EP1811853A1

EP1811853A1 - Protection of bioactive food ingredients by means of encapsulation

Info

Publication number: EP1811853A1
Application number: EP05803119A
Authority: EP
Inventors: Sophie Vaslin; Anne Le Guillou; Baya Hannoucene; Thierry Saint Denis
Original assignee: Gervais Danone SA
Current assignee: Gervais Danone SA
Priority date: 2004-10-22
Filing date: 2005-10-21
Publication date: 2007-08-01
Also published as: CN101065018A; WO2006042861A1; JP2008516623A; FR2876875A1; FR2876875B1; BRPI0516996A; MX2007004879A; CA2584621A1; US20080050355A1

Abstract

The invention relates to a food product containing one or more live micro-organisms and at least one bioactive food ingredient of interest, in which the bioactive food ingredient(s) of interest is protected by means of encapsulation in a fat, such as to reduce the metabolisation thereof by the aforementioned live micro-organisms.

Description

Protection of bioactive food ingredients by encapsulation

The present invention relates to a food product containing one or more live microorganisms and at least one bioactive food ingredient of interest, wherein the living microorganism (s) and the bioactive food ingredient (s) of interest are used in such a way as to reduce the metabolisation of said bioactive ingredient (s) by said microorganism (s).

The market for food ingredients, in particular peptides, bioactive or functional (ie, having a beneficial activity for the consumer either locally in the digestive tract, or remotely in the body, after having passed into the circulatory system) is, since a few years, booming.

Bioactive peptides are defined sequences of amino acids that are inactive in their original protein, but which have particular properties once released by enzymatic action. They are also called functional peptides. These bioactive peptides are capable of exerting, inter alia, an effect on the digestive system, the defenses of the body (for example, an antimicrobial or immunomodulatory effect), the cardiovascular system (in particular an antithrombotic or antihypertensive effect), and or the nervous system (such as a sedative and analgesic effect of the opioid type) (see Tables 1 and 2 below). Table 1 below lists the main functional peptides released by the hydrolysis of the proteins of human milk and cow's milk. Table 1

( ^* ) the amino acid sequences are not exactly the same

( ^** ) H: human milk / V: cow's milk

Table 2 below lists the main physiological activities of functional peptides from milk known to date. Table 2

iaoieau <- (more)

Table 2 (continued)

O \

These peptides are most often obtained by hydrolysis of plant proteins (for example soy proteins) or animal proteins (for example, caseins or serum proteins of milk), hydrolysis generated by enzymatic and / or fermentative processes, most often accompanied by a concentration of the active fraction, a step usually necessary to provide the targeted "health benefit". The manufacture and use of these peptides for a health benefit is the subject of an abundant literature (see in particular the Danone World Newsletter N ° 17 of September 1998). Among the food vectors that can accommodate such ingredients, fermented milk products are in a good position because of their health benefits due to the presence of ferments and fermentation products (ie the molecules resulting from the transformation , by lactic acid bacteria, substrates present in the milk). Until now, the scientific community has mainly taken into account the properties of enzymes. Researchers have recently begun to take an interest in fermentation products, among which certain peptides occupy a special place because they are numerous and specific biological messengers. Fermented milk products therefore seem particularly suitable as vectors for hydrolysates of bioactive peptides obtained, for example, from dairy substrates such as caseins or serum proteins. A major problem then arises: the microorganisms and, in particular, the lactic acid bacteria used in the manufacture of fresh dairy products (such as yogurt, fermented dairy specialties, fermented milk-based beverages, etc.), are generally capable of consuming the peptides to meet their nutritional needs and, more particularly, their nitrogen requirements. We will talk about this in the following of "peptide metabolism". Lactic bacteria are indeed endowed with several systems of degradation and / or transport allowing them to metabolize the peptides, thus making them disappear from the medium:

1 / a proteolytic system (PRT wall proteases) which cuts proteins and large peptides to facilitate their assimilation ("extracellular metabolism system"),

2 / in-cell transport systems, one of which is specific for oligo-peptides of a size close to 10 amino acids, the other being suitable for the transport of di- and tri-peptides (the lactobacilli possess an additional system of tri-peptide permeases) ("system (s) of transport to the interior of the cell"), and

3 / an intracellular enzymatic system capable of degrading the peptides into amino acids (comprising about fifteen endo- and exopeptidases) ("intracellular metabolization system").

Since the amount of peptides naturally present in milk is generally too low compared to the needs of lactic acid bacteria, it is common to accelerate their growth by providing a supplement of peptides. These are then totally consumed during fermentation.

Ultimately, because of: (i) the nitrogen requirement of lactic acid bacteria, whose peptides are the main source in milk, (ii) the ability of these bacteria to consume peptides efficiently, and (iii) survival. of a large population of lactic acid bacteria in milk-based fermented products, until the expiry date (DLC), the implementation of functional peptide ingredients in fermented milk products is difficult, even impossible because these ingredients are most often consumed by lactic acid bacteria, during fermentation, or even during storage of products up to DLC.

In addition, not only is this problem of degradation by "untimely" metabolization of the peptides by the bacteria is not specific to a given peptide, but it is also not specific to a ferment (or microorganism, preferably bacteria, capable of to ferment) particular.

This is a general problem, which arises regardless of the peptide (s) and the microorganism (s) considered. By way of example, mention may be made of the bioactive peptide αS1 [91-100] (see European patent EP 0 714 910, peptide with relaxing properties contained in the hydrolyzate of milk proteins marketed in particular by the company Ingredia: 51-53, Avenue Fernand Lobbedez BP 946 62033 ARRAS Cedex, France, under the name Lactium®). The Applicant has thus observed that the population of living lactic acid bacteria in the finished product continues to metabolize the bioactive peptide during storage of the finished product, so that after only 10 days (for fresh products with a DLC of 28 days) approximately 35-55% of the peptide αS1 [91 -100] has disappeared, which is completely unacceptable to guarantee a "health" effect in the consumer (data not shown).

