Methods for preparing bacterial cultures

Abstract

The present invention relates to a method for preparing a bacterial culture and the definition of the bacterial culture obtained thereby. The present invention also relates to a method for reducing the loss of viability and / or activity during freezing and / or drying of a bacterial culture, and to a method for increasing the storage stability of frozen and / or dried bacterial cultures.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to a method for preparing a bacterial culture and the definition of the bacterial culture obtained thereby. The present invention also relates to a method for reducing the loss of viability and / or activity during freezing and / or drying of a bacterial culture, and to a method for increasing the storage stability of frozen and / or dried bacterial cultures. [Background technology]

[0002] 2. Background of the Invention Prior to inoculation of the bacterial culture into a product, such as a food product, the bacteria are cultivated to provide a suspension containing a large amount of bacteria. Fermentative bacterial cultures are typically produced by a fermentation process at an acidic pH, typically in the range of pH 3.0 to pH 6.5. After completion of the fermentation process, the suspension is usually concentrated. This concentration step is often followed by freezing the bacterial concentrate as a frozen product in liquid nitrogen and optionally further freeze-drying the frozen bacterial concentrate for bacteria preservation and / or storage. Alternatively, this concentration step is often followed by drying the bacterial concentrate before storage.

[0003] However, downstream processing steps, especially freezing and / or drying, can hinder industrial production of storable viable bacteria due to cell damage and loss of viable cells during these steps: freeze-drying can take several days, while other processing steps typically require a few hours.

[0004] Thus, there is an unmet need to improve the robustness of bacterial cultures so that they can better withstand downstream processing steps after fermentation is terminated.

[0005] It would therefore be beneficial to provide improved methods for preparing bacterial cultures, and the bacterial cultures obtained thereby, In particular, it would be beneficial to provide methods that reduce viability and / or activity loss during freezing and / or drying of bacterial cultures, as well as methods that increase the storage stability of frozen and / or dried bacterial cultures. Summary of the Invention

[0006] Summary of the Invention The present invention relates to a method for preparing a bacterial culture, the pH of which is adjusted to a pH in the range of pH 6.0 to pH 8.0 at a stage after the end of fermentation. In particular, the present invention discloses a bacterial culture prepared by such a method. Accordingly, the present invention provides a method for preparing a bacterial culture, a method for reducing viability loss and / or activity loss during freezing and / or drying of a bacterial culture, a method for increasing the storage stability of frozen and / or dried bacterial cultures, and bacterial cultures produced by such methods.

[0007] The inventors have discovered that increasing the pH of the bacterial culture after the end of fermentation or after downstream processing steps of concentrating the bacterial culture or adding protective compounds to the concentrated bacterial culture results in bacterial cells that exhibit better activity and / or viability during subsequent downstream processing steps than bacterial cells processed from bacterial cultures at a pH below 6.0. As shown herein, the methods of the present invention allow for improved overall cell robustness and reduced loss of viability and / or activity during freezing and freeze-drying. For example, these bacterial cells exhibit increased maintenance of cell integrity and active metabolism. Furthermore, freeze-dried products with adjusted pH after fermentation have improved stability during storage storage at elevated temperatures or water activities greater than 0.1.

[0008] Therefore, for the first time, the inventors have surprisingly shown that increasing the pH of a bacterial culture at the end of fermentation stage is an effective means of improving the overall robustness of the cells.

[0009] This novel invention is inexpensive to implement and applicable to a wide variety of cultures of fermenting bacteria, and therefore can be applied to improve the quality and performance of bacterial cultures, including frozen, freeze-dried and dried forms.

[0010] Accordingly, a first aspect of the present invention relates to a method for preparing a bacterial culture, the method comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) optionally concentrating the bacterial culture by a one-step enrichment method to obtain an enriched bacterial culture; and (c) optionally further comprising the step of adding a protective compound to the enriched bacterial culture of step (b); Here, the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0.

[0011] A second aspect of the invention relates to a bacterial culture obtained by the method of the first aspect of the invention.

[0012] A third aspect of the present invention relates to a method for reducing viability and / or activity loss during freezing and / or drying of a bacterial culture, comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

[0013] A fourth aspect of the present invention relates to a method for increasing the storage stability of a frozen and / or dried bacterial culture, comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture. [Brief description of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the loss of viable cells in freeze-dried granules of Lactobacillus acidophilus bacterial culture over a 16-week storage period in aluminum pouches at 30° C., as measured by the loss of colony forming units (CFU) on a logarithmic scale. A comparison was made between freeze-dried granules from bacterial culture with a post-concentration pH increase to pH 6.5 and freeze-dried granules without a post-concentration pH increase (maintained at pH < 5.0). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The invention will be explained in more detail below.

[0016] Detailed Description of the Invention A first aspect of the present invention provides a method for preparing a bacterial culture, the method comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) optionally concentrating the bacterial culture by a one-step enrichment method to obtain an enriched bacterial culture; and (c) optionally further comprising the step of adding a protective compound to the enriched bacterial culture of step (b); Here, the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0.

[0017] In the present context, the term "bacterial culture" refers to a population of bacteria.

[0018] "Fermentation" in the present process is to be understood as the conversion of sugars to alcohols or acids through the action of microorganisms. Preferably, fermentation in the present process comprises the conversion of lactose to lactic acid.

[0019] "Fermentative bacteria" should be understood as bacteria capable of fermentation.

[0020] "Fermentation medium" should be understood as a liquid medium for the growth of fermentative bacteria. Such a medium will typically contain essential nutrients for the growth of fermentative bacteria. Preferably, the fermentation medium contains nutrients suitable for the particular fermentative bacteria to be cultured. A wide variety of fermentation media are well known in the art, including fermentation media appropriate for a given bacterial species to be cultured.

[0021] Methods for culturing fermenting bacteria in fermentation media are also well known in the art, as are culture vessels for use in such methods, for example sealed bottles or conical flasks, optionally shaken by a rotary shaker, or industrial fermenters or bioreactors.

[0022] "End of fermentation" is understood to mean the point at which the fermentation process ends. This is because, for example, the bacterial culture may go through a holding period (e.g., when the bacteria reach a stationary growth phase or when the carbohydrates present in the fermentation medium have been metabolized) or may be treated as a non-fermentation process, such as concentrating, freezing, drying, etc. Typically, the optical density (OD) measured at a wavelength of 600 nm is 600 ) is at least 1, preferably OD 600 It will be appreciated that the fermentation is terminated after reaching 4-60. Suitable methods and devices for measuring optical density or turbidity are well known in the art, for example spectrophotometers.

[0023] According to the invention, fermenting bacteria are cultured and the final pH of the fermentation broth at the end of the fermentation is a pH lower than pH 6.0, such as pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, pH 4.0, pH 3.9, pH 3.8, pH 3.7, pH 3.6, pH 3.5, pH 3.4, pH 3.3, pH 3.2, pH 3.1, pH 3.0 or lower. It may be understood that culturing a fermenting bacterium at a pH lower than pH 6.0 at the end of fermentation includes (i) the use of a means of pH control to achieve an essentially constant pH level in the culture during the fermentation process, (ii) starting at a pH greater than or equal to pH 6.0 and not limiting natural acidification of the medium by the bacteria during the fermentation process, or (ii) starting at a pH lower than pH 6.0 and not limiting natural acidification of the medium by the bacteria during the fermentation process.

[0024] For example, from the start to the end of the fermentation in step (a), the pH decreases to pH 6.0 or below, such as pH 5.5 or below, pH 5.0 or below, pH 4.5 or below, pH 4.0 or below, pH 3.5 or below, pH 3.0 or below, and / or decreases by at least 0.1 pH units, preferably by at least 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 pH units.

