The present invention relates to lactic bacteria and bifidobacteria, preferably multilayer microencapsulated bacteria with probiotic activity, and the use thereof to prepare a food product, supplement product, medical device or pharmaceutical composition or water- and/or fruit-based beverage. Moreover, the present invention relates to a process for preparing lactic bacteria and bifidobacteria, preferably multilayer microencapsulated bacteria with probiotic activity. In particular, the present invention relates to a food product selected from among water, water- and/or fruit-based beverages, milk, fresh whole milk, partially skimmed milk, powdered milk, cheese, fresh cheese, aged cheese, grated cheese, butter, margarine, yogurt, cream, milk- and chocolate- based custards, custards for sweets, jams and oily suspensions comprising bacteria, preferably multilayer microencapsulated lactic bacteria and bifidobacteria with probiotic activity.

The presence on the market of food products such as, for example, chocolate and yogurt, or supplement products such as, for example, nutritional supplements or products in the form of an oily suspension, all containing probiotic bacteria, is well known. However, said finished products exhibit some drawbacks which limit their effectiveness and use.

A first drawback relates to the stability of the bacteria present within a finished product. In practical terms, the lactic bacteria or bifidobacteria present, for example, in a finished food product suffer from low or reduced stability. The low or reduced stability is due to the environment in which the bacteria are situated. The low or reduced stability in general causes a decline in the concentration of bacteria present in the finished product over time. In practical terms, a given initial concentration of bacteria declared at tO for a given finished product declines over time because of the low stability of the bacteria themselves within said finished product .

Therefore, the initial concentration of bacteria present (at t(0), initial time of manufacture of the finished product) in the finished product does not correspond, already after a certain relatively brief interval of time after the manufacturing date, to the concentration of bacteria declared on the label, due to the decline in the concentration of bacteria that occurs over said interval of time.

To increase stability it has been proposed to coat or microencapsulate (cover) the bacteria with a coating.

However, there does not exist at present a coating or microencapsulation or covering technology capable of imparting stability to the bacteria irrespective of the nature of the finished product they will be added to, the chemical and physical properties of the finished product, the water content present in the ingredients, excipients and additives used to formulate the finished product, and the physical state of the finished product, which can be, for example, solid, in powder or granular form, liquid or in a suspension.

Therefore, it would be desirable to have a technology for coating or microencapsulating or covering the bacteria which enables the coated or microencapsulated (covered) bacteria to be prepared in such a way that said coated or microencapsulated bacteria can be used to prepare any finished product irrespective of the nature of the finished product they will be added to, the chemical and physical properties of the finished product, the water content present in the ingredients, excipients and additives used to formulate the finished product, and the physical state of the finished product, which can be, for example, solid, in powder or granular form, liquid or in a suspension.

A second drawback relates to the nature of the ingredients, excipients and additives used to formulate and prepare the finished product, such as, for example, the pH value, the free water content and the chemical composition from a qualitative and quantitative viewpoint. All of these factors, besides influencing the viability of the bacteria, can condition/modify their effectiveness once administered into the body (in vivo viability and functionality) and, consequently, prejudice their ability to colonize the intestine. In this regard it is very important to stress that the bacteria must be protected during gastric and duodenal transit, otherwise they will arrive in the intestine in a greatly reduced number and in a hardly viable state for multiplying in sufficient number.

Therefore, it would be desirable to have a technology for coating or microencapsulating (covering) bacteria which enables the coated or microencapsulated (covered) bacteria to be prepared in such a way that said coated or microencapsulated bacteria can acquire the necessary resistance enabling them to pass through gastric and duodenal transit intact.

A third drawback relates to the fact that the coatings used to coat or cover the bacteria are not such as to ensure a sufficient endurance or resistance to mechanical friction stresses that occur during mixing of the bacteria with the ingredients, excipients and additives used in the formulation of the final product. In practical terms, it often occurs that the coatings used to coat the bacteria suffer from mechanical stresses or friction that are created during the processing steps, for example during mixing of the bacteria with the various ingredients, excipients and additives necessary to formulate a finished product, be it a food product, a supplement product, a medical device or a pharmaceutical composition. The consequence is that an erosion occurs which weakens the coating, causing it to lose consistency and structure. Moreover, micro fractures (cracks) are created on the outer part of the coating, which allows the passage of humidity and substances that are toxic for the bacteria. The consequence is a loss of stability and viability and a low or reduced colonization.

Finally, there are also several considerations to be made concerning the stability of the food product itself. In practical terms, after a certain time interval, the lactic bacteria placed within a food product can give rise to precipitation phenomena and/or aggregation phenomena with the subsequent formation of a bacterial aggregate or a precipitate. These phenomena can alter the shelf life of the food product.

Therefore, there remains a need to be able to have a finished product (food product or a medical device or a supplement product or a pharmaceutical composition) comprising lactic bacteria or bifidobacteria, preferably bacteria with probiotic activity, having an improved shelf stability compared to the finished products present on the market.

