Bifidobacterium strains with improved stability

Abstract

The present invention relates to a mutant strain of Bifidobacterium longum (Bifidobacterium longum) having improved properties (e.g., stability). Furthermore, the present invention relates to a method for producing a mutant strain of Bifidobacterium longum and the use of these strains for improving intestinal barrier function and / or eliciting an anti-inflammatory immune response.

Description

Technical Field

[0001] The present invention relates to mutant strains of Bifidobacterium longum having improved properties, such as stability.

[0002] Furthermore, the present invention relates to methods for producing mutant strains of Bifidobacterium longum, and the use of these strains for improving intestinal barrier function and / or eliciting anti-inflammatory immune responses.

[0003] background

[0004] Bifidobacteria are natural inhabitants of the gastrointestinal tract and have genetic adaptations that allow them to colonize this harsh and complex habitat. Bifidobacteria interact with key elements of intestinal function and contribute to the maintenance of homeostasis. Recent scientific advances have shown that bifidobacteria can reduce mucosal antigen load, improve the intestinal barrier, and induce modulation of local and systemic immune responses through strain-dependent interactions with the host.

[0005] Bifidobacterium is used as probiotics due to its recognized benefits to human health. Probiotics are "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (FAO / WHO, 2001). About 12 strains of Bifidobacterium with clinically proven effects are available on the market. Half of them are strains of Bifidobacterium animalissubsp.lactis, and the rest are strains of Bifidobacterium longumsubsp.longum, B.longum subsp.infantis, B.adolescentis or Bifidobacterium breve.

[0006] EP 0 768 375 describes specific strains of Bifidobacterium subspecies that are able to colonize the intestinal flora and to competitively exclude pathogenic bacteria from adhering to intestinal cells. It is reported that these bifidobacteria contribute to immunomodulation and thus maintain the health of the individual. The immunomodulatory effects of bifidobacteria can even be passed on to unborn children. For example, WO 01 / 97822 et al. describe the ingestion of strains of Bifidobacterium animalis by mothers during their pregnancy. Reduces the incidence of atopic diseases in children. In addition, WO 03 / 099037 describes strains of Bifidobacterium animalis According to Masco et al. (2004), strains of Bifidobacterium animalis Bifidobacterium animalis subsp. lactis

[0007] Bifidobacterium animalis subsp. lactis Relatively stable. This makes the strain conveniently incorporated into many products.

[0008] However, there is still a need for further Bifidobacterium strains having high stability. SUMMARY OF THE INVENTION

[0010] The present invention relates to bifidobacterium variants having single nucleotide polymorphisms in their multifunctional microbial type I fatty acid synthase (FAS) genes. Such mutations are associated with variants that lack a fatty acid source (e.g., polysorbate / 80) and contributes to the improved robustness and stability of the variants compared to their parental strains.

[0011] The variant may be a strain of Bifidobacterium longum, the mutation in whose gene results in an amino acid substitution at position 407 of the protein sequence. The wild-type arginine at this position is replaced by another small uncharged amino acid (e.g., glycine or serine).

[0012] According to a first aspect, there is provided an isolated strain of Bifidobacterium longum, characterized in that the arginine in the FAS gene is substituted with an amino acid, glycine or serine.

[0013] In one embodiment, the isolated strain of Bifidobacterium longum is characterized in that the FAS gene sequence comprises SEQ ID NO: 1; and it is capable of growing in the absence of an exogenous fatty acid source.

[0014] In one embodiment, the isolated strain of Bifidobacterium longum is characterized in that the FAS protein sequence comprises SEQ ID NO: 2; and it is capable of growing in the absence of an exogenous fatty acid source.

[0015] In one embodiment, the isolated strain has a low ratio of unsaturated fatty acids to saturated fatty acids compared to the wild-type strain, such as about 1:1.

[0016] In one embodiment, the strain is selected from Bifidobacterium longum subsp. longum BB-46, Bifidobacterium longum subsp. longum AF13-34 or Bifidobacterium longum subsp. longum AF13-41.

[0017] In one embodiment, the strain is Bifidobacterium longum subsp. longum BB-46.

[0018] According to a second aspect, there is provided a probiotic product comprising the isolated strain according to any one of claims 1 to 6 and a cryoprotectant.

[0019] In one embodiment, the cryoprotectant is a sugar or a sugar alcohol.

[0020] According to a third aspect, an isolated strain according to the first aspect or a probiotic product according to the second aspect is provided for preventing, alleviating the symptoms of and / or treating intestinal inflammatory diseases such as IBD and IBS, liver diseases such as NAFLD, NASH, cirrhosis or alcohol-related liver disease, metabolic disorders such as metabolic syndrome, insulin resistance, type 2 diabetes, obesity, cardiovascular atherosclerosis, autoimmune diseases such as celiac disease, type 1 diabetes, multiple sclerosis or rheumatoid arthritis, and / or mental disorders such as major depression, affective disorders, cognitive disorders, chronic fatigue syndrome or anxiety.

