Technical field of the invention

The present invention relates to a preparation comprising an encapsulated microbial culture as well as methods and apparatus for producing these. In particular, the present invention relates to a method of simultaneously introducing a microbial culture and a salt solution into drying gas to provide a matrix encapsulated microbial culture comprising a thin exterior layer.

Background of the invention

In human and animal bodies, microbial cultures, such as lactic acid bacteria (LAB), are the part of normal microbiota. LAB are mostly used as starter cultures in fermented dairy foods and beverages as they can help to improve the nutritional and organoleptic characteristics, as well as extend the shelf life. Some strains of LAB have been reported to exhibit health benefits to human and animals and may thereby be referred to as probiotic strains.

However, these vital microbial cultures are often subject to attacks by bacteriophages (or phages in short), a type of virus that infects and replicates in bacteria. As other viruses, bacteriophages are in need of a suitable host in which they can develop their progeny by hijacking the biosynthetic machinery and the genetic material of the specific bacterium. Bacteria infected by bacteriophages will not contribute to acidification, ultimately causing fermentation failure and development of products of low quality that are microbially unsafe as contaminants and pathogens do not have to compete with the microbial culture protecting the product. This problem is a continuous cause of economic losses for the dairy industry.

Bacteriophages are in the environment and can enter the production facilities through the raw material, such as the large volumes of raw milk that contains both native lactic bacteria and their specific bacteriophages. Consequently, it is not possible to prevent the presence of bacteriophages in the production facilities. To protect the production line and minimise the risk of fermentation failures of e.g. cheese starter cultures, it is common practice to apply thermal treatments, mixed-strain cultures and culture rotation regimes.

These strategies can mitigate the impact of bacteriophages. Unfortunately, fighting bacteriophages pose a daunting task as they are diverse population of viruses with up to 10% of lactic acid production batches being estimated to battle specific bacteriophages. Many of these bacteriophages have been shown to be thermo resistant and prone to rapid evolution. Accordingly, the present strategies to handle bacteriophages are not only cumbersome and expensive but also insufficient to prevent the presence of bacteriophages in industrial production lines. Given the inevitable presence of bacteriophages in industrial facilities, there is a great unmet need for phage resistant microbial cultures that does not serve as a platform for propagation of the unwelcomed bacteriophages.

Hence, it would be advantageous to provide a microbial culture with improved resistance against bacteriophages as well as suitable methods and equipment for its production. In particular, such microbial cultures may reduce the risk of fermentation failure and improve the overall efficiency of the fermentation process.

Summary of the invention

The present invention relates to a method for a matrix encapsulation of a microbial culture, such as lactic acid bacteria (LAB). The matrix protects the embedded microbial culture from bacteriophage attacks and leaves a microbial culture that is easy to handle. The method is based on a spray freezing approach enacted by a custom designed apparatus and enables matrix encapsulation of live microbial culture with minimal loss of cell viability. The obtained encapsulated microbial cultures are well suited for production of dairy products and limits the need for use of mixed- strain cultures and excessive culture rotation regimes.

Thus, an object of the present invention relates to the provision of methods and equipment for preparing a microbial culture that may be utilized to reduce the risk of fermentation failure in large scale production.

In particular, it is an object of the present invention to provide an encapsulated microbial culture, such as lactic acid bacteria (LAB), with improved resistance against bacteriophages.

Thus, an aspect of the present invention relates to a method for providing a preparation comprising an encapsulated microbial culture, said method comprising the following steps of: i) providing a first solution comprising a microbial culture and one or more matrix components, ii) providing a second solution comprising a salt comprising a divalent metal cation, iii) spraying simultaneously said first and second solutions into a drying gas in an upper spray chamber to produce atomized particles comprising an encapsulated microbial culture, iv) contacting the atomized particles comprising an encapsulated microbial culture with a cryogenic gas in a lower spray chamber to produce frozen atomized particles comprising an encapsulated microbial culture, and v) collecting the frozen atomized particles comprising an encapsulated microbial culture, thereby providing a preparation comprising an encapsulated microbial culture.

Another aspect of the present invention relates to use of a method as described herein to increase phage resistance of a preparation comprising a microbial culture.

A further aspect of the present invention relates to a preparation comprising an encapsulated microbial culture obtainable by the method as described herein.

An even further aspect of the present invention relates to a preparation comprising an encapsulated microbial culture in the form of particles, wherein the particles comprise: i) an interior core comprising a microbial culture and one or more matrix components, and ii) an exterior layer comprising a salt comprising a divalent metal cation, wherein the thickness of said exterior layer is less than 7% of the particle radius.

Yet another aspect of the present invention relates to a composition comprising a preparation comprising an encapsulated microbial culture as described herein.

Still another aspect of the present invention relates to a kit-of-parts comprising: i) one or more distinct preparations comprising an encapsulated microbial culture as described herein; ii) one or more components selected from the group consisting of foodgrade ingredients, pharmaceutical ingredient, excipients and combinations thereof; iii) optionally, instructions for use. An even further aspect of the present invention relates to use of a preparation comprising an encapsulated microbial culture or a composition as described herein in a product selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

A still further aspect of the present invention relates to a product comprising a preparation comprising an encapsulated microbial culture or a composition as described herein, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

Yet another aspect of the present invention relates to an apparatus for producing frozen particles comprising a first upper chamber (11) and a second lower chamber (12), wherein the upper chamber comprises: i) a 3-fluid nozzle for atomizing at least two suspensions or solutions (5); ii) an inlet for a drying gas (1); iii) means (3) for heating the drying gas to a temperature in the range 20°C to 250°C; and wherein the lower chamber comprises: iv) an inlet for a cryogenic gas (4); v) an outlet for the frozen particles, said outlet being connected to a cyclone (14); and wherein the upper chamber is arranged so the particles descend into the lower chamber for subsequent freezing.

Brief description of the figures

Figure 1 shows an example of a 3-fluid nozzle wherein a side-on view (A) as well as a cross-sectional view (B) is given. The cross-sectional view show that the 3-fluid nozzle is configured with an inner, intermediate and outer gaps making up concentric annular rings (black rings in figure).

Figure 2 shows a microtiter plate assay to probe robustness of an encapsulated formulation (SF-DVS) and a non-encapsulated/normal formulation (F-DVS) of Streptococcus thermophilus against bacteriophage attack in a fermentation of milk at 37°C without addition of rennet. The two rows on top show the acidification curves of SF-DVS at 5g I 100L and F-DVS at 0.6 g/lOOL, respectively. The bottom row shows the difference in acidification between the SF-DVS and the F-DVS acidification curves. Each column, from left to right, shows the effect of increasing the addition of phages, i.e. the numbers represent the multiples of the lowest amount of phage added (1). 0 represents no phage added. The inoculation doses were chosen to obtain similar acidification rates between formats when no phages were present.

Figure 3 shows a microtiter plate assay to probe robustness of an encapsulated formulation (SF-DVS) and a non-encapsulated/normal formulation (F-DVS) of Streptococcus thermophilus against bacteriophage attack in a fermentation of milk at 37°C with addition of rennet at 600 IMCU per ml. The two rows on top show the acidification curves of SF-DVS at 5g I 100L and F-DVS at 1.25 g/lOOL, respectively. The bottom row shows the difference in acidification between the SF-DVS and the F- DVS acidification curves. Each column, from left to right, shows the effect of increasing of the addition of phages, i.e. the numbers represent the multiples of the lowest amount of phage added (1). 0 represents no phage added. The inoculation doses were chosen to obtain similar acidification rates between formats when no phages were present.

Figure 4 shows a microtiter plate assay to probe robustness of an encapsulated formulation (SF-DVS) and a non-encapsulated/normal formulation (F-DVS) of Lactococcus lactis against bacteriophage attack in a fermentation of milk at 30°C with addition of rennet at 600 IMCU per ml. The two rows on top show the acidification curves of SF-DVS at 10g I 100L and F-DVS at 5 g/lOOL, respectively. The bottom row shows the difference in acidification between the SF-DVS and the F-DVS acidification curves. Each column, from left to right, shows the effect of increasing of the addition of phages, i.e. the numbers represent the multiples of the lowest amount of phage added (1). 0 represents no phage added. The inoculation doses were chosen to obtain similar acidification rates between formats when no phages were present.

Figure 5 shows an example of a configuration of a spray freezing apparatus for producing frozen particles as described herein. Each part of the system is as follows;

(I) Drying gas supply, (2) supply fan, (3) heater, (4) cryogenic gas supply, (5) 3- fluid nozzle (with gas supply shown), (6a) liquid feed of first solution comprising microbial culture, (6b) liquid feed of second solution comprising a salt, (7) liquid feed tank, (8) solution comprising microbial culture, (9) water inlet, (10) liquid feed pump,

(II) upper drying chamber, (12) lower freezing chamber, (13) frozen powder discharge, (14) cyclone, (15) exhaust fan, (16) exhaust gas, (T) temperature regulator, (P) pressure regulator, (F) drying gas regulator. The present invention will in the following be described in more detail.

Detailed description of the invention

Definitions

Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

Microbial culture

In the present context, the term "microbial culture" refers to a population of microorganisms. Microorganisms include all unicellular organisms, such as archaea and bacteria, but also many multicellular organisms, such as fungi and algae. Microbial cultures as referred to herein does not include unwanted microorganisms that contribute to spoilage or risk of disease.

Microbial cultures may comprise one or more different microorganism strains, such as strains with equivalent phenotype but different genotype.

Probiotic culture

In the present context, the terms "probiotic" or "probiotic culture" refers to microbial cultures which, when ingested in the form of viable cells by humans or animals, confer an improved health condition, e.g. by suppressing harmful microorganisms in the gastrointestinal tract, by enhancing the immune system or by contributing to the digestion of nutrients. Probiotics may also be administered to plants. Probiotic cultures may comprise bacteria and/or fungi.

Lactic acid bacteria (LAB)

In the present context, the term "lactic acid bacteria (LAB)" refers to a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria that ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria include, but are not limited to, Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, Bifidobacteria, i.e. Bifidobacterium spp. which are frequently used as food starter cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria. Even certain bacteria of the genus Staphylococcus e.g. S. carnosus, S. equorum, S. sciuri, S. vitulinus and S. xyloSus') have been referred to as LAB (Seifert & Mogensen (2002)).

Matrix components

In the present context, the term "matrix components" refers to components that are added in a first solution together with the microbial culture to form the interior core of the particles of encapsulated microbial culture. The first solution or interior core may comprise one or more matrix components.

Preferably, all matrix components of the interior core are food graded, such as complying with the Food Chemicals Codex (FCC) and/or Generally Recognized as Safe (GRAS) ingredients. Matrix components include, but are not limited to, antioxidants, cryoprotectants, carbohydrates, proteins, alone or in combination between each other.

Encapsulated microbial culture

In the present context, the term "encapsulated" refers to the state of an entity, which is secluded from the surrounding environment. Thus, an encapsulated microbial culture is a microbial culture which are compartmentalized into distinct entities separated from each other and the medium into which they are dispersed.

Drying gas

In the present context, the term "drying gas" refers to a gas into which one or more solutions are sprayed to break the bulk liquids into small droplets that are subsequently dried. As the solutions are atomized in the encounter with the drying gas, the terms "drying gas" and "atomization gas" are used interchangeably herein.

Cryogenic gas

In the present context, the term "cryogenic gas" refers to a gas that originates from a cryogenic liquid. These cryogenic liquids are in gas phase at normal temperature and pressure but can be kept in liquid state at very low temperatures. The cryogenic gases herein may be derived from cryogenic liquids with boiling points below -150°C

Examples of cryogenic gases include, but are not limited to, nitrogen, helium, neon, argon, carbon dioxide, and methane.

Atomized particles In the present context, the term "atomized particles" refers to droplets with a diameter ranging from sub-micrometer to a few hundred micrometers. Atomization is achieved by conversion of bulk liquid into a dispersion of small droplets by creation of a high velocity difference between the liquid and the surrounding gas.

3-fluid nozzle

In the present context, the term "3-fluid nozzle" refers to a nozzle with separate inner, intermediate, and outer gaps. The gaps are arranged with the inner gap as centre liquid core (or inner annular ring), and the intermediate and outer gaps making up concentric annular rings.