There remains a need in particular to be able to have a finished product (food product or a medical device or a supplement product or a pharmaceutical composition) comprising lactic bacteria or bifidobacteria, preferably bacteria with probiotic activity, in which the concentration of bacteria initially present is not subject to a decline over time such as to lead to a drastic reduction in the concentration of bacteria initially estimated at time t(0), the time of the product's manufacture.

Finally, it is necessary for the food product containing the probiotic bacteria to be prepared in such a way as to maintain the bacteria in a good state of viability and functionality in order to ensure sufficient colonization also if the coated bacteria are placed in contact, in the formulation, with substances of a toxic character or antibiotics.

After intense research activity, the Applicant has provided an answer to the above-mentioned needs by developing a technology for coating or microencapsulating (covering) bacteria which makes it possible to produce coated or microencapsulated (covered) bacteria that do not exhibit the drawbacks of the prior art.

The subject matter of the present invention relates to multilayer coated or microencapsulated bacteria, as claimed in the appended claim.

The subject matter of the present invention relates to a method for preparing the multilayer coated or microencapsulated bacteria, as claimed in the appended claim.

The subject matter of the present invention relates to a finished product (food product or a medical device or a supplement product or a pharmaceutical composition) comprising the multilayer coated or microencapsulated bacteria, as claimed in the appended claim.

The subject matter of the present invention relates to the use of the multilayer coated or microencapsulated bacteria to prepare a finished product (food product or a medical device or a supplement product or a pharmaceutical composition) , as claimed in the appended claim.

Preferred embodiments of the present invention are set forth in the detailed description that follows, which is presented by way of example, therefore without limiting the scope of the invention .

The Applicant has found that the coating to be applied externally to the bacteria (bacterial cells) must not be formed of a single coating (or covering) layer but, on the contrary, it must be formed of at least two coating layers. The formation of a coating consisting of a single layer does not fall within the context of the present invention. The coating layers that are formed on the bacteria are in a number ^w n" comprised from 2 to 10; preferably "n" is comprised from 3 to 9; advantageously "n" is equal to 3 , or 4 , or 5 , or 6, or 7, or 8. A given amount "X" by weight of bacteria, having a concentration expressed in CFU/g, is coated or microencapsulated with a given amount "Y" by weight of a coating material comprising lipids of vegetable origin. The amount by weight Y can be less than, equal to or greater than X. The ratio by weight Y:X, relative to the final weight of the coated bacteria, can be, for example, 1:1, or 1,25:1, or 1,50:1, or 1,75:1, or 2:1. The amount Y is applied in a number "n" of layers or coatings, where in each layer or coating the amount by weight applied is equal to Y/n.

For example, 100 grams of bacteria ("X") having a concentration of 200xl0 ⁹ CFU/g can be coated or microencapsulated with 100 grams ("Y") of coating material. In this case Y is equal to X. The 100 grams ( "Y" ) of coating material are not applied to the bacteria in a single coating or microencapsulation step in order to yield bacteria with a single layer or coating (mono-coated bacteria) . On the contrary, the 100 grams ("Y") of coating material are applied on the bacteria in a number "n" of coating layers . For each coating layer that is formed, the amount of coating material applied is equal to Y/n. The coated or microencapsulated bacteria that are obtained are multilayer coated or multi- coated bacteria. The value of "n" is fixed a priori according to the properties it is desired to impart to the coated bacteria, which depends on the chemical and physical properties of the finished product they will be added to, the type of processing necessary to formulate the finished product, the water content present in the finished product, ingredients, excipients and additives used to formulate the finished product, the physical state of the finished product or the presence of toxic or antibiotic substances.

The types of lipids to be used also depend on the chemical and physical properties of the finished product the coated bacteria will be added to, the type of processing necessary to formulate the finished product, the water content present in the finished product, ingredients, excipients and additives used to formulate the finished product, the physical state of the finished product or the presence of toxic or antibiotic substances .

In this example, the 100 grams ("Y") of coating material can be applied on the bacteria in two steps (n=2) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 50 grams. The 100 grams ( "Y" ) of coating material can be applied on the bacteria as follows: 60 grams (first layer) + 40 grams (second layer) or, alternatively, 80 grams (first layer) + 20 grams (second layer) . At the end of the coating or microencapsulation process 200 grams of bacteria coated with two layers will be obtained, at a concentration of lOOxlO ⁹ CFU/g..

In this example, the 100 grams ("Y") of coating material can be applied on the bacteria in three steps (n=3). Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 33.3 grams. The 100 grams ("Y") of coating material can also be applied on the bacteria as follows: 40 grams (first layer) + 40 grams (second layer) + 20 grams (third layer) or, alternatively, 50 grams (first layer) + 25 grams (second layer) + 25 grams (third layer) . At the end of the coating or microencapsulation process 200 grams of bacteria coated with three layers will be obtained, at a concentration of lOOxlO ⁹ CFU/g.

