Therefore, the fermentation medium preferably does not contain any milk derived from cows, sheep, goats, buffalo, camels, llamas, horses, or reindeer and / or extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, sesame, or sunflower seeds.

[0039] Further details regarding the fermentation medium are provided below. More preferably, as described below, the fermentation medium is a solution, suspension, or dispersion, wherein acetate ions are present as dissociated or non-dissociated calcium acetate salts and / or dissociated or non-dissociated ammonium acetate salts, most preferably as dissociated or non-dissociated ammonium acetate salts. Preferably, the fermentation medium is stirable, more preferably a stirable liquid or slurry, and preferably, the medium is not a solid or gel. Furthermore, further details regarding the fermentation conditions are provided below.

[0040] Conveniently, step (a) can produce or obtain fermentation broth. As mentioned above, such fermentation broth can be appropriately removed from the fermenter before applying step (b).

[0041] glucose Preferably, the culture medium, also referred to herein as a fermentation medium, is a glucose-containing medium. Glucose may be added as a solid or as a solution. Preferably, glucose is added to the culture medium (also referred to herein as a fermentation medium) in the form of an aqueous solution, aqueous suspension, or aqueous dispersion containing glucose.

[0042] For example, glucose may be added to the culture medium (also referred to herein as fermentation medium) before fermentation begins and / or during fermentation. If glucose is added during fermentation, it is preferably added continuously, for example by online addition, such as via a circulating reactor. Preferably, for example at the start of fermentation and / or during fermentation, glucose is present in the culture medium at a concentration from equal to or greater than 20 g / kg, preferably equal to or greater than 60 g / kg, more preferably equal to or greater than 80 g / kg, even more preferably equal to or greater than 100 g / kg, and still more preferably equal to or greater than 110 g / kg to equal to or less than 600 g / kg, more preferably equal to or less than 400 g / kg, even more preferably equal to or less than 300 g / kg, even more preferably equal to or less than 200 g / kg, still more preferably equal to or less than 170 g / kg, and most preferably equal to or less than 150 g / kg, where g / kg refers to the weight of glucose in grams / the total weight of the culture medium in kilograms. Within the aforementioned range, the preference for a specific concentration in gr / kg can be interchanged with the corresponding preference in gr / liter.

[0043] That is, preferably, for example, at the start of fermentation and / or during fermentation, glucose is present in the culture medium at a concentration of equal to or greater than 2% w / w, preferably equal to or greater than 6% w / w, more preferably equal to or greater than 8% w / w, even more preferably equal to or greater than 10% w / w, and still more preferably equal to or greater than 11% w / w to equal to or less than 60% w / w, more preferably equal to or less than 40% w / w, even more preferably equal to or less than 30% w / w, even more preferably equal to or less than 20% w / w, even more preferably equal to or less than 17% w / w, and most preferably equal to or less than 15% w / w, based on the total weight of the culture medium. When glucose is added as a solution, suspension, or dispersion, the above ranges apply to the weight of the amount of glucose "as such" therein, not the weight of the solution, suspension, or dispersion as a whole.

[0044] calcium Preferably, the fermentation in step (a) is further carried out in the presence of calcium or calcium salt.

[0045] Calcium can exist as elemental calcium or as a salt. If calcium exists as a salt, it can exist in either a dissociated or non-dissociated (i.e., associated) form. That is, calcium can, for example, exist as Ca... 2+ Cations are present.

[0046] Preferably, calcium is present or provided to the culture medium (also referred to herein as a fermentation medium) as a dissociated or non-dissociated calcium salt. Thus, preferably, the fermentation medium contains calcium or a calcium salt, suitably a dissociated or non-dissociated calcium salt.

[0047] Preferably, the calcium salt is a calcium halide salt, an organic acid salt of calcium, or calcium hydroxide. In a preferred embodiment, the calcium salt is a calcium halide salt, more preferably calcium chloride, calcium iodide, or calcium bromide, or a mixture thereof. Most preferably, the calcium halide salt is calcium chloride. In another preferred embodiment, the calcium salt is an organic acid salt of calcium, preferably an organic acid salt containing 1 to 6 carbon atoms, more preferably an organic salt containing 1 to 4 carbon atoms. Preferably, the organic acid salt of calcium is selected from the group consisting of calcium carbonate, calcium acetate, calcium propionate, calcium butyrate, calcium citrate, calcium gluconate, or mixtures thereof. Most preferably, the organic acid salt of calcium is calcium carbonate.

[0048] Most preferably, calcium is present or provided to the culture medium as a dissociated or non-dissociated calcium chloride salt and / or a dissociated or non-dissociated calcium carbonate salt and / or a dissociated or non-dissociated calcium hydroxide salt.

[0049] Preferably, calcium is added to the culture medium in the form of an aqueous solution, aqueous suspension, or aqueous dispersion containing calcium, optionally as Ca 2+ The cation may be present as a dissociated or non-dissociated salt in other ways. Therefore, preferably, the fermentation medium comprises an aqueous solution, aqueous suspension, or aqueous dispersion containing calcium or a dissociated or non-dissociated calcium chloride salt and / or a dissociated or non-dissociated calcium carbonate salt and / or a dissociated or non-dissociated calcium hydroxide salt, most preferably a dissociated or non-dissociated calcium chloride salt.