Since the consumption of the bioactive peptide is due to the metabolic activity of the ferments, it could be envisaged to reduce this phenomenon by destroying all or part of the microorganisms, for example by means of an appropriate heat treatment (thermization or pasteurization). In this case, it is possible to preserve the peptide αS1 [91-100] (for example after heating at 75 ° C. for about 1 min). However, such a solution has many disadvantages:

the thermisation of a fermented milk mass involves the use of stabilizers added before the heat treatment (pectins, starches, carrageenans, etc.), which complicates the process and significantly increases the cost of the formula;

the industrial manufacturing line is more complex and requires a higher specific investment;

the product no longer benefits from names related to products containing live ferments (yogurt type) and in fact loses the benefits associated with the consumption of lactic ferments; and

- The organoleptic impact, generally negative, is significant.

There is therefore a need for a food product containing both living microorganisms, for example a yoghurt, and one or more bioactive food ingredients of interest, wherein these bioactive food ingredients of interest would be protected against metabolism by said living microorganisms, while preserving the organoleptic qualities of the food product. By the present invention, the Applicant provides a solution that satisfies the existing need.

The present invention is therefore concerned with a food product containing one or more living microorganisms and at least one bioactive food ingredient of interest, characterized in that the living microorganism (s) and the bioactive food ingredient (s) of interest are placed in to reduce metabolism of said bioactive ingredient (s) by said living microorganism (s). Thus, the Applicant has been able to show that one or more bioactive food ingredients of interest could be effectively protected against metabolism by living microorganisms, provided that the conditions of implementation with each other are appropriate. Such appropriate implementation conditions may involve a variety of means, including: (a) the use of live microorganisms with reduced ability to metabolize bioactive ingredients; and / or (b) the use of lure food ingredients that are deliberately "fed" to living microorganisms; and / or c) the implementation of a physical protection of the bioactive ingredients, in particular by encapsulation of the latter.

It should be noted in this regard that one or more of, or all, these means can be advantageously combined within the same food product.

The Applicant therefore proposes a food product containing one or more living microorganisms and at least one bioactive food ingredient of interest, characterized in that the one or more bioactive food ingredients of interest are protected:

physically, the one or more bioactive food ingredients of interest being preferably encapsulated; and or

- By means of at least one food ingredient decoy contained in said food product, so that their metabolism by the living microorganism or microorganisms is reduced.

More specifically, an object of the present invention relates to a food product containing one or more living microorganisms and at least one bioactive food ingredient of interest, characterized in that the one or more bioactive food ingredients of interest are physically protected by encapsulation in a fat, so that their metabolism by the living microorganism (s) is reduced. As briefly indicated in the above general description, by "metabolized" or "metabolized" is meant according to the present invention the transformation or degradation of a substance by one or more living microorganisms, aiming at its consumption as a source of nutrients, and having as a final consequence its disappearance, more or less complete, of the medium.

For the purposes of the invention, the metaboiization of an ingredient is "reduced" if it is less than the metaboiization of the same ingredient when the latter ingredient is not protected by at least one of the means provided in the context of this invention. invention.

Advantageously, and ideally, this reduced metaboiization tends to, or is worth, zero, which amounts to little, almost no, or not, metaboiization of said ingredient.

According to a particular embodiment of the present invention, the residual amount of bioactive food ingredient (s) of interest in said food product is, 3 weeks after its preparation, between 50 and 100% approximately relative to the amount of bioactive food ingredient (s) of interest present in the product just after preparation.

Preferably, said residual amount is between about 80 and 100%. By "residual amount of bioactive food ingredient (s) of interest in said food product" is meant according to the present invention the percentage of bioactive food ingredient (s) d interest present in said food product when the latter is maintained under suitable storage conditions (for example, of the order of 4 to 10 ⁰ C for a fresh product) for 3 weeks, relative to the percentage of ingredient (s) ) bioactive food (s) of interest present at the start, that is to say right after manufacture of the product. During the implementation of the present invention, the one or more bioactive food ingredients of interest will be chosen, in particular from:

proteins, peptides,

- vitamins, - micronutrients,

analogues or derivatives thereof, and

- their combinations.

Preferably, the bioactive food ingredient of interest will be chosen from:

- proteins,

peptides,

analogues or derivatives thereof, and their combinations.

Preferably, the bioactive food ingredient of interest is chosen from: the peptide αS1 [91-100] (see European patent EP 0 714 910), the peptide C6-α _s i 194-199 (see US Pat. 514 _941), the C7-β177-183 peptide (see US patent No. 6,514,941), the peptide C12 α _s - | 23-34 (see the US patent US 6,514,941), the caseinophosphopeptides, the α -casomorphine, α-exorphin casein, casokinin, β-casomorphine, CaséinoMacroPeptides (CMP) also called GlycoMacroPeptides (GMP) or CaséinoGlycoMacroPeptides (CGMP), casoxin, casoplatellins, fragments 50-53, β-lactorphines, lactoferroxine, the Val-Pro-Pro peptides (see European patent EP 0 583 074), Lys-Val-Leu-Pro-Val-Pro-GIn (see application EP 0 737 690), Tyr-Lys-Val- Pro-GIn-Leu (see Application EP 0 737 690), Tyr-Pro (see Application EP 1 302 207 and Patent EP 0 821 968), He-Pro-Pro (see Nakamura et al., 1995; Japanese Patent JP 6 197 7 86), fragments, analogs, derivatives thereof, proteins and / or peptides containing them, and their combinations (for a review, see in particular Danone World Newsletter N ⁰ 17 September 1998). Even more preferably, the bioactive food ingredient of interest is chosen from: αS1 peptide [91-100], fragments, analogs, derivatives thereof, proteins and / or peptides containing them, and combinations thereof.