Alternatively, for example, the 100 grams ("Y") of coating material can be applied on the bacteria in four steps (n=4) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 25 grams. The 100 grams ("Y") of coating material can also be applied on the bacteria as follows: 30 grams (first layer) + 20 grams (second layer) + 30 grams (third layer) + 20 grams (fourth layer), or, alternatively, 40 grams (first layer) + 20 grams (second layer) + 20 grams (third layer) + 20 grams (fourth layer) . At the end of the coating or microencapsulation process 200 grams of bacteria coated with four layers will be obtained, at a concentration of lOOxlO ⁹ CFU/g. Alternatively, for example, the 100 grams ("Y") of coating material can be applied on the bacteria in five steps (n=5) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 20 grams. The 100 grams ( "Y" ) of coating material can also be applied on the bacteria as follows: 40 grams (first layer) + 15 grams (second layer) + 15 grams (third layer) + 15 grams ( ourth layer) + 15 (fifth layer), or, alternatively, 30 grams (first layer) + 20 grams (second layer) + 20 grams (third layer) + 15 grams (fourth layer) + 15 grams (fifth). At the end of the coating or microencapsulation process 200 grams of bacteria coated with five layers will be obtained, at a concentration of lOOxlO ⁹ CFU/g.

For example, 100 grams of bacteria ("X") having a concentration of 200xl0 ⁹ CFU/g can be coated with 150 grams ("Y") of coating material. In this case Y is greater than X. The 150 grams ("Y") of coating material can be applied on the bacteria in a number "n" of coating layers, for example, n=3 , or 4 , or 5.

With n=2 , for example, the 150 grams ("Y") of coating material can be applied on the bacteria in two steps (n=2) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 75 grams. The 150 grams ("Y") of coating material can also be applied on the bacteria as follows: 100 grams (first layer) + 50 grams (second layer) or, alternatively, 80 grams (first layer) + 70 grams (second layer) . At the end of the coating or microencapsulation process 250 grams of bacteria coated with two layers will be obtained, at a concentration of 80xl0 ⁹ CFU/g.

With n=3 , for example, the 150 grams ("Y") of coating material can be applied on the bacteria in three steps (n=3) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 50 grams. The 150 grams ("Y") of coating material can also be applied on the bacteria as follows: 75 grams (first layer) + 50 grams (second layer) + 25 grams (third layer) or, alternatively, 60 grams (first layer) + 60 grams (second layer) + 30 grams (third layer) . At the end of the coating or microencapsulation process 250 grams of bacteria coated with three layers will be obtained, at a concentration of 80xl0 ⁹ CFU/g.

With n=4, for example, the 150 grams ( "Y" ) of coating material can be applied on the bacteria in four steps (n=4) . Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 37.5 grams. The 150 grams ("Y") of coating material can also be applied on the bacteria as follows: 50 grams (first layer) + 50 grams (second layer) + 25 grams (third layer) + 25 grams (fourth layer), or, alternatively, 60 grams (first layer) + 30 grams (second layer) + 30 grams (third layer) + 30 grams (fourth layer) . At the end of the coating or microencapsulation process 250 grams of bacteria coated with four layers will be obtained, at a concentration of 80xl0 ⁹ CFU/g.

With n=5, for example, the 150 grams ( "Y" ) of coating material can be applied on the bacteria in five steps (n=5). Therefore, in each layer or coating the amount applied is equal to Y/n, i.e. 30 grams, the 150 grams ("Y") of coating material can also be applied on the bacteria as follows: 50 grams (first layer) + 25 grams (second layer) + 25 grams (third layer) + 25 grams (fourth layer) + 25 (fifth layer) , or, alternatively, 40 grams (first layer) + 30 grams (second layer) + 30 grams (third layer) + 25 grams (fourth layer) + 25 grams (fifth) . At the end of the coating or microencapsulation process 250 grams of bacteria coated with five layers will be obtained, at a concentration of 80xl0 ⁹ CFU/g.

The lactic , bacteria and bifidobacteria are preferably probiotic bacteria. Probiotic bacteria are live bacteria capable of assuring a beneficial effect to the consumer when taken in large amounts and for an adequate amount of time.

The bacteria are coated or microencapsulated with a coating comprising or, alternatively, consisting of at least one lipid of vegetable origin. The coating is formed of a number of coating layers comprised from 2 to 10, in order to yield a multilayer coating or covering. Advantageously, n is equal to 3 , or 4 , or 5 , or 6.

The probiotic bacteria used in preparing the finished product, in accordance with the present invention, are selected from the group comprising the species: L. acidophilus, L. crispatus, L. gasseri, group L. delbrueckii, L. salivarius, L. casei, L. paracasei , group L. plantaru , L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, L. Johnsonii , B. adolescentis, B. angulatum, B. bifidu , B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudolongum, B. pseudocatenulatum and S. thermophilus.

The bacteria to be coated or microencapsulated can be in solid form, in particular in powder, granular, dehydrated powder or lyophilized form.

The bacteria are coated or microencapsulated with a coating material comprising or, alternatively, consisting, of at least one lipid of vegetable origin, using techniques and processes known to those skilled in the art.