[0050] For example, calcium may be added to the culture medium (also referred to herein as fermentation medium) before fermentation begins and / or during fermentation. If calcium is added during fermentation, it is preferably added continuously, for example by online addition, such as via a circulating reactor. Preferably, for example at the start of fermentation and / or during fermentation, calcium is added at a rate from equal to or greater than 10 mg / kg, preferably equal to or greater than 20 mg / kg, more preferably equal to or greater than 30 mg / kg, even more preferably equal to or greater than 40 mg / kg to equal to or less than 10 gr / kg, more preferably equal to or less than 5 gr / kg, even more preferably equal to or less than 1 gr / kg, even more preferably equal to or less than 500 mg / kg, even more preferably equal to or less than 300 mg / kg, and most preferably equal to or less than 200 mg / kg, wherein gr / kg or mg / kg refers to the weight of calcium in grams or milligrams per kilogram of total weight of culture medium. That is, preferably, for example, at the start of fermentation and / or during fermentation, calcium is present in the culture medium at a concentration of equal to or greater than 10 ppmw (parts per million by weight), preferably equal to or greater than 20 ppmw, more preferably equal to or greater than 30 ppmw, even more preferably equal to or greater than 40 ppmw to equal to or less than 10% w / w, more preferably equal to or less than 5% w / w, even more preferably equal to or less than 1.0% w / w, even more preferably equal to or less than 500 ppmw, even more preferably equal to or less than 300 ppmw, and most preferably equal to or less than 200 ppmw, based on the total weight of the culture medium. Very high amounts of calcium may be less preferred because, depending on the circumstances, this may lead to precipitation. The risk of precipitation is highest when calcium carbonate is used. Unfortunately, calcium carbonate has very low solubility in pure water (about 15 mg / L at 25°C, equivalent to about 0.0015% w / w). Therefore, most preferably, any calcium salt is not present in the form of calcium carbonate. Preferably, any calcium salt is calcium chloride. Most preferably, for example at the start of fermentation and / or during fermentation, calcium may be added and / or present in the culture medium at a concentration equal to or greater than 0.0001% w / w, more preferably equal to or greater than 0.001% w / w to equal to or less than 0.1% w / w, more preferably equal to or less than 0.05% w / w, based on the total weight of the culture medium. That is, most preferably, the fermentation medium contains calcium in the range of equal to or greater than 0.0001% w / w, more preferably equal to or greater than 0.001% w / w to equal to or less than 0.1% w / w, more preferably equal to or less than 0.05% w / w, based on the total weight of the culture medium.

[0051] When calcium is added as a salt or in a solution, suspension or dispersion, the above range applies to the weight of the amount of calcium "as such" therein, not the weight of the salt, solution, suspension or dispersion as a whole.

[0052] Acetate In step (a), fermentation is carried out in the presence of acetate or acetate.

[0053] The acetate ion preferably exists in the form of a salt. If the acetate ion exists as a salt, it can exist in either a dissociated or non-dissociated (i.e., associated) form. That is, the acetate ion can exist, for example, as an acetate anion. This acetate anion can be represented, for example, by the chemical formula CH3CO. 2. C2H3O 2. or CH3COO express.

[0054] Preferably, the acetate ion is present or provided to the culture medium (also referred to herein as a fermentation medium) as a dissociated or non-dissociated acetate. Thus, preferably, the fermentation medium contains acetate, suitably a dissociated or non-dissociated acetate.

[0055] In a preferred embodiment, the acetate is an alkali metal acetate or an alkaline earth metal acetate. Preferably, such an alkali metal acetate or alkaline earth metal acetate is selected from the group consisting of sodium acetate, potassium acetate, calcium acetate, or mixtures thereof. Most preferably, the alkali metal acetate or alkaline earth metal acetate is calcium acetate. In another preferred embodiment, the acetate ion is present in the form of ammonium acetate. This ammonium acetate can be represented, for example, by the chemical formula NH4CH3CO2.

[0056] More preferably, the acetate ion is present or provided to the culture medium as a dissociated or non-dissociated calcium acetate salt and / or a dissociated or non-dissociated ammonium acetate salt, most preferably a dissociated or non-dissociated ammonium acetate salt.

[0057] Preferably, acetate is added to the culture medium in the form of an aqueous solution, aqueous suspension, or aqueous dispersion containing acetate, optionally as an acetate anion or otherwise as a dissociated or non-dissociated salt. Therefore, preferably, the fermentation medium comprises an aqueous solution, aqueous suspension, or aqueous dispersion containing ammonium acetate, suitably dissociated or non-dissociated.

[0058] For example, acetate ions can be added to the culture medium (also referred to herein as fermentation medium) before fermentation begins and / or during fermentation. If acetate ions are added during fermentation, they are preferably added continuously, for example by online addition, such as via a circulating reactor. Preferably, for example at the start of fermentation and / or during fermentation, acetate is present in the culture medium at a concentration ranging from equal to or greater than 0.4 g / kg, preferably equal to or greater than 1.0 g / kg, more preferably equal to or greater than 2.0 g / kg, even more preferably equal to or greater than 3.0 g / kg, and still more preferably equal to or greater than 4.0 g / kg to equal to or less than 60 g / kg, preferably equal to or less than 40 g / kg, more preferably equal to or less than 30 g / kg, even more preferably equal to or less than 20 g / kg, even more preferably equal to or less than 10 g / kg, still more preferably equal to or less than 8.0 g / kg, and most preferably equal to or less than 6.0 g / kg, where g / kg refers to the weight of acetate in grams divided by the total weight of the culture medium in kilograms. Within the above ranges, the preference for a specific concentration in g / kg can be interchanged with a corresponding preference in g / L.

[0059] That is, preferably, for example, at the start of fermentation and / or during fermentation, acetate is present in the culture medium at a concentration of equal to or greater than 0.04% w / w, preferably equal to or greater than 0.1% w / w, more preferably equal to or greater than 0.20% w / w, even more preferably equal to or greater than 0.30% w / w, and still more preferably equal to or greater than 0.40% w / w to equal to or less than 6% w / w, preferably equal to or less than 4% w / w, more preferably equal to or less than 3% w / w, even more preferably equal to or less than 2% w / w, even more preferably equal to or less than 1% w / w, still more preferably equal to or less than 0.8% w / w, and most preferably equal to or less than 0.60% w / w, based on the total weight of the culture medium. Acetates with low solubility in water (e.g., vitamin A acetate or tocopherol acetate) may be less preferred, as this may lead to precipitation, depending on the circumstances. More preferred are acetates with good solubility in water, such as ammonium acetate, potassium acetate, and / or sodium acetate. Ammonium acetate is the most preferred. Without wishing to be bound by any type of theory, it is believed that the use of ammonium acetate can have a smaller osmotic effect than that of sodium acetate or potassium acetate. Most preferably, the fermentation medium contains acetate ions in the range of equal to or greater than 0.10% w / w, more preferably equal to or greater than 0.20% w / w to equal to or less than 2.0% w / w, more preferably equal to or less than 1.0% w / w, based on the total weight of the medium, preferably in the form of ammonium acetate.

[0060] When acetate is added as a salt, solution, suspension or dispersion, the above range applies to the weight of the amount of acetate "as such" therein, not the weight of the salt, solution, suspension or dispersion as a whole.