In one configuration, the inner gap supplies the first solution comprising a microbial culture, the intermediate gap supplies the second solution comprising a salt solution, and the outer gap supplies a drying gas (or atomization gas). This configuration is termed an "external mixing 3-fluid nozzle".

Example of a 3-fluid nozzle is shown in figure 1, wherein a side-on view as well as a cross-sectional view is given.

The terms "3-fluid nozzle" and "coaxial nozzle" is used interchangeably herein.

Antioxidant

In the present context, the term "antioxidant" refers to a compound that inhibit oxidation. The antioxidant may be industrial chemicals or natural compounds. As used herein, antioxidants include, but are not limited to, trisodium citrate, vitamin C, vitamin E, glutathione, ascorbate, ascorbyl palmitate, quercetin, gallic acid, and tocotriene.

It is to be understood that antioxidants as used herein include mineral salts of vitamin C, such as sodium ascorbate. Also, the vitamin E is to be understood as including all variants of tocopherols and tocotrienols (alpha, beta, gamma, delta).

Food-grade ingredient

In the present context, the term "food-grade ingredient" refers to any compound that is non-toxic and safe for consumption and comply with the Food Chemicals Codex (FCC) and/or Generally Recognized as Safe (GRAS) ingredients. Food-grade ingredients include, but are not limited to, compounds that can alter attributes such as aroma, flavour, acidity, colour, viscosity and texture, as well as preservatives, nutrients, thickeners, sweeteners and emulsifiers.

Pharmaceutical ingredient

In the present context, the term "pharmaceutical ingredient" refers to an ingredient in a pharmaceutical formulation that is not an active ingredient.

Pharmaceutical ingredients include, but are not limited to, calcium carbonate, sodium carboxymethyl cellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

Excipient

In the present context, the term "excipient" refers to a natural or synthetic substance formulated alongside the active ingredient or pharmaceutical ingredient (an ingredient that is not the active ingredient) of a medication, included for the purpose of stabilization, bulking, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, enhancing solubility, adjusting tonicity, mitigating injection site discomfort, depressing the freezing point, or enhancing stability.

Examples of excipients include, but are not limited to, microcrystalline cellulose, titanium dioxide and aluminium silicate.

Feed

In the present context, the term "feed" refers to a food given to domestic animals. Domesticated animals include, but are not limited to, pets, such as dogs, cats, rabbits, hamsters and the like, livestock, such as cattle, sheep, pigs, goats and the like, and beast of burden, such as horses, camels, donkeys and the like.

Feed may be blended from various raw materials and additives and specifically formulated according to the requirements of the recipient animal. Feed may be provided e.g. in the form of mash feed, crumbled feed or pellet feed.

The term "feed" includes also premixes, which are composed of ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and combinations thereof. Premixes are typically added as a nutritional supplement to the feed given to the domestic animals. Water activity

In the present context, the term "water activity" refers to the partial vapour pressure of water in a substance divided by the standard state partial vapour pressure of water. The water activity is denoted Aw. Specifically, Aw of a food is the ratio between the vapor pressure of the particles of encapsulated microbial culture and/or preparation itself, when in a completely undisturbed balance with the surrounding air media, and the vapor pressure of distilled water under identical conditions.

As a general note, water migrates from areas of high Aw to areas of low Aw. The storage stability of food products can typically be extended by formulating the product with low Aw.

Water activity as referred to herein is measured using a Rotronics water activity analyzer with HC2-AW probe. This probe is fitted with a HYGROMER® WA-1 1 Pt-100, 1/3 DIN Class B humidity sensor and the psychrometric calculations are done by the dew or frost point method.

Briefly, a sample is placed into a sample cup and filled up to within 3 mm of the rim while ensuring as less air in the container as possible to ensure a faster equilibration time. Next, the measurement head is placed on the sample holder ensuring a tight seal. The water activity is the measured using a predictive model of the Rotronic water activity analyser.

The water activity is calculated using the following formulae: Aw= p/p _s and %ERH = 100 X Aw, where, p=partial pressure of water vapor at the surface of the product; p _s = saturation vapor/ partial pressure of water vapor above pure water at the product temperature; ERH=Equilibrium Relative Humidity.

Dry preparation

In the present context, the term "dry preparation" refers to a preparation which is substantially devoid of any solvent, such as liquid water. The moisture content of the dry preparation can be related to the water activity of the dry preparation through the moisture sorption isotherm. Thus, a dry preparation may be characterized as a preparation with a water activity of e.g. less than 0.8.

Microencapsulated microbial cultures, compositions comprising the same and methods for their production Bacteriophages ("phages") are ubiquitous natural organisms viruses attacking bacteria that exist in high numbers in solid food matrices, such as cheese. Phages constitute a problem in the dairy industry because they attack and kill the bacteria used to make dairy products. Specifically, by attacking the various strains in a dairy culture, phages can cause the fermentation process to fail. Fermentation failure comes with a financial cost to the customer as it may affect the quality, slow the fermentation process or ultimately prevent the product from being produced.

Phages are often strain-specific, meaning that they will only attack a very specific strain of bacteria. Thus, the conventional way to ensure that fermentation is not adversely affected by phages is to use compositions of one or more cultures each comprising multiple bacterial strains that provide the same benefit (/.e. having the same phenotype) but are genetically different from each other. However, the frequent substitution of phage prone strains from such compositions is time consuming and very expensive. Specifically, to prevent and mitigate the effects of serious phage attacks it is necessary to constantly finding or constructing new phage robust strains as well as designing, producing, and stocking new cultures.

Herein are provided microbial cultures with improved phage resistance and methods and equipment for providing these microbial cultures. The methods are based on matrix encapsulation of the microbial culture. The matrix is based on interaction of one or more matrix components with a salt comprising divalent metal cations, wherein a protective matrix is formed in which the amount of salt decreases gradually going from the surface to the core of the resulting particle. The resulting matrix comprises an exterior layer comprising mainly the salt and protects the microbial culture from phage attacks at the surface of the particle. Moreover, entrapment establishes a dense population of the microbial culture, which effectively behaves as a seed colony. Without being bound by theory, the establishment of these seed colonies increase phage robustness because the growth of the seed colony (scaling with the volume of the particle comprising the microbial culture) progress faster than the phage is capable of killing cells on the surface of the particle (scaling with the surface area of the particle comprising the microbial culture).

The production of such preparation comprising the encapsulated microbial culture is however technically challenging because conditions must be in place which enables formation of the particles (coating, crosslinking, non-aggregation). Described herein are methods that establishes conditions for formation of matrix encapsulated microbial cultures with a thin exterior layer that display up to 100-fold increased phage resistance compared to non-encapsulated microbial cultures.

The encapsulated microbial cultures described herein are well suited for production of dairy products and limits the need for use of mixed-strain cultures and excessive culture rotation regimes.

Thus, an aspect of the present invention relates to a method for providing a preparation comprising an encapsulated microbial culture, said method comprising the following steps of: i) providing a first solution comprising a microbial culture and one or more matrix components, ii) providing a second solution comprising a salt comprising a divalent metal cation, iii) spraying simultaneously said first and second solutions into a drying gas in an upper spray chamber to produce atomized particles comprising an encapsulated microbial culture, iv) contacting the atomized particles comprising an encapsulated microbial culture with a cryogenic gas in a lower spray chamber to produce frozen atomized particles comprising an encapsulated microbial culture, and v) collecting the frozen atomized particles comprising an encapsulated microbial culture, thereby providing a preparation comprising an encapsulated microbial culture.

The method relies on the simultaneous spraying of the first and second solution into a drying gas which ensures that the particles obtained contains an even amount of exterior layer. Upon spraying the liquid solutions into the drying gas, disturbances lead to disintegration of the bulk liquids into atomized particles in the form of liquid droplets. This atomization occurs as the magnitude of the disruptive force exceeds the consolidating surface tension force. Disruptive forces include turbulence in the liquid, cavitation in the spray nozzle, and aerodynamic interaction with the surrounding drying gas. The formed atomized particles comprise an interior core comprising substantially microbial culture and matrix component(s) and a thin exterior layer comprising substantially the salt within the second solution.

When descending in an upper spray chamber, the atomized particles (liquid droplets) are dried to a low content of moisture and are then brought into contact with a cryogenic gas in a lower spray chamber to freeze the atomized particles. The gradual transfer from the drying zone to the freezing zone ensures that matrix components and salt have time to crosslink and establish a protected environment for the microbial culture. If the encapsulated microbial culture freezes too quickly crosslinking will be insufficient to provide the microbial culture with adequate protection.

The matrix encapsulation protects the microbial culture against phage attacks and prevents lumping of the particles after processing. Lumped particles are more difficult to handle a do not distribute well when used in preparation of a final product, such as a dairy product. Upon charging of the preparation comprising encapsulated microbial culture to a product in the making, the protected microbial culture will proliferate faster than any potential bacteriophage attack can eliminate the microbial culture. As the microbial culture proliferate, the matrix will eventually disintegrate, leaving a densely populated seed colony with great phage resilience.

The simultaneous spraying of the first and second solutions into the drying gas can advantageously be enacted by the use of a 3-fluid nozzle configured with an inner gap as centre liquid core (or inner annular ring), and intermediate and outer gaps making up concentric annular rings (see figure 1). The nozzle with three separate outlets is suited for handily controlling the relative proportions of liquid solutions and drying gas. Moreover, by using a 3-fluid nozzle system, it is possible to run the method for providing the preparation comprising the encapsulated microbial culture continuously in contrast to other competing methods that are run in batch-mode.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the first solution, second solution and drying gas is sprayed or ejected into the upper chamber via a 3-fluid nozzle.

The 3-fluid nozzle may preferably be configured as an external mixing 3-fluid nozzle, wherein the inner gap supplies the first solution comprising a microbial culture and matrix component(s), the intermediate gap supplies the second solution comprising a salt, and the outer gap supplies a drying gas (or atomization gas).

Thus, an embodiment of the present invention relates to the method as described herein, wherein the 3-fluid nozzle is an external mixing 3-fluid nozzle. Another embodiment of the present invention relates to the method as described herein, wherein the external mixing 3-fluid nozzle comprises separate inner, intermediate, and outer gaps arranged as three concentric annular rings.

A further embodiment of the present invention relates to the method as described herein, wherein the first solution is sprayed via the inner gap, the second solution is sprayed via the intermediate gap, and the drying gas is ejected via the outer gap.

The first solution comprises one or more matrix components that contribute to crosslinking with the salt of the second solution and enables full matrix encapsulation of the microbial culture. Additionally, matrix components may be included to tailor the preparation to the expected application of the final consumer product. If the preparation is intended for an application for which extended storage is expected, it may for instance be preferable to increase the content of antioxidants. The content of antioxidants may also be adjusted depending on the conditions of storage, e.g. the degree of exposure to air. While the specific matrix component(s) may be selected on a product-by-product basis, the method as described herein is not limited to a specific type of microbial culture but is a general encapsulation concept to improve phage resilience. Thus, it is contemplated that any type of microbial culture may advantageously be processed as described herein.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the one or more matrix components are selected from the group consisting of carbohydrates, proteins, antioxidants, and combinations thereof

Another embodiment of the present invention relates to the method as described herein, wherein the carbohydrates are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

Polymers are molecules that are suitable for combination with water-soluble salts to provide the full matrix encapsulation of the microbial culture. Different polymers, such as polysaccharides, can used with preferred option including naturally occurring polymer such as alginate or milk protein such as caseinate.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the one or more matrix components comprise polymers. Another embodiment of the present invention relates to the method as described herein, wherein the polysaccharides are selected from the group consisting of alginate, pectin, cellodextrin, gums, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan, and combinations thereof.

A further embodiment of the present invention relates to the method as described herein, wherein the one or more matrix components comprise anionic polymers.

Alginates are salts of alginic acid which is a linear copolymer with homopolymeric blocks of (1— >4)-linked p-D-mannuronate (M) and o-L-guluronate (G) residues. Alginates are commonly used for industrial purposes including food, animal food, fertilisers, and pharmaceuticals. Metal salts of alginic acid includes, but are not limited, sodium alginate, calcium alginate and potassium alginate.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the one or more matrix components comprises an alginate. Another embodiment of the present invention relates to the method as described herein, wherein the alginate is selected from sodium alginate, calcium alginate and potassium alginate.