[0025] The invention involves adjusting the pH of the bacterial culture to a less acidic, neutral or slightly alkaline pH at a stage subsequent to the end of the fermentation selected from step (a), step (b) or step (c). According to the invention, the pH is adjusted to within the range of pH 6.0 to pH 8.0. For example, the pH may be adjusted to pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH 8.0. The pH adjustment is typically carried out by the addition of an alkali or base to the bacterial culture when it is in the form of a suspension of bacterial cells. Any suitable alkali or base may be used. Preferably, the alkali or base is non-toxic not only to bacteria but also to eukaryotic organisms, such as mammals, so that it can be used safely without damaging the bacterial cultures or any subsequent uses thereof.

[0026] Therefore, most preferably, the pH adjustment after steps (a), (b) or (c) is by addition of a base selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide and sodium carbonate.

[0027] In one embodiment, the adjustment of the pH is performed after the end of the fermentation (i.e., after step (a)), but before any additional operational steps. In one embodiment, a holding period follows immediately after the end of the fermentation, and the adjustment of the pH is performed at the end of this holding period. A "holding period" should be understood as the period between the end of the fermentation and the start of downstream processing.

[0028] In some embodiments, the method further comprises the step (b): concentrating the bacterial culture by a one-step enrichment method to obtain an enriched bacterial culture. In some of these embodiments, the adjustment of pH is performed on the enriched bacterial culture instead of being performed after step (a).

[0029] An "enriched bacterial culture" should be understood as a bacterial culture that, as a result of the enrichment process, has a higher optical density of cells suspended therein compared to the bacterial culture prior to carrying out the enrichment process, without increasing the total number of cells. In other words, the number of cells remains essentially constant, but the volume of suspension of the bacterial culture is reduced. Enriched bacterial cultures are typically characterized by an OD 600 50-800. Concentrated bacterial cultures typically have a dry matter content of 8%-28%. Concentrated bacterial cultures typically have a dry matter content of 1×10 total cells per gram of concentrated bacterial culture. 9 ~1×10 13 "One-step enrichment method" is to be understood as an enrichment process which produces an enriched bacterial culture in one step and in which the cells are maintained in suspension during the entire enrichment process. For the avoidance of doubt, a one-step enrichment method does not include a two-step process: (i) forming a pellet of bacterial cells, and (ii) resuspension of the cell pellet. The use of a one-step enrichment method is advantageous, inter alia, in that it prevents changes to the cells resulting from their suspension state in the culture medium, as well as the procedure being capable of being carried out on an industrial scale.

[0030] Preferably, step (b) is carried out by a technique selected from the group consisting of centrifugation, vacuum evaporation and filtration.

[0031] In some embodiments, the method further comprises step (c): adding a protective compound to the concentrated bacterial culture of step (b). A "protective compound" should be understood as any compound that provides a protective effect to the bacterial culture during further steps of industrial processing and storage. In some of these embodiments, the adjustment of pH is performed on the concentrated bacterial culture containing the protective compound instead of performing it after either of steps (a) and (b).

[0032] The bacterial culture of the present invention may be provided in several forms. It may be in frozen, dried, lyophilized or liquid form. It may be a powder, pellet or tablet. Thus, in one embodiment, the composition is in frozen, dried, lyophilized or liquid form.

[0033] In some embodiments, the method comprises steps (a) and (b), or steps (a), (b) and (c), and further comprises the step (d): freezing the concentrated bacterial culture to obtain a frozen bacterial culture. Thus, in such embodiments, the method may comprise steps (a), (b) and (d); or steps (a), (b), (c) and (d).

[0034] Freezing of the concentrated bacterial culture can be performed by any suitable method known in the art. Typically, the concentrated bacterial culture is frozen by pelleting the cell concentrate in liquid nitrogen. The frozen concentrated bacterial culture is typically stored at about -55°C. Preferably, the concentrated bacterial culture contains a cryoprotectant as the protective compound added in step (c).

[0035] In some embodiments, the method further comprises the step (e): removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture. Thus, in such embodiments, the method may comprise steps (a), (b) and (e); steps (a), (b), (c) and (e); steps (a), (b), (d) and (e); or steps (a), (b), (c), (d) and (e).

[0036] The removal of water can be carried out by any suitable method known in the art. Preferably, step (e) is carried out by a technique selected from the group consisting of spray drying, spray freezing, vacuum drying, air drying, freeze drying, shallow plate drying and vacuum shallow plate drying.

[0037] The dried bacterial culture may be in the form of granules and / or powder, for example the dried bacterial culture may be in the form of a spray-dried powder, freeze-dried granules or freeze-dried powder.

[0038] In some embodiments, the method further comprises the step (f): packaging the frozen bacterial culture obtained in step (d) or the dried bacterial culture obtained in step (e).Thus in such embodiments, the method comprises steps (a), (b), (d) and (f); steps (a), (b), (c), (d) and (f); steps (a), (b), (e) and (f); steps (a), (b), (c), (e) and (f); steps (a), (b), (d), (e) and (f); or steps (a), (b), (c), (d), (e) and (f).

[0039] The method of the invention is broadly applicable. Thus, in one embodiment, the bacterial culture comprises or consists of the following fermentative bacteria: From the phylum Firmicutes, e.g.: Lactic acid bacteria (LAB), preferably Streptococcus (e.g., Streptococcus thermophilus, etc.), Lactococcus (e.g., Lactococcus lactis, etc.), Oenococcus (e.g., Oenococcus oeni), Leuconostoc (e.g., species such as Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides), Lactobacillus, Rimosilactobacillus, Lacticaseibacillus, Lidilactobacillus, Lacticaseibacillus, Lactipranchibacillus, Rimosilactobacillus, Lidilactobacillus, Lactilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus, genera selected from the group consisting of Shirasu and Lactiprunchbacillus; Eubacterium (e.g., Eubacterium limosum, Eubacterium agregans, Eubacterium barkeri, Eubacterium lentum, etc.), Roseburia (Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faeces and Roseburia sesicola), Faecalibacterium (e.g., species such as Faecalibacterium prausnitzii), Anaerostipes (e.g., Anaerostipes caccae, etc.), Anaerobutyricum (e.g., species such as Anaerobutyricum hallii and Anaerobutyricum souengenii, etc.), etc. From the phylum Actinobacteria, for example the genera Bifidobacterium (e.g. species Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, etc.), Propionibacterium (e.g. species Propionibacterium freudenreici, etc.), and Cutibacterium (e.g. Cutibacterium acnes, etc.), from the phylum Bacteroidetes, for example the genera Bacteroides (e.g. species Bacteroides fragilis, Bacteroides xylansorbens, etc.), Prevotella (e.g. species Prevotella copri, etc.) or Alistipes, and / or From the phylum Verrucomicrobium, e.g., Ackermansia (e.g., species such as Ackermansia muciniphila).

[0040] In particular, the bacterium can be one or more of the following: Rimosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ridilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Rimosilactobacillus fermentum, Ridilactobacillus animalis, Retactobacillus boekenrei, Latiliactobacillus culbar bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus and Lactobacillus acidophilus, Lactobacillus jensenii, and Lactobacillus inellus.

[0041] The method is applicable to vegetative cells of non-spore-forming bacteria from the domain "Bacteria". The invention is relevant to a wide spectrum of non-spore-forming bacteria used in the food- and feed-production industries, agriculture, medicine, the production of biofuels and bio-based chemicals.