[0061] Other components in the fermentation medium As described above, the fermentation medium is preferably a solution, suspension, or dispersion, more preferably an aqueous solution, suspension, or dispersion, wherein acetate ions are present as dissociated or non-dissociated calcium acetate salts and / or dissociated or non-dissociated ammonium acetate salts, and most preferably as dissociated or non-dissociated ammonium acetate salts.

[0062] In addition to the components already mentioned above, the culture medium (also referred to herein as fermentation medium) may contain one or more additional components. In other words, optionally, the fermentation in step (a) may be carried out in the presence of one or more additional components.

[0063] Preferably, the culture medium contains additional components, such as: - Nutrients, such as yeast; and / or - pH adjusters, such as ammonium hydroxide, sodium hydroxide, or potassium hydroxide; and / or - Minerals, such as manganese, magnesium and / or zinc and / or any associated or dissociated salts thereof.

[0064] More preferably, the culture medium contains at least one or more nutrients (e.g., yeast) and / or pH adjusters (e.g., ammonium hydroxide, sodium hydroxide, and / or potassium hydroxide).

[0065] Most preferably, the culture medium (also referred to herein as fermentation medium) contains sodium hydroxide and / or potassium hydroxide, preferably as a pH adjuster. More preferably, ammonia (NH3) is not added to or otherwise supplied to the culture medium. In other words, ammonia (NH3) is more preferably not used as a pH adjuster. Most preferably, sodium hydroxide is used as a pH adjuster. As described, for example, in European Patent EP2398890B1, the use of ammonia as a titrant is understood to result in an undesirable pink / red tinge observed on the surface of the dried lactic acid bacteria composition during storage. Without being bound by any kind of theory, it is believed that the use of NaOH and KOH bases to control pH (as described in EP2398890B1) may have a negative impact on cell counts. It has been advantageously found that this negative impact can be at least partially offset by the application of ammonium acetate during fermentation.

[0066] Preferably, the culture medium is an aqueous culture medium. Therefore, in addition to the above, the culture medium preferably also contains water.

[0067] Fermentation conditions, pH and temperature Preferably, fermentation is carried out at a temperature equal to or greater than 10°C, more preferably equal to or greater than 15°C, even more preferably equal to or greater than 20°C, even more preferably equal to or greater than 25°C, even more preferably equal to or greater than 28°C, even more preferably equal to or greater than 32°C, and most preferably equal to or greater than 35°C. Simultaneously, fermentation is preferably carried out at a temperature equal to or less than 46°C, more preferably equal to or less than 42°C, even more preferably equal to or less than 40°C, even more preferably equal to or less than 39°C, even more preferably equal to or less than 38°C, and most preferably equal to or less than 37°C.

[0068] Preferably, fermentation is carried out at a pH point, or preferably towards a final pH point, i.e., equal to or greater than pH 3.0, more preferably equal to or greater than pH 3.5, even more preferably equal to or greater than pH 4.2, even more preferably equal to or greater than pH 4.6, even more preferably equal to or greater than pH 4.8, and most preferably equal to or greater than pH 5.0. Simultaneously, fermentation is preferably carried out at a pH point, or preferably towards a final pH point, i.e., equal to or less than pH 6.2, more preferably equal to or less than pH 6.0, even more preferably equal to or less than pH 5.8, even more preferably equal to or less than pH 5.6, even more preferably equal to or less than pH 5.5, and most preferably equal to or less than pH 5.4.

[0069] pH can be conveniently controlled by adding a titrant (preferably selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, and combinations thereof), as described herein. Suitably, using sodium hydroxide and / or potassium hydroxide as a titrant eliminates the need for using ammonia (NH3) as a titrant, and thus allows for reduced powdering. Therefore, most preferably, the titrant does not contain ammonia (NH3). Most preferably, the titrant contains or consists of sodium hydroxide. Preferably, the titrant is supplied to the fermenter as an aqueous solution of such a titrant. That is, preferably, an aqueous solution of potassium hydroxide and / or sodium hydroxide, and most preferably, an aqueous solution of sodium hydroxide, is supplied to the fermenter.

[0070] In this document, "fermentation" is preferably understood as the process until the fermentation broth is removed from the fermenter. Fermentation can be continuous, semi-continuous, or batch fermentation. If fermentation is carried out in batches, it is preferably stopped by cooling, pH reduction, and / or depletion of the carbon source used as feed, after which the fermentation broth can be conveniently removed from the fermenter.

[0071] In a preferred embodiment, during fermentation in step (a), the pH is maintained in the range of 5.4 or greater to 5.6 or less, while the temperature is maintained in the range of 37°C or greater to 39°C or less.

[0072] In a first preferred embodiment, the pH of the fermentation medium is maintained at 5.6, and the temperature is maintained at 39°C. In a second embodiment, the pH is maintained at 5.4, and the temperature is maintained at 39°C. In a third embodiment, the pH is maintained at 5.6, and the temperature is maintained at 39°C. In a fourth embodiment, the pH is maintained at 5.4, and the temperature is maintained at 37°C.

[0073] Step (b) In step (b) of the method according to the invention, shearing is applied to the fermentation broth. As described above, shearing is appropriately applied to the fermentation broth that has been removed from the fermenter. The method according to the invention may include an additional step between step (a) and step (b). However, preferably, step (b) is performed on the fermentation broth produced and / or obtained by step (a).

[0074] Shear stress can advantageously be applied by mechanically treating the fermentation broth. This mechanical treatment may, for example, involve subjecting the fermentation broth to a volumetric power input of 1-500 kW / m³, more preferably 1-200 kW / m³, and even more preferably 1-100 kW / m³, preferably for a duration of 0.1-60 min, more preferably 1-30 min, and even more preferably 1-10 min. The mechanical treatment may preferably include treatment with a mixer and / or with a homogenizer and / or with a grinder and / or with a centrifuge (including, for example, a continuous centrifuge with or without differential gravity) or combinations thereof. More preferably, step (b) includes applying and / or adding mechanical shear stress to the fermentation broth or combinations thereof, preferably by one of the exemplary mechanical treatments described above.

[0075] In mechanics, shear force is a common phenomenon. Shear force is understood to exist when there is a first force acting on (partially) the composition along a first direction and a second force acting on (partially) the composition, with the second force either stationary or moving in a second misaligned direction.