A further embodiment of the present invention relates to the method as described herein, wherein the polysaccharide is alginate and wherein said alginate is preferably sodium alginate.

It is contemplated that the preparation will be especially suited for use in the production of dairy products, such as cheese. Thus, a variant of the method involves matrix components that are milk proteins to gain optimal compatibility with the product.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the protein is a milk protein.

Another embodiment of the present invention relates to the method as described herein, wherein the milk protein is caseinate, such as sodium caseinate.

Oxidation is the loss of electrons of an atom or ion. In the present context, oxidation refers to oxidation of molecular oxygen and means that oxygen is metabolised to unstable free radicals, which can pry away electrons from other molecules. Oxidation may therefore lead to damaging of cell membranes and other cellular components, such as proteins, lipids and DNA. To avoid damage to the encapsulated microbial culture, one or more antioxidants may be included in the encapsulation matrix to prevent oxidation. The antioxidants may be of either natural or synthetic origin.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the antioxidants are selected from the group consisting of citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienol, glutathione, and combinations thereof.

Another embodiment of the present invention relates to the method as described herein, wherein the antioxidants are selected from vitamin C and/or vitamin E. It is to be understood that antioxidants as used herein include mineral salts of vitamin C, such as sodium ascorbate. Also, the vitamin E is to be understood as including all variants of tocopherols and tocotrienols (alpha, beta, gamma, delta).

Cryoprotecting agents can be used to improve the ability of microbial culture to survive against the harmful effect of freezing, frozen storage and freeze-drying. Therefore, for some microbial cultures it may be preferable to include one or more cryoprotecting agents in the first solution in addition to the one of the matrix components. Preferably, these cryoprotecting agents should not be metabolized by the microbial strain to produce acids as it may cause a loss of viability due to damage to ATPase membrane-bound enzymes, [3-galactosidase, and cell membrane fluidity. In general, cryoprotecting agents that are not producing acids are more effective in improving the survival rate of freeze-dried microbial cultures. One preferred category of cryoprotecting agents is carbohydrates and the associated subgroups thereof.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the first solution further comprises one or more cryoprotecting agents.

The person skilled in the art can contemplate suitable cryoprotecting agents that are suitable for protecting the microbial culture. Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the one or more cryoprotecting agents are selected from the group consisting of disaccharides, such as sucrose, lactose or trehalose, maltodextrin, sodium ascorbate and protein peptone, such as casein peptone, pea protein peptone or potato protein peptone, and combinations thereof. Another embodiment of the present invention relates to the method as described herein, wherein the cryoprotecting agents are selected from a disaccharide, maltodextrin, sodium ascorbate, protein peptone and combinations thereof. A further embodiment of the present invention relates to the method as described herein, wherein the cryoprotecting agents are a combination of a disaccharide, maltodextrin, sodium ascorbate, and a protein peptone, preferably in a weight ratio of 7:3:3: 1.

A still further embodiment of the present invention relates to the method as described herein, wherein the ratio (wt%/wt%) of microbial culture to cryoprotecting agent(s) in the first solution is in the range of 2: 1 to 1 :5.

It is desired to include a high amount of microbial culture to optimise the beneficial effects of the microbial culture of the preparation, such as lactic acid bacteria. However, it is also necessary to include a sufficient amount of matrix components to ensure full matrix encapsulation of the microbial culture. Therefore, the first solution may be designed to accommodate the specific microbial culture by adjusting the ratio of microbial culture to matrix component(s).

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the ratio (wt%/wt%) of microbial culture to matrix component(s) is in the range of 10: 1 to 1 :5, such as in the range of 7: 1 to 1:4, preferably in the range of 4: 1 to 1 :3, more preferably 2: 1 to 1 :2.

For many microbial cultures it may be sufficient to include only a single matrix component in the first solution to achieve full matrix encapsulation.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the first solution comprises only a single matrix component.

In a preferred embodiment of the present invention the first solution comprises only a single matrix component selected from alginate and caseinate, preferably sodium alginate.

The exterior layer that shields the microbial culture from the surrounding environment can be made from a variety of second solutions comprising different divalent metal cations that are capable of interacting with the one or more matrix component(s). Thus, an embodiment of the present invention relates to the method as described herein, wherein the divalent metal cation is selected from the group consisting of Ca ²⁺ , Zn ²⁺ and Mg ²⁺ .

A preferred embodiment of the present invention relates to the method as described herein, wherein the divalent metal cation is Ca ²⁺ . Another embodiment of the present invention relates to the method as described herein, wherein the divalent metal cation is Zn ²⁺ .

The divalent metal cation may be combined with any anionic counterion. Preferably, the salt is a chloride salt.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the salt comprises an anion selected from the group consisting of Cl’, Br, OH’, SO ₄ ² ’, CO ₃ ² ’, CH ₃ COO’ and HCOO’.

Another embodiment of the present invention relates to the method as described herein, wherein the salt is a chloride salt.

A further embodiment of the present invention relates to the method as described herein, wherein the salt is selected from CaCI?, ZnCI? and MgCI?, preferably CaCI?.

The concentration of salt in the second solution can differ depending on the choice of divalent metal cation to modulate the interaction with the one or more matrix components. Therefore, an embodiment of the present invention relates to the method as described herein, wherein the concentration of salt in the second solution is in the range of 0.05 - IM.

A preferred embodiment of the present invention relates to the method as described herein, wherein the second solution comprises CaCI? in a concentration of 0.2-0.8 M, such as 0.4-0.6 M, preferably approximately 0.5 M.

Another embodiment of the present invention relates to the method as described herein, wherein the second solution comprises MgCI? in a concentration of 0.6-1.4 M, such as 0.8-1.2 M, preferably approximately 1 M. A further embodiment of the present invention relates to the method as described herein, wherein the second solution comprises ZnCI? in a concentration of 0.01-0.10 M, such as 0.03-0.07 M, preferably approximately 0.05 M.

The exterior layer surrounding the interior core comprising the microbial culture may be described as a thin shell. To obtain this thin shell the second solution is only provided in low amount relative to the first solution.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the ratio (% wv /%wv) of microbial culture to second solution is in the range of 5: 1 to 10: 1.

The encapsulated microbial culture may in some variants also comprise a boosting agent. Including a boosting agent can promote proliferation of the beneficial microbial culture and in some cases also depress growth of unwanted microorganisms. In some instances, the use of boosting agents relies on supplying pre-cursors necessary for the internal production of nucleotides required for proliferation. Examples of boosting agents includes, but are not limited to, organic acids.

Thus, an embodiment of the present invention relates to the method as described herein, wherein either the first solution and/or the second solution comprises a boosting agent. Another embodiment of the present invention relates to the method as described herein, wherein the boosting agent is an organic acid. A further embodiment of the present invention relates to the method as described herein, wherein the organic acid is inosinic acid. Inosinic acid may also be termed inosine monophosphate (IMP) thus, inosinic acid and IMP is used herein interchangeably.

The boosting agent can be included as salt with a monovalent cation. Therefore, an embodiment of the present invention relates to the method as described herein, wherein the first or second solution further comprises a monovalent cation.

Another embodiment of the present invention relates to the method as described herein, wherein the monovalent cation is Na ⁺ or NH ₄ ⁺ , preferably Na ⁺ .

A further embodiment of the present invention relates to the method as described herein, wherein the second solution comprises the sodium salt of inosinic acid. The formation of the encapsulated microbial culture is driven by the simultaneous spraying of the first solution comprising the microbial culture and the second solution comprising the divalent metal cation. Thus, without being bound by theory, it is contemplated that the microbial culture itself plays a passive role in the encapsulation process. Accordingly, the method as described herein is not limited to a specific type of microbial culture but may be applied as a general encapsulation concept. Thus, it is envisioned that a broad variety of microbial cultures may advantageously be encapsulated by the method described herein to enhance their resistance to bacteriophages.

One type of microorganism of great importance in e.g. the dairy industry is bacteria. They are used widely in fermented food, feed mixes and nutritional supplements, wherein their health benefits are well-documented.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture is a bacterium.

A further embodiment of the present invention relates to the method as described herein, wherein the microbial culture is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Propionibacterium, Oenococcus and Bacillus.

Probiotic cultures are cultures of live microorganisms, which upon ingestion by a subject provide health benefits to the subject. Products comprising probiotic cultures include dairy products, animal feed and beverages. Thus, it is to be understood that the preparations comprising encapsulated microbial cultures described herein may be administered not only to humans but also animals, and even plants.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture is or comprises a probiotic culture.

Another embodiment of the present invention relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is according to the present invention is Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus easel, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii, the genus Bifidobacterium, such as the Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis, and the like.

In a particular embodiment of the invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii.

In a particular embodiment of the invention, the probiotic Lactobacillus strain is selected from the group consisting of a Lacticaseibacillus rhamnosus strain and a Lacticaseibacillus paracasei strain.

In a particular embodiment of the invention, the probiotic strain is Lacticaseibacillus rhamnosus strain LGG® deposited as ATCC53103.

In a particular embodiment of the invention, the probiotic strain is Lacticaseibacillus paracasei strain CRL 431 deposited as ATCC55544.

In a particular embodiment of the invention, the probiotic Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis.

In a particular embodiment of the invention, the probiotic Bifidobacterium probiotic strain is Bifidobacterium animalis subsp. lactis BB-12 deposited as DSM15954.

Of particular interest are lactic acid bacteria (LAB) that are an order of Gram-positive bacteria sharing common metabolic and physiological characteristics. LAB may be probiotic microorganism as defined above and all share the ability to produce lactic acid as the major metabolic outcome of carbohydrate fermentation. Ever since it was discovered that acidification by food fermentation could preserve food by inhibiting growth of spoilage agents, LAB has been utilized purposefully in food fermentation. However, since efficient food fermentation requires high quality viable microorganisms, the development of fermented foods has been halted for producers do not have advanced facilities and/or procedures in place to efficiently prevent spoilage of products due to fermentation failures caused by bacteriophage attacks. The method and resulting preparation described herein can accommodate these challenges by providing microbial cultures with enhanced resistance bacteriophages.

Hence, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture is or comprises a lactic acid bacterium (LAB) culture.

Another embodiment of the present invention relates to the method as described herein, wherein the lactic acid bacterium (LAB) is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

It will be appreciated that the Lactobacillus genus taxonomy was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be cohered to herein if nothing else is noticed. For the purpose of the present invention, Table 1 presents a list of new and old names of some Lactobacillus species relevant to the present invention.

Table 1. New and old names of some Lactobacillus species relevant to the present invention The microbial culture is provided as part of a first solution. The first solution is of a viscosity that allows transfer and spraying of the microbial culture without any clogging or aggregation of the microbial culture.

An embodiment of the present invention relates to the method as described herein, wherein the microbial culture of step (i) is provided as a solution or suspension.

To safeguard against production failure, it may for some applications be preferable to include two or more bacterial strains of similar phenotype but different genotype in the microbial culture to reduce the risk that any bacteriophage attack will result in complete production failure. Using a blend of microbial strains may be preferable to simply blending preparations after the encapsulation process.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the microbial culture comprises two or more different microbial strains, such as two different microbial strains, such as three different microbial strains, such as four different microbial strains.

Another embodiment of the present invention relates to the method as described herein, wherein the microbial culture comprises at least 5 different microbial strains, such as at least 10 different microbial strains, such as at least 15 different microbial strains.

Different microbial strains may have different phenotype and/or genotype. Thus, microbial strains with same phenotype but different genotype are considered to be different.

Thus, a further embodiment of the present invention relates to the method as described herein, wherein the microbial culture comprise two or more microbial strains of similar phenotype but different genotype.

A further embodiment of the present invention relates to the method as described herein, wherein the first solution comprises two or more microbial strains of different genotype and phenotype.

The microbial culture is provided in a concentration that is sufficiently high to result in a preparation comprising encapsulated microbial culture with adequate amounts of viable microorganisms to efficiently promote fermentation. Thus, an embodiment of the present invention relates to the method as described herein, wherein the first solution comprises a concentration of microbial culture of at least 1.0E+07 CFU/ml, such as at least 1.0E+08 CFU/ml, preferably at least 1.0E+09 CFU/ml.