[0021] According to a fourth aspect, there is provided a method for producing a Bifidobacterium longum strain, wherein the growth medium is free of exogenous fatty acid sources (e.g. 80) culture medium.

[0022] According to a fifth aspect, a method for preventing, alleviating the symptoms of and / or treating intestinal inflammatory diseases such as IBD and IBS, liver diseases such as NAFLD, NASH, cirrhosis or alcohol-related liver disease, metabolic disorders such as metabolic syndrome, insulin resistance, type 2 diabetes, obesity, cardiovascular atherosclerosis, autoimmune diseases such as celiac disease, type 1 diabetes, multiple sclerosis or rheumatoid arthritis, and / or psychiatric disorders such as major depression, affective disorders, cognitive disorders, chronic fatigue syndrome or anxiety, the method comprising administering to an individual in need thereof a therapeutically effective dose of an isolated strain according to the first aspect or a probiotic product according to the second aspect.

[0023] Details

[0024] The present invention relates to an isolated strain of Bifidobacterium longum, such as Bifidobacterium longum subsp. longum, characterized in that a) the strain comprises a nucleic acid sequence according to SEQ ID NO: 1; or b) a sequence encoding a protein having at least 90% identity to SEQ ID NO: 2 and having an amino acid mutation at position 407 of SEQ ID NO: 2.

[0025] In a specific embodiment, the strain comprises a nucleic acid sequence encoding a protein having at least 90% identity to SEQ ID NO: 2, such as a sequence encoding a protein having at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 2. In one embodiment, the strain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0026] In a specific embodiment, the amino acid at position 407 of SEQ ID NO: 2 is glycine or serine.

[0027] For the purpose of the present invention, the sequence alignment method of Clustal Omega is used to perform pairwise alignment of nucleotide sequences (DNA) or amino acid sequences (protein) to determine the degree of sequence identity between two nucleotide sequences or two amino acid sequences, respectively.

[0028] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged while growing or multiplying. This includes multiple identical bacteria. A "wild-type strain" refers to a non-mutated form of a bacterium found in nature.

[0029] In this context, the term "derivative strain" is understood to be a strain derived from a wild-type strain by, for example, genetic engineering, radiation and / or chemical treatment and / or selection, adaptation, screening, etc. In a specific embodiment, a derivative strain is a mutant that is functionally equivalent, for example, a mutant having substantially the same or improved properties as the parent strain (for example, about probiotic properties). Such derivative strains are part of the present invention. The term "derivative strain" includes a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenic treatment (including chemical mutagens, UV light treatments such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG)) or a spontaneously generated mutant.

[0030] "Mutant bacteria" or "mutant strains" refer to natural (spontaneous, naturally occurring) mutant bacteria or induced mutant bacteria, which contain one or more mutations in their genome (DNA) that are not present in wild-type DNA. It can also be a single nucleotide polymorphism (SNP). "Induced mutants" are bacteria in which mutations are induced by human treatment, such as any conventionally used mutagenic treatment, including treatment with chemical mutagens, such as selected from the following chemical mutagens: (i) mutagens that bind to or incorporate into DNA, such as base analogs, such as 2-aminopurine or chelators such as ICR-191, (ii) mutagens that react with DNA, including alkylating agents, such as nitrosoguanidine or hydroxylamine, or ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV radiation or gamma radiation, etc. In contrast, "spontaneous mutants" or "naturally occurring mutants" have not been artificially induced.

[0031] Variant strains such as mutants may have been subjected to several mutagenesis treatments (a single treatment is understood to be a mutagenesis step followed by a screening / selection step), but typically no more than 20, no more than 10, or no more than 5 treatments are performed. In specific embodiments of derivative strains (such as mutants), less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome are altered (e.g., by substitution, insertion, deletion, or a combination thereof) compared to the parent strain.

[0032] As described above the mutant bacteria are non-GMO, i.e. have not been genetically modified by recombinant DNA technology. As an alternative to the preferred method of providing mutants by random mutagenesis described above, such mutants may also be provided by site-directed mutagenesis, for example by using appropriately designed PCR techniques or by using transposable elements that can be integrated into bacterial replicons or by genome editing.

[0033] When mutants were provided as spontaneously arising mutants, the selection step was performed on the wild type strain as described above, but without any previous mutagenesis treatment.

[0034] Any mutant strain of the Bifidobacterium longum strain having accession number DSM 15955 can be obtained by subjecting the strain to the mutagenesis treatment described above to obtain a mutant strain and selecting a mutant strain having the desired properties. Alternatively, spontaneously generated mutants can be selected.