[0076] Shear stress (usually represented by the Greek symbol "tau" or "tau") τ” The stress component is coplanar with the cross-section of the material. It is generated by shear force, and more specifically, by the component of the force vector parallel to the cross-section of the material. The mean shear stress is the force applied per unit area and can be calculated using the following formula (I): τ = F / A (I) in: τ =Mean shear stress; F = The force applied; A= The cross-sectional area of ​​the material having a region parallel to the applied force vector.

[0077] Shear rate is the rate at which progressive shear deformation is applied. In simpler cases, this can be the velocity gradient in the flowing material. Shear rate is expressed in reciprocals of a second, i.e., in seconds (s). 1 "express.

[0078] In their article "The determination of viable counts in probiotic cultures microencapsulated by spray-coating," published in Food Microbiology, volume 24, 2010, pages 1104–1111, Champagne et al. described the application of high-shear homogenization (HSH) as part of an analytical method for determining viable cell counts (CFU) in freeze-dried and dried microencapsulated (ME) probiotic cultures. Microencapsulation was performed by spraying dried Lactobacillus rhamnosus R0011 or Bifidobacterium longum ATCC 15708 cultures with lipids. They concluded that HSH reduced the variability of CFU results for both free cells and ME. However, in their subsequent article, “Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices,” published in *Int. J. Food Microbioly*, volume 149, 2011, pages 185–193, Champagne et al. warned that excessive homogenization could actually kill cells. This is consistent with other publications in which high-pressure or high-shear homogenizers are actually touted as destroying cells.

[0079] In view of the above, the beneficial effects discovered by the inventors regarding the currently claimed method are surprising.

[0080] In the method according to the invention, the fermentation broth is sheared after it has been removed from the fermenter, and preferably before it has been optionally frozen, freeze-dried or encapsulated.

[0081] Preferably, the applied shear rate is within the range of 0.5 s⁻¹. -1 Preferably equal to or greater than 1.0 s -1 More preferably equal to or greater than 10 s -1More preferably equal to or greater than 100 s -1 More preferably equal to or greater than 500 s -1 , equal to or less than 500,000 s -1 Preferably equal to or less than 100,000 s -1 More preferably equal to or less than 50,000 s -1 More preferably equal to or less than 10000 s -1 More preferably equal to or less than 5000 s -1 .

[0082] Preferably, the applied shear stress is within the range of 1.10. -9 Pascal, preferably equal to or greater than 1.10 -8 Pascal, more preferably equal to or greater than 1.10 -7 Pascal, more preferably equal to or greater than 1.10 -6 Pascal, or more preferably equal to or greater than 1.10 -5 Pascal, equal to or less than 1.10 -1 Pascal, more preferably equal to or less than 1.10 -2 Pascal, or more preferably equal to or less than 1.10 -3 Pascal, or more preferably equal to or less than 1.10 -4 Pascal.

[0083] Therefore, shearing can be applied to the fermentation broth in every suitable manner known to those skilled in the art. Preferably, shearing is applied to the fermentation broth by homogenizing and / or centrifuging. More preferably, shearing is applied by subjecting the fermentation broth to a centrifugation step. That is, preferably, step (b) comprises or consists of centrifuging the fermentation broth. Centrifugation advantageously allows for the simultaneous concentration of the fermentation broth. It may be advantageous to apply a further concentration step after centrifugation, for example, the fermentation broth may be filtered after centrifugation. However, advantageously, such filtration is not necessary, and step (b) can be performed without filtration.

[0084] As described above, in a preferred embodiment, step (b) may or may not include homogenization of the fermentation broth or may consist of homogenization therein. Preferably, step (b) includes or consists of homogenizing the fermentation broth. If homogenization is applied, it can be applied to the fermentation broth before or after any optional concentration. Preferably, any homogenization is applied before any optional (subsequent) concentration. When step (b) includes homogenizing or comprising (preferably, unconcentrated) fermentation broth, the homogenization is preferably carried out at a rate ranging from equal to or greater than 1000 rpm, more preferably from equal to or greater than 3000 rpm, even more preferably from equal to or greater than 5000 rpm, even more preferably from equal to or greater than 8000 rpm, even more preferably from equal to or greater than 10000 rpm, even more preferably from equal to or greater than 11000 rpm, most preferably from equal to or greater than 12000 rpm to equal to or less than 50000 rpm, more preferably from equal to or less than 30000 rpm, even more preferably from equal to or less than 23000 rpm, even more preferably from equal to or less than 18000 rpm, and most preferably from equal to or less than 16000 rpm. When step (b) includes homogenizing or comprising (preferably, unconcentrated) fermentation broth, the homogenization is preferably carried out for a period of time (also referred to as duration) within the range of 0.5 minutes or more preferably 1 minute or more preferably 2 minutes or more preferably 3 minutes or more preferably 4 minutes or more preferably 30 minutes or less preferably 20 minutes or more preferably 10 minutes or more preferably 7 minutes or less preferably 5 minutes or less.

[0085] In a preferred embodiment, step (b) includes homogenizing or comprising the unconcentrated fermentation broth, wherein the homogenization is performed for a period of time (also referred to as duration) at a rate greater than 10,000 rpm, more preferably equal to or greater than 11,000 rpm, most preferably equal to or greater than 12,000 rpm to equal to or less than 50,000 rpm, more preferably equal to or less than 30,000 rpm, the duration being in the range of equal to or greater than 1 minute, more preferably equal to or greater than 2 minutes and even more preferably equal to or greater than 4 minutes, even more preferably equal to or greater than 4 minutes to equal to or less than 30 minutes, more preferably equal to or less than 20 minutes, even more preferably equal to or less than 10 minutes, even more preferably equal to or less than 7 minutes and most preferably equal to or less than 5 minutes.

[0086] In another preferred embodiment, step (b) may or may not include centrifugation of the fermentation broth or consist thereof. When step (b) includes centrifugation of the fermentation broth or consists thereof, the centrifugation is preferably performed at a centrifugation rate in the range of 500 rpm or greater, more preferably 800 rpm or greater, even more preferably 1000 rpm or greater, even more preferably 1500 rpm or greater, most preferably from 2000 rpm or greater to 30000 rpm or less, more preferably 20000 rpm or less, even more preferably 16000 rpm or less, even more preferably 10000 rpm or less, and most preferably 8000 rpm or less. When step (b) includes centrifuging the fermentation broth or any component thereof, the centrifugation is preferably carried out for a period of time (also referred to as duration) within the range of 0.5 minutes or more preferably 1 minute or more preferably 2 minutes or more preferably 3 minutes or more preferably 30 minutes or less preferably 20 minutes or more preferably 10 minutes or less preferably 7 minutes or less preferably 5 minutes or less.

