WO2014128071A1

WO2014128071A1 - Encapsulated plasmolysed micro-organism particles

Info

Publication number: WO2014128071A1
Application number: PCT/EP2014/052991
Authority: WO
Inventors: Pierre-Etienne Bouquerand; Nathalie Vivien Castioni; Anupama KOORAPATI; Serge MAIO; François Meyer
Original assignee: Firmenich Sa
Priority date: 2013-02-25
Filing date: 2014-02-17
Publication date: 2014-08-28
Also published as: BR112015020306A2; EP2958440A1; RU2015140808A; US20160015061A1; JP6494530B2; JP2016514951A; MX2015010541A; EP2958440B1; RU2654748C2; CN111011802A; US11202463B2; CN105072924A

Abstract

Provided herein are formulations of flavors and fragrances encapsulated in plasmolysed micro-organisms, as well as glass particles or beads comprising these encapsulated flavors and fragrances. Also provided herein are methods of making formulations of encapsulated flavors and fragrances.

Description

ENCAPSULATED PLASMOLYSED MICRO-ORGANISM PARTICLES Technical Field

The technical field relates to formulations of encapsulated flavors in glassy particles and plasmolysed organisms.

Background of the Invention

Delivery systems for the controlled release of flavor or fragrance compounds and methods of making them have been reported. Microcapsules made of organisms used to encapsulate flavors or fragrances are known. These product and processes have, amongst other limitations, limitations in the amount of the flavor or fragrance that can be encapsulated.

Summary of the Invention

Provided herein is a composition comprising a homogenous paste comprising:

a) a plasmolysed micro-organism;

b) a flavor or fragrance; and

c) water wherein the micro-organism to water ratio is provided in an amount, by weight, in the range of about 1.5: 1 to 4: 1 and the flavor or fragrance is encapsulated in the plasmolysed organism.

Also provided herein is a method of making a plasmolysed micro-organism encapsulated flavor or fragrance comprising combining a flavor or fragrance, a plasmolysed micro-organism and water for a time sufficient to form a viscous paste or cake wherein the ratio of the micro-organism to the water, by weight, is in the range of about 1.5: 1 to about 4: 1.

Further provided herein is a glass particle or bead comprising:

a) a plasmolysed micro-organism encapsulated flavor or fragrance wherein the microorganism encapsulated flavor or fragrance comprises a viscous cake formed from the mixture of the plasmolysed micro-organism, the flavor or fragrance, and water wherein the ratio of the micro-organism to the water, by weight, is in the range of about 1.5: 1 to about 4: 1 and wherein the cake is provided in an amount, by weight, of up to about 55% of total weight of the particle; and

b) a carrier comprising a polymeric water soluble emulsifier and optionally a carbohydrate wherein the emulsifier is provided in an amount, by weight, of about 5% up to about 100% of the total weight of the carrier and the carrier is provided in an amount, by weight, of about up to about 80% of the total weight of the particle.

Brief Description of the Drawings

Figure 1 is directed to the comparison of the Menthol release from different Menthol + cooling agent flavored chewing gums (Iso-Load) by APCI-MS nose test.

Figure 2 shows the comparison of the Lemon Flavor Intensity from chewing gums (Iso- load) by APCI-MS nose test.

Figure 3 shows the comparison of the Menthol release from Mint flavored chewing gums (Iso-dosage) by APCI-MS nose test.

Figure 4 shows the comparison of the Menthol release for the Menthol & Cooling Agent (CA) flavored chewing gums (Iso-dosage) APCI-MS nose test.

Description of the Invention

For the Summary, Description and Claims, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of".

In carrying out particular embodiments provided herein, a plasmolysed microorganism is selected from the group consisting of fungi, a bacteria, algae, protozoa, or mixtures of two or more of these. In a particular embodiment the micro-organism is a fungus or a bacterium, more particularly it may comprise a yeast. The micro-organism may be pre-treated to increase its permeability or to remove undesired odors or aromas. Such pre-treatments are disclosed in US 5,521,089, col. 2, line 58 to col. 4, line 63 and WO 93/11869. A peroxygen bleaching of micro-organisms for removing odor and lightening the color of micro-organisms is disclosed. A micro-organism that has at least a part of its endogenous intracellular material removed is defined herein as a "plasmolysed micro-organism."

The plasmolysed micro-organism cake provided herein comprises a mixture of a micro-organism and water wherein the ratio, by weight, ranges from about 1.5: 1 up to about 1.5:4. In another aspect, the mixture of the micro-organism and water is provided in a ratio that ranges from about 1.5: 1 up to about 2.5: 1. In a particular embodiment, the microorganism and water mixture is provided in a ratio, by weight, of about 2: 1.

In some embodiments, the plasmolysed micro-organism cake, and the process for making it, may provide a flavor or fragrance load in the cake in an amount, by weight, of up to about 50%, up to about 55% and further up to about 60% of the total weight of the cake.

Further provided herein is a method of making a glass particle or bead comprising:

1) blending

(a) a plasmolysed micro-organism encapsulated flavor or fragrance wherein the plasmolysed micro-organism encapsulated flavor or fragrance comprises a viscous cake formed from a mixture of water, the flavor or fragrance, and the plasmolysed micro-organism;

(b) a carrier comprised of a polymeric water soluble emulsifier and optionally a carbohydrate;

2) heating the blend in an extruder to a temperature sufficient to form a molten mass;

3) extruding the molten mass;

4) cutting the extruded molten mass into granules; and

5) allowing the granules to cool to form a glassy particle.