"Analogous" is understood to mean any modified version of an initial compound, here a protein or a peptide, said modified version possibly being natural or synthetic, in which one or more atoms, such as carbon atoms, d Hydrogen, oxygen, or heteroatoms such as nitrogen, sulfur, or halogen, have been added or removed from the structure of the parent compound to obtain a new molecular compound. A "derivative" within the meaning of the invention is any compound that has a resemblance or a structural unit in common with a reference compound (protein or peptide). Also included in this definition are compounds which, alone or with other compounds, may be precursors or intermediates in the synthesis of a reference compound, by one or more chemical reactions, and on the other hand compounds which can be formed from said reference compound, alone or with other compounds, via one or more chemical reactions. Thus, the definition of "derivatives" above covers at least the hydrolysates, in particular tryptic hydrolysates, of proteins and / or peptides, the hydrolyzate fractions, as well as the mixtures of hydrolysates and / or of hydrolysate fractions.

In addition, the terms "analog" and "derivative of a peptide or protein" mentioned above cover, for example, a glycosylated or phosphorylated peptide or protein or which has undergone any group grafting. chemical.

In another embodiment of the present invention, the bioactive food ingredient of interest may in particular be a sugar or a fatty acid. In the context of the present invention, only the one or more bioactive food ingredient (s) of interest is (are) encapsulated; live microorganisms are not.

By "encapsulated" or "encapsulation" is meant to designate according to the present invention the implementation of a method of protecting an active ingredient in a microparticle type vehicle to allow controlled release of this active ingredient. In this case, the active ingredient is constituted by one or more bioactive food ingredients of interest. Advantageously, encapsulation overcomes the drawbacks of the solutions of the state of the art, in that it prevents the metabolization of bioactive food ingredients of interest by living microorganisms.

In addition, encapsulation allows the bioactive food ingredients of interest to reach the level of the intestine, without being degraded, and to cross the intestinal barrier without damage in order to produce their effects.

In addition, the Applicant observes that the encapsulation of substances is quite original in the case of fermented food products, in particular fermented dairy products. Finally, it should be emphasized that, in a very interesting way, encapsulation makes it possible to obtain an organoleptically more acceptable final product, for example by masking the more or less strong bitterness of certain bioactive ingredients, in particular certain peptides. Therefore, according to a particularly preferred embodiment, the present invention relates to a food product as described above, the bitterness is reduced.

For the purposes of the invention, the bitterness of a food is "reduced" if a food is considered less bitter after a pair test (Directional Paired Comparison Method, Sensory Evaluation of Food, Harry T. Lawless, Hildegarde Heymann (1990)). This test can be performed on a panel (from 10 people) bringing together different people having integrated well what is the notion of bitterness (a learning of this notion is realized by the tasting of products containing a bitter molecule (eg, caffeine), these products being made more or less bitter according to the concentration of the said molecule) . The product according to the present invention is then blinded (panel members do not know which product is presented to them first) to the panel members, and they compare it with a product not in accordance with the invention. Of course, the two products presented to the panel differ only in that one was made in accordance with the invention, and not the other. The order of presentation of the two products from one person of the panel to another obeys the laws of chance, the number of people receiving first the product realized according to the invention being equal to the number of people receiving the other product first . Each person must indicate, for each repetition of the test, which is the most bitter product among the two products tasted.

In the context of the invention, the one or more bioactive food ingredients of interest are encapsulated in a fat. The encapsulation in a fat can in particular be carried out by dispersing the bioactive food ingredient (s) of interest in a fatty phase, where it (they) is (are) finally encapsulated (s), ( ie, emphsonné (s), retained (s)) in very fine lipid droplets. It will be noted that, in contrast with known dispersion methods and used in particular in other technical fields such as cosmetology, the implementation of such a dispersion does not require any subsequent drying step.

According to the usual meaning, "fat" or "fat" means any substance containing one or more lipids. It may be, for example, an oil, a fat, a butter, etc. This fat may be natural, then existing in various forms in animals, plants, and in their products (that is, in products derived from their metabolism), or synthetic.

A criterion for selecting the fat that can be used in the context of the present invention is related to its melting temperature. Indeed, in order to achieve a dispersion as envisaged supra, it is appropriate to use a concrete fat, that is to say solid at room temperature. Suitable oils include palm oil and its fractions, coconut oil and its fractions, palm kernel oil and its fractions, cocoa-type vegetable butter oils, margarines, hydrogenated, or partially hydrogenated vegetable oils, and the like.

The choice of the concrete fat will be done taking into account also its nutritional qualities. In this respect, for example, fractionated fats will be preferred to hydrogenated or partially hydrogenated fats.

If it is desired to disperse the bioactive food ingredient more effectively or even solubilize, the encapsulation will preferably be implemented within a multiple water / oil / water emulsion. In this case, the use of fluid fluids at room temperature (oils) may be more appropriate (rape, oleic rapeseed, soybean, sunflower, oleic sunflower, fish oils, seaweed oils, etc.). In this regard, the Applicant emphasizes the fact that, by the choice of a concrete fat, it takes advantage of its recrystallization after fusion, resulting in imprisonment and physical protection of the bioactive food ingredient.

