Producing a bifidobacterium strain

Abstract

The present invention relates to a process for producing a Bifidobacterium strain with low viability, and a Bifidobacterium strain obtained by the process.

Description

[0001] PRODUCING A BIFIDOBACTERIUM STRAIN

[0002] REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

[0003] This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see last paragraph of the description.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a process for producing a Bifidobacterium strain with low viability and enhanced functional properties, and a Bifidobacterium strain obtained by the process.

[0006] BACKGROUND OF THE INVENTION

[0007] Bifidobacterium is a gram-positive, nonmotile, anaerobic bacteria genus commonly found in the gastrointestinal tract of mammals, including humans. They are a significant part of the gastrointestinal microbiota and are also present in the vagina and mouth of mammals. Certain strains of bifidobacteria, such as Bifidobacterium longum, are utilized as probiotics.

[0008] Bifidobacterium plays a significant role in maintaining gut health by aiding in the balance of gut microbiota, supporting digestion, and contributing to colon health through the production of, e.g., short-chain fatty acids (SCFAs). Furthermore, its potential in modulating the gut-brain axis and influencing mental well-being, as well as its interaction with the immune system to regulate anti-inflammatory responses, underscores its diverse health-promoting effects.

[0009] Environmental factors, such as temperature, pH, and oxygen sensitivity, can impact the viability and functionality of Bifidobacterium, leading to reduced health benefits of the strain when used as a probiotic. To address these challenges, the utilization of postbiotics derived from Bifidobacterium is proposed, offering enhanced stability, safety, and functional integrity, which makes them ideal for incorporation into supplements or functional foods to meet the growing demand for convenient, health-enhancing supplement or food options.

[0010] Obtaining a Bifidobacterium strain with a sufficiently low viability can be challenging, as many of the common methods for reducing the viability of bacterial cells can also damage or denature the beneficial effects of the cells. For example, heat treatment is a commonly used method for reducing the viability of bacterial cells, but it can also denature proteins and enzymes that are important for the beneficial effects of the postbiotic. Similarly, chemical treatments, such as acids or oxidizing agents, can disrupt the bacterial cell membrane and cause the release of cellular components that may be harmful to the host. Other methods such as radiation treatment or pressure treatment may be effective in reducing the viability of Bifidobacterium cells without damaging the beneficial features, but these methods can also be expensive, difficult to scale up, or may require specialized equipment.Thus, there remains a need for improved methods for reducing the viability of Bifidobacterium strains in a way that retains their functional features while ensuring a low CFU count.

[0011] SUMMARY OF THE INVENTION

[0012] The invention provides a process for producing a Bifidobacterium strain. In an aspect, the process comprises the steps of:

[0013] (a) obtaining a composition comprising a Bifidobacterium strain;

[0014] (b) adjusting the pH of the composition of step (a) to a pH in the range of pH 3.5-10.0 to obtain a pH adjusted composition;

[0015] (c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for at least 60 minutes to obtain a heat-treated composition;

[0016] (d) incubating the heat-treated composition of step (c) at a temperature in the range of 4-20°C for at least 4 hours to obtain the Bifidobacterium strain.

[0017] In a preferred embodiment, the Bifidobacterium strain is Bifidobacterium longum. For example, the Bifidobacterium longum may be Bifidobacterium longum deposited as NCI MB 41676, also referred to herein as Bifidobacterium longum 1714, Bifidobacterium longum 1714™, ProbioBrain™ orMindAble™ 1714™.

[0018] Bifidobacterium longum 1714 is available commercially as a dried spore composition, manufactured and sold, e.g., by Novonesis or Novozymes OneHealth (Part of Novonesis). A commercially available spore composition of strain Bifidobacterium longum 1714 is available and sold by Novonesis (previously Novozymes A / S, Bagsvaerd, Denmark) under the tradename MindAble™ 1714™. Also, commercially available spore composition of strain Bifidobacterium longum 1714 is available and sold by Novozymes OneHealth (Part of Novonesis) under the trademarks Bifidobacterium longum 1714™ and ProbioBrain™.

[0019] In an aspect, the process further comprises adding the obtained Bifidobacterium strain to a composition to form a composition comprising a low amount of living Bifidobacterium cells.

[0020] In another or yet further aspect, the obtained Bifidobacterium strain may be added to a food product to obtain a functional food. Such functional food may be a beverage or a nutriment in solid and / or dried form. For example, the obtained Bifidobacterium strain may be added to a food selected from the group comprising dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparations.

[0021] Also comprised herein are Bifidobacterium strains obtained by the process.

[0022] BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 shows log CFU / g reduction for Bifidobacterium longum across different pH levels and treatment times, with and without cooled incubation.Figure 2 shows cytokine levels (TNF-a, IL-12p70, and IL-10) in Peripheral Blood Mononuclear Cells (PBMC) exposed to different samples of Bifidobacterium longum prepared across different pH levels and treatment times. For each of pH 6.5, 7.5, 8 and 8.5 conditions on the x-axis, boxes indicate cytokine levels after Oh, 1 h, and 2h treatment at 60°C, respectively, from left to right.

[0024] Figure 3 shows the mean Iog10 CFU / g reduction data from the scaling-up of the pH 8.0 treatment from 40L to 450L after spray drying. Previously generated 40L reference data and newly generated 450L scale-up confirmation data are shown for untreated material and material adjusted to pH 8.0, incubated for 12 h at 10°C prior to spray drying. In the 40 L reference experiment, untreated control samples showed viable counts of 1.73 x 1OA8 and 1.65 x 1OA8 CFU / g, whereas the corresponding pH 8.0-treated samples showed viable counts of 1, 180, and 203 CFU / g. In the 450 L scale-up dataset, untreated spray-dried material retained measurable viable counts ranging from 9.20 x ioA4 to 4.47 x 10A5 CFU / g, with a mean of 2.90 x 10A5 ± 1.44 x 10A5 CFU / g (5.40 ± 0.28 Iog10 CFU / g, n = 5). In contrast, the pH 8.0-treated 450 L material yielded no detectable colonies in any plated determination and is reported as ND I <LOD.

[0025] DEFINITIONS

[0026] As used herein, the verb “comprise” as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

[0027] Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”). The term "% (w / v)" refers to the concentration of a substance in a solution, expressed as a percentage by weight per unit volume.

[0028] Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X". When used in combination with measured values, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and can include a range of plus or minus two standard deviations around the stated value.

[0029] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the invention and are not to be considered as limitation thereto.

[0030] Furthermore, it will be appreciated that embodiments described in connection with one of the aspects described herein may equally be applied to the other aspects, unless otherwise stated.Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] Bacterial count: The term "bacterial count" and "bacterial load" are used interchangeably and have their normal meaning which is well known and understood by those of skill in the art. In the context of the invention, the bacterial count is given in the measures viable bacterial colony forming units per grams or milliliters, abbreviated CFU / g and CFU / mL, respectively. The bacterial count may be determined by any method known to the skilled person such as, but not limited to, by viable bacterial colony forming unit (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live-dead staining, Propidium Monoazide qPCR (PMA-qPCR), microscopy, a metabolic assay, spectrophotometry, or any combination thereof. Methods for determining bacterial count, such as those disclosed herein, are common and would be apparent to a person of skill in the art.

[0032] Colony: The term “colony”, referred to in connections with a “clone” or “isolate” or “cell”, means a visible cluster or grouping of microbial cells. The colony may be grown and obtained from a solid medium, such as agar.

[0033] Composition: In the context of the invention and unless otherwise clearly stated, the term "composition" encompasses any kind of compositions comprising a Bifidobacterium strain of the invention. In the context of the invention, the composition may be a liquid composition or a dry composition. In an embodiment, the composition comprising Bifidobacterium is obtained or derived from a fermentation of Bifidobacterium.

[0034] Effective amount / concentration / dosage: The terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the strain and / or other ingredients sufficient to achieve a desired biological result, such as reducing, preventing, or treating a disease or condition and / or inducing a particular beneficial effect. The actual effective dosage in absolute numbers depends on factors including: the state of health of the consumer in question, other ingredients present. The “effective amount”, “effective concentration”, or “effective dosage” of the strain and / or other ingredients may be determined by routine assays known to those skilled in the art. In some embodiments, the effective amount is the amount provided in a single serving functional food where consumption of multiple servings is required over time for the beneficial effect to be observed.

[0035] Freeze drying: The terms “freeze drying”, “lyophilization” and “cryodesiccation” are terms known to the person skilled in the art and are used interchangeably herein..

[0036] Isolated: The term “isolated” means that the one or more microorganisms, such as bacterial strains, described herein are in a form or environment which does not occur in nature, that is, the one or more microorganisms are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.Probiotic: The term “probiotic” refers to microorganisms that may have a beneficial effect on the health of a subject, such as on the health of a human subject, upon consumption. Typically, probiotics are commonly defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host,” such as restoring or improving the composition of intestinal microflora. See, e.g., KQ)N,'J\-\Q), Guidelines for the evaluation of probiotics in food, London, Ontario, Canada (2002), incorporated by reference herein in its entirety.