A lubricant may be added to the blend.

The carrier provided herein is a carrier comprising a water soluble polymeric emulsifier and optionally a carbohydrate. The carrier is provided in the formulations described herein in an amount that ranges, by total weight of the formulation, from about 40 up to about 80%, by weight, of the total weight of the particle provided herein. In a further embodiment, the carrier is provided in an amount of about 50 up to about 75%, more particularly from about 55 up to about 70%, by weight, of the total weight of the particle provided herein.

In a particular embodiment, provided herein is a carrier comprising a carbohydrate or carbohydrate derivative which can be readily processed through extrusion techniques to form a dry extruded solid. Particular examples of suitable materials include those selected from the group consisting of sucrose, glucose, lactose, maltose, fructose, ribose, dextrose, isomalt, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, Trehalose ^®, hydrogenated corn syrup, maltodextrin, agar, carrageenan, gums, polydextrose and derivatives and mixtures thereof. Other suitable carrier ingredients are cited in reference texts such as H. Scherz, Hydrokolloide : Stabilisatoren, Dickungs- und Geliermittel in Lebensmittel, Band 2 der Schriftenreihe Lebensmittelchemie, Lebensmittelqualitat, Behr's VerlagGmbH & Co, Hamburg, 1996. In a particular embodiment provided herein comprises a maltodextrin having a dextrose equivalent not above twenty (< 20 DE).

Particularly, the carbohydrate may comprise a non-emulsifying water soluble material such as, but not limited to, maltodextrins. In a particular embodiment, the carbohydrate is a maltodextrin with a dextrose equivalent (DE) of about 1 to about 20. In a particular embodiment, the maltodextrin is selected from a maltodextrin with a DE of about 10 up to about 18 DE. In another embodiment, the carbohydrate comprises corn syrup with a DE from 21 up to 49. Any carbohydrate can be used that is made by the hydrolysis of starches from different origins such as, but not limited to, maize, wheat, potato or rice. In another embodiment, the carbohydrate is a hydrogenated starch hydrolysate (e.g., HSPolyols), fructose oligosacharides (e.g., but not limited to Inuline from Orafit), soluble fibers such as for example but not limited to Nutriose (Roquette) and pregelatinized starch.

In another aspect, the carrier comprises a polymeric emulsifier which comprises a water soluble material such as Capsul (National starch). In another embodiment, the emulsifier is an akenyl succinated starch, more particularly and ocytenyl succinated starch (OSS). Some particular example, but not limited to, are N-lock (Emcap), Purity gum (Cargill). In another embodiment, the emulsifier is gum Arabic. In a particular embodiment, the carrier is provided in an amount that ranges, by weight, of about 40% to about 80% of the total weight of the particle or bead. Particularly the carrier is provided in an amount, by weight of about 50% to about 75% and more particularly from about 55% to about 70.

In a particular embodiment, the emulsifier may be provided in an amount, by weight, from about 5% to about 100%, particularly from about 5% to about 40%, even more particularly from about 10% to about 40%, and more particularly at about 10% of the total weight of the carrier.

In another embodiment, a lubricant is provided herein. While not wishing to be bound to any theory it is believed that the lubricant reduces shear and expansion of the molten mass at the exit die. In some embodiments, the lubricant may comprise a medium chain triglyceride (MCT). In another embodiment, the lubricant comprises a micellar surfactant like lecithin or a fatty acid ester (e.g., citric, tartaric, acetic), DATEM, CITREM or mixtures of the above. In a particular embodiment, the lubricant is may be provided in an amount, by weight, up to about 5%, particularly about 0.2 up to about 5%, more particularly from about 0.8% up to about 2% and even more particularly about 1% of the total weight of the particle.

Extruded glassy particles comprising plasmolysed micro-organism cake may be obtained following the general methods set forth in US Patent No. 6,607,771, the whole of which is incorporated by reference herein. In a particular embodiment a carbohydrate, and at least one water soluble emulsifier are mixed and a non plasticizing flavor or fragrance is dispersed into the mix. In one embodiment, water is added to the mix until a free flowing powder is formed. Ideally the water is added without forming lumps. The components of the mixture described above can be premixed for example in a bowl or even stepwise into an extruder. The components may be mixed prior to being added to the extruder or alternatively, added stepwise or as a mixture of one or more of the components that make up the particle.

In some embodiments, the water acts as a plasticizer. The amount of water may be adjusted to obtain a glass transition temperature (Tg) between 30 and 100°C, more particularly at about 50°C. In a particular embodiment, the encapsulation of the plasmolysed micro-organism cake may be done without the addition of any further water than that which is provided by the content of the water in the plasmolysed micro-organism cake. In another embodiment, the water may be added to the process for making the glass transition in a desired range. In a particular embodiment the cake and carrier are blended in the substantial absence of added water, for example less than 2%, more particularly less than 1%, even more particularly less than 0.5%. In yet a further particular embodiment, no additional water is added than that which is included in the plasmolysed micro-organism cake.

The blended powder may then be extruded. In a particular embodiment, the powder may be extruded at a throughput of 500g/h through a 0.7mm die hole using for example, but not limited to a Thermo Prism 16 mm twin-screw lab extruder equipped with a cutter knife in order to granulate the melt at the die exit. In a particular embodiment, the screws are configured so that they have two mixing zones. In a further embodiment the temperature profile may be 80°C-100-105-108°C in a first mixing zone to the die plate. The melt temperature may range from about 80°C and up to about 120°C. In a particular embodiment, the temperature of the mix is about 108°C.

