WO2025114446A1

WO2025114446A1 - Formulations of heat resistant microencapsulated microbial culture

Info

Publication number: WO2025114446A1
Application number: PCT/EP2024/083933
Authority: WO
Inventors: Hamed NASRI LARI; Zuzana Mladenovska; Esben Ulrich VULFF; Surender Kumar DHAYAL
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-11-28
Filing date: 2024-11-28
Publication date: 2025-06-05
Anticipated expiration: 2026-05-28

Abstract

The present disclosure relates to a microencapsulated microbial culture, methods for producing microencapsulated microbial cultures and their uses.

Description

FORMULATIONS OF HEAT RESISTANT MICROENCAPSULATED MICROBIAL CULTURE

Technical field of the invention

The present disclosure relates to microencapsulated microbial cultures with increased survivability under harsh conditions, such as high temperature, high acidity and high water activity.

Technical background

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. The typical process for the production of LAB is through fermentation followed by concentration and freezing of cell biomass. When it comes to applications of LAB, dried powder form produced using freeze drying (FD) is often desired. The dried powders are frequently kept for extended periods of time before utilized in a final application.

However, it is widely known that when dried LABs are subjected to high temperatures above 30 °C, their viability reduces significantly. The viability loss becomes more dramatic at higher temperatures above 70 °C, where holding times of several seconds, results in survivability of lower than 1%.

This reduction in viability is even more pronounced in compositions, foods, feeds, and beverages, where there is a high water activity and high acidity, as high temperatures exacerbates the adverse effects of high water activity and acidic environment and as a result overburden the LABs. This limits the application of LABs as living cultures in foods and beverages with low acidity and/or high water content prior to pasteurization, for example to post-pasteurized yogurts, pasteurized and ultra-high temperature (UHT) treated milks, pasteurized beverages, etc.

The issue of temperature generally recognized and a number of possible solutions have been suggested in the prior art.

WO2011/004375 reported production of a multi-layer coated probiotic which can withstand heat treatment during baking products. This disclosure relates to production of encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum in four steps by means of fluidized bed spray coating including: (1) absorption of microorganism on microcrystalline cellulose substrate, (2) coating with hydrogenated vegetable oil, (3) coating with ethyl cellulose as an enteric coating, and (4) coating with calcium alginate as the heat-resistant polymer. All three layers were coated using fluidized bed spray coating technique.

CN105228457B also described a probiotic product containing heat and moisture resistant probiotic in the granule forms for liquid infant food applications. A Starchbased polymer was used as coating layer in a multi-layer arrangement.

US10561161B2 (YUAN et al., 2019) produced an encapsulated probiotic with resistance to heat and acidic conditions for beverage application. The probiotic cells were encapsulated using a wet-coating technique such that bacteria was entrapped within a gelled matrix consists of alginate and denatured protein.

However, the prior art does not adequately solve the problems associated with providing the features required for obtaining microencapsulated microbial cultures that can survive at high temperatures, high water concentrations, high water activity and high acidity.

Hence, there is a need for the provision of microbial cultures with improved heatresistance, acid tolerance and/or high humidity resistance that can withstand and survive harsh treatments used for preparing and conserving foods, such as pasteurization processes and methods for producing such microbial cultures.

Detailed description

Microbial cultures, such as lactic acid bacteria (LAB), play key parts in many fermented products, in which they add nutritional value to the product and improve the organoleptic and textural profile of e.g. food products. The microbial cultures are typically acquired separately as powdered compositions and mixed with additional ingredients to yield a final product. Thus, the powdered composition comprising the microbial culture need as a minimum to maintain viability from the point of becoming a dried granulate to the point at which the powdered microbial cultures is included in a final product. However, it is widely known that when dried LABs are subjected to high temperatures above 30 °C, their viability reduces significantly.

The present disclosure provides microencapsulated microbial cultures and methods for these microencapsulated microbial cultures that can withstand the harsh conditions of pasteurization used in the food and feed industry. The microencapsulation of microbial cultures secludes the microbial culture from the surrounding environment. This seclusion from the environment protects the microbial cultures from at least environments with high water content, high acidity, high temperatures and high water activity, leading to increased survivability of the microbial cultures under these conditions.

As demonstrated in examples 4, 4A and 4B, a micro-encapsulated Bifidobacterium animalis subsp. lactis showed an excellent superior survivability along with excellent reproducibility when exposed to pasteurization methods in dairy and beverage applications. The micro-encapsulation of Bifidobacterium animalis subsp. Lactis bacteria resulted in a survival rate of on average about 50% when supplied to yoghurt prior to pasteurization at 74°C for 18 seconds, whereas the survival rate of unencapsulated bacteria was below 1%. When the micro-encapsulated bacteria were instead added to vegurt prior to pasteurization, they had a survival rate of 70% (example 4A; figure 3).

When the micro-encapsulated Bifidobacterium animalis subsp. lactis was instead added to orange juice in example 4 and subjected to a harsher pasteurization method with temperatures reaching 84°C for 4 seconds, the survival rate of the encapsulated bacteria reached 27%, whereas un-encapsulated bacteria (control) had a survival rate of 0% (Example 4B; figure 4).

These characteristics are very useful for production of foodstuff where pasteurization processes are applied to avoid and/or reduce spoilage. This includes foods such as yoghurt and vegurt, feeds for animals, beverages such as juices and dairy products such as milk, cream, and cheese, but could in general be used in any food products that contain microbes and undergo pasteurization processes.

In particular, such products may have increased longevity in distant regions where constant refrigeration and cold storage and electricity can be an issue, but also in regions prone to vast heat waves or floodings, which may compromise conditions otherwise considered optimal storage conditions for foods, feeds and beverages comprising microbial components.

Additionally, these microencapsulated microbial cultures are produced using only plant-based materials, thus providing a more sustainable product, which is also suitable for consumption by individuals who would like to avoid animal food sources.

Thus, as the micro-encapsulation technology disclosed herein both confers heat resistance and increased survival rates under high temperature stress conditions onto the microencapsulated microbial cultures, the cost-effectiveness of using microbial cultures as additives is increased, the handling of the microencapsulated microbial culture is simplified as it can be added prior to pasteurization (thus, also undergoing pasteurization), improved ease of use with products undergoing harsh disinfection treatments such as pasteurization e.g. in food, feed, beverage and dairy applications.

The materials used in the present disclosure for preparation of the microencapsulated microbial culture also have the advantage that they enable simple decapsulation of the microencapsulated microbial culture, following e.g. pasteurization, which enables easy estimation of the survival rate of the microbial culture following pasteurization.

A further advantage of the microencapsulated microbial culture and the materials used to produce it as disclosed herein, is that the microencapsulated microbial culture is readily decapsulated, e.g. in the human or animal body in the gastro-intestinal tract, leading to release of an increased ratio of live and active microbial culture in the gastro-intestinal tract of its host. In the case of probiotics, this increased ratio of live and active microorganisms, leads to a more robust probiotic effect in the host and thus leads to a more robust and effective probiotic product.

Additionally, the microencapsulated microbial culture as disclosed herein can also be easily decapsulated mechanically, which is useful for evaluation and determination of survival rate and metabolic activity of the microencapsulated microbial culture following treatment under harsh conditions, such as pasteurization in environments having high water content and high water activity.

Accordingly, the micro-encapsulated microbial cultures disclosed herein and the methods for producing these micro-encapsulated microbial cultures represent a great leap forward in terms of providing the food and feed industry with microbial cultures that can be produced easily and readily applied across a wide range of products that are traditionally used together with standard pasteurization techniques to help increase longevity, preservation and conservation of various food and feed products.

Micro-encapsulated microbial cultures

The heat resistant micro-encapsulated microbial cultures of the present disclosure comprise three coating layers that encapsulate a core material, wherein the core material comprises a microbial culture.

The three coating layers consist of a first coating layer, a second coating layer and a third coating layer. The first coating layer and the third coating layer comprise a plant- based food grade polymer and the second coating layer comprises a vegetable wax admixture.

Therefore, a first aspect of the present disclosure relates to a micro-encapsulated microbial culture comprising i. a core material comprising a microbial culture, ii. a first coating layer, a second coating layer, and a third coating layer encapsulating the core material, wherein, iii. the first coating layer comprises a plant-based food grade polymer, the second coating layer comprises a food grade admixture of vegetable wax, and the third coating layer comprises a plant-based food grade polymer.

The core material of the micro-encapsulated microbial culture is coated in the first coating layer, the first coating layer is coated in the second coating layer and the second coating layer is coated in the third coating layer.

In this way, the coating layers seclude the core material from the surrounding environment and helps protect the microbial culture comprised in the core material from the conditions of the surrounding environment.

In particular, the coating layers insulate the microbial culture comprised in the core material from high water content, high acidity, high temperature and high water activity in the surrounding environment.

The first coating layer protects the bacterial cells in the core from heat damage during the application of a second layer of fat/wax coating, such as through a hot-melt process. This dual-layer system minimizes process losses, ensuring the viability and integrity of the microbial culture.

The second coating layer

The second coating layer of the micro-encapsulation comprises an admixture of vegetable wax and vegetable waxes are known to melt with increasing temperatures, such as for example during pasteurization processes. Therefore, to provide structural support and maintain the integrity of the second coating layer when the temperature of the surrounding environment increases, the second coating layer is sandwiched between the first coating layer and the third coating layer.

In this configuration, the plant-based food grade polymer of the first coating layer and the third coating layer ensures that even when the temperature of the surrounding environment increases and the admixture of vegetable wax melts, the vegetable wax remains in place between the first coating layer and the third coating layer and continually acts as a heat sink against the high temperatures of the environment, without coming into direct contact with the microbial culture of the core material, thereby reducing direct heat transfer from the vegetable wax to the microbial culture. Additionally, in environments having high water content and water activity, the vegetable wax of the second coating layer acts as a hydrophobic barrier against the high water content and water activity of the environment.

The admixture of vegetable wax in the second coating layer protects the core material from increasing temperatures in the surrounding environment by melting, i.e., by absorbing the heat of the environment and subsequently undergoing a phase transition from solid to liquid. It is believed that this feature is highly important for the protective effect against high temperatures, high water activity and acidity of the micro-encapsulation as demonstrated in the present disclosure (see figures 3-4 and examples 5-6).

However, the composition of the vegetable wax admixture is also important for the protective effects vegetable wax admixtures composed of medium melting point vegetable wax and high melting point wax produce the desired protective effects when mixed. As demonstrated in example 2 and shown in figure 1, it was found that the ratio of medium melting point vegetable wax to high melting point vegetable wax in the admixture of vegetable wax is an important factor in achieving protection from heat, high water activity and acidity (see figure 1 and example 4). It is currently believed that the mixture of a medium melting temperature vegetable wax and a high temperature improves the ability of the vegetable wax in the second coating layer to act as heat sink and insulating layer, as the vegetable wax admixture will undergo gradual phase transition as temperature increases and reaches the melting point of each of the vegetable wax components, thereby providing an insulating effect on the core material that is active over a greater range of temperatures than if the second coating layer consisted of only either medium melting temperature vegetable wax or high melting temperature vegetable wax.