[0037] Prebiotic: In the context of the invention, the term “prebiotic” has its normal meaning which is well known and understood by those of skill in the art. The term "prebiotic" as used herein refers to any compound, nutrient or additional microorganism used to support or enhance a desired health effect or to assist the growth and / or activity of probiotic microorganism.

[0038] Postbiotic: In the context of the invention, the term “postbiotic” has its normal meaning which is well known and understood by those of skill in the art. The term postbiotic, as defined by the International Scientific Association of Probiotics and Prebiotics (ISAPP), refers to a preparation of inanimate microorganisms and / or their components that confers a health benefit on the host. The term “component” refers to cellular biomass in the form of cell fragments or disrupted cells. In the context of the invention, the postbiotic may further comprise metabolites which may also confer a health benefit or contribute to the overall well-being of the host. The term “metabolites” as used herein, refers to substances produced by microbes that may be found within the cell or may be excreted and therefore external to the cell. Example of such metabolites are SCFAs, vitamins, or bacteriocins.

[0039] Probiotics contain a range of cellular structures that contribute to their health benefits, and it is recognized that many of these structures retain their biological activity after cell death. Thus, the process of inactivating a live microbe and the production of a postbiotic product can also result in a mixture of potentially functional cell components. For example, cell wall structures, such as lipoteichoic acid, may play a role in immunomodulation.

[0040] In the context of the invention, the Bifidobacterium strain obtained according to the herein disclosed processes is a postbiotic. In a preferred embodiment, the Bifidobacterium strain is a postbiotic composition comprising less than 1E+4 CFU / g Bifidobacterium cells.

[0041] Specific activity: The term is herein used for activity of strains that is important for the function of the strain. In the context of the invention, the term "activity" may refer to the specific biological functions and health-promoting properties exhibited by the strain within the body. This may include the strain’s ability to influence gut microbiota balance, aid in digestion, produce beneficial metabolites such as SCFAs, modulate the gut-brain axis, interact with the immune system, and contribute to overall well-being and health maintenance in the host.

[0042] Stable: When used in the context of postbiotics and functional foods, "stable" refers to the ability of the product to maintain its integrity, viability, and functionality overtime and under various environmental conditions, including processing, storage, and transportation. Stability encompasses factors such as resistance to environmental changes, maintenance of health-promoting properties, and consistent performance, ensuring that the product retains its efficacy and safety throughout its intended shelf life and usage.

[0043] Synbiotic: The term "synbiotics" refers to a combination of probiotics and prebiotics that work together to confer a health benefit on the host, such as improving the composition of intestinal microflora and promoting overall gut health. This synergistic combination aims to enhance the survival and colonization of beneficial microorganisms in the gastrointestinal tract, thereby providing additional health benefits beyond those offered by either probiotics or prebiotics alone.

[0044] Viable: As used herein with respect to the Bifidobacterium strain, the terms “viable” or “viability” is understood as the ability of the Bifidobacterium cells to propagate on or in a suitable growth medium or substrate when conditions, such as, e.g., temperature, moisture, nutrient availability, pH, etc., are favourable for microbial growth at a given period of time.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] Herein described is a process for the production of Bifidobacterium strains, in particular for the production of Bifidobacterium strains with reduced viability.

[0047] With the present invention, the challenge of reducing viable cell counts in Bifidobacterium strains is addressed without losing the activity of the strains. By developing a process that effectively reduces viable cell counts while maintaining strain activity, regulatory requirements for reduced cell viability are met without compromising the effectiveness of the product.

[0048] In particular, this invention discloses a tailored process for inactivating Bifidobacterium strains, such as Bifidobacterium longum 1714, and specifically enhancing its immunomodulatory effect. The herein disclosed process combines pH modulation (adjusting pH to a pH in the range of pH 3.5-10.0, preferably in the range of pH 4.0-9.0) with heat treatment at 50-70°C for at least 1 hour, followed by cooled incubation at 4-20°C for at least 4 hours. This combination effectively reduces viable cells to below 104CFU / g while preserving key bioactive properties.

[0049] Designed for Bifidobacterium strains, in particular B. longum 1714, the process not only ensures regulatory compliance but also enhances the strains’ ability to modulate the immune system. Even after inactivation, the Bifidobacterium cells retain their structural integrity, which boosts their interaction with the host immune system, promoting anti-inflammatory and immune-regulating effects (see Figure 2). This positions the postbiotic as a potent alternative to live probiotics, for anti-inflammatory benefits with added stability.

[0050] The flexibility to apply the pH and heat treatment at various stages in the process, such as post-fermentation, post-separation, or before drying the Bifidobacterium strain, provides adaptability in manufacturing.

[0051] The herein obtained Bifidobacterium strain is stable, cost-effective, and energy-efficient, making it ideal for use in functional foods and supplements. Overall, the herein disclosed and claimed process enables obtaining a superior, stable inactivated strain, i.e., postbiotic, withenhanced health benefits, specifically tailored to optimize the immunomodulatory activity of Bifidobacterium strains, such as Bifidobacterium longum strains.

[0052] The inventors of the present invention have identified the optimal process conditions for producing a Bifidobacterium strain having reduced viability while the specific activity, such as the immunomodulatory activity, is retained. The process for producing a Bifidobacterium strain comprises the steps of:

[0053] a) obtaining a composition comprising a Bifidobacterium strain;

[0054] b) adjusting the pH of the composition of step (a) to a pH in the range of pH 3.5-10.0 to obtain a pH adjusted composition;

[0055] c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for at least 60 minutes to obtain a heat-treated composition; d) incubating the heat-treated composition of step (c) at a temperature in the range of 4-20°C for at least 4 hours to obtain the Bifidobacterium strain.

[0056] Without the wish of being bound by any particular theory, the inventors believe that the combination of specific steps, including pH adjustment, heat treatment, and cooled incubation, offers a range of benefits. The pH manipulation stresses the bacterial cells, making them more susceptible to inactivation, heat treatment further reduces the viable cell count, and the step of cooled incubation ensures that the bacterial cells are effectively inactivated without losing their bioactive properties.

[0057] The Bifidobacterium strains produced in the herein disclosed processes may be any Bifidobacterium strains. In an embodiment, the Bifidobacterium strain is Bifidobacterium longum. In a preferred embodiment, the Bifidobacterium longum is Bifidobacterium longum deposited as NCI MB 41676. In an embodiment, the Bifidobacterium strain is Bifidobacterium longum deposited as NCIMB 41676, also referred to as Bifidobacterium longum 1714, Bifidobacterium longum 1714™, ProbioBrain™ or MindAble™ 1714™.

[0058] The composition of step (a) is preferably obtained by fermenting the Bifidobacterium strain. In an embodiment, the composition in step (a) is obtained by fermenting the Bifidobacterium strain at a pH that is suitable for ensuring growth of the Bifidobacterium strain. In an embodiment, the pH used for fermenting the Bifidobacterium strain is at least pH 5.0. In a further embodiment, the pH used for fermenting the Bifidobacterium strain is between pH 5.0 and pH 7.0. In a further embodiment, the pH used for fermenting the Bifidobacterium strain is between pH 5.5 and pH 6.9. In yet a further embodiment, the pH used for fermenting the Bifidobacterium strain is between pH 6.2 and pH 6.8.

[0059] The temperature used for fermenting the Bifidobacterium strain may be any temperature suitable for ensuring growth of the Bifidobacterium strain. In an embodiment, the temperature used for fermenting the Bifidobacterium strain is between 35-40°C. In an embodiment, the temperature used for fermenting the Bifidobacterium step is about 37°C.The fermentation of Bifidobacterium may be carried out in any suitable growth medium containing suitable nutrients such as e.g. one or more carbon sources, one or more nitrogen sources, one or more amino acids, one or more vitamins and / or one or more minerals. In an embodiment, the growth medium is selected from the group consisting of: de Man, Rogosa and Sharpe broth (MRS broth); Tryptic Soy Broth (TSB broth); and Bifidobacterium Selective Medium (BSM broth).

[0060] Preferably, the Bifidobacterium strain is grown under controlled conditions to maximize biomass production. Pre-cultures may preferably be used to ensure the Bifidobacterium cells are in the optimal growth phase for large-scale fermentation.

[0061] The fermentation may be carried out as long as needed to obtain a satisfactory growth and / or cell count of the Bifidobacterium strain. In one aspect, step (a) is a step of fermenting Bifidobacterium and is carried out for 7 to 20 hours, such as for 7 to 15 hours. In a further aspect, step (a) is a step of fermenting Bifidobacterium and is carried out for about 10 hours.