[0042] Non-spore-forming bacteria can be identified within the phyla Firmicutes, Actinobacteria and Bacteroidetes. The invention is particularly applicable to homo- and hetero-fermentative lactic acid bacteria of the phylum Firmicutes, as well as to Bifidobacteria and Propionibacteria of the phylum Actinobacteria. The invention is also applicable to obligate anaerobes of the class Clostridia of the phylum Firmicutes, which represent the core microflora of the human intestinal tract and are candidates for the next generation of probiotic bacteria, such as fermentative butyrate-producing bacteria of the genera Roseburia (e.g., Roseburia hominis and Roseburia inulinivorans), Anaerobutyricum hallii, Anaerobutyricum souengenii, Eubacterium (e.g., Eubacterium limosum), Anaerostipes (e.g., Anaerostipes caccae) and Faecalibacterium (e.g., F. prausnitzii).

[0043] In this context, the term "lactic acid bacteria (LAB)" refers to a group of Gram-positive, catalase-negative, non-motile, microaerophilic or anaerobic bacteria that ferment sugars with the production of acids, including lactic, acetic, formic and propionic acids as the main acids produced. The most industrially useful lactic acid bacteria include, but are not limited to, Lactococcus spp., Streptococcus spp., Lactobacillus spp. (including all those classified as Lactobacillus until 2020), Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. In addition, lactic acid-producing bacteria belonging to the group of strict anaerobes, bifidobacteria, i.e. Bifidobacterium spp., which are frequently used as food starter cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria. Certain bacteria from the genus Staphylococcus (e.g., S. carnosus, S. aequorum, S. scrib, S. vitulinus and S. xylosus) have also been referred to as LAB (Mogensen et al (2002)).

[0044] It will be understood (and mentioned above) that the Lactobacillus taxonomy was revised in 2020. This new taxonomy is disclosed in Zheng et al. (2020) and will be summarized herein without any indication to the contrary. For the purposes of the present invention, Table 1 provides a list of the old and new names of some Lactobacillus species relevant to the present invention. [Table 1]

[0045] The lactic acid bacteria is preferably of a genus selected from the group consisting of Lactobacillus, Rimosilactobacillus, Lacticaseibacillus, Rizilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactipranchibacillus, Rimosilactobacillus, Rizilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactipranchibacillus. In particular, these may be Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Rizilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Rizilactobacillus animalis, Lentilactobacillus boekenrai, Latilactobacillus culvertus, Companilactobacillus fussai, Latilactobacillus sakei subsp. sakei, and / or Lactiplantibacillus pentosus. Others include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar diacetylactis, Streptococcus thermophilus, Enterococcus such as Enterococcus faecium, Bifidobacterium spp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

[0046] The composition may comprise one or more strains of lactic acid bacteria, which may be selected from the group including: BB-12® (Bifidobacterium animalis subsp. lactis BB-12®, DSM 15954), ATCC 29682, ATCC 27536, DSM 13692, DSM 10140, LA-5 (Lactobacillus acidophilus LA-50®, DSM 13241), LGG® (Lactobacillus rhamnosus LGG®, ATCC 53103), GR-1® (Lactobacillus rhamnosus GR-1®, ATCC 55826), RC-14® (Lactobacillus reuteri RC-14®, ATCC 54111), ATCC 54112, ATCC 54113, ATCC 54114, ATCC 54115, ATCC 54116, ATCC 54117, ATCC 54119 ... 55845), L. casei 431® (Lactobacillus paracasei subsp. paracasei, L. casei 431®, ATCC 55544), F19® (Lactobacillus paracasei F19®, LMG P-17806_), TH-4 (Registered Trademark) (Streptococcus thermophilus TH-4®, DSM 15957), PCC® (Lactobacillus fermentum PCC®, NM02 / 31074), LP-33® (Lactobacillus paracasei subsp. paracasei, LP-33®), Lactococcus lactis DSM 21404, Rizilactobacillus animalis DSM 33570, Bifidobacterium animalis subsp. lactis DSM 33868, and CCTCCM204012.

[0047] The LAB culture may be a "mixed LAB culture" or a "pure LAB culture". The term "mixed LAB culture" or "LAB" culture means a mixed culture containing two or more different LAB species. The term "pure LAB culture" means a pure culture containing only a single LAB species. Thus, in a preferred embodiment, the LAB culture is a LAB culture selected from the group consisting of these cultures.

[0048] Preferably, the LAB cells are probiotic cells.

[0049] It will be understood that any suitable fermentation medium can be used to cultivate the fermenting bacteria in step (a). For example, modified Difco™ M17 broth (Becton, Dickinson and Company, France) contains peptone and meat derivatives as carbon, nitrogen, vitamin and mineral sources; yeast extract, which provides vitamin B-complex to stimulate bacterial growth; disodium β-glycerophosphate, which buffers the medium during acid production from lactose fermentation; and ascorbic acid, which stimulates the growth of lactic streptococci. Difco™ Lactobacillus MRS broth powder contains peptone and dextrose, which provide nitrogen, carbon and other elements essential for growth; polysorbate 80, acetate, magnesium and manganese, which provide growth factors for culturing a wide variety of lactobacilli.

[0050] It is highly preferred that the fermentation medium contains at least one carbohydrate, preferably one or more of the following: monosaccharides such as glucose, fructose, galactose or mannose; disaccharides such as sucrose, trehalose, maltose or lactose; sugar alcohols such as inositol; trisaccharides such as maltotriose or raffinose; oligosaccharides such as fructooligosaccharides; or maltodextrins such as DE3-20; glucose syrup with DE21-39; and polysaccharides such as starch or inulin.

[0051] Fructo-oligosaccharides (FOS), also known as oligofructose or oligofructans, are a mixture of oligosaccharides and fructans and are typically prepared by the transfructosyl action of Aspergillus niger or Aspergillus spp. β-fructosidase on sucrose.

[0052] Inulin is a heterogeneous collection of fructose polymers and is commercially available in a variety of forms, such as granules and powder, which may be utilized in the present invention.

[0053] Maltodextrins are polysaccharides consisting of D-glucose units connected in chains of various lengths. The glucose units are linked mainly by α(1→4) glycosidic bonds. Maltodextrins are typically composed of a mixture of chains varying in length from 3 to 17 glucose units. Maltodextrins are classified by DE (dextrose equivalent), having a DE between 3 and 20, preferably between 10 and 20. A higher DE value translates to shorter glucose chains, higher sweetness, higher solubility, and lower heat resistance.

[0054] For growth of LAB, the fermentation medium preferably contains simple carbohydrates, such as monosaccharides.

[0055] Preferably, the total concentration of carbohydrates in the fermentation medium in step (a) is 1 to 15% w / w, for example, 2 to 14% w / w, for example, 3 to 13% w / w, 4 to 12% w / w, for example, 5 to 11% w / w, 6 to 10% w / w, for example, 7 to 9% w / w, 8 to 10% w / w, more preferably 1 to 10% w / w.

[0056] Preferably, the fermentation medium in step (a) contains at least one nitrogen source, such as peptone, yeast extract, one or more amino acids or one or more ammonia salts.

[0057] Preferably, the fermentation medium in step (a) contains one or more yield enhancing agents selected from the group consisting of purine bases, pyrimidine bases, nucleosides, nucleotides and derivatives thereof.

[0058] The term "purine base" herein is intended to cover a cyclic nitrogen-containing base having a purine core structure. Thus, in this context, the term "purine base" is intended to mean an optionally substituted purine. Specific examples of purine bases include adenine, guanine, xanthine, and hypoxanthine. Similarly, the term "pyrimidine base" is intended to cover a cyclic nitrogen-containing base having a pyrimidine core structure. Thus, in this context, the term "pyrimidine base" is intended to mean an optionally substituted pyrimidine. Specific examples of pyrimidine bases include cytosine, thymine, and uracil.