The individual coating layers are applied/ formed with a multilayer coating or microencapsulation or multi-covering technique that envisages the formation of separate layers. The process efficiency for applying/forming a single coating layer is at least 70%, but it is usually comprised from 80 to 90%.

For example, bacteria in lyophilized form can be coated or microencapsulated using a fluid bed technique (for example, top spray or bottom spray) in which the coating material, represented by lipids of vegetable origin, is applied externally on the bacteria after being heated and turned into a liquid state. The coated probiotic bacteria are then added, using known techniques, to the finished product (food product, a supplement product, a medical device or a pharmaceutical composition), for example a food product. The food product is selected from the group comprising milk, whole fresh milk, partially skimmed milk, powdered or freeze-dried milk, cheese, fresh cheese, aged cheese, grated cheese, butter, margarine, yogurt, cream, milk- and chocolate-based custards, custards for sweets, jams and oily suspensions. The food product can also be represented by drinking water or a non-alcoholic beverage. The water or beverage can contain the coated bacteria of the present invention. For example, the coated probiotic bacteria in solid form are gradually added, under stirring, to the finished product, avoiding the formation of lumps and agglomerates. When the addition of bacteria has ended, the product is kept under stirring for a time comprised from 1 to 20 minutes at a temperature comprised from 4 to 18°C. Alternatively, the coated bacteria can be, for example, accommodated in an undercap of a bottle containing water or a beverage, for example orange-flavoured or ruit-flavoured in general. At the time of need, the undercap can be opened and the coated bacteria contained in it will fall into the beverage contained in the bottle. The bacteria can be mixed by simple stirring with water or with the beverage, which may be orange-flavoured for example.

The coating material comprises or, alternatively, consists of at least one lipid of vegetable origin. The lipids are selected from the group comprising or, alternatively, consisting of saturated vegetable fats having a melting point comprised from 35°C to 85 °C, preferably comprised from 45 to 70°C. Advantageously, from 50 to 60°C.

In a preferred embodiment, saturated vegetable fats having a certain degree of hydrophilicity and/or hydrophobicity can be used; these can be selected from the group comprising mono- and di-glycerides of saturated fatty acids, polyglycerols esterified with saturated fatty acids and free saturated fatty acids.

The saturated fatty acids can be selected from the group comprising from 8 to 32 carbon atoms, preferably 12 to 28 carbon atoms, even more preferably 16 to 24 carbon atoms. Advantageously, the lipid of natural origin is selected from the group comprising or, alternatively, consisting of:

- (i) Glyceryl dipalmitostearate E471, INCI (PCPC) : glyceryl stearate, CAS: 85251-77-0 (or 1323-83-7), EINECS: 286-490-9 (or 215-359-0). Example of a commercial product: Biogapress Vegetal BM 297 ATO -Gattefosse SAS - lipid (i);

(ii) Polyglyceryl palmitostearate E475, INCI: polyglyceryl-6-distearate, CAS: 61725-93-7. Example of a commercial product: Plurol Stearique WL 1009 -Gattefosse SAS -lipid (ii) ;

(iii) a mixture of esters of glycerol and fatty acids C16-C18, CAS: 68002-71-1, EINECS: 268-084-3. Example of a commercial product Precirol Ato 5 -Gattefosse SAS -lipid

(iii) ;

(iv) a hydrogenated vegetable fat of non-lauric origin, having a content of free fatty acids calculated as a % of oleic acid, max. 0.20%, a peroxide value of max. 0.20 meqC /Kg of saturated fatty acids, a minimum solid fat percentage at 20°C of 94% and a solid fat percentage at 40°C ranging from a minimum of 94% to a maximum of 99%. Example of a commercial name: Revel C -Loders Croklaan B.V. -lipid (iv) .

The type and chemical nature of the lipid used in the coating layer depend on the chemical and physical properties of the finished product, the water content present in the finished product the coated bacteria are added to, the ingredients, excipients and additives used to formulate the finished product, the physical state of the finished product, for example it can be a finished product in an aqueous solution (for example, milk), a finished product in powder or granular form (for example a powdered milk or a grated cheese or butter) or an oily suspension.

In the context of the present invention, "first coating layer" means the coating layer applied externally on surface of the bacteria, whereas "second coating layer" means the coating layer applied externally on said first layer and so forth for the other layers that follow.

The coated bacteria of the present invention are coated or microencapsulated (covered) with a coating comprising or, alternatively, consisting of at least one lipid of vegetable origin. Said coating is a multilayer coating formed of a number of coating layers "n" comprised from 2 to 10. When n=2 , the first and the second coating layer comprise or, alternatively, consist of a lipid of vegetable origin which is the same between them; or else when n=2 , the first and second coating layer comprise or, alternatively, consist of a lipid of vegetable origin which differs between them; said different lipid is lipid (i) .

When n is comprised from 3 to 10, the coating layers comprise or, alternatively, consist of at least one lipid of vegetable origin which is the same or differs between them.