[0062] The fermentation method used to obtain Bifidobacterium may be any suitable method, such as, e.g., batch-fermentation, fed-batch fermentation, continuous fermentation, and two-stage fermentation.

[0063] In an embodiment, the composition of step (a) comprises at least 1E+9 CFU / g Bifidobacterium cells. In a further embodiment, the composition of step (a) comprises between 1E+9 and 1E+12 CFU / g Bifidobacterium cells.

[0064] In one aspect, the invention provides for a process for producing a Bifidobacterium strain, the process comprising the steps of:

[0065] (a) fermenting a Bifidobacterium strain at a pH of at least pH 5.0, such as at a pH in the range of 5.0-7.0, to obtain a fermentation composition comprising the Bifidobacterium strain;

[0066] (b) adjusting the pH of the fermentation composition of step (a) to a pH in the range of pH 3.5-10.0 to obtain a pH adjusted composition;

[0067] (c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for at least 60 minutes to obtain a heat-treated composition;

[0068] (d) incubating the heat-treated composition of step (c) at a temperature in the range of 4-20°C for at least 4 hours to obtain the Bifidobacterium strain.

[0069] In the processes of the invention, pH is adjusted for the composition comprising Bifidobacterium strain to a pH in the range of pH 3.5-10.0. In an embodiment, the pH is adjusted to a pH in the range of pH 4.0-9.0, such as to a pH of about pH 4.5-8.5. In an embodiment, the pH is adjusted to a pH in the range of pH 6.5-8.5, such as about pH 7.0-8.0. In an embodiment, the pH is adjusted to a pH of about 4.0-4.5, or about 6.0-7.0, or about 7.0-8.0, or about 7.0-7.5. In a preferred embodiment, the pH is adjusted to a pH in the range of 7.0-8.0. In a preferred embodiment, the pH is adjusted to a pH in the range of 7.0-7.5.

[0070] Following pH adjustment, the composition comprising Bifidobacterium strain is heat-treated at a temperature in the range of 50-70°C for at least 60 minutes. In an embodiment, theheat treatment is at a temperature in the range of about 55-65°C. In an embodiment, the heat treatment is at a temperature of about 60°C. The temperature set in step (c) is kept for at least 60 minutes. In an embodiment, the heat treatment in step (c) is between 1 hour and 10 hours, such as between 1 hour and 4 hours. In a preferred embodiment, the heat treatment in step (c) is at a temperature in the range of 55-65°C, such as at a temperature of about 60°C, for at least 1 hour, such as for 1.5-4 hours.

[0071] In the processes of the invention, the pH of step (b) and the temperature of step (c) are kept for at least 1 hour. In an embodiment, the pH of step (b) and the temperature in step (c) are kept for between 1 to 10 hours, such as between 1 and 8 hours, between 1 and 6 hours, between 1 and 5 hours, or between 1 and 4 hours. In a further embodiment, the pH of step (b) and the temperature in step (c) are kept for about 1.5 hours. In a further embodiment, the pH of step (b) and the temperature in step (c) are kept for about 1 hour.

[0072] Following steps (b) and (c), the composition comprising Bifidobacterium is incubated, in step (d), at a temperature in the range of 4-20°C for at least 4 hours. In the context of the invention, this is also referred to as a step of cooled incubation or overnight incubation. In an embodiment, the incubation in step (d) is at a temperature in the range of about 5-15°C, such as a temperature of about 10°C, for at least 8 hours, such as at least 10 hours, such at least 12 hours, such as at least 15 hours, such as at least 20 hours. In a preferred embodiment, the incubation in step (d) is at a temperature of about 5-15°C for at least 10 hours.

[0073] In some embodiments, a separation step is performed before step (b) or after step (d). In an embodiment, the separation step is performed before step (b). In an embodiment, the separation step is a centrifugation, a microfiltration or an ultrafiltration step. In a preferred embodiment, the separation step is a centrifugation step. In a preferred embodiment, the separation step is a centrifugation step which is performed before step (b). For example, the step of centrifugation may be a step of disc stack centrifugation. Including a step of separation, such as, e.g., centrifugation or filtration, after fermentation of the Bifidobacterium strain, aids in concentrating the biomass to increase the cell density for further processing.

[0074] In some embodiments, a carrier is added before step (b) and / or after step (d). In a preferred embodiment, the carrier is added before step (b). The carrier may be any carrier deemed suitable by a person of skill in the art. In an embodiment, the carrier is selected from the group consisting of dextrin, maltodextrin, polydextrin, maize dextrin, potassium chloride, starch, lactose, Nutriose, sucrose, glucose, whey, gelatine, casein and / or albumin. In a preferred embodiment, the carrier is maltodextrin, dextrin, polydextrin or a combination thereof. In a further preferred embodiment, the carrier is maltodextrin. When a stabilizing carrier, such as maltodextrin, is added to the Bifidobacterium composition (such as, e.g., before step (b) of the process), it may help protecting the bacterial cell integrity during the inactivation and optional drying processes.In preferred embodiments, after step (d), the Bifidobacterium strain is dried. Drying may be carried out by any method known to the person skilled in the art and may be carried out using any suitable apparatus. In an embodiment, the drying is selected from the group consisting of spray drying, freeze drying, air drying, drum drying, vacuum drying, fluidized bed drying, extrusion drying, or any combination thereof. In a preferred embodiment, the drying is spray drying, fluidized bed drying, or freeze drying. In a specific embodiment, drying after step (d) is spray drying.

[0075] The processes for producing a Bifidobacterium strain provides Bifidobacterium strains for use in a composition or product comprising a low amount of living Bifidobacterium cells. In an embodiment, provided is a Bifidobacterium strain for use in a composition or product comprising less than 1E+8 CFU / g Bifidobacterium cells, such as less than 1E+7 CFU / g, 1E+6 CFU / g, 1E+5 CFU / g, 1E+4 CFU / g, 1E+3 CFU / g, 1E+2 CFU / g or 1E+1 CFU / g Bifidobacterium cells. In an embodiment, the Bifidobacterium strain is for use in a composition or product comprising less than 1E+4 CFU / g Bifidobacterium cells. In an embodiment, the Bifidobacterium strain is for use in a composition or product comprising less than 1E+3 CFU / g Bifidobacterium cells. In an embodiment, the Bifidobacterium strain is for use in a composition or product comprising less than 1E+2 CFU / g Bifidobacterium cells. In an embodiment, the Bifidobacterium strain is for use in a composition or product comprising less than 1E+1 CFU / g Bifidobacterium cells. In an embodiment, the Bifidobacterium strain is for use in a composition or product comprising no measurable live Bifidobacterium cells. In another or further embodiment, the Bifidobacterium strain is for use as a postbiotic. In another or yet further embodiment, the Bifidobacterium strain is for use in a food, such as for use in a beverage.

[0076] Use of Bifidobacterium cells produced by the production method

[0077] Bifidobacterium cells produced by the process of the invention can be used in a pharmaceutical supplement, a dietary supplement, a nutraceutical, a food and / or a dietary composition comprising a physiologically effective dose of the obtained Bifidobacterium cells and optionally a physiologically compatible carrier.

[0078] The pharmaceutical compositions are compositions which serve solely therapeutic or prophylactic purposes.

[0079] Supplement and dietary supplement compositions are human health compositions that are intended to supplement the diet and promote overall health and well-being.

[0080] Nutraceutical compositions have health benefits beyond basic nutrition and are typically marketed as foods or dietary supplements.

[0081] Dietary compositions within the meaning of the present invention are compositions which, in addition to the Bifidobacterium cells produced according to the invention, comprise a food or foodstuff (see, for example, not exhaustively, EU Directive 2002 / 46 / EC of June 10, 2002) and / or dietary supplement, optionally comprising adjuvants and additives.The invention further relates to the use or application of Bifidobacterium cells produced according to the invention in producing a pharmaceutical or dietary composition, or a pharmaceutical product, supplement, nutraceutical, food, e.g., a functional food, or a dietary supplement, comprising the Bifidobacterium cells or a pharmaceutical, supplement, dietary supplement, nutraceutical, food or dietary composition, in particular for use in:

[0082] prophylaxis and / or treatment of undesirable gastrointestinal inflammatory activity such as inflammatory bowel disease, e.g., Crohn’s disease or ulcerative colitis, irritable bowel syndrome, pouchitis, or post infection colitis;

[0083] reducing the levels of pro-inflammatory cytokines, such as by antagonising and excluding pro-inflammatory microorganisms from the gastrointestinal tract; improving or sustaining vitality and / or reducing mental fatigue in humans; counter-regulating stress responses and / or negative emotions in humans; and / or improving sleep in humans.