In particular, it will be possible to choose an appropriate fat from animal fats, in particular dairy or fish fats, and vegetable fats.

It should be noted that the fats extracted from fish are particularly interesting for their high content of Omega 3 polyunsaturated fatty acids. Suitable vegetable fats include palm oil, rapeseed oil and / or fat extracted from an algae.

According to a particular embodiment of the present invention, the living microorganism (s) have an intact or reduced capacity for metabolizing the one or more bioactive food ingredients of interest.

According to the present invention, a "reduced metabolic capacity" is such that the amount of bioactive ingredients of interest metabolized during fermentation (which therefore disappears from the medium) is less than or equal to 40% of the initial amount of ingredients ( before fermentation).

This is mathematically translated by:

Q _r > 0.6 Q ₀ (I)

Or :

Q _r : quantity of residual bioactive ingredients (present in the medium after fermentation)

Q _o : quantity of initial bioactive ingredients

The amount of residual bioactive ingredients Q _r can be measured by an HPLC liquid chromatography method coupled to an MS / MS type detector. An example of an experimental procedure is given in the examples below.

The living microorganism or microorganisms will preferably be bacteria, more preferably lactic acid bacteria, living. More particularly, living bacteria will be chosen from: Streptococcus spp., Preferably Streptococcus thermophilus; o Lactobacillus spp. o Lactococcus spp. o Bifidobacterium spp.

In a preferred manner, the living bacteria will be chosen from: - Streptococcus thermophilus, deposited at the CNCM (National Collection of Cultures of Microorganisms (Institut Pasteur, Paris, France)) on 24/01/02 under number I-2774;

Streptococcus thermophilus, deposited at the CNCM on October 24, 1995 under the number 1-1630;

- Streptococcus thermophilus, deposited at the CNCM on the 10/05/04 under the number 1-3211;

Streptococcus thermophilus, deposited at the CNCM on September 16, 2004 under the number 1-3301; and Streptococcus thermophilus, deposited at the CNCM on September 16, 2004 under the number I-3302.

More preferably, said living bacteria are S. thermophilus bacteria deposited at the CNCM on 05/05/04 under the number 1-3211.

Advantageously, the food product contains at least the living bacteria S. thermophilus and Lactobacillus spp.

Preferably, said living bacteria Streptococcus thermophilus are chosen from S. thermophilus deposited at the CNCM on 24/01/02 under the number I-2774, S. thermophilus deposited at the CNCM on October 24, 1995 under the number 1-1630, S. thermophilus deposited at the CNCM on May 10, 2004 under the number 1-3211, S. thermophilus deposited at the CNCM on September 16, 2004 under the number 1-3301, and S. thermophilus deposited at the CNCM on March 16 / 09/04 under number I-3302. The content of the food product according to the invention in live microorganisms may vary and will be chosen by those skilled in the art in the light of his general knowledge in the field. In practice, a standard overall content, for example of the order of 10 ⁷ to 10 ⁹ bacteria per gram of food product, will preferably be sought. According to a particular embodiment, a food product according to the invention also contains at least one lure food ingredient. The term "lure food ingredient" is intended to denote according to the present invention a food ingredient (preferably a peptide, a protein, an analogue or a derivative thereof, and combinations thereof) capable of serving as a source of nutrients (especially nitrogen source) for living microorganisms, and intended to be preferentially metabolized by said microorganisms, so as to divert the latter from the bioactive ingredients of interest that are of course intended to be preserved as a priority. Thus, the lure ingredient represents a nutrient source for microorganisms, which is sacrificed deliberately to save as much bioactive ingredients of interest as possible. In this respect, the lure food ingredient acts as a competitive inhibitor of the transport of the bioactive ingredients of interest.

By "food product" is meant here a product for animal feed and / or human, preferably human. This product can be in any consumable form. It can be a drink such as water, vegetable juices (fruits and / or vegetables and / or cereals ...), milk, yoghourts to drink, mixtures of these. It can also be solid products or intermediate products, more or less wet. According to a particular embodiment, the food product according to the invention is a water.

Preferably, the food product according to the present invention is a fermented product. More preferably, the fermented food product is a dairy or vegetable product.

By "dairy product" is meant according to the present invention, in addition to milk, products derived from milk, such as cream, ice cream, butter, cheese, yogurt; secondary products, such as whey, casein; and any prepared food containing, as the main ingredient, milk or milk constituents. "Plant product" means, inter alia, products obtained from a vegetable base such as, for example, fruit juices and vegetable juices, including soya juice, oat juice or the rice juice. In addition, the above definitions of "dairy product" and "plant product" each cover any product made from a mixture of dairy and vegetable products, such as a mixture of milk and fruit juice, for example. The present invention also relates to a method for encapsulating at least one bioactive food ingredient of interest in a fat.

In this respect, to the extent that the addition of a bioactive food ingredient in a melted concrete fat causes an acceleration of the kinetics of crystallization thereof, it is important that the fat is completely melted before adding of the ingredient. Thus, the gradual addition and stirring of an ingredient in an aqueous phase itself with stirring allows both an induced and slow recrystallization around the ingredient, and the formation of oil droplets of suitable sizes, which the distribution is homogeneous. For the introduction of the bioactive food ingredient into a white mass, preferably fermented, it will be preferred to prepare an aqueous intermediate syrup type. This syrup will then be introduced into the white mass. The addition of the bioactive food ingredient in the aqueous medium must be done under certain conditions:

(i) total recrystallization of the fat should be avoided prior to mixing the bioactive food ingredient and the aqueous medium to ensure gradual and homogeneous addition; (ii) the temperature of the aqueous medium must be close to the melting temperature of the fat encapsulation, so as to prevent it recrystallizes on contact with the aqueous medium; and

(iii) in order to ensure a homogeneous mixture of the bioactive food ingredient and the syrup, a strong stirring (for example, using an ultraturax type stirring motor) is necessary; white emulsion having the texture of a thick cream; in addition, the light consistency of this system is easy to pump when the temperature remains on the order of the melting temperature of the encapsulation fat.