Therefore, suitable vegetable wax admixtures comprise vegetable wax admixtures, wherein the vegetable wax admixtures consist essentially of a ratio of medium melting point vegetable wax to high melting point vegetable wax as defined in any one of a)- e) a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, and e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72- 78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer consists essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax.

The medium melting point vegetable waxes that are suitable for use herein include, but are not limited to palm wax and rapeseed wax.

A non-vegetable alternative to the medium melting temperature vegetable wax, that is suitable for use in non-vegan products is bees wax.

The high melting point vegetable waxes that are suitable for use herein include, but are not limited to candelilla wax and carnauba wax.

In one or more exemplary embodiments, the medium melting point vegetable wax is selected as one of palm wax and rapeseed wax and the high melting point vegetable wax is selected as one of candelilla wax and carnauba wax.

In one or more exemplary embodiments, the high melting point vegetable wax is carnauba wax. In one or more exemplary embodiments, the high melting point vegetable wax is candelilla wax. In one or more exemplary embodiments, the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is carnauba wax, or, the medium melting point vegetable wax is palm wax and the high melting point vegetable wax is carnauba wax, or, the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is candelilla wax, or, the medium melting point vegetable wax is palm wax and the high melting point vegetable wax is candelilla wax

In one or more exemplary embodiments, the medium melting point vegetable wax of a), b), c), d) and e) is selected as one of palm wax and rapeseed wax, and the high melting point vegetable wax of a), b), c), d) and e) is selected as one of candelilla wax and carnauba wax.

In one or more exemplary embodiments, the medium melting point vegetable wax of a), b), c), d) and e) is rapeseed wax and the high melting point vegetable wax of a), b), c), d) and e) is carnauba wax.

In one or more exemplary embodiments, the medium melting point vegetable wax of a), b), c), d) and e) is rapeseed wax and the high melting point vegetable wax of a), b), c), d) and e) is candelilla wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is palm wax and the high melting point vegetable wax of a), b), c), d) and e) is carnauba wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is palm wax and the high melting point vegetable wax of a), b), c), d) and e) is candelilla wax.

The first coating layer and the third coating layer

Each of the first coating layer and the third coating layer comprises a plant-based food grade polymer and the role of these layers is to providing a sandwich structure surrounding the second coating layer that ensures that when the vegetable waxes disposed in the second coating layer gradually melts under increasing temperatures, the vegetable waxes will remain in place in the second coating layer, instead of leaking into the surrounding environment. Another important feature of this sandwich structure is that the vegetable wax of the second coating layer is not in direct contact with the core material, and therefore does not directly transfer heat to the core material, thereby adding to the protective effect obtained by the melting of the second coating layer as temperature increases.

The plant-based food grade polymer of the first coating layer and the third coating layer can be the same plant-based food grade polymer or different plant-based food grade polymers.

In one or more exemplary embodiments of the method as disclosed herein, the plantbased food grade polymer of the first coating layer and the third coating layer is the same plant-based food grade polymer.

In one or more exemplary embodiments of the method as disclosed herein, the plantbased food grade polymer of the first coating layer and the third coating layer are different plant-based food grade polymers.

In one or more exemplary embodiments of the method as disclosed herein, the plantbased food grade polymer of the first coating layer and the third coating layer is a blend of different plant-based food grade polymers.

In one or more exemplary embodiments, the first coating layer and the third coating layer comprises at least 70%, 75%, 80%, 85,% 90%, 95%, 98% or 99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer comprises at least 90%, 95%, 98% or 99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer comprises at least 95%, 98% or 99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer comprises at least 98% or 99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer thus comprise a concentration of between 70-99%, such as between 80- 99%, between 90-99% or 95-99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer comprise a concentration of between 80-99%, 90-99% or 95-99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer comprise a concentration of between 90-99% or 95-99% plant-based food grade polymer. In one or more exemplary embodiments, the first coating layer and the third coating layer thus comprise a concentration of between 95-99% plantbased food grade polymer. In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of cellulose, alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose in the first coating layer and third coating layer, and the second coating layer comprises a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70- 80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose and a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72- 78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of ethyl cellulose, methyl cellulose, and carboxymethyl cellulose in the first coating layer and third coating layer, and the second coating layer comprises a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises ethyl cellulose and the second coating layer comprises a vegetable wax admixture consisting essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material coated and a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises ethyl cellulose and the second coating layer comprises a vegetable wax admixture consisting essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments the micro-encapsulated microbial culture comprises a core material coated and a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprising ethyl cellulose and the second coating layer comprises a vegetable wax admixture consisting essentially 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, a non-vegetable alternative to the medium melting point vegetable waxes, is bees wax.

In one or more exemplary embodiments, the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of cellulose, alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose in the first coating layer and third coating layer, and the second coating layer comprises a wax admixture consisting essentially of 50-90% bees wax and 10-50% high melting point vegetable wax, or 65-85% bees wax and 15-35% high melting point vegetable wax, or 70-80% bees wax and 20-30% high melting point vegetable wax, or 72-78% bees wax and 22-28% high melting point vegetable wax, or 75% bees wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose in the first coating layer and third coating layer, and the second coating layer comprises a wax admixture consisting essentially of 50-90% bees wax and 10-50% carnauba wax or candelilla wax, or 65-85% bees wax and 15-35% carnauba wax or candelilla wax, or 70-80% bees wax and 20-30% carnauba wax or candelilla wax, or 72-78% bees wax and 22-28% carnauba wax or candelilla wax, or 75% bees wax and 25% carnauba wax or candelilla wax.

In one or more exemplary embodiments, the micro-encapsulated microbial culture comprises a core material encapsulated by a first coating layer, second coating layer and third coating layer as defined herein, wherein the first and third coating layers comprises a plant-based food grade polymer selected as one of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose in the first coating layer and third coating layer, and the second coating layer comprises a wax admixture consisting essentially of 50-90% bees wax and 10-50% carnauba wax, or 65-85% bees wax and 15-35% carnauba wax, or 70-80% bees wax and 20-30% carnauba wax, or 72-78% bees wax and 22-28% carnauba wax, or 75% bees wax and 25% carnauba wax.

Mass ratio (coating index/CI) of core material to coating layers

The micro-encapsulated microbial cultures as defined herein is further defined by the mass-ratio of the individual coating layer to the core material, or by the mass-ratio of an individual coating layer to the core material plus one or more coating layers.

Thus, the mass-ratio of the first coating layer to core material is in one case selected as a mass ratio of 0.3, 0.4, 0.5, 0.6 or 0.7. the mass-ratio of the first coating layer to core material is in one case selected as a mass ratio of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1. The mass ratio of the first coating layer to the core material can therefore be selected within a range of 0.2-1.

Alternatively, the mass-ratio of the first coating layer to the core material is selected as a mass ratio selected from the group consisting of 0.3, 0.4, 0.5, 0.6 and 0.7.

In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.3-0-7. In one or more exemplary embodiments, the ratio of the first coating layer to the core material is selected as 0.4-0.6. In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.5. In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.3. In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.7.

In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as a mass ratio selected from the group consisting of 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 1.5-5. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 1.5-4. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2.5-4.5. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2.5-3.5. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 3. In one or more exemplary embodiments, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 4.

In one or more exemplary embodiments, the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25- 0.75. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25-0.5. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.35. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.5. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.75. In one or more exemplary embodiments, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 1.

In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is selected within the range of 0.25-0.5. In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third layer to the core material coated in the first coating layer and the second coating layer is 0.25.

In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third coating layer to core material coated in the first coating layer and the second coating layer to the third coating layer is 0.5.

In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.4-0.6, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 2.5-3.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5.

In one or more exemplary embodiments, the mass ratio of the first coating layer to the core material is selected as 0.3-0.7, the mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer is selected as 1.5-4.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-1.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of between 0.3-0.7, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of between 1.5-4.5, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of between 0.25-1, wherein the first coating layer and the third coating layer comprises a plant-based food grade polymer and the second coating layer comprises a wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax. In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of between 0.3-0.7, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of between 1.5-4.5, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of between 0.25-1, wherein the first coating layer and the third coating layer comprises a plant-based food grade polymer and the second coating layer comprises a wax admixture consisting essentially of 50-90% rapeseed wax and 10-50% carnauba wax, or 65-85% rapeseed wax and 15-35% carnauba wax, or 70- 80% rapeseed wax wax and 20-30% carnauba wax, or 72-78% rapeseed wax and 22- 28% carnauba wax, or 75% rapeseed wax and 25% carnauba vegetable wax.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of between 0.3-0.7, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of between 2.5-3.5, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of between 0.25-1, wherein the first coating layer and the third coating layer comprises a plant-based food grade polymer and the second coating layer comprises a wax admixture consisting essentially of 50-90% rapeseed wax and 10-50% carnauba wax, or 65-85% rapeseed wax and 15-35% carnauba wax, or 70- 80% rapeseed wax wax and 20-30% carnauba wax, or 72-78% rapeseed wax and 22- 28% carnauba wax, or 75% rapeseed wax and 25% carnauba vegetable wax.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of between 0.3-0.7, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of between 1.5-4.5, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of between 0.25-1, wherein the first coating layer and the third coating layer comprises ethyl cellulose and the second coating layer comprises a wax admixture consisting essentially of 50-90% rapeseed wax and 10-50% carnauba wax, or 65-85% rapeseed wax and 15-35% carnauba wax, or 70-80% rapeseed wax wax and 20-30% carnauba wax, or 72-78% rapeseed wax and 22-28% carnauba wax, or 75% rapeseed wax and 25% carnauba vegetable wax.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of 0.5, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of 2, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of 0.25-0.5, wherein the first coating layer and the third coating layer comprises ethyl cellulose and the second coating layer comprises a wax admixture consisting essentially of 50-90% rapeseed wax and 10-50% carnauba wax, or 65-85% rapeseed wax and 15-35% carnauba wax, or 70-80% rapeseed wax wax and 20-30% carnauba wax, or 72-78% rapeseed wax and 22-28% carnauba wax, or 75% rapeseed wax and 25% carnauba vegetable wax.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of 0.5, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of 3, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of 0.25-0.5, wherein the first coating layer and the third coating layer comprises ethyl cellulose and the second coating layer comprises a wax admixture consisting essentially of 75% rapeseed wax and 25% carnauba vegetable wax.

In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of 0.5, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of 2, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of 0.25-0.5, wherein the first coating layer and the third coating layer comprises ethyl cellulose and the second coating layer comprises a wax admixture consisting essentially of 75% rapeseed wax and 25% carnauba vegetable wax. In one or more exemplary embodiments, a micro-encapsulated microbial culture as defined herein comprises a mass ratio of the first coating layer to the core material of 0.5, a mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer of 3, and a mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer of 0.25-0.5, wherein the first coating layer and the third coating layer comprises ethyl cellulose and the second coating layer comprises a wax admixture consisting essentially of 50% rapeseed wax and 50% carnauba vegetable wax.

Methods for Production of micro-encapsulated microbial cultures

The micro-encapsulated microbial cultures of the present application are produced in several steps, these steps comprise providing a core material comprising a microbial culture, and encapsulating the core material in a first coating layer a second coating layer and a third coating layer, where the first coating layer and the third coating layer comprises a plant-based food grade polymer and the second coating layer comprises an admixture of food grade vegetable wax.

