Method for producing stable cultures

Abstract

The present invention relates to the field of frozen or dried compositions directed against prokaryotes, in particular fermentative bacteria such as lactic acid bacteria, to a process for the preparation of frozen or dried prokaryote compositions with improved stability, and to the compositions that can be prepared by said process.

Description

[0001] The present invention relates to the field of frozen or dried compositions targeting certain bacteria, in particular fermentative bacteria such as lactic acid bacteria, to a method for preparing a frozen or dried bacterial composition and to a composition that can be prepared by said method. Background of the Invention

[0003] Fermentative bacteria are anaerobic bacteria in which organic compounds (rather than oxygen) are the terminal electron (or hydrogen) acceptors in their metabolism.

[0004] According to the pattern of products formed in the fermentation, bacteria are divided into homofermentation and heterofermentation. Lactic acid bacteria (LAB) with homofermentative metabolism produce lactic acid as the main product or the only product of sugar fermentation. Examples of homofermentative lactic acid bacteria are species of Lactococcus lactis, Lactobacillus delbrueckii subsp.bulgaricus or Streptococcus thermophilus. Heterofermentative bacteria produce various products from the fermentation of sugar, and the end product depends on the type of sugar used in the fermentation. Heterofermentative lactic acid bacteria, such as species of Oenococcus, Leuconostoc and some Lactobacillus, for example Lactobacillus reuteri, in addition to fermenting sugar into lactate, CO2 and ethanol, also ferment it into acetate and polyols. The present invention is applicable to both types of fermenting bacteria.

[0005] Fermentative bacteria are involved in many industrially relevant processes. For example, bacterial cultures (especially those usually classified as LAB) are essential in the production of all fermented dairy products, cheese and butter. Cultures of such bacteria may be called starter cultures, and they impart specific characteristics to various dairy products by performing a variety of functions.

[0006] Many lactic acid bacteria are known to have probiotic properties (i.e., they have beneficial health effects on humans and animals when ingested). Probiotics are widely used in dried form. In most cases, after long-term storage of the dried product, the microorganisms must remain viable in order for them to exert their beneficial effects.

[0007] It is well known in the art that bacteria can easily lose vitality when exposed to various stresses, so it is common practice to use protective agents in the industrial production of bacterial cultures. These protective agents are considered to protect cells in different steps of the production process and later during the shelf life of dried bacteria. Bacteria to be frozen or dried (e.g., spray drying, freeze drying, vacuum drying) are mixed with protective agents as a cell suspension and then processed in a series of different technical steps. The role of the protective agent is to protect the bacterial cell composition during freezing (so-called cryoprotectants), drying or freeze drying (so-called lyoprotectants). However, some damage to cells in these processes is inevitable (Coulibaly et al. (2018) ARRB 24 (4): 1-15).

[0008] Bacterial products can be formulated as frozen products. For example, commercial starters can be distributed as frozen cultures. Highly concentrated frozen cultures, especially when prepared as granules, are very useful commercially because these cultures can be directly inoculated into fermentation media (such as milk or meat) without intermediate transfer. In other words, this highly concentrated frozen culture contains a certain amount of bacteria that makes the end user's internal mother starter too much. "Bulk starter" is defined herein as a starter that is proliferated in a food processing plant for inoculation into a fermentation medium. Highly concentrated cultures can be referred to as direct-dump starter (DVS)-cultures. In order to contain enough bacteria for the end user to use as a DVS culture, the concentrated frozen culture must generally have a weight of at least 50 g and at least 10 9 Viable bacterial content in colony forming units (CFU). WO 2005 / 080548 (Chr. Hansen) discloses pelleted frozen lactic acid bacteria (LAB) cultures which are stabilized with, for example, a mixture of trehalose and sucrose and which do not form lumps on storage.

[0009] The prior art discloses that in all intermediate steps of the process, including in the step of preparing the cell concentrate with a protective agent, the cell culture is kept at 4°C in order to limit cell degradation reactions. (Fonseca et al. (2015) Chapter 24: Freeze-Drying of Lactic Acid Bacteria in Wolkers & Oldenhof (Eds), Cryopreservation and Freeze-drying Protocols, 3rd edition).

[0010] In the prior art process, concentrated bacterial cultures are obtained by known methods of growing the bacteria in a growth medium and then concentrating the culture, e.g. by centrifugation, while the bacteria are separated from the growth medium. The concentrated culture is then mixed with the desired preservative and shortly thereafter, the resulting mixture is frozen or dried.

[0011] The microbial cell surface has a very complex composition and plays a key role in the interaction between microorganisms and the surrounding environment (Burgain J, et al (2014) Advances in Colloid and Interface Science 213, 21–35).

