Method for improving stability of cells by mechanical treatment

Abstract

The present invention relates to the field of prokaryotic products with improved stability, in particular fermentative bacteria such as lactic acid bacteria, a method for preparing products with improved stability and compositions which may be prepared by said method.

Description

[0001] METHOD FOR IMPROVING STABILITY OF CELLS BY MECHANICAL TREATMENT

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of bacteria, such as probiotic bacteria, and in particular fermentative bacteria such as lactic acid bacteria, a method for preparing bacterial compositions and compositions which may be prepared by said method.

[0004] TECHNICAL BACKGROUND

[0005] Bacteria, such as lactic acid bacteria, are known to have probiotic properties (i.e. they have a beneficial health effect on humans and animals when ingested). Probiotics are widely applied in dry form. Besides, there is an increasing demand for food cultures, e.g., for dairy application, which are able to maintain acidification activity during storage at ambient temperatures, at 20°C and higher. In most cases, it is imperative that the microorganisms remain viable after prolonged storage of products, in order for these to impart their beneficial effect. Since it is well known that bacteria can easily lose viability upon exposure to various stresses, it is a general practice in industrial production of bacterial cultures to use protectants. These protectants are supposed to protect cells during different steps of a production process and later on during shelf storage of dried bacteria. Bacteria that are to be frozen or dried, for example spray-dried, freeze-dried, vacuum-dried, are mixed as a cell suspension with protectants and then processed in a sequence of various technological steps. The role of the protectant is to protect the bacterial cell composition during freezing (so called cryo-protectants), drying or freeze- drying (so called lyo-protectants). However, certain damage of cells during these processes cannot be avoided (Coulibaly et al. (2018) ARRB 24 (4) : 1-15).

[0006] Bacterial products can also be formulated as frozen products. For example, commercial starter cultures may be distributed as frozen cultures. Highly concentrated frozen cultures, particularly when prepared as pellets, are commercially very useful since such cultures can be inoculated directly into the fermentation medium (e.g. milk or meat) without intermediate transfer. In other words, such highly concentrated frozen cultures comprise bacteria in an amount that makes in-house bulk starter cultures at the endusers superfluous. A "bulk starter" is defined herein as a starter culture propagated at the food processing plant for inoculation into the fermentation medium. Highly concentrated cultures may be referred to as direct vat set (DVS)-cultures. In order to comprise sufficient bacteria to be used as a DVS-culture at the end-users, a concentrated frozen culture generally has to have a weight of at least 50 g and a content of viable bacteria of at least 109colony forming units (CFU) per g. W02005 / 080548 (Chr. Hansen) discloses pellet-frozen lactic acid bacteria (LAB) cultures that are stabilised with, for example, a mixture of trehalose and sucrose and do not form clumps when stored.

[0007] In prior art processes, a concentrated bacterial culture is obtained by known methods of culturing the bacteria in a growth medium and then concentrating the culture, for example by centrifugation or microfiltration, with the bacteria being separated from the growth medium. The concentrated culture is then admixed with the desired preservative(s) and, shortly thereafter, the resulting mixture is frozen or dried.

[0008] However, there is still a need to develop ways to ensure improved stability of the bacteria, increased shelf life of products containing said bacteria, and viability of the bacteria.

[0009] SUMMARY

[0010] The present invention is derived from the unexpected observation that cells with a certain cell surface hydrophobicity show improved tolerance to long-term storage, if they are treated either mechanically to increase cell surface hydrophobicity. Existing manufacturing processes can easily be modified to accommodate the treatment.

[0011] According to a first aspect of the invention, a method of preparing a frozen or dried product comprising an asporogenous prokaryote, the method comprising the steps of: i. growing the prokaryote cells by fermentation; II. treating the cells by mechanical manipulation, to obtain a more stable cell product; ill. optionally adjusting pH in the fermentate to be in the range pH 4 to pH 8; iv. concentrate the cells by separation of fermentation broth, thereby obtaining a cell product; v. optionally combining cell concentrate with a medium containing a protective compound, to obtain a preprocessing composition; vi. optionally preserving the cell product by (a) freezing the composition to form a frozen prokaryote product; (b) drying the composition to form a dried prokaryote product; or (c) freezing the composition to form a frozen prokaryote intermediate product and then lyophilising the intermediate product to form a freeze- dried prokaryote product, (d) optionally storing the freeze-dried procaryote product at -20°C, +5°, +25°, +30°C and +37°C, wherein the hydrophobicity of the cell surface is at least at least 20%, 30% or 40%, 50%, 60%, 70%, 80%, 90% or 100%,, as measured by the MATH method at 22°C and expressed as [(Initial OD600 - Final OD600) / Initial OD600]*100 when measured with a <t> [VH / VB] at at least one point between 0.01 and 1.0 and the initial OD600 (nm) is 0.5 and wherein the potency of product is 1E+08 to 1E+ 13 CFU / g. In one embodiment the mechanical manipulation in step ii) of the cells is done prior to concentrating the cells in step iv. It may be advantageous to perform the mechanical manipulation of the cells prior to concentrating the cells because the separation of the cells from the fermentation broth is improved following the mechanical manipulation resulting in a more stabile product.

[0012] In one embodiment, the protective compound is one or more of: a monosaccharide such as glucose, fructose, galactose or mannose; a disaccharide such as sucrose, trehalose, maltose or lactose; a sugar alcohol such as inositol; a trisaccharide such as maltotriose or raffinose; an oligosaccharide such as a fructooligosaccharide or such as a maltodextrin with DE 3-20; a polysaccharide such as starch or inulin; a cryoprotectant and / or a lyoprotectant and / or a storage stabiliser, such as gum arabic, a maltodextrin, starch, pectin, cellulose, xylan, or a polyol such as glycerol, sucrose, trehalose or maltose, a protein such as gelatin, a peptide such as are supplied by yeast extract, an amino acid such as proline or a sugar alcohol such as sorbitol, mannitol or inositol, an antioxidant, such as sodium ascorbate, sodium citrate.

[0013] In one embodiment, the frozen prokaryote product or the frozen prokaryote intermediate product has a dry weight ratio of the final protectant to cell concentrate of between 10: 1 and 0.1 : 1, preferably between 3:1 and 0.5: 1 and most preferably between 2: 1 and 1 : 1.

[0014] In one embodiment, the mechanical manipulation step (ii) is performed through homogenization to produce a uniform and stable composition. This process reduces particles, droplets, or components into smaller, uniform sizes, ensuring consistent texture, appearance, and functionality. Homogenization may be carried out using a homogenizer, which operates with electric motors to drive the mechanical forces required, such as high-pressure pumps, rotors, or ultrasonic generators.

[0015] In another embodiment, the homogenization is conducted by applying pressure within a range of 60 bar at the inlet and 30 bar at the outlet to 1000 bar at the inlet and 500 bar at the outlet, including, for example, 500 bar at the inlet and 250 bar at the outlet, or 200 bar at the inlet and 100 bar at the outlet. Applying a pressure of 60 bar at the inlet and 30 bar at the outlet during homogenization is also described in here using the format inlet value-outlet value (bar), i.e. 60-30 bar, 100-50 bar or 200-100 bar.