[0059] In the present context, the term "nucleotide" refers to a 2-deoxyribose (DNA) or ribose (RNA) monomer that is linked to a purine or pyrimidine base through its 1-position carbon atom. This DNA or RNA monomer is further linked to a phosphate group through its 5-position carbon atom. As used herein, the term "nucleoside" is intended to refer to a 2-deoxyribose (DNA) or ribose (RNA) monomer that is linked to a purine or pyrimidine base through its 1-position carbon atom. In the present context, the term "derivative", when used in conjunction with the term "nucleotide" or "nucleoside", is intended to mean that the nucleotide or nucleoside in question is modified in its sugar (i.e., 2-deoxyribose or ribose) unit, or that the nucleotide or nucleoside in question is modified in its cyclic nitrogen-containing base, or that the nucleotide or nucleoside in question is modified in both its sugar unit and its cyclic nitrogen-containing base.

[0060] Preferably, the fermentation medium in step (a) contains one or more vitamins.

[0061] The fermentation medium can also contain a source of fatty acids, such as oleic acid in polysorbate 80.

[0062] As discussed above, optional step (c) provides for the further addition of protective compounds to the concentrated bacterial culture of step (b). The protective compounds may be selected from the group consisting of cryoprotectants, lyoprotectants, antioxidants, nutrients, bulking agents, flavorings, and mixtures thereof. The bacterial culture obtained according to the present invention may thus contain one or more cryoprotectants, lyoprotectants, antioxidants, and / or nutrients, more preferably cryoprotectants, lyoprotectants, and / or antioxidants, and most preferably cryoprotectants or lyoprotectants, or both. The concentrated bacterial culture to which one or more protective compound(s) have been added is sometimes referred to herein as a formulated bacterial culture.

[0063] The use of protective substances such as cryoprotectants and lyoprotectants is known to those skilled in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri- and polysaccharides (e.g., glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (gum acacia) and the like), polyols (e.g., erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (e.g., proline, glutamic acid, etc.), complex substances (e.g., skim milk, peptone, gelatin, yeast extract) and inorganic compounds (e.g., sodium tripolyphosphate, etc.).

[0064] In one embodiment, the bacterial culture of the present invention contains one or more cryoprotectants or lyoprotectants selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and derivatives of any such compounds. Antioxidants suitable for use in the present invention include ascorbic acid, citric acid and their salts, gallates, cysteine, vitamin E, β-carotene and other carotenoids. Nutrients suitable for use in the present invention include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (e.g., vitamin B-family, vitamin C, etc.). The bacterial culture may optionally further contain substances including bulking agents (eg, lactose, maltodextrin or milk powder) and / or flavoring agents.

[0065] In a further aspect, the bacterial culture comprises or contains an ammonium salt (e.g., an ammonium salt of an organic acid (e.g., ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g., a growth booster or an acidification booster) for bacterial cells, e.g., (substantially) urease-negative bacterial cells, e.g., cells belonging to the species S. thermophilus. The terms "ammonium salt", "ammonium formate", and the like, should be understood as a source of a combination of salts or ions. For example, the "source" of the term "ammonium formate" or "ammonium salt" refers to a compound or mixture of compounds that, when added to a culture of cells, provides ammonium formate or an ammonium salt. In some embodiments, the source of ammonium releases ammonium into the growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, the ammonium is not provided by the dairy substrate. It should of course be understood that ammonia may be added instead of an ammonium salt. Thus the term ammonium salt refers to ammonia (NH3), NH4OH, NH4 + , and the like.

[0066] The amount of protective compound or mixture of protective compounds added to the bacterial culture is typically calculated as the dry weight of the protective compound(s) as a percentage of the dry weight of the formulated bacterial culture, i.e. the total dry weight of the biomass of the concentrated bacterial culture and the protective compound(s). It should be noted that the protective compound(s) are typically used in the form of a solution that is added to the concentrated bacterial culture (in the form of a suspension of bacterial cells). Thus, for example, the addition of protective compound(s) at 50% w / w means the addition of the same amount of dry weight of protective compound(s) as the dry weight of the concentrated bacterial culture, i.e. in a 1:1 ratio. Preferably, the concentration of protective compound in the formulated bacterial culture in step (c) is 5-90% w / w evaluated as dry weight, more preferably 25-75% w / w evaluated as dry weight, more preferably 50-75% w / w evaluated as dry weight. It should be appreciated that for all embodiments of the invention described herein, these ranges may be increments of the described ranges.

[0067] It will be understood that the parameters of the fermentation in step (a) may vary, since the present invention is broadly applicable. In some embodiments, the fermentation in step (a) lasts for about 4 hours to about 7 days.

[0068] Preferably, the temperature of fermentation in step (a) will be particularly suitable for the growth of the fermenting bacteria. For example, the temperature may be the optimum temperature known for the growth of the fermenting bacteria. In some embodiments, the fermentation in step (a) is at a temperature of about 25°C to about 50°C; for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C.

[0069] In some embodiments, termination of the fermentation in step (a) is immediately followed by a holding period of from about 1 minute to about 10 hours, during which the bacterial culture is preferably held at a temperature between 4°C and 45°C, more preferably between 5°C and 37°C, 6°C and 25°C, or even more preferably between 7°C and 15°C.

[0070] In some embodiments, the dried bacterial culture obtained in step (e) has a water activity (a) in the range of 0.01 to 0.8. w ), preferably in the range of 0.05 to 0.6, and more preferably in the range of 0.05 to 0.4.

[0071] In this context, the term "water activity" refers to the partial vapor pressure of water in a substance divided by the partial vapor pressure of water at standard conditions. w Specifically, the a of the product (e.g., dried bacterial culture) is w is the ratio between the vapor pressure of the dried bacterial culture itself, when in perfect undisturbed equilibrium with the surrounding air medium, and the vapor pressure of distilled water under the same conditions.

[0072] For example, as in the Examples provided herein, the water activity (a w ) may be measured at room temperature using a Rotronic HYGROMER® AwVC (Rotronic Instrument Corp., Huntington, NY, USA). The range of measurements achievable using this instrument corresponds to a water activity in the range of 0.03 to 1, thus having an instrumental limit of detection (LOD) of 0.03 water activity.

[0073] In some embodiments, the frozen bacterial culture comprises 10 4 ~10 12 In some embodiments, the dried bacterial cultures range from 10 4 ~10 13 CFU / g dried bacterial culture. For example, frozen and / or dried bacterial cultures range from 108 ~10 12 CFU / g, or 10 9 ~10 11 CFU / g, or 10 9 ~10 10 CFU / g range.

[0074] In the present context, and as shown in the present Examples, the robustness and viability of bacterial cells in a bacterial culture can be determined by at least the following methods:

[0075] (1) Total cell and viable cell counts can be performed by flow cytometry. As disclosed in the present "Examples", sample preparation and flow cytometry assays can be performed according to the disclosure of EP1891436B1 (Worm et al.), paragraphs

[0059] to

[0061] . Viable cells are characterized by the integrity of the cell wall, the activation of the cell metabolism, and their ability to maintain the cell membrane potential. Such results are a good indicator of the degree of cell preservation.

[0076] (2) Enumeration of viable cells by colony counting. In this context, the terms "survival" and "viability" refer to the ability of bacteria to replicate. Viability can therefore be assessed by viability assays, such as the determination of colony forming units. This can be performed with any bacterial culture, but for the purposes of this "Example" it was determined on frozen and dried products, respectively, after pelleting in liquid nitrogen and freeze-drying. A standard pour plate method is used. The materials are suspended in sterile peptone saline diluent and homogenized by stomaching. After 30 minutes of revitalization, the stomaching is repeated and the cell suspension is serially diluted in peptone saline diluent. The dilutions are plated in duplicate on suitable agar. The agar plates are incubated anaerobically for 3 days at the optimal growth temperature of the tested bacteria. Plates with 30 to 300 colonies are selected for counting colony forming units (CFU). Results are reported as the average CFU / g sample, calculated from duplicates.