Thus, in a second aspect, the present disclosure relates to a method for producing a micro-encapsulated microbial culture, the method comprising ai. providing a core material comprising a microbial culture, bi. encapsulating said core material in a first coating layer, a second coating layer, and a third coating layer, ci. Wherein the first coating layer comprises a plant-based food-grade polymer, the second coating layer comprises an admixture of vegetable wax, and the third coating layer comprises a plantbased food-grade polymer, thereby providing a micro-encapsulated microbial culture.

Encapsulating the core material in the first coating layer, the second coating layer and the third coating layer in step comprises the coating of the core material in a first coating layer, the coating of the first coating layer in the second coating layer, and the coating of the second coating layer in the third coating layer.

Thus, the encapsulation step, step bi, for producing micro-encapsulated microbial culture is achieved by performing the steps

-coating the core material in the first coating layer,

-coating the first coating layer in the second coating layer, and

-coating the second coating layer in the third coating layer.

To ensure the core material is properly encapsulated the coating process as described in step bi is performed in three successive rounds of coating, starting with the coating of the first coating layer onto the core material, followed by coating of the second coating layer onto the first coating layer and completed with the coating of the third coating layer onto the second coating layer.

This process of encapsulation ensures that the second coating layer is sandwiched between the first coating layer and the third coating layer, thereby providing structural support for the second coating layer.

As previously stated this structural support is believed to be important for the function of the encapsulation, as the structural support ensures that the second coating layer remains in place when temperatures in the environment increases and the admixture of food grade vegetable wax of the second coating layer melts, e.g. during a pasteurization process.

The coating process can be performed using several methods including spray drying, spray congealing, extrusion, emulsification, and fluidized bed spray coating.

Thus, in one or more exemplary embodiments, the encapsulation of the core material is performed using a method selected from spray drying, spray congealing, extrusion, emulsification, and fluidized bed spray coating.

A desirable process is fluidized bed spray coating as this is a very convenient and easy way of performing the coating process and thereby providing the micro-encapsulated microbial culture of the present disclosure. This method is desirable because it is a dry method that leaves no need for specialized recovery steps or downstream purification of the product following completion of the coating procedure.

In one or more exemplary embodiments, the encapsulation of the core material in step bi is performed using fluidized bed spray coating.

However, even though it is not strictly necessary to include recovery or downstream purification following fluidized bed spray coating, there can still be cases where it will be advantageous to employ such method steps anyway. This is also the case for other coating methods than fluidized bed spray coating.

Therefore, the method for producing a microencapsulated microbial culture as disclosed herein may further comprise a recovery step, following completion of the third round of coating in the encapsulation step b, the recovery step comprising recovering the microencapsulated microbial culture from the coating process mixture.

In one or more exemplary embodiment of the present disclosure, the encapsulation step, step bi, further comprises a recovery step for recovering the microencapsulated microbial culture after applying the third coating layer, thus comprising the steps

-coating the core material in the first coating layer,

-coating the first coating layer in the second coating layer,

-coating the second coating layer in the third coating layer, and

-recovering the microencapsulated microbial culture following application of the third coating layer in the third coating round.

It is further contemplated that the method for producing microencapsulated microbial culture, may comprise a recovery step following each of the three successive rounds of coating, the recovery steps comprising recovering a coated product following each of the three successive rounds of coating rounds, wherein the first coated product that is recovered following the first coating round is the core material coated in a first coating layer, the second coated product that is recovered following the second coating round is the core material coated in a first coating layer and a second coating layer, and finally, the third coated product recovered following the third round of coating is the microencapsulated product as defined herein. In one or more exemplary embodiments, the encapsulation step, step bi, further comprises a recovery step after each of the three successive coating rounds, thus comprising the steps

-coating the core material in the first coating layer,

-recovering the core material coated in the first coating layer, thereby obtaining a first recovered product,

-coating the first recovered product in a second coating layer,

-recovering the first coated product coated in the second coating layer, thereby obtaining a second recovered product,

-coating the second recovered product in the third coating layer, and

-recovering a second recovered product coated in the third coating layer, thereby obtaining the microencapsulated microbial culture.

It is envisioned that the microencapsulated microbial culture can be recovered using any standard method for recovery of coated materials, such recovery methods comprising, but not being limited to size exclusion, size separation, centrifugation, and/or filtration-based methods.

Following a recovery step it is further contemplated that a purification step, a washing step or both a purification step may be performed. However, in particular after the third coating round, a purification step, a washing step, or both a purification step and a washing step is contemplated.

In one or more exemplary embodiments of the present disclosure, the methods for producing a micro encapsulated microbial culture as disclosed herein further comprises a purification step and/or a washing step immediately after recovery step following the third coating round of the encapsulation step.

In one or more exemplary embodiments of the present disclosure, the methods for producing a micro encapsulated microbial culture as disclosed herein further comprises a purification step and/or a washing step immediately after each of the recovery steps following the three successive coating rounds of the encapsulation step.

Producing the second coating layer

It is particularly important in the methods disclosed herein that the second coating layer is applied and that a suitable vegetable wax admixture is used, so that the second coating layer can properly absorb heat from the environment by undergoing a phase transition from solid to liquid and thereby improve the survivability of the microencapsulated microbial culture.

To achieve this objective (as was demonstrated by the different vegetable admixtures used in example 2), a suitable vegetable admixture to be used in the second coating layer in the methods disclosed herein is a vegetable admixture consisting essentially of medium melting point vegetable wax and high melting point vegetable wax.

Vegetable wax admixtures that are suitable for use in the methods herein comprise vegetable wax admixtures consisting essentially of vegetable waxes as defined in a)- e) a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, and e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22- 28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax. In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax.

In one or more exemplary embodiments, the vegetable wax admixture of the second coating layer used in the methods disclosed herein consists essentially of 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

The medium melting point vegetable waxes that are suitable for use in the methods disclosed herein include, but are not limited to palm wax and rapeseed wax. The high melting point vegetable waxes that are suitable for use in the methods disclosed herein include, but are not limited to candelilla wax and carnauba wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax is selected as one of palm wax and rapeseed wax, and the high melting point vegetable wax is selected as one of candelilla wax and carnauba wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is carnauba wax, or, the medium melting point vegetable wax is palm wax and the high melting point vegetable wax is carnauba wax, or, the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is candelilla wax, or, the medium melting point vegetable wax is palm wax and the high melting point vegetable wax is candelilla wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is selected as one of palm wax and rapeseed wax, and the high melting point vegetable wax of a), b), c), d) and e) is selected as one of candelilla wax and carnauba wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is rapeseed wax and the high melting point vegetable wax of a), b), c), d) and e) is carnauba wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is rapeseed wax and the high melting point vegetable wax of a), b), c), d) and e) is candelilla wax.

In one or more exemplary embodiments of the methods disclosed herein, the medium melting point vegetable wax of a), b), c), d) and e) is palm wax and the high melting point vegetable wax of a), b), c), d) and e) is candelilla wax. Producing the first coating layer and the third coating layer

In one or more exemplary embodiments of the methods disclosed herein, the first coating layer and the third coating layer is obtained by applying a plant-based food grade polymer, thereby providing a sandwich structure surrounding the second coating layer and ensuring that when the vegetable waxes disposed in the second coating layer gradually melts under increasing temperatures, the vegetable waxes will remain in place in the second coating layer, instead of leaking into the surrounding environment. Another important feature of this sandwich structure is that the vegetable wax of the second coating layer is not in direct contact with the core material, and therefore does not directly transfer heat to the core material, thereby adding to the protective effect obtained by the melting of the second coating layer as temperature increases.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% plant-based food grade polymer, when applied in the methods disclosed herein.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises at least 90%, 95%, 98%, or 99% plantbased food polymer, when applied in the methods disclosed herein.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises at least 95%, 98%, or 99% plant-based food polymer, when applied in the methods disclosed herein.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises between 70-99%, such as between 80-99%, between 90-99% or 95-99% plant-based food grade polymer.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises between 90-99% plant-based food grade polymer.

In one or more exemplary embodiments, the first coating layer and the third coating layer is obtained by applying a composition having a dry weight material content of plant-based food grade polymer that results in the deposition of a first coating layer and/or third coating layer that comprises between 95-99% plant-based food grade polymer.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of a plant-based food grade polymer selected from the list consisting of cellulose, alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax. In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of a plant-based food grade polymer selected from the list consisting of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22- 28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of a plant-based food grade polymer selected from the list consisting of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22- 28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of ethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of ethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax, as the second coating layer.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of ethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax as the second coating layer.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to use of ethyl cellulose in the first coating layer and third coating layer, and use of a vegetable wax admixture consisting essentially of 75% medium melting point vegetable wax and 25% high melting point vegetable wax as the second coating layer.

In one or more exemplary embodiments, the methods for producing micro- encapsulated microbial culture as disclosed herein relate to the use of a non-vegetable alternative in place of the medium melting point vegetable waxes, wherein the nonvegetable wax is bees wax.

To achieve a particular coating index (Cl/mass ratio) of the three coating layers relative to each other and relative to the core material, the amount of materials introduced in each of the respective coating steps of the methods as disclosed herein can be varied to obtain a microencapsulated microbial culture with a specific coating index (Cl/mass ratio).

Mass ratio (coating index/CI) of core material to coating layers

The micro-encapsulated microbial cultures produced by the methods disclosed herein are further defined by the mass-ratio of the core material to an individual coating layer, or by the mass-ratio of the core material plus one or more coating layers to an individual coating layer. In short, in order to control the make-up and thickness of the microencapsulated microbial culture as produced herein, the amount of material used in each of the coating steps can be varied.

For example in the methods disclosed herein, the mass-ratio of the first coating layer to core material is selected as a mass ratio of 0.3, 0.4, 0.5, 0.6 or 0.7. Alternatively, the mass-ratio of the first coating layer to core material is selected as a mass ratio of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1. Therefore, the mass ratio of the first coating layer to the core material can be selected within a range of 0.2-1.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.3-0-7. In one or more exemplary embodiments of the methods disclosed herein, the ratio of the first coating layer to the core material is selected as 0.4-0.6. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.5. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.3. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.7.

In one or more exemplary embodiments of the methods disclosed herein, the massratio of the second layer to the core material coated in the first coating layer is selected as a mass ratio selected from the group consisting of 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5.

In one or more exemplary embodiments of the methods disclosed herein, the massratio of the second layer to the core material coated in the first coating layer is selected as 1.5-5. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 1.5-4. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2.5-4.5. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2.5-3.5. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 2. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 3. In one or more exemplary embodiments of the methods disclosed herein, the mass-ratio of the second layer to the core material coated in the first coating layer is selected as 4.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25-0.75. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25-0.5. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.25. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.35. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.5. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 0.75. In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the core material coated in the first coating layer and the second coating layer to the third coating layer is 1.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is selected within the range of 0.25-0.5.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third layer to the core material coated in the first coating layer and the second coating layer is 0.25.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.5, the mass ratio of the second layer to the core material coated in the first coating layer is selected as 3, and the mass ratio of the third coating layer to core material coated in the first coating layer and the second coating layer to the third coating layer is 0.5.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.4-0.6, the mass ratio of the second coating layer to the core material coated in the first coating layer is selected as 2.5-3.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5.