The use of a food additive such as an emulsifier during the encapsulation of the bioactive food ingredient provides a more homogeneous "population" of fat globules. Thus, in the absence of emulsifier, it has been possible to observe a majority of large fat globules (with a diameter of about 25 μm and more), and a relatively low dispersion of the peptide in the product. On the other hand, in the presence of emulsifier, the dispersion is higher and the smaller globules (with a maximum diameter of about 10 μm). According to the present invention, the encapsulation process comprises at least: a) the preparation of a water / fat mixture, to be maintained at a temperature close to the melting point of said fat, preferably within a range of ± 10 ⁰ C around said melting temperature; b) the gradual incorporation, with stirring, preferably gentle, of the bioactive ingredient of interest in the mixture obtained in step a), keeping the assembly at a temperature close to the melting temperature of said material fat, and preferably within a range of ± 10 ⁰ C around said melting temperature; and c) optionally, adding one or more food additives such as emulsifiers, thickeners, etc., and mixing, preferably strong, all while maintaining a temperature close to the melting temperature of said fat, and preferably within a range of ± 1O ⁰ C around said melting temperature.

The temperature of implementation of steps a), b) and, optionally, c) above may slightly vary from one step to another, while remaining preferably in the range of ± 1 O ⁰ C around the melting temperature of the fat. In practice, the temperature of implementation of all of these steps will advantageously be substantially the same.

The person skilled in the art may, if necessary, adapt this process according to the fat that he will use, with the help of his general knowledge and, where appropriate, by implementing simple routine experiments.

Then, for the purpose of preparing a food product according to the invention, such as a dairy product, the mixture thus obtained, maintained at a temperature close to the melting temperature of the fat, is incorporated, quite conventional, in a white mass, especially through pump systems and, preferably, after the heat treatment and fermentation steps thereof. The present invention also relates to a process for the preparation of a food product as described above, in which one or more bioactive food ingredients of interest are encapsulated, for example according to the method described above, before being added to the mixture intended to constitute said food product. According to a particular embodiment of the present invention, the encapsulated bioactive food ingredient (s) of interest are added to said mixture with stirring. When the food product is fermented, the encapsulated bioactive food ingredient (s) of interest can be added to the mixture before or after fermentation.

However, it will be preferred to add the bioactive ingredients encapsulated after fermentation to preserve as much as possible the integrity of the fat particles vis-à-vis the possible heat treatment steps. According to one embodiment, the preparation method according to the present invention is such that the living microorganism (s) and the encapsulated bioactive food ingredient (s) of interest are added one after the other in the mixture intended to constitute said food product.

Alternatively, the living microorganisms and encapsulated bioactive food ingredients of interest are added simultaneously to the mixture intended to constitute said food product. The culture conditions of the microorganisms depend on said microorganisms and are known to those skilled in the art. For example, it will be specified that the optimum growth temperatures for S. thermophilus are generally between about 36 ° and 42 ° C. they are between 42 ° and 46 ⁰ C for L. delbrueckii spp. bulgahcus (which is typically found in yoghurt).

As a general rule, the fermentation stoppage, which depends on the desired pH, is obtained by rapid cooling, which slows down the metabolic activity of the microorganisms. The present invention further relates to the use of a food product as described above as a functional food.

By "functional food" is meant a food product that advantageously affects one or more target functions of the body, regardless of its nutritional effects. This can result in an improvement of the state of health and / or well-being and / or a reduction of the risks of occurrence of diseases in a consumer who ingests normal amounts of said product. As examples of activities of a "Functional food" may include anti-cancer, immunostimulatory, bone health promoting, anti-stress, opioid, antihypertensive, calcium bioavailability enhancing, or antimicrobial (Functional Food Science in Europe) activities. , 1998).

Such functional foods may be intended for humans and / or animals.

The present invention also relates to the use of a fat to encapsulate at least one bioactive food ingredient of interest intended to be incorporated into a food product containing one or more living microorganisms.

The present invention is illustrated, without being limited, by the following figures:

Figure 1: Chromatogram LC-MS illustrating the disappearance of the bioactive peptide αS1 [91-100] included in the ingredient Lactium® during lactic fermentation. The MS / MS detector is set to display only the signal of ions of m / z = 634.5 Da (mass of peptide αS1 [91-100] doubly charged) which, after fragmentation, have ions of z = 991.5 Da; 771.5 Da; 658.3 Da (characteristic fragments of the peptide αS1 [91-100]).

Figure 2: Identification and quantification of the main peptides of the Lactium® ingredient by LC-MS / MS before and after fermentation of the dairy "mix" with a ferment consisting of a mixture of strains I-2783 (deposited at the CNCM on 24 / 01/02), I-2774 (filed at the CNCM on 24/01/02), I-2835 (filed at the CNCM on 04/04/02) and 1-1968 (filed at the CNCM on 14/01 / 98). After fermentation, these peptides are only found in trace amounts and merge with the baseline. "? Means that the identification of the sequence was not possible or is not certain; only the mass of the peptide is then reported. FIG. 3: Compared peptide profiles (LC-MS / MS chromatograms) of a dairy "mix" containing 1.5 g / L of DMV C 12® hydrolyzate, before (1) and after fermentation (2) up to pH 4.7 by the Hansen YC380 lactic ferment. Almost all the peptides of the hydrolyzate, including the bioactive C12 peptide (fragment αS1 [23-34]), disappeared following metabolism by the ferment strains.