[0012] The cell wall of Gram-positive bacteria consists of a peptidoglycan layer in which teichoic acid, lipoteichoic acid and cell wall polysaccharides are embedded. The peptidoglycan layer can be covered by a protein S layer and modified by various polysaccharides (Zeidan et al 2017, FEMSMicrobiology Reviews 41: 168-200). The surface of Gram-negative bacteria is different. It is made of capsular polysaccharides, which are modified by various polymeric substances such as carbohydrates, lipooligosaccharides and lipopolysaccharides. This complex composition of the cell surface can be captured by physicochemical analyses, such as measuring cell-surface interactions by hydrophobicity analysis and determining cell surface charge by zeta potential. In particular, the combination of these two assays with advanced microscopy techniques has helped to provide a deeper characterization of the cell wall of lactic acid bacteria ( and Ubbink (2003) Biophysical Journal 85, 4076-4092). Hydrophobicity analysis was originally developed by Rosenberg et al. (1980, FEMS Microbiology Letters 9, 29-33) to measure the adhesion of bacterial cells to liquid hydrocarbons. and Ubbink (op. cit.) improved it to the determination of the interfacial adhesion curve, which reflects the partitioning of bacteria from the aqueous phase to the organic phase in hexadecane. Based on the interfacial adhesion curve and the pattern of the zeta potential, the main components of the cell surface can be distinguished. It was found that the presence of surface proteins is associated with the high isoelectric point and high hydrophobicity of the surface. Teichoic acid makes the surface hydrophobic and strongly negatively charged. A large amount of polysaccharides makes the cell surface hydrophilic and weakly charged.

[0013] The characterization of cell surface properties and their association with bacterial growth conditions has been the subject of many studies ( et al (2005) AEM 71, 8165–8173; and Millsap KW et al (1996) J. Microbiol. Methods 27, 239-242). These studies showed that the composition of the growth medium during fermentation has a significant effect not only on cell yield but also on the cell surface properties of lactic acid bacteria.

[0014] In the industrial process of producing beneficial bacteria, it is important that the bacterial cells show a high degree of robustness in several steps of the downstream process and remain viable after fermentation. The connection between the physicochemical properties of cells and cell viability in downstream processes has only been described in a study by Zupancic et al. (Pharmaceutics 2019, 11, 483; doi: 10.3390 / pharmaceutics11090483). In this work, it was demonstrated that lactic acid bacteria with hydrophobic cell surfaces survived better during electrospinning than bacteria with hydrophilic cell surfaces. However, Shakirova et al. J Ind Microbiol Biotechnol (2013) 40: 85-93, showing an inverse relationship between cell surface hydrophobicity and the viability of cells subjected to subsequent conditions such as long-term storage. SUMMARY OF THE INVENTION

[0016] The present invention arises from the unexpected observation that cells with a certain cell surface hydrophobicity show improved tolerance to long term storage if certain protectant compounds are added in downstream processing.The present invention will now be described in more detail.

[0017] The present invention provides a method for preparing a frozen, dried or lyophilized product comprising a non-spore-forming prokaryotic organism, the method comprising the following steps:

[0018] i. Cultivating prokaryotic cells by fermentation;

[0019] ii. Concentrating the cells by separating the fermentation broth;

[0020] iii. mixing the cell concentrate with a medium containing a protective compound to obtain a pretreatment composition;

[0021] iv. maintaining the pretreatment composition under conditions that do not induce metabolic activity;

[0022] v. Preservation of the composition by

[0023] (a) freezing the composition to form a frozen prokaryotic product;

[0024] (b) drying the product to form a dried prokaryotic product; or

[0025] (c) freezing the composition to form a frozen prokaryotic intermediate product and then lyophilizing the intermediate product to form a lyophilized prokaryotic product,

[0026] When Φ[V H / V B ] when measured at least at one point between 0.1 and 1.0, measured by the MATH method at 22°C and expressed as [(initial OD 600 -Final OD 600 ) / initial OD 600 ]*100, and the initial OD 600 (nm) is 0.5, the cell surface hydrophobicity of cells in the product is at least 40%, and the efficacy of the product is 1E+08 to 1E+13 CFU / g.

[0027] In terms of the increase in hydrophobicity, the starting and ending values ​​should be at the same Φ[V H / V B ] value.

[0028] The fermentation broth typically has 5E+08 to 1E+11 total cells / g of fermentation broth, where "total cells" refers to both viable and non-viable cells, and the weight of the fermentation broth includes cells suspended therein. The concentration of cells in the liquid can be measured by standard techniques, such as the Petroff Hausser counting chamber method or flow cytometry.

[0029] The concentrated culture ("cell concentrate") is typically formed by separating cells from the fermentation broth with a concentration factor of 2x to 90x, typically 5x to 60x, such as 10x to 50x or 20x to 40x. Thus, the total concentration of cells in the cell concentrate will be in the range of 1E+09 prokaryotic cells / g to 9E+12 prokaryotic cells / g, preferably 2.5E+09 prokaryotic cells / g to 3E+12 prokaryotic cells / g, 1.3E+10 prokaryotic cells / g to 2E+12 prokaryotic cells / g, 2E+10 prokaryotic cells / g to 1.3E+12 prokaryotic cells / g, 3E+10 prokaryotic cells / g to 2.5E+11 prokaryotic cells / g or 4.5E+10 prokaryotic cells / g to 1E+11 prokaryotic cells / g.

[0030] For example, the protective compound can be one or more of: 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 oligofructose or, for example, maltodextrin with DE 3-20; polysaccharides, such as starch or inulin; cryoprotectants and / or lyoprotectants and / or storage stabilizers, such as gum arabic, maltodextrin, starch, pectin, cellulose, xylan or polyols such as glycerol, sucrose, trehalose or maltose, proteins such as gelatin, peptides such as peptides provided by yeast extract, amino acids such as proline or sugar alcohols such as sorbitol, mannitol or inositol, antioxidants such as sodium ascorbate, sodium citrate.