In one or more exemplary embodiments of the methods disclosed herein, the mass ratio of the first coating layer to the core material is selected as 0.3-0.7, the mass ratio of the second coating layer to the core material coated in the first coating layer to the second layer is selected as 1.5-4.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-1.

The provision of microencapsulated microbial culture with a particular coating index as defined herein will amount to only routine work for the person skilled in the art upon the instructions provided herein.

Exemplary modes of fluidized bed spray coating

A fluid bed spray coater was used for micro-encapsulating the core material. Nitrogen gas was used as fluidizing and atomizing gas. The powders of core material were fluidized at fluidizing velocity of 1-1.5 m/s and a temperature of 35-50 °C followed by spraying the coating material at given spraying rate. The pressure of atomizing gas was sat to 0.8 bar (P_g). Also, in the case of spraying wax, the temperature of atomizing gas was set to 110 °C to avoid solidification of wax in the line or the nozzle sets. The temperature of the fluidized bed, and pressure of atomizing gas, and spraying rate depends on the coating material and are considered as key parameters in determining the morphology of the coating and eventually the performance of the coating. The layers were deposited and formed a solid protective layer through a rapid solidification or drying step depending on the precursor. Each layer was deposited in one single coating batch and therefore a triple layer microencapsulated prototype was produced in three successive separate coating batches.

The application of the three coating layers onto the core material was performed in three successive coating batches, starting with the first coating layer, followed by the second coating layer and completed with the third coating layer. The separation of the coating process into three successive coating batches ensured that the first coating layer and the third coating layer consist of a food grade plant-based biopolymer, and the second coating layer comprises a food grade admixture of vegetable wax, wherein the second coating layer is sandwiched between the first coating layer and the third coating layer.

In one or more exemplary embodiments of the present disclosure, the core material comprising a microbial culture is coated in a first layer consisting of ethyl cellulose, a second layer composed of 75 rapeseed wax and 25% carnauba wax and a third layer consisting of ethyl cellulose as described herein. The mass ratio (CI) of the first, second and third coating layers is a mass ratio (CI) of 0.5:3:0.25 and the coating process is performed by fluidized bed spray coating of the first, second and third coating layers onto the core material.

To microencapsulate 50 g core material in a microcapsule consisting of a first layer of ethyle cellulose, a second layer consisting of 75% rapeseed wax and 25% carnauba wax, and a third layer of ethyl cellulose, three successive coating steps can be performed using fluidized bed spray coating. For the first coating step, 50 grams of core material (e.g. freeze dried powder comprising Bifidobacterium animalis subsp. Lactis) is coated with 100 grams 25% ethyl cellulose aqueous solution in a fluid bed spray coater with maximum product volume of 0.5 L. To apply the 25% ethyl cellulose aqueous solution, the core material is fluidized using a dried inert gas at fluidizing velocity of 0.5-1 m/s and fluidizing gas temperature of 35-50 C (preferably 45 C). When the bed temperature stabilizes, the 25% ethyl cellulose aqueous solution is sprayed in the fluidizing chamber via a two-fluid nozzle. A dried inert gas helps to atomize the coating material at atomizing pressure of 0.5-1 bar (preferably 0.7 bar) and spraying rate of 0.5-4 g/min (preferably 2 g/min). When all the coating material is sprayed, the fluidization and atomization are stopped, and materials is retrieved from the fluidized bed chamber. For the second step of coating, 50 grams of retrieved powder from the first step is coated with 150 grams of molten vegetable wax admixture consisting of 75% rapeseed wax and 25% carnauba wax. To this end, the retrieved powder from the first step is fluidized at fluidizing velocity of 1-2 m/s and fluidizing gas temperature of < 50 C (preferably at ambient temperature). Then, the molten wax admixture is sprayed in the fluidized bed chamber at atomizing pressure of 0.5-1 bar (preferably 0.7 bar) and spraying rate of 5-30 g/min (preferably 15 g/min). The minimum temperature of the molten wax at injection point is required to be higher than the solidification point of the molten wax blend to ensure no solidification occurs in the line. For this purpose, the molten wax admixture is sprayed at minimum temperature of 90 C (this temperature depends on the type of waxes), and the atomizing gas temperature at the minimum temperature of 110 C. When all the coating material is sprayed, the fluidization and atomization are stopped, and materials is retrieved from the fluidized bed chamber.

For depositing the third layer, 50 grams of retrieved powder from the second step is coated with 50 grams 25% ethyl cellulose aqueous solution in the same fluid bed spray coater system. The coating material is applied under the same condition as the first step. The retrieved coated powder from the third step is considered as final ready-to-be-used microencapsulated microbial culture product without needing any further treatment.

After the first and third step, a post-drying step is preferable to guarantee of having a dried final product. Post-drying is performed by extending the fluidization time after completing the spraying until the water activity of the coated powder reaches < 0.10 (preferably < 0.05) at ambient temperature. Post-drying can be done at the same fluidizing velocity and fluidizing temperature of 40 C for extended times.

The same process can be used for coating a core material with three coating layers in a mass ratio of 0.5:3:0.5 instead of 0.5:3:0.25 by adjusting the relative amount of the component used in the third coating layer according to the change in mass ratio. Similarly, changes in the mass ratio of any of the other components may be adjusted in the same way. General

Microencapsulation

In the present context, the term "microencapsulated" refers to an entity, which on a micrometric scale is secluded from the surrounding environment. Thus, a microencapsulated microbial culture is a microbial culture which is compartmentalized into a distinct entity and thereby separated from the medium and/or surrounding environment into which such entities are dispersed. In the context of the present disclosure a microencapsulated core material is achieved by coating of a core material in three coating layers as described by the methods as described herein. The terms micro-encapsulated and microencapsulated are used interchangeably herein.

Core material

In the present context, the term "core material" refers to a preparation comprising a microbial culture.

In particular, such preparations of a microbial culture refer to a powder or granulate comprising a microbial culture, but is not considered to be limited to this type of preparation. Any type of preparation that provides a microbial culture that can be micro-encapsulated in a coating process as described herein, is considered as a suitable preparation of a microbial culture to be used as a core material.

To provide powdered or granulated core material, a microbial culture can be prepared as either a powder or a granulate by pelletization of the followed by grinding.

In order to provide a freeze-dried core material in a powder form, the core material is pelletized in liquid nitrogen and ground using a grinder, such as pilot-scale grinder to provide a micron sized powder.

Therefore, in one or more exemplary embodiments, the core material is a micron sized powder produced from a mixture of a liquid cell concentrate of a microbial culture and a plant-based protein cryoprotectant.

However, as the viability of the of microbial culture can be affected by freezing, freeze drying and extended periods of storage even at elevated temperatures, in some cases it is advantageous to also add a plant-based food grade cryoprotectant or plant-based food grade stabilizer to the core material. In one or more exemplary embodiments, the core material is a micron sized powder produced by formulation of a liquid cell fermentate together with a plant-based cryoprotectant, followed by pelletization in liquid nitrogen and grinding of the frozen pellet.

A suitable plant-based protein cryoprotectant is for example a cryoprotectant that is mainly composed of one or more plant proteins, one or more dextrins, and/or one or more plant derived gums.

For example, such a cryoprotectant composition is mainly composed of isolated pea and/or potato protein together with maltodextrin and gum arabic.

In one or more exemplary embodiments, a suitable plant-based protein cryoprotectant is mainly composed of one or more plant proteins, one or more dextrins, and/or one or more plant derived gums.

In one or more exemplary embodiments, a suitable plant-based protein cryoprotectant is mainly composed of isolated pea and/or potato protein together with maltodextrin and gum arabic.

In one or more exemplary embodiments, the core material is a micron sized powder produced by pelletization in liquid nitrogen followed by grinding of a mixture of a liquid cell fermentate of a microbial culture and a plant-based protein cryoprotectant composed of isolated pea protein, maltodextrin and gum arabic.

In one or more exemplary embodiments, the core material is a micron sized powder produced by pelletization in liquid nitrogen and grinding of a mixture of a liquid cell concentrate of a microbial culture and a plant-based protein cryoprotectant composed of isolated potato protein, maltodextrin and gum arabic.

In one or more exemplary embodiments, the core material comprises trehalose when the microencapsulated microbial culture is intended for use in a non-vegan food, feed, beverage, composition or product. In one or more exemplary embodiments, A desirable plant-based protein cryoprotectant for use in non-vegan products is mainly composed of isolated pea protein, maltodextrin, gum arabic, and trehalose.

In one or more exemplary embodiments, a core material intended for use with non- vegan products is a micron sized powder produced by pelletization in liquid nitrogen and grinding of a mixture of a liquid cell concentrate of a microbial culture and a plant-based protein cryoprotectant composed of isolated pea protein, maltodextrin, gum arabic, and trehalose. Microbial culture

In the present context, the term "microbial culture" refers to one or more microorganism(s) that is/are cultivated in a industrial fermentation process and recovered from the fermentation medium at the end of the industrial fermentation process. Generally speaking a microbial culture can for example be recovered, isolated or separated from an industrial fermentation process by way of centrifugation, filtration or size separation methods.

Desirable microbial cultures include for example probiotic microorganisms, Lactic acid bacteria (LAB) and microorganisms belonging to a genus selected from Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Propionibacterium, Brevibacterium, Staphylococcus, Bacillus and Saccharomyces.

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)).

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 3 presents a list of new and old names of some Lactobacillus species relevant to the present invention.

Table 3. New and old names of some Lactobacillus species relevant to the present invention

Bacteria of the Lactobacillus genus, as well as the related newly updated genera, have for a long time been known to constitute a significant component of the microbiota in the human body, such as in the digestive system, urinary system and genital system. For this reason, these bacteria have been heavily utilized in in health and/or nutritional products aimed at aiding, maintaining or restoring the natural balance of microbiota in the human body. Examples of application of Lactobacillus include treatment or amelioration of diarrhea, vaginal infections, and skin disorders such as eczema.

In one or more exemplary embodiments, the microbial culture comprises a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus.

In one or more exemplary embodiments, the microbial culture is a lactic acid bacteria (LAB) of a genus selected from the group consisting of Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lacticaseibacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Lentilactobacillus, Latilactobacillus, Companilactobacillus, Latilactobacillus and Lactiplantibacillus. In one or more exemplary embodiments, the microbial culture as described herein comprises or comprises a species of Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp., Lactiplantibacillus pentosus, Lactobacillus acidophillus, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus delbrueckii.

In one or more exemplary embodiments, the microbial culture as described herein is a species of Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Ligilactobacillus salivarius, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum subsp. plantarum, Limosilactobacillus fermentum, Ligilactobacillus animalis, Lentilactobacillus buchneri, Latilactobacillus curvatus, Companilactobacillus futsaii, Latilactobacillus sakei subsp., Lactiplantibacillus pentosus, Lactobacillus acidophillus, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus delbrueckii.

In one or more exemplary embodiments, the microbial culture as described herein comprises a species of lactic acid bacteria (LAB) of a genus selected from the group consisting of 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, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

In one or more exemplary embodiments, the microbial culture as described herein is a species of lactic acid bacteria (LAB) of a genus selected from the group consisting of 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, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus. In one or more exemplary embodiments, the microbial culture as described herein is a lactic acid bacteria (LAB) of a genus selected from Lactobacillus and Bifidobacterium.

