EP1802652A1

EP1802652A1 - Ami-deficient streptococcus thermophilus strains with reduced post-acidification

Info

Publication number: EP1802652A1
Application number: EP05801557A
Authority: EP
Inventors: Peggy Garault; Jean-Michel Faurie; Jérôme MENGAUD; Anne Druesne; Gaëlle QUERE-CHASSANG
Original assignee: Gervais Danone SA
Current assignee: Gervais Danone SA
Priority date: 2004-10-22
Filing date: 2005-10-21
Publication date: 2007-07-04
Also published as: FR2877012B1; JP2008516624A; FR2877012A1; WO2006042862A1; BRPI0516230A; CN101103041A; MX2007004880A; US20070292561A1; CA2584625A1

Abstract

The invention relates to Ami-deficient wild-type strains of Streptococcus thermophilus with reduced post-acidification. The invention also relates to the use of said strains for the preparation of fermented food products and to the fermented food products thus prepared.

Description

STREPTOCOCCUS THERMOPHILUS AMI DEFICIENT STRAINS WITH REDUCED POST-ACIDIFICATION

The present invention relates to the field of lactic acid bacteria useful in the food industry.

More specifically, the invention relates to natural strains of Streptococcus thermophilus, or natural variants of such strains, said strains or variants being AMI deficient in reduced post-acidification. The invention also relates to the use of these strains for preparing fermented food products, as well as the fermented food products themselves.

The choice of lactic acid bacteria for the production of food products, especially dairy products, ferments involves various criteria, among which the acidifying activity and the formation of aromatic components which are responsible for the organoleptic properties of the product, as well as the production of thickening agents which play a role on texture and smoothness.

The acidifying activity is essentially characterized by three parameters: (i) the kinetics of acidification; (ii) the titratable acidity or the final fermentation pH which conditions the organoleptic properties of the product and its suitability for preservation; and (iii) the post-acidification which develops during the conservation of the product. A high acidification speed makes it possible to reduce the period during which the preparation is sensitive to contaminants, and thus to reduce the risk of bacterial contamination.

Increasing the rate of acidification also improves the economics of the process by increasing the productivity and flexibility of industrial equipment. The post-acidification properties of the strains are particularly important for the preservation of the products. In fact, the fresh fermented products are generally stored at temperatures between approximately 4 ^° C. and 8 ^° C. for a period not usually exceeding 4 weeks. However, if the metabolic activity of bacteria is reduced by cold storage, it is not blocked. This remanent activity leads to the production of lactic acid from lactose, which results in a decrease in pH and an increase in acid taste which degrade the organoleptic properties of the product.

In addition, in addition to the criteria retained for their contribution to the quality of the product, other elements more specifically linked to the process are involved in the choice of strains, for example the fermentation temperature, the rate of acidification and the resistance to phages.

Phage resistance is a very important criterion in the choice of strains in order to reduce the risk of phage incidents in production, which are likely to block the whole production for decontamination, over more or less long periods.

French patent FR 2 725 212 describes a strain of S. thermophilus, deposited in the National Collection of Cultures of Microorganisms (CNCM, Institut Pasteur, Paris, France) under the number I-1477, which exhibits rapid acidification kinetics allowing to reach a high degree of acidity, and which does not post-acidify during the conservation of fresh fermented dairy products.

Another strain of S. thermophilus (deposited on December 30, 1994 with the CNCM under the number 1-1520) is described in French patent FR 2 771 600 as having a reduced post-acidification capacity. However, it is not advantageous to use such strains to manufacture functional foods (ie, foods which have a beneficial effect on one or more target functions of the organism) comprising in particular bioactive food ingredients of interest based proteins and / or peptides. These strains remain in fact capable of metabolizing (that is to say degrading in order to use as nitrogen sources) the proteins and / or peptides, including the functional proteins and / or peptides of interest, that l 'we obviously intend to preserve. This is why it is important for the food industry to have fermentative bacteria which have both: (i) excellent viability; (ii) a very weak or almost zero post-acidifying activity; and (iii) an ability to metabolize proteins and / or peptides, also very weak or almost zero. The food industry must also take into account a fourth constraint when developing products, intended for human and / or animal food, in which microorganisms are incorporated, more particularly living microorganisms. Genetically modified organisms (here microorganisms) (GMOs or mutants) generally arouse fear and apprehension in consumers. This negative image from which GMOs suffer is such that the public tends to "boycott" foods containing GMOs. Also, in a context where consumers are always demanding more transparency as to the content of the food products offered to them and as to the origin of the ingredients that these products contain, manufacturers prefer to offer GMO-free products. It is therefore essential that food products from industry and containing microorganisms be prepared using exclusively natural strains or natural variants of natural strains. It is precisely to all the constraints mentioned above that the present invention meets, by proposing natural strains of S. thermophilus, or natural variants of such strains, said strains or variants being AMI deficient with reduced post-acidification. .