The bacteria can be coated or microencapsulated with a coating comprising lipids of vegetable origin. Said coating is formed of a number of coating layers "n" comprised from 2 to 10. When "n" is 2, there are two coating layers. In practical terms, a double coating (two layers) is produced in succession, with two lipids differing from or the same as each other.

When "n" is equal to 2, the first and second coating layer comprise or, alternatively, consist of at least one lipid of vegetable origin which is the same between them. The lipid is selected from the group comprising or, alternatively, consisting, of lipids (i), (ii), (iii) and (iv).

The bacteria can be coated with a first coating layer comprising or, alternatively, consisting of lipid (i) and a second coating layer comprising or, alternatively, consisting of lipid (i) . The ratio by weight between said first and second coating layer is comprised from 1:3 to 3:1, preferably 1:2 to 2:1, or 1:1. The bacteria can be coated with a first coating layer comprising or, alternatively, consisting of lipid (ii) and a second coating layer comprising or, alternatively, consisting of lipid (ii) . The ratio by weight between said first and second coating layer is comprised from 1:3 to 3:1, preferably 1:2 to 2:1, or 1:1.

When "n" is equal to 2, the first and second coating layer comprise or, alternatively, consist of at least one lipid of vegetable origin which differs between them. In this case said different lipid is lipid (i) . Whereas the second lipid is selected from the group comprising or, alternatively, consisting, of lipids (ii), (iii) and (iv) .

The bacteria can be coated with a first coating layer comprising or, alternatively, consisting of lipid (i) and a second coating layer comprising or, alternatively, consisting of lipid (ii), or (iii), or (iv) . The ratio by weight between said first and second coating layer is comprised from 1:3 to 3:1, preferably 1:2 to 2:1, or 1:1.

The bacteria can be coated with a first coating layer comprising or, alternatively, consisting of lipid (ii), or (iii), or (iv) and a second coating layer comprising or, alternatively, consisting of lipid (i). The ratio by weight between said first and second coating layer is comprised from 1:3 to 3:1, preferably 1:2 to 2:1, or 1:1.

Irrespective of the specific type of lipid used, the two lipids are sprayed onto the lyophilized bacteria in succession, i.e. a double covering is applied on the lyophilizate , consisting of a first coating layer (the coating layer applied externally on the surface of the bacteria) and a second coating layer (the coating layer applied externally on said first layer) . Between said first and said second coating layer, a pause is made in order to let the bacteria with the first coating layer cool and enable the coating to solidify. Subsequently, the second coating layer is applied. The lipid to be applied is heated to the melting temperature in order to obtain a sprayable liquid form and, at that temperature, is applied on the lyophilized bacteria.

The bacteria can be coated or microencapsulated with three coating layers. In practical terms, a coating with three lipids different from or the same as each other (triple coating or triple layer) is produced in succession.

The bacteria can be coated with a first and second layer of lipid (i) and then a third layer of lipid (ii) , or with a first and second layer of lipid (ii) and a third layer of lipid (i) .

Advantageously, the stability that is achieved is maintained over time with the coated bacteria of the present invention; in particular, in an environment that is highly unfavourable to bacteria, such as that represented by water or very moist powders, it enables water- or water and fruit-based beverages to be successfully prepared.

Moreover, the coated bacteria of the present invention enable the probiotic bacteria to be formulated in an intimate mixture with antibiotics so as to prepare, for example, a capsule containing coated probiotic bacteria and antibiotics for simultaneous administration. In this manner we are able to assure that the bacteria resist the gastric barrier and the presence of antibiotics and are able to arrive intact in the intestine and colonize so as to restore the balance of bacterial flora devastated by the effect of the antibiotic.

The subject matter of the present invention also relates to a pharmaceutical composition comprising the coated lyophilized bacteria of the present invention and at least one pharmaceutical active ingredient with antibiotic activity; preferably an antibiotic can be selected from the group comprising, among others, ciprofloxacin, erythromycin or ampicillin . The Applicant conducted a series of experimental trials, the results of which are reported below.

Table A shows, by way of example, a group of microorganisms that have valid application in the context of the present invention. All of the strains were deposited in accordance with the Budapest Treaty and are made accessible to the public, on request, by the competent Depositing Authority.

The Applicant conducted experimental trials in vivo and in vitro in order to evaluate the stability and resistance to gastric juices, pancreatic juices and bile salts of the bacteria coated with two, three and four coating layers comprising the above-mentioned lipids (i), (ii), (iii) and (iv) . The tests conducted confirm that the coated bacteria (gastro-protected) are capable of withstanding the attack of gastric and pancreatic juices and bile salts and are therefore capable of arriving in the intestine live and viable and at a concentration identical to the initial one present in the product at the time of preparation.

1) Stability analysis of 3 bacterial samples of Lactobacillus rhamnosus GG (ATCC53103) in water at 25°C for 4 days, Table 1.