[0035] The term "probiotic product" refers to any product comprising probiotics. Probiotic products comprising the strains according to the invention can be administered in the form of food or dietary supplements. For example, Bifidobacterium longum can be incorporated into dairy products, such as milk, and in particular fermented dairy products, optionally in combination with other lactic acid bacteria, for example with yogurt starters, or into other foods such as snack bars, or beverages such as juices.

[0036] The probiotic product comprising Bifidobacterium longum may also be provided as a dietary supplement in the form of a powder, tablet, such as a lozenge or effervescent tablet, lozenge, capsule, chewing gum, in individual sachets, or as a component of a more general composition, such as an oil drop, emulsion or paste, or any other suitable carrier determined by a person skilled in the art to be an effective carrier of live microorganisms.

[0037] Probiotics are live microorganisms, and this can be a challenge during the formulation and storage of probiotic products. Probiotics are particularly sensitive to temperature, moisture content, and oxygen and other ingredients in the formulation matrix. It is preferred that the bacteria of the present invention remain viable after long-term storage so that when the probiotic product of the present invention is administered to an individual in need thereof, the bacteria are able to impart its beneficial effects.

[0038] The term "viable" means that the cells are alive and are able to form colonies in the culture dish during pouring or spreading. The number of viable probiotics is determined as the number of colony forming units (CFU) by pouring or spreading the plate method and incubating under conditions suitable for the growth of the probiotic strain. By this method, cells that are able to grow and form colonies are counted. When numerical values ​​are given in this specification and claims, they should be understood as CFU / mL unless the context indicates otherwise. In some embodiments, the probiotic product of the present invention contains at least 10 CFU / mL at the end of shelf life (EOS). 9 CFU / unit. The end of shelf life may be at least 3 months, such as at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 24 months.

[0039] The probiotics used in the probiotic products of the present invention are usually frozen or freeze-dried. In order to obtain a high survival rate, the bacteria are mixed with a cryoprotectant before freezing or freeze-drying.

[0040] The term "cryoprotectant" refers to a substance that can increase the survival rate and improve the storage stability of bacteria during freezing and / or drying. The cryoprotectant used herein generally includes a sugar or a sugar alcohol (such as glycerol).

[0041] Sugar can be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide, or a mixture of at least two sugars. The composition can even include three, four or more sugars. In some embodiments, the composition includes a mixture of at least one monosaccharide or a disaccharide and at least one oligosaccharide. In other embodiments, the composition includes a mixture of at least one monosaccharide or a disaccharide and at least one polysaccharide.

[0042] Monosaccharides useful in the probiotic products of the present invention include glucose (also known as dextrose), fructose, ribose and galactose. Disaccharides useful in the probiotic products of the present invention include sucrose, trehalose, maltose and lactose. The composition may comprise one or more monosaccharides or disaccharides, such as one, two or three or even more different sugars.

[0043] In some embodiments, the probiotic product of the present invention comprises at least one oligosaccharide. Oligosaccharides are carbohydrate polymers containing three to nine monosaccharides. Fructooligosaccharides (FOS) are present in many vegetables and consist of short-chain fructose molecules. Galactooligosaccharides (GOS) are also naturally occurring and consist of short-chain galactose molecules. The human body can only partially digest these compounds. The composition can comprise one, two or even more different oligosaccharides.

[0044] In some embodiments, the probiotic product of the present invention comprises at least one polysaccharide. Polysaccharides are polymeric carbohydrate molecules composed of more than ten monosaccharide units bound together by glycosidic bonds, and hydrolyzed to produce component monosaccharides or oligosaccharides. Their structures vary from linear to highly branched. Examples of polysaccharides used in the probiotic product of the present invention are maltodextrin, cyclodextrin, alginate, pectin, chitosan, starch and inulin. The composition can comprise one, two, three or even more different polysaccharides.

[0045] For example, the cryoprotectant may comprise a mixture of a disaccharide (such as sucrose or glucose) and a polysaccharide (such as maltodextrin).

[0046] Addition of oligo- or polysaccharides such as FOS, GOS, inulin and other polysaccharides may help to reduce the water activity and have the additional advantage that oligo- and polysaccharides are not as sweet as mono- and disaccharides, and they further add fiber to the composition.

[0047] Polyols (sugar alcohols) have the general formula HOCH2(CHOH) n CH2OH. They are often added to foods because they are lower in calories and less sweet than sugar. In addition, they are not broken down or metabolized into acid by bacteria in the mouth and therefore do not cause tooth decay.

[0048] Said composition can also comprise at least one polyol, such as erythritol, inositol, isomalt, mannitol, maltitol, sorbitol or xylitol, or its mixture.Preferred polyol is xylitol, sorbitol and mannitol.Said composition can comprise one, two, three or even more different polyols.