[0016] In one embodiment, the prokaryote is a fermentative bacterium from the phylum Firmicutes, such as: a lactic acid bacterium (LAB), preferably of a genus selected from the group consisting of Streptococcus / such as Streptococcus thermophilus), Lactococcus (such as Lactococcus lactis), Oenococcus (such as Oenococcus oeni), Leuconostoc (such as species Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides), Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilacto- bacillus, Latilactobacillus and Lactiplantibacillus; Eubacterium (such as Eubacterium limosum, Eubacterium aggregans, Eubacterium barken, Eubacterium ientum), Roseburia (Roseburia intestinalis, Roseburia horn inis, Roseburia inulinivorans, Roseburia faecis and Roseburia cecicola), Faecalibacterium (such as species Faecalibacterium prausnitzii), Anaerostipes (such as Anaerostipes cacccae), Anaerobutyricum (such as species Anaerobutyricum hallii, Anaerobutyricum soehngenii) from the phylum Actinobacteria, such as genus Bifidobacterium (such as species Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve'), genus Propionibacterium (such as species Propionibacterium freudenreichii), Cutibacterium (such as Cutibacteriun acnes) from the phylum Bacteroidetes, such as genera Bacteroides (such as species Bacteroides fragilis, Bacteroides xylanisolvens), genus Prevotella (such as species Prevotella copri) or Alistipes, or from the phylum Verrucomicrobia, such as an Akkermansia (such as species Akkermansia muciniphila).

[0017] In one embodiment, the prokaryote is one or more of: Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp. sakei, Lactiplantibacillus pentosus, Levilactobacillus brevis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus and Lactobacillus acidophilus, Lactobacillus jensenii, and Lactobacillus iners.

[0018] In a preferred embodiment is Steptococcus thermophilus such as S. thermophilus strains nos. DSM 35049 or DSM 34676.

[0019] Many prokaryotes, especially those involved in fermentation (e.g., Lactobacillus spp, Bifidobacterium spp), produce EPS and / or CPS that are highly hydrophilic. These polysaccharides contain numerous hydroxyl (-OH) groups, which can form hydrogen bonds with water molecules, making the surface more hydrophilic.

[0020] The prokaryotes may produce capsular polysaccharides (CPS) or exopolysaccharides (EPS), which form gel-like layers around the bacterial cells, helping to retain water and promote enhanced cell-to-cell interactions within biofilms or during interactions with host tissues. In one embodiment, the prokaryotes or lactic acid bacteria are CPS-producing. CPS are high-molecular-weight carbohydrates consisting of complex sugar molecules that form a protective capsule around the bacterial cell. Typically composed of repeating sugar units like glucose, galactose, rhamnose, or mannose, these polysaccharides serve critical biological functions, including enhancing bacterial survival and facilitating interactions with the surrounding environment. They are secreted by bacteria, forming a hydrated gel-like layer known as the capsule, which provides protection and supports various microbial processes, such as adhesion and biofilm formation.

[0021] In another embodiment, the prokaryotes may produce exopolysaccharides (EPS), which are similar high-molecular-weight polysaccharides secreted into the surrounding environment. EPS are composed of repeating sugar units, which can vary depending on the bacterial strain, and may include sugars such as glucose, fructose, rhamnose, and glucuronic acid. EPS play important roles in microbial ecology, providing structural support in biofilms, protecting bacteria from environmental stresses, enhancing nutrient retention, and facilitating microbial interactions with their environment. Like CPS, EPS also form a gel-like matrix around bacterial cells, but unlike CPS, EPS are often secreted into the surrounding medium rather than being tightly bound to the bacterial surface, but may bind weakly to the surface by electrostatic interaction.

[0022] In one embodiment, the EPS producing lactic acid bacteria may be S. thermophilus strains nos. DSM 34679 or DSM 34290 (mutant of DSM 34676). Reference is made to W02024 / 008845 Althat DSM 34290 is mainly producing EPS.

[0023] In yet another embodiment, the procaryotes or lactic acid bacteria are both EPS and CPS producing.

[0024] In yet another embodiment, the EPS and CPS producing lactic acid bacteria may be S. thermophilus strains nos. DSM 35049 or DSM33982 (mutant of DSM 35049). Reference is made to W02023 / 094430 Al or W02024 / 008845 Al showing that DSM33982 is producing both CPS and EPS.

[0025] CPS and EPS are thus both polysaccharides, but their differences in location (attached to the cell vs. secreted into the environment) may lead to differences in their measurement. EPS is typically measured in the culture medium, often through simple carbohydrate quantification, viscosity measurements, or more advanced sugar composition analysis (HPLC, GC). CPS requires extraction from the bacterial surface, and its measurement often involves methods including capsule staining, immunoassays, or microscopy.

[0026] EPS produced by bacterial cultures may be measured phenol-sulfuric acid assay or chromatography including High-Performance Liquid Chromatography (HPLC). CPS-producing strain and / or EPS-producing strains may be identified by PCR by the amplification of specific gene clusters associated with polysaccharide biosynthesis. The gene cluster encoding for the enzymes responsible for the production of CPS and / or EPS is commonly referred to as the eps gene cluster. These genes are often specific to the organism and the type of capsule it produces.

[0027] In one embodiment the procaryote or lactic acid bacterium strain comprise an active eps gene cluster.

[0028] In yet another embodiment, the active eps gene cluster has a sequence identity of at least 95% or 99% with SEQ ID NO: 1 or SEQ ID NO:2.

[0029] For CPS-producing strains, primers designed to target genes within the capsular polysaccharide synthesis may be used. These genes may include, but are not limited to, those encoding capsular polymerases, glycosyltransferases, and capsule-specific transporters (e.g., cpsA, cpsB, cap, or species-specific orthologs). For EPS-producing strains, primers targeting genes associated with the synthesis and secretion of extracellular polysaccharides are employed, including but not limited to genes such as epsA, epsB or epsC, or species-specific orthologs.

[0030] These methods are all well-known to the skilled person in the field of microbiology, biochemistry, and biotechnology, especially those working with bacterial polysaccharides or in industrial fermentation processes.

[0031] According to a second aspect of the invention, a frozen or dried product comprising an asporogenous prokaryote, obtainable by a method according to the first aspect, is provided.

[0032] In one embodiment, the frozen or dried product has a potency of 1E+08 to 1E+13 CFU / g.

[0033] According to a third aspect, a composition comprising a frozen or dried product according to the second aspect is provided, wherein potency of the bacteria is 1E+05 to 1E+12 CFU / g.

[0034] In one embodiment, the product is a food, feed, agricultural product, dietary supplement or pharmaceutical product.

[0035] According to a fourth aspect, a method of manufacturing a food, feed, agricultural product, dietary supplement or pharmaceutical product is provided, said method comprising addition of a frozen or dried product according the third aspect.

[0036] BRIEF DESCRIPTION OF THE SEQUENCE LIST

[0037] SEQ ID NO: 1 sets out the complete sequence of the eps gene cluster of S. thermophilus strain DSM35049.

[0038] SEQ ID NO:2 sets out the complete sequence of the eps gene cluster of S. thermophilus strain DSM34676.

[0039] BRIEF DESCRIPTION OF THE FIGURES

[0040] Figure 1 are graphs (A and B) showing measurement of sedimentation velocities according to an embodiment. Measurement of sedimentation velocities for fermentate of DSM 35049 exposed for depolymerization and different intensity of homogenization.