[0077] (3) Determination of metabolic activity in foods. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or freeze-dried cultures retain their viability, but they may lose a significant part of their metabolic activity, e.g. cultures may lose their acid-producing (acidifying) activity if they are kept in storage even for a relatively short period of time. Viability and metabolic activity should therefore be assessed by different assays. Viability is typically assessed by a viability assay, such as the determination of colony forming units, whereas metabolic activity is assessed by quantification of the relative metabolic activity of the bacterial culture. For example, metabolic activity may be measured as the acidification rate of milk inoculated with the bacterial culture. The term "metabolic activity" refers to the oxygen scavenging activity of the culture, its acid-producing activity, i.e. its metabolite-producing activity, e.g. the production of lactic acid, acetic acid, formic acid and / or propionic acid, or the production of aroma compounds such as acetaldehyde (a-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0078] In the present context, and as shown in the Examples, the storage stability of a sample (e.g. liquid or dry) is assessed by counting colony-forming units (CFU) per gram using the viability assay described above.

[0079] Thus, in some embodiments, the dried bacterial culture is dried to remove viable fermenting bacterial content at 30° C. and w After 16 weeks of storage at 0.3, 4 ~10 13 Contains in the range of CFU / g dried bacterial culture.

[0080] In some embodiments, the loss of viability of the dried bacterial culture, measured in CFU / g, is greater than or equal to 30° C. and 100% at 30° C. w = 0.3, after 16 weeks of storage, is less than 4 log units, preferably less than 3 log units, more preferably less than 2 log units, even more preferably less than 1 log unit or less than 0.5 log units. Most preferably, there is no loss of viability, i.e., 0 log units.

[0081] The viability of dried bacterial cultures (such as freeze-dried bacterial cultures) can be measured over time using methods such as those described below and demonstrated in the stability demonstration experiments of this "Example" to determine storage stability. Bacterial cultures were sampled immediately at the start and at selected time points during the storage stability study. Stability of the bacterial cultures was assessed by the difference in CFU / g measured at time 0 of the storage stability demonstration experiment and at specific sampling time points during the stability study period. Loss of viability was quantified as CFU log loss.

[0082] A second aspect of the invention provides a bacterial culture obtained by a method according to the first aspect of the invention.

[0083] A third aspect of the invention provides a method for reducing viability and / or activity loss during freezing and / or drying of a bacterial culture, the method comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

[0084] The features of the third aspect of the invention follow the corresponding features of the first aspect of the invention.

[0085] In the present context, the term "viability loss" refers to a decrease in the viability of a bacterial culture. For example, this may be expressed as a decreased number of colony forming units per gram of bacterial culture. Thus, in the present context, "reducing viability loss" means a decrease in the proportion of bacterial cells that become non-viable as a result of one or more processing steps, especially during freezing and / or drying of the bacterial culture.

[0086] In the present context, the term "loss of activity" refers to a decrease in the cell number of a bacterial culture that is viable. Thus, in the present context, "reducing loss of activity" means a decrease in the proportion of bacterial cells that are no longer viable as a result of one or more processing steps, especially during freezing and / or drying of the bacterial culture.

[0087] A fourth aspect of the present invention provides a method for increasing the storage stability of a frozen and / or dried bacterial culture, the method comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

[0088] The features of the fourth aspect of the present invention follow the corresponding features of the first aspect of the present invention.

[0089] In this context, the term "storage stability" refers to the ability of a bacterial culture to maintain viability when stored for an extended period of time. Typically, storage stability tests are performed at ambient temperature (25°C) for periods up to two years. Accelerated storage stability tests are performed at elevated water activity (a w ), elevated temperature, or elevated a w and temperature combinations. The breakdown of bacterial cells is expected to be faster under accelerated storage conditions. Examples of elevated temperatures are 30° C. or 37° C., as well as elevated a w is higher than 0.1 w For example, storage stability is at a temperature of 30° C. and w ≦0.15, for a period of 4 weeks or 8 weeks, or at a temperature of 30°C and w =0.30 and may be measured for periods up to 24 weeks (such as 8, 12, 16, 20 or 24 weeks).

[0090] Storage stability can be determined by analyzing how the count of viable bacterial cells develops over time. The viability of the bacterial culture is measured by determining the CFU / g as described herein. Thus, a measurement of the storage stability of a bacterial culture may be determined by evaluating the CFU / g of the dried bacterial culture at time 0 (immediately after drying) and after storage at accelerated storage conditions, for example after 4 weeks.

[0091] Briefly, the storage stability of dried bacterial cultures, including those demonstrated in the Examples herein, may be examined as follows: (1)a w The freeze-dried granules having a CFU <0.15 are sealed in aluminum pouches and incubated at a suitable temperature for a desired period of time, and CFU / g is determined for the samples. (2)a w The freeze-dried granules having a CFU <0.15 are aliquoted but not sealed into aluminum pouches and then incubated open to the atmosphere at a suitable temperature and relative humidity for a desired period of time and CFU / g are determined for those samples. (3) The freeze-dried granules are ground into a powder and diluted to a specific water activity (a w The samples are formulated in microcrystalline cellulose equilibrated to 0.5% CO2. The samples are placed in aluminum bags and the bags are sealed. The bags are stored at a suitable temperature for the desired period of time and CFU / g is determined for the samples.

[0092] The listing or discussion of an apparently prior-published document in this specification does not necessarily constitute an admission that the document is part of the state of the art or is common general knowledge.

[0093] Preferences, alternatives and embodiments with respect to a given aspect, feature or parameter of the invention shall be considered as being expressly set forth in combination with any and all preferences, alternatives and embodiments with respect to all other aspects, features and parameters of the invention, unless the context dictates otherwise. Embodiments and features of the invention are also outlined in the following clauses. EXAMPLES

[0094] The invention will now be described in further detail in the following non-limiting examples.

[0095] Working Example Example 1: Increased activity of Lactococcus lactis cultures by increasing post-fermentation pH This "Example" involves the cultivation and downstream processing of a Lactococcus lactis strain deposited by Chr. Hansen A / S on April 23, 2008 at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7 B, D-38124 Braunschweig, Germany under deposit number DSM 21404. This strain is well known to those skilled in the art and is commercially available from Chr. Hansen A / S.

[0096] method: Lactococcus lactis DSM 21404 was grown by fermentation in modified Difco™ M17 broth (Becton, Dickinson and Company, France), which contains 5 g / L casein pancreatic digest, 5 g / L soy peptone, 5 g / L beef extract, 2.5 g / L yeast extract, 0.5 g / L ascorbic acid, 0.25 g / L magnesium sulfate, 19 g / L disodium β-glycerophosphate, and 25 g / L lactose in purified water. The inoculum for the fermentation was prepared by growing the strain in a sealed bottle of modified M17 broth under sterile conditions and at 30° C. without pH control. The incubation period was 16 hours. The fermentation was started by inoculation of a bottle of modified M17 broth with 1% of the pre-culture. The fermentation was carried out under the same conditions as described for the cultivation of the pre-culture. The fermentation was stopped after 16 hours. The fermentation broth was divided into aliquots for the adjustment of the pH of the fermentation broth in the post-fermentation process. The pH was adjusted to either 5.0; or 5.5; or 6.0: or 6.5: or 7.0: or 7.5: or 8.0 with 12% aqueous ammonia. Subsequently, the aliquots of the pH-adjusted fermentation broth were cooled to 4° C. and processed by one-step centrifugation at 4° C. in a laboratory centrifuge to produce cell concentrates. The cell concentrates were prepared by 26× concentration of this pH-adjusted fermentation broth. Each cell concentrate was used for the production of a corresponding frozen pellet by freezing a droplet of the cell concentrate in liquid nitrogen.