[0031] Preferably, step (ii) lasts from 0.25 hours to 16 hours and is best carried out at 4°C to 20°C, preferably below 10°C.

[0032] Preferably, the dry weight ratio of the final protectant to the cell concentrate of the frozen prokaryotic product or frozen prokaryotic intermediate product is from 10:1 to 0.1:1, preferably from 3:1 to 0.5:1, and most preferably from 2:1 to 1:1.

[0033] The method of the present invention is widely applicable. Prokaryotes can be fermentative bacteria such as

[0034] From the phylum Firmicutes, for example:

[0035] Lactic acid bacteria (LAB), preferably selected from the group consisting of Streptococcus (e.g., Streptococcus thermophilus), Lactococcus (e.g., Lactococcus lactis), Oenococcus (e.g., Oenococcus oeni), Leuconostoc (e.g., species of Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, pseudomesenteroides) species), Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactaseibacillus, Lactiplantibacillus, Lactobacillus mucilaginosus, Lactobacillus united, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Lactaseibacillus, and Lactiplantibacillus;Eubacterium (e.g., Eubacterium limosum, Eubacterium aggregans, Eubacterium barkeri, Eubacterium lentum), Roseburia (Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faecis, and Roseburia cecicola), Faecalibacterium (e.g., Faecalibacterium prausnitzii), prausnitzii) species), anaerobic Corynebacterium (such as Anaerostipes saccae), anaerobic butyricum (such as species of Anaerobutyricum hallii, species of Anaerobutyricum soehngenii);

[0036] From the Actinobacteria, such as Bifidobacterium (e.g.

[0037] The following species: Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve), Propionibacterium (such as species of Propionibacterium freudenreichii), Cutibacterium (such as Cutibacterium acnes);

[0038] From the phylum Bacteroidetes, such as the genus Bacteroides (such as the following species:

[0039] Bacteroides fragilis, Bacteroides xylanisolvens, Prevotella (such as species of Prevotella copri) or Alistipes;

[0040] or from the phylum Verrucomicrobia, such as the genus Akkermansia (eg species Akkermansia muciniphila).

[0041] In particular, the prokaryotic organism may be one or more of the following: Limosilactobacillus reuteri, Lactobacillus rhamnosus, Ligilactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sake subsp. sakei subsp. sakei), Lactiplantibacillus pentosus, Levilactobacillus brevis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus jensenii, and Lactobacillus iners.

[0042] The present invention also provides a frozen or dried product comprising non-spore-forming prokaryotes obtainable by the above method.

[0043] The potency of frozen or dried products can be 1E+08 CFU / g–1E+13 CFU / g.

[0044] The method is applicable to vegetative cells of prokaryotic microorganisms from the domains of bacteria and archaea.The present invention relates to a broad spectrum of non-spore-forming microorganisms in food and feed production, agriculture, medicine, for the production of biofuels and bio-based chemicals.

[0045] Non-spore-forming bacteria can be identified within the phyla Firmicutes, Actinobacteria and Bacteroidetes. The present invention is particularly applicable to homofermentative and heterofermentative lactic acid bacteria within the phylum Firmicutes, and Bifidobacteria and Propionibacteria within the phylum Actinobacteria. The present invention is also applicable to obligate anaerobes of the class Clostridium of the phylum Firmicutes, such as fermentative, butyrate-producing bacteria of the genus Roseburia (e.g., Roseburia hominis and Roseburia inulinivorans), Anaerobutyricum hallii, Anaerobutyricum soehngenii, Eubacterium (e.g., Eubacterium limosum), Anaerostipes (e.g., Anaerostipes caccae), and Faecalibacterium (e.g., F. prausnitzii), which represent the core microbiota of the human intestine and are candidates for the next generation of probiotics.

[0046] The most industrially useful lactic acid bacteria are found in species of the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus (including all species classified as Lactobacillus before 2020), Leuconostoc, Oenococcus, Bifidobacterium, Propionibacterium and Pediococcus. Therefore, in a preferred embodiment, the lactic acid bacteria are selected from the group consisting of these lactic acid bacteria.

[0047] The lactic acid bacteria are preferably selected from the genus consisting of Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus. Specifically, they can be Limosilactobacillus rueteri, 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 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 animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium longum longum, Bifidobacterium adolescentis, Bifidobacterium breve, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

[0048] The composition may comprise one or more strains of lactic acid bacteria, which may be selected from the group consisting of: (Bifidobacterium animalis subsp lactis ), DSM15954; ATCC 29682, ATCC 27536, DSM 13692, DSM 10140, (Lactobacillus acidophilus )、DSM 13241、 (Lactobacillus rhamnosus )、ATCC 53103、 (Lactobacillus rhamnosus )、ATCC 55826、 (Lactobacillus reuteri ), ATCC 55845, Lactobacillus casei (L.casei )(Lactobacillus paracaseisubsp.paracasei L.casei) Lactobacillus casei )、ATCC 55544、 (Lactobacillus paracasei )、LMG-17806、 (Streptococcus thermophilus )、DSM15957、 (Lactobacillus fermentum ), NM02 / 31074 and (Lactobacillus casei subsp. casei ), CCTCC M204012.