Sample 1: 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with a coating layer consisting of 100 grams of lipid (ii) . Ratio by weight of lyophilized bacteria : lipid (ii)= 1:1 - (mono coating).

Sample 2: 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with a coating layer consisting of 100 grams of lipid (i) . Ratio by weight lyophilized bacteria : lipid (i) = 1:1 - (mono coating).

Sample 3: 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers: the first coating layer consists of 50 grams of lipid (i), whereas the second coating layer consists of 50 grams of lipid (ii) - (double coating) . Ratio by weight of lyophilized bacteria : lipid (i)+(ii) = 1:1 - (double coating).

The coated bacteria (samples 1, 2 and 3) were placed in water in a quantity such as to ensure a concentration of 5xl0 ⁹ CFU/10 ml. The suspensions obtained were stored at 25°C in glass vials .

Table 1

BLN/g %

Sample 3

4 mortality LGG + lipid (i) + (ii) BLN/g expected BLN/g obtained

days 4 days Double coating

25°C 25°C

Total 0.62 0.63 0.58 6.45

Free 0.12 0.15 0.09 25.00

Coated 0.5 0.48 0.49 2.00 coating % 81 76 84 Table 1 shows that with an equal amount of coating material used (Y) , in this case 100 grams, the formation of two coating layers surprisingly imparts a higher stability to the cells of the coated bacteria.

2) Stability analysis of a bacterial sample of Lactobacillus rhamnosus GG (ATCC53103) in water at 25°C for 14 days, Table 2.

A sample like the one in the above trial, sample 3, was tested in water at 25°C for 14 days. Sample 3: 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers: the first coating layer consists of 50 grams of lipid (i) , whereas the second coating layer consists of 50 grams of lipid (ii) (double coating) . Ratio by weight of lyophilized bacteria : lipid (i)+(ii) = 1:1 - (double coating).

The coated bacteria (sample 3) were placed in water in a quantity such as to ensure a concentration of 5xl0 ⁹ CFU/10 ml. The suspensions obtained were stored at 25°C in glass vials.

Table 2

Table 2 shows that in an aqueous environment (highly unfavourable to bacteria) , the double coating imparts excellent stability, also for a very long period of time such as 14 days. The results of Table 2 confirm those shown in Table 1. 3) Stability analysis of a sample of powdered milk supplemented with bacteria of the strain Bifidobacterium breve BR03 (DSM 16604) at 25°C for 60 days, Table 3.

Sample a: Powdered milk + uncoated lyophilized bacteria ("naked" cells) of the strain Bifidobacterium breve BR03 (DSM 16604) -NR.

Sample b: Powdered milk + bacteria of Bifidobacterium breve BR03 (DSM 16604) . 100 grams of the strain Bifidobacterium breve BR03 (DSM 16604) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (i) , whereas the second coating layer consists of 50 grams of lipid (i) - (double coating) . Ratio by weight of lyophilized bacteria : lipid (i)+(i) = 1:1 - (double coating) . The coated bacteria (sample (b) ) were mixed with powdered milk and stored at 25°C for 60 days.

Table 3

4) Stability analysis of a sample of fresh milk supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 4°C for 7 and 14 days, Table 4 and Figure 1.

Sample 4a: Fresh milk + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) - NR.

Sample 4b: Fresh milk + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103). 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (i), whereas the second coating layer consists of 50 grams of lipid (i) -(double coating). Ratio by weight of lyophilized bacteria : lipid (i)+(i) = 1:1 - (double coating). The coated bacteria (sample (4b) ) were mixed with fresh milk and stored at 4°C for 7 and 14 days.

Table 4

5) Stability analysis of a sample of butter supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 4°C for 20, 50 and 150 days, Table 5 and Figure 2.

Sample 5a: Butter + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) - NR.

Sample 5b: Butter + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103). 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (ii), whereas the second coating layer consists of 50 grams of lipid (ii) -(double coating) . Ratio by weight of lyophilized bacteria : lipid (ii)+(ii) = 1:1 - (double coating) . The coated bacteria (sample (5b) ) were mixed with fresh butter and stored at 4°C for 20, 50 and 150 days. Table 5

6) Stability analysis of a sample of grated cheese supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 4°C for 20, 50 and 150 days, Table 6 and Figure 3.

Sample 6a: Grated cheese + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) -NR.

Sample 6b: Grated cheese + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103). 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (ii) , whereas the second coating layer consists of 50 grams of lipid (ii) (double coating) . Ratio by weight of lyophilized bacteria : lipid (ii)+(ii) = 1:1 - (double coating). The coated bacteria (sample 6(b)) were mixed with a grated cheese and stored at 4°C for 20, 50 and 150 days.

Table 6

7) Stability analysis of a sample of milk-flavoured custard for filling sweets supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 25°C for 30, 90 and 180 days, Table 7 and Figure 4.

Sample 7a: Milk-flavoured custard for filling sweets + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) -NR.