To orchestrate the sequence of steps of drying and freezing of the microbial culture, the upper spray chamber is adjusted to sustain temperatures well above the freezing point of the first and second solutions and the lower chamber is arranged for injection of a cryogenic gas to accommodate freezing of the partially dehydrated atomized particles descending from the upper chamber. The method is designed so that the atomized particles spend enough time in both the upper chamber and lower chamber to achieve full matrix encapsulation of the microbial culture and yield a frozen preparation thereof. The residence time (or retention time) of the atomized particles may be altered by adjusting the flow rate through the chamber system.

Thus, an embodiment of the present invention relates to the method as described herein, wherein step iii) is performed for a time sufficient for achieving the desired degree of drying and crosslinking and step iv) is performed for a time sufficient for a complete freezing can be achieved.

The residence time is defined as the time spent by the atomized particles in the chamber system and can be calculated as the chamber system volume divided by the volume flow rate, which is the volume of material which passes per unit time (m ³ /s). As an example, a process with a chamber system of 1.1 m ³ and a volume flow rate of 0.031 m ³ /s yields a retention time of 35 seconds. As will be appreciated from this definition, the residence time will be scale dependant and it is expected that residence times will vary between pilot scale chamber systems and production scale chamber systems. By adjusting the volume flow rate an adequate residence time can be found for chamber systems of different sizes.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the volume flow is adjusted to obtain a residence time sufficient for drying and freezing the atomized particles.

Another embodiment of the present invention relates to the method as described herein, wherein the residence time of the atomized particles in the drying gas and/or cryogenic gas is in the range of 10 to 300 seconds. A further embodiment of the present invention relates to the method as described herein, wherein the atomized particles are transferred from the upper spray chamber into the lower spray chamber by means of the gravity.

The drying gas (or atomization gas) is utilized to promote atomization of the first and second solutions as they are sprayed into the upper spray chamber. Beyond facilitating fragmentation of the solutions into droplets (or atomized particles), the drying gas should interact as little as possible with the first and second solutions. To this end, certain types of drying gas are suitable.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the drying gas is selected from the group consisting of an inert gas, a noble gas, carbon dioxide, an alkane gas, and combinations thereof.

Another embodiment of the present invention relates to the method as described herein, wherein the drying gas is selected from nitrogen, helium, neon, argon, carbon dioxide, methane, and combinations thereof.

A preferred embodiment of the present invention relates to the method as described herein, wherein the drying gas is nitrogen.

Preferably, the content of oxygen in the drying gas is kept at a minimum. Thus, an embodiment of the present invention relates to the method as described herein, wherein the drying gas comprises less than 5% oxygen, such as less than 2%.

Beyond serving as a means for atomizing the first a second solution, the drying gas can in heated form improve removal of liquid from the first and second solutions before the atomized particles are frozen. The pre-drying step result in partially dehydrated atomized particles which after freezing have a higher bulk density than comparable frozen microbial cultures prepared by conventional freeze-drying wherein a porous particle is formed as water sublimates. The high density is advantageous because it yields a preparation of low porosity and therefore improved product stability. Accordingly, it is favourable to heat the drying gas.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the drying gas has an inlet temperature in the range from 20°C to 250°C, preferably in the range from 100°C to 200°C. Another embodiment of the present invention relates to the method as described herein, wherein the drying gas has an inlet temperature of at most 300°C.

To obtain the favourable higher bulk density of the final product, it was found that the rate of dehydration of the atomized particles in the drying step did not have to be very high. Thus, the decrease of moisture content (or water content) of the atomized particles before freezing are in the range of 0.5-50 wt%, depending on the drying gas rate and temperature as well as the flow rate of the first and second solutions.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the water content of the atomized particles is reduced by at least 0.5 wt% compared to the first solution, such as at least 1 wt%, such as at least 2 wt%, such as at least 5 wt%, such as at least 10 wt%, such as at least 20 wt%, before contacting the cryogenic gas. Another embodiment of the present invention relates to the method as described herein, wherein the water content of the atomized particles is reduced by an amount in the range of 0.5-20 wt% before contacting the cryogenic gas.

The cryogenic gas should be capable of freezing the descending atomized particles before they enter the bottom of the lower chamber and are recovered. However, the descending atomized particles should not be frozen too quickly either since the salt of the second solution would in that case not crosslink sufficiently with the matrix component(s) of the first solution to give optimal protection of the microbial culture. The present method introduces the cryogenic gas in the lower chamber to form a "cold mist" in which the descending atomized particles are frozen upon entering. Preferably, the cryogenic gas does not interact inadvertently with the atomized particles.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the cryogenic gas is selected from the group consisting of an inert gas, a noble gas, carbon dioxide, an alkane gas, and combinations thereof.

Another embodiment of the present invention relates to the method as described herein, wherein the cryogenic gas is selected from nitrogen, helium, neon, argon, carbon dioxide, methane, and combinations thereof.

Traditionally, liquid nitrogen is used for instant-freezing of solutions or suspensions of microbial culture to prepare frozen preparations that are easy to store and handle. The cryogenic gas of the method described herein may be nitrogen as well. However, the method described herein can be a continuous process in contrast to conventional batch instant-freeze processes using liquid nitrogen. This is great advantage from a production point of view. Additionally, the present method has less consumption of liquid nitrogen and is therefore not only cheaper but also leaves a smaller foot print than conventional methods for producing frozen preparations of microbial cultures.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the cryogenic gas is nitrogen.

Another embodiment of the present invention relates to the method as described herein, wherein the cryogenic gas comprises less than 5% oxygen, such as less than 2%.

For the purposes of optimizing the delicate balance between drying and freezing of the microbial culture, the inlet temperature of the cryogenic gas may be adjusted to suit the exact composition of the first and second solutions.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the cryogenic gas has an inlet temperature in the range from -20 to -250°C, such as in the range of -50 to -160°C, such as in the range of - 80 to -120°C.

If desired, it is possible to adjust the pressure of the drying step and/or the freezing steps of the method. This can be achieved by adding a means for regulation of the pressure of the upper and/or lower chambers. The skilled person would understand how to accomplish this. However, the present method may also be worked at ambient pressure.

Thus, an embodiment of the present invention relates to the method as described herein, wherein step iii) and/or iv) takes place under a pressure between 60 and 200 kPa, such as between 80 and 150 kPa, such as between 90-110 kPa.

The morphology, such as particle size and thickness of the exterior layer of salt, can be controlled by adjusting the flow ratio between the first and second solutions and the gas:feed ratio. The latter is defined as the ratio between mass/hour of drying gas and the combined mass/hour of the first and second solutions. The morphology of the particles within the preparation may therefore be tailored to the specific application of the encapsulated microbial culture. Thus, an embodiment of the present invention relates to the method as described herein, wherein the gas:feed ratio is adjusted to control the size of the atomized particles, wherein the gas:feed ratio is defined as the ratio between the mass/hour of drying gas and the combined mass/hour of the first and second solutions.

Another embodiment of the present invention relates to the method as described herein, wherein the gas:feed ratio is in the range of 0.8 to 3, such as 1 to 2, such as 1.2 to 1.8.

A further embodiment of the present invention relates to the method as described herein, wherein the diameter of the atomized particles is in the range of 5-50 pm, such as 5-25 pm, preferably 10-15 pm measured as Dv50 according to ISO 13320:2020.

A still further embodiment of the present invention relates to the method as described herein, wherein the flow ratio between the first solution and the second solution is adjusted to control the thickness of an exterior layer of salt of the second solution.

Yet another embodiment of the present invention relates to the method as described herein, wherein the flow ratio between the first solution and the second solution is in the range of 20: 1 to 2: 1, such as in the range of 10: 1 to 5: 1, preferably approximately 7: 1.

Liquid from the first and second solutions evaporate as the two solutions are simultaneously sprayed into the drying gas in the upper chamber. This produces partially dehydrated atomized particles. It was found that the evaporated liquid did not form particles on their own but instead shock froze to small ice crystals that could be separated from the desired frozen atomized particles together with the cryogenic gas.

The separation may be of the desired frozen atomized particles may be achieved by utilizing a cyclone or an electrostatic filter. All material can be transferred pneumatically to the means of separation. Thus, an embodiment of the present invention relates to the method as described herein, wherein the frozen atomized particles are collected by means of a cyclone or an electrostatic filter, preferably by means of a cyclone.

Another embodiment of the present invention relates to the method as described herein, wherein the cyclone is operated with a with a maximum differential pressure drop across the cyclone in the range of 10mm to 300mm water column, such as 50 to 200mm water column, or approximately 100 mm water column.

After separation and collection of the frozen atomized particles the resulting frozen preparation may, if desired, be subjected to a downstream freeze-drying step. This downstream step may further improve the storage stability of the encapsulated microbial culture. The freeze-drying step may be performed according to common general practice, wherein parameters such as pressure and temperature is adjusted to achieve efficient drying without subjecting the microbial culture to unnecessary stress. A person skilled in the art would be knowledgeable of appropriate conditions for freeze-drying. Microbial cultures processed according to the present method exhibit high viability and activity after downstream freeze-drying, presumably due to the full matrix encapsulation with a protective exterior layer.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein step v) is followed by a step vi) of freeze drying the frozen atomized particles.

Another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at reduced pressure, such as at 0.1 to 0.4 mbar.

A further embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at a sublimation temperature in the range of -60°C to 45°C until complete water removal.

A still further embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed at a pressure of 0.3 mbar with the sublimation temperature increasing from -55°C to 32°C with 1.5°C/min. Yet another embodiment of the present invention relates to the method as described herein, wherein said freeze drying is performed until the water activity (Aw) of the frozen atomized particles are below 0.20, preferably below 0.10.

To obtain a high-quality product that will efficiently encourage fermentation, it is favourable to maintain a high content of microbial culture in the preparation. Thus, although components different from the microbial culture are included in the method to facilitate the encapsulation and protection of the microbial culture, the main constituent of the preparation is the microbial culture.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein said preparation substantially consists of the encapsulated microbial culture.

The preparation comprising encapsulated microbial culture produced by the present method has several advantages compared to conventional freeze-drying of heat- labile microbial material prepared by liquid nitrogen pelletizing. Firstly, the encapsulated microbial culture exhibit improved viability with more active material. Secondly, the obtained preparation has easier applicability as a free-flowing powder is obtained in contrast to the conventional powders that tend to be slightly aggregated (looking like small "peas"). The high homogeneity of the powder improves dispersibility in aqueous solution or suspension, such as milk.

After separation and collection of the preparation, and any other post-processing steps such as freeze-drying, the final product may be further packaged as part of the method. Packaging is done to ensure that the preparation will reach the end user without any spoilage of the encapsulated microbial culture e.g. due to competition from unwanted microorganism. Thus, it is preferred to keep the preparation in an airtight and/or moisture-tight sealed package to shield the preparation from the environment. A person skilled in the art is capable of selecting a conventional means for suitable packaging material for achieving the required closing.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein said method as a final step comprises packaging of the preparation comprising an encapsulated microbial culture.

Another embodiment of the present invention relates to the method as described herin, wherein packaging is in an air-tight and/or moisture-tight package. For some applications it may be desired by the end user to utilize two or more different microbial cultures that contribute to different characteristics of their product, e.g. aroma, flavour, texture and others. Conveniently, preparations of different microbial cultures may therefore be packaged together, or the preparation may be packaged together with a microbial culture prepared by conventional means, e.g. by freeze-drying.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the preparation comprising an encapsulated microbial culture is packaged together with a microbial culture of a different strain.

The method described herein is based on the simultaneous spraying of the first and second solutions into the drying gas which yields the atomized particles with full matrix encapsulated microbial culture comprising a thin protective exterior layer. The method can be performed using a custom-designed apparatus also described herein.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the method is performed by means of an apparatus as described herein.

The present method is especially suitable for conferring microbial cultures with resistance against attacks by bacteriophages. By employing the present method, it is thus possible to obtain a preparation comprising viable encapsulated microbial culture that can be used in fermentation production with reduced risk of fermentation failure.

Accordingly, an aspect of the present invention relates to use of a method as described herein to increase phage resistance of a preparation comprising a microbial culture.

Another aspect of the present invention relates to a preparation comprising an encapsulated microbial culture obtainable by the method as described herein.