[0049] The LAB culture may be a "mixed lactic acid bacteria (LAB) culture" or a "pure lactic acid bacteria (LAB) culture". The term "mixed lactic acid bacteria (LAB) culture" or "LAB" culture refers to a mixed culture comprising two or more different LAB species. The term "pure lactic acid bacteria (LAB) culture" refers to a pure culture comprising only a single LAB species. Therefore, in a preferred embodiment, the LAB culture is a LAB culture selected from the group consisting of these cultures.

[0050] The LAB cultures may or may not be washed prior to mixing with the protectant.

[0051] Preferably, the LAB cells are probiotic cells.

[0052] The frozen or dried cells can be mixed with any suitable excipients to prepare mixtures, such as human food and animal feed compositions.

[0053] The frozen or dried product comprising non-spore-forming prokaryotes obtainable by the above-described process can be used to produce various types of compositions wherein the potency of the bacteria is between 1E+05 CFU / g and 1E+12 CFU / g.

[0054] The composition may be a food, a feed, an agricultural product, a dietary supplement or a pharmaceutical product.

[0055] The frozen or dried product comprising non-spore-forming prokaryotes obtainable by the above method may also be used in a method for manufacturing a food, feed, agricultural product, dietary supplement or pharmaceutical product, the method comprising adding the frozen or dried product. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Freeze-dried Bifidobacterium animalis subsp. lactis without a protectant (FD 0) and with a protectant (FD 1) are shown. Interfacial adhesion curves of BCSH assay. All data points are the mean and standard deviation of three measurements.

[0057] Figure 2 Freeze-dried Lactobacillus acidophilus without a protective agent (FD 0) and with a protective agent (FD 1) are shown. Interfacial adhesion curves of BCSH assay. All data points are the mean and standard deviation of three measurements.

[0058] Figure 3 Freeze-dried Lactobacillus reuteri without a protectant (FD 0) and with a protectant (FD 1) are shown. Interfacial adhesion curves of the BCSH assay. All data points are the mean and standard deviation of three measurements.

[0059] Figure 4 Interfacial adhesion curves from the BCSH assay of freeze-dried Lactobacillus animalis LA51 without protectant (FD 0) and with protectant (FD 1) are shown. All data points are the mean and standard deviation of three measurements.

[0060] Figure 5 Freeze-dried Streptococcus thermophilus without a protectant (FD 0) and with a protectant (FD 1) are shown. Interfacial adhesion curves of BCSH assay for HA. All data points are the mean and standard deviation of three measurements.

[0061] Figure 6Interfacial adhesion curves of the BCSH assay of freeze-dried Lactococcus lactis R-607-1 without protectant (FD 0) and with protectant (FD 1) are shown. All data points are the mean and standard deviation of three measurements.

[0062] Figure 7. (A) Relationship between viability loss and cell surface hydrophobicity of lyophilized products without protective agents in 37°C storage stability test. (B) Relationship between viability loss and cell surface hydrophobicity of lyophilized products with protective agents in 37°C storage stability test.

[0063] definition

[0064] Fructooligosaccharides (FOS), also known as oligofructose or oligofructan, are a mixture of oligosaccharides. FOS can be produced by the degradation of inulin or polyfructose, which is a polymer of D-fructose residues linked to terminal α-linked D-glucose via β(2→1) bonds. The degree of polymerization of inulin ranges from 10 to 60. Inulin can be degraded enzymatically or chemically into oligofructose residues with the general structure Glu–Fru n (Abbreviated as GF n ) and Fru m (F m ) with n and m ranging from 1 to 7. This process also occurs to a certain extent in nature, and these oligosaccharides can be found in a large number of plants, especially in Jerusalem artichoke, chicory and blue agave plants. The main components of commercial products are gnatsose (GF2), gnatsose (GF3), fructosylnastose (GF4), rye bi-oligosaccharides (GF3), inulinose (F2), inulinose (F3) and inulinose (F4). The second type of FOS is prepared by the transfructosylation of sucrose by β-fructosidase from Aspergillus niger or Aspergillus. The resulting mixture has the general formula GF n , where n ranges from 1 to 5. In contrast to inulin-derived FOS and the β(1→2) binding, other linkages do exist, but in limited numbers. In this patent application, "FOS" and related terms are used to describe the second type of FOS. Example

[0065] The examples relate to the strains listed in Table 1. All strains have been deposited with a depository which has been granted the status of an international depository under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institut DSMZ-German Collection of Microorganisms, Inhofenstrasse 7B, 38124 Braunschweig, Germany. The accession numbers are given in Table 1.

[0066] The applicant requests that, in accordance with existing regulations of industrial property offices of contracting states of the Budapest Treaty, samples of the deposited microorganisms described below may only be provided to experts before the date of grant of the patent.

[0067] Table 1. Overview of strains used in the examples

[0068]

[0069] The recipe of the preservative composition was adapted from the book (Cryopreservation and Freeze-drying Protocols) by Wolkers & Oldenhof (eds.), 3rd edition (2015) Chapter 24: Freeze-Drying of Lactic Acid Bacteria, Fernanda Fonseca, Stéphanie Cenard and Stéphanie Passot, page 480, with the following modifications: 200 g / l sucrose was replaced with 150 g / l trehalose and 50 g / l gum arabic, 9 g / l NaCl was retained, and 5 g / l sodium ascorbate was increased to 10 g / l sodium ascorbate in demineralized water.