Sample 7b: Milk-flavoured custard for filling sweets + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103). 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (ii), whereas the second coating layer consists of 50 grams of lipid (ii) -(double coating) . Ratio by weight of lyophilized bacteria : lipid (ii)+(ii) = 1:1 - (double coating). The coated bacteria (sample 7(b)) were mixed with a milk-flavoured custard for filling sweets and stored at 25°C for 30, 90 and 180 days.

Table 7

Custard TO 30 days 90 days 180 days for sweets Viable cells (Billions BLN/CFU/g)

Sample 1.00 0.95 0.86 0.75

7(b)

Sample 1.00 0.22 0.05 0.02

7 (a) 8) Stability analysis of a sample of chocolate-flavoured custard for filling sweets supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 25°C for 30, 90 and 180 days, Table 8 and Figure 5.

Sample 8a: Chocolate-flavoured custard for filling sweets + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) -NR.

Sample 8b: Milk-flavoured custard for filling sweets + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103). 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (ii) , whereas the second coating layer consists of 50 grams of lipid (ii) -(double coating). Ratio by weight of lyophilized bacteria : lipid (ii)+(ii) = 1:1 - (double coating). The coated bacteria (sample 8(b)) were mixed with a chocolate-flavoured custard for filling sweets and stored at 25°C per 30, 90 and 180 days.

Table 8

9) Stability analysis of a sample of apricot-flavoured jam supplemented with bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) at 25°C for 30, 90 and 180 days, Table 9 and Figure 6.

Sample 9a: Apricot-flavoured jam + uncoated lyophilized bacteria ("naked" cells) of the strain Lactobacillus rhamnosus GG (ATCC53103) -NR. Sample 9b: Apricot-flavoured jam + bacteria of the strain Lactobacillus rhamnosus GG (ATCC53103) . 100 grams of Lactobacillus rhamnosus GG (ATCC53103) at a concentration of 200 CFU/g are coated with two coating layers -R: the first coating layer consists of 50 grams of lipid (i) , whereas the second coating layer consists of 50 grams of lipid (i) (double coating) . Ratio by weight of lyophilized bacteria : lipid (i)+(i) = 1:1 - (double coating). The coated bacteria (sample 9(b)) were mixed with an apricot-flavoured jam and stored at 25°C for 30, 90 and 180 days.

Table 9

In Tables 10, 11, 12 and 13, the following expressions are used:

T= Total

R= Coated

NR= Uncoated

Days= number of days

BLN= Billion

CFU= Colony forming units

LGG Lactobacillus rhamnosus GG (ATCC53103)

BR03 Bifidobacterium breve BR03 (DSM16604)

BS01 Bifidobacterium lactis BS01 (LMG P-21384)

LR04 Lactobacillus casei ssp. rhamnosus LR04 (DSM 16605)

LR06 Lactobacillus rahmnosus LR06 (DSM 21981)

LA02 Lactobacillus acidophilus LA02 (LMG P-21382)

LP01 Lactobacillus plantarum LP 01 (LMG P-21021)

Table 10: Survival analysis of coated bacteria (R) in contact with toxic elements.

Table 11: Survival analysis of uncoated bacteria (NR) in contact with toxic elements.

Viable cells

Apricot

Time zero Orange flavour Copper sulphate

jam

NR

%

BLN/g BLN/g % mortality BLN/g mortality BLN/g mortality

LGG 120 0.3 99.8 0.02 99.98 0.09 99.93

BR03 130 0.6 99.5 0.016 99.99 0.08 99.94

BS01 100 0.15 99.9 0.032 99.97 0.032 99.97

LR04 150 0.03 100.0 0.049 99.97 0.056 99.96

LR06 120 0.2 99.8 0.032 99.97 0.023 99.98

LA02 112 0.3 99.7 0.022 99.98 0.016 99.99

LP01 116 0.16 99.9 0.026 99.98 0.018 99.98

Table 12 : Survival analysis of coated bacteria (R) in contact with antibiotics: Ciprofloxacin (10 ug/ml) -Al,

Erythromycin (0.5 ug/ml) -A2 and Ampicillin (1 ug/ ml for lactobacilli and 0.5 ug/ml for bifidobacteria) -A3. T= Total, R= Coated, NR= Uncoated.

Table 13: Survival analysis of coated lyophilized bacteria

(NR) in contact with antibiotics:

Ciprofloxacin (10 ug/ml) -Al, Erythromycin (0.5 ug/ml) -A2 and Ampicillin (1 ug/ ml for lactobacilli and 0.5 ug/ml for bifidobacteria) -A3. T= Total,

R= Coated, NR= Uncoated.