[0084] The invention further relates to the use or application of Bifidobacterium cells produced according to the invention in producing a pharmaceutical or dietary composition, or a pharmaceutical product, supplement, nutraceutical, food, e.g. a functional food, or a dietary supplement, for prophylaxis and / or the treatment of diseases arising from gastrointestinal conditions. These include in particular gastritis, stomach ulcers and stomach cancer. Also covered are diseases and symptoms, such as abdominal discomfort, in particular discomfort of the upper abdomen, gastric heaviness, gastric spasms, stomach pain, pain or pressure in the upper abdomen, burning sensation in the upper abdomen, chronic-recurrent abdominal disorders, permanent feeling of fullness, lack of appetite, fasting pain, bloating, heartburn, diarrhea, irregular bowel movements, indisposition, nausea, sickness and vomiting, food intolerance, malabsorption, upset stomach (functional dyspepsia), gastritis, damage to the mucous membrane, or gastroduodenal ulcer.

[0085] The invention further relates to the use or application of Bifidobacterium cells produced according to the invention in producing a pharmaceutical or dietary composition, or a pharmaceutical product, supplement, nutraceutical, food, e.g. a functional food, or a dietary supplement, for use in the preparation of anti-inflammatory biotherapeutic agents or functional foods for reducing the levels of pro inflammatory cytokines.

[0086] Chronic inflammation is a recognized contributor to the development and progression of for example metabolic diseases, the cytokine-modulating effect showed herein indicates that the strain and compositions comprising it may also be useful in the prevention, management, amelioration, or treatment of metabolic disorders. In some embodiments, the anti-inflammatory effect is for the prophylaxis and / or the treatment of a metabolic disorder selected from: obesity, metabolic syndrome, insulin resistance, prediabetes, type 2 diabetes, dyslipidemia, and / or fatty liver disease.The invention also relates to the use or application of Bifidobacterium cells produced according to the invention as an anti- infective postbiotic.

[0087] The reference quantity here is a unit of administration, for example a tablet. The composition is preferably prepared for oral administration.

[0088] The galenic preparation of a pharmaceutical, supplement, nutraceutical, food or dietary composition, in particular of a supplement, dietary supplement or pharmaceutical product (drug), can be carried out in a way that is common practice in the art. Suitable solid or liquid galenic forms of preparation include, for example, granules, powders, sugar-coated tablets, tablets (micro)capsules, hard capsules, suppositories, syrups, juices, suspensions or emulsions, in the production of which conventional adjuvants such as excipients, disintegrants, binders, coating agents, swelling agents, glidants, lubricants, flavour additives, sweetening agents and solubilizers, are employed. Adjuvants that should be mentioned include magnesium stearate, sodium chloride, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum, milk protein, gelatine, starch, cellulose and the derivatives thereof, animal and vegetable oils such as cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycols and solvents, such as sterile water, and monohydric or polyhydric alcohols, such as glycerine. The pharmaceutical composition can be produced by mixing cells of at least one Bifidobacterium strain in a defined dosage with a pharmaceutically suitable and physiologically compatible carrier, and optionally with further suitable active ingredients, additives or adjuvants having defined dosages, and preparing the desired form of administration. Possible carriers include in particular substances that are selected from the group consisting of dextrin, polydextrin, maltodextrin, microcrystalline cellulose, starch, in particular corn starch, levulose, lactose, dextrose, and mixtures of such substances. The composition may comprise or consist of 0.1 to 95% by weight carrier and 5 to 99.9% by weight spray-dried Bifidobacterium cells, based on the total quantity of cells and carrier.

[0089] The invention further relates to a method for producing a pharmaceutical and / or dietary composition according to the invention, in particular a supplement, dietary supplement or pharmaceutical product (drug), wherein the Bifidobacterium cells obtained by the process disclosed herein are mixed with the physiologically compatible carrier and are preferably prepared for oral administration.

[0090] The administration can take place over a limited period of time, for example 1 to 30 weeks, or without limitations in terms of time. In particular the latter is suited for permanent prophylaxis and for the prevention of relapses.

[0091] Postbiotics derived from Bifidobacterium strains offer several advantages for incorporation into functional foods. These advantages include enhanced stability, as postbiotics are more resistant to environmental changes, ensuring stability during processing, storage, and transportation. Additionally, with their non-viable nature, the postbiotics of Bifidobacterium poses no risk of infection or overgrowth, making them especially suitable for immunocompromisedindividuals. Despite inactivation, the postbiotics retain their immunomodulatory and other beneficial health properties, providing similar benefits to live probiotics. Furthermore, their improved shelf-life, not requiring refrigeration or special handling, makes them ideal for use in various functional food products. The stability of postbiotics also ensures consistent health benefits over time, regardless of temperature fluctuations during processing and storage, ultimately meeting the growing consumer demand for convenient, health-enhancing food options.

[0092] Thus, the invention further relates to a method for producing a functional food, wherein the Bifidobacterium cells produced by the herein disclosed process are mixed with the food, optionally in the presence of a physiologically compatible carrier. The reference quantity is a unit of administration, for example a packaging unit of a foodstuff for sale to an end consumer. The physiologically compatible carrier will generally be a food, which in particular may be selected from the group consisting of dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparations. Suitable foods or foodstuffs, including water are those as defined, not exhaustively, for example, in (EC) Regulation No. 178 / 2002 of January 28, 2002.

[0093] Additionally, an aspect of the present invention relates to a functional food comprising the Bifidobacterium strain obtainable by a process as embodied herein. In an embodiment, the functional food is selected from the list comprising infant formula, follow-on formula, baby food formula, infant cereals formula, growing-up milk, infant or child's food supplement, fruit juice, fruit drink, flavoured water, soda, sports drink, tea-based beverage, coffee-based beverage, kombucha-based beverage, dairy-based yogurt beverage, dairy alternative yogurt beverage, dairy-based kefir beverage, dairy alternative kefir beverage, dairy-based milk beverages, dairy alternative beverages, clinical nutrition beverage, dairy-based stirred yogurt, dairy alternative stirred yogurt, dairy-based set-type yogurt, dairy alternative set-type yogurt, dairy-based strained yogurt, dairy alternative strain yogurt, dairy-based ice cream, dairy alternative ice cream, protein bar, snack bar, meal replacement bar, cookie, biscuit, cracker, breakfast cereals, muesli, powdered milk, instant coffee, instant chocolate, instant cocoa, instant tea, instant soup, instant porridge, instant noodles, baby food, instant baby food, instant sauce mix, instant gravy, instant mashed potatoes, instant pudding, instant curry, instant tofu powder, powdered meals, field rations, and any suitable mixtures thereof.

[0094] In a preferred embodiment, the functional food is a food selected from the group comprising dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparation.

[0095] In another preferred embodiment, the functional food is a beverage selected from the group comprising a fruit juice, fruit drink, flavoured water, soda, sports drink, kombucha-based beverage, yogurt-based beverage, tea-based beverage, or coffee-based beverage. In an embodiment, the functional food is a beverage selected from a fruit juice, fruit drink, flavoured water, soda, sports drink, kombucha-based beverage, or yogurt-based beverage.In an embodiment, the functional food is a kombucha-based beverage. For example, the beverage may be kombucha-based beverage that is not alcoholic.

[0096] In another preferred embodiment, the functional food is a carbonated beverage.

[0097] LIST OF EMBODIMENTS

[0098] Embodiment 1. A process for producing a Bifidobacterium strain, the process comprising the steps of:

[0099] (a) obtaining a composition comprising a Bifidobacterium strain;

[0100] (b) adjusting the pH of the composition of step (a) to a pH in the range of pH 3.5-10.0 to obtain a pH adjusted composition;

[0101] (c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for at least 60 minutes to obtain a heat-treated composition;

[0102] (d) incubating the heat-treated composition of step (c) at a temperature in the range of 4-20°C for at least 4 hours to obtain the Bifidobacterium strain.

[0103] Embodiment 2. The process of the preceding embodiment, wherein the Bifidobacterium strain is Bifidobacterium longum.

[0104] Embodiment 3. The process of any of the preceding embodiments, wherein the Bifidobacterium strain is Bifidobacterium longum deposited as NCIMB 41676.

[0105] Embodiment 4. The process of any of the preceding embodiments, wherein the pH in step (b) is adjusted to a pH in the range of pH 4.0-9.0, such as to a pH of about pH 4.5-8.5.

[0106] Embodiment 5. The process of any of the preceding embodiments, wherein the pH in step (b) is adjusted to pH of about pH 6.5-8.5, such as about pH 7.0-8.0.

[0107] Embodiment 6. The process of any of embodiments 1-4, wherein the pH in step (b) is adjusted to pH 4.0-5.0.

[0108] Embodiment 7. The process of any of embodiments 1-4 or 6, wherein the pH in step (b) is adjusted to pH of about 4.0-4.5.