[0097] result: The fermentation broth of L. lactis DSM 21404 had a pH of 4.59 and an OD 600nm The cell count was performed by flow cytometry and showed 5.04E+09 total cells / g fermentation broth, 4.66E+09 active cells / g fermentation broth, and the ratio of (active cell count / g) / (total cell count / g) was equal to 92.4%.

[0098] Cell counts were quantified in all frozen pellets (Table 2). Similar counts of total cells indicated that the products were homogenous. The increase in flow cytometry activity of cells in frozen pellets following an increase in pH of the post-fermentation broth revealed that neutralization of the fermentation broth improved cell activity upon freezing of the cell concentrate in liquid nitrogen. [Table 2]

[0099] Conclusion: This example demonstrates that increasing the pH of a bacterial culture after completion of fermentation of the bacterial culture, prior to concentration and freezing, resulted in the maintenance of an increased proportion of viable cells in the frozen bacterial culture.

[0100] Example 2: Increased activity of Ridilactobacillus animalis cultures by increasing pH after fermentation This "Example" concerns the cultivation and downstream processing of the resiliently Lactobacillus animalis strain (formally known as Lactobacillus animalis), deposited on July 8, 2020 by Chr. Hansen A / S at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7 B, D-38124 Braunschweig, Germany, under deposit number DSM 33570.

[0101] method: Ridilactobacillus animalis DSM 33570 was grown in a complex fermentation medium by a batch fermentation process with pH control at a pH set point of 5.5. The fermentation was stopped when the cells reached stationary growth phase and all sugars were utilized. The pH 5.5 fermentation broth (i.e., bacterial culture after fermentation) was divided into four aliquots and subjected to the following post-fermentation treatments: (1) adjustment of pH to 4.0 by addition of phosphoric acid; (2) no adjustment, i.e., pH was maintained at 5.5; (3) adjustment of pH to 7.0 by addition of ammonium hydroxide base; and (4) adjustment of pH to 8.5 by addition of ammonium hydroxide base. The post-fermentation broth was centrifuged by one-step centrifugation in a laboratory centrifuge to produce a cell concentrate. The cell concentrate was formulated with additives containing antioxidants as well as cryoprotective compounds and lyoprotectants. The formulated cell concentrate was processed into granules that were frozen by pelleting in liquid nitrogen. The frozen granules were freeze-dried to freeze-dried granules.

[0102] result: Flow cytometry was used to determine cell quantification and viable cells in the post-fermentation broth, intermediate product and freeze-dried granules (Table 3). Products at a particular processing step were found to be similar in the parameter total cells / g product. The only exception within the freeze-dried granule group was the lower total cell count in the freeze-dried granules at post-fermentation broth pH 4.0. Analysis of cell activity throughout the process showed that the optimum conditions for recovery of active cells in the product were obtained with a post-fermentation broth at pH 7.0. Test conditions of post-fermentation pH 5.5 and post-fermentation pH 8.5 correlated with reduced activity of cells in the intermediate and final products. Adjustment of the post-fermentation broth to pH 4.0 was detrimental to cell activity. [Table 3]

[0103] Conclusion: This example demonstrates that increasing the pH of the bacterial culture to pH 7.0 after completion of fermentation of the bacterial culture and prior to concentration, formulation with a protective compound, freezing and freeze-drying resulted in the maintenance of an increased proportion of viable cells at each step in the process.

[0104] Example 3: Increased viability and storage stability of Lactobacillus acidophilus cultures with increased pH after concentration This "Example" involves the culture and downstream processing of a Lactobacillus acidophilus strain.

[0105] method: Lactobacillus acidophilus strains were grown in a complex fermentation medium by a batch fermentation process with pH control at a pH set point <5.0. The fermentation was stopped when the cells reached stationary growth phase and all sugars were utilized. The fermentation broth (i.e., bacterial culture after fermentation) at pH <5.0 was centrifuged in one step in an industrial centrifuge to produce a cell concentrate. The dry matter of the cell concentrate was 18%. The cell concentrate was divided into two aliquots and subjected to the following post-fermentation treatments: (1) no pH adjustment, i.e., maintained at pH <5.0; (2) adjustment of pH to 6.5 by addition of ammonium hydroxide base. The cell concentrate at pH <5.0 contained 2E+11 total cells / g with 85% active cells / g and the cell concentrate at pH = 6.5 contained 2E+11 total cells / g with 88% active cells / g. Both concentrates were formulated with additives including cryoprotective compounds and lyoprotectants in addition to antioxidants. The formulated cell concentrates were processed into frozen granules. The frozen granules were freeze-dried into freeze-dried granules.

[0106] result: Cell counts were analyzed in the freeze-dried products by flow cytometry and CFU (Table 4). pH adjustment of the cell concentrate to pH 6.5 showed flow cytometry activity of cells in the freeze-dried products comparable to that in freeze-dried products made from concentrates with pH<5.0. However, CFU analysis pointed to better survival of freeze-drying and higher cell viability when the freeze-dried products were made from cell concentrates at pH 6.5. [Table 4]

[0107] The freeze-dried granules were sealed in aluminum pouches and tested for storage stability at 30° C. Cell viability in the freeze-dried granules was measured by CFU analysis over the course of the 16 week stability demonstration (FIG. 1). In addition to starting with higher cell viability as determined by CFU (see Table 4 above), the freeze-dried product made from the cell concentrate at pH 6.5 was also significantly more stable than the freeze-dried product made from the cell concentrate at pH <5.0 (FIG. 1).

[0108] Therefore, although the percentage of viable cells measured by flow cytometry was the same for both test conditions before storage, CFU analysis indicated better survival of freeze-drying and higher cell viability when the freeze-dried product was prepared from a cell concentrate at pH 6.5.

[0109] Conclusion: This example demonstrates that increasing the pH of the bacterial culture after the concentration step and prior to freezing and freeze-drying of the bacterial culture resulted in increased viability counts and increased storage stability of the freeze-dried product.

[0110] Example 4: Enhanced storage stability of Bifidobacterium animalis subsp. lactis cultures by increasing post-fermentation pH This "Example" concerns the cultivation and downstream processing of the Bifidobacterium animalis subsp. lactis strain deposited by Chr. Hansen A / S on May 26, 2021 at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7 B, D-38124 Braunschweig, Germany, under deposit number DSM 33868.

[0111] method: Bifidobacterium animalis subsp. lactis DSM 33868 was grown in a complex fermentation medium by a batch fermentation process with pH control at a pH set point of 6.0±0.1. The fermentation was stopped when the cells reached stationary growth phase and all sugars were utilized. The fermentation broth (i.e., bacterial culture after fermentation) at pH 6.0±0.1 was divided into five aliquots and subjected to the following post-fermentation treatments: (1) pH adjustment to pH 4.0 with phosphoric acid; (2) pH adjustment to pH 5.0 with phosphoric acid; (3) no pH adjustment, i.e., pH was maintained at pH 6.0; (4) pH adjustment to pH 7.0 with sodium hydroxide base; and (5) pH adjustment to pH 8.0 with sodium hydroxide base. The post-fermentation broth was processed into a cell concentrate, which was formulated with additives and subsequently frozen into frozen granules. The frozen granules were freeze-dried into freeze-dried granules. The freeze-dried product was transferred into an aluminum bag and incubated in the open aluminum bag, ie exposed to the atmosphere, at 30° C. and 30% relative humidity.