In a further embodiment, the temperature ranges at the die exit ranges from about 90 to about 130°C and particularly at about 98°C. In a particular embodiment, the pressure is maintained below 100 bar. Particularly, the temperature at the die exit may be around 50°C higher than the expected Tg.

The softening or glass transition temperature is preferably kept above 40°C to guarantee the free flowing nature of the produced powder at ambient temperature. A low water content to guarantee that the carrier's glass transition temperature is above room temperature and preferably above 40°C may be added to the mixture. The glass transition temperature of the flavor or fragrance/carbohydrate mixture depends on the amount of water added to the initial mixture. The Tg decreases when the proportion of water increases. Ideally, the proportion of water added to the mixture will be low, i.e., such that the glass transition temperature of the resulting mixture is substantially equal to the glass transition temperature desired for the final flavor or fragrance delivery system, i.e., the extruded product. In one embodiment a glass transition temperature Tg is provided significantly above the temperature at which the particle will be stored and subsequently used. Ideally, the temperature should be at least above room temperature and preferably above 40°C. The proportions in which water is employed may therefore vary in a wide range of values which the skilled person is capable of adapting and choosing as a function of the carbohydrate glass used in the matrix and the required Tg of the final product. For instance, for a carbohydrate glass having a DE (dextrose equivalent) of 18, proportions from 5 to 10% of water in the mixture can be used.

In some embodiments, the size (diameter) of the particle or bead provided herein ranges in size from about 0.4 mm up to about 5 mm, particularly from about 0.5mm up to about 2 mm, more particularly from about 0.5 mm up to about 1.4 mm, more particularly from 0.5 mm up to 1 mm and even more particularly at about 0.6. , 0.7 or 1.4 mm.

The above processes of using making a plasmolysed micro-organism cake and then further encapsulating the cake to make a glass particle or bead provides for a high flavor or fragrance load in the particle. In some embodiments the particle comprises a flavor or fragrance in an amount of up to about, by weight, 20%, up to 25%, particularly up to 30% and more particularly up to 33% of the total weight of the particle. In some embodiments, the particle comprises a flavor or fragrance in an amount of greater than about, by weight, 12% up to about 25% of the total weight of the particle.

The flavors and fragrances that have particular applicability here are those that are difficult to entrap in large amount using twin-screw extrusion but which can diffuse inside a plasmolysed micro-organism, in particular those flavors and fragrances that have a Log P of >2 with a Mw<600 Daltons.

By "flavor or flavoring composition", it is meant here a flavoring ingredient or a mixture of flavoring ingredients, solvents or adjuvants of current use for the preparation of a flavoring formulation, i.e. a particular mixture of ingredients which is intended to be added to an edible composition or chewable product to impart, improve or modify its organoleptic properties, in particular its flavor and/or taste. Flavoring ingredients are well known to a person skilled in the art and their nature does not warrant a detailed description here, which in any case would not be exhaustive, the skilled flavorist being able to select them on the basis of his general knowledge and according to the intended use or application and the organoleptic effect it is desired to achieve. Many of these flavoring ingredients are listed in reference texts such as in the book by S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, N.J., USA, or its more recent versions, or in other works of similar nature such as Fenaroli's Handbook of Flavor Ingredients, 1975, CRC Press or Synthetic Food Adjuncts, 1947, by M. B. Jacobs, van Nostrand Co., Inc. Solvents and adjuvants of current use for the preparation of a flavoring formulation are also well known in the art.

In a particular embodiment, the flavor is a mint flavor. In a more particular embodiment, the mint is selected from the group consisting of peppermint and spearmint.

In a further embodiment, the flavor is a cooling agent or mixtures thereof.

In another embodiment, the flavor is a menthol flavor.

Flavors that are derived from or based on fruits where citric acid is the predominant, naturally- occurring acid include but are not limited to, for example, citrus fruits (e.g. lemon, lime), limonene, strawberry, orange, and pineapple. In one embodiment, the flavors food is lemon, lime or orange juice extracted directly from the fruit. Further embodiments of the flavor comprise the juice or liquid extracted from oranges, lemons, grapefruits, key limes, citrons, Clementines, mandarins, tangerines, and any other citrus fruit, or variation or hybrid thereof. In a particular embodiment, the flavor comprises a liquid extracted or distilled from oranges, lemons, grapefruits, key limes, citrons, Clementines, mandarins, tangerines, any other citrus fruit or variation or hybrid thereof, pomegranates, kiwifruits, watermelons, apples, bananas, blueberries, melons, ginger, bell peppers, cucumbers, passion fruits, mangos, pears, tomatoes, and strawberries.

In a particular embodiment, the flavor comprises a composition that comprises limonene, in a particular embodiment, the composition is a citrus that further comprises limonene.

In another particular embodiment, the flavor comprises a flavor selected from the group comprising strawberry, orange, lime, tropical, berry mix, and pineapple.

The phrase flavor includes not only flavors that impart or modify the smell of foods but include taste imparting or modifying ingredients. The latter do not necessarily have a taste or smell themselves but are capable of modifying the taste that other ingredients provides, for instance, salt enhancing ingredients, sweetness enhancing ingredients, umami enhancing ingredients, bitterness blocking ingredients and so on. In a further embodiment, suitable sweetening components may be included in the particles described herein. In a particular embodiment, a sweetening component is selected from the group consisting of sugar (e.g., but not limited to sucrose), a stevia component (such as but not limited to stevioside or rebaudioside A), sodium cyclamate, aspartame, sucralose, sodium saccharine, and Acesulfam K or mixtures thereof.