In one or more exemplary embodiments, the microbial culture as described herein is a lactic acid bacteria (LAB) of the genus Bifidobacterium.

In one or more exemplary embodiments, the microbial culture as described herein is of the species Bifidobacterium animalis.

In one or more exemplary embodiments, the microbial culture as described herein is of the subspecies Bifidobacterium animalis subsp. Lactis.

In one or more exemplary embodiments, the microbial culture as described herein is of the subspecies Bifidobacterium animalis subsp. Lactis. strain no. DSM 15954.

In one or more exemplary embodiments, the microbial culture as described herein is of the species is a lactic acid bacteria (LAB) of the genus Lactobacillus.

Microbial cultures as referred to herein does not include unwanted microorganisms that contribute to spoilage or risk of disease.

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.

In one or more exemplary embodiments of the present disclosure relates to the microencapsulated microbial culture as described herein, wherein the microbial culture is a probiotic culture.

Another embodiment of the present disclosure relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is of a genus selected from the group consisting of Lactobacillus or Bifidobacterium.

Another embodiment of the present disclosure relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is of the Bifidobacterium genus. Yet another embodiment of the present disclosure relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is selected from the group consisting of Lacticaseibacillus rhamnosus, Ligilactobacillus animalis and Bifidobacterium animalis subsp. Lactis.

Another embodiment of the present disclosure relates to the microencapsulated microbial culture as described herein, wherein the probiotic culture is the Bifidobacterium subspecies Bifidobacterium animalis subsp. lactis.

In one or more exemplary embodiments a probiotic microorganism is a LAB.

Products comprising probiotic cultures include dairy products, animal feed and beverages. Thus, it is to be understood that the microencapsulated microbial cultures described herein may be administered to humans, animals and even plants. Additionally, it is to be understood that the microencapsulated microbial cultures described herein may be consumed by not only humans but also animals.

The microencapsulated microbial culture may be utilized for many different types of applications spanning from e.g. health products, nutritional supplement, and animal feeds to pharmaceuticals. Thus, a composition encompassing the microencapsulated microbial culture may in some cases comprise additives.

Therefore, 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, feed-grade ingredients, pharmaceutical ingredients and excipients.

Plant-based food grade polymers

The term plant-based food grade polymers (which is used interchangeably with food grade plant-based polymers herein) as used in the present disclosure relates to polymers that are sourced directly from plant sources (e.g. cellulose) and which are suitable for use in food, feed and beverages. The term plant-based food grade polymers is also intended to include chemically modified polymers of polymers that are sourced from plants (e.g. ethyl cellulose), as long as the chemically modified polymers are suitable for use in food, feed and beverages and does not contain undesirable chemicals from the modification process that make them unsuitable for use as ingredients in food, feed and beverages.

Plant-based food grade polymers that are considered suitable for use herein include the naturally occurring polymers cellulose and alginate and polymers based on these polymers. Plant-based food grade polymers based on these biopolymers may be selected from polymers comprising ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose and other plant-based polymers that are suitable for use in foods, feed and beverages.

In one or more exemplary embodiments, the plant-based food grade polymer is a fibrous material sourced from a plant that is suitable for use in food, feed and beverages.

In one or more exemplary embodiments, the plant-based food grade polymer is selected as one of cellulose, alginate, calcium alginate, sodium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose. In one or more exemplary embodiments, the plant-based food grade polymer is selected as one of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate, and carboxymethyl cellulose.

In one or more exemplary embodiments, the plant-based food grade polymer is selected as one of ethyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose. In one or more exemplary embodiments, the plant-based food grade polymer is selected as one of ethyl cellulose and hydroxyethyl cellulose.

In one or more exemplary embodiments, the plant-based food grade polymer is ethyl cellulose.

Food grade vegetable wax

A Food grade vegetable wax (also referred to as vegetable wax herein) within the context of the present disclosure is a wax or oil that is isolated from a plant source, and which is suitable for use in a food product.

High melting point vegetable wax

A high melting point wax in the context of the present disclosure is a vegetable wax or vegetable oil that has a melting point of above 65 °C. Examples of such vegetable waxes include candelilla wax and carnauba wax, but there is no particular limitation regarding which vegetable wax or vegetable oil is used as long as the vegetable wax or vegetable oil is suitable for use as a food ingredient and has a melting temperature of above 65 °C.

In one or more exemplary embodiments, the high melting point vegetable wax is selected from carnauba wax and candelilla wax. In one or more exemplary embodiments, the high melting point vegetable wax is candelilla wax. In one or more exemplary embodiments, the high melting point vegetable wax is carnauba wax.

Medium melting point vegetable wax

A medium melting point wax in the context of the present disclosure is a vegetable wax or vegetable oil that has a melting point of 55-65 °C. Examples of such waxes include palm wax and rapeseed wax, but is not particularly limited to these examples as long as the vegetable wax or vegetable oil is suitable for use as a food ingredient and has a melting temperature of 55-65 °C.

Within the present disclosure, the term wax is considered as being synonymous with oil, for example in the case of rapeseed, hydrogenated rapeseed oil is considered to be equivalent to rapeseed wax.

In one or more exemplary embodiments, the medium melting point vegetable wax is selected from palm and rapeseed wax. In one or more exemplary embodiments, the medium melting point vegetable wax is palm wax. In one or more exemplary embodiments, the medium melting point vegetable wax is rapeseed wax.

In one or more exemplary embodiments, a non-vegetable food grade wax alternative with a medium melting point is bees wax. In one or more exemplary embodiments, bees wax is used as a non-vegetable medium melting point wax in food, feed, beverages, compositions and products that are not intended for vegan consumption or use.

Foods

While it is envisioned that the microencapsulated microbial culture as defined herein can be useful in any food that comprise a microbial additive, the microencapsulated microbial culture is particularly well suited for use in foods that have high water content, high acidity and high water activity, and which are exposed to pasteurization processes in order to preserve and conserve the food for later use.

The practice of pasteurization is used extensively within the food industry, but remains particularly relevant to the beverage and dairy industries. Accordingly, the microencapsulated microbial cultures as disclosed herein are particularly useful in the preparation of beverages and dairy products, as they can survive the pasteurization process and therefore be added to the product in question prior to pasteurization, increasing the preservation and stability of the product, even while ensuring the product contains a suitable dose of viable microorganisms after pasteurization in the form of the microencapsulated microbial culture. In one or more exemplary embodiments, the dairy product is one of a yoghurt, milk, cream, cheese or cream-cheese. In one or more exemplary embodiments, the dairy product is one of a yoghurt, milk, or cheese. In one or more exemplary embodiments, the dairy product is a yoghurt. In one or more exemplary embodiments, the dairy product is a milk. In one or more exemplary embodiments, the dairy product is a cheese. In one or more exemplary embodiments, the dairy product is cream. In one or more exemplary embodiments, the dairy product is a cheese. In one or more exemplary embodiments, the food product is a vegurt.

In one or more exemplary embodiments, the beverage is an alcoholic beverage or a non-alcoholic beverage. In one or more exemplary embodiments, the beverage is a juice, mocktail or a tea. In one or more exemplary embodiments, the beverage is a juice. In one or more exemplary embodiments, the beverage is a mocktail. In one or more embodiments, the beverage is orange juice. In one or more embodiments, the beverage is a tea. In one or more embodiments, the beverage is orange juice. In one or more exemplary embodiments, the beverage is a beer or a cocktail. In one or more embodiments, the beverage is a beer. In one or more embodiments, the beverage is a cocktail.

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.

An exemplary embodiment of the present disclosure relates to the composition, product or dairy product as described herein, wherein the one or more food-grade ingredients are selected from the group consisting of compounds that can alter attributes such as aroma, flavour, acidity, colour, viscosity and texture, as well as preservatives, nutrients, thickeners, sweeteners, emulsifiers, and combinations thereof.

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, casein, corn starch, dietary fibres, gums and gelatine. Feed and feed ingredients

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 comprises 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.

Decapsulation of a microencapsulated microbial culture

In order to accurately estimate the viability of the microencapsulated microbial cultures in a standard plate counting assay, it was necessary to first decapsulate the microbial culture.

This decapsulation is performed by adjusting the pH to 6.0-6.5 and subjecting the microencapsulated core material to either or soft mechanical treatment. High-shear mechanical force can be applied for example using a high-shear homogenizer at minimum speed of 11000 rpm. Soft mechanical treatment can be applied for example using a stomacher at minimum normal speed.

The pH of 6.0 - 6.5 for the decapsulation step is selected because it is considered the optimum pH range for survival of Bifidobacterium animalis subsp. lactis. In cases of microencapsulation of other species, the decapsulation pH can be adjusted accordingly by the skilled person, without the exercise of inventive skills and merely as a routine task.

Survival rate

In the present context, the term "survival rate" refers to living cells in a product comprising a culture post pasteurization. Thus, the survival rate of a cell culture may be determined by measuring the number of colony forming units (CFU) in a product that has been pasteurized. CFU refers to the number of individual colonies of any microbe that grow on a plate of media. This value in turn represents the number of bacteria or fungi capable of replicating as they have formed colonies on the plate.

Viable cell counts are determined in freeze-dried sampled immediately after freeze- drying and at selected time points during the stability studies. A standard pour-plating method is used. In brief, a known amount of sample is homogenized with a specific volume of diluent (1: 100), using a stomacher, the solution is then resuspended by using a vortex mixer and is then subjected to decimal dilutions in peptone saline diluent (also referred to as 'maximum recovery diluent (MRD)'). MRD comprises peptone, NaCI and demineralised water. Dilutions are poured on the plates, mixed with MRS Agar (Hi-media, M641) and incubated anaerobically for three days at 37 °C. After incubation, colonies are counted manually. The result is reported as average CFU/g freeze-dried sample, calculated from the duplicates.

Mass ratio/coating index(CI)

Formulation of coating layers according to a mass ratio (Cl) of 0.5:3:0.25

This section describes how mass ratio should be understood in connection with the heat-resistant hydrophobic microencapsulation that is produced by application of coating layers using e.g. ethyl cellulose and an admixture of vegetable fat/wax consisting of a hydrogenated rapeseed oil and carnauba wax. The coating is generally applied using a fluid bed spray coater in three separate batches, such that each batch deposits a layer of coating material on the surface of particles with given thickness of the layer.

The thickness of each coating layer is represented by mass ratio which can also be termed as coating index (CI), which, for the first coating layer is defined as the mass ratio of the coating formulation to the core material. For example, if 100 g of powder is coated with 100 g of a 25% w/w suspension of ethyl cellulose (25 g ethyl cellulose), it corresponds to a Cl/mass ratio of 0.25. The calculation of CI for coating layer 2 is calculated in relation to the weight of the first coating layer plus the weight of the core material and the CI for coating layer 3 is calculated as in relation to the weight of the core material plus the weight of the first coating layer plus the weight of the second coating layer.