It is to be understood that the embodiments of the present invention described herein in relation to the method are equally compatible with these two aspects of the invention. Yet another aspect of the present invention relates to a preparation comprising an encapsulated microbial culture in the form of particles, wherein the particles comprise: i) an interior core comprising a microbial culture and one or more matrix components, and ii) an exterior layer comprising a salt comprising a divalent metal cation, wherein the thickness of said exterior layer is less than 7% of the particle radius.

The thickness of the exterior layer may be calculated based on the liquid feed flows supplying the first and second solutions. The thickness of the exterior layer, t, may be determined as the difference between the total radius of the particle, ETOT, and the radius of the interior core, E _C ORE- Accordingly, t = E _T OT - ECORE-

By example the two liquid feed flows may be supplied to the 3-fluid nozzle in a ratio of 7: 1, with the second solution having the lower feed rate. Thus, the first media flow comprising the microbial culture, V _C ORE, and the second media flow comprising the salt solution, v _E xr, are related as V _E XT = (1/7)V _C ORE- The media flows scale proportionally with their volumetric contribution to the particle.

Accordingly, the contribution of the two media flows to the total volume of the particle, VTOT, is given by the relation VTOT = VCORE + V _E xr = VCORE + ( /7) V _C ORE = (8/7)V _C ORE- From this relation it is possible to express E _C ORE as function of E _T OT, namely as ECORE = ETOT ³ (7 /8).

The thickness of the exterior layer, t, can therefore be expressed in terms of the radius of the particle, E _T OT, as t = ETOT- E _T OT ³ (7/8) = (1 - ³ 7/8))E _T OT = 0.0435 ETOT. For the specific liquid feed ratio of 7: 1 between the first and second solutions, respectively, the thickness of the exterior layer will be approximately 4.35% of the total particle radius.

Operating the spray freezing apparatus at different liquid feed flow ratios will change the thickness of the exterior layer according to the calculation outline above. Thus, it is possible to tailor the particles to suit different needs.

In practice, not all of the salt provided in the second solution is used to crosslink with the matrix component(s). In some variants of the method described herein, it is estimated that less than 0.1 % of the divalent metal salt is taken up by the interior core and the vast majority of the salt is comprised in the exterior layer with fast gradual decline in direction towards the center of the particle. However, provision of high concentrations of divalent metal cations may be advantageous to speed up the formation of particles.

The particles of the preparation are micrometer-sized and comprise a thin protective exterior layer that is thick enough to provide protection against phage attacks but thin enough to disintegrate as the microbial culture starts to proliferate and reach a critical mass of a seed colony to withstand phage attacks during fermentation. The actual size of the particles can be tailored by adjusting the pressure of the drying gas and thereby affecting the atomization process.

Therefore, an embodiment of the present invention relates to the preparation as described herein, wherein the size of the particles is in the range of 5-50 pm, such as 5-25 pm, preferably 10-15 pm measured as Dv50 according to ISO 13320:2020.

Another embodiment of the present invention relates to the preparation as described herein, wherein the thickness of said exterior layer is less than 5% of the particle radius, preferably less than 4.5% of the particle radius.

The particles of the preparation can be produced by the method described herein. It is therefore to be understood that the interior core can comprise a microbial culture and matrix component(s) as described for the present method. Likewise, the exterior layer can comprise a salt comprising a divalent cation as described for the present method.

The exterior layer may further comprise a boosting agent, e.g. added as a salt of a monovalent cation. The boosting agent is typically an organic acid, such as inosinic acid.

Thus, an embodiment of the present invention relates to the preparation as described herein, wherein the exterior layer further comprises a boosting agent.

Another embodiment of the present invention relates to the preparation as described herein, wherein the exterior layer further comprises a monovalent salt.

Yet another embodiment of the present invention relates to the preparation as described herein, wherein the monovalent cation is Na ⁺ or NH ₄ ⁺ . A further embodiment of the present invention relates to the preparation as described herein, wherein the exterior layer comprises a sodium salt of inosinic acid.

The morphological properties of the preparation play an important role in how easy it is to handle and store the preparation without loss of microbial activity. Notably, the preparation is highly dispersible in aqueous solution or suspension which is an advantage when it is utilized in production of dairy foods. The preparation may be further processed, e.g. by freeze-drying to reduce water activity, and thereby further enhance storage stability and create unfavourable conditions for unwanted microorganisms to contaminate the preparation.

Thus, an embodiment of the present invention relates to the preparation as described herein, wherein the preparation is a dry preparation.

Another embodiment of the present invention relates to the preparation as described herein, wherein the preparation is in the form of a powder and/or a granulate.

Yet another embodiment of the present invention relates to the preparation as described herein, wherein the preparation is a freeze-dried preparation.

A further embodiment of the present invention relates to the preparation as described herein, wherein the water activity (Aw) of the preparation is in the range from 0.01-0.8, preferably in the range from 0.05-0.6, most preferably in the range from 0.1-0.4.

A still further embodiment of the present invention relates to the preparation as described herein, wherein the preparation substantially consists of the encapsulated microbial culture.

The preparation may be used on its own without any further additives e.g. for fermentation of dairy products. However, the preparation may also be part of a composition comprising one or more additives that supplements the properties of the microbial culture. The additives may be relevant for application of the preparation in e.g. nutraceuticals or health related products as well as pharmaceutical products.

Thus, an aspect of the present invention relates to a composition comprising a preparation comprising an encapsulated microbial culture as described herein An embodiment of the present invention relates to the composition as described herein, wherein the composition further comprises one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients and excipients.

Another embodiment of the present invention relates to the composition as described herein, wherein the food-grade ingredients are selected from the group consisting of lactose, maltodextrin, whey protein, corn starch, dietary fibres, gums and gelatine.

A further embodiment of the present invention relates to the composition as described herein, wherein the pharmaceutical ingredients are selected from the group consisting of calcium carbonate, sodium carboxymethyl cellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

Yet another embodiment of the present invention relates to the composition as described herein, wherein the excipients are selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminium silicate.

It is to be understood that the composition may be of a form as described also for the preparation, e.g. a powder or granulate, optionally freeze-dried to reduce the water activity.

The preparation can also be provided as a kit-of-parts together with one or more additives to add flexibility at the point of the end user. The end user (or manufacturer) can then decide independently for which application to use the preparation.

Accordingly, an aspect of the present invention relates to a kit-of-parts comprising: i) one or more distinct preparations comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29; ii) one or more components selected from the group consisting of foodgrade ingredients, pharmaceutical ingredient, excipients and combinations thereof; iii) optionally, instructions for use.

Another aspect of the present invention relates to use of a preparation comprising an encapsulated microbial culture or a composition as described herein in a product selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

The preparation or composition comprising encapsulated microbial culture will inevitably be present also in the final product that it is used to produce. However, the use itself of the preparation or composition for preparing such a product is certainly within the scope of the present invention.

The preparation or composition may especially find advantageous use in production of dairy products in which the risk of production failure caused by bacteriophage attacks is elevated.

Thus, an embodiment of the present invention relates to the use as described herein, wherein the food product is a dairy product.

Another embodiment of the present invention relates to the use as described herein, wherein the dairy product comprises a solid or semi-solid food matrix, preferably a solid food matrix.

A preferred embodiment of the present invention relates to the use as described herein, wherein the dairy product comprising a solid food matrix is a cheese.

A variety of products and production lines that can benefit from the encapsulated microbial culture provided herein may be contemplated. Especially, the dairy industry is battling the damaging effects of phage attacks on their production lines but other end user such as producers of animal feed, health products and pharmaceuticals could benefit from the properties of the encapsulated microbial culture provided herein.

Therefore, an aspect of the present invention relates to a product comprising a preparation comprising an encapsulated microbial culture or a composition as described herein, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

Another aspect of the present invention relates to a dairy product comprising a preparation comprising an encapsulated microbial culture or a composition as described herein. An embodiment of the present invention relates to the dairy product as described herein, wherein said dairy product is selected from the group consisting of yoghurt, cheese, butter, inoculated sweet milk and liquid fermented milk products.

Another embodiment of the present invention relates to the dairy product as described herein, wherein the dairy product comprises a solid or semi-solid food matrix, preferably a solid food matrix.

A preferred embodiment of the present invention relates to the dairy product as described herein, wherein the dairy product comprising a solid food matrix is a cheese.

The preparation comprising an encapsulated microbial culture can be produced using a custom-designed apparatus comprising upper and lower chambers for atomization/drying and freezing of particles, respectively, and a 3-fluid nozzle for simultaneous spraying of two solutions into a drying gas.

Thus, an aspect of the present invention relates to an apparatus for producing frozen particles comprising a first upper chamber (11) and a second lower chamber (12), wherein the upper chamber comprises: i) a 3-fluid nozzle for atomizing at least two suspensions or solutions (5); ii) an inlet for a drying gas (1); iii) means (3) for heating the drying gas to a temperature in the range 20°C to 250°C; and wherein the lower chamber comprises: iv) an inlet for a cryogenic gas (4); v) an outlet for the frozen particles, said outlet being connected to a cyclone (14); and wherein the upper chamber is arranged so the particles descend into the lower chamber for subsequent freezing.

3-fluid nozzles, also known as coaxial nozzles, exist mainly in two overall categories; external mixing 3-fluid nozzle and internal mixing 3-fluid nozzle. Both types of nozzles can be depicted as shown in figure 1 with a centre core surrounded by two annular rings. These hollow spaces in the nozzle can also be referred to as gaps. The external mixing 3-fluid nozzle is arranged with inner gap being for a first liquid, the intermediate gap being for a second liquid, and the outer gap being for the atomization/drying gas. The reversed configuration with the atomization/drying gas in the inner gap, and the first and second liquid solutions in the intermediate and outer gap, respectively, is referred to as an internal mixing 3-fluid nozzle.

An embodiment of the present invention relates to the apparatus as described herein, wherein the 3-fluid nozzle (5) is an external mixing 3-fluid nozzle.

Another embodiment of the present invention relates to the apparatus as described herein, wherein the 3-fluid nozzle (5) comprises separate inner, intermediate, and outer gaps arranged as three concentric annular rings.

A further embodiment of the present invention relates to the apparatus as described herein, wherein the inner gap is adapted for receiving a first solution or suspension, the intermediate gap is adapted for receiving a second solution or suspension, and the outer gap is adapted for receiving the drying gas.

The apparatus can be configured to receive hot drying gas and cold cryogenic gas. A person skilled in the art knows how to provide secure inlet for such gases by conventional means. The apparatus may be more or less directly connected to the source of drying gas and/or cryogenic gas.

Therefore, an embodiment of the present invention relates to the apparatus as described herein, wherein the inlet for a cryogenic gas (4) is adapted for a gas having a temperature in the range -50 to -250°C.

Another embodiment of the present invention relates to the apparatus as described herein, wherein the apparatus comprises a tank for storing the cryogenic gas.

Yet another embodiment of the present invention relates to the apparatus as described herein, wherein the upper chamber (11) is connected to a heater (3) for heating the drying gas.

The apparatus comprises a cyclone separator (or simply cyclone) capable of separating fine particulate material from a gaseous stream. Herein, the frozen particles are separated from the cryogenic gas and small ice crystals formed as the drying gas coincides with the cooled and moist drying gas. Preferably, there is a maximum differential pressure drop across the cyclone of approximately 100 mm water column. As indicated by the terminology used herein, the apparatus can be a spray tower with the upper chamber (11) positioned on top of the lower chamber (12). This configuration allows transfer of the descending atomized particles from the upper chamber (11) into the lower chamber (12) by means of gravity.

Accordingly, an embodiment of the present invention relates to the apparatus as described herein, wherein the apparatus is a spray tower, wherein the upper chamber (11) is placed over the lower chamber (12).

The absolute and relative dimensions of the apparatus can be designed to optimize residence time of the atomized particles in the upper (11) and lower (12) chambers with the goal of ensuring some dehydration of the atomized particles before entering the lower chamber (12). It is to be understood that the pilot scale dimensions may differ to some extent from the production scale apparatus.

Therefore, an embodiment of the present invention relates to the apparatus as described herein, wherein the upper chamber (11) has a height that allows at least 5% of the liquid in the atomized particles of the solution or suspension to evaporate during the passage, and wherein the lower chamber (12) has a height that allows a complete freezing of the atomized particles entering from the upper chamber (11).