[0049] The cryoprotectant may further comprise peptides, proteins, protein hydrolysates or mixtures thereof. Examples of peptides and proteins used herein are casein, pea, whey, albumin, soy protein, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives may also be present, for example antioxidants such as ascorbate, sodium citrate, propyl gallate.

[0050] The present invention also relates to a probiotic product comprising the isolated strain according to the present invention and a cryoprotectant such as a sugar.

[0051] Combinations of several probiotic species or strains may be used, i.e. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or even more of the species and strains listed herein. In a currently preferred embodiment, only one, two, three, four or five different strains are present in the probiotic product according to the invention.

[0052] In addition to probiotics, one or more other active ingredients, for example one, two, three, four or more active ingredients selected from vitamins (such as vitamin A, D, E, K2, C, B2, B6, B12, biotin, niacin, folic acid), minerals (such as zinc, selenium, chromium, copper, calcium, chloride), and plant extracts, such as cranberry extract / juice, royal jelly can be included in the probiotic product.

[0053] It is envisaged that in order to achieve a therapeutic effect, probiotic products should be administered at a dose equivalent to at least 10 6 CFU, for example at least 10 7 CFU, preferably at least 10 8 CFU, usually 10 9 CFU to 10 12 The amount of CFU of Bifidobacterium longum is administered daily for at least one week, and advantageously longer, such as at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 9 weeks, preferably at least 12 weeks.

[0054] In this study, the probiotic product contained Bifidobacterium longum as an active ingredient. Bifidobacterium longum can be used as the only active ingredient. Alternatively, the probiotic product as described herein can contain other compounds of interest, such as other bacterial strains, vitamins, prebiotics, fibers, or other compounds that may have a beneficial health effect.

[0055] The other bacterium may be selected from Bifidobacterium lactis, Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Bifidobacterium longum, Lactobacillus lactis, lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckiisubsp.bulgaricus and Lactobacillus acidophilus.

[0056] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a, an" and "said / the" and similar references should be interpreted as covering the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including, but not limited to,"). Unless otherwise indicated herein, the description of the numerical range herein is intended only as a shorthand method for referring to each individual numerical value within the range individually, and each individual value is incorporated into the specification as if it were described separately herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise required. Any language in the specification should not be interpreted as indicating that any unrequired element is essential to the practice of the present invention.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Yes Display and a graph of BB-46's growth and vitality.

[0059] Figure 2 is displayed in CDM-reference and CDM-no Tween Figure 3 Fatty acid profiles of FAS mutants of BB-46 in CDM-reference and BB-46 in CDM-no Tween.

[0060] Preservation and Expert Solutions

[0061] 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 until the date of grant of the patent.

[0062] Table 1 : The applicant has made the following deposit with a depositary institution which has been granted the status of an international depositary institution 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.

[0063]

[0064] Sequence Listing

[0065] The nucleotide and protein sequences of the multifunctional microbial type I fatty acid synthase genes in BB-46 (BL / wild type) and BL mutants are given in Table 2.

[0066] Table 2. Sequence Listing

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] In the context of the present invention, a mutation in a gene (gene mutation) is understood to be a change in the nucleotide sequence of the genome of an organism, which leads to a change in the phenotype of the organism, wherein the change can be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide or a frameshift.

[0086] In the present specification and claims, the conventional single letter codes for nucleotides are used following a similar principle to the amino acid nomenclature described above.

[0087] Algorithms for aligning sequences and determining the degree of sequence identity between them are well known in the art. For the purposes of the present invention, the method of nucleotide sequence alignment using blastn provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov using standard parameters can be used. Example

[0088] Example 1 - Medium composition affects the stability of Bifidobacterium

[0089] Bacterial strains

[0090] The following strains: Bifidobacterium animalis subsp. lactis (also referred to herein as BA, DSM 15954) and B. longum subsp. longum BB-46 (also referred to herein as BL, DSM 15955) were obtained from the Chr. Hansen culture collection.

[0091] Strain maintenance

[0092] The 20% (v / v) glycerol stock solution of the strain was stored at -80°C. The glycerol stock solution was prepared by -1 The cells were prepared as anaerobic batch cultures in chemically defined medium (CDM) with sucrose as the carbon source.

[0093] Batch fermentation

[0094] The fermentation was carried out in a DASGIP parallel bioreactor system (Eppendorf, Hamburg, Germany) with anaerobic pH control (pH 6.5) in a 10 g L -1 The strains were cultured in triplicate in 200 mL of sucrose-free medium at 37 °C. Two different medium compositions were tested in this study, using unmodified Bifidobacterium CDM as the reference medium. CDM-reference contained 1 mL L -1 80 (CAS nr.: 9005-65-6). In addition, in the original CDM (CDM-reference), in the absence of The cells were cultured in CDM (CDM-without Tween) at 80%.