The dry particles provided herein may be suitable for conveying flavors to beverages, fluid dairy products, condiments, baked goods, frostings, bakery fillings, candy, chewing gum and other food products.

Beverages include, without limitation, carbonated soft drinks, including cola, lemon- lime, root beer, heavy citrus ("dew type"), fruit flavored and cream sodas; powdered soft drinks, as well as liquid concentrates such as fountain syrups and cordials; coffee and coffee- based drinks, coffee substitutes and cereal-based beverages; teas, including dry mix products as well as ready-to-drink teas (herbal and tealeaf based); fruit and vegetable juices and juice flavored beverages as well as juice drinks, nectars, concentrates, punches and "ades"; sweetened and flavored waters, both carbonated and still; sport/energy/health drinks; alcoholic beverages plus alcohol-free and other low-alcohol products including beer and malt beverages, cider, and wines (still, sparkling, fortified wines and wine coolers); other beverages processed with heating (infusions, pasteurization, ultra high temperature, ohmic heating or commercial aseptic sterilization) and hot-filled packaging; and cold-filled products made through filtration or other preservation techniques.

Fluid dairy products include, without limitation, non-frozen, partially frozen and frozen fluid dairy products such as, for example, milks, ice creams, sorbets and yogurts.

Condiments include, without limitation, ketchup, mayonnaise, salad dressing, Worcestershire sauce, fruit-flavored sauce, chocolate sauce, tomato sauce, chili sauce, and mustard.

Baked goods include, without limitation, cakes, cookies, pastries, breads, donuts and the like.

Bakery fillings include, without limitation, low or neutral pH fillings, high, medium or low solids fillings, fruit or milk based (pudding type or mousse type) fillings, hot or cold make-up fillings and nonfat to full-fat fillings. In one embodiment, the encapsulated flavors provide an initial burst of flavor followed by a sustained release of the flavors.

In another aspect, a plasmolysed micro-organism cake and glass particles encapsulating the cake contain a higher load of flavor or fragrance over what is typically achieved for example through typical extrusion processes.

Particular fragrances that may be used herein are selected from the group consisting of l-Pentyl-2-propenyl acetate, Hexylcinnamic aldehyde, 8,12-epoxy-13,14,15,16- tetranorlabdane, Tricyclo[5.2.1.0(2,6)]dec-3,4-en-l-yl acetate , Coumarine, 2-Pentyl-l- cyclopentanol, Cyclamen aldehyde, a-Damascone, Dihydromyrcenol, pentadecenolide, methyl ionone, Lilial , Linalol, cis-4-( 1,1 -dimethyl)- 1-cyclohexyl acetate, 3-methyl-(4,5)- cyclopentadecen- 1-one, Tetrahydro-2-isobutyl-4-methyl-4(2H)-pyranol, γ-methyl-benzene pentanol, Hexyl salicylate, and Vertofix Coeur.

The following Examples are illustrative only and are not meant to limit the scope of the claims, the Summary or any invention presented herein.

EXAMPLES

Example 1

Production of Plasmolysed Organism Encapsulated Lemon Flavor Yeast Cake

About 50 g of dried plasmolysed yeast (Ohly washed williams) was dispersed within 75g of lemon flavor with a spatula within a one liter glass jar. About 25 g of distilled water was added and gently stirred with a spatula to obtain a yeast cells to water ratio about 2: 1. The jar was then sealed with a screw cape at left to stand for 12 hours at room temperature so that the liquid dispersion was observed to be a homogeneous thick paste. The dispersion was roughly mixed with a spatula and the paste was left to stand for another 12 hours period at room temperature. After sitting for the second 12 hours, the dispersed paste was observed to be a brown cake ("yeast cake"). In summary, the cake had about 50% lemon flavor, 33% empty cells and about 17% water. Example 2

Production of Glass particles Comprising Encapsulated Lemon Flavor Yeast Cake About 150g of the "cake" made according to Example 1 was blended with 35 g of Capsul (National starch), 310g of 18 DE maltodextrin (Roquette) and 5 g of lubricant (Firmenich) within a blade blender (Cut-o-Matic, Switzerland) and blended for less than 10 seconds at full speed. The resulting powder was poured into a Brabender (Switzerland) gravimetric feeder and feed into an extruder having two mixing zones separated by a relaxing zone with a twin-screw configuration. The temperature profile was about 108°C at the level of the mixing zone and about 98°C at the die zone. The pressure at the die zone was kept below 20 bars and the blend was mixed in a mixing zone. The mixture was extruded at a throughput of 0.5 kg/h through a 0.7 mm die hole using 16 mm a Thermo Prism twin-screw laboratory extruder (Thermo Electron Germany). The extruder was equipped with a cutterknife used to chop the melt at the die exit while it is still plastic. The extruded granules were collected and conveyed by air cyclone system (Thermo electron (Germany)) and allowed to cool at ambient temperature. The resulting particles have a glass transition higher than 40 °C and a size (diameter) of about 0.6 mm. In summary, the extruded particle comprises about 30% cake, about 69% carrier and about 1% lubricant. This corresponds to final flavor load of 15%. More than 90% of the flavor was retained as measured by hydro-distillation. To evaluate the proportion of the flavor which was present within the yeast cells and not just entrapped within the extrusion carrier, a differential gravimetric measurement was performed on the cake. From the thermograms 60% of the lemon was entrapped within the yeast. One conclusion is that within 15% load of extruded yeast cake, 9% of lemon was double encapsulated and 6% was dispersed within the carrier.