Another embodiment of the present invention relates to the apparatus as described herein, wherein the upper chamber (11) is substantially cylindrical and has a diameter in the range of 0.5 to 5 m and a height in the range of 1 to 4 times the diameter.

A further embodiment of the present invention relates to the apparatus as described herein, wherein the lower chamber (12) is substantially cylindrical and has a diameter in the range of 0.5 to 5 m and a height in the range of 1 to 2 times the diameter.

A still further embodiment of the present invention relates to the apparatus as described herein, wherein the lower chamber (12) has a structure comprising a first part that is substantially cylindrical and a second part that is substantially conical, wherein the first a second parts are connected at the interface between the bottom of the cylinder and the base of the cone. Yet another embodiment of the present invention relates to the apparatus as described herein, wherein the upper (11) and lower (12) chambers are connected so the upper chamber (11) is the upper part of a substantially cylindrical structure, and the lower chamber (12) is the lower part of said substantially cylindrical structure, wherein said substantially cylindrical structure comprises a diameter in the range of 0.5 to 5 m and a total height in the range of 2 to 6 times the diameter.

It may be envisioned that some applications would require a change of pressure in the upper (11) and/or lower (12) chamber. Thus, the apparatus may be equipped with a means for adapting the pressure (P) to operate the apparatus at a pressure different from ambient pressure.

Thus, an embodiment of the present invention relates to the apparatus as described herein, wherein the apparatus comprises means for lowering or increasing the pressure (P) in the upper chamber (11) and/or in the lower chamber (12).

Another embodiment of the present invention relates to the apparatus as described herein, wherein said means for lowering or increasing the pressure (P) is adapted for adjusting the pressure in the upper chamber (11) and/or in the lower chamber (12) to a pressure below 900 kPa or a pressure above 1100 kPa.

The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for providing the preparation comprising an encapsulated microbial culture and all its features, which may readily be part of the preparation comprising an encapsulated microbial culture perse as described herein. Embodiments and features of the present invention are also outlined in the following items.

Items

XI. A method for providing a preparation comprising an encapsulated microbial culture, said method comprising the following steps of: i) providing a first solution comprising a microbial culture and one or more matrix components, ii) providing a second solution comprising a salt comprising a divalent metal cation, iii) spraying simultaneously said first and second solutions into a drying gas in an upper spray chamber to produce atomized particles comprising an encapsulated microbial culture, iv) contacting the atomized particles comprising an encapsulated microbial culture with a cryogenic gas in a lower spray chamber to produce frozen atomized particles comprising an encapsulated microbial culture, and v) collecting the frozen atomized particles comprising an encapsulated microbial culture, thereby providing a preparation comprising an encapsulated microbial culture.

X2. The method according to item XI, wherein the first solution, second solution and drying gas is sprayed or ejected into the upper chamber via a 3-fluid nozzle.

X3. The method according to item X2, wherein the 3-fluid nozzle is an external mixing 3-fluid nozzle.

X4. The method according to item X3, wherein the external mixing 3-fluid nozzle comprises separate inner, intermediate, and outer gaps arranged as three concentric annular rings.

X5. The method according to item X4, wherein the first solution is sprayed via the inner gap, the second solution is sprayed via the intermediate gap, and the drying gas is ejected via the outer gap.

X6. The method according to any one of the preceding items, wherein the one or more matrix components are selected from the group consisting of carbohydrates, proteins, antioxidants, and combinations thereof

X7. The method according to item X6, wherein the carbohydrates are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

X8. The method according to any one of the preceding items, wherein the one or more matrix components comprise polymers. X9. The method according to any one of items X7 or X8, wherein the polysaccharides are selected from the group consisting of alginate, pectin, cellodextrin, gums, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan, and combinations thereof.

X10. The method according to any one of items X7-X9, wherein the polysaccharide is alginate and wherein said alginate is preferably sodium alginate.

XI 1. The method according to any one of items X6-X10, wherein the protein is a milk protein.

X12. The method according to item XI 1, wherein the milk protein is caseinate, such as sodium caseinate.

X13. The method according to any one of items X6-X12, wherein the antioxidants are selected from the group consisting of citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienol, glutathione, and combinations thereof.

X14. The method according to any one of the preceding items, wherein the first solution further comprises one or more cryoprotecting agents.

X15. The method according to item X14, wherein the one or more cryoprotecting agents are selected from the group consisting of disaccharides, such as sucrose, lactose or trehalose, maltodextrin, sodium ascorbate and protein peptone, such as casein peptone, pea protein peptone or potato protein peptone, and combinations thereof.

X16. The method according to any one of the preceding items, wherein the ratio (wt%/wt%) of microbial culture to matrix component(s) is in the range of 10: 1 to 1 :5, such as in the range of 7: 1 to 1 :4, preferably in the range of 4: 1 to 1:3, more preferably 2: 1 to 1 :2.

X17. The method according to any one of the preceding items, wherein the first solution comprises only a single matrix component. X18. The method according to any one of the preceding items, wherein the divalent metal cation is selected from the group consisting of Ca ²⁺ , Zn ²⁺ and Mg ²⁺ .

X19. The method according to any one of the preceding items, wherein the salt comprises an anion selected from the group consisting of Cl’, Br’, OH’, SO4 ² ’, COs ² ’, CH ₃ COO’ and HCOO’.

X20. The method according to any one of the preceding items, wherein the salt is a chloride salt.

X21. The method according to any one of the preceding items, wherein the salt is selected from CaCh, ZnCI? and MgCh, preferably CaCI?.

X22. The method according to any one of the preceding items, wherein the concentration of salt in the second solution is in the range of 0.05 - IM.

X23. The method according to any one of the preceding items, wherein the ratio (% wv /%wv) of microbial culture to second solution is in the range of 5: 1 to 10: 1.

X24. The method according to any one of the preceding items, wherein the first or second solution further comprises a monovalent cation.

X25. The method according to any one of the preceding items, wherein the monovalent cation is Na ⁺ or NH ₄ ⁺ , preferably Na ⁺ .

X26. The method according to any one of the preceding items, wherein the microbial culture is a bacterium.

X27. The method according to any one of the preceding items, wherein the microbial culture is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Propionibacterium, Oenococcus and Bacillus.

X28. The method according to any one of the preceding items, wherein the microbial culture is or comprises a probiotic culture.

X29. The method according to any one of the preceding items, wherein the microbial culture is or comprises a lactic acid bacterium (LAB) culture. X30. The method according to item X29, wherein the lactic acid bacterium (LAB) is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

X31. The method according to any one of the preceding items, wherein the microbial culture of step (i) is provided as a solution or suspension.

X32. The method according to any one of the preceding items, wherein the microbial culture comprises two or more different microbial strains, such as two different microbial strains, such as three different microbial strains, such as four different microbial strains.

X33. The method according to any one of the preceding items, wherein the first solution comprises a concentration of microbial culture of at least 1.0E+07 CFU/ml, such as at least 1.0E+08 CFU/ml, preferably at least 1.0E+09 CFU/ml.

X34. The method according to any one of the preceding items, wherein step iii) is performed for a time sufficient for achieving the desired degree of drying and crosslinking and step iv) is performed for a time sufficient for a complete freezing can be achieved.

X35. The method according to any one of the preceding items, wherein the volume flow is adjusted to obtain a residence time sufficient for drying and freezing the atomized particles.

X36. The method according to any one of the preceding items, wherein the residence time of the atomized particles in the drying gas and/or cryogenic gas is in the range of 10 to 300 seconds. X37. The method according to any one of the preceding items, wherein the atomized particles are transferred from the upper spray chamber into the lower spray chamber by means of the gravity.

X38. The method according to any one of the preceding items, wherein the drying gas is selected from the group consisting of an inert gas, a noble gas, carbon dioxide, an alkane gas, and combinations thereof.

X39. The method according to any one of the preceding items, wherein the drying gas is selected from nitrogen, helium, neon, argon, carbon dioxide, methane, and combinations thereof.

X40. The method according to any one of the preceding items, wherein the drying gas is nitrogen.

X41. The method according to any one of the preceding items, wherein the drying gas comprises less than 5% oxygen, such as less than 2%.

X42. The method according to any one of the preceding items, wherein the drying gas has an inlet temperature in the range from 20°C to 250°C, preferably in the range from 100°C to 200°C.

X43. The method according to any one of the preceding items, wherein the drying gas has an inlet temperature of at most 300°C.

X44. The method according to any one of the preceding items, wherein the cryogenic gas is selected from the group consisting of an inert gas, a noble gas, carbon dioxide, an alkane gas, and combinations thereof.

X45. The method according to any one of the preceding items, wherein the cryogenic gas is selected from nitrogen, helium, neon, argon, carbon dioxide, methane, and combinations thereof.

X46. The method according to any one of the preceding items, wherein the cryogenic gas is nitrogen.

X47. The method according to any one of the preceding items, wherein the cryogenic gas comprises less than 5% oxygen, such as less than 2%. X48. The method according to any one of the preceding items, wherein the cryogenic gas has an inlet temperature in the range from -20 to -250°C, such as in the range of -50 to -160°C, such as in the range of -80 to -120°C.

X49. The method according to any one of the preceding items, wherein step iii) and/or iv) takes place under a pressure between 60 and 200 kPa, such as between 80 and 150 kPa, such as between 90-110 kPa.

X50. The method according to any one of the preceding items, wherein the gas:feed ratio is adjusted to control the size of the atomized particles, wherein the gas:feed ratio is defined as the ratio between the mass/hour of drying gas and the combined mass/hour of the first and second solutions.

X51. The method according to item X50, wherein the gas:feed ratio is in the range of 0.8 to 3, such as 1 to 2, such as 1.2 to 1.8.

X52. The method according to any one of the preceding items, wherein the diameter of the atomized particles is in the range of 5-50 pm, such as 5-25 pm, preferably 10-15 pm measured as Dv50 according to ISO 13320:2020.

X53. The method according to any one of the preceding items, wherein the flow ratio between the first solution and the second solution is adjusted to control the thickness of an exterior layer of salt of the second solution.

X54. The method according to any one of the preceding items, wherein the flow ratio between the first solution and the second solution is in the range of 20: 1 to 2: 1, such as in the range of 10: 1 to 5: 1, preferably approximately 7: 1.

X55. The method according to any one of the preceding items, wherein the frozen atomized particles are collected by means of a cyclone or an electrostatic filter, preferably by means of a cyclone.

X56. The method according to item X55, wherein the cyclone is operated with a with a maximum differential pressure drop across the cyclone in the range of 10mm to 300mm water column, such as 50 to 200mm water column, or approximately 100 mm water column. X57. The method according to any one of the preceding items, wherein step v) is followed by a step vi) of freeze drying the frozen atomized particles.

X58. The method according to item X57, wherein said freeze drying is performed at reduced pressure, such as at 0.1 to 0.4 mbar.

X59. The method according to any one of items X57 or X58, wherein said freeze drying is performed at a sublimation temperature in the range of -60°C to 45°C until complete water removal.

X60. The method according to any one of items X57-X59, wherein said freeze drying is performed at a pressure of 0.3 mbar with the sublimation temperature increasing from -55°C to 32°C with 1.5°C/min.

X61. The method according to any one of items X57-X60, wherein said freeze drying is performed until the water activity (Aw) of the frozen atomized particles are below 0.20, preferably below 0.10.

X62. The method according to any one of the preceding items, wherein said preparation substantially consists of the encapsulated microbial culture.

X63. The method according to any one of the preceding items, wherein said method as a final step comprises packaging of the preparation comprising an encapsulated microbial culture.

X64. The method according to item X63, wherein packaging is in an air-tight and/or moisture-tight package.

X65. The method according to any one of items X63 or X64, wherein the preparation comprising an encapsulated microbial culture is packaged together with a microbial culture of a different strain.

X66. The method according to any one of the preceding items, wherein the method is performed by means of an apparatus according to items T1-T17.

El. Use of a method according to any one of items X1-X66 to increase phage resistance of a preparation comprising a microbial culture. Yl. A preparation comprising an encapsulated microbial culture obtainable by the method according to any one of the items X1-X66.

Zl. A preparation comprising an encapsulated microbial culture in the form of particles, wherein the particles comprise: i) an interior core comprising a microbial culture and one or more matrix components, and ii) an exterior layer comprising a salt comprising a divalent metal cation, wherein the thickness of said exterior layer is less than 7% of the particle radius.