[0095] Viability Assessment During Short-Term Storage

[0096] The adaptability and stability of BA and BL were evaluated during short-term storage trials under suboptimal conditions. For each replicate, 5 mL of cell culture samples were harvested at 11,000 rpm and 5°C for 10 min in the stationary growth phase. To avoid cell loss, only 4 mL of cell supernatant was discarded. After adding 4 mL of peptone saline solution (8.5 g L -1 NaCl, 1 g L -1 Peptone, pH 7) and then resuspend the cell pellet. After 28 days of storage at 2°C-4°C in the dark, the cell viability was evaluated. After 14 days, the cells were vortexed at 24,000 rpm for 1 min to avoid cell aggregation. Before and after 28 days of storage, the number of viable cells was determined by colony forming units (CFU) using the pour culture method. The loss of viable cells was calculated as log10 (CFU 储存前 / CFU 储存后 ) and used as a measure of strain viability.

[0097] Fatty acid analysis

[0098] The relative fatty acid composition of BA and BL in the stationary phase was evaluated using a gas chromatography-mass spectrometry (GC-MS) system (ISQ-LT, Thermo Scientific, USA) equipped with a ZB-FAME column (20 m x 0.18 mm x 0.15 μm, Phenomenex, Torrance, CA, USA). As a standard mixture, a bacterial acid methyl ester (BAME) mixture (47080-U, Sigma-Aldrich) was used. In order to test the significant difference of the mean value of fatty acids compared with the curve observed in CDM-Ref, a multiple t-test was applied ( Methods, unpaired, Welch correction). Statistical analysis was performed using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA).

[0099] Growth curve

[0100] The optical density of the culture solution at 600 nm (OD600) was measured using an Ultrospec 10 spectrophotometer (GE Healthcare, USA).

[0101] Genome sequencing and SNP analysis

[0102] Genome sequencing according to M, Gaspar P, Neves AR, et al (2021) Identifying the essential nutritional requirements of the probiotic bacteria Bifidobacterium animalis and Bifidobacterium longum through genome-scale modeling. npj Syst Biol Appl 7: 1–15. Single nucleotide polymorphism (SNP) analysis was performed in CLC Genomics Workbench (22.0.1), with the minimum frequency set to 85%.

[0103] Growth characteristics. The metabolic and physiological characteristics of BA and BL were characterized in CDM with and without exogenous fatty acid sources. The growth and survival of both strains were found to be affected by the medium formulation ( Figure 1 ). The culture was carried out under anaerobic conditions (20% CO2 and 80% N2) at 37°C and pH 6.5. CDM-reference: containing 1 mL L -1 80 Reference Chemically Defined Medium, CDM-Tween-free: None 80 CDM.

[0104] When cultured in CDM-Tween-free medium, BL exhibited a very long lag phase of approximately 20 h ( Figure 1 A). This suggests that BL were fed exogenous fatty acids in pre-culture and were challenged to switch to de novo fatty acid synthesis.

[0105] Sequencing of BL grown in CDM-Tween-free. Due to the long stasis period of BL in CDM-Tween-free, the cells cultured in this condition were subjected to genome sequencing. The genome sequencing revealed the presence of a single nucleotide polymorphism (SNP) in its microbial type I fatty acid synthase (FAS) gene (I3242_09035). At position 1219 of the nucleotide sequence of this gene, cytosine was replaced by adenine. FAS is a multifunctional enzyme responsible for de novo fatty acid synthesis [Schweizer E, Hofmann J (2004) Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiol Mol Biol Rev 68:501–17, table of contents.]. The gene mutation resulted in the replacement of arginine by serine at position 407 of the protein sequence. Multiple sequence analysis showed that the FAS gene of BA (BIF_00783) had a glycine at the corresponding position, which, like serine, is a small amino acid. The surrounding amino acids in the FAS gene of BA and BL were found to be highly similar.

[0106] To assess whether the mutation was recurrent, the culture of BL in CDM-Tween-free was repeated three times and the genome of cells from all replicates was sequenced. In all replicates, cells showed the same SNP in the FAS gene as described above. In addition, when the derivatives were subsequently grown in the presence of The mutation showed genetic stability when cultured in CDM-Ref at 80%.

[0107] Hereinafter, derivatives of BL are referred to as FAS mutants of BL.

[0108] The formulation of the growth medium was found to affect the fitness and stability of BA and BL, which was evaluated in a short-term storage trial ( Figure 1 B). Colony forming unit (CFU) counts were used to assess the viability of the strains before and after storage (aerobic conditions, pH = 7, 2°C–4°C, 28 days). 0: CFU counts before storage, 28: CFU counts after 28 days of storage. The values ​​on the bar graph correspond to the logarithmic loss of viable cells during storage measured in CFU.