The growth of S. thermophilus in milk is broken down into three growth phases:

- a first phase of exponential growth, which does not depend on milk caseins; - a second phase of growth known as "transition"; and

- a third phase of exponential growth, no doubt limited by the transport of peptides (Letort et al., 2002).

The oligopeptide transport system in S. thermophilus is an ABC transporter (ATP-Binding Cassette) made up of a translocon of four proteins (AmiC, AmiD, AmiE and AmiF) and three binding proteins very homologous to each other (AmiA1, AmiA2 and AmiA3). The growth in milk of mutants in which the transport system of the oligopeptides is altered is no longer optimal, which shows the importance of this transport system (Garault et al., 2002). In the context of the present invention, the Applicant has for the first time demonstrated natural strains and natural variants of strains of S. thermophilus, in which the oligopeptide transport system is at least partially altered. The Applicant has also shown that such strains and variants have particularly advantageous milk acidification properties.

On the one hand, the pH stabilizes at the end of fermentation for deficient AMI variants. In fact, the first phase of growth in milk is not affected, and these variants can retain a first phase of relatively rapid acidification while having a second phase of much slower acidification. They can present an acidification curve having a strong slowing down at a pH higher than that for which acidification by a wild functional AMI strain is slowed down. This “acidification tray”, at fermentation temperature, is interesting insofar as it allows the fermented mass to be stored longer at fermentation temperature, without excessively acidification. This gives appreciable flexibility in the implementation of industrial processes. In fact, the time taken for the fermentation tanks to decut off is long, given their volume. The use of deficient AMI variants therefore makes it possible to limit the evolution of the pH of the fermented milk during the racking stage (cutting with cooling or direct packaging).

On the other hand, the pH threshold for which the acidification strongly slows down depends on the quantity of free amino acids and di- / tripeptides which can be transported by the AMI system and present in milk. It is therefore possible to control the stop pH of fermentation by modifying the content (quantitative and / or qualitative) of milk peptides.

The subject of the present invention is therefore a natural strain of S. thermophilus AMI deficient with reduced post-acidification, and its natural variants AMI deficient with similar milk acidification characteristics, as well as their cultures and fractions of biologically pure cultures.

For the purposes of the present invention, the term “deficient natural AMI strain” or “deficient natural AMI variant” means a strain or variant whose growth is stimulated by a mixture of free amino acids, and not by a mixture consisting solely oligopeptides. The term “natural variants having similar milk acidification characteristics” is intended to denote strains obtained without genetic manipulation, from a natural reference strain, the strains thus obtained being, like the reference strain, post- reduced acidification. In the present context, the terms “variant” or “natural variant” will be used to denote a strain obtained mainly by mutation and selection. from the reference strain; while the term “mutant” will be more specifically reserved for a strain obtained by site-directed mutagenesis techniques, in particular by genetic transformation using vectors, applied to the reference strain. According to one embodiment, the natural strain and / or the natural variant are resistant to a toxic oligopeptide analog transported by the AMI oligopeptide transport system.

In particular, said toxic analog may be chosen from aminopterin, triornithine and trilysine. This analog will preferably be aminopterin.

We will describe here as “resistance to aminopterin”, the fact that, in the presence of a paper disc soaked with 200 μg of aminopterin, an inhibition diameter less than or equal to about 2 cm is observed, preferably less than or equal to about 1.8 cm, on a box seeded with 100 μl of saturated bacterial suspension.

Such a definition will be extended mutatis mutandis by those skilled in the art to other toxic analogs of oligopeptides transported by the AMI system in the light of their general knowledge. Thus, a person skilled in the art will be able to vary the maximum values of the inhibition diameters appropriately as a function of the toxic analogs considered.

Preferably, a strain in accordance with the present invention is chosen from:

- the strain deposited at the CNCM on May 10, 2004 under the number 1-3211; - the strain deposited at the CNCM on 09/16/04 under the number I-3301;

- the strain deposited at the CNCM on 09/16/04 under the number I-3302;

- the strain deposited at the CNCM on 24/01/02 under the number I-2774.