Thus, a microencapsulated microbial culture comprises a core material and three coating layers, wherein the three coating layers have the following mass ratios/coating indexes (CI): First coating layer: Ethyl cellulose with CI = 0.5

Second coating layer: Admixture of carnauba wax and hydrogenated rapeseed oil with ratio of 1:3, respectively, with CI = 3

Third coating layer: Ethyl cellulose with CI = 0.25

The encapsulated particle described above is written as having a mass ratio/CI of 0.5:3:0.25. For each mass ratio (Cl/coating index) value (0.5, 3 and 0.25), the CI value represents the mass ratio of a coating layer to the material to be coated. In the case of the first CI value, the mass ratio (in dry weight) of 0.5 indicates that half as much dry weight of the first coating layer compared to the dry weight of the core material, is applied as the first coating layer. The second CI value indicates that 3 times as much dry weight of the second coating layer compared to the dry weight of the core material coated in the first coating layer, is applied as the second coating layer. The third CI value indicates that 0.25 times as little dry weight of the third coating layer compared to the dry weight of the core material coated in the first coating layer and second coating layer, is applied as the third coating layer.

Thus, for example, when the core material weighs 100 g, the ethyl cellulose of the first coating layer weighs 50 g (mass ratio/CI of 1:0.5 relative to the weight of the core particle, i.e. 0.5 times the weight of the core particle, 0.5 x 100 g = 50 g).

The second coating layer has a weight ratio/CI of 1 :3, i.e., 3 times the weight of the core material plus the weight of the first coating layer, 3x(100 g + 50 g) = 450 g.

The third coating layer has a weight ratio/CI of 1 :0.25. i.e., 0.25 times the weight of the core material plus the weight of the first coating layer plus the weight of the second coating layer, i.e., 0.25x(100 g + 50 g + 450 g) = 150 g ethyl cellulose.

Any Coating index (CI) value, mass ratio or weight ratio mentioned herein relates only to dry weight of the materials that make up the core material and coating layers of the microencapsulated microbial culture as defined and produced herein.

Comprising

The term "comprising" as used herein when understood in the broadest possible meaning generally refers to a concept or item containing the specific component and is not particularly limited in regards to what other components may be present. However, the term comprising is also considered to include in one or more exemplary embodiments the terms "essentially consisting" and "consisting", referring in these cases to a concept or item consisting essentially of the specific component or a concept or item consisting of the specific component.

In relation to compositions, the term "essentially consisting" is to be understood as not having any other major component(s) other than what has been specified. This means that impurities or low concentrations of by-products, e.g. from the manufacturing process, may be present.

Compositions

The present disclosure also relates to compositions comprising the microencapsulated microbial cultures as defined herein or produced as defined herein and their use in a food, feed, beverage or other product. Such compositions include for example probitic compositions and pharmaceutical compositions.

In the present context a probiotic composition is to be understood as a composition that comprises microencapsulated microbial culture(s), wherein the microencapsulated microbial culture(s) is probiotic microbial culture(s).

In one or more exemplary embodiments, the present disclosure further relates to products comprising a composition, a probiotic composition and/or a pharmaceutical composition as defined herein.

Pharmaceutical compositions

In one or more exemplary embodiments, the present disclosure relates to a pharmaceutical composition.

In the context of the present disclosure, a pharmaceutical composition is a composition that comprises the microencapsulated microbial culture as disclosed herein or produced by the methods disclosed herein, formulated together with one or more pharmaceutical ingredients and/or excipients.

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. Thus, in one or more embodiments, the present disclosure relates to a pharmaceutical 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. 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.

In one or more exemplary embodiments, the present disclosure relates to the pharmaceutical composition as described herein, wherein the excipients are selected from the group consisting of microcrystalline cellulose, titanium dioxide and aluminium silicate.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Fig. 1 shows a comparison of the survival rate of microencapsulated microbial cultures, for different thickness and composition of the second coating layer (wax layer). The sample Pl is a microencapsulated microbial culture wherein the second coating layer has mass ratio (CI) of 2, and the composition of the second coating layer is a 1 : 1, the sample P2 is a microencapsulated microbial culture wherein the second coating layer has mass ratio (CI) of 3, and the composition of the second coating layer is a 1 : 1, whereas the sample P3 is a microencapsulated microbial culture wherein the second coating layer has mass ratio (CI) of 3, and the composition of the second coating layer is a 1:3 ratio of carnauba wax and hydrogenated rape seed oil. The left bar for each sample signifies survival rate prior to a pasteurization process and the right bar with a percentage above signifies the survival rate for each sample post pasteurization.

Figure 2

Fig. 2 shows two different pasteurization profiles, profile A and profile B, where profile A maintains a temperature of 74 °C for 18 seconds and profile B maintains a temperature of 85 °C for seconds.

Figure 3

Fig. 3 shows the survival rate of microencapsulated Bifidobacterium produced according to example 3 in different yoghurts and vegurt prior to (left bar) and after (right bar) pasteurization and a control sample. The survival rate after pasteurization is indicated in percent above the relevant bar.

Figure 4

Fig. 4 shows the survival rate of microencapsulated Bifidobacterium produced according to example 3 in orange juice prior to (left bar) and after (right bar) pasteurization and a control sample. The survival rate after pasteurization is indicated in percent above the relevant bar. The microorganism Bifidobacterium animalis subsp. lactis has been deposited under the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), located at InhoffenstraBe 7B, 38124 Braunschweig, Germany. The deposit was made on 30 September 2003 and is assigned the accession number DSM 15954.

Bifidobacterium animalis subsp. lactis strain no. DSM 15954 was used in the following examples.

EXAMPLES

Example 1 - Preparation of a freeze-dried core material

Bifidobacterium animalis subsp. lactis were formulated with a cryo-additive consisting of a plant-based protein, sugar, polysaccharides, and an antioxidant and pelletized in liquid nitrogen. The frozen pellets were then dried in a freeze dryer and subjected to a grinding step to produce a freeze dried Bifidobacterium powder. This freeze dried powder was subsequently used as core material for coating steps.

The cell count of the freeze dried powder was measured to be 4.98E+11 CFU/g powder with a very low water activity (aw) of 0.01, representing an efficient drying without formation of lumps or meringues. The freeze-dried powder was kept in freezer at -55 °C until further use as a core material.

The freeze dried powder may be sieved before coating to improve the fluidizability, which may impact the coating quality and uniformity. The size fraction of powder between 200 and 500 um is considered to have the best fluidizability.

Example 2: Effect of wax layer composition and wax layer mass ratio (CI) on the survival rate of microencapsulated microbial cultures during pasteurization

To understand the effect of the composition and the thickness of the wax mixture, three prototypes were prepared with two wax blend ratios and different thickness of wax mixture of the second coating layer, but similar thickness of ethyl cellulose in the other layers, i.e., the first coating layer and the third coating layer (Table 1).

Table 1: Variations in wax layer thickness and composition

The prototypes were dispersed in yogurt and heat tested using heat profile A. The CFU count before and after pasteurization was plotted in Fig 1. The percentage values on the bars indicates the survival rate of cells. Comparing Pl and P2 showed that the thicker coating (coating with higher CI) has significant effect on the survival rate of Bifidobacterium after pasteurization, increasing the survival rate from 8% to 27%. Additionally, comparing P2 and P3 revealed that changing the composition of the wax layer from a 1 : 1 (50/50%) ratio of carnauba wax to hydrogenated rapeseed oil to a 1 :3 (25%/75%) ratio of carnauba wax to hydrogenated rapeseed oil also has a significant effect on the survival rate, further increasing the survival rate of the bacteria to 47%.

Thus, it is clear the composition and thickness of the second coating layer is an important feature to consider when attempting to optimize survivability of microencapsulated microbial cultures.

Example 3: Production of a micro-encapsulated microbial culture with a mass ratio of 0.5:3:0.25 by fluidized bed spray coating

A microencapsulated microbial culture having a triple layer coating with dry mass ratio (CI) of 0.5:3:0.25 was produced in three successive coating steps. For the first coating step, 50 grams of freeze-dried powder comprising Bifidobacterium animalis subsp. Lactis was coated with 100 grams 25% ethyl cellulose aqueous solution in a fluid bed spray coater with maximum product volume of 0.5 L.

To apply the 25% ethyl cellulose aqueous solution, the freeze-dried powders are fluidized using a dried inert gas at fluidizing velocity of 0.5-1 m/s and fluidizing gas temperature of 45°C. When the bed temperature stabilizes, the 25% ethyl cellulose aqueous solution is sprayed in the fluidizing chamber via a two-fluid nozzle. A dried inert gas helps to atomize the coating material at atomizing pressure of 0.8 bar and a spraying rate of 2 g/min. When all the coating material is sprayed, the fluidization and atomization are stopped, and materials is retrieved from the fluidized bed chamber.

For the second step of coating, 50 grams of retrieved powder from the first step is coated with 150 grams of molten vegetable wax admixture consisting of 75% rapeseed wax and 25 carnauba wax. To this end, the retrieved powder from the first step is fluidized at fluidizing velocity of 1-2 m/s and fluidizing gas temperature of < 50 °C. Then, the molten wax admixture is sprayed in the fluidized bed chamber at atomizing pressure of 0.8 bar and spraying rate of 15 g/min. The minimum temperature of the molten wax at injection point is required to be higher than the solidification point of the molten wax blend to ensure no solidification occurs in the line. For this purpose, the molten wax admixture is sprayed at minimum temperature of 90°C, and the atomizing gas temperature at the minimum temperature of 110°C. When all the coating material is sprayed, the fluidization and atomization are stopped, and materials is retrieved from the fluidized bed chamber.

After the first and third step, a post-drying step is performed to guarantee of having a dried final product. Post-drying is performed by extending the fluidization time after completing the spraying until the water activity of the microencapsulated microbial culture powder reached < 0.5 at ambient temperature.

Example 4 - Testing heat tolerance of microencapsulated Bifidobacterium

The heat tolerance of the micro-encapsulated Bifidobacterium as produced in example 3 in yoghurt vegurt and juice was tested by pasteurization using two heat profiles, profile A and profile B (see fig. 2), in three different types of carriers including commercial yogurt (0.5% fat, Aria), vegurt (oat/soy based and produced internally), and commercial orange juice from Rynkeby Foods A/S. The yogurt and vegurt were tested using pasteurization profile A (74 °C for 18 s, see fig. 3), and orange juice was tested using pasteurization profile B (85 °C for 4 s, see fig 4). In each heat test trial, 4 Kg of carrier was uniformly mixed with given amount of micro-encapsulated Bifidobacterium to reach a theoretical dose of about 1E+8 CFU/g of food product.

Then, three samples, each of about 100 g, were sampled and designated "BEFORE", and the rest were undergoing a pasteurization process through a pilot-size tubular heat exchanger (OMVE HT122).

The heat profile was established before feeding the liquid food, and three pasteurized samples, each of about 100 g, were sampled under a steady-state temperature profile. These samples were designated "AFTER".

The number of viable colonies (CFU) of the samples designated "BEFORE" and "AFTER" were measured using a standard plate counting method. However, since the Bifidobacterium cells are micro-encapsulated, a step of decapsulation was performed prior to the plate counting step, involving adjustment of pH and high shear mechanical treatment.

Example 4A - Viability of coated core material in yoghurt and vegurt following pasteurization at 74°C.