FIG. 4: Curves illustrating the evolution of the residual content of bioactive peptide αS1 [91-100] in a finished product constituted of 95% fermented mass by the ferment containing strains I-2783, I-2774, I-2835 and 1-1968, and 5% flavored sugar syrup containing the peptide αS1 [91-100], during storage at 10 ^° C. The experiment was carried out at the rate of 4 independent tests E1, E2, E3 and E4 . FIG. 5: Curves illustrating the evolution of the residual content of bioactive peptide αS1 [91-100], added after fermentation in a fermented product then thermised at 75 ° C. for 1 minute, and stored at 10 ^° C. until the date consumption limit (DLC).

Figure 6: Illustration of the evolution of the content of bioactive ingredient encapsulated over time (storage at 4 ⁰ C). FIG. 7: Compared Peptide Profiles (LC-MS / MS Chromatograms) of a milk mix containing 1.5 g / L of Lactium® provided in emulsion form in palm oil according to the present invention, immediately after addition to the fermented mass (1) and after 14 days storage at 10 ⁰ C (2). Other features and advantages of the present invention will appear on reading the following examples, given purely by way of illustration.

Examples

Example 1: Implementation of Bioactive Ingredients of Interest Without Applying the Claimed Invention 1.1) Example with the bioactive peptide αS1 [91-100] contained in the hydrolyzate Lactium®

The implementation of ingredients of the peptide or protein type, often made in the form of powders, is simpler when they are added during the step of preparation of the dairy "mix" (powdering of the milk), before the treatment thermal sanitation (ie, 95 ° C, 8 min) and, therefore, before fermentation. In this case, the risk of metabolizing the active peptide is very high. For the example, this is the case when using a functional ingredient such as Lactium® (Ingredia, France) containing a bioactive peptide (fragment 91-100 of casein αS1). Protocol: The medium was prepared by hydration of a skimmed milk powder at 120 g / L, supplemented with 1.5 g / L of Lactium® ingredient (corresponding to about 30 mg / L of bioactive peptide αS1 [91-100] ), then pasteurized at 95 ° C for 8 minutes.

The lactic ferment was added at a rate of 0.02%, and the fermentation was conducted at the optimum temperature of the selected ferment (between 37 and 42 ° C) until a pH of 4.70 was reached. The analysis of the residual peptides, and in particular that of the bioactive peptide αS1 [91-100], was carried out using a liquid chromatography method HPLC coupled to an MS / MS type detector as described below:

the sample was prepared by dilution of the fermented medium in a mixture of water, methanol and trifluoroacetic acid (50/50 / 0.1%) in a ratio of 1 to about 6. The supernatant after centrifugation constituted the representative sample of the peptide content of the fermented medium.

This sample was injected into an Agilent 1100 HPLC chromatography system (Agilent Technologies France, 1 rue Galvani 91745 Massy Cedex, France), equipped with a column adapted to the analysis of peptides, Waters Symetry® type (5 .mu.m 2.1 x 150 mm, WAT056975, Water France, 5, Rue Jacques Monod, 78280 Guyancourt) at a temperature of 40 ⁰ C, flow rate of 0.25 ml / min. The peptides were eluted conventionally with an increasing gradient of solvent B (Acetonitrile + 0.100% of formic acid) in the solvent A (Water + 0.106% formic acid) over a period of 40 minutes to 2 hours depending on the desired resolution.

The detection was carried out using a specific MS / MS type detector, for example with an ion trap device such as IΕsquire3000 + (Bruker Daltonique, rue de l'Industrie, 67166

Wissembourg Cedex), set for either the global analysis of the peptide content (MS-MS mode), or for the precise and specific quantification of a peptide from its characteristic fragments. For example, the peptide αS1 [91-100]) was isolated from its mass (doubly loaded ion of mass 634.5 Da) and quantified from the intensity of its characteristic son ions after fragmentation (ions of m / z of 991.5 Da, 771.5 Da and 658.3 Da). Even more precisely, an internal standard consisting of the same deuterated synthetic peptide twice (characteristic fragment of 993.5 Da) made it possible to take into account and eliminate any interferences related to the matrix.

The results are illustrated in Figure 1.

When it is used at this stage (before fermentation with a ferment consisting of a mixture of I-2783 strains (deposited at the CNCM on 24/01/02), I-2774 (deposited at the CNCM on 24/01) 02), I-2835 (filed at the CNCM on 04/04/02) and 1-1968 (filed at the CNCM on 14/01/98), or a ferment such as YC380 (Hansen SA, Le Moulin d 'Aulnay, BP64, 91292 ARPAJON Cedex France), it has been demonstrated that more than 95% of the bioactive peptide αS1 [91-100] were consumed after fermentation.These observations show that the incorporation of bioactive peptides in accordance with the above is not applicable as it stands obtaining food products, especially dairy products, supplemented with quantities of peptides and / or bioactive proteins sufficiently stable over time to observe the desired effect on the consumer.

1. 2) Examples with other bioactive peptides of interest

The results are illustrated in Figures 2 and 3.

The Lactium® ingredient contains many other peptides, some of which potentially have biological activity (such as αS1 casein fragment 23-34, which is also commercially available in DMV International's C12 ingredient). It is interesting to note that almost all the peptides provided by the addition of Lactium® are widely consumed during fermentation.