Example 3

Production of Glass particles Comprising Encapsulated Mint Flavor Yeast Cake

Examples 1 and 2 were repeated but with the following ingredients and proportions: The yeast cake had about 55% mint flavor, 30% empty cells and about 15% added water. The extruded particles comprised about 45% cake, + 54% carrier, and about 1% lubricant. Increasing the proportions of the cake means increasing the water addition up to 6.8% and decreasing the carrier fraction. The carrier was made of 90 % 10 DE maltodextrin (Roquette- France) and 10 % Capsul™ octenyl succinated starch. The glass transition of the extruded particles was greater than 40°C to guarantee their physical stability. A flavor load about 25 % was obtained with encapsulation efficiency about 100%.

Example 4

Production of Glass particles Comprising Encapsulated Cooling-mix Flavor Yeast Cake

Examples 1 and 2 were repeated but with the following ingredients and proportions: The yeast cake had about 55% of a cooling mix composed of equal amount of menthol and cooling compound (WS 23 n° 927878), 30% empty cells and about 15% added water. The extruded particles comprised about 45% cake, + 54% carrier, and about 1% lubricant.

Like example 3, the carrier was based on 90 % 10 DE maltodextrin (Roquette-France) and 10% Capsul™ octenyl succinated starch (National Starch-USA) to guarantee a glass transition higher than 40°C (52°C). A flavor load about 22 % was obtained with encapsulation efficiency about 88% mixture.

Example 5

Loading Procedure of Plasmolysed Yeast Dispersed in Water

In a closed double-jacketed vessel equipped with an agitator, 113.2 g of plasmolysed yeast was dispersed within 649.1 g of warm water. The dispersion was constantly stirred and kept at 40°C for 20 minutes to completely hydrate the yeast. Lemon flavor (37.7 g) was added to the dispersion and the whole mixture was stirred within the closed vessel at 40°C for 2 hours. After this infusion step, the dispersion was dried using a mini-Buchi lab spray-drier with an inlet temperature of about 175°C and an outlet temperature of about 85°C. The resulting powder had a flavor content of about 24 % which corresponds to an encapsulation yield of 96%. The same process was carried out with mint and the Menthol - cooling mix flavors.

Example 6

A dry blended formulation of about 500g was prepared by admixing the following ingredients:

Ingredients Parts by weight

Maltodextrin 18 DE (Roquette) 79.5

Capsul™ (National Starch) 9.0 Lecithin 1.0

Lemon flavor 6.0

Tap water 4.5

100.0

Extruded glassy particles were obtained following the general methods set forth in WO01/17372 Al. In a bowl mixer 397.5 g of 18 DE maltodextrins was blended with 45g of OctenylSuccinated Starch (Capsul™) and 5g of lecithin. Then 30g of lemon flavor was homogeneously dispersed into the mix. Finally 22.5g of water was carefully added to the blend without forming lumps or increasing too much the temperature by the shear. The resulting mix was a free flowing powder. The powder was extruded at a through-put of 500g/h through a 0.7mm die hole using Thermo Prism 16 mm twin-screw lab extruder equipped with a cutterknife allowing to granulate the melt at the die exit. The screws configuration has two mixing zones. From the first mixing zone to the die plate, the temperature profile was 80°C-100-105-108°C.

The resulting particles have a Tg >40°C and a flavor retention >95 . The same process was carried with mint and cooling mix flavors. Example 7

Chewing gum preparation

An unflavored chewing gum base was prepared having the following ingredients in the amounts shown (Table 1). Table 1 : List of ingredients with weight by percent

Ingredient Amount (wt%)

Solsona T Gum Base (1) 12.44

Vega Gum Base (1) 12.44

Crystalline sorbitol P60W 56.50

Maltitol Syrup 11.50

Glycerin 7.00 Aspartame 0.08

Acesulfame K 0.04

(1) ex Cafosa

A Sigma-blade mixer was pre-heated to 45°C-50°C and half of the polyols (Crystalline sorbitol P60W and Maltitol Syrup) were added. The gum base was pre-heated to 60°C-65°C and added to the mixer. Mixing was carried out for approximately 4 minutes. Finally, the remaining polyols, sweeteners (Aspartame and Acesulfame K ) and humectants (Glycerin ) were added and mixing continued for 4 minutes. Example 8

Gum Preparation with Menthol/Cooling Flavor

The following samples were prepared to attain 0,5% by weight Menthol (50%) + Cooling Mix (50%) flavor of the total weight of the flavored gum formulation for each Sample (Iso- load).

Sample #1: 0.5%, by weight, the liquid flavor; 0.5%, by weight, mint; and 99% unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs. Sample #2: 8.3%, by weight, of the encapsulated flavor of Example 6; 1%, by weight, Neobee, 0.5% mint and 90.2 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs. Sample #3: 1.9%, by weight, of the encapsulated flavor of Example 5; 1%, by weight, Neobee, 0.5% mint and 96.6 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs. Sample #4: 3.3 %, by weight, of the encapsulated flavor of Example 4; 1%, by weight, Neobee, 0.5% mint and 95.2 % , by weight, unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gums was laminated and cut into slabs.

The following samples were prepared to attain 1.5%, by weight, liquid or encapsulated, Menthol (50%) + Cooling Mix (50%) flavor of the total weight of the flavored gum formulation for each Sample (Iso-dosage).