A comparison of the CFU numbers of the Bifidobacterium per gram of yogurt/vegurt, before and after pasteurization at 74 C for 18 seconds in a carrier medium is shown in figure 3 and the survival rates in percent are listed in table 2. The number above the "AFTER pasteurization" bar in figure 3 indicates the survival rate in terms of percent compared to CFU numbers before pasteurization. The results showed that the uncoated sample, which served as "control", had survival rate of lower than 1%, while the micro-encapsulated Bifidobacterium had an average survival rate of 50%. The micro-encapsulated Bifidobacterium even reached a superior survival rate post pasteurization of 70% in the vegurt carrier.

Table 2: survival rate of encapsulated microbial cultures after pasteurization in a carrier medium.

These results clearly demonstrates that the micro-encapsulation of microbial cultures as disclosed herein, provides heat tolerance to and dramatically improves survivability during pasteurization of the micro-encapsulated microbial culture under conditions with high acidity and high water activity (i.e. in yoghurts and vegurt).

Example 4B - Viability of coated core material in orange juice following harsh heat treatment at 85°C.

A comparison of the CFU numbers of the Bifidobacterium per gram of orange juice, before and after pasteurization at 85 °C for 4 seconds in a carrier medium is shown in figure 4. The micro-encapsulated Bifidobacterium showed an enhanced survival rate of 27%, whereas the non-encapsulated control had a survival rate of 0% post-treatment with Heat Profile-B at higher temperature of 85 °C for 4 seconds (see fig. 4).

This result clearly demonstrates that the micro-encapsulation of microbial cultures as disclosed herein, also provides heat tolerance to and dramatically improves survivability during harsh heat-treatment processes in beverages, where the high acidity and high water activity of the carrier (i.e. the orange juice) are further exacerbated by the high acidity and the extremely high water activity and concentration.

In conclusion, it is clear that the micro-encapsulated microbial cultures as disclosed herein have increased heat tolerance and resistance to both high acidity and high water activity even when these conditions are exacerbated by high temperatures. The micro-encapsulated cultures disclosed herein and produced by the methods disclosed herein are suitable for applications where high acidity and water activity during pasteurization have previously prevented preservation of such microbial cultures.

These characteristics are very useful for production of foodstuff where pasteurization processes are applied to avoid and/or reduce spoilage. This includes foods such aas yoghurt and vegurt, feeds for animals, beverages such as juices and dairy products such as milk, cream, and cheese, but could in general be used in any food products that contain microbes and undergo pasteurization processes. In particular, such products may have increased longevity in distant regions where constant refrigeration and cold storage and electricity can be an issue, but also in regions prone to vast heat waves or floodings, which may compromise otherwise optimal storage options for foods, feeds and beverages comprising microbial components.

Example 5 - Evaluation of a fat/wax layer as second coating layer Freeze-dried powder of Bifidobacterium animalis subsp. lactis DSM 15954 was used as core material for the following coating steps.

The composition of the fat/wax admixture was evaluated for its efficacy in protecting Bifidobacterium animalis subsp. lactis probiotic bacteria in a yogurt matrix during pasteurization heat treatment. The composition, comprising 25% glycerol monostearate and 75% hydrogenated rapeseed oil, was applied as a second layer of coating to the probiotic bacteria. The first and third coating layer remained unchanged as compared to Example 3.

The presence of the middle fat layer resulted in a 50% recovery of Bifidobacterium animalis subsp. lactis in the pasteurized yogurt samples. In comparison, Bifidobacterium animalis subsp. lactis without any microencapsulation (coating) did not survive the pasteurization treatment.

Conclusion:

The results demonstrate that the composition of the second fat layer effectively protects Bifidobacterium animalis subsp. lactis in the yogurt matrix during pasteurization, highlighting its potential for enhancing the viability of probiotic bacteria in yogurt products. Additionally, it indicates that diverse compositions of fat/waxes can be used depending on the application and level of heat treatment.

ITEMS

1. A micro-encapsulated microbial culture comprising i. a core material comprising a microbial culture, ii. a first coating layer, a second coating layer, and a third coating layer encapsulating the core material, wherein, iii. the first coating layer comprises a plant-based food grade polymer, the second coating layer comprises an admixture of vegetable wax, and the third coating layer comprises a plant-based food grade polymer.

2. The micro-encapsulated microbial culture according to item 1, wherein the core material is coated in the first coating layer, the first coating layer is coated in the second coating layer and the second coating layer is coated in the third coating layer.

3. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the admixture of vegetable wax comprises a vegetable wax having a medium melting point mixed with a vegetable wax having a high melting point.

4. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the admixture of vegetable wax essentially consists of a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax. 5. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the medium melting point vegetable wax is selected from the list consisting of palm wax and rapeseed wax.

6. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the medium melting point vegetable wax is palm wax.

7. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the medium melting point vegetable wax is rapeseed wax.

8. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the medium melting point wax is a non-vegetable wax and the non-vegetable wax is bees wax.

9. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the high melting point vegetable wax is selected as one of carnauba wax and candelilla wax.

10. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the high melting point vegetable wax is carnauba wax.

11. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the high melting point vegetable wax is candelilla wax.

12. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is carnauba wax.

13. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based polymer of the first coating layer and the third coating layer are the same plant-based food grade polymer.

14. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based polymer of the first coating layer and the third coating layer are different plant-based food grade polymers.

15. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based polymer of the first coating layer and the third coating layer is a blend of different food grade plant-based polymers.

16. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is selected from the list consisting of cellulose, alginate, calcium alginate, sodium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose and carboxymethyl cellulose. 17. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is selected from the list consisting of calcium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose and carboxymethyl cellulose.

18. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is selected from the list consisting of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose and carboxymethyl cellulose.

19. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is selected from the list consisting of ethyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose.

20. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is selected from the list consisting of ethyl cellulose and hydroxyethyl cellulose.

21. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the food grade plant-based food grade polymer is ethyl cellulose.

22. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprises ethylcellulose and the second coating layer comprises 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22- 28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

23. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprises ethylcellulose and the second coating layer comprises 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

24. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprises ethylcellulose and the second coating layer comprises 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

25. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprises ethylcellulose and the second coating layer comprises 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

26. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprises ethylcellulose and the second coating layer comprises 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

27. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprise a concentration of between 70-99%, between 80-99%, between 90-99% or between 95-99% plant-based food grade polymer.

28. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprise a concentration of between 80-99%, 90-99% or 95-99% plant-based food grade polymer.

29. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprise a concentration of between 90-99% or 95-99% plant-based food grade polymer.

30. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the first coating layer and the third coating layer comprise a concentration of between 95-99% plant-based food grade polymer.

31. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.2-1.

32. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.3-0.7.

33. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of first coating layer to the core material is 0.4- 0.6. 34. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.

35. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.3 or 0.4 or 0.5 or 0.6 or 0.7.

36. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.4.

37. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material 0.5.

38. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.6.

39. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.8.

40. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 1.

41. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the second coating layer to the core material coated in the first coating layer is 3.

42. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the third coating layer to the core material coated in both the first coating layer and the second coating layer is 0.25-0.5.

43. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.3-0.7, and the mass ratio of the second layer to the core material coated in the first coating layer and the second coating layer is 1.5-4.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-1.

44. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.4-0.6, and the mass ratio of the second coating layer to the core material coated in the first coating layer is 2.5-3.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5.

45. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the mass ratio of the first coating layer to the core material is 0.5, and the mass ratio of the second coating layer to the core material coated in the first coating layer and the second coating layer is 1 :3, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB). The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of 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, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp., Propionibacterium spp., and Bifidobacterium spp. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of Lactobacillus spp. and Bifidobacterium spp. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to the genus of Lactobacillus spp. The micro-encapsulated microbial culture according to any one of the preceding items, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to the genus of Bifidobacterium spp. A method for producing a micro-encapsulated microbial culture, the method comprising ai. providing a core material comprising a microbial culture, bi. encapsulating said core material in a first coating layer, a second coating layer, and a third coating layer, ci. Wherein the first coating layer comprises a plant-based food-grade polymer, the second coating layer comprises an admixture of vegetable wax, and the third coating layer comprises a plant-based food-grade polymer, thereby providing a micro-encapsulated microbial culture. . The method according to item 52, wherein encapsulating the core material in step bi further comprises the steps

-coating the core material in the first coating layer,

-coating the first coating layer in the second coating layer, and

-coating the second coating layer in the third coating layer. . The method according to any one of items 52-53, wherein the admixture of vegetable wax comprises a vegetable wax having a medium melting point mixed with a vegetable wax having a high melting point. . The method according to any one of items 52-54, wherein the admixture of vegetable wax essentially consists of a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

56. The method according to any one of items 52-55, wherein the medium melting point vegetable wax is selected from the list consisting of palm wax and rapeseed wax.

57. The method according to any one of items 52-56, wherein the medium melting point vegetable wax is palm wax.

58. The method according to any one of items 52-57, wherein the medium melting point vegetable wax is rapeseed wax.

59. The method according to any one of items 52-58, wherein the medium melting point wax is a non-vegetable wax and the non-vegetable wax is bees wax.

60. The method according to any one of items 52-59, wherein the high melting point vegetable wax is selected as one of carnauba wax and candelilla wax.

61. The method according to any one of items 52-60, wherein the high melting point vegetable wax is carnauba wax.

62. The method according to any one of items 52-61, wherein the high melting point vegetable wax is candelilla wax.

63. The method according to any one of items 52-62, wherein the medium melting point vegetable wax is rapeseed wax and the high melting point vegetable wax is carnauba wax.

64. The method according to any one of items 52-63, wherein the plant-based food grade polymer used in the first coating layer is the same as the plant-based food grade polymer used in the third coating layer.

65. The method according to anyone of items 52-64, wherein the plant-based food grade polymer used in the first coating layer is a different plant-based food grade polymer than the plant base food grade polymer used in the third coating layer.

66. The method according to any one of items 52-65, wherein the plant-based food grade polymer of the first coating layer and the third coating layer is a blend of different plant-based food grade polymers.

67. The method according to any one of items 52-66, wherein the plant-based food grade polymer is selected from the list consisting of cellulose, alginate, calcium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate and carboxymethyl cellulose. The method according to any one of items 52-67, wherein the food grade plantbased food grade polymer is selected from the list consisting of calcium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate and carboxymethyl cellulose. The method according to any one of items 52-68, wherein the plant-based food grade polymer is selected from the list consisting of ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate and carboxymethyl cellulose. The method according to any one of items 52-69, wherein the plant-based food grade polymer is selected from the list consisting of ethyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose. The method according to any one of items 52-70, wherein the plant-based food grade polymer is selected from the list consisting of ethyl cellulose and hydroxyethyl cellulose. The method according to any one of items 52-71, wherein the plant-based food grade polymer is ethyl cellulose. The method according to any one of items 52-72, wherein the plant-based food grade polymer used for the first coating layer and for the third coating layer is ethyl-cellulose and the second coating layer comprises 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22- 28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax. The method according to any one of items 52-73, wherein the plant-based food grade polymer used for the first coating layer and for the third coating layer is ethyl-cellulose and the second coating layer comprises 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax. The method according to any one of items 52-74, wherein the plant-based food grade polymer used for the first coating layer and for the third coating layer is ethyl-cellulose and the second coating layer comprises 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

76. The method according to any one of items 52-75, wherein the plant-based food grade polymer used for the first coating layer and for the third coating layer is ethyl-cellulose and the second coating layer comprises 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

77. The method according to any one of items 52-76, wherein the plant-based food grade polymer used for the first coating layer and for the third coating layer is ethyl-cellulose and the second coating layer comprises 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

78. method according to any one of items 52-77, wherein the first coating layer and the third coating layer comprise a concentration of between 70-99%, between 80-99%, between 90-99% or between 95-99% plant-based food grade polymer.