[0109] Of the two media used for proliferation, BA showed a higher survival rate than BL ( Figure 1 B). BA showed similar survival when produced in CDM-reference and CDM-no Tween ( Figure 1B). In contrast, FAS mutants of BL grown in CDM-no Tween showed better survival than BL generated in CDM-reference ( Figure 1 B).

[0110] Fatty acid profiles. Another cell characteristic compared in both media was the cell membrane fatty acid composition of BA, BL, and the FAS mutant of BL. After entering stationary phase, the fatty acid composition of the strains was evaluated. For comparison, the fatty acid profile of the strain in CDM-reference was used as a reference. In CDM-no-Tween, significant changes in the fatty acid composition of the FAS mutant of BA and BL were observed ( Figure 2 ).

[0111] Figure 2 A shows The contents of various fatty acids in the cell membranes of BB-46 and BB-46. Each value gives the average of three replicates. Figure 2 B shows Fatty acid profiles of BB-46 and BB-50. Each value gives the mean ± SD of three replicates. UFA / SFA: ratio of unsaturated fatty acids to saturated fatty acids. Cyclic fatty acids were not considered in the calculation of UFA / SFA. The significance of the differences in means was tested using multiple unpaired t-tests with CDM-reference as reference. One asterisk: p 调整 <0.05: two asterisks: p 调整 <0.005: ** .

[0112] Thus, in CDM-Tween-free, in the absence of an exogenous fatty acid source, significantly lower oleic acid (C18:1) and significantly higher palmitic acid (C16:0) contents were detected in the cell membranes of BA and the cell membranes of the FAS mutant of BL ( Figure 2 A). The relative increase in palmitic acid content in the cell membrane of BA was only about 8%, while the relative increase in palmitic acid content in the cell membrane of the FAS mutant of BL was more than 20%. In addition, the myristic acid (C14:0) content of both strains was significantly higher in CDM-no Tween ( Figure 2 ). The FAS mutant of BL further exhibited a higher percentage of linoleic acid (C18:2) but a lower percentage of cis-9,10-methylenehexadecanoate (C17:0 cyclic) compared to BL in the CDM-reference ( Figure 2 A). Other notable differences in the BA fatty acid profiles between CDM-No Tween and CDM-Reference included higher nonadecanoic acid (C19:0) and lower 15-methylhexadecanoate (iso-C17:0) contents ( Figure 2 A).

[0113] Although the cell membrane composition of BA in CDM-no Tween was significantly different compared to CDM-reference, the fatty acid profile of this strain was always dominated by oleic acid and palmitic acid. This is consistent with the ubiquitous presence of these two fatty acids in the cell membrane of other bifidobacterial strains [Ruiz L, Sánchez B, Ruas-Madiedo P, et al (2007) Cell envelope changes in Bifidobacterium animalis subsp. lactis as a response to bile. FEMS Microbiol Lett 274: 316–322., and Louesdon S, Charlot-Rougé S, Tourdot-Maréchal R, et al (2015) Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri and Bifidobacterium longum. Microb Biotechnol 8: 311–318.]. In contrast, the fatty acid profile of BL in CDM-Ref was clearly dominated by oleic acid (>60%), with palmitic acid content less than 7.5% ( Figure 2 A) Due to The major fatty acid in 80 is oleic acid (product information sheet, Sigma, CAS nr.: 9005-65-6), and the fact that oleic acid was the major component in the BL cell membrane under these conditions indicated a large utilization of exogenous fatty acids.

[0114] In addition, significant differences were observed in the fatty acid profiles of BA and BL in CDM-Ref. The comparison showed significant differences in the percentages of seven fatty acids in the cell membranes of the two strains. In contrast, the fatty acid profile of the FAS-mutant of BL in CDM-Tweenless was more similar to that of BA in CDM-Ref, showing only a significantly lower C16:0 content. However, this difference (25%) was significantly smaller than the differences when BL was cultured in other media (80%-96%) ( Figure 2 A).

[0115] Overall, the changes in the fatty acid profiles of both strains observed in CDM-no Tween compared to the CDM-reference confirm that the cell membrane composition of both strains is affected by the supply of exogenous fatty acids in the medium. The smaller changes observed in the fatty acid profile of BA when propagated in CDM-no Tween suggest that the strain synthesizes a high proportion of cell membrane fatty acids de novo, regardless of the presence of fatty acids in the medium. In contrast, the long stasis phase of the FAS mutant and the Natural selection in a culture medium with a concentration of 80% indicated that BL could not grow without an exogenous fatty acid source. Conversely, mutations in the FAS gene are likely responsible for the ability of BL derivatives to grow without an exogenous fatty acid source. 80% CDM. Bifidobacterium FAS has not been characterized, but protein sequence analysis revealed that the mutation in the BL derivative is located in the enoyl reductase domain (COG4981) of the yeast-type FAS subunit β. The functional consequences of the mutation remain to be investigated.