Viable cells

A1 A2

Time zero A3

(10ug/ml) (0.5ug/ml)

NR

% /o %

BLN/g BLN/g BLN/g BLN/g

mortality mortality mortality

LGG 120 0.1 99.9 0.09 99.93 0.11 99.91

BR03 130 1.02 99.2 1.3 99.00 0.103 99.92

BS01 100 0.102 99.9 1.23 98.77 0.111 99.89

LR04 150 0.12 99.9 0.01 99.99 0.023 99.98

LR06 20 0.13 99.9 0.13 99.89 0.106 99.91

LA02 112 0.2 99.8 0.12 99.89 0.16 99.86

LP01 116 0.1 99.9 0.16 99.86 0.12 99.90

Table A

The present invention relates to the following points:

1. Bacteria coated with a coating comprising lipids of vegetable origin characterized in that said coating is a multilayer coating formed of a number of coating layers n comprised from 2 to 10, and in that:

- when n=2, a first coating layer, formed on the outer surface of the bacteria, and a second coating layer, formed on the outer surface of said first coating layer, said first and second layer comprise or, alternatively, consist of a lipid of vegetable origin which is the same between them, or

- when n=2 , a first coating layer, formed on the outer surface of the bacteria, and a second coating layer, formed on the outer surface of said first coating layer, said first and second layer comprise or, alternatively, consist of a lipid of vegetable origin represented by a glyceryl dipalmitostearate E471 -lipid (i) , said lipid (i) being present in said first layer or, alternatively, in said second layer, but not in both layers, and in that:

- when n is comprised from 3 to 10, the coating layers comprise or, alternatively, consist of at least one lipid of vegetable origin.

2. The bacteria according to point 1, wherein the lipids are selected from the group comprising the saturated vegetable fats having a melting point comprised from 35°C to 85 °C, preferably comprised from 45 to 70°C.

3. The bacteria according to point 1 or 2 , wherein the lipids are selected from the group comprising mono- and di-glycerides of saturated fatty acids, polyglycerols esterified with saturated fatty acids and free saturated fatty acids; preferably they are selected from the group comprising a glyceryl dipalmitostearate E471 -lipid (i), a polyglyceryl palmitostearate E475 -lipid (ii), a mixture of esters of glycerol and fatty acids C16-C18 -lipid (iii) and a hydrogenated vegetable fat of non-lauric origin-lipid (iv). 4. The bacteria according to any one of points 1-3, wherein when n is 2, a first and second coating layer comprise or, alternatively, consist of at least one lipid of vegetable origin which is the same between them and selected from the group comprising or, alternatively, consisting of lipids (i), (ii) , (iii) and (iv) ; preferably said first coating layer comprising or, alternatively, consisting of lipid (i) and said second coating layer comprising or, alternatively, consisting of lipid ( i ) .

5. The bacteria according to any one of points 1-3, wherein when n is 2, a first and second coating layer comprise or, alternatively, consist of at least one lipid of vegetable origin which is the same between them and selected from the group comprising or, alternatively, consisting of lipids (i),

(ii) , (iii) and (iv); preferably said first coating layer comprising or, alternatively, consisting of the lipid (ii) and said second coating layer comprising or, alternatively, consisting of lipid (ii).

6. The bacteria according to any one of points 1-3, wherein when n is 2, a first coating layer comprises or, alternatively, consists of lipid (i) and a second coating layer comprises or, alternatively, consists of lipid (ii) ,

( iii ) and ( iv) .

7. The bacteria according to any one of points 1-3, wherein when n is 2, a first coating layer comprises or, alternatively, consists of lipid (ii) and a second coating layer comprises or, alternatively, consists of lipid (i),

( iii ) and ( iv) .

8. The bacteria according to any one of points 1-3, wherein when n is 3 a first and second coating layer comprise or, alternatively, consist of lipid (i) and a third coating layer comprises or, alternatively, consists of lipid (ii) , (iii) and

(iv) or, alternatively, a first and second coating layer comprise or, alternatively, consist of lipid (ii) and a third coating layer comprises or, alternatively, consists of lipid (i) , (iii) and (iv) .

9. The bacteria according to any one of points 1-8, wherein said coated bacteria have a concentration comprised from 1x10 to lxlO ¹¹ CFU/g, preferably lxlO ⁷ to lxlO ¹⁰ CFU/g, even more preferably lxlO ⁸ to lxlO ¹⁰ CFU/g.

10. A food product or a medical device or a supplement product comprising the coated bacteria according to any one of points 1-9.

11. The food product according to point 10, wherein the coated bacteria, preferably at a concentration comprised from lxlO ⁶ to lxlO ¹¹ CFU/g or lxlO ⁷ to lxlO ¹⁰ CFU/g or lxlO ⁸ to lxlO ¹⁰ CFU/g, are introduced into a food selected from the group comprising:

- powdered milk, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product; fresh milk, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

- butter or margarine, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

- cream or yogurt, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

- grated cheese, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

- milk-flavoured custard for filling sweets, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

chocolate-flavoured custard for filling sweets, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product;

- apricot-flavoured jam, in a quantity comprised from 0.1 to 20% by weight, preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, relative to the weight of the food product .

12. A pharmaceutical composition comprising the coated bacteria according to any one of points 1-9 and at least one pharmaceutical active ingredient with antibiotic activity; preferably an antibiotic selected from the group comprising ciprofloxacin, erythromycin and ampicillin.