Whatever their origin (from different caseins αS1, αS2,

K, β) and their size (from 2 to 3 residues up to 12 residues and more), all the peptides are generally consumed during the fermentation process.

1.3) Implementation of the bioactive peptide αS1 [91-100] (Lactium®) with other enzymes

In order to verify that this phenomenon was not peculiar to the two ferments used in paragraph 1.1) above, the main industrial ferments, as well as various pure strains included in the composition of these ferments, were tested on the basis of the same test: reconstituted milk from milk powder, to which Lactium® was added at a dose of 1.5 g / L, was fermented under standard conditions (optimum temperature of the ferment between 37 and 42 ° C, stop fermentation at pH 4.7, 2 repetitions). Analysis of the level of bioactive peptide αS1 [91-100] was then performed on the sample before and after fermentation. The results obtained on the pure strains are presented in Table 3 below:

Table 3

In Table 3 above, which reflects the consumption of the bioactive peptide αS1 [91-100] by different ferments and industrial strains during the fermentation of a milk mix containing 1.5 g / L of Lactium®, the pure strains were identified by their respective number and filing date with the CNCM (Institut Pasteur, Paris, France).

Table 3 shows that all the enzymes and strains tested metabolize from 94 to 100% of the bioactive peptide αS1 [91-100] during the fermentation of a standard dairy mix. The implementation of this ingredient is therefore impossible under conventional conditions to produce food products, including dairy, containing peptides and / or bioactive proteins in amounts sufficiently stable over time to produce an effect in the consumer.

In addition, to verify that this phenomenon was not specific to the ingredient Lactium®, several combinations of enzymes and other ingredients based on bioactive peptides were studied using the same test (reconstituted milk + ingredient). to test at a dose of 1, 5 g / L, fermented under standard conditions, fermentation stop at pH 4.7, 2 repetitions). The various combinations tested are reported in Table 4 below. Table 4

The C12 and CPP ingredients produced by DMV International are hydrolysates of milk proteins containing bioactive peptides targeting the control of hypertension and the assimilation of minerals respectively.

On the whole of the experiments, it appeared that all the ferments tested have an important capacity of metabolizing peptides, whatever their nature and their size.

1.4) Addition after fermentation

A logical alternative to the procedure studied above is to introduce the functional ingredient after fermentation ("delayed differentiation" type process), for example with the syrup used to aromatize the fermented mass. The implementation of the same amount of Lactium ® ingredient according to this protocol leads to the results illustrated in FIG. 4.

As shown in FIG. 4, even added cold (4 ° C.) after fermentation, the active peptide (provided by the equivalent of 1.5 g of Lactium® per kg of finished product) is rapidly degraded during storage, so that leave only 30 to 40% of the initial quantity at the use-by date (DLC). Thus, the population of living lactic acid bacteria in the finished product continues to metabolize the bioactive peptide during storage of the finished product, so that after only 10 days (for fresh products with a 28-day DLC), between 35 and 50% of the peptide αS1 [91-100] have disappeared, which remains unacceptable to obtain the desired effect in the consumer.

1.5) Thermal treatment of the fermented dairy product containing the bioactive ingredient of interest In this case, it is possible to ensure the stability of the peptide αS1 [91-100] (Figure 5), but to the detriment of the overall quality of the product finished. This solution has indeed many disadvantages:

the thermization of a fermented milk mass involves the use of stabilizers added before the heat treatment (pectins, starches, carrageenans, etc.), which complicates the process and substantially increases the cost of the formula; the industrial manufacturing line is more complex and requires a higher specific investment; - the product no longer benefits from names related to products containing live ferments (yogurt type) and in fact loses the benefits associated with the consumption of lactic ferments; - the organoleptic impact (generally negative) is significant.

Example 2: Implementation of bioactive ingredients of interest by applying the claimed invention

2.1) Step 1: encapsulation of the bioactive food ingredient Selected encapsulation oil: palm oil (melting point = 37 ° C). Suppliers of this type of oil are numerous (eg, Cargill). The oil was melted at 50 ⁰ C for a total absence of crystals, and the Lactium ^® containing the bioactive food ingredient αS1 [91-100] was added gradually with magnetic stirring (maintained at 50 ° c).

2.2) Step 2: manufacture of the aqueous medium of the syrup type

A kind of syrup was made from water and premix palm oil / bioactive food ingredient. The bioactive food ingredient was gradually added to water (30 ^° C.) with stirring with ultraturax (22,000 rpm), the palm content in the solution being arbitrarily set at 30%.

The emulsion obtained was easily pumpable at 30 ^° C.-35 ^° C., but became increasingly firm as soon as the temperature was below 25 ° C.-30 ° C. (gradual recrystallization of palm oil). The use of a food emulsifier (eg, Lactem® supplied by Danisco) at a level of 0.5% / MG resulted in a less heterogeneous population of fat globules.

2.3) Step 3: addition of the emulsion in the fermented white mass.

As illustrated by Figures 6 and 7, the bioactive ingredient content is stable over time, indicating that a bioactive food ingredient can be effectively encapsulated in accordance with the present invention.

Thus, in addition to the peptide αS1 [91-100] whose stability is shown in FIG.

6, other peptides, such as the bioactive peptide αS1 [23-34] (present in ingredient C12 from DMV International), are also well protected (Figure 7). The disappearance of peptides other than those of interest

(Fig.7, (1), symbolized by the arrows) contributes to the decrease of the overall peptide load and, therefore, presumably to the loss of bitterness of the finished product. REFERENCES

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Claims

1. Food product containing one or more live microorganisms and at least one bioactive food ingredient of interest, characterized in that said bioactive food ingredient(s) of interest are physically protected by encapsulation in a fatty material, so that their metabolism by said live microorganism(s) is reduced.