[0116] Correlating the fatty acid profiles with the viability of the two strains under different growth conditions showed that the almost equal content of palmitic acid and oleic acid together with a UFA / SFA ratio of about 1 contributed to the enhanced fitness and stability of the two Bifidobacterium strains ( Figure 1 and Figure 2 ). It is believed that a high content of SFA in bacterial cell membranes helps reduce membrane fluidity, which may promote the strain's tolerance to stressors [Louesdon S, Charlot-Rougé S, Tourdot-Maréchal R, et al (2015) Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri and Bifidobacterium longum. Microb Biotechnol 8: 311–318., Beales N (2004) Adaptation of microorganisms to cold temperatures, weak acid preservatives, low pH, and osmotic stress: A review. Compr Rev Food Sci Food Saf 3: 1–20.]. The cell membrane of BA showed the above characteristics in both growth media, which were not observed in BL, but were observed in the FAS mutant of BL in CDM-no Tween.

[0117] In conclusion, our results highlight that exogenous fatty acids can have adverse effects on the fatty acid profile of Bifidobacterium strains, potentially reducing strain fitness and stability. To avoid variations in fatty acid supply in complex production media due to batch-to-batch variations in undefined medium composition, the use of CDM may be advantageous in industrial-scale production.

[0118] According to our results, cell membrane composition plays a key role in stress tolerance of Bifidobacterium. Previously, the fatty acid profiles of Bifidobacterium strains were found to change in adaptive laboratory evolution experiments and in response to stress exposure, accompanying the acquisition of stress tolerance by the strains [Ruiz L, Sánchez B, Ruas-Madiedo P, et al (2007) Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response tobile. FEMS Microbiol Lett 274: 316–322., Louesdon S, Charlot-Rougé S, Tourdot-Maréchal R, et al (2015) Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri and Bifidobacterium longum. Microb Biotechnol 8: 311–318, Yang X, Hang X, Zhang M, et al (2015) Relationship between acid tolerance and cell membrane in Bifidobacterium,revealed by comparative analysis of acid-resistant derivatives and their parental strains grown in medium with and without Tween 80.Appl MicrobiolBiotechnol99:5227–5236.,Wei Y,Gao J,Liu D,et al(2019)Adaptational changes inphysiological and transcriptional responses of Bifidobacterium longuminvolvedin acid stress resistance after successive batches cultures.Microb Cell Fact18:156.] For example, the bile-resistant derivative, Bifidobacterium animalis subsp. lactis IPLA 4549, showed a different fatty acid profile compared to the parent strain, with lower UFA / SFA in the absence of bile and higher UFA / SFA in the presence of bile [Ruiz L, Sánchez B, Ruas-Madiedo P, et al (2007) Cell envelope changes in Bifidobacterium animalis subsp. lactis as aresponse to bile. FEMS Microbiol Lett 274: 316–322]. In contrast to our findings, when the acid-tolerant derivatives Bifidobacterium longum JDY1017dpH and Bifidobacterium breve BB8dpH were in. When grown in the presence of Tween 80, the acid tolerance of these strains was particularly enhanced, and compared with the parent strain, the oleic acid content and UFA / SFA were increased. However, the acid-resistant strains showed reduced membrane fluidity, most likely due to the presence of cyclic fatty acids and higher average fatty acid chain length in the cell membrane of the derivatives [Yang X, Hang X, Zhang M, et al (2015) Relationship between acid tolerance and cell membrane in Bifidobacterium, revealed by comparative analysis of acid-resistant derivatives and their parental strains grown in medium with and without Tween 80. Appl Microbiol Biotechnol 99: 5227–5236.]. In addition, in Bifidobacterium longum R0175, the increased viability of stable phase cells during freezing compared to exponential phase cells is believed to be related to the decrease in membrane fluidity caused by the relative increase in saturated fatty acids in the strain membrane [Louesdon S, Charlot-Rougé S, Tourdot-Maréchal R, et al (2015) Membrane fatty acid composition and fluidity are involved in thesistance to freezing of Lactobacillus buchneri and Bifidobacterium longum. Microb Biotechnol 8: 311–318.].