[0109] Embodiment 8. The process of any of the preceding embodiments, wherein the heat treatment in step (c) is between 1 hour and 10 hours.

[0110] Embodiment 9. The process of any of the preceding embodiments, wherein the heat treatment in step (c) is between 1 hour and 4 hours.

[0111] Embodiment 10. The process of any of the preceding embodiments, wherein the heat treatment in step (c) is at a temperature in the range of 55-65°C, such as about 60°C.

[0112] Embodiment 11. The process of any of the preceding embodiments, wherein the incubation in step (d) is carried at a temperature in the range of 5-15°C, such as at a temperature of about 10°C.

[0113] Embodiment 12. The process of any of the preceding embodiments, wherein the incubation in step (d) is for at least 8 hours.Embodiment 13. The process of any of the preceding embodiments, wherein the incubation in step (d) is for at least 10 hours.

[0114] Embodiment 14. The process of any of the preceding embodiments, wherein the incubation in step (d) is for at least 12 hours.

[0115] Embodiment 15. The process of any of the preceding embodiments, wherein the composition in step (a) is obtained by fermenting the Bifidobacterium strain at a pH suitable for ensuring growth of the Bifidobacterium strain.

[0116] Embodiment 16. The process of any of the preceding embodiments, wherein the composition in step (a) is obtained by fermenting the Bifidobacterium strain at a pH of at least 5.0, such as at a pH of between 5.0-7.0.

[0117] Embodiment 17. The process of any of the preceding embodiments, wherein the composition in step (a) is obtained by fermenting the Bifidobacterium strain at a pH in the range of 5.5-6.9, such as at a pH in the range of 5.9-6.8, such as at a pH in the range of 6.2-6.6.

[0118] Embodiment 18. The process of any of the preceding embodiments, wherein the composition in step (a) is obtained by fermenting the Bifidobacterium strain at a temperature in the range of 35-40°C, such as at a temperature of about 37°C.

[0119] Embodiment 19. The process of any of the preceding embodiments, wherein the composition in step (a) has a temperature in the range of 35-40°C.

[0120] Embodiment 20. The process of any of the preceding embodiments, wherein the composition in step (a) has a temperature of about 37°C.

[0121] Embodiment 21. The process of any of the preceding embodiments, wherein the composition in step (a) comprises at least 1E+9 CFU / g Bifidobacterium cells, optionally wherein the composition in step (a) comprises between 1E+9 and 1E+12 CFU / g Bifidobacterium cells.

[0122] Embodiment 22. The process of any of the preceding embodiments, wherein a separation step is performed before step (b) or after step (d).

[0123] Embodiment 23. The process of the preceding embodiment, wherein the separation step is a centrifugation, a microfiltration or an ultrafiltration step.

[0124] Embodiment 24. The process of any of embodiments 22-23, wherein the separation step is a centrifugation step.

[0125] Embodiment 25. The process of any of the preceding embodiments, wherein a carrier is added before step (b) and / or after step (d).

[0126] Embodiment 26. The process of any of the preceding embodiments, wherein a carrier is added before step (b).

[0127] Embodiment 27. The process of any of embodiments 25-26, wherein the carrier is selected from the group consisting of dextrin, polydextrin, maltodextrin, maize dextrin, potassium chloride, starch, lactose, Nutriose, sucrose, glucose, whey, gelatine, casein and / or albumin.

[0128] Embodiment 28. The process of any of embodiments 25-27, wherein the carrier is maltodextrin.Embodiment 29. The process of any of the preceding embodiments, wherein after step (d), the Bifidobacterium strain is dried.

[0129] Embodiment 30. The process of any of the preceding embodiments, wherein after step (d), the Bifidobacterium strain is dried, wherein the drying is spray drying, freeze drying, air drying, drum drying, vacuum drying, fluidized bed drying, extrusion drying, or any combination thereof.

[0130] Embodiment 31. The process of any of the preceding embodiments, wherein after step (d), the Bifidobacterium strain is dried by spray drying, fluidized bed drying, or freeze drying.

[0131] Embodiment 32. The process of any of the preceding embodiments, wherein after step (d), the Bifidobacterium strain is dried by spray drying.

[0132] Embodiment 33. The process of any of the preceding embodiments, further comprising adding the Bifidobacterium strain to a composition to obtain a composition characterized by having a low amount of living Bifidobacterium cells.

[0133] Embodiment 34. The process of any of the preceding embodiments, further comprising adding the Bifidobacterium strain to a composition to obtain a composition comprising less than 1E+8 CFU / g Bifidobacterium cells.

[0134] Embodiment 35. The process of any of embodiments 33-34, wherein the composition comprises less than 1E+8 CFU / g, 1E+7 CFU / g, 1E+6 CFU / g, 1E+5 CFU / g, 1E+4 CFU / g, 1E+3 CFU / g, 1E+2 CFU / g or 1E+1 CFU / g Bifidobacterium cells.

[0135] Embodiment 36. The process of any of embodiments 33-35, wherein the composition comprises less than 1E+4 CFU / g, such as less than 1E+3 CFU / g, 1E+2 CFU / g or 1E+1 CFU / g Bifidobacterium cells.

[0136] Embodiment 37. The process of any of embodiments 33-36, wherein the composition comprises no measurable live Bifidobacterium cells.

[0137] Embodiment 38. The process of any of embodiments 33-37, wherein the composition comprising the Bifidobacterium strain is a postbiotic.

[0138] Embodiment 39. The process of any of the preceding embodiments, further comprising adding the Bifidobacterium strain or the composition comprising Bifidobacterium cells to a food product to obtain a functional food.

[0139] Embodiment 40. The process of the preceding embodiment, wherein the functional food is selected from the list comprising infant formula, follow-on formula, baby food formula, infant cereals formula, growing-up milk, infant or child's food supplement, fruit juice, fruit drink, flavoured water, soda, sports drink, tea-based beverage, coffee-based beverage, kombucha-based beverage, dairy-based yogurt beverage, dairy alternative yogurt beverage, dairy-based kefir beverage, dairy alternative kefir beverage, dairy-based milk beverages, dairy alternative beverages, clinical nutrition beverage, dairy-based stirred yogurt, dairy alternative stirred yogurt, dairy-based set-type yogurt, dairy alternative set-type yogurt, dairy-based strained yogurt, dairy alternative strain yogurt, dairy-based ice cream, dairy alternative ice cream, protein bar, snack bar, meal replacement bar, cookie, biscuit, cracker, breakfast cereals, muesli, powdered milk,instant coffee, instant chocolate, instant cocoa, instant tea, instant soup, instant porridge, instant noodles, baby food, instant baby food, instant sauce mix, instant gravy, instant mashed potatoes, instant pudding, instant curry, instant tofu powder, powdered meals, field rations, and any suitable mixtures thereof.

[0140] Embodiment 41. The process of any of the preceding embodiments, further comprising adding the Bifidobacterium strain or the composition comprising Bifidobacterium cells to a food selected from the group comprising dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparations.

[0141] Embodiment 42. The process of any of the preceding embodiments, further comprising adding the Bifidobacterium strain or the composition comprising Bifidobacterium cells to a milk based food product, such as yoghurt.

[0142] Embodiment 43. The process of any of the preceding embodiments, wherein the process is for production of a composition comprising a low amount of living Bifidobacterium cells.

[0143] Embodiment 44. The process of any of the preceding embodiments, wherein the process is for production of a composition comprising less than 1 E+4 CFU / g Bifidobacterium cells.

[0144] Embodiment 45. The process of any of the preceding embodiments, wherein the process is for production of a composition comprising less than 1 E+3 CFU / g, 1 E+2 CFU / g or 1 E+1 CFU / g Bifidobacterium cells.

[0145] Embodiment 46. The process of embodiment 45, wherein the composition comprises less than 1 E+3 CFU / g Bifidobacterium cells.

[0146] Embodiment 47. The process of embodiment 45, wherein the composition comprises less than 1E+2 CFU / g Bifidobacterium cells.

[0147] Embodiment 48. The process of embodiment 45, wherein the composition comprises no measurable live Bifidobacterium cells.

[0148] Embodiment 49. The process of any of embodiments 43-48, wherein the composition is a food product.

[0149] Embodiment 50. The process of any of embodiments 43-49, wherein the composition is a food selected from the group comprising dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparation.

[0150] Embodiment 51. The process of any of the preceding embodiments, wherein the steps are:

[0151] (a) obtaining a composition comprising a Bifidobacterium strain;

[0152] (b) adjusting the pH of the composition of step (a) to a pH of about 4.5-8.5;

[0153] (c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for about 60-120 minutes to obtain a heat-treated composition;

[0154] (d) incubating the heat-treated composition of step (c) at a temperature in the range of 5- 15°C for at least 4 hours to obtain the Bifidobacterium strain.Embodiment 52. The process of the preceding embodiment, wherein the incubation in step (d) is for at least 10 hours, such as at least 12 hours.