[0041] Figure 2 is a graph showing zeta potential curves according to an embodiment. Zeta potential curves were measured for fermentate of DSM 35049 exposed for depolymeriza-tion and different intensity of homogenization (Untreated / unhomogenized Fermentate, 60 / 30 bar equals low intensity homogenization, 100 / 50 bar equals medium intensity homogenization and 200 / 100 bar equals high intensity homogenization)

[0042] Figure 3 is a graph showing hydrophobicity measurements according to an embodiment. Hydrophobicity measurement for fermentate of DSM 35049 exposed for depolymerization and different intensity of homogenization.

[0043] Figure 4 are a graph showing measurement of sedimentation velocities according to an embodiment.

[0044] Figure 5 is a graph showing zeta potential curves according to an embodiment.

[0045] Figure 6 is a graph showing hydrophobicity measurements according to an embodiment.

[0046] Figure 7 show graphs with stability data according to an embodiment where the DSM 35049 culture is subjected to intensity of homogenization (pH after 4 hours).

[0047] Figure 8 show graphs with stability data according to an embodiment where the DSM 35049 culture is subjected to intensity of homogenization (pH after 6 hours).

[0048] Figure 9 show graphs with stability data according to an embodiment, where the DSM 35049 culture is subjected to intensity of homogenization (Ta values).

[0049] Figure 10 are graphs showing measurement of sedimentation velocities according to an embodiment where the DSM 34676 culture were subjected to different intensity of homogenization and different pH (pH 5, 6 and 7, respectively). For cultures that were not homogenized or treated with a low intensity homogenization, the pH was 6.

[0050] Figure 11 are graphs (A and B) showing zeta potential curves according to an embodiment, where in (A) the DSM 34676 culture is subjected to high and low mechanical treatments at pH 6, where in (B) the DSM 34676 culture is subjected to high mechanical treatments at pH 5, 6 and 7.

[0051] Figure 12 are graphs showing hydrophobicity measurements according to an embodiment. The graphs are BCSH curves of mechanical treatments of the DSM 34676 culture including pH adjustments. Vertical lines indicate error bars from the triplicate measurements.

[0052] Figure 13 show graphs with stability data according to an embodiment (pH after 4 hours). Figure 14 show graphs with stability data according to an embodiment (pH after 6 hours).

[0053] Figure 15 show graphs with stability data according to an embodiment (Ta values).

[0054] DETAILED DESCRIPTION

[0055] Microbial cultures of lactic acid bacteria (LAB) are produced by fermentation processes with acidic pH. Typically, pH during these fermentation processes is in range 3,5 - 6,5. After fermentation, the downstream processing of the LAB begins and the first step is to harvested the biomass using e.g. a centrifugation step followed by addition of protective agents. The subsequently steps consist mainly of freezing the biomass in liquid nitrogen followed by freeze dried.

[0056] The cell wall of Gram-positive bacteria consists of a peptidoglycan layer with embedded teichoic, lipoteichoic acid and cell wall polysaccharides. The peptidoglycan layer can be covered by a proteinaceous S-layer and decorated by various polysaccharides (Zeidan et aL, 2017, FEMS Microbiology Reviews 41 : 168-200). The surface of Gram-negative bacteria is different. It is made of capsular polysaccharides which are decorated with various polymeric substances such as carbohydrates, lipo-oligosaccharides and lipopolysaccharides. This complex composition of cell surface can be captured by physicochemical analyses such as measurement of cell surface interactions by hydrophobicity analysis and cell surface charge determined by zeta potential. Particularly, the combination of these two assays with advanced microscopy techniques has contributed to a more profound characterization of the cell wall of lactic acid bacteria (Schar-Zammaretti and Ubbink (2003) Biophysical Journal 85, 4076-4092).

[0057] Several cultures of Streptococcus thermophilus (ST) and LAB produce polysaccharides that occur as cell wall constituents (peptidoglycan) that are released from the cell. The latter are either permanently attached to the surface of the microbial cell in form of capsules (capsular polysaccharide, CPS) or secreted into the environment as exopolysaccharides (EPS) (Chapot-Chartier et al. 2011). Depending on its nature free EPS can be bound loosely to the cell surface by e.g electrosatic forces. It is well known that capsular polysaccharide producing LAB can be difficult to handle during the downstream centrifugation process due to the fact that the capsular polysaccharide binds high amount of water creating a small density difference between the bacteria and the surrounding supernatant which is one of the driving forces to get an efficient separation. Besides being difficult to separate the CPS producing bacteria also result in less compact biomass (high PCV values) taking up extra Freeze-Dried (FD) capacity. One way to increase the compactness of the biomass and accommodate the small density differences is to add an extra unit operation downstream before centrifugation where the CPS layer is partly or completely removed from the surface of the bacteria. The treatment of the post fermentation broth can be either mechanical such as homogenization using high pressure.

[0058] The inventors of the present invention found that mechanical homogenization of the CPS producing Streptococcus (S.) thermophilus cultures were found to be an efficient way to increase the surface hydrophobicity of S. thermophilus cultures. The higher homogenization pressure used the more hydrophobic surface was obtained. Stability studies (CPH analysis) of the mechanical treated S. thermophilus culture also revealed a direct correlation between surface hydrophobicity and stability - i.e. the more hydrophobic surface the better stability.

[0059] DEFINITIONS

[0060] The term "packed cell volume" or "PCV" is determined by exposing fermentates and concentrates to the same degree of g-force for an equal amount of time it is possible to compare the compressibility of different fermentation cultures and their packability, assuming equal behaviour of the liquids and cells. The PCV can be used as an indicator for how well cells may be separated from supernatant. It is calculated by (pellet volume) / (total sample volume), and can be multiplied with 100 express in %.

[0061] The term "Zeta potential" or "<( potential" is a measure of the surface charge of particles in a solution. The surface of a charged particle will attract opposite charges. This forms a thin liquid layer called the Stern layer. On top of the Stern layer more ions are attracted and loosely attach themselves as well. The zeta potential itself is the electric potential in the interfacial double layer, compared to a point in the continuous phase. So the zeta potential is the difference in potential of the dispersion medium attached to the particle and the freely moving dispersion medium.

[0062] The term "hydrophobicity" is the attraction between non-polar or slightly polar molecules, particles, or cells when immersed in water. The hydrophobicity is highly driven by the hydrogen bonding free energy of cohesion of water molecules. If the forces on the particle surface are responsible for the orientation of the water molecules adsorbed to the surface of the particle then water molecules on the surface of the particle may repel other particles with the same orientation. This may be the case when the particle surface is highly monopolar or asymmetrical. Water molecules on the surface may also orient in a way that particles will approach one another due to their Lifschitz-van der Waals attraction. On bacteria, the hydrophobicity is mainly determined by the cell surface composition, which may contain compounds useful for adhesion.

[0063] The term "homogenization" means mechanical mixing of cell mass. Specifically, homogenization can be achieved by applying a high pressure a bacterial suspension is forced through a small gap. This process can lyse microbial cells at high pressures, typically in the range of 20-120 MPa (S.T. Harrison, "Cell Disruption," Comprehensive Biotechnology, Second Edition, vol. 2, pp. 619-640, Jan. 2011). The cells flow through the narrow opening, where the pressure is quickly released. Different factors affect cell disruption in high-pressure homogenisation, such as the rate and magnitude of the pressure release, solid surface impact, cavitation, turbulence, and shear stress. However, the pressure release and the impact of the cells are seen as the most important mechanisms in cell disruption. However, the microbes that are treated using high-pressure homogenisation may be affected differently conditions in the homogenizer. It has been shown that the degradation of polysaccharides is possible through the use of high-pressure homogenisation, thereby also reducing the viscosity of the liquid (A. Villay, F. Lakkis de Filippis, L. Picton, D. Le Cerf, C. Vial, and P. Michaud, "Comparison of polysaccharide degradations by dynamic high-pressure homogenization," Food Hydrocolloids, vol. 27, no. 2, pp. 278-286, Jun. 2012). It was shown that branched structures were nearly unaffected by homogenisation, whereas stiff linear polysaccharides underwent depolymerisation. Thus it could be possible to find homogenisation pressures where the polysaccharides may be degraded while the cells remain relatively unaffected.