[0119] Consistent with our results, when When grown in medium with 80% CO, higher palmitic acid and lower oleic acid contents were detected in the cell membranes of the hydrogen peroxide (H2O2)-resistant Bifidobacterium animalis subsp. lactis strain BL-04 compared to a genetically closely related, publicly available, H2O2-sensitive Bifidobacterium animalis subsp. lactis strain [Oberg TS, Steele JL, Ingham SC, et al (2011) Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species. J Ind Microbiol Biotechnol 38: 1947–1953., and Oberg TS, Ward RE, Steele JL, Broadbent R (2013) Genetic and physiological responses of Bifidobacterium animalis subsp. lactis to hydrogen peroxide stress. J Bacteriol 195: 3743–3751.]. When this H2O2-sensitive strain was grown in the absence of any fatty acid source, stress tolerance increased and de novo fatty acid synthesis resulted in higher palmitic acid and lower oleic acid content in the strain's cell membranes. The higher cyclic fatty acid content in the cell membrane of BL-04 was the determining factor for the higher H2O2 tolerance of this strain compared with the H2O2-sensitive strain in the presence of 80. However, this conclusion contradicts our results, which showed that the cyclic acid content in the cell membrane of the stress-sensitive strain BL was higher than that of the cell membrane of BA ( Figure 2 B). In addition, the 54 bp deletion (erroneously stated as a 45 bp deletion in the reference) in the gene encoding long-chain fatty acid CoA ligase in the BL-04 genome is thought to limit the ability of the H2O2-sensitive strain to utilize exogenous fatty acids compared to the strain. However, this conclusion may be questioned. First, like BA and BL, both BL-04 and the H2O2-sensitive strain contain four long-chain fatty acid CoA ligase genes, which may facilitate the utilization of exogenous fatty acids. In addition, in our study, the fatty acid profile of BA in the growth medium did not change as significantly as that of BL, which possessed exactly the same long-chain fatty acid CoA ligase genes as the H2O2-sensitive strain, but did not have the gene with the 54 bp deletion detected in BL-04. Both findings are inconsistent with the proposed hypothesis.

Claims

1. An isolated strain of Bifidobacterium longum, characterized in that The arginine in the wild-type fatty acid synthase (FAS) gene is replaced with an amino acid selected from glycine and serine.

2. An isolated strain of Bifidobacterium longum, characterized in that a. The FAS gene sequence comprises SEQ ID NO: 1; and b. It has the ability to grow in the absence of exogenous fatty acid sources.

3. An isolated strain of Bifidobacterium longum, characterized in that a. the FAS protein sequence comprises SEQ ID NO: 2; and b. It has the ability to grow in the absence of exogenous fatty acid sources.

4. The isolated strain according to claim 1, which has a low ratio of unsaturated fatty acids to saturated fatty acids, such as 1:1, compared to the wild-type strain.

5. The isolated strain according to any one of claims 1 to 4, which is selected from Bifidobacterium longum subsp. longum (B. longum subsp. longum) BB-46, Bifidobacterium longum subsp. longum AF13-34 or Bifidobacterium longum subsp. longum AF13-41.

6. The isolated strain according to claim 5, which is Bifidobacterium longum subsp. longum BB-46 deposited as DSM 15955.

7. A probiotic product comprising the isolated strain according to any one of claims 1 to 6 and a cryoprotectant.

8. The probiotic product of claim 7, wherein the cryoprotectant is a sugar.

9. An isolated strain according to any one of claims 1 to 6 or a probiotic product according to claim 7 or 8, for preventing, alleviating the symptoms of and / or treating intestinal inflammatory diseases such as IBD and IBS, liver diseases such as NAFLD, NASH, cirrhosis or alcohol-related liver disease, metabolic disorders such as metabolic syndrome, insulin resistance, type 2 diabetes, obesity, cardiovascular atherosclerosis, autoimmune diseases such as celiac disease, type 1 diabetes, multiple sclerosis or rheumatoid arthritis, and / or psychiatric disorders such as major depression, affective disorders, cognitive disorders, chronic fatigue syndrome or anxiety.

10. A method for producing a Bifidobacterium longum strain according to any one of the preceding claims, wherein the growth medium is free of exogenous fatty acid sources such as 80% culture medium.

11. A method for preventing, alleviating the symptoms of and / or treating intestinal inflammatory diseases such as IBD and IBS, liver diseases such as NAFLD, NASH, cirrhosis or alcohol-related liver disease, metabolic disorders such as metabolic syndrome, insulin resistance, type 2 diabetes, obesity, cardiovascular atherosclerosis, autoimmune diseases such as celiac disease, type 1 diabetes, multiple sclerosis or rheumatoid arthritis, and / or psychiatric disorders such as major depression, affective disorders, cognitive disorders, chronic fatigue syndrome or anxiety, the method comprising administering to an individual in need thereof a therapeutically effective dose of an isolated strain according to any one of claims 1 to 6 or a probiotic product according to claim 7 or 8.

Images