[0155] Embodiment 53. The process of any of embodiments 51-52, wherein the process is for production of a composition comprising less than 1E+2 CFU / g Bifidobacterium cells.

[0156] Embodiment 54. The process of any of embodiments 51-53, wherein after step (d), the Bifidobacterium strain is dried by spray drying.

[0157] Embodiment 55. The process of any of the preceding embodiments, wherein the steps are:

[0158] (a) obtaining a composition comprising a Bifidobacterium strain;

[0159] (b) adjusting the pH of the composition of step (a) to a pH of about 6.5-8.5;

[0160] (c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for about 60 minutes to obtain a heat-treated composition;

[0161] (d) incubating the heat-treated composition of step (c) at a temperature in the range of 5-15°C for at least 4 hours to obtain the Bifidobacterium strain.

[0162] Embodiment 56. The process of the preceding embodiment, wherein the incubation in step (d) is for at least 10 hours, such as at least 12 hours.

[0163] Embodiment 57. The process of any of embodiments 55-56, wherein the process is for production of a composition comprising less than 1E+1 CFU / g Bifidobacterium cells, preferably wherein the composition comprises no measurable live Bifidobacterium cells.

[0164] Embodiment 58. The process of any of embodiments 55-57, wherein after step (d), the Bifidobacterium strain is dried by spray drying.

[0165] Embodiment 59. A Bifidobacterium strain obtained by the process of any of the preceding embodiments.

[0166] Embodiment 60. A functional food comprising the Bifidobacterium strain obtainable by the process of any of embodiments 1-58.

[0167] Embodiment 61. The functional food of embodiment 60, characterized by having a low amount of living Bifidobacterium cells.

[0168] Embodiment 62. The functional food of embodiment 60-61, characterized by comprising less than 1E+4 CFU / g Bifidobacterium cells.

[0169] Embodiment 63. The functional food of any of embodiments 60-62, wherein the functional food is a food selected from the group comprising dairy products, fermented dairy products, milk, yogurt, cheese, cereals, granola bars, baked goods, beverages and infant food preparations.

[0170] Embodiment 64. The functional food of any of embodiments 60-63, wherein the functional food is a milk based food product such as yoghurt.

[0171] Embodiment 65. The process, the strain or the functional food of any of the preceding embodiments, wherein the Bifidobacterium cells obtained by the process of any of the preceding embodiments lead to increased IL-10 levels as measured by a PBMC assay, such as the PBMC assay described herein, compared to the IL-10 levels obtained by a live Bifidobacterium cellscomposition such as a composition comprising at least 1E+9 CFU / g Bifidobacterium cells, such as a composition comprising between 1E+9 and 1E+12 CFU / g Bifidobacterium cells, as measured by said assay.

[0172] Embodiment 66. The process, the strain or the functional food of embodiment 65, wherein the IL-10 levels are increased by at least 50%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 250%, such as at least 300%, compared to the IL-10 levels obtained from the live Bifidobacterium cells composition, such as a composition comprising at least 1E+9 CFU / g Bifidobacterium cells, such as a composition comprising between 1E+9 and 1E+12 CFU / g Bifidobacterium cells.

[0173] EXAMPLES

[0174] Materials and Methods

[0175] Cell Storage: Bifidobacterium longum deposited as NCIMB 41676 was stored in a frozen state at -80°C. For storage, 1 mL of culture, grown to the stationary phase in MRS (55 g / L, pH 6.5; Difco, USA) medium supplemented with 0.5 g / L L-Cysteine, was mixed with 500 pL of a 50% (v / v) sterile glycerol solution.

[0176] Cultivation: Bifidobacterium longum deposited as NCIMB 41676: Cultured in MRS medium supplemented with 0.5 g / L L-Cysteine and 30 g / L glucose monohydrate at 37°C under anaerobic conditions until the stationary phase was reached, as indicated by the OD600 measurements.

[0177] Fermentation of Bifidobacterium longum-. Conducted in MRS medium supplemented with 0.5 g / L L-Cysteine and 30 g / L glucose monohydrate under anaerobic conditions at 37°C. The fermentation process was monitored and controlled for pH, temperature, and monitored for OD600.

[0178] Centrifugation: Following the fermentation, B. longum cultures were centrifuged at 5000g for 15 minutes to concentrate the bacterial cells. The concentrate was washed with deionized water and centrifuged again (5000g for 15 minutes). After discarding the supernatant, the concentrate was used for further processing.

[0179] Carrier Addition and Preparation for Spray Drying: After the centrifugation, Nutriose® (maltodextrin) was added to the concentrated bacterial cells at a 1 :3 ratio based on dry cell weight. The treated bacterial suspensions were then subjected to spray drying using standard parameters (inlet temperature of 140-180°C, outlet temperature of 60-90°C) (Buchi Labortechnik AG, Flawil, Switzerland).

[0180] CFU Counting: Samples were prepared for CFU counting by dissolved and serial diluted in sterile saline solution. The diluted samples were plated using a Spiral Plater (Eddy Jet, IUL, SA, Spain) to ensure uniform distribution and accuracy. The agar plates were then incubated fora period of 48 to 72 hours at a constant temperature of 37°C. The CFU per gram of the original sample was calculated based on the number of colonies, taking into account the dilution factor and the volume of the sample that was plated.

[0181] PBMC Assay and Quantitative Analysis: The PBMC assay was conducted following established methodologies as described in Foligne et al. (2007), Cuffaro et al. (2021), and STAR Protocols (2021), with modifications tailored to the experimental requirements. The critical steps are outlined below, with references to the corresponding published protocols for detailed methodology.

[0182] Materials and Equipment:

[0183] LEGENDplex™ HU Essential Immune Response Panel (13-plex) w / VbP (Cat. No.

[0184] 740930): Used for the quantification of cytokines, including IL-10, IL-12p70, and TNF-a.

[0185] Attune NxT Flow Cytometer: Employed for precise and high-throughput cytokine analysis during the PBMC assay.

[0186] References:

[0187] Foligne, B., et al. (2007). Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. Journal of Applied Microbiology. DOI: 10.1111 / j.1365-2672.2006.03139.x

[0188] Cuffaro, B., et al. (2021). Identification of new potential biotherapeutics from human gut microbiota-derived bacteria. Frontiers in Microbiology. DOI: 10.3389 / fmicb.2021.682732 STAR Protocols (2021). Isolation and functional analysis of human peripheral blood mononuclear cells. STAR Protocols. DOI: 10.1016 / j.xpro.2021.100286

[0189] Bacterial Preparation: Preparation of the bacterial test strains was performed according to the protocols detailed in Foligne et al. (2007), with the following modifications:

[0190] Strain: Bifidobacterium longum deposited as NCIMB 41676 was utilized as the test strain.

[0191] Cultivation: Cultivation was in MRS medium supplemented with 0.5 g / L L-Cysteine and 30 g / L glucose monohydrate at 37°C under anaerobic conditions until reaching the stationary phase (OD600).

[0192] Storage: Post-cultivation, bacterial cells were stored at -80°C in a sterile 50% (v / v) glycerol solution.

[0193] PBMC Isolation: PBMC isolation was performed following the procedure described in STAR Protocols (2021), utilizing a density gradient separation with Histopaque. The key steps included:

[0194] 1. Dilution of buffy coat with PBS containing 2% FCS at a 1:1 ratio.

[0195] 2. Layering of the diluted buffy coat over Histopaque in SepMate tubes, followed by centrifugation at 1200g for 20 minutes at room temperature.

[0196] 3. Collection of the mononuclear cell (MNC) layer and subsequent washing with PBS + 2% FCS to remove residual plasma and platelets.4. Final resuspension of PBMCs in RPMI-1640 medium with 10% heat-inactivated FCS to achieve a concentration of 2x106cells / mL.

[0197] Co-lncubation of Bacteria and PBMCs: The co-incubation procedure was carried out as described in Foligne et al. (2007) and Cuffaro et al. (2021), with the following adjustments:

[0198] 1. PBMCs were seeded in 96-well tissue culture plates at a volume of 130 pL (1.54x106cells / mL).

[0199] 2. Bacterial suspensions (108cells / mL) were prepared as described above and added to the wells at a volume of 20 pL.

[0200] 3. Each well was supplemented with 50 pL RPMI-1640 medium containing 10% FCS to ensure a total volume of 200 pL per well.

[0201] 4. Plates were incubated for 20 hours at 37°C with 5% CO2.

[0202] Supernatant Collection: The supernatant collection was performed in accordance with STAR Protocols (2021), with the following additions:

[0203] 1. Post-incubation, plates were centrifuged at 300g for 10 minutes.

[0204] 2. 180 pL of supernatant was transferred to new 96-well polypropylene plates and centrifuged at 3500 rpm for 10 minutes to remove debris.