[0070] The various single protective agents can be derived, for example, from the following sources: glucose (dextrose monohydrate, Roquette Freres, France), lactose (lactose monohydrate, Arla Food Ingredients Group P / S, Denmark), IT12 (trade name of maltodextrin DE 12, Roquette Freres, France), oligofructose (FOS, oligofructose 950P, Beghin-Meiji, France), trehalose (trehalose dihydrate, Cargill, Germany), inositol (Zhucheng, China, Haotian Pharmaceutical Co., Ltd.), Pectin YM-115-H (CP Kelco, Denmark) and gum arabic (Willy Benecke GmbH, Natural Gums, Germany). FOS is a mixture of sugars with chain lengths varying between 1 and 5 sugar units, 31 g-43 g GF2 / 100 g; 47 g-59 g GF3 / 100 G and 4-16 g GF4 / 100 g FOS. IT 12 contains oligomers with 11-14 dextrose equivalents (97%); glucose (1%) and disaccharides (2%).

[0071] The carbohydrates were autoclaved for 20 min at 121° C. Sodium ascorbate was prepared by sterile filtration and mixed with the autoclaved carbohydrates immediately before use.

[0072] Viable cells were counted using the standard pour plate method. Lactobacillus animalis LA51, Lactobacillus reuteri The viable cell count was performed in duplicate. The lyophilized material was suspended in sterile peptone saline diluent and homogenized by slapping homogenization. After 30 minutes of resuscitation, the slapping homogenization was repeated and the cell suspension was serially diluted in peptone saline diluent. The dilutions were plated in duplicate on MRS agar (BD Difco TM Lactobacillus MRS agar, Fisher Scientific). Agar plates were incubated anaerobically at 37°C for three days. Plates with 30-300 colonies were selected for colony forming unit (CFU) counting. Results were reported as average CFU / g lyophilized sample, calculated based on quadruplicate.

[0073] Bifidobacterium animalis subsp. lactis was determined by the same method as for Lactobacillus The difference is that MRS agar (BD Difco TM Lactobacillus MRS agar, Fisher Scientific) supplemented with 0.5 g / l cysteine ​​hydrochloride.

[0074] Streptococcus thermophilus The viable cell assay for HA was similar to that for Lactobacillus with the following modifications: M-17 agar (Oxoid, England) was used instead of MRS agar. Incubations were performed at 37°C under aerobic conditions.

[0075] The determination of viable cells of Lactococcus lactis R-607-1 was similar to that of Lactobacillus, but with the following modifications: M-17 agar (Oxoid, England) was used instead of MRS agar. The incubation was carried out at 30°C under aerobic conditions.

[0076] Stability Assessment. The stability of the cells was assessed based on the difference between the CFU / g measured at time 0 of the stability test and at specific sampling points during the stability test. Viability loss was quantified as the loss in log CFU / g.

[0077] The MATH method was used to measure the cell surface hydrophobicity and determine the interfacial adhesion curve. &Ubbink ((2003) Biophysical Journal 85, 4076-4092)) was applied with modified buffer strength and using cell wash in the initial step of the procedure: 0.2 g of lyophilized pellet 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 of ≤10°C. The supernatant was removed and the cells were washed twice with 100 mM sodium phosphate buffer. The washed cell pellet was resuspended in 100 mM sodium phosphate buffer to an optical density OD600nm of 0.5±0.05. The suspension was mixed and 3 ml aliquots were pipetted into plastic tubes. Hexadecane (99% purity, Sigma-Aldrich) was added to the cell suspension in the following volumes: 10 μl, 30 μl, 100 μl, 200 μl, 400 μl, 800 μl, 1400 μl and 2000 μl of hexadecane. Each combination of hexadecane and cell suspension in buffer was prepared, Φ[V H / V B ] in triplicate. Cap the tubes tightly and vortex each mixture at maximum speed for 30 seconds. Repeat vortexing for 30 seconds for the entire sample series. Let the samples stand for 5 minutes. Transfer 2 ml of the aqueous phase to a cuvette for OD 600nm The bacterial cell surface hydrophobicity (BCSH) was calculated from the proportion of bacteria adhering to the hexadecane / water interface according to the following formula:

[0078] BCSH (%) = [(initial OD600 - final OD600) / initial OD600] * 100

[0079] If the cell partitions to a BCSH < 20%, the cell surface is classified as non-hydrophobic, i.e., hydrophilic. Hydrophobic cell surfaces are characterized by a cell partition of BCSH > 50%, while moderately hydrophobic surfaces have a BCSH in the range of 20%-50% (Lee and Yii (1996) Letters in Applied Microbiology 23:343-346).