Sample #5: 1.5%, by weight (1.5%, by weight, flavor), of the liquid flavor and 98.5% unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #6: 1.5%, by weight (0.01%, by weight, flavor), of the encapsulated flavor of Example 6; 1%, by weight, Neobee; and 97.5% unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #7: 1.5%, by weight (0.35%, by weight, flavor), of the encapsulated flavor of Example 5; 1%, by weight, Neobee; and 97.5 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #8: 1.5%, by weight (0.33%, by weight, flavor), of the encapsulated flavor of Example 4; 1%, by weight, Neobee; and 97.5%, by weight, unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Example 9

Gum prepared with Mint Flavor by adding 1.5%, by weight, of the various flavor formulations (Iso-dosage)

Sample #9: 1.5%, by weight (1.5%, by weight, flavor), of the liquid flavor and 98.5% unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs (Iso-dosage).

Sample #10: 1.5%, by weight (0.01%, by weight, flavor), of the encapsulated flavor of Example 6; 1%, by weight, Neobee; and 97.5% unflavored gum from Example 8 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #11: 1.5%, by weight (0.31%, by weight, flavor), of the encapsulated flavor of Example 5; 1%, by weight, Neobee; and 97.5 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #12: 1.5%, by weight (0.38%, by weight, flavor), of the encapsulated flavor of Example 3; 1%, by weight, Neobee; and 97.5%, by weight, unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Example 10

Gum Preparation with Lemon Flavor

The following samples were prepared to attain 0.7% by weight, Lemon flavor of the total weight of the flavored gum formulation for each Sample (Iso-load).

Sample #13: 0.7%, by weight, the liquid flavor; 1%, by weight, citric acid; and 98.3% unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Sample #14: 11.7%, by weight, of the encapsulated flavor of Example 6; 1%, by weight, Neobee; 1%, by weight, citric acid; and 86.3 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs. Sample #15: 2.9%, by weight, of the encapsulated flavor of Example 5, 1%, by weight, Neobee; 1.%, by weight, citric acid; and 95.1 % unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gums was laminated and cut into slabs.

Sample #16: 5.4%, by weight, of the encapsulated flavor of Example 4 1%, by weight, Neobee; 1.%, by weight, citric acid; and 92.6 %, by weight, unflavored gum from Example 7 were combined and mixed for approximately 2 minutes to obtain a homogenous distribution. The final resulting flavored gum was laminated and cut into slabs.

Example 11

Sensory Evaluation

Twelve trained panelists evaluated each chewing gum sample for flavor intensity. Samples 1-4 and 13-16 (Menthol + cooling mix and Lemon having the same amount of flavor 0.5% and 0.7% respectively) were evaluated on a scale of 0 to 10 where 0 denotes no flavor and 10 denotes very strong flavor effect. Samples 5-12 (mint/cooling and mint) were evaluated a scale of 0 to 5 where 0 denotes no flavor and 5 denotes very strong flavor effect. Statistical Analysis was done using ANOVA with Post Hoc Duncan's test. The results for Samples 1-4 are displayed in Table 1.

Table 1

Samples 2 & 4 both had significantly more mint flavor intensity than Sample 1 after 1, 5, 10 and 15 minutes of chew (at 90% confidence). Sample 3 had more mint flavor intensity than Sample 1 after 10 and 15 minutes of chew (at 90% confidence). There was no significant difference in mint flavor intensity between any of the samples after 20 minutes of chew at 90% confidence. The results for Samples 13- 16 (Lemon Flavor) are presented in Table 2.

Table 2

There was no significant difference in lemon flavor intensity between the samples at any of the time points throughout the 20 minutes of chew (at 90% confidence).

The results for Samples 9- 12 (Mint Flavor) are presented in Table 3.

Table 3

* significantly different at 95%, ** at 99% and *** at 99.9% from Duncan comparison test The results for Samples 5-8 (Cooling Flavor) are presented in Table 4.

Table 4

*** significantly different at 99.9% from Duncan comparison test

Sample 12 out performs all the encapsulation systems up to 5 minutes with regard to the Mint flavor (Table 3) and up to 3 minutes with the Menthol + Cooling Agent mix (Table 4). Sample 8 was statistically found to be significantly different at 99.9% up to 3 minutes when compared to samples 5, 6 and 7.

Example 12

Evaluation of Chewing Gums with Atmospheric Pressure Chemical Ionization - Mass Spectrometer (APCI-MS Nose Test Samples were evaluated using APCI-MS (atmospheric pressure chemical ionization mass spectroscopy) nose in real time. The flavor load analysis of technologies was performed by the following methods:

1) Solvent extraction with diethylethenpentane 2: 1

2) Steam distillation with a Clevenger type apparatus

3) Quantization with GC-FID (using internal standard and relative response factors)

Each sample was chewed for five minutes while breath was sampled for Menthol release from the nose using the APCI-MS nose interface.

Samples 1-4 (Iso-Load Cooling + menthol (with mint) Flavor) were tested using APCI-NS nose in real time and the results are presented in Table 5 and Figure 1.