[0087] Preferably, step (b) further includes concentrating the fermentation broth. Therefore, concentration can be carried out by any suitable means known to those skilled in the art. Preferably, concentration is carried out by filtration and / or centrifugation. That is, preferably, step (b) includes filtration and / or centrifugation. When concentrating, the fermentation broth is preferably concentrated at a concentration factor equal to or greater than 2, more preferably equal to or greater than 5, even more preferably equal to or greater than 7, even more preferably equal to or greater than 8, still more preferably equal to or greater than 10, and most preferably equal to or greater than 12, or until the concentration factor reaches a factor equal to or greater than 2, more preferably equal to or greater than 5, even more preferably equal to or greater than 7, even more preferably equal to or greater than 8, still more preferably equal to or greater than 10, and most preferably equal to or greater than 12. The fermentation broth can be suitably concentrated at a factor equal to or less than 1000, more preferably equal to or less than 200, and even more preferably equal to or less than 100. Most preferably, step (b) includes concentrating the fermentation broth, most preferably by centrifugation, and the fermentation broth is concentrated until a concentration factor equal to or greater than 10, more preferably equal to or greater than 12 is reached.

[0088] In another preferred embodiment, step (b) may include homogenizing the fermentation broth, optionally filtering the fermentation broth, and subsequently centrifuging the (appropriately homogenized) fermentation broth. The preferred methods of homogenization and centrifugation are as described above. When step (b) includes or consists of homogenizing and centrifuging the fermentation broth, centrifugation preferably allows for further concentration of the fermentation broth as described above. Most preferably, such further concentration is carried out until a concentration factor equal to or greater than 8, more preferably equal to or greater than 10, and even more preferably equal to or greater than 12 is reached.

[0089] Step (b) may appropriately produce a sheared and / or concentrated fermentation broth, more preferably a homogenized and / or filtered and / or centrifuged fermentation broth.

[0090] Preferably, the *Lactobacillus rhamnosus* cells produced, obtained, or available through step (b) are *Lactobacillus rhamnosus* cells, wherein bacterial particles with a particle size (diameter) 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, even 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 (diameter) equal to or less than 8.0 micrometers (μm) as determined by laser diffraction particle size analysis, preferably equal to or less than 7.5 micrometers, more preferably equal to or less than 7.0 micrometers, even more preferably equal to or less than 6.5 micrometers, and most preferably equal to or less than 6.0 micrometers, preferably equal to or less than 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 micrometers, and the cells preferably have a particle size equal to or greater than 2.00 × 10⁻¹⁰. 10 CFU / g cell viability, more preferably equal to or greater than 2.10·10 10 CFU / g cell viability, or more preferably equal to or greater than 1.50 × 10⁻⁶. 11 The cell viability is CFU / g, and most preferably equal to or greater than 2.00 × 10⁻⁶. 11 CFU / gram of cell viability, for example, by applying a concentration step.

[0091] In view of the above, step (b) preferably produces a composition containing *Lactobacillus rhamnosus* cells, wherein the bacterial particles have a particle size (diameter) of 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, even 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, as determined by laser diffraction particle size analysis. This particle size distribution is preferably equal to or less than 8.0 μm, preferably equal to or less than 7.5 μm, more preferably equal to or less than 7.0 μm, even more preferably equal to or less than 6.5 μm, and most preferably equal to or less than 6.0 μm, preferably equal to or less than 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 μm, and the composition preferably contains equal to or greater than 2.00 μm. 10 CFU / g cell viability, more preferably equal to or greater than 2.10·10 10 CFU / g cell viability, or more preferably equal to or greater than 1.50 × 10⁻⁶. 11 The cell viability is CFU / g, and most preferably equal to or greater than 2.00 × 10⁻⁶. 11 CFU / gram of cell viability, for example, by applying a concentration step.

[0092] Other process steps As noted above, step (b) may suitably produce a sheared and / or concentrated fermentation broth, more preferably a homogenized and / or filtered and / or centrifuged fermentation broth.

[0093] In addition to steps (a) and (b), the method preferably includes one or more additional method steps. More preferably, the method includes freezing and / or drying steps.

[0094] Therefore, the present invention also provides a method for producing Lactobacillus rhamnosus, Lactobacillus rhamnosus cells, or compositions comprising Lactobacillus rhamnosus or Lactobacillus rhamnosus cells, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shearing to the fermentation broth and concentrate the fermentation broth; and (c) Freeze and / or dry the concentrated fermentation broth.

[0095] Methods for freezing and / or drying optionally concentrated fermentation broth are known in the art. Suitable drying methods include, for example, vacuum drying, infrared convection drying, microwave drying, freeze drying, and / or spray drying. Combinations of these may also be used.

[0096] Preferably, step (c) comprises spray drying or freeze-drying the concentrated fermentation broth. If step (c) includes freeze-drying, then step (c) preferably includes freezing and subsequently freeze-drying the sheared and concentrated fermentation broth caused or produced or obtained by step (b). Preferably, step (b) results in or produces homogenized and / or filtered and / or centrifuged fermentation broth. Therefore, preferably, step (c) comprises spray drying; freezing; or freezing and freeze-drying the homogenized and / or filtered and / or centrifuged fermentation broth caused or produced or obtained by step (b).

[0097] During the above method, preferably after step (a) but before step (c), one or more additives, preferably cryoprotectants and / or stabilizers, may be added. More preferably, such cryoprotectants and / or stabilizers are added to the sheared and concentrated fermentation broth, preferably to the homogenized and / or filtered and / or centrifuged fermentation broth. This sheared and concentrated fermentation broth (preferably homogenized and / or filtered and / or centrifuged fermentation broth) is conveniently caused, generated, or obtained by step (b). Therefore, it is preferable that any cryoprotectant and / or stabilizer is added after step (b) and before step (c). However, if desired, any cryoprotectant and / or stabilizer may also be added after step (a) and before step (b).

[0098] Preferred cryoprotectants or stabilizers include, but are not limited to, glucose, lactose, raffinose, sucrose, trehalose, edetol, starch maltodextrin, glycerol, mannitol sorbitol, polypropylene glycol, polyethylene glycol, ribitol alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, monosodium glutamate, glycine, betaine, glycogen, hypotaurine, skim milk peptone, polyvinylpyrrolidone, taurine, nucleosides, nucleotides, and any combination thereof.