[0064] The term "Encapsulation Index" (El) refers to the weight-to-weight (wt / wt) ratio of dry lyoprotectant to dry biomass, expressed in grams per gram..

[0065] The term "depolymerisation" means breaking of polymers into one or more monomers using chemical means. For example, Streptococcus thermophilus is a producer of exopolysaccharides, which are polymers that are possible to depolymerise. Many different reactive oxygen and nitrogen species exist that are able to depolymerise polysaccharides, such as; ozone, hypochlorite, hydroxyl radicals, peroxynitrite, nitrous acid and nitric oxide. Of these, the hydroxyl radical is the most reactive oxygen species. It is able to abstract hydrogen atoms at most sites on carbohydrates except the C-2 of N-acetyl hexosamine. This abstraction leads to a p-scission reaction which results in the breakdown of polysaccharide chains. However, if the polysaccharides are sulfated, they are more resistant to the hydroxyl radical attack. Although hydroxyl radicals are able to break down polymers, they are also capable of causing oxidative damage to many other chemical structures and can, in fermentation media, cause oxidative stress to the cells. In the case where bacteria are manufactured as a commercial product, it is important that product remains viable. Cells exposed to radicals may experience damage to their cell structure and membrane or important molecules such as lipids, proteins and DNA. Damage to the cell membrane might cause a change in cell viability or activity.

[0066] The preservation procedure used in the following examples are freeze drying and can be performed according to to classical procedures well known by the skilled person.

[0067] DEPOSIT AND EXPERT SOLUTION

[0068] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0069] Table A: The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0070] Table A:

[0071] EXAMPLES

[0072] Overview of Examples 1-3

[0073] A series of experiments were performed to evaluate the impact of homogenization on surface properties and long term stability of lactic acid bateria. Overview of each batches tested and analysis applied to characterize surface properties are summaried in Table B below. Table B. Overview of Examples 1-3

[0074] Materials and methods

[0075] Example 1: Streptococcus thermophilus (ST) production batch (TD-DVS 01)

[0076] Non homogenize post fermentation broth of Streptococcus thermophilus patent deposit no. DSM 33982, herein also denoted DSM 35049 was manufactured in 100m3scale at Chr. Hansen A / S. Non homogenized post fermentation broth was sampled at the end of the manufacturing. The sample was frozen and thawed before used. The production batches was subjected to three different levels of intensity of homogenization, i.e. low 60-30 bar (60 bar in and 30 bar out), medium 100-50 bar and high 200-100 bar using a small lab homogensator. Besides homogenization a sample was subjected to depolymerization. The production batch was primary use to evaluate the effect of mechanical and chemical treatment on the surface structure of the ST bacteria.

[0077] Example 2: Streptococcus thermophilus (ST) production batch (TD-DVS 02)

[0078] Streptococcus thermophilus strain with patent deposit no. DSM 33982 (herein denoted as DSM 35049) was manufactured in 450 L pilot scale at Chr. Hansen A / S.

[0079] Part of the fresh post fermentation material was homogenized on a pilot plant homogenisator using a pressure of 75-50 bar. The remaining non-homogenized material was divided in 4 fractions which subsequently were treated with different intensities i.e. non-homogenized, low 60-30 bar, Medium 100-500 bar and High 200-100 bar using a small lab homogensator. All samples - total 5 - were concentrated on lab centrifuge to the same concentration factor and formulated to same encapsulation index (ratio between dry lyoprotectant (g) to dry biomass (g )) followed by freezing in liquid nitrogen and finally freeze dried using a safe FD profile. The freeze dried products were subsequently prepared for stability study that was followed up to one month at 37°C.

[0080] Example 3 -.Streptococcus thermophilus (ST) production batch (TD-DVS 03)

[0081] Streptococcus thermophilus strain with patent deposit no. DSM 34676 (herein denoted as DSM 34676) was manufactured in 450 L pilot scale at Chr. Hansen A / S.

[0082] The non-homogenized material of DSM 34676 (pH 6) was divided in 2 fractions which subsequently were treated with different intensities i.e. non-homogenized, low 60-30 bar and high 200-100 bar using a small lab homogensator. The high homogenized sample was divided in 3 fractions and adjusted to pH 5, pH 7 and the last sample was kept at the original pH at pH 6. All five types of samples were concentrated on a lab centrifuge to the same concentration factor and formulated to same encapsulation index followed by freezing in liquid nitrogen and finally freeze dried using a safe FD profile. The freeze dried products were subsequently prepared for stability study that was followed up to two months at 37°C.

[0083] Stability study for (TD-DVS 02 and FD-DVS 03)

[0084] After freeze drying all samples were placed in closed alu bags that subsequently were stored at 37°C. Samples were taken out time zero and up to 2 months for CPH (CINAC) analysis to evaluate their acidification activity. In order to get as much valid results the CPH analysis was performed using different 3 standard inoculation percentages i.e. 0,001 %, 0,003 % and 0,006 % respectively for FD DSM 35049 and 0,0006 %, 0,0012 % and 0,0024 %, respectively for FD DSM 34676. In order to compare the performance of the freeze dried cultures subjected to the different shearing conditions, the same amount of active cells (measured by flow cytometry) were dosed for each of the shearing conditions adjusting the inoculation percentage accordingly. Performance of FD DSM 35049 and FD DSM 34676 were evaluated by measuring Ta values and their ability to acidify (pH) after 4 and 6 hrs, respectively.

[0085] Analysis used to characterize surface properties

[0086] Bacterial Cell Surface Hydrophobicity (BCSH):

[0087] Bacterial cell surface hydrophobicity (BCSH) is performed by mixing an organic solvent (here hexadecane) with the bacterial suspension at different ratio Vhex / Vceii. The mixture is then rigorously vortexed for a set time and allowed to settle into the aqueous and organic phase. Hydrophilic bacteria will then settle in the aqueous phase, and hydrophobic bacteria will then settle in the organic phase. Any change in bacteria surface hydrophobicity due to either mechanical or chemical treatment would lead to changes in the BCSH curves. Bacterial cell surface characteristics, such as bacterial cell surface hydrophobicity and zeta potential, were determined for concentrated cells after fermentation.