[0080] Example 1

[0081] Bifidobacterium animalis subsp. lactis The cells were cultured in MRS medium (BD Difco) supplemented with 0.5 g / l cysteine ​​hydrochloride and 1 g / l sodium carbonate. TMLactobacillus MRS broth, Fisher Scientific). The inoculum for fermentation was prepared by growing the strain in a closed bottle with the above specified growth medium at 37°C under static conditions and without pH control. The incubation period was 16 hours. Fermentation was performed in a bench-scale fermentor by first inoculating 1% of the preculture into the above specified MRS medium. Fermentation was performed under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 37°C, and the pH set point was controlled at 6.0 by adding 24% aqueous ammonia. Fermentation was completed within 16 hours, at which time All sugars are utilized. The fermentation broth is cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by a concentration factor of 30x has a dry matter of 14% (w / w). The cell concentrate is kept at a temperature of <10°C during further processing. The cell concentrate is divided into two equal parts: 1 / a crude concentrate without any protective agent and 2 / a cell concentrate for preparation containing a protective agent. The protective agent contains 150g / l trehalose (trehalose dihydrate, Cargill, Germany), 50g / l gum arabic (Willy Benecke GmbH, Germany), 9g / l NaCl and 10g / l sodium ascorbate in demineralized water. The dry matter of the protective agent is 22% (w / w). The protective agent is added to the cell concentrate in an amount such that the ratio of the dry matter of the protective agent to the dry matter of the cell concentrate is 1:1, i.e., 0.64 g of the protective agent with a dry matter of 22% (w / w) is added to every 1 g of the cell concentrate with a dry matter of 14%. After the protective agent and the cell concentrate are mixed, the prepared concentrate is frozen by granulation in liquid nitrogen within 15 minutes. At the same time, the crude concentrate is granulated in liquid nitrogen. The frozen material is freeze-dried into products without protective agent and with protective agent, respectively. FD 0 and FD 1.

[0082] From freeze-dried products FD 0 and The cell surface hydrophobicity of cells of FD 1 was characterized. The interface adhesion curve ( Figure 1 )show The cell surface composition is highly stable and is not affected by the presence of protective agents. As the volume of hexadecane increases, the partitioning of cells into the hexadecane phase increases, which confirms the presence of hydrophobic molecules on the cell surface. H / V B )=0.1, FD 0 and The cell surface hydrophobicity of FD 1 was 44% ± 1% and 42% ± 2%, respectively. These values ​​will The cell surface is classified as moderately hydrophobic.

[0083] Example 2

[0084] Lactobacillus acidophilus In MRS medium (BD Difco TM Lactobacillus was grown in MRS broth (Fisher Scientific). The inoculum for fermentation was prepared by growing the strain in a closed bottle with MRS medium at 37°C under static conditions and without pH control. The incubation period was 16 hours. Fermentation was performed in a laboratory scale fermentor by first inoculating 1% of the preculture into MRS medium. Fermentation was performed under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 37°C, and the pH set point was controlled at 5.5 by adding 24% aqueous ammonia. Fermentation was completed in 9 hours, at which time All sugars were utilized. The fermentation broth was cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by concentration factor 30x had a dry matter content of 11% (w / w). The cell concentrate is further processed as described. The cell concentrate is prepared by The cell concentrate was prepared by adding 0.5 g of a preservative containing 22% dry matter (w / w). FD 0 and FD 1.

[0085] Freeze-dried products FD 0 and The cell surface hydrophobicity of FD 1 cells was characterized. Figure 2 The interfacial adhesion curves were compared. Although the composition of the cell surfaces with and without the protectant was slightly different, both types of cell surfaces can be classified as hydrophobic because FD 0 and FD 1 in Φ(V H / V B )=0.1, the cell surface hydrophobicity was 64%±3% and 51%±3%, respectively.

[0086] Example 3

[0087] Lactobacillus reuteri In MRS medium (BD Difco TMLactobacillus was grown in MRS broth (Fisher Scientific). The inoculum for fermentation was prepared by growing the strain in a closed bottle with MRS medium at 37°C under static conditions and without pH control. The incubation period was 16 hours. Fermentation was performed in a laboratory scale fermentor by first inoculating 1% of the preculture into MRS medium. Fermentation was performed under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 37°C, and the pH set point was controlled at 5.5 by adding 24% ammonia water. Fermentation was completed in 9 hours, at which time All sugars were utilized. The fermentation broth was cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by concentration factor 30x had a dry matter content of 8.6% (w / w). The cell concentrate is further processed as described. The cell concentrate is prepared by The cell concentrate was prepared by adding 0.39 g of a protective agent at 22% (w / w) dry matter. FD 0 and FD 1.

[0088] Freeze-dried products FD 0 and The cell surface hydrophobicity of FD 1 cells was characterized. Figure 3 The interfacial adhesion curve is shown. The shape of the curve reflects that the cells have highly partitioned toward the hexadecane phase when the minimum volume of hexadecane is used. This finding is related to the high abundance of hydrophobic molecules. The use of a protective agent further enhances the formation of hydrophobic structures, as shown by the increased response in the middle region of the interfacial adhesion curve. FD 0 and FD 1 in Φ(V H / V B )=0.1, the cell surface hydrophobicity was 64%±3% and 83%±3% respectively. The cell surface is classified as highly hydrophobic.

[0089] Example 4

[0090] Lactobacillus animalis In MRS medium (BD Difco TMLactobacillus MRS broth, Fisher Scientific). The inoculum for fermentation was prepared by growing the strain in closed bottles with MRS medium at 37°C under static conditions and without pH control. The incubation period was 7 hours. Fermentation was carried out in a laboratory scale fermentor by first inoculating 1% of the preculture into MRS medium. Fermentation was carried out under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 37°C and the pH set point of 5.5 was controlled by adding 24% ammonia water. Fermentation was completed within 7 hours when all the sugars were utilized by LA51. The fermentation broth was cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by a concentration factor of 30x had a dry matter of 13% (w / w). The cell concentrate was further processed as described. The formulation of LA51 cell concentrate was completed by adding 0.6 g of a protective agent containing 22% dry matter (w / w) per 1 g of LA51 cell concentrate. The freeze-dried materials LA51 FD 0 and LA51 FD 1 were produced.