[0099] Composition The present invention further provides the following compositions: Lactobacillus rhamnosus bacterial particles or compositions containing Lactobacillus rhamnosus bacterial particles are preferably obtained or can be obtained by the methods described above.

[0100] Lactobacillus rhamnosus bacterial particles, wherein preferably, the bacterial particles have a particle size 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, even 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, as appropriately determined by laser diffraction particle size analysis, equal to or less than 8.0 μm, preferably equal to or less than 7.5 μm, more preferably equal to or less than 7.0 μm, even more preferably equal to or less than 6.5 μm, and most preferably equal to or less than 6.0 μm, preferably equal to or less than 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 μm, and the bacterial particles preferably have a particle size equal to or greater than 2.00 × 10⁻¹⁰ μm. 10 CFU / g, more preferably equal to or greater than 2.10·10 10 CFU / g, or more preferably equal to or greater than 1.50·10 11 CFU / gram and the optimal value is equal to or greater than 2.00·10 11 CFU / gram of cell viability, or live cell count.

[0101] A composition comprising *Lactobacillus rhamnosus* bacterial particles, wherein preferably, the bacterial particles have a particle size of 90% w / w or greater, preferably 95% w / w or greater, more preferably 99% w / w or greater, even more preferably 99.5% w / w or greater, still more preferably 99.9% w / w or greater, and most preferably 100.0% w / w or greater, as appropriately determined by laser diffraction particle size analysis, equal to or less than 8.0 μm, preferably equal to or less than 7.5 μm, more preferably equal to or less than 7.0 μm, even more preferably equal to or less than 6.5 μm or most preferably equal to or less than 6.0 μm, preferably equal to or less than 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 μm, and the composition preferably contains 2.00 μm or greater. 10 CFU / g, more preferably equal to or greater than 2.10·10 10 CFU / g, or more preferably equal to or greater than 1.50·10 11 CFU / gram and the optimal value is equal to or greater than 2.00·10 11 CFU / gram of cell viability, or live cell count.

[0102] In a particularly preferred embodiment, the *Lactobacillus rhamnosus* bacterial particles have a particle size distribution appropriately determined by laser diffraction particle size analysis, wherein the particle size distribution is bimodal. That is, preferably, such a particle size distribution has two peaks: a first peak (“peak 1”) in particles with a particle size equal to or less than 2.0 micrometers and a second peak (“peak 2”) in particles with a particle size equal to or greater than 2.0 micrometers. Specifically, the *Lactobacillus rhamnosus* bacterial particles preferably have a bimodal particle size distribution appropriately determined by laser diffraction particle size analysis, wherein the first peak (“peak 1”) is located before 2.0 micrometers and the second peak (“peak 2”) is located after 2.0 micrometers. More preferably, the ratio of the peak area of ​​peak 1 to the peak area of ​​peak 2 is 3:1 to 5:1, more preferably about 4:1. That is, the Lactobacillus rhamnosus bacterial particles preferably have a bimodal particle size distribution appropriately determined by laser diffraction particle size analysis, wherein 75% or more, preferably 76% or more, more preferably 77% or more, even more preferably 78% w / w, even more preferably 79% or more, even more preferably 80% or more, even more preferably 81% or more, even more preferably 82% or more, and most preferably 83% of the particles have a particle size of 2.0 micrometers or less. Preferably, the remaining Lactobacillus rhamnosus bacterial particles with a bimodal particle size distribution (i.e., particles with a particle size greater than 2.0 micrometers with a bimodal particle size distribution) have a particle size distribution in which 90% or more, preferably 95% or more, more preferably 99% or more, even more preferably 99.5% or more preferably 99.9% or more preferably 100.0% have a particle size equal to or less than 8.0 micrometers, preferably 7.5 micrometers, more preferably 7.0 micrometers, more preferably 6.5 micrometers, and most preferably 6.0 micrometers or less, as appropriately determined by laser diffraction particle size analysis.

[0103] Preferably, the above composition further comprises one or more additives, preferably cryoprotectants and / or stabilizers. Preferred cryoprotectants or stabilizers include, but are not limited to, glucose, lactose, raffinose, sucrose, trehalose, edetol, starch maltodextrin, glycerol, mannitol sorbitol, polypropylene glycol, polyethylene glycol, ribitol alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulfoxide, monosodium glutamate, glycine, betaine, glycogen, hypotaurine, skim milk peptone, polyvinylpyrrolidone, taurine, nucleosides, nucleotides, and any combination thereof.

[0104] Preferably, the composition is lactose-free; more preferably, the composition is milk-free. Preferably, the composition may further contain acetate or acetate.

[0105] use This invention further provides the following uses and applications: A composition for animal and / or human consumption, preferably a pharmaceutical, food, or beverage product, comprising: - As described above, Lactobacillus rhamnosus bacterial particles; or - As described above, a composition containing Lactobacillus rhamnosus bacterial particles.

[0106] Use of Lactobacillus rhamnosus bacterial particles as described above, or of compositions containing Lactobacillus rhamnosus bacterial particles as described above, in the production of food or beverage products.

[0107] Lactobacillus rhamnosus bacterial particles as described above, or compositions containing Lactobacillus rhamnosus bacterial particles as described above, are used in probiotic compositions and / or for medical purposes and / or in pharmaceuticals or as pharmaceuticals, preferably as pharmaceuticals for the treatment or prevention of diseases or conditions in or related to the gastrointestinal tract of animals or humans.

[0108] In the context of this invention, the term "food product" is intended to cover any consumable substance. Therefore, it can be a product intended for human consumption, but the term also includes products intended for animal consumption.

[0109] In a preferred embodiment, the food or beverage product is a dairy product, preferably yogurt, cheese, butter, buttermilk, quark, sour cream, kefir, twarog, fermented whey-based beverage, koumiss, milk beverage, yogurt beverage, fermented milk, mature cream, fresh fromage frais, milk, dairy residue, processed cheese, farm cheese, cream dessert, or infant formula.

[0110] In another preferred embodiment, compositions obtained or available by the methods described herein, or compositions as described herein, can be added to other components to produce compositions that can be used as probiotics and / or prebiotics. These can be used as direct-feeding microorganisms in food or feed for humans or animals. Probiotics and / or prebiotics can be used as food or feed additives for humans or animals.