Table 5

Real Time of Mean S-l S-2 S-3 S-4

0.00 15325 33193 16199 19600

0.10 34437 126579 29918 62107

0.20 66178 513986 75796 173489

0.30 58629 464897 93880 143624

0.40 64374 576885 110467 293767

0.50 83456 464319 125386 356419

0.60 59556 353207 95024 454963

0.70 70561 388068 115510 286355

0.80 81526 508539 102981 417149

0.90 71152 356615 154268 384195

1.00 74986 337498 128230 623998

1.10 85456 271937 122149 474969

1.20 52036 327260 154188 389751

1.30 65883 208649 155303 433173

1.41 68783 311525 125640 393586

1.61 82082 274529 113067 353088 1.81 81253 276458 124382 294639

2.01 96678 222588 139492 250775

2.21 89453 196469 122277 298173

2.41 85395 191747 122978 212318

2.61 100801 194920 124752 210989

2.81 84183 158540 134458 177682

3.01 116119 166028 122710 174714

3.22 99027 175597 149538 161494

3.42 93004 165134 156012 136310

3.62 92336 167693 144995 157513

3.82 108386 141261 146647 176701

4.02 115774 135730 137757 133659

4.22 102779 174306 122294 129244

4.42 123768 158515 123280 163581

4.63 120417 169421 139239 153643

4.83 110966 132622 118658 145596

The comparison of Sample 4 vs. the other samples indicates that it outperformed Sample 1 and Sample 3 with regard to menthol intensity. There was no real difference in maximum intensity, but there was a significant temporal delay between Samples 4 and 2.

Samples 13-16 (Iso-Load Lemon Flavor) were tested by APCI-MS nose as described above. The results are presented in Table 6 and Figure 2.

Table 6

Real Time of Mean S-13 S-14 S-15 S-16

0.00 91705 1630725 2803373 71782

0.10 170521 2433294 8318229 157863

0.20 258557 8310137 7395482 300697

0.31 189610 12882806 13950902 400768

0.42 136250 9687738 15767027 601877

0.52 179421 7618496 6846614 318643 0.63 188389 7888296 9629829 413752

0.74 248113 8232934 6289378 298229

0.85 369036 4623950 5132223 272027

0.95 267698 5637191 4236606 418773

1.06 129626 3621578 4404041 813572

1.17 307259 2836960 3986527 608490

1.28 179712 5439182 3864269 636669

1.38 168140 5228974 3771433 484203

1.59 298634 3990712 2834125 537814

1.79 219515 3194645 2970111 601395

1.99 328298 3182276 1613010 463445

2.19 217799 2039083 803963 778406

2.40 232928 1711547 1276520 383546

2.60 341409 1157455 656502 531310

2.80 159318 1016572 625198 416038

3.01 310650 892351 842987 497130

3.21 274822 1318322 798625 393953

3.41 340903 1365171 679638 802513

3.61 361533 957773 1217057 467975

3.81 370735 1046363 768057 721767

4.02 542680 620693 1002660 222997

4.22 264350 682728 956522 431933

4.42 313121 931698 459631 686584

4.63 408859 1236326 454522 434805

The comparison of the lemon flavor systems shows that Sample 16 at the Iso-flavor load outperformed Sample 13 & Sample 15. The performance is slightly less in terms of maximum intensity (not believed to be sensorially significant) and there is no significant temporal difference in flavor release between Samples 14 and 16.

Samples 9-12 (Iso-Dosage - Mint Flavor) were tested (APCTMS nose) and the results are presented in Table 7 and Figure 3. Table 7

Real Time of Mean S-9 S-10 S-ll S-12

0 2754.0039 1798.8472 2165.268 2123.442

0.098 3586.9892 2284.1277 2692.447 2178.58

0.2 8278.9221 7805.9241 9241.938 11673.07

0.3 20971.008 54181.325 13846.81 42336.18

0.402 33652.717 89499.721 19197.74 113435.7

0.502 38751.305 74948.581 18248.18 246173.5

0.602 58473.9 86309.443 16028.13 297600.1

0.702 50013.651 53287.448 23060.27 235075.4

0.804 76858.371 74333.903 17517.3 267618.2

0.904 49916.392 63245.689 22165.7 308683

1.004 82578.163 49545.518 22643.25 233639.2

1.104 56196.316 48659.562 18710.4 164987.4

1.206 54813.334 42294.784 20076.04 220251.7

1.306 62236.797 35995.757 22893.53 244416.6

1.408 45193.136 32370.921 27123.18 183486.8

1.509 57245.774 32064.59 23807.19 192343.3

1.608 60649.732 29869.09 20029.18 141986.2

1.81 51812.301 24220.157 22825.35 125398.9

2.01 56132.64 20171.186 21054.47 105836.1

2.212 53112.853 18284.819 21132.39 91987.67

2.412 61042.881 15105.945 22769.56 74108.18

2.614 55464.305 15336.906 19935.24 66342.11

2.814 68984.06 14036.1 17893.31 63776.11

3.016 85972.706 12242.436 18280.08 56730.9

3.218 83123.57 12844.893 18839.57 53627.8

3.421 82085.875 11119.893 20111.29 49724.71

3.621 84351.457 10282.869 18876.98 41752.29

3.823 84170.4 9544.4467 17086.43 44314.06 4.023 77381.46 9971.295 17065.84 35992.99

4.225 84876.798 9204.4398 19422.72 32774.69

4.427 96940.151 8778.2854 18717.49 33300.94

4.627 83931.417 8705.8635 16385.89 33126.71

4.829 81786.744 8791.7421 16711.95 28136.72

5.029 99726.348 7987.4875 17816.13 27075.34

Samples 5-8 (Iso-Dosage Menthol + Cooling agent) were tested (APCI-MS nose) and results are displayed in Table 8 and Figure 4.