[0088] Cell surface hydrophobicity was measured by the MATH method, and interfacial adhesion curves were determined. The method of Schar-Zammaretti & Ubbink ((2003) Biophysical Journal 85, 4076-4092)) was applied with modified buffer strength and the use of a cell wash in the initial step of the procedure: 1 g of cell concentrate or 0,2 g of freeze-dried granulate was resuspended in 10 ml of 100 mM sodium phosphate buffer (pH 7.0). The cell suspension was centrifuged at 5000 g for 10 minutes at a temperature < 10°C. Supernatant was removed and cells were washed twice with the 100 mM sodium phosphate buffer. The washed cell pellet was resuspended in the 100 mM sodium phosphate buffer to optical density OD600 nm of 0.5 ± 0.05. The suspension was mixed and aliquots of 3 ml were pipetted into plastic tubes. Hexadecane (99% purity, Sigma Aldrich) was added to the cell suspension in the following volumes : 10 pl, 30 pl, 100 pl, 200 pl, 400 pl, 800 pl, 1400 pl and 2000 pl hexadecane. Each combination of hexadecane and cell suspension in the buffer, <t> [VH / VB], was prepared in triplicate. The tubes were closed and the mixtures were vortexed one by one for 30 seconds at highest speed. Vortexing was repeated for 30 seconds once again for the whole sample series. The samples were left to rest for 5 minutes. 2 ml of aqueous phase was transferred to a cuvette for measurement of the OD600 nm. Bacterial cell surface hydrophobicity (BCSH) was calculated from the fraction of bacteria which adhered to the hexadecane / water interface according to the formula : BCSH (%) = [(Initial OD600 - Final OD600) / Initial QD600]*100 A cell surface is classified as non-hydrophobic, i.e. hydrophilic, if partitioning of cells gives BCSH < 20%. A hydrophobic cell surface is characterized by partitioning of cells with BCSH > 50%, and a moderately hydrophobic surface has a BCSH in the range 20-50% (Lee and Yii (1996) Letters in Applied Microbiology 23: 343-346).

[0089] Zeta potential:

[0090] Zeta potential curves show the surface charged of the bacteria as a function of pH. Any change in bacteria surface due to either mechanical or chemical treatment would lead to changes in the zeta potential curves- however it's not possible to conclude from our data what constituents at the surface that have been removed (i.e. CPS, S-layer proteins, peptide glycans or teichoic acid) - just that changes at the surface have happen. However, since the CPS or free EPS bound to the surface of the bacteria are located at the outer surface of the lactic acid bacteria it is also most likely the first to be sheared off. Zeta potential can be performed according to to classical procedures well known by the skilled person.

[0091] Zeta potential measurement:

[0092] 1 g of cell concentrate or 0,2 g of freeze-dried granulate was suspended in 1 mM NaCI and washed twice by centrifugation at 5000 g for 10 minutes at < 10 °C . The washed cell pellet was resuspended in 1 mM NaCI to OD600 nm = 0.5 ± 0.05. Aliquots of the cell suspension were pH adjusted with 20 mM HCI or 20 mM NaOH to create a serial of samples within the pH range 2,70 to 8,45. The Zeta potential of samples was measured in apparatus SZ-100Z (Horiba Scientific, France). Measurement was done in triplicate for each pH. The results are presented as average of triplicates with standard deviation. The pH at which the Zeta potential becomes zero is called the isoelectric point (pl).

[0093] Measurement of water activity:

[0094] Measurement of water activity (Aw) of the FD material prepared from fermentate subjected to the different chemical / mechanical treatment. CPS structure or free EPS bound to the surface of the bacteria can bind water but since it is removed the Aw will be reduced.

[0095] Measurement of the sedimentation velocity:

[0096] Measurement of the sedimentation velocity and velocity distribution for a bacteria using LUMISizer. The theoretical fundamentals for calculating sedimentation velocities and velocity distributions, are described by Stokes' law. The settling velocity of a spherical particle or bacteria in a gravitational field can be expressed as: where pPand pf are the particle and fluid density, d is the particle diameter, q is the dynamic viscosity of the fluid and g the gravitational acceleration.

[0097] CPS producing bacteria or bacteria producing free EPS bound to the surface of the bacteria can be difficult to separate on a centrifuge since the polysaccharides bind high amount of water creating a small density difference between the bacteria and the surrounding supernatant. Removing the polysaccharide layer will increase the density difference between the particle and the surrounding liquid and thus increases the sedimentation velocity of the bacteria when measure with the LUMISizer. Measurement of sedimentation velocity can be performed according to to classical procedures well known by the skilled person.

[0098] Packed bed volume:

[0099] Packed bed volume (PCV) measurements for cell dispersions are a standardized method of measuring the packing efficiency and compressibility of the cells in a given fermentate. 100 CPS producing bacteria or bacteria producing free EPS bound to the surface of the bacteria can be difficult to handle during the downstream centrifugation process due to the polysaccharide bind high amount of water creating a small density difference between the bacteria and the surrounding supernatant. One way to increase the compactness of the biomass and thus to recuce the PCV value is to remove the polysaccharides exposing the post fermentation broth to either mechanical / chemical treatment.

[0100] Flow cytometry :

[0101] Procedure used to measure total cells and number of active cells was flow cytrometry and can be performed according to to classical procedures well known by the skilled person. Samples were taken out for Flow cytometry analysis as described in WO 2006 / 125446.

[0102] Continuous pH measurement (CpH)

[0103] The acidification activity in the freeze-dried culture was measured according to the International standard ISO 26323:2009 (IDF 213: 2009) : "Milk products - Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH)".

[0104] Acidification activity is qualified by the following parameters:

[0105] Ta is the time it takes to start acidifying the standardized milk, i.e. the time in which the pH drops 0.08 pH units from the initial pH. The time Ta is measured in minutes from the time of inoculation, t=0. pH-6h: The pH that is reached after 6 hours at 30°C for this particular starter culture. pH-4h: The pH that is reached after 6 hours at 30°C for this particular starter culture.

[0106] The higher Ta and pH-4hr, pH-6h are, the longer the latency phase and, thus, the lower the acidification activity (Fernanda et al . 2004).

[0107] Acidification activity

[0108] In the present context, acidification activity refers to the rate at which lactic acid bacteria produce acids, mainly lactic acid, and thereby influences the pH of the immediate environment, such as for example of a fermentation medium, incubation medium, dairy product, food product and / or fermented product. Thus, the acidification activity is related to the metabolism and metabolic fitness of the lactic acid bacteria and can therefore also, when determined after a storage period and compared to a determined value prior to storage, be used as an indication of the metabolic activity, metabolic fitness and loss of acidification activity of the lactic acid bacteria post storage. However, there are many ways of determining a value of one or more parameters that is representative of the acidification activity of lactic acid bacteria and only a few of the most useful are exemplified in the present disclosure. Generally, the skilled person will understand which parameters to look for and whether a change in parameter value represents an improvement or a decline of acidification activity. In the present context, particularly useful ways of determining a value that is representative of the acidification activity include determining Ta or pH of the incubation medium. These parameters are described in detail below.

[0109] Determination of Ta - time to obtain a reduction in pH of 0.08

[0110] In the present disclosure, the ta is measured in terms of the number of minutes it takes to lower the pH of the incubation medium by 0.08. Thus, a higher number of minutes, i.e. a higher ta, represents a lower acidification activity, as it takes longer for the lactic acid bacteria to reduce pH by 0.08 points. Conversely, a lower number of minutes, i.e. a lower ta represents a higher acidification activity, as the lactic acid bacteria takes less time to reduce pH by 0.08 points, ta can for example be measured using an iCinac system (KPM; AMS Alliance), but generally any piece of equipment that is capable of accurately measuring pH in a liquid medium may be used. In the present context ta is measured in terms of minutes unless otherwise specified. pH of the incubation medium

[0111] Methods for measuring the pH of a solution are generally known to the skilled person and can be easily applied for measuring the pH in the incubation medium of the incubation step c). A non-limiting example of this is the use of a pH-meter to measure the pH of the incubation medium at various intervals. The iCinac system (KPM; AMS Alliance) can also be applied for this purpose. pH is determined by performing these standard assays of incubated dried lactic acid bacteria prior to and following storage. Like the acidification activity, these pH measurements correspond directly to the metabolic activity of the lactic acid bacteria, and can then be used to determine if the pH of the incubation medium is a lower pH, when compared to a reference value. This difference in pH (if any) is a direct measure of the difference in metabolic activity of the lactic acid bacteria prior to and following storage at ambient temperature.