[0205] 3. Clarified supernatants were aliquoted into storage plates, sealed with extreme sealing foil, and stored at -80°C for subsequent analysis.

[0206] Results

[0207] The supernatants were analyzed for cytokine production (e.g., IL-10 and IL-12) to assess the immunomodulatory effects of the bacterial strains on PBMCs.

[0208] Control Strains: Ligilactobacillus salivarius Ls-33 served as a positive control (high IL-10 / IL-12 ratio), and Lactobacillus plantarum Lp-115 served as a negative control (low I L-10 / IL-12 ratio).

[0209] Experimental Samples: The commercial powder of live Bifidobacterium longum (Sample ID: Comm._1714) and spray-dried test samples demonstrated distinct cytokine response profiles, which were quantified and compared against the two control strains.

[0210] Example 1: Effect of pH Treatment on Viable Cell Count and Immunomodulatory Activity This example evaluates the effect of varying pH conditions on the viability and immunomodulatory activity of Bifidobacterium longum.

[0211] Procedure: The pH of the bacterial suspension was adjusted to 4.37, 6.55, 7.0, 7.5, 8.0, or 8.5 using sterile NaOH or HCI solutions. Samples were then incubated at 37°C without additional heat treatment or cooled incubation.

[0212] Results:

[0213] CFU Analysis:pH 4.37 and 6.55 significantly reduced CFU counts compared to untreated controls but did not achieve complete inactivation (CFU counts remained above 104).

[0214] pH 7.0 and 7.5 showed moderate reductions, with CFU counts exceeding 106.

[0215] pH 8.0 and 8.5 exhibited minimal impact on CFU viability without additional treatments.

[0216] PBMC Assay:

[0217] IL-10 levels increased slightly at pH 7.0 and 7.5 compared to the live control (Comm_1714), indicating mild enhancement of anti-inflammatory activity.

[0218] Pro-inflammatory cytokines (TNF-a and I L- 12p70) remained comparable to the live control across all pH levels.

[0219] Conclusion: pH adjustments alone are insufficient to achieve significant CFU reduction or enhance immunomodulatory activity, highlighting the need for additional treatments.

[0220] Example 2: Effect of Heat Treatment on Viable Cell Count and Immunomodulatory Activity This example assesses the impact of heat treatment at 60°C for varying durations on CFU and PBMC responses.

[0221] Procedure: Bacterial suspensions were incubated at 60°C for 1 or 2 hours without pH adjustment or cooled incubation.

[0222] Results:

[0223] CFU Analysis:

[0224] Heat treatment alone reduced CFU counts but did not achieve complete inactivation (CFU counts remained above 104).

[0225] Longer durations of the heat treatment resulted in greater CFU reduction, with diminishing returns after 2 hours.

[0226] PBMC Assay:

[0227] IL- 10 production increased marginally after 2 hours of heat treatment, while TNF-a and I L-12p70 levels remained stable compared to untreated samples.

[0228] Conclusion: Heat treatment alone reduces CFU counts but is not sufficient for complete inactivation or significant enhancement of anti-inflammatory activity.

[0229] Example 3: Combined Effect of pH and Heat Treatment

[0230] This example investigates the synergistic impact of pH adjustment and heat treatment on CFU reduction and PBMC responses.Procedure: The pH of the bacterial suspension was adjusted to 4.37, 6.55, 7.0, 7.5, 8.0, or 8.5. Samples were then heated at 60°C for 1 or 2 hours.

[0231] Results:

[0232] CFU Analysis:

[0233] pH 7.5 combined with 1 hour of heat treatment achieved significant CFU reduction (below 104CFU / g).

[0234] At pH 4.37 and 6.55, complete inactivation required 2 hours of heat treatment.

[0235] Higher pH levels (8.0 and 8.5) were less effective, with residual CFU counts exceeding 106CFU / g.

[0236] PBMC Assay:

[0237] IL-10 levels increased significantly at pH 7.5 and 8.0 after 2 hours of heat treatment, with levels surpassing those of the live control.

[0238] Pro-inflammatory cytokine levels (TNF-a and I L-12p70) remained consistent with the live control.

[0239] Conclusion: The combination of pH adjustment and heat treatment enhances CFU reduction and improves anti-inflammatory cytokine production, particularly at pH 7.5 and 8.0.

[0240] Example 4: Effect of Cooled Incubation on Viable Cell Count and Immunomodulatory Activity

[0241] This example evaluates the role of cooled incubation in enhancing the effects of pH and heat treatment.

[0242] Procedure: Treated samples from Example 3 were incubated at 10°C for 12 hours.

[0243] Results:

[0244] CFU Analysis:

[0245] Cooled incubation significantly enhanced CFU reduction across all pH conditions.

[0246] At pH 7.5, complete inactivation (below 104CFU / g) was achieved already after 1 hour of heat treatment followed by cooled incubation.

[0247] Without cooled incubation, residual CFU counts remained above 104.

[0248] PBMC Assay:

[0249] IL-10 levels increased further with cooled incubation, particularly at pH 7.5 and 8.0, with values reaching 300% of the live control.

[0250] TNF-a and I L-12p70 levels remained unaffected.Conclusion: Cooled incubation is effective in achieving sufficient CFU inactivation and enhancing anti-inflammatory cytokine production.

[0251] Example 5: Combined Effect of pH, Heat Treatment, and Cooled Incubation

[0252] This example demonstrates the cumulative effect of pH adjustment, heat treatment, and cooled incubation on CFU reduction and PBMC responses.

[0253] Procedure: The pH of the bacterial suspension was adjusted to 7.0, 7.5, 8.0, or 8.5. Then, samples were heated at60°C for 1 or 2 hours, followed by cooled incubation at 10°C for 12 hours.

[0254] Results:

[0255] CFU Analysis:

[0256] Complete inactivation (below 104CFU / g) was consistently achieved at pH 7.5 and 8.0 with 1 hour of heat treatment and cooled incubation.

[0257] Residual CFU counts remained higher without the cooled incubation, highlighting its importance in the process.

[0258] PBMC Assay:

[0259] IL-10 levels were maximized under combined conditions, with pH 8.0 showing the highest enhancement (300% increase compared to the live control).

[0260] TNF-a and I L-12p70 levels remained comparable to the live control across all conditions.

[0261] Conclusion: The combination of pH adjustment, heat treatment, and cooled incubation provides optimal conditions for achieving complete CFU inactivation and significantly enhancing anti-inflammatory cytokine production.

[0262] Log CFU / g Reduction for Bifidobacterium longum Across Different pH Levels and Treatment Times with and without Cooled Incubation:

[0263] Figure 1 illustrates the effect of treatment time and pH on the viable cell count (log CFU / g) of Bifidobacterium longum, highlighting the importance of both pH adjustment and the cooled incubation after the heat treatment in achieving significant cell inactivation. The x-axis represents the treatment time (hours) at different pH levels, both with and without cooled incubation, while the y-axis shows the log CFU / g values.

[0264] Figure 1 shows that successful reduction of B. longum to below log 4 CFU / g is achieved under specific pH conditions and treatment times, with the critical addition of cooled incubation. The most effective pH levels were pH 7.0 and pH 7.5, which achieved complete cell inactivation (log 0 CFU / g) within 2 hours at pH 7.0 and as guickly as 1 hour at pH 7.5. Both conditions relied on the further step of cooled incubation to be fully effective.Additionally, pH 4.37 and pH 6.55 also achieved log CFU / g reductions below 4, but required longer treatment times, with log CFU / g values ranging from 1.8 to 2.5 after 1 to 4 hours of treatment. Cooled incubation after the heat treatment was necessary for these conditions as well, without which the reductions were not as effective.

[0265] In conclusion, the parameters that consistently result in successful inactivation of B. longum were pH 4.37, pH 6.55, pH 7.0, and pH 7.5 with heat treatment followed by cooled incubation, with pH 7.0 and 7.5 being the most efficient, achieving full inactivation in the shortest time. These findings define the optimal conditions for producing a postbiotic product with reliable and significant reduction of viable cell counts.

[0266] Assessment of Immunomodulatory activity:

[0267] Figure 2 presents the cytokine levels (TNF-a, I L-12p70, and IL- 10) in PBMCs (Peripheral Blood Mononuclear Cells) exposed to postbiotic samples derived from Bifidobacterium longum. Cytokine responses are displayed across a range of pH levels (6.5, 7.0, 7.5, 8.0, and 8.5) and heating durations (0, 1 and 2 hours at60°C), with "Comm_1714" (ProbioBrain, the live B. longum 1714) and a "Blank" (no postbiotic) serving as controls. The horizontal dashed line represents the baseline (as positive control) cytokine levels from the live commercial control, "Comm_1714".