[0111] In another aspect, the present invention provides probiotics comprising or composed of: Lactobacillus rhamnosus bacterial particles as described herein, or compositions comprising or composed of Lactobacillus rhamnosus bacterial particles, and preferably acetate or acetate.

[0112] In the context of this invention, the term "probiotic" is intended to refer to any microorganism that is desired to be consumed because it may have any beneficial effects on its consumer.

[0113] In a preferred embodiment, the composition obtained or obtainable by the methods described herein or the composition described herein is a pharmaceutical composition, in which the composition optionally contains a pharmaceutically acceptable excipient.

[0114] In another aspect, compositions or probiotics obtained or available by means of the methods described herein, or as described herein, are used as medicines, preferably for treating gastrointestinal disorders and / or improving gut health, such as improving symptoms of irritable bowel syndrome and / or improving digestive health and / or enhancing the immune system.

[0115] In another aspect, the present invention provides the use of a composition obtained or obtainable by the methods described herein, or the composition described herein, in a food or beverage product.

[0116] The present invention is illustrated below by way of non-limiting examples.

[0117] Example As shown in the examples below, the application of acetate to the culture medium during fermentation and the application of shear to the fermentation broth after fermentation resulted in a beneficial synergistic effect, leading to small particle size and high CFU / g count, which in turn resulted in high bioavailability of fimbriae.

[0118] Materials and methods Method for determining the number of CFU (colony forming units) per gram.

[0119] The pour plate technique was used to calculate the CFU / gram count of Lactobacillus rhamnosus GG.

[0120] In the first step, weigh 1 gram of fermentation broth and mix it with 9 grams of sterile PSW (Peptone Salt Water, PSW, obtained from Bio Trading). After generating the initial suspension, pipette 1 mL + / - 0.01 mL of the initial suspension into a tube containing 9 mL of sterile PSW solution using a sterile disposable 2 mL serological pipette to obtain a decimal dilution range. Use a new pipette for each dilution step. Ensure there are no air bubbles and no droplets falling or sticking to the side of the pipette. Then, mix everything by vortexing until a homogeneous suspension is obtained. Repeat the above steps until the desired dilution is achieved.

[0121] For the pour plate technique, transfer 1 mL (+ / - 0.01 mL) of the desired dilution to a ridged 90 mm sterile Petri dish. Then, pour 10–20 mL of melted and tempered agar (MRS with glucose) (46 °C + / - 1 °C) into the dish. Rotate the dish to evenly distribute the inoculated material throughout the agar. Melt the agar (this can be done, for example, by using a microwave oven, a 99 °C water bath, or an autoclave) and temper the agar to 46 + / - 1 °C for at least 30 minutes before use. Allow the agar to solidify and incubate the dish upside down at 37 °C under appropriate conditions for 2–3 days. Protect the plates from dehydration during incubation. Obtain anaerobic conditions by incubating the Petri dishes in an anaerobic flask using anaerobic pre-packets (Oxoid) according to the manufacturer's instructions. Validate by adding a CO2 indicator strip.

[0122] After incubation, count the cells on plates containing 25 to 250 colony-forming units (CFU). Viable cell counts are expressed as CFU / ml or CFU / g accordingly. Only CFU counts from plates are used below.

[0123] Methods for determining bacterial particle size distribution According to the provided manual, the bacterial particle size distribution of the fermentation samples was analyzed using a laser diffraction particle size analyzer (Beckman Coulter LS 13320). If needed, the Fraunhofer optical model could be used to determine the bacterial particle size (μm). For all samples, the bacterial particle size in the range of 0.4–20 µm was recorded, and the results were expressed as volume (%), with all particle sizes summed to 100%. In each case, 0.65 mL of fermentation broth was added to the sample port, followed by sample analysis. The container contained 0.85% NaCl. Graphs were generated, where cell size is represented on the x-axis, ranging from 0.4 to 20 µm, and volume (%) is represented on the y-axis for all sample fractions.

[0124] Example 1 Lactobacillus rhamnosus GG strain (derived from the ATCC 53103 collection described above) was added to several (separate) fermenters. In each fermenter, Lactobacillus rhamnosus GG strain was fermented in an aqueous fermentation medium containing the components listed in Table 1 under the conditions listed in Table 1. During fermentation, glucose as a (carbohydrate) feed was used, and Lactobacillus rhamnosus GG was allowed to ferment the glucose. When the glucose was depleted, samples were taken. At the time of sampling, the samples were divided into two portions: (i) Freeze the first fraction of each sample in liquid nitrogen; (ii) Apply shear to the second fraction of each sample and then freeze it in liquid nitrogen. Apply shear by mixing the sample fractions at 13,000 rpm for 4 minutes in an Ultra-Turrax mixer.

[0125] For each fraction of each sample, the CFU / g count was determined as described under “Materials and Methods”. The results are provided in Table 2. As shown in Table 2, application of acetate alone resulted in a count of 0.33 × 10⁻⁶ CFU / g. 10 The improvement in CFU / gr (ΔCFU / g) and the effect of applying shear alone resulted in 0.48·10 10 The improvement (ΔCFU / g) was observed. An increased synergistic effect was obtained when acetate and shear were applied together, with an improvement (ΔCFU / g) of 0.97·10⁻⁶. 10 CFU / gram.

[0126] In addition, bacterial particle size distribution was determined for each fraction of each sample, as described under "Materials and Methods". Results are provided in Figure 1 In. Figure 1 The image shows the bacterial particle size distribution for fractions without shearing (light gray lines) and fractions with shearing applied (dark gray).

[0127] from Figure 1 As can be seen in the reflection on fermenter B1, there is a significant change in bacterial particle size distribution (light gray line) without the application of acetate and shear force. However, after the application of acetate and UltraTurrax treatment, the bacterial particle size distribution has decreased. After the application of acetate and shear, the total bacterial particle size is much smaller, which allows for a higher amount of bacterial particles per gram.

[0128] Table 1: Fermentation medium components and fermentation medium conditions.

[0129] *If calcium is present in the fermentation medium, add it at the start of fermentation via an aqueous solution at a concentration of approximately 55-56 mg calcium / kg. Add the same solution for each fermenter.