[0112] PVC measurements:

[0113] PCV measurements for cell dispersions are a standardized method of measuring the lacking efficiency and compressibility of the cells in a given fermentate or concentrate sample. The measurement consists of exposing a sample to a set amount of centrifugal force for a set amount of time (e.g 5000 rpm and 10 min). Assuming standard sedimentation behavior - i.e., no shear thickening or thinning of the fermentate deviating from the behavior of the suspension liquid, different cultures or sample conditions can be compared, given comparable initial concentrations. Different cultures or sample conditions can be compared, given comparable initial concentrations.

[0114] PCV [%] = Pellet Volume / Total Sample Volume)*100

[0115] LUMiSizer measurements:

[0116] The primary technology utilized by the LUMiSizer® is known as STEP, which stands for Space and Time resolved Extinction Profiles. This technology enables the analysis of various types of separations, including sedimentation and creaming. By measuring the change in transmission over time across the spatial dimension of the cuvette observation area, extinction profiles are generated to characterize and quantify demixing behavior. The transmission is measured by illuminating the samples with either NIR light (870nm) or blue light (410nm) during centrifugation and detecting the amount of light reaching the sensors beneath the cuvettes. These curves are then overlapped to create qualitative profiles known as Fingerprints.

[0117] General measurements run on the LUMiSizer® equipment were performed using the following standard conditions:

[0118] Fermentate:

[0119] Temperature: 25°C

[0120] Centrifuge speed : 3000 rpm Measure interval: 5 seconds No. of profiles: 200 Wavelenght: 870nm Light factor: 1 The cuvette type used for all LUMISizer® measurements was the 2x8 mm polycarbonate cuvette. The sample volume was ~400-420 pL.

[0121] When performing measurements, one uses the accompanying software SEPView to setup a Standard Operating Procedure (SOP) which serves as the "recipe" for the given centrifugation and can be reviewed and reused later if necessary. The SOP includes choosing the physical parameters for the measurement as mentioned above, but also the number and names of the samples and their allocation in the 12 possible channels. The first step after creating the SOP is for the centrifuge compartment to reach the desired temperature during an initial spin. Once that temperature is reached, the LUMiSizer® perform a normalization measurement of the empty channels. After this, the samples can be loaded in their respective channels and the final sedimentation measurement can be initiated.

[0122] Results

[0123] Example 1.

[0124] Production batch / DSM 35049 (FD-DVS 01) LUMiSizer study:

[0125] Figure 1 A and B and table 1 show the measured sedimentation velocities (median velocity and harmonic mean both in pm / s) from the LUMiSizer study from the production batch of DSM 35049. The median velocity represents the mean value from the Gaussian distribution and is chosen as the most appropriate value for comparison of velocities between the different shearing conditions since the samples show a low measuring noise - i.e mainly one peak is observed from each sample conditions. For samples exhibiting significant measuring noise (e.g. partly agglomeration of two or more bacteria), the harmonic mean velocity can be a more representative value and represent an overall average sedimentation velocity. Figure IB is for illustration and shows the same graphs and results as in figure 1A. Table 1 lists the measured median velocity data and harmonic mean. DSM 35049 represents untreated or non homogenized fermentate, DSM 35049A represents a low intensity homogenization 60-30 bar fermentate, DSM 35049B represents medium intensity homogenization 100-50 bar fermentate, DSM 35049C represents a high intensity homogenization 200-100 bar fermentate and DSM 35049D represents depolymerized fermentate. The median velocities increase with increasing mechanical forces subjected to the fermentate of DSM 35049 and fermentate subjected to depolymerization gave the highest median sedimentation velocity. Thus, with regard to the median sedimentation velocity DSM 35049 < DSM 35049A < DSM 35049B < DSM 35049C < DSM 35049D. The correlation in sedimentation velocity with increasing shearing is likely relating to removal of CPS from the surface of the bacteria creating a higher density difference between the bacteria and the surrounding supernatant.

[0126] Table 1. Median velocity data and harmonic mean for different intensity homogenization of DSM 35049, production batch FD-DVS 01.

[0127] Production batch / DSM 35049 {FD-DVS 01) - Zeta potential measurements:

[0128] Figure 2 summarizes the Zeta potential measurement for the fermentate of DSM 35049 from the production batch, exposed for depolymerization and different intensity of homogenization compared to the non treated fermentate. The different treatment shows no clear picture and no trends is evident when e.g. the intensity of homogenization is increased. However, the results clearly shows a change in surface charge of DSM 35049 for the different treatments compared to the non treated biomass suggesting that part of the CPS has been sheared off. Furthermore it can be seen that the different treatments have resulted in an increase of the isoelectric point (pl) corresponding from 0,2 - 0,4 pH units compare to the untreated fermentate.

[0129] Production batch Surface Hydrophobicity measurements (BCSH) for DSM 35049 FD-DVS 01

[0130] Figure 3 summarizes the hydrophobicity measurement for the fermentate of DSM 35049 from the production batch exposed for depolymerization and different intensity of homogenization compare to the non treated fermentate. The picture for the fermentate expose to mechanical treatment is very evident and the trend is apparent. Increasing the intensity of homogenization it's possible to increase the surface hydrophobicity compare to the non treated fermentate. Depolymerisation reduce the surface hydrophiobicity of DSM 35049. The mechanism here is unknown but besides removing CPS it's likely that the depolymerization treatment also damage the hydrophobic components located in the cell wall of DSM 35049. Example 2.

[0131] Pilot batch / DSM 35049 {FD-DVS 02) LUMISizer study, Zeta potential and hydrophobicity measurements:

[0132] Table 2, Figure 4, 5 and 6 summarize the measured sedimentation velocities from the LUMISizer study, Zeta potential and hydrophobicity measurements for fermentate of FD-DVS 02 / DSM 35049 produced in pilot plant exposed for different intensity of shearing.

[0133] Table 2 Median velocity data and harmonic mean for different intensity homogenization of DSM 35049, pilot batch FD-DVS 02.

[0134] The observations and conclusions are very similar to what is describe above for the production batch of FD-DVS 01 / DSM 35049.

[0135] LUMISizer study: The (median) sedimentation velocities increase as expected with increasing mechanical forces exposed to the FM of FD-DVS 02 / DSM 35049- i.e. DSM 35049 non homogenized FM < DSM 35049 low intensity homogenization 60-30 bar < DSM 35049 / medium intensity homogenization 100-50 bar (= DSM 35049 homogenized Pilot Plant 75-50 bar) < DSM 35049 high intensity homogenization 200-100bar.

[0136] Sedimentation velocity for fermentate homogenized on Pilot plant homogenizer (75- 50bar) fits the sedimentation velocity obtained on the lab scale homogenizer when using 100-50 bar.