[0268] The postbiotic form of B. longum showed a favourable anti-inflammatory profile, particularly at pH 8.0 and 8.5 with extended heating times. At these conditions, IL-10 levels increase by 250% to 300% compared to the commercial control, reaching concentrations between 1500 and 3000 pg / ml. This significant boost in IL-10 suggests a heightened anti-inflammatory response while maintaining pro-inflammatory cytokine levels (TNF-a and IL-12p70) similar to those in the live commercial control (“Comm_1714”).

[0269] In summary, the postbiotic treatment as claimed and disclosed herein not only inactivates Bifidobacterium effectively, reducing viable cell counts to below 104CFU / g, but also enhances anti-inflammatory activity, positioning Bifidobacterium postbiotics, such as Bifidobacterium longum postbiotics, as an ideal candidate for immune-modulating applications aimed at reducing inflammation and promoting immune regulation.

[0270] Example 5: Scale-Up Confirmation of pH 8.0 Treatment and Spray Drying at 450 L

[0271] Methods are as described in the Material and Methods section of the Examples, unless otherwise indicated hereafter.

[0272] Materials and Methods

[0273] Cell Storage and Cultivation: Bifidobacterium longum deposited as NCIMB 41676 was stored at -80°C. The strain was cultivated in MRS medium (55 g / L, pH 6.5; Difco, USA) supplementedwith 0.5 g / L L-cysteine and 30 g / L glucose monohydrate at 37°C under anaerobic conditions until stationary phase.

[0274] Fermentation: Fermentation was carried out in the same medium at 37°C under anaerobic conditions with monitoring of pH, temperature, and OD600. A reference experiment was first performed at 40L. The process was then transferred to 450 L to confirm scale-up feasibility. Centrifugation: After fermentation, the culture was concentrated by pilot-scale centrifugation. At 450L scale, the centrifugation step provided an approximately 17-fold concentration on a mass: mass basis. The biomass was washed with deionized water and centrifuged again.

[0275] pH Treatment and Incubation: The concentrated biomass was divided into an untreated control and a treated condition. For the treated condition, the biomass was adjusted to pH 8.0 using sterile NaOH (27%) and incubated for 12 h at 10°C. The untreated control was processed in parallel without pH adjustment and incubated for the same period. pH 8.0 was selected as a proof-of-concept condition for downstream evaluation.

[0276] Carrier Addition and Spray Drying: Nutriose® was added at a 1:3 ratio based on dry cell weight. Samples were spray dried using a custom-designed pilot-scale spray drier. The inlet temperature was kept in the range of 140 to 180°C and the outlet temperature in the range of 60 to 90°C. The average outlet temperature was 70°C. Feed dry matter was 20 to 30%.

[0277] CFU Counting: Spray-dried powders were dissolved in sterile saline, serially diluted, and plated for viable count determination. Plates were incubated at 37°C for 48 to 72 h. Results were expressed as CFU / g powder. Samples with no detectable colonies were reported as ND or <LOD.

[0278] Results

[0279] In the 40 L reference experiment, untreated control samples showed viable counts of 1.73 x 10A8 and 1.65 x 1OA8 CFU / g. The corresponding pH 8.0-treated samples showed viable counts of 1, 180, and 203 CFU / g.

[0280] In the 450 L scale-up experiment, untreated spray-dried material remained culturable, with viable counts ranging from 9.20 x 1OA4 to 4.47 x 1OA5 CFU / g. The mean value was 2.90 x 1OA5 ± 1.44 x 1OA5 CFU / g, corresponding to 5.40 ± 0.28 Iog10 CFU / g (n = 5). In contrast, the pH 8.0-treated 450 L material yielded no detectable colonies in any plated determination and was therefore below the detection limit of the assay.

[0281] The data confirms that the pH 8.0 treatment can be transferred from 40L to 450L while maintaining reduction of viable cells after spray drying.Deposit of Biological Material

[0282] The following biological material has been deposited under the terms of the Budapest Treaty with the National Collections of Industrial and Marine Bacteria Limited (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, United Kingdom, and given the following accession number:

[0283] Deposited strain Accession Number Depositor Date of Deposit Alimentary Health Ltd

[0284] Bifidobacterium Building 2800 Cork

[0285] 5 November longum strain NCIMB 41676 Airport Business Park

[0286] 2009 AH1714 Kinsale Road Cork

[0287] Ireland

[0288] The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Claims

CLAIMS1. A process for producing a Bifidobacterium strain, the process comprising the steps of:(a) obtaining a composition comprising a Bifidobacterium strain;(b) adjusting the pH of the composition of step (a) to a pH in the range of pH 3.5-10.0 to obtain a pH adjusted composition;(c) heat-treating the pH adjusted composition of step (b) at a temperature in the range of 50-70°C for at least 60 minutes to obtain a heat-treated composition;(d) incubating the heat-treated composition of step (c) at a temperature in the range of 4-20°C for at least 4 hours to obtain the Bifidobacterium strain.

2. The process according to claim 1, wherein the Bifidobacterium strain is Bifidobacterium longum.

3. The process according to any one of the preceding claims, wherein the Bifidobacterium strain is Bifidobacterium longum deposited as NCI MB 41676.

4. The process according to any one of the preceding claims, wherein the pH in step (b) is adjusted to a pH in the range of pH 4.0-9.0, such as a pH in the range of about pH 4.5-8.5.

5. The process according to any one of the preceding claims, wherein the pH in step (b) is adjusted to pH of about pH 6.5-8.5, such as about pH 7.0-8.0.

6. The process according to any one of the preceding claims, wherein the heat treatment in step (c) is between 1 hour and 10 hours, preferably between 1 hour and 4 hours.

7. The process according to any one of the preceding claims, wherein the incubation in step (d) is at a temperature in the range of 5-15°C, optionally wherein the incubation is for at least 8 hours, such as at least 10 hours, such as at least 12 hours.

8. The process according to any one of the preceding claims, wherein the composition of step (a) is obtained by fermenting the Bifidobacterium strain at a pH of at least pH 5.0, such as at a pH in the range of 5.0-7.0, optionally wherein the composition of step (a) comprises at least 1 E+9 CFU / g Bifidobacterium cells.

9. The process according to any one of the preceding claims, wherein a separation step is performed before step (b) or after step (d), optionally wherein the separation step is a centrifugation, a microfiltration or an ultrafiltration step.

10. The process according to any one of the preceding claims, wherein a carrier is added before step (b) and / or after step (d), optionally wherein the carrier is selected from the group consisting of dextrin, polydextrin, maltodextrin, maize dextrin, potassium chloride, starch, lactose, Nutriose, sucrose, glucose, whey, gelatine, casein and / or albumin.

11. The process according to any one of the preceding claims, wherein after step (d), the Bifidobacterium strain is dried, optionally wherein the drying is spray drying, freeze drying, air drying, drum drying, vacuum drying, fluidized bed drying, extrusion drying, or any combination thereof, preferably wherein the drying is spray drying.

12. The process according to any one of the preceding claims, further comprising adding the Bifidobacterium strain to a composition to obtain a composition characterized by having a low amount of living Bifidobacterium cells.

13. The process according to claim 12, wherein the composition characterized by having a low amount of living Bifidobacterium cells comprises less than 1E+8 CFU / g, 1E+7 CFU / g, 1E+6 CFU / g, 1E+5 CFU / g, 1E+4 CFU / g, 1E+3 CFU / g, 1E+2 CFU / g or 1E+1 CFU / g Bifidobacterium cells.

14. The process according to any one of the preceding claims, wherein the process is for production of a composition comprising less than 1E+4 CFU / g Bifidobacterium cells, such as less than 1E+3 CFU / g, 1E+2 CFU / g, or 1E+1 CFU / g Bifidobacterium cells.

15. The process according to any one of the preceding claims, further comprising adding the Bifidobacterium strain or the composition comprising Bifidobacterium cells to a food product to obtain a functional food.

16. A Bifidobacterium strain obtained by the process according to any one of the preceding claims.

17. A functional food comprising the Bifidobacterium strain obtainable by the process according to any one of claims 1 to 15.

18. The functional food according to claim 17, characterized by having a low amount of living Bifidobacterium cells.

19. The process, the strain or the functional food according to any one of the preceding claims, wherein the Bifidobacterium cells obtained by the process of any of the preceding claims lead to increased IL-10 levels as measured by a PBMC assay, such as the PBMC assay described herein, compared to the IL-10 levels obtained from a live Bifidobacterium cells composition such as a composition comprising at least 1E+9 CFU / g Bifidobacterium cells, such as a composition comprising between 1E+9 and 1E+12 CFU / g Bifidobacterium cells, as measured by said assay.

20. The process, the strain or the functional food according to claim 19, wherein the IL- 10 levels are increased by at least 50%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 250%, such as at least 300%, compared to the IL-10 levels obtained from the live Bifidobacterium cells composition.

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