[0112] result: The viability of B. animalis subsp. lactis DSM 33868 during storage stability testing was measured by CFU analysis over a period of 24 weeks (Table 5). Freeze-dried products made from acidic post-fermentation broths with pH 4.0 and pH 5.0 had poor storage stability. Among the freeze-dried products made from post-fermentation broths with pH 6.0-8.0, pH 7.0 resulted in the most stable freeze-dried product. [Table 5]

[0113] Conclusion: This example demonstrates that increasing the pH of the bacterial culture after completion of fermentation and prior to concentration, formulation, freezing and freeze-drying of the bacterial culture results in increased storage stability of the freeze-dried product.

[0114] References Mogensen et al (2002) Bulletin of the International Dairy Federation, 377, 10-19.

[0115] Deposits and expert resolution Applicants request that samples of the deposited microorganisms referenced in Table 6 below be made available to the public only until the date this patent is granted. [Table 6]

[0116] Terms The present invention relates to the following provisions:

[0117] Clause 1. A method for preparing a bacterial culture, the method comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of the fermentation and obtaining a bacterial culture; (b) optionally concentrating the bacterial culture by a one-step enrichment method to obtain an enriched bacterial culture; and (c) optionally further comprising the step of adding a protective compound to the enriched bacterial culture of step (b); A method wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0.

[0118] Clause 2. The method comprises steps (a) and (b), or steps (a), (b) and (c), and further comprises: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture.

[0119] Clause 3. The method further comprises: 3. The method of claim 1 or 2, comprising: (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

[0120] Clause 4. The method further comprises: 4. The method according to claim 2 or 3, comprising: (f) packaging the frozen bacterial culture obtained in step (d) or the dried bacterial culture obtained in step (e).

[0121] Clause 5. The method of any one of clauses 1 to 4, wherein step (b) is carried out by a technique selected from the group consisting of centrifugation, vacuum evaporation and filtration.

[0122] Clause 6. The method according to any one of clauses 3 to 5, wherein step (e) is carried out by a technique selected from the group consisting of spray drying, spray freezing, vacuum drying, air drying, freeze-drying, shallow plate drying and vacuum shallow plate drying.

[0123] Clause 7. The bacterial culture comprises a fermenting bacterium: From the phylum Firmicutes, e.g.: Lactic acid bacteria (LAB), preferably Streptococcus (e.g., Streptococcus thermophilus, etc.), Lactococcus (e.g., Lactococcus lactis, etc.), Oenococcus (e.g., Oenococcus oeni), Leuconostoc (e.g., species such as Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides), Lactobacillus, Rimosilactobacillus, Lacticaseibacillus, Ridilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactipranchibacillus, Rimosilactobacillus, Ridilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and genera selected from the group consisting of Eubacterium (e.g., Eubacterium limosum, Eubacterium agregans, Eubacterium barkeri, Eubacterium lentum, etc.), Roseburia (Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faeces and Roseburia sesicola), Faecalibacterium (e.g., species such as Faecalibacterium prausnitzii), Anaerostipes (e.g., Anaerostipes caccae), Anaerobutyricum (e.g., species such as Anaerobutyricum hallii and Anaerobutyricum souengenii), etc.; From the phylum Actinobacteria, for example the genera Bifidobacterium (e.g. species Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, etc.), Propionibacterium (e.g. species Propionibacterium freudenreici, etc.), and Cutibacterium (e.g. Cutibacterium acnes, etc.), from the phylum Bacteroidetes, for example the genera Bacteroides (e.g. species Bacteroides fragilis, Bacteroides xylansorbens, etc.), Prevotella (e.g. species Prevotella copri, etc.) or Alistipes, and / or From the phylum Verrucomicrobium, e.g. Ackermansia (e.g. species such as Ackermansia muciniphila): 7. The method according to any one of clauses 1 to 6, comprising or consisting of:

[0124] Clause 8. The method of any one of clauses 1 to 7, wherein the fermentation medium contains at least one carbohydrate.

[0125] Clause 9. The method of clause 8, wherein the carbohydrate 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 fructooligosaccharide; or, for example, maltodextrin with DE3-20; glucose syrup with DE21-39; and a polysaccharide, such as starch or inulin.

[0126] Clause 10. The method of any one of clauses 1 to 9, wherein the fermentation medium contains at least one nitrogen source, such as peptone, yeast extract, one or more amino acids or one or more ammonia salts.

[0127] Clause 11. The method of any one of clauses 1 to 10, wherein the fermentation medium contains one or more yield enhancing agents selected from the group consisting of purine bases, pyrimidine bases, nucleosides, nucleotides and derivatives thereof.

[0128] Clause 12. The method of any one of clauses 1 to 11, wherein the fermentation medium contains one or more vitamins.

[0129] Clause 12a. The method of any one of clauses 1-11, wherein the fermentation medium contains one or more fatty acids.

[0130] Clause 13. The method according to any one of clauses 1 to 12, wherein the total concentration of carbohydrates in the fermentation medium is 1 to 15% w / w, preferably 1 to 10% w / w.

[0131] Clause 14. The method of any one of clauses 1 to 13, wherein the protective compound is selected from the group consisting of cryoprotectants, lyoprotectants, antioxidants, nutrients, bulking agents, flavoring agents, and mixtures thereof.

[0132] Clause 15. The method according to any one of clauses 1 to 14, wherein the concentration of the protective compound in the concentrated bacterial culture in step (c) is from 5 to 90% w / w, assessed on dry weight, preferably from 25 to 75% w / w, assessed on dry weight, more preferably from 50 to 75% w / w, assessed on dry weight.

[0133] Clause 16. The method of any one of clauses 1 to 15, wherein the fermentation in step (a) lasts for about 4 hours to about 7 days.

[0134] Clause 17. The method of any one of clauses 1 to 16, wherein the fermentation in step (a) is at a temperature of about 25°C to about 50°C.

[0135] Clause 18. The method of any one of clauses 1-17, wherein termination of the fermentation in step (a) is immediately followed by a holding time of from about 1 minute to about 10 hours.

[0136] Clause 19. The method of clause 18, wherein during said holding period the bacterial culture is held at a temperature between 4°C and 45°C, preferably between 5°C and 37°C, between 6°C and 25°C, or more preferably between 7°C and 15°C.

[0137] Clause 20. The method according to any one of clauses 1 to 19, wherein from the start to the end of the fermentation in step (a), the pH decreases to below 6.0, such as below 5.5, below 5.0, below 4.5, below 4.0, below 3.5, below 3.0, and / or decreases by at least 0.1 pH units, preferably at least 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 pH units.

[0138] Clause 21. The process of any one of clauses 1 to 20, wherein the pH adjustment following step (a), (b) or (c) is by addition of a base selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide and sodium carbonate.

[0139] Article 22. The dried bacterial culture has a water activity (a w 22. The method according to any one of clauses 3 to 21, wherein the ratio of the above-mentioned stoichiometric ratio to the total molecular weight of the compound is in the range of 0.01 to 0.8, preferably in the range of 0.05 to 0.6, and more preferably in the range of 0.05 to 0.4.

[0140] Clause 23. The method of any one of clauses 3 to 22, wherein the dried bacterial culture is in the form of granules and / or powder.

[0141] Article 24. Frozen bacterial cultures are 4 ~10 12 The method of any one of clauses 2 to 23, wherein the CFU / g of frozen bacterial culture is in the range.

[0142] Article 25. The dried bacterial culture is 4 ~10 13 The method of any one of clauses 3 to 24, wherein the CFU / g dried bacterial culture is in the range.