[0137] Zeta-Potential study: The different shear treatments show no clear picture and no trends is evident when e.g. the intensity of homogenization is increased. However, the results clearly show a change in surface charge of DSM 35049 for the different treatments (change in pl values and positive and negative net charge) compare to the non treated biomass suggesting that part of the CPS likely has been sheared off.

[0138] Hydrophobicity study: The tendency in Figure 6 for the fermentate expose to different mechanical treatment is very evident and the trend is apparent. Increasing the intensity of shearing it is possible to increase the surface hydrophobicity compare to the non treated fermentate - same as observed for the production batch FD-DVS 01 / DSM 35049.

[0139] Stability study with freeze dried DSM 35049 FD-DVS 02

[0140] Packed cell volume (%PCV), concentration factor over the centrifuge and water activity (Aw) and flow numbers for the FD samples are summarized in Table 3. The encapsulation index (El) for all the FD samples were kept constant.

[0141] Table 3. %PCV, concentration over the centrifuge of the FM, Aw and flow numbers for the freeze-dried (FD) samples of DSM 35049, FD-DVS 02 exposed to different degree of shearing.

[0142] As highlighted in Table 3 the start materials for the stability study for all the homogenized samples all started out with more or less the same amount of active cells.

[0143] From Table 3 it is evident that the different intensities of homogenisation have i) reduced the % PCV significantly due to removal of the CPS layer and ii) have not had any damaging effect on the bacteria. According to Table 3 more active cells are measured after shearing the fermentate and the recovery (mass balance based on active cells) over the freeze drying step is in general 10 % higher than the untreated sample.

[0144] All the samples subjected to different intensity of shearing also show the lowest water activity (Aw) compared to the non-sheared sample - suggesting that some part of the CPS structure has been sheared off.

[0145] Stability studies for freeze dried DSM 35049 exposed for different intensity of homogenisation were performed as described under the above method paragraph. CPH results from the stability study after one month at 37°C are summarized below in Figure 7 - 9 for three different inoculation % - i.e 0,001 %; 0,003 % and 0,006 %. To evaluate performance of the culture, pH was measured after 4 hrs (Figure 7) and 6 hrs (Figure 8) and the time it takes to obtain a pH drop on 0,08 pH units i.e. Ta value (Figure 9).

[0146] The trend from the stability study is very apparent and shows that the non-sheared sample has lost most of its ability to acidify after 0,5 months. The sheared samples, however are still capable to acidify after 1 months of storage at 37°C. Furthermore the stability data also suggest that the ability to acidify is correlated to the intensity of shearing - i.e. the more shearing the better stability is obtained. Best performance with regard to pH after 4 and 6 hrs and Ta values were obtained for fermentates exposed to the highest shearing during homogenisation compare to the non-sheared sample. This trend is especially evident when looking at the Ta values for the different measured inoculation percentages.

[0147] Example 3.

[0148] Pilot batch / DSM 34676 (FD-DVS 03) LUMISizer study, Zeta potential and hydrophobicity measurements:

[0149] Figure 10, 11 A & B, 12 and table 4 summarize the measured median sedimentation velocities from the LUMISizer study of DSM 34676, Zeta potential and hydrophobicity measurements for fermentate of DSM 34676 produced in pilot plant were subjected to different intensity of homogenization.

[0150] Table 4. Sedimentation velocities - Median velocity data and harmonic mean for different pH values of DSM 34676, pilot batch FD-DVS 03. The observations and conclusion are very similar to what is describe above for the production batch of DSM 35049 (FD-DVS 01) and pilot plant production of DSM 35049 (FD-DVS 02).

[0151] The results of the LUMISizer study for DSM 34676 is shown in figure 10. It is observed that the (median) sedimentation velocities increase with increasing mechanical forces subjected to the fermentate (FM) of DSM 34676, i.e. DSM 34676 (non homogenized fermentate, pH 6) < DSM 34676 (low intensity homogenization 60-30 bar of fermentate, pH 6) < DSM 35049 (High intensity homogenization 200-100 bar of fermentate, pH 6). The effect on the sedimentation velocity by changing pH of the high intensity sheared sample to either pH 5 or pH 7 has an insignificant effect on the sedimentation velocity.

[0152] The results of the Zeta-Potential study for the DSM 34676 is shown in Figure 11 A and B. The different shear treatments (Figure 11 A; constant pH 6) show no clear picture and no trends is evident when the intensity of homogenization is increased. No changes can be observed for the isoelectric point of the high and low sheared samples compare to the non treated sample. For the low intensity homogenized samples there seems to be some changes to the non treated samples at towards the alkaline pH extreme. For the high intensity homogenized sample there seems to be significant changes in the zeta potential at both ends of the pH extremes compart to the non treated sample. However, the results show a change in surface charge of DSM 34676 for the different treatments compare to the non treated biomass suggesting that "loosely" EPS has been sheared off.

[0153] For the pH adjusted high sheared samples (Figure 11B) there seems to be some changes in the zeta potential at both pH extremes, most significantly in the acidic end compare to the non sheared sample suggesting that the pH itself contribute to the change at the surface of in the DSM 34676.

[0154] The results of the hydrophobicity curves for the DSM 34676 is shown in Figure 12. Vertical lines indicate error bars from the triplicate measurements.

[0155] The tendency for the fermentate (pH 6) expose to mechanical treatment is again very evident and the trend is apparent. Increasing the intensity of shearing it's possible to increase the surface hydrophobicity compare to the non treated fermentate - same as observed for the production batch FD-DVS 01 / DSM 35049 and FD-DVS 02 / DSM 35049. The high sheared pH 7 adjusted sample shows little overall change in BCSH% compared to the high non-adjusted condition (pH 6), except for a more linear slope and a decrease at the very extreme end. Observing the high sheared pH 5 adjusted sample, the BCSH% has been drastically reduced compared to the high non-adjusted (pH 6). It now overlaps comparatively with the non-homogenized sample condition - likely related to conformational changes of the proteins located at the surface of the stripped bacteria.

[0156] Stability study for DSM 34676 (FD-DVS 03) with homogenized fermentate:

[0157] % packed cell volume (% PCV), concentration factor over the centrifuge and water activity (Aw) and flow numbers for the FD samples are summarized in Table 5. The encapsulation index (El) for all the FD samples were kept constant.

[0158] Table 5: % PCV, concentration over the centrifuge of the fermentate (FM), Aw and flow numbers for the freeze-dried (FD) samples exposed to different degree of shearing.

[0159] From Table 5 it is evident that the different intensities of homogenisation have i) reduced the % PCV significantly. Reduction in % PCV is limited from low to high intensity suggesting a more more "loose" bound polysaccharides compare to DSM 35049 (FD- DVS 01) and DSM 35049 (FD-DVS02). Furthermore ii) the shearing has not had any damaging effect on the bacteria. According to Table 5 more active cells are measured after shearing and adjusting the pH of the fermentate.

[0160] All the samples subjected to different intensity of shearing also show the lowest water activity (Aw) compare to the non-sheared sample - suggesting that some part of loosely bound EPS has been sheared off.