[0130] Table 2: Results of CFU / gram count Predictive Example 2 The following is a predictive example. *Lactobacillus rhamnosus* GG strain (derived from the aforementioned deposit ATCC53103) was added to several (separate) fermenters. In each fermenter, *Lactobacillus rhamnosus* GG was fermented in an aqueous fermentation medium containing glucose as the carbohydrate feed, and fermentation of this glucose was permitted. The effect of acetate type in combination with the titrant sodium hydroxide (NaOH) or potassium hydroxide (KOH) was investigated in pH-controlled fermentation of LbrGG at pH 5.6 and 39°C. The effects of both ammonium acetate (NH4-acetate) and sodium acetate (Na-acetate) were evaluated. pH was controlled by the titrant, and fermentation was stopped upon carbon depletion. Subsequently, the pH in the fermenter was increased to 6.2, and the temperature in the fermenter was decreased to 7°C. Upon reaching this temperature, samples were collected and shear force was applied, followed by freezing in liquid nitrogen. Shear force was applied by mixing the samples at 13,000 rpm for 4 minutes in an Ultra-Turrax mixer.

[0131] Cell counts of viable, damaged, and dead cells in samples were determined by flow cytometry (FCM). It was found that viable cell counts were consistently lower without an acetate source compared to samples with an acetate source. Compared to samples without an acetate source, viable cell counts increased by approximately 35% when ammonium acetate was used as the titrant with NaOH, and by approximately 15% when sodium acetate was used as the acetate source. Similar effects were observed with KOH as the titrant, and here ammonium acetate produced a higher number of viable cells than sodium acetate.

Claims

1. A method for producing Lactobacillus rhamnosus, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free culture medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shear to the fermentation broth and optionally concentrate the fermentation broth.

2. A method for producing a composition comprising Lactobacillus rhamnosus, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free culture medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shear to the fermentation broth and optionally concentrate the fermentation broth.

3. The method according to claim 1 or 2, wherein the acetate is ammonium acetate.

4. The method according to any one of the preceding claims, The fermentation is carried out in the presence of ammonium acetate, and The fermentation is carried out at a pH point, or the fermentation is carried out toward 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. The method according to any one 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 a dissociated or non-dissociated ammonium acetate salt.

6. The method according to any one of the preceding claims, wherein the fermentation medium has a volume equal to or greater than 50 liters.

7. The method according to any one of the preceding claims, wherein step (b) comprises homogenizing the unconcentrated fermentation broth, wherein the homogenization is performed at a speed in the range of greater than 10,000 rpm to equal or less than 50,000 rpm for a duration in the range of equal or greater than 1 minute to less than 5 minutes.

8. The method according to any one of the preceding claims, wherein the applied shear rate is in the range of 0.5 s⁻¹ or greater. -1 Preferably equal to or greater than 1.0 s -1 More preferably equal to or greater than 10 s -1 More preferably equal to or greater than 100 s -1 More preferably equal to or greater than 500 s -1 , equal to or less than 500,000 s -1 Preferably equal to or less than 100,000 s -1 More preferably equal to or less than 50,000 s -1 More preferably equal to or less than 10000 s -1 More preferably equal to or less than 5000 s -1 .

9. The method according to any one of the preceding claims, wherein the applied shear stress is within the range of 1.

10. -9 Pascal, preferably equal to or greater than 1.10 -8 Pascal, more preferably equal to or greater than 1.10 -7 Pascal, more preferably equal to or greater than 1.10 -6 Pascal, or more preferably equal to or greater than 1.10 -5 Pascal, equal to or less than 1.10 -1 Pascal, more preferably equal to or less than 1.10 -2 Pascal, or more preferably equal to or less than 1.10 -3 Pascal, or more preferably equal to or less than 1.10 -4 Pascal.

10. The method according to any one of the preceding claims, wherein the shearing is applied by subjecting the fermentation broth to homogenization and / or centrifugation steps.

11. The method according to any one of the preceding claims, wherein the method comprises the following steps: (a) Fermenting the Lactobacillus rhamnosus in a fermenter in a lactose-free, preferably milk-free culture medium, and removing the fermentation broth from the fermenter, wherein the fermentation is carried out in the presence of acetate or acetate; (b) Apply shearing to the fermentation broth and concentrate the fermentation broth; and (c) Freeze and / or dry the concentrated fermentation broth.

12. The method according to any one of the preceding claims, wherein the method comprises spray drying or freeze drying the concentrated fermentation broth.

13. The method according to any one 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. Lactobacillus rhamnosus bacterial particles or compositions comprising Lactobacillus rhamnosus bacterial particles, which are obtained or can be obtained by the method according to any one of the preceding claims.

15. Lactobacillus rhamnosus bacterial particles, wherein the bacterial particles having a particle size 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, even 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 equal to or less than 8.0 μm, preferably equal to or less than 7.5 μm, more preferably equal to or less than 7.0 μm, even more preferably equal to or less than 6.5 μm, and most preferably equal to or less than 6.0 μm, as determined by laser diffraction particle size analysis, and the bacterial particles preferably have a particle size equal to or greater than 2.00·10⁻⁶. 10 CFU / g, more preferably equal to or greater than 2.10·10 10 CFU / g, or more preferably equal to or greater than 1.50·10 11 CFU / gram and the optimal value is equal to or greater than 2.00·10 11 CFU / gram of cell viability, or live cell count.

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, even 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 equal to or less than 8.0 μm, preferably equal to or less than 7.5 μm, more preferably equal to or less than 7.0 μm, even more preferably equal to or less than 6.5 μm, and most preferably equal to or less than 6.0 μm, as determined by laser diffraction particle size analysis, and the composition preferably contains equal to or greater than 2.00·10⁻⁶ μm. 10 CFU / g, more preferably equal to or greater than 2.10·10 10 CFU / g, or more preferably equal to or greater than 1.50·10 11 CFU / gram and the optimal value is equal to or greater than 2.00·10 11 CFU / gram of cell viability, or live cell count.

17. A composition for animal and / or human consumption, preferably a pharmaceutical, food, or beverage product, comprising: - Lactobacillus rhamnosus bacterial granules according to claim 14 or 15; or - The composition comprising Lactobacillus rhamnosus bacterial particles according to claim 14 or claim 16.

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 17, in the production of food or beverage products.

19. The *Lactobacillus rhamnosus* bacterial granules according to claim 14 or 15, or the composition containing *Lactobacillus rhamnosus* bacterial granules 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 prevention of diseases or conditions in or related to the gastrointestinal tract of animals or humans.

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