[0091] The cell surface hydrophobicity of cells from the freeze-dried products LA51 FD 0 and LA51 FD 1 was characterized. Figure 4 The interfacial adhesion curves are shown. The shape of the curve for LA51 FD 0 reflects the weaker partitioning of cells into hexadecane, which is associated with a reduced abundance of hydrophobic molecules on the cell surface. 1 The hydrophobicity of the cell surface increases, which can be seen from the LA51FD 1 cells in Φ(V H / V B )>0.1. H / V B )=0.1, the cell surface hydrophobicity was 29%±4% and 23%±2%, respectively. These values ​​classify the cell surface of LA51 as moderately hydrophobic.

[0092] Example 5

[0093] Streptococcus thermophilus HA in MRS medium (BDDifco TMLactobacillus MRS broth, Fisher Scientific). The inoculum for fermentation was prepared by growing the strain in a closed bottle with MRS medium at 40°C under static conditions and without pH control. The incubation period was 16 hours. The fermentation was carried out in a laboratory scale fermentor by first inoculating 1% of the preculture into MRS medium. The fermentation was carried out under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 40°C, and the pH set point was controlled at 6.0 by adding 24% ammonia water. The fermentation was completed within 18 hours, at which time HA utilized all sugars. The fermentation broth was cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by concentration factor 30x had a dry matter content of 7.8% (w / w). The cell concentrate is further processed as described. HA cell concentrate is formulated by HA cell concentrate was prepared by adding 0.35 g of a protective agent containing 22% dry matter (w / w). HA FD 0 and HA FD 1.

[0094] Freeze-dried products HA FD 0 and The cell surface hydrophobicity of HA FD 1 cells was characterized. Figure 5 The interfacial adhesion curves are shown. The cell surface properties of cryopreserved cells without and with protectants are similar. HA FD 0 and HA FD 1 in Φ(V H / V B )=0.1, the hydrophobicity is 27%±3% and 22%±2% respectively. These values ​​will The cell surface of HA is classified as moderately hydrophobic.

[0095] Example 6

[0096] Lactococcus lactis R-607-1 was grown in Difco TMThe strain was grown in M-17 broth (Becton, Dickinson and Company, USA). The lactose concentration was increased to 20 g / l. The inoculum for fermentation was prepared by growing the strain in a closed bottle with modified M-17 medium at 30°C under static conditions and without pH control. The incubation period was 16 hours. Fermentation was carried out in a laboratory-scale fermentor, and 1% of the preculture was first inoculated into the modified M-17 medium. The fermentation was carried out under anaerobic conditions with nitrogen in the headspace. The contents of the fermentor were stirred continuously at 300 rpm, the temperature was maintained at 30°C, and the pH set point of 6.0 was controlled by adding 24% aqueous ammonia. The fermentation was completed within 8 hours, at which time R-607-1 utilized all the sugars. The fermentation broth was cooled to 10°C and concentrated by centrifugation at 4°C. The cell concentrate prepared by a concentration factor of 30x had a dry matter of 12.5% ​​(w / w). As described in Example 1 The cell concentrate was further processed as described above. The R-607-1 cell concentrate was prepared by adding 1 g of R-607-1 The HA cell concentrate was completed by adding 0.57 g of a protective agent containing 22% dry matter (w / w). Lyophilized materials R-607-1FD 0 and R-607-1FD 1 were produced.

[0097] The cell surface hydrophobicity of cells of the lyophilized products R-607-1FD 0 and R-607-1FD 1 was characterized. Figure 6 The interfacial adhesion curves are shown. The shape of the curve reflects the lower partitioning of the cells into hexadecane. The cell preparations containing the protectant altered the hydrophobic structure of the cell surface. R-607-1FD 0 and R-607-1FD 1 showed a significant difference in Φ(V H / V B ) = 0.1, the cell surface hydrophobicity was 7% ± 2% and 19% ± 7%, respectively. These values ​​classify the cell surface of R-607-1 as non-hydrophobic.

[0098] Example 7

[0099] The freeze-dried particles FD 0 and FD 1 produced in Examples 1 to 6 were equally divided into aluminum bags, which were sealed and subjected to an accelerated storage stability test at 37° C. Cell viability was determined after incubation at 37° C. for 2 weeks, and the results are summarized in Table 2.

[0100] Table 2. CFU counts of freeze-dried products of Bifidobacterium, Lactobacillus, Streptococcus and Lactococcus for storage stability study at 37°C.

[0101]

[0102]

[0103] In general, the freeze-dried product FD 0 without any protective agent showed poorer stability compared to the freeze-dried product FD 1 containing a protective agent.

[0104] The correlation between the loss of viability and cell surface hydrophobicity of six representative lactic acid bacteria in storage stability tests was studied. Fig. 7A and Figure 7B The logarithmic loss of CFU / g FD products (see Table 2) and the number of lyophilized cells at Φ(V H / V B ) = 0.1. These values ​​have been described in detail in Examples 1 to 6.

[0105] Fig. 7A : It was found that the loss of storage stability in the FD product group without a protective agent (FD 0 product) was directly related to the cell surface hydrophobicity. The linear regression equation is as follows: y = 0.0257x + 0.8173; R 2 = 0.3545. It was found that the lyophilized, unprotected strain with a higher cell surface hydrophobicity was not as stable as the lyophilized, unprotected strain with a lower cell surface hydrophobicity.