[0143] Clause 26. The dried bacterial culture is heated at 30°C and w After 16 weeks of storage at 10 4 ~10 13 26. The method of any one of clauses 3 to 25, comprising a content of viable fermentative bacteria in the range of CFU / g dried bacterial culture.

[0144] Article 27. 30°C and a w 27. The method of any one of clauses 3 to 26, wherein the loss of viability of the dried bacterial culture measured by CFU / g after 16 weeks of storage at CFU / g = 0.3 is less than 4 log units, preferably less than 3 log units, more preferably less than 2 log units, even more preferably less than 1 log unit or less than 0.5 log units.

[0145] Clause 28. A bacterial culture obtained by the method of any one of clauses 1 to 27.

[0146] Clause 29. A method for reducing loss of viability and / or activity during freezing and / or drying of a bacterial culture, comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 until the end of fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

[0147] Clause 30. A method for increasing the storage stability of a frozen and / or dried bacterial culture, comprising: (a) cultivating a fermenting bacterium in a fermentation medium with a pH lower than pH 6.0 until the end of fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step enrichment method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the enriched bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

Claims

1. 1. A method for preparing a bacterial culture, the method comprising: (a) cultivating a fermentative bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of fermentation and obtaining a bacterial culture; (b) optionally concentrating the bacterial culture by a one-step concentration method to obtain a concentrated bacterial culture; and (c) optionally further comprising the step of adding a protective compound to the concentrated bacterial culture of step (b); A method wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.

0.

2. The method comprises steps (a) and (b), or steps (a), (b) and (c), and further comprises: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or 10. The method of claim 1, comprising: (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

3. 3. The method of claim 1, wherein step (b) is carried out by a technique selected from the group consisting of centrifugation, vacuum evaporation, and filtration.

4. 3. The method of claim 2, wherein step (e) is carried out by a technique selected from the group consisting of spray drying, spray freezing, vacuum drying, air drying, freeze-drying, shallow tray drying, and vacuum shallow tray drying.

5. The bacterial culture comprises a fermenting bacterium: From the phylum Firmicutes, for example: Lactic acid bacteria (LAB), preferably Streptococcus (e.g., Streptococcus thermophilus), Lactococcus (e.g., Lactococcus lactis), Oenococcus (e.g., Oenococcus oeni), Leuconostoc (e.g., species such as Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides), Lactobacillus, Rimosilactobacillus, Lacticazeibacillus, Ridilactobacillus, Lacticazeibacillus, Lacticazeibacillus, Lactiprantibacillus, Rimosilactobacillus, Ridilactobacillus, Retinactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and genera selected from the group consisting of Eubacterium (e.g., Eubacterium limosum, Eubacterium agregans, Eubacterium barkeri, Eubacterium lentum, etc.), Roseburia (Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faeces and Roseburia sesicola), Faecalibacterium (e.g., species such as Faecalibacterium prausnitzii), Anaerostipes (e.g., Anaerostipes caccae), Anaerobutyricum (e.g., species such as Anaerobutyricum hallii and Anaerobutyricum souengenii), etc.; from the Actinobacteria phylum, for example, the genera Bifidobacterium (e.g., species such as Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium breve), Propionibacterium (e.g., species such as Propionibacterium freudenreichii), and Cutibacterium (e.g., Cutibacterium acnes), from the phylum Bacteroidetes, for example from the genera Bacteroides (e.g. species such as Bacteroides fragilis, Bacteroides xylansorbens), Prevotella (e.g. species such as Prevotella copri) or Alistipes, and / or From the Verrucomicrobium phylum, for example, Ackermansia (e.g., species such as Ackermansia muciniphila):

2. The method of claim 1, comprising or consisting of:

6. The fermentation medium comprises: (i) at least one carbohydrate; (ii) at least one nitrogen source, such as peptone, yeast extract, one or more amino acids, or one or more ammonia salts; (iii) one or more yield enhancers selected from the group consisting of purine bases, pyrimidine bases, nucleosides, nucleotides, and derivatives thereof; (iv) one or more vitamins; and / or 10. The method of claim 1, further comprising: (v) one or more fatty acids.

7. (i) the carbohydrate 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 a maltodextrin with a DE of 3-20; a glucose syrup with a DE of 21-39; and a polysaccharide such as starch or inulin; and / or (ii) The process according to claim 6, wherein the total concentration of carbohydrates in the fermentation medium is 1 to 15% w / w, preferably 1 to 10% w / w.

8. The protective compound is selected from the group consisting of cryoprotectants, lyoprotectants, antioxidants, nutrients, bulking agents, flavoring agents, and mixtures thereof.

2. The method of claim 1, optionally wherein the concentration of the protective compound in the concentrated bacterial culture in step (c) is 5 to 90% w / w, measured on dry weight, preferably 25 to 75% w / w, measured on dry weight, more preferably 50 to 75% w / w, measured on dry weight.

9. (i) the fermentation in step (a) lasts from about 4 hours to about 7 days; (ii) the fermentation in step (a) is at a temperature of about 25°C to about 50°C; (iii) completion of the fermentation in step (a) is immediately followed by a hold time of from about 1 minute to about 10 hours, optionally wherein during the hold time the bacterial culture is held at 4°C to 20°C, preferably 5°C to 37°C, 6°C to 25°C, or more preferably 7°C to 15°C; and / or 4. The method of claim 1, wherein (iv) from the beginning to the end of the fermentation in step (a) the pH is reduced to 6.0 or less, such as 5.5 or less, 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, 3.0 or less, and / or reduced by at least 0.1 pH units, preferably by at least 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 pH units.

10. 10. The method of claim 1, wherein the pH adjustment following steps (a), (b), or (c) is by the addition of a base selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide, and sodium carbonate.

11. The dried bacterial culture comprises: (i) Water activity (a ｗ ) in the range of 0.01 to 0.8, preferably in the range of 0.05 to 0.6, more preferably in the range of 0.05 to 0.4; (ii) in the form of granules and / or powder; (iii) 10 ４ ~10 １３ containing dried bacterial cultures in the range of CFU / g; (iv) 30°C and a ｗ After 16 weeks of storage at 10 ４ ~10 １３ and / or containing viable fermentative bacteria in the range of CFU / g of dried bacterial culture; (v) 30°C and a ｗ = 0.3 after 16 weeks of storage, the method of claim 2, wherein the viability loss measured by CFU / g is less than 4 log units, preferably less than 3 log units, more preferably less than 2 log units, even more preferably less than 1 log unit or less than 0.5 log units.

12. The frozen bacterial culture ４ ~10 １２ 3. The method of claim 2, comprising frozen bacterial cultures in the range of CFU / g.

13. A bacterial culture obtainable by the process of claim 1.

14. 1. A method for reducing viability and / or activity loss during freezing and / or drying of a bacterial culture, the method comprising: (a) cultivating a fermentative bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step concentration method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the concentrated bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

15. 1. A method for increasing the storage stability of a frozen and / or dried bacterial culture, the method comprising: (a) cultivating a fermentative bacterium in a fermentation medium with a pH lower than pH 6.0 at the end of fermentation and obtaining a bacterial culture; (b) concentrating the bacterial culture by a one-step concentration method to obtain a concentrated bacterial culture; and (c) optionally adding a protective compound to the concentrated bacterial culture of step (b); wherein the pH is adjusted after step (a), (b) or (c) to a pH in the range of pH 6.0 to pH 8.0; and further comprising: (d) freezing the concentrated bacterial culture to obtain a frozen bacterial culture; and / or (e) removing water from the concentrated or frozen bacterial culture to obtain a dried bacterial culture.

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