2. Food product according to claim 1, characterized in that the residual quantity of bioactive food ingredient(s) of interest in said food product is, 3 weeks after its preparation, between 50 and 100 approximately % compared to the quantity of bioactive food ingredient(s) of interest present in the product just after its preparation.

3. Food product according to claim 2, characterized in that said residual quantity is between approximately 80 and 100% relative to said quantity of bioactive food ingredient(s) of interest present in the product just after its preparation.

4. Food product according to any one of claims 1 to

3. characterized in that the said bioactive food ingredient(s) of interest are chosen from:

- proteins,

- peptides, - vitamins, micronutrients,

- analogues or derivatives thereof, and - their combinations.

5. Food product according to claim 4, characterized in that the said bioactive food ingredient(s) of interest are chosen from: - proteins,

- peptides,

- analogues or derivatives thereof, and

- their combinations.

6. Food product according to claim 4 or 5, characterized in that the said bioactive food ingredient(s) of interest are chosen from: the peptide αS1 [91-100], the peptide C6-α _s i 194-199, the peptide Cl- β177-183, C12-α peptide _s i 23-34, caseinophosphopeptides, α-casomorphin, α-casein exorphin, casokinin, β-casomorphin, caseinomacropeptides and glycomacropeptides, casoxin, casoplatellins , fragments 50-53, β-lactorphins, lactoferroxin, peptides Val-Pro-Pro, Lys-Val-Leu-Pro-Val-Pro-GIn, Tyr-Lys-Val-Pro-Gln-Leu, Tyr -Pro, Ile-Pro-Pro, fragments, analogues, derivatives thereof, proteins and/or peptides containing them, and combinations thereof.

7. Food product according to any one of claims 1 to 6, characterized in that it has reduced bitterness.

8. Food product according to any one of claims 1 to 7, characterized in that said fat is chosen from animal fats, in particular dairy or fish, and vegetable fats.

9. Food product according to claim 8, characterized in that said vegetable fat is chosen from palm oil, rapeseed oil and a fat extracted from an algae.

10. Food product according to any one of claims 1 to

9, characterized in that the said living microorganism(s) have an intact or reduced capacity for metabolizing the said bioactive food ingredient(s) of interest.

11. Food product according to any one of claims 1 to

10, characterized in that the said living microorganism(s) are bacteria, preferably live lactic acid bacteria.

12. Food product according to claim 11, characterized in that said living bacteria are chosen from:

- Streptococcus spp, preferably Streptococcus thermophilus; - Lactobacillus spp;

- Lactococcus spp;

- Bifidobacterium spp.

13. Food product according to claim 12, characterized in that it contains at least the live bacteria S. thermophilus and Lactobacillus spp.

14. Food product according to claim 12 or 13, characterized in that said live bacteria S. thermophilus are chosen from: - Streptococcus thermophilus, deposited at the CNCM on 01/24/02 under number I-2774;

- Streptococcus thermophilus, deposited with the CNCM on 10/24/95 under number 1-1630;

- Streptococcus thermophilus, deposited with the CNCM on 05/10/04 under number 1-3211; - Streptococcus thermophilus, deposited with the CNCM on 09/16/04 under number 1-3301;

- Streptococcus thermophilus, deposited with the CNCM on 09/16/04 under number I-3302.

15. Food product according to claim 14, characterized in that said live bacteria are S. thermophilus bacteria deposited at the CNCM on 05/10/04 under number 1-3211.

16. Food product according to any one of claims 1 to 15, characterized in that it also contains at least one lure food ingredient.

17. Food product according to any one of claims 1 to 16, characterized in that it is a drink, preferably water.

18. Food product according to any one of claims 1 to

17, characterized in that it is a fermented product.

19. Food product according to any one of claims 1 to

18, characterized in that it is a dairy or vegetable product.

20. Process for encapsulating at least one bioactive food ingredient of interest in a fat, comprising at least:

a) the preparation of a water/fat mixture, to be maintained at a temperature close to the melting temperature of said fat, preferably within an interval of + 10 ⁰ C around said melting temperature; and b) the gradual incorporation and with stirring of the bioactive ingredient of interest into the mixture obtained in step a), while maintaining the whole at a temperature close to the melting temperature of said fat, preferably within an interval of ± 10 ⁰ C around said melting temperature.

21. Method according to claim 20, further comprising adding one or more food additives such as emulsifiers, thickeners, etc., and mixing the whole, while maintaining a temperature close to the temperature of melting of said fatty material, preferably within an interval of ± 10°C around said melting temperature.

22. Process for preparing a food product according to any one of claims 1 to 19, characterized in that said bioactive food ingredient(s) of interest are encapsulated before being added to the mixture intended to constitute said food product.

23. Method according to claim 22, characterized in that said bioactive food ingredient(s) of interest are encapsulated in accordance with the method according to claim 19 or 20.

24. Method according to claim 22 or 23, characterized in that said encapsulated bioactive food ingredient(s) of interest are added to said mixture with stirring.

25. Method according to any one of claims 22 to 24, characterized in that:

- said food product is fermented; And

- said encapsulated bioactive food ingredient(s) of interest are added to said mixture before or after, preferably after, fermentation.

26. Use of a food product according to any one of claims 1 to 19 as a functional food.

27. Use of a fat to encapsulate at least one bioactive food ingredient of interest intended to be incorporated into a food product containing one or more live microorganisms.