Z2. The preparation according to item Zl, wherein the size of the particles is in the range of 5-50 pm, such as 5-25 pm, preferably 10-15 pm measured as Dv50 according to ISO 13320:2020.

Z3. The preparation according to any one of items Zl or Z2, wherein the thickness of said exterior layer is less than 5% of the particle radius, preferably less than 4.5% of the particle radius.

Z4. The preparation according to any one of items Z1-Z3, wherein the divalent metal cation is selected from the group consisting of Ca ²⁺ , Zn ²⁺ and Mg ²⁺ .

Z5. The preparation according to any one of items Z1-Z4, wherein the salt comprises an anion selected from the group consisting of Cl’, Br’, OH’, SO4 ² ’, COs ² ’, CH ₃ COO’ and COO’.

Z6. The preparation according to any one of items Z1-Z5, wherein the salt is a chloride salt.

Z6. The preparation according to any one of items Z1-Z6, wherein the salt is selected from CaCh, ZnCI? and MgCh, preferably CaCI?.

Z7. The preparation according to any one of items Z1-Z6, wherein the exterior layer further comprises a monovalent salt.

Z8. The preparation according to any one of items Z1-Z7, wherein the monovalent cation is Na ⁺ or NH ₄ ⁺ . Z9. The preparation according to any one of items Z1-Z8, wherein the one or more matrix components are selected from the group consisting of carbohydrates, proteins, antioxidants, and combinations thereof.

Z10. The preparation according to item Z9, wherein the carbohydrates are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and combinations thereof.

Zll. The preparation according to any one of items Z1-Z10, wherein the one or more matrix components comprise polymers.

Z12. The preparation according to any one of items Z10 or Zll, wherein the polysaccharides are selected from the group consisting of alginate, pectin, cellodextrin, gums, starch, glycogen, cellulose, chitin, inulin, dextran, carrageenan, chitosan, and combinations thereof.

Z13. The preparation according to any one of items Z10-Z12, wherein the polysaccharide is alginate and preferably sodium alginate.

Z14. The preparation according to any one of items Z9-Z13, wherein the protein is a milk protein.

Z15. The preparation according to item Z14, wherein the milk protein is caseinate, such as sodium caseinate.

Z16. The preparation according to any one of items Z9-Z15, wherein the antioxidants are selected from the group consisting of citrate, ascorbate, tocopherol, ascorbyl palmitate, quercetin, gallic acid, tocotriene, tocotrienol, glutathione, and combinations thereof.

Z17. The preparation according to any one of items Z1-Z16, wherein the first solution further comprises one or more cryoprotecting agents.

Z18. The preparation according to item Z17, wherein the one or more cryoprotecting agents are selected from the group consisting of disaccharides, such as sucrose, lactose or trehalose, maltodextrin, sodium ascorbate and protein peptone, such as casein peptone, pea protein peptone or potato protein peptone, and combinations thereof. Z19. The preparation according to any one of items Z1-Z18, wherein the ratio (wt%/wt%) of microbial culture to matrix component(s) is in the range of 1 :0.5 to 1 :5.

Z20. The preparation according to any one of items Z1-Z19, wherein the microbial culture is a bacterium.

Z21. The preparation according to any one of items Z1-Z20, wherein the microbial culture is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Propionibacterium, Oenococcus, and Bacillus.

Z22. The preparation according to any one of items Z1-Z21, wherein the microbial culture is or comprises a probiotic culture.

Z23. The preparation according to any one of items Z1-Z22, wherein the microbial culture is or comprises a lactic acid bacterium (LAB).

Z24. The preparation according to item Z23, wherein the lactic acid bacterium (LAB) is of a genus selected from the group consisting of Lactococcus, Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

Z25. The preparation according to any one of items Z1-Z24, wherein the preparation comprises a concentration of viable microbial culture of at least 1.0E+07 CFU/g, such as at least 1.0E+08 CFU/g, preferably at least 1.0E+09 CFU/g.

Z26. The preparation according to any one of items Z1-Z25, wherein the preparation is in the form of a powder and/or a granulate.

Z27. The preparation according to any one of items Z1-Z26, wherein the preparation is a freeze-dried preparation. Z28. The preparation according to any one of items Z1-Z27, wherein the water activity (Aw) of the preparation is in the range from 0.01-0.8, preferably in the range from 0.05-0.6, most preferably in the range from 0.1-0.4.

Z29. The preparation according to any one of items Z1-Z28, wherein the preparation substantially consists of the encapsulated microbial culture.

QI. A composition comprising a preparation comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29.

Q2. The composition according to item QI, wherein the composition further comprises one or more additives selected from the group consisting of food-grade ingredients, pharmaceutical ingredients and excipients.

Q3. The composition according to item Q2, wherein the food-grade ingredients are selected from the group consisting of lactose, maltodextrin, whey protein, corn starch, dietary fibres, gums and gelatine.

Q4. The composition according to any one of items Q2 or Q3, wherein the pharmaceutical ingredients are selected from the group consisting of calcium carbonate, sodium carboxymethyl cellulose, talc, polydimethylsiloxane, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

Q5. The composition according to any one of items Q2-Q4, wherein the excipients are selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminium silicate.

Q6. The composition according to any one of items Q1-Q5, wherein the composition is in the form of a powder and/or a granulate.

Q7. The composition according to any one of items Q1-Q6, wherein the composition is a freeze-dried composition.

Q8. The composition according to any one of items Q1-Q7, wherein the water activity (Aw) of the composition is in the range from 0.01-0.8, preferably in the range from 0.05-0.6, most preferably in the range from 0.1-0.4. Pl. A kit-of-parts comprising: i) one or more distinct preparations comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29; ii) one or more components selected from the group consisting of foodgrade ingredients, pharmaceutical ingredient, excipients and combinations thereof; iii) optionally, instructions for use.

Wl. Use of a preparation comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29 or a composition according to any one of items Q1-Q8 in a product selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

W2. The use according to item Wl, wherein the food product is a dairy product.

W3. The use according to item W2, wherein the dairy product comprises a solid or semi-solid food matrix, preferably a solid food matrix.

W4. The use according to item W3, wherein the dairy product comprising a solid food matrix is a cheese.

Rl. A product comprising a preparation comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29 or a composition according to any one of items Q1-Q8, wherein the product is selected from the group consisting of a feed, a plant health product, a food, a beverage, a nutraceutical and a pharmaceutical product.

R2. A dairy product comprising a preparation comprising an encapsulated microbial culture according to any one of items Y1 or Z1-Z29 or a composition according to any one of items Q1-Q8.

R3. The dairy product according to item R2, wherein said dairy product is selected from the group consisting of yoghurt, cheese, butter, inoculated sweet milk and liquid fermented milk products.

R4. The dairy product according to any one of items R2 or R3, wherein the dairy product comprises a solid or semi-solid food matrix, preferably a solid food matrix. R5. The dairy product according to item R4, wherein the dairy product comprising a solid food matrix is a cheese.

Tl. An apparatus for producing frozen particles comprising a first upper chamber (11) and a second lower chamber (12), wherein the upper chamber comprises: i) a 3-fluid nozzle for atomizing at least two suspensions or solutions (5); ii) an inlet for a drying gas (1); iii) means (3) for heating the drying gas to a temperature in the range 20°C to 250°C; and wherein the lower chamber comprises: iv) an inlet for a cryogenic gas (4); v) an outlet for the frozen particles, said outlet being connected to a cyclone (14); and wherein the upper chamber is arranged so the particles descend into the lower chamber for subsequent freezing.

T2. The apparatus according to item Tl, wherein the 3-fluid nozzle (5) is an external mixing 3-fluid nozzle.

T3. The apparatus according to any one of items Tl of T2, wherein the 3-fluid nozzle (5) comprises separate inner, intermediate, and outer gaps arranged as three concentric annular rings.

T4. The apparatus according to item T3, wherein the inner gap is adapted for receiving a first solution or suspension, the intermediate gap is adapted for receiving a second solution or suspension, and the outer gap is adapted for receiving the drying gas.

T5. The apparatus according to any one of items T1-T4, wherein the inlet for a cryogenic gas (4) is adapted for a gas having a temperature in the range -50 to - 250°C.

T6. The apparatus according to any one of items T1-T5, wherein the apparatus comprises a tank for storing the cryogenic gas.

T7. The apparatus according to any one of items T1-T6, wherein the upper chamber (11) is connected to a heater (3) for heating the drying gas. T8. The apparatus according to any one of items T1-T7, wherein the apparatus is a spray tower, wherein the upper chamber (11) is placed over the lower chamber (12).

T9. The apparatus according to any one of items T1-T8, wherein the upper chamber (11) has a height that allows at least 5% of the liquid in the atomized particles of the solution or suspension to evaporate during the passage, and wherein the lower chamber (12) has a height that allows a complete freezing of the atomized particles entering from the upper chamber (11).

T10. The apparatus according to any one of items T1-T9, wherein the upper chamber (11) is substantially cylindrical and has a diameter in the range of 0.5 to 5 m and a height in the range of 1 to 4 times the diameter.

Til. The apparatus according to any one of items T1-T10, wherein the lower chamber (12) is substantially cylindrical and has a diameter in the range of 0.5 to 5 m and a height in the range of 1 to 2 times the diameter.

T12. The apparatus according to any one of items Tl-Tll, wherein the lower chamber (12) has a structure comprising a first part that is substantially cylindrical and a second part that is substantially conical, wherein the first a second parts are connected at the interface between the bottom of the cylinder and the base of the cone.

T13. The apparatus according to any one of items T1-T12, wherein the upper (11) and lower (12) chambers are connected so the upper chamber (11) is the upper part of a substantially cylindrical structure, and the lower chamber (12) is the lower part of said substantially cylindrical structure, wherein said substantially cylindrical structure comprises a diameter in the range of 0.5 to 5 m and a total height in the range of 2 to 6 times the diameter.

T14. The apparatus according to any one of items T1-T13, wherein the apparatus comprises means for lowering or increasing the pressure (P) in the upper chamber (11) and/or in the lower chamber (12).

T15. The apparatus according to item T14, wherein said means for lowering or increasing the pressure is adapted for adjusting the pressure (P) in the upper chamber (11) and/or in the lower chamber (12) to a pressure below 900 kPa or a pressure above 1100 kPa. The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1: Production of preparation comprising encapsulated microbial culture

The purpose of this experiment was to demonstrate production of frozen particles using the method described herein.

Methods

To a 5 L blue cap flask 2.304 kg of reverse osmosis (RO)-water was added and then 0.096 kg of sodium alginate (FMC with Kosher/Halal certificate) was slowly added and mixed with a high shear mixer. The resulting 4 % (w/w) aqueous alginate solution was autoclaved at 121°C for 60 min and allowed to slowly cool down to room temperature over 24 hours.

About 2.4 kg frozen "Direct Vat Set" (F-DVS) of the dairy culture LACIa 10675 {Lactococcus lactis subspecies lactis, Chr. Hansen GIN No. 697876) was slowly thawed in a 15°C water bath and then mixed with the 2.4 kg autoclaved 4 % (w/w) alginate solution. The mixture was placed in an ice-water bath in order to keep the mixture below 5°C during operation of the spray freezer.

In parallel, a 0.7 kg 0.1 M CaCI2 solution in RO-water was prepared as the salt solution.

The custom-made spray freezer as described herein was used for the experiment.

The spray freezer has an 0.8 m internal diameter and a total internal vertical length of 2.75 m, and is divided into a 1.9 m long drying section with an internal diameter ranging from 0.35 m to 0.8 m and a 0.85 m long cooling/freezing section with an internal diameter ranging from 0.8 m to 0.05 m - the latter being the diameter of the outlet duct to the downstream "Buhler MGXE" cyclone with an internal diameter of 0.28 m.

The spray freezer is equipped with a number of flat spray nozzles (Lechler - FU1) placed 0.75 m over the bottom outlet with at total liquid nitrogen atomization capacity of approx. 180 kg/h at 1 bar. At the top of the spray freezer the drying gas distributor surrounding the Schlick 3-fluid nozzle arrangement is supplying approx. 85 kg/h drying gas to the drying section.