[0106] Figure 7B : Trend analysis of the freeze-dried product containing the protective agent (FD 1 product) surprisingly showed the opposite trend. The linear regression equation is as follows: y = -0.003768x + 0.5319; R 2 =0.1867. It was found that the loss of storage stability in the FD 1 product group was negatively correlated with the cell surface hydrophobicity. The freeze-dried strains containing the protective agent with higher cell surface hydrophobicity showed higher storage stability than the strains with lower cell surface hydrophobicity.

Claims

1. A method for preparing a frozen or dried product containing non-spore-forming prokaryotes, the method comprising the following steps: i. culturing the prokaryotic cells by fermentation; ii. concentrating the cells by separating the fermentation broth; iii. mixing the cell concentrate with a medium containing a protective compound to obtain a pretreatment composition; iv. maintaining the pretreatment composition under conditions that do not induce metabolic activity; v. Preserving the composition by (a) freezing the composition to form a frozen prokaryotic product; (b) drying the product to form a dried prokaryotic product; or (c) freezing the composition to form a frozen prokaryotic intermediate product and then lyophilizing the intermediate product to form a lyophilized prokaryotic product, When Φ[V H / V B ] is measured at at least one point between 0.1 and 1.0, measured by the MATH method at 22°C and expressed as [(initial OD 600 - Final OD 600 ) / initial OD 600 ]*100 and initial OD 600 The hydrophobicity of the cell surface of the cells in the product is at least 40%, and the potency of the product is 1E+08 to 1E+13 CFU / g.

2. The method according to claim 1, wherein the protective compound is one or more of the following: 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 oligofructose or, for example, maltodextrin with DE 3-20; a polysaccharide, such as starch or inulin; a cryoprotectant and / or a lyoprotectant and / or a storage stabilizer, such as gum arabic, maltodextrin, starch, pectin, cellulose, xylan or a polyol, such as glycerol, sucrose, trehalose or maltose, a protein, such as gelatin, a peptide, such as a peptide provided by a 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.

3. The method according to any one of claims 1 or 2, wherein the dry weight ratio of the final protective agent to the cell concentrate of the frozen prokaryotic product or frozen prokaryotic intermediate product is 10:1 to 0.1:1, preferably 3:1 to 0.5:1, and most preferably 2:1 to 1:

1.

4. The method according to any one of claims 1 to 3, wherein step (iii) lasts from 0.25 hours to 16 hours.

5. The process according to any one of claims 1 to 4, wherein step (iii) is carried out at 4°C to 20°C, preferably below 10°C.

6. The method according to any one of claims 1 to 5, wherein the prokaryotic organism is a fermenting bacterium From the phylum Firmicutes, for example: Lactic acid bacteria (LAB), preferably selected from the group consisting of Streptococcus Streptococcus (such as Streptococcus thermophilus), Lactococcus (such as Lactococcus lactis), Oenococcus Oenococcus (e.g., Oenococcus oeni), Leuconostoc spp. (Leuconostoc) (such as species of Leuconostoc mesenteroides, species of Leuconostoc pseudomesenteroides), Lactobacillus Lactobacillus, Limosilactobacillus, Lactobacillus casei Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus, Combined Lactobacillus, Lentilactobacillus, Lactobacillus (Latilactobacillus), Companilactobacillus, Lactobacillus and Lactobacillus; Eubacterium (such as Eubacterium mucosum Eubacterium limosum, Eubacterium aggregans, Eubacterium barkeri, Eubacterium lentum), Roseburia (enteric Roseburia Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia fecal (Roseburia faecis) and Roseburia cecicola, Faecalibacterium (such as species of Faecalibacterium prausnitzii), anaerostipes (such as Anaerostipes caccae), Anaerobutyricum (such as species of Anaerobutyricum hallii, species of Anaerobutyricum soehngenii); from the phylum Actinobacteria, such as Bifidobacterium (e.g. species: Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve), Propionibacterium (e.g. species of Propionibacterium freudenreichii), Cutibacterium (e.g. Propionibacterium acnes); from the phylum Bacteroidetes, such as the genus Bacteroides (e.g. species: Bacteroides fragilis, Bacteroides xylanisolvens), the genus Prevotella (e.g. species Prevotella copri) or the genus Alistipes, or - From the phylum Verrucomicrobia, such as the genus Akkermansia (eg species Akkermansia muciniphila).

7. The method according to claim 6, wherein the prokaryotic organism is one or more of the following: Limosilactobacillus reuteri, Lactobacillus rhamnosus, Ligilactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus flexus 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, Lactobacillus acidophilus, Lactobacillus jensenii, and Lactobacillus iners.

8. A frozen or dried product comprising non-spore-forming prokaryotes obtainable by a method according to any one of the preceding claims.

9. The frozen or dried product according to claim 8, wherein the potency of the product is 1E+08 CFU / g to 1E+13 CFU / g.

10. A composition comprising the frozen or dried product according to claim 8 or 9, wherein the bacterium has a potency of 1E+05 CFU / g to 1E+12 CFU / g. The composition according to claim 10 , which is a food, feed, agricultural product, dietary supplement or medicine.

12. A method of manufacturing a food, feed, agricultural product, dietary supplement or medicine comprising adding the frozen or dried product according to claim 8 or 9.

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