79. method according to any one of items 52-78, wherein the first coating layer and the third coating layer comprise a concentration of between 80-99%, 90-99% or 95-99% plant-based food grade polymer.

80. method according to any one of items 52-79, wherein the first coating layer and the third coating layer comprise a concentration of between 90-99% or 95-99% plant-based food grade polymer.

81. method according to any one of items 52-80, wherein the first coating layer and the third coating layer comprise a concentration of between 95-99% plant-based food grade polymer.

82. The method according to any one of items 52-81, wherein the mass ratio of the first coating layer to the core material is 0.2-1.

83. The method according to any one of items 52-82, wherein the mass ratio of the first coating layer to the core material is 0.3-0.7.

84. The method according to any one of items 52-83, wherein the mass ratio of the first coating layer to the core material is 0.4-0.6.

85. The method according to any one of items 52-84, wherein the mass ratio of the first coating layer to the core material is 0.2, or 0.3 or 0.4 or 0.5 or 0.6 or 0.7 or 0.8 or 0.9 or 1. 86. The method according to any one of items 52-85, wherein the mass ratio of the first coating layer to the core material is 0.3 or 0.4 or 0.5 or 0.6 or 0.7.

87. The method according to any one of items 52-86, wherein the mass ratio of the first coating layer to the core material is 0.4.

88. The method according to any one of items 52-87, wherein the mass ratio of the first coating layer to the core material is 0.5.

89. The method according to any one of items 52-88, wherein the mass ratio of the first coating layer to the core material is 0.6.

90. The method according to any one of items 52-89, wherein the mass ratio of the first coating layer to the core material is 0.8.

91. The method according to any one of items 52-90, wherein the mass ratio of the first coating layer to the core material is 1.

92. The method according to any one of items 52-91, wherein the mass ratio of the second coating layer to the core material coated in the first coating layer is 1:3.

93. The method according to any one of items 52-92, wherein the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5.

94. The method according to any one of items 52-93, wherein the mass ratio of the first coating layer to the core material is 0.3-0.7, and the mass ratio of the second coating layer to the core material coated in the first coating layer is 1.5- 4.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-1. 5. The method according to any one of items 52-94, wherein the mass ratio of the first coating layer to the core material is 1 :0.4-0.6, and the mass ratio of the second coating layer to the core material coated in the first coating layer is 2.5- 3.5, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer is 0.25-0.5.

96. The method according to any one of items 52-95, wherein the mass ratio of the first coating layer to the core material is 1 :0.5, and the mass ratio of the second coating layer to the core material coated in the first coating layer is 1 :3, and the mass ratio of the third coating layer to the core material coated in the first coating layer and the second coating layer 0.25-0.5.

97. The method according to any one of items 52-96, wherein the microbial culture is a lactic acid bacteria (LAB).

98. The method according to any one of items 52-97, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of 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, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

99. The method according to any one of items 52-98, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp., Propionibacterium spp., and Bifidobacterium spp.

100. The method according to any one of items 52-99, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of Lactobacillus spp. and Bifidobacterium spp.

101. The method according to any one of items 52-100, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to the genus of Lactobacillus spp.

102. The method according to any one of items 52-101, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to the genus of Bifidobacterium spp.

103. The method according to any one of items 52-102, wherein the coating process is performed using a method selected from spray drying, spray congealing, extrusion, emulsification, and fluidized bed spray coating.

104. The method according to any one of items 52-103, wherein the coating process is performed using fluidized bed spray coating.

105. The method according to any one of items 52-104, wherein the step bi comprises the steps

-coating the core material in the first coating layer,

-coating the first coating layer in the second coating layer,

-coating the second coating layer in the third coating layer, and recovering the microencapsulated microbial culture following the third coating round.

106. The method according to any one of the items 52-105, wherein step bi comprises the steps

-coating the core material in the first coating layer,

-coating the first recovered product in a second coating layer,

-coating the second recovered product in the third coating layer, and recovering the microencapsulated microbial culture.

107. The method as defined in any one of items 52-106, wherein the recovery step comprises size exclusion, size separation, centrifugation, and/or filtration-based methods.

108. The method according to any one of items 52-107, further comprising a purification step and/or a washing step immediately after each recovery step.

109. A product produced by the methods as defined in any one of items 52-108.

110. A composition comprising a microencapsulated microbial culture according to any one of items 1-51, a microencapsulated microbial culture produced by a method according to any one of items 52-108, or a product according to item 109.

111. The composition according to item 110, wherein the composition is a dairy product.

112. The composition according to item 110, wherein the composition is a yoghurt.

113. The composition according to item 110, wherein the composition is a vegurt.

114. The composition according to item 110, wherein the composition is a beverage.

115. The composition according to item 110, wherein the composition is a juice.

116. A probiotic composition comprising a microencapsulated probiotic microbial culture according to any one of items 1-51, a microencapsulated probiotic microbial culture produced by a method according to any one of items 52-108 or a product according to item 109.

117. A pharmaceutical composition comprising a microencapsulated microbial culture according to any one of items 1-51, a microencapsulated microbial culture produced according to a method of any one of items 52-108, a product according to item 109, a composition according to item 110, and/or a probiotic composition according to item 116.

118. Use of a microencapsulated microbial culture according to any one of items 1- 51, a microencapsulated microbial culture produced by a method according to any one of items 52-108, a product according to item 109 or a composition according to any one of items 110-116 as a probiotic.

119. Use of a microencapsulated microbial culture according to any one of items 1- 51, a microencapsulated microbial culture produced by a method according to any one of items 52-108, a product according to item 109, a composition according to any one of items 110-116, or a probiotic composition according to item 116, in a feed, food or beverage.

120. The use according to item 119, wherein the use is use in a feed.

121. The use according to item 119, wherein the use is use in a food.

122. The use according to item 121, wherein the food is a yogurt.

123. The use according to item 121, wherein the food is a vegurt.

124. The use according to item 121, wherein the food is a dairy product.

125. The use according to item 121, wherein the food is a cheese.

126. The use according to item 119, wherein the use is use in a beverage.

127. The use according to item 126, wherein the beverage is a dairy product.

128. The use according to item 127, wherein the dairy product is milk.

129. The use according to item 126, wherein the beverage is a juice.

130. The use according to item 129, wherein the juice is orange juice.

131. A product comprising a microencapsulated microbial culture according to any one of items 1-51, a micro-encapsulated microbial culture as produced by a method according to any one of items 52-108, a composition according to item 110, or a probiotic composition according to item 116.

132. The product according to item 131, wherein the product is a food, a feed and/or a beverage.

133. The product according to item 132, wherein the product is a food. 134. The product according to item 133, wherein the food is a cheese, yoghurt or vegurt.

135. The product according to item 134, wherein the food is a yoghurt.

136. The product according to item 134, wherein the food is a vegurt.

137. The product according to item 134, wherein the food is a cheese.

138. The product according to item 132, wherein the product is a feed.

139. The product according to item 132, wherein the product is a beverage.

140. The product according to item 139, wherein the beverage is a dairy product.

141. The product according to item 140, wherein the dairy product is milk.

142. The product according to item 139, wherein the beverage is a juice.

143. The product according to item 142, wherein the juice is orange juice.

(Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)

FOR RECEIVING OFFICE USE ONLY

FOR INTERNATIONAL BUREAU USE ONLY -5 This form was re international Bur -5-1 Authorized officer

Claims

2. The micro-encapsulated microbial culture according to claim 1, wherein the core material is coated in the first coating layer, the first coating layer is coated in the second coating layer and the second coating layer is coated in the third coating layer.

3. The micro-encapsulated microbial culture according to any one of the preceding claims, wherein the admixture of vegetable wax comprises a vegetable wax having a medium melting point mixed with a vegetable wax having a high melting point.

4. The micro-encapsulated microbial culture according to any one of the preceding claims, wherein the admixture of vegetable wax essentially consists of a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

5. The micro-encapsulated microbial culture according to any one of the preceding claims, wherein the medium melting point vegetable wax is selected from the list consisting of palm wax and rapeseed wax and the high melting point vegetable wax is selected as one of carnauba wax and candelilla wax.

6. The micro-encapsulated microbial culture according to any one of the preceding claims, wherein the food grade plant-based food grade polymer is selected from the list consisting of cellulose, alginate, calcium alginate, sodium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose and carboxymethyl cellulose.

7. The micro-encapsulated microbial culture according to any one of the preceding claims, wherein the microbial culture is a lactic acid bacteria (LAB) belonging to a genus selected from the list consisting of 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, Lactococcus, Streptococcus, Enterococcus, Bifidobacterium, Brevibacterium, and Staphylococcus.

8. A method for producing a micro-encapsulated microbial culture, the method comprising ai. providing a core material comprising a microbial culture, bi. encapsulating said core material in a first coating layer, a second coating layer, and a third coating layer, ci. Wherein the first coating layer comprises a plant-based food-grade polymer, the second coating layer comprises an admixture of vegetable wax, and the third coating layer comprises a plant-based food-grade polymer, thereby providing a micro-encapsulated microbial culture.

9. The method according to claim 8, wherein encapsulating the core material in step bi further comprises the steps

-coating the core material in the first coating layer,

-coating the first coating layer in the second coating layer, and

-coating the second coating layer in the third coating layer.

10. The method according to any one of claims 8-9, wherein the admixture of vegetable wax comprises a vegetable wax having a medium melting point mixed with a vegetable wax having a high melting point.

11. The method according to any one of claims 8-10, wherein the admixture of vegetable wax essentially consists of a) 50-90% medium melting point vegetable wax and 10-50% high melting point vegetable wax, or b) 65-85% medium melting point vegetable wax and 15-35% high melting point vegetable wax, or c) 70-80% medium melting point vegetable wax and 20-30% high melting point vegetable wax, or d) 72-78% medium melting point vegetable wax and 22-28% high melting point vegetable wax, or e) 75% medium melting point vegetable wax and 25% high melting point vegetable wax.

12. The method according to any one of claims 8-11, wherein the medium melting point vegetable wax is selected from the list consisting of palm wax and rapeseed wax and the high melting point vegetable wax is selected as one of carnauba wax and candelilla wax.

13. The method according to any one of claims 8-12, wherein the plant-based food grade polymer is selected from the list consisting of cellulose, alginate, calcium alginate, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose hydroxypropyl cellulose, hydroxypropyl methylcellulose acetate succinate and carboxymethyl cellulose.

14. A composition comprising a microencapsulated microbial culture according to any one of claims 1-7, or a microencapsulated microbial culture produced according to a method of any one of claims 8-13.

15. Use of a microencapsulated microbial culture according to any one of claims 1-8, a microencapsulated microbial culture produced by a method according to any one of claims 9-13, or a composition according to claim 14 in a feed, food or beverage.