The standard 3-fluid nozzle from Schlick (model No. 946S15 version 1.0) was modified to fit into the atomizer arrangement pit of the gas distributor and the centre bore for the liquid product formulation had been increased to 1.0 mm to accommodate higher viscosity fluids.

Before product start-up of the spray freezer, the lower section of the spray freezer was cooled down with approx. 180 kg/h liquid nitrogen and the upper section was heated with approx. 85 kg/h nitrogen gas with a temperature of approx. 40°C. After 30 min a steady state outlet temperature of -140°C had been reached. Finally, the Schlick 3-fluid nozzle was supplied with 12.8 kg/h nitrogen gas at room temperature.

A Watson Marlow 30S/L peristaltic pump with a 505L pump head was used for both the liquid product formulation and the 0.1 M calcium chloride solution as they were pumped to the 3-fluid nozzle assembly simultaneously using a 4.8 mm bore hose for the liquid product solution and a 2.4 mm bore hose for the calcium chloride solution. Using the same pump head for both hoses yielded seamless synchronization of the two liquid flows to the 3-fluid nozzle at a ratio of 1:6.85 between the 0.7 kg 0.1 M calcium chloride solution (led to the middle gap of the 3-fluid nozzle ) and the 4.8 kg liquid product formulation (led to the centre bore of the 3-fluid nozzle).

The nozzle was operated using a liquid formulation mass flow of approx. 7.78 kg/h and a 0.1 M calcium chloride solution mass flow of 1.135 kg/h, which resulted in a gas to liquid ratio of approx. 1.44 (= 12.8 I (7.78+1.135)), ensuring a cloud of droplets below 30 pm in general.

The total equipment volume is about 1.08 m ³ and the (180+85+12.8) kg/h nitrogen mass flow is equivalent to approx. 108 m ³ /h gas flow at -140°C, which in turn gives an average residence time of approx. 36 seconds. At -130°C, the gas flow is increased to 116 m ³ /h, which in turn gives an average residence time of approx. 33.4 seconds. Accordingly, during experimentation, the average residence time was in the range of 33-36 seconds.

Results About 4.62 kg SF-DVS was recovered. About 0.1 kg normally pelletized reference sample was made, which corresponds to a mass yield of approx. 86 % of the 4.8+0.7- 0.1= 5.4 kg liquids sprayed

The obtained spray frozen material had a bulk density of approx. 0.37 kg/L and after storage at -55°C for 9 months the frozen powder was still free-flowing and showed no signs of lumping.

The recovered product comprised a sufficient amount of viable encapsulated microbial culture for subsequent evaluation of resistance to phage attacks (see example 2).

Conclusions

The obtained mass yield was good for a sub-hour test on a spray-based type of equipment. Moreover, the stability of the recovered product was excellent and retained the desired morphology and handling characteristics due to the outer frozen salt layer staying intact upon storage and serving as an efficient ant-lumping agent.

Example 2: Evaluation of robustness against bacteriophage attack

The purpose of this example was to demonstrate the phage robustness of frozen particles using the method described herein compared to a conventional frozen product in milk.

Methods

Strains

The strains used were from an internal culture collection:

1) Streptococcus thermophilus CH-Q-5

2) Lactococcus lactis subsp. Lactis LACIa 10675

These strains, in whatever format used, is termed the bacterial "host" of the phage in the following.

Strain formats

Two set of frozen particles were produced using the method described herein:

1) SF-DVS CH-Q-5

2) SF-DVS LACIa 10675

They were produced from and compared with the conventional product formats:

3) F-DVS ESP CH-Q-5 (GIN no 620981) 4) F-DVS LACIa 10675 (GIN no 697876)

The frozen pellets using the method of the invention were produced as described in example 1 and termed "SF-DVS" (Spray Frozen - Direct Vat Set) in the following. The conventional product format is denoted "F-DVS" (Frozen - Direct Vat Set) in the following.

Phages

Phages were from an internal phage collection specific to each of the hosts:

1) Cos bacteriophage specific against CH-Q-5

2) C2 bacteriophage specific against LACIalO75

Phage propagation

The phage was propagated on the host to ensure a high titer of the phage prior to the experiments.

Two tubes were prepared:

1) 100 pl lysate (or a single plague) + 10 ml M17 broth + 2% lactose + 10 mM CaCI? + 1% host strain

2) 10 ml M17 broth + 2% lactose + 10 mM CaCI? + 1% host strain

After a few hours of incubation (at 30°C for Lactococcus lactis and 37°C for Streptococcus thermophilus), tube one (1) was lysed and tube two (2) had bacterial growth. Half of tube two with bacterial growth was subseguently transferred into the lysed tube one and further incubated until tube one lysed again. This latter step was repeated until all of tube two had been transferred and lysed. The obtained fresh lysate in tube one was filtered on a 0.45 pm filter to remove the host and kept refrigerated until use.

Preparation of Indicator Milk

The following indicators were prepared:

1) Bromcresol purple (Sigma Inc.): 50 mg in 1 ml 0.02N NaOH plus MQW to 25 ml

2) Bromcresol green (Sigma Inc.): 50 mg in 1 ml 0.02N NaOH plus MQW to 25 ml

2.5 ml of each indicator was added to 50 ml whole milk (Aria® 24 Frisk Dansk Sodmaelk 3,5% 1 L) to make the indicator milk (I-milk). Preparation of Microtiter Plates

Experiments were conducted according to table 1:

Table 1. Sample setup.

In each experiment, a microtiter plate (MTP) (96 wells, Nunc), with eight rows (A to H) and 12 columns (Cl to C12) was prepared.

The final volume in each well in the MTP was 315 pL, comprising:

1) 270 pL solution of I-milk and product format (SF-DVS or F-DVS)

2) 15 pL phage solution

3) 15 pL coagulant solution (or sterile water if no coagulant was added)

Preparation of product format solution

In each plate, the host was inoculated in I-milk to a total of 270 pL, with decreasing final concentrations in the well as follows: 20, 10, 5, 2.5, 1.25, 0.6, 0.3, and 0.15 g sample (F-DVS or SF-DVS) per 100L. Concentrations are based on the final volume of 315 pL. All required pre-dilutions were done in the same milk type as the milk being the base for the I-milk preparation.

The inoculation layout of a plate is shown in table 2 below. Table 2. Inoculation layout of a 96-well microtiter plate. A-H corresponds to rows. Cl- C12 corresponds to columns.

Preparation of the phage solution

A 10-fold, 5 times dilution, of the phage lysate was made in a Ringer solution (Merck Millipore, 96724). The most diluted sample was marked 5° = 1 and the undilute sample was marked 5 ¹⁰ ~ 1E+07. Each number indicating the increasing strength of the solutions: 1, 5, 25, 125, 640, 3200, 16000, 80000, 4E+05, 2E+06, and 1E+07 where 1 is the most diluted and 1E+07 the least diluted.

15 pL of each dilution was added in turn to columns C2 (strength 1) to C12 (strength 1E+07), reserving column Cl as the reference column without any phage added (strength 0).

The phage titer layout of a plate is shown in table 3 below.

Table 3. Phage titer layout of a 96-well microtiter plate. A-H corresponds to rows. Cl- C12 corresponds to columns.

Preparation of the coagulant solution

In the experiments with added chymosin (chymax plus), 15 pL solution was added to a final strength of 0.6 International Milk Clotting Unit (IMCU) per well.

In the experiments without added chymosin, 15 pL sterile water was added instead.

Determination of activity

Each plate was placed on a flatbed scanner (Epson V39 scanner, connected to a computer with custom-made software: pH MultiScan, HNH Consult ApS) and incubated for 16 hours at a fixed temperature: 1) Lactococcus lactis at 30°C

2) Streptococcus thermophilus at 37°C

While incubating, the change in hue was recorded over time (pH MultiScan, HNH Consult ApS). The time-series thus obtained correlates with pH as described in detail in WO 2005/068982 Al.

Comparing the different formats

In each experimental set (/.e. 1 (A and B), 2 (A and B), 3 (A and B)), the reference acidification curves (/.e. the curves with no phage added (Cl)) for F-DVS (A) and SF- DVS (B) were compared.

The row with matching reference (Cl) curves, i.e. the rows with inoculation doses at which the two product formats would yield the same activity in the absence of phages, were identified and compared for the effect of increasing phage titer.

Results

Experiment 1 (figure 2): The effect of the product format for CH-O-5 without coagulant

Figure 2 shows the results of experiment 1 (A and B) as outlined in table 1. The figure displays 36 panels.

The columns represent the strength of the phage solution added. The first (left) column is the reference column where no phage has been added. In the other columns the titer increases 5-fold from left to right. The number in the column headings indicates the relative strength of the of the phage solution tested; the higher the more potent.

The two top rows represent the two different formats ("SF-DVS" and "F-DVS") and the curves indicate the acidification activity of the two formats. Decreasing hue values corresponds to decreasing pH values. The "-R" indicates that no coagulant was added.

The last row ("delta") shows the difference in activity between formats; If delta is below zero then the SF-DVS format is more active, if above zero then the F-DVS format is more active. The data compared in the two top rows were selected from the MTP experiments in such a manner that the activity in the reference column of the two formats are comparable.

In figure 2, row 1 shows the curves of SF-DVS at 5g/100L and row 2 F-DVS at 0.6 g/lOOL, it is clear from the left column (phage strength 0) that the two formats under these conditions have comparable acidification activities, when phages are absent.

Having the same activity when phages are absent, it is possible to directly compare how the two sample formats cope with increasing phage pressure, moving from left to right across the figure. Notably, the sample formats have roughly the same activity up to a phage strength of 640. Somewhere between 640 and 3200 F-DVS breaks down under the increasing pressure of phages while SF-DVS continues to be active until at least a phage strength of 16000. At a phage pressure of 80000, both formats are approximately equally inhibited.

The results of experiment 1 results demonstrate that given the same initial acidification activity, the SF-DVS CH-Q-5 format is about 16000/640 = 25 times more phage robust than F-DVS CH-Q-5 in milk without rennet.

Experiment 2 (figure 3): The effect of the product format for CH-Q-5 with coagulant Figure 3 shows the results of experiment 2 (A and B) as outlined in table 1. The figure displays 36 panels. The overall layout of the experiment is the same as in experiment 1. The "+R" indicates that coagulant was added.

In figure 3, row 1 shows the curves of SF-DVS at 5g/100L and row 2 F-DVS at 1.25 g/lOOL, it is clear from the left column (phage strength 0) that the two formats under these conditions have comparable acidification activities, when phages are absent.

The sample formats have roughly the same activity up to a phage strength of 3200. Somewhere between 3200 and 16000 F-DVS breaks down under the increasing pressure of phages while SF-DVS continues to be active until at least a phage strength of 80000. At a phage pressure of 4E+05, both formats are approximately equally inhibited. The results of experiment 2 demonstrate that given the same initial acidification activity, the SF-DVS CH-Q-5 format is about 80000/3200 = 25 times more phage robust than F-DVS CH-Q-5 in milk with coagulant.

Experiment 3 (figure 4): The effect of the product format for LaCla 10675 with coagulant

Figure 4 shows the results of experiment 3 (A and B) as outline in table 1. The figure displays 36 panels. The overall layout of the experiment is the same as in experiments 1 and 2. The "+R" indicates that coagulant was added.

In figure 4, row 1 shows the curves of SF-DVS at lOg/lOOL and row 2 F-DVS at 5 g/lOOL, it is clear from the left column (phage strength 0) that the two formats under these conditions have comparable acidification activities, when phages are absent.

The sample formats have roughly the same activity up to a phage strength of 130. Somewhere between 130 and 650 F-DVS breaks down under the increasing pressure of phages while SF-DVS continues to be active until at least a phage strength of 650. At a phage pressure of 3200, both formats are approximately egually inhibited.

The results of experiment 3 demonstrate that given the same initial acidification activity, the SF-DVS LACIa 10675 format is about 650/130 = 5 times more phage robust than F-DVS LACIalO675 in milk with rennet.

Conclusion

Together, experiments 1-3 demonstrate that the new SF-DVS format offers an improved protection against phages, regardless of the strain used.

References

• Seifert & Mogensen (2002), Bulletin of the IDF, 377, 10-19

• Zheng et al. (2020), Int. J. Syst. Evol. Microbiol., 70, 2782-2858

• WO 2005/068982 Al.