[0161] Stability study for DSM 34676 was performed as described under the above method paragraph. CPH results from the stability study after two months at 37°C are summarized below in Figure 13 - 15 for three different inoculation % - i.e 0,0006 %; 0,0012 % and 0,0024 %. To evaluate performance of the culture, pH was measured after 4 hrs (Figure 13) and 6 hrs (Figure 14) and the time it takes to obtain a pH drop on 0,08 pH units i.e. Ta value (Figure 15). The trend from the stability study of DSM 34676 (no treatment, low and high shearing / pH 6) support the data obtained for DSM 35049 (FD-DVS 02) and shows that the non-sheared samples has lost most of its ability to acidify after 1 month. Stability is improved after shearing and both the low and high samples, are still capable to acidify after 1 months of storage at 37°C. DSM 34676 however, seems to be more sensitive to shearing compare to DSM 35049 (FD-DVS 02). Significant improvement of the stability is already obtained after using the low shearing conditions. No further improvement in stability is obtained from low to high shearing conditions (pH 6). Changing pH of the high sheared fermentate also has a significant positive effect on stability. Best performance with regard to pH after 4 and 6 hrs and Ta values were obtained for the high sheared fermentate adjusted to pH 7. This trend is especially evident when looking at the Ta values for the different measured inoculation percentages.

[0162] Conclusion

[0163] Factors that e.g. have influence on the stability of a freeze-dried bacteria are the presence of water and temperature. The cultures that after mechanical shear shows an increase in surface hydrophobicity would likely be more resistant and repulsive to water. This makes them more robust against enzymatic degradation reactions taking place at the surface of the bacteria, since any enzymatic reaction requires water. The overall result indicate an improvement in the stability of the freeze-dried bacteria.

Claims

28CLAIMS1. A method of preparing a frozen or dried product comprising an asporogenous prokaryote, the method comprising the steps of: i. growing the prokaryote cells by fermentation to obtain a fermentate;II. treating the fermentate by mechanical manipulation, to obtain a more stable cell product; ill. optionally adjusting pH in the fermentate to be in the range of from pH 4 to PH 8; iv. concentrate the cells of the fermentate by separation of fermentation broth, thereby obtaining a cell product; v. optionally combining the cell product with a medium containing a protective compound, to obtain a preprocessing composition; vi. optionally preserving the cell product by(a) freezing the composition to form a frozen prokaryote product;(b) drying the composition to form a dried prokaryote product; or(c) freezing the composition to form a frozen prokaryote intermediate product and then lyophilising the intermediate product to form a freeze- dried prokaryote product,(d) optionally storing the freeze-dried procaryote product at -20°C, +5°, + 25°, +30°C and +37°C, wherein the hydrophobicity of the cell surface is at least 20%, 30% or 40%, 50%, 60%, 70%, 80%, 90% or 100%, as measured by the MATH method at 22°C and expressed as [(Initial ODeoo - Final OD6oo) / Initial OD6oo]*100 when measured with a <t> [VH / VB] at at least one point between 0.01 and 1.0 and the initial ODeoo (nm) is 0.5 and wherein the potency of product is in the range of from 1E+08 to 1E+13 CFU / g.

2. The method according to Claim 1, wherein the mechanical manipulation of the cells in step II is done prior to concentrating the cells in step iv.

3. The method according to Claims 1 or 2, wherein the protective compound is one or more of: a monosaccharide such as glucose, fructose, galactose or mannose; a disaccharide such as sucrose, trehalose, maltose or lactose; a sugar alcohol such as inositol; a trisaccharide such as maltotriose or raffinose; an oligosaccharide such as afructooligosaccharide or such as a maltodextrin with DE 3-20; a polysaccharide such as starch or inulin; a cryoprotectant and / or a lyoprotectant and / or a storage stabiliser, such as gum arabic, a maltodextrin, starch, pectin, cellulose, xylan, or a polyol such as glycerol, sucrose, trehalose or maltose, a protein such as gelatin, milk powder, a peptide such as are supplied by yeast extract, an amino acid such as proline or a sugar alcohol such as sorbitol, mannitol or inositol, an antioxidant, such as sodium ascorbate, sodium citrate.

4. The method according to any of Claims 1 to 3 wherein the frozen prokaryote product or the frozen prokaryote intermediate product has a dry weight ratio of the final protectant to cell concentrate of between 10: 1 and 0.1 : 1, preferably between 3: 1 and 0.5: 1 and most preferably between 2: 1 and 1 : 1.

5. The method according to any of Claims 1 to 4 wherein step (ii) is homogenisation.

6. The method according to any of Claim 5, wherein the homogenization is performed by applying a pressure in the ranges from 60 bar at the inlet and 30 bar at the outlet to 1000 bar at the inlet and 500 bar at the outlet, such as 500 bar at the inlet and 250 bar at the outlet, or 200 bar at the inlet and 100 bar at the outlet.

7. A method according to any of Claims 1 to 6 wherein the prokaryote is a fermentative bacterium• from the phylum Firmicutes, such as: a lactic acid bacterium (LAB), preferably of a genus selected from the group consisting of Streptococcus / such as Streptococcus thermophilus), Lactococcus (such as Lactococcus lactis), Oenococcus (such as Oenococcus oeni), Leuconostoc (such as species Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides), Lactobacillus, Limo- silactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus; Eubacterium / such as Eubacterium limosum, Eubacterium aggregans, Eubacterium barken, Eubacterium ientum), Roseburia (Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faecis and Roseburia cecicola), Faecalibacterium (such as species Faecalibacterium prausnitzii), Anaerostipes (such as Anaerostipes cacccae), Anaerobutyricum (such as species Anaerobutyricum hallii, Anaerobutyricum soehngenii)• from the phylum Actinobacteria, such as genus Bifidobacterium (such as species Bifidobacterium animaiis, Bifidobacterium iongum, Bifidobacterium adolescentis, Bifidobacterium breve'), genus Propionibacterium (such as species Propionibacterium freudenreichii), Cutibacterium (such as Cutibacteriun acnes)• from the phylum Bacteroidetes, such as genera Bacteroides (such as species Bacteroides fragilis, Bacteroides xylanisolvens), genus Prevotella (such as species Prevotella copri) or Alistipes, or• from the phylum Verrucomicrobia, such as an Akkermansia (such as species Akkermansia muciniphila).

8. A method according to Claim 7 wherein the prokaryote is one or more of: Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animaiis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp. sakei, Lactiplantibacillus pentosus, Levilactobacillus brevis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus and Lactobacillus acidophilus, Lactobacillus jensenii, and Lactobacillus iners.

9. The method according to any of Claims 1 to 8, wherein the procaryote or lactic acid bacterium strain comprise an active eps gene cluster.

10. The method according to claim 9, wherein the procaryote or lactic acid bacterium is CPS- and / or EPS-producing.

11. The method according to claim 9, wherein the active eps gene cluster has a sequence identity of at least 95% or 99% with SEQ ID NO: 1 or SEQ ID NO: 2.

12. A frozen or dried product comprising an asporogenous prokaryote, obtainable by a method according to any of the preceding claims.

13. A frozen or dried product according to Claim 12 wherein the potency of the product is 1E+08 to 1E+ 13 CFU / g.

14. A composition comprising a frozen or dried product according to claims 12 or 13, wherein potency of the bacteria is 1E+05 to 1E+12 CFU / g.

15. The composition according to claim 14, which is a food, feed, agricultural product, dietary supplement or pharmaceutical product.

16. A method of manufacturing a food, feed, agricultural product, dietary supplement or pharmaceutical product, comprising addition of a frozen or dried product according to claims 12 or 13.

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