Table 8

Real Time of Mean S-5 S-6 S-7 S-8

0 5965.665 2760.892 2048.927 2778.232

0.098 3308.576 2408.959 3817.735 2849.723

0.2 10725.24 5924.178 11023.61 14386.88

0.3 14172.65 19298.74 10677.15 136895.6

0.402 17779.86 47314.66 12437.3 631121.1

0.502 21423.72 36528.77 11051.98 1207012

0.602 24402.97 43111.42 13828.64 1145425

0.702 18784.91 36531.9 14652.88 1022564

0.804 22835.98 35765.47 11565.82 1310693

0.904 22184.49 35239.92 14717.47 1147059

1.004 20159.43 35142.34 12879.64 666752.8

1.104 20221.49 42480.65 11715.3 693166.3

1.206 20924.65 24955.64 15356.38 638327.8

1.306 24549.87 33877.81 12374.33 507965.6

1.408 21872.85 37063.38 13878.28 480303.5

1.509 20404.23 28958.32 13226.73 574695

1.608 22895.44 26983.42 12697.85 468330.5

1.81 23700.63 25224.34 13272.82 391311.4

2.01 20071.76 21016.1 12857.81 265352.8 2.212 23613.49 21027.4 12621.37 219009.7

2.412 22459.44 18501.1 12722.82 181844.6

2.614 21222.55 17140.44 11127.51 138183.5

2.814 23368.64 14570.6 11001.66 121370.7

3.016 21828.26 14213.46 11934.97 107509.8

3.218 21499.24 14188.37 12059.99 94210.78

3.421 23999.16 13230.6 12311.71 76880.46

3.621 24753.14 12595.31 12075.22 62396.25

3.823 27280.1 10580.84 12354.06 63710.81

4.023 26388.81 10652.98 12196.49 61931.75

4.225 26198.03 9805.38 11997.76 52243.09

4.427 26537.27 8634.474 12207.72 49084.03

4.627 28714.66 8586.885 12440.02 45000.51

4.829 28234.18 9495.796 12988.75 46287.41

5.029 30059.37 9305.313 13457.37 49188.47

In both cases of iso-dosage (Mint flavor - table 7, figure 3 and Menthol - cooling agent mix, Table 8 & Figure 4), Samples 12 and 8 had higher menthol intensity and had a later time to maximum intensity than the other samples at approximately 30 seconds.

Claims

1. A composition comprising a homogenous cake comprising:

a) a plasmolysed micro-organism;

b) a flavor or fragrance; and

c) water wherein the micro-organism to water ratio in the cake is provided in an amount, by weight, of about 1.5: 1 to 1:4 and the flavor or fragrance is encapsulated in the plasmolysed micro-organism.

2. The composition as recited in claim 1 wherein the plasmolysed microorganism is yeast.

3. A glass particle or bead comprising:

a) a plasmolysed micro-organism encapsulated flavor or fragrance wherein the plasmolysed micro-organism encapsulated flavor or fragrance comprises a viscous cake formed from a mixture of water, the flavor or fragrance, and wherein the ratio of the micro-organism to the water, by weight, is in the range of about 1.5: 1 to about 4: 1 and wherein the cake is provided in an amount, by weight, of up to about 55% of the total weight of the particle;

b) a carrier comprising a polymeric water soluble emulsifier and optionally a carbohydrate wherein the polymeric water soluble emulsifier is provided in an amount, by weight, of about 5% up to about 100% of the total weight of the carrier and the carrier is provided in an amount, by weight, of about up to about 80% of the total weight of the particle.

4. The composition as recited in claim 3 wherein the composition is a granule having a size greater than or equal to about 400 nanometers.

5. The composition as recited in claim 4 wherein the granule has a size in a range of about 400 nm up to about 5 mm (millimeters).

6. The composition as recited in any one of claims 3-5 wherein the flavor or fragrance is provided in an amount, by weight, of about up to about 33% of the total weight of the particle.

7. The composition as recited in any one of claims 6 wherein the flavor or fragrance is provided in an amount, by weight, of about 12% up to about 25%.

8. The composition as recited in any of claims 1-7 wherein the microorganism is yeast.

9. The composition as recited in any one of claims 1-8 wherein the flavor or fragrance comprises a flavor.

10. A food composition comprising a composition as recited in claim 9.

11. The food composition as recited in claim 10 wherein the food composition is selecting from the group consisting of beverages, chewing gum, baked goods, candies, savory goods, and cereals.

12. The food composition as recited in claim 11 wherein the food is composition is chewing gum.

13. The composition as recited in any one of claims 3-12 wherein the flavor or fragrance comprises a cooling component.

14. The composition as recited in any one of claims 1-13 wherein the flavor is selected from the group consisting of menthol and mint.

15. A method of making a plasmolysed micro-organism encapsulated flavor or fragrance comprising: I) Combining a flavor or fragrance, a plasmolysed micro-organism and water for a time sufficient to form a viscous cake wherein the ratio of the organism to the water, by weight, is in the range of about 1.5: 1 to about 2.5: 1.

16. A method of making a glass composition comprising:

i) blending in an extruder the plasmolysed micro-organism encapsulated flavor or fragrance of claim 1 with:

a. a carrier comprised of a carbohydrate a lubricant; and

b. an emulsifier;

ii) heating the blend to a temperature sufficient to form a molten mass;

iii) extruding the molten mass;

iv) cutting the molten mass into granules; and

v) allowing the granules to cool to form a glassy particle.