By way of example of interesting acidifying characteristics within the meaning of the present invention, and as shown in the following examples, it will be specified that the AMI-deficient strain I-3302

(variant of the functional AMI mother strain I-3299 deposited at the CNCM 09/16/04) allows a pH variation of 0.29 between the pH at the end of incubation and the pH of the product at the end of life, while the functional AMI mother strain I-3299 gives result in a pH variation of 0.56 between these two times. In this case, the advantage of using an AMI strain deficient compared to a functional AMI strain therefore lies in a reduction of 0.27 in the pH variation between the end of incubation and the end of life of the product.

As indicated above, the deficient AMI variants are obtained from a reference mother strain. Thus, in the context of the present invention, these will be natural variants obtained for example by mutation and selection according to their acidifying properties. The techniques for doing this are known, as are the tests making it possible to control the acidifying properties (in particular Spinnier H. E., Corrieu G., 1989). Unlike strains 1-3211, 1-3301, and I-3302, which are natural AMI variants deficient in functional AMI mother strains (see the experimental part below), strain I-2774 was isolated and identified by the Applicant as being naturally AMI deficient. The strains according to the invention are particularly advantageous for the preparation of fermented food products.

Preferably, such fermented food products are dairy or vegetable products.

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; as well as any prepared food containing, as a main ingredient, milk or milk constituents (eg infant formula). By “plant product” is meant, inter alia, products obtained from a plant base such as, for example, fruit juices and vegetable juices including soybean juice, oat juice or rice juice.

In addition, the above definitions of "dairy product" and "vegetable product" each work any product based on a mixture of dairy and vegetable products, such as a mixture of milk and fruit juice, for example.

Thus, the present invention also relates to a process for the preparation of a fermented food product in which a substrate is fermented with at least one living strain as described above.

It is advantageous to provide, in this method, the use of a combination of one or more bacterial strains as described above, with in particular at least one other strain of living bacteria. Advantageously, said other strain of bacteria is a strain of lactic acid bacteria, such as Streptococcus spp. ; Lactobacillus spp., Including L. bulgaricus, L. acidophilus and L. casei; Lactococcus spp. and Bifidobacterium spp.

Preferably, the substrate to be used in the context of the aforementioned process will be chosen from: milk substrates, in particular milk; substrates based on vegetable raw materials, such as fruits, cereals and soybeans. In particular, we can consider:

- if the substrate is milk, natural or reconstituted milk, skimmed or not, or media based on milk or milk product (s). ; and

- if the substrate is based on vegetable raw materials, fruit juices and / or vegetable juices.

The substrate may also advantageously include elements commonly used in the food industry for the preparation of fermented products, in particular milk desserts, in particular solid elements such as fruits, nuggets. chocolate or cereals for example, but also additional sweet or liquid chocolate products.

The present invention further relates to fermented food products, the organoleptic properties of which are preserved during storage. The expression “organoleptic properties retained during storage” means here that the organoleptic properties of the products do not vary (or not significantly for the consumer) over time, at least until the expiry date (DLC) ) products, or even advantageously beyond this date.

According to one embodiment, such products comprise at least one living strain according to the invention.

According to another embodiment, the fermented food products according to the invention are capable of being obtained by the process described above.

Preferably, the fermented food products according to the invention contain at least one bioactive food ingredient (or functional food ingredient), in particular chosen from proteins, peptides, analogs or derivatives thereof. The expression “bioactive or functional food ingredient” designates an ingredient which advantageously affects one or more target functions of the organism, independently of its nutritional effects. Thus, the effect of such an ingredient can be an improvement in the state of health and / or well-being and / or a reduction in the risks of occurrence of diseases in a consumer who ingests normal amounts of said ingredient.

This definition can be extended to the food or product containing such an ingredient, said food or product can therefore itself be qualified as bioactive or functional. The term “analog” means any modified version of an initial compound, here a protein or a peptide, said modified version may be natural or synthetic, in which one or more atoms, such as carbon, hydrogen, oxygen, or heteroatoms such as nitrogen, sulfur or halogen, have been added or removed from the structure of the initial compound, so as to obtain a new molecular compound.

A “derivative” within the meaning of the invention is any compound which presents a resemblance or a structural motif in common with a reference compound (protein or peptide). This definition also includes, on the one hand, the compounds which, alone or with other compounds, may be precursors or intermediate products in the synthesis of a reference compound, by means of one or more chemical reactions, and on the other hand the compounds which can be formed from said reference compound, alone or with other compounds, via one or more chemical reactions. Are thus covered by the definition of “derivatives” above at least hydrolysates, in particular tryptic, of proteins and / or peptides, hydrolysate fractions, as well as mixtures of hydrolysates and / or hydrolysate fractions.

Among the bioactive food ingredients capable of being used in the fermented food products according to the present invention, there may be mentioned, by way of nonlimiting examples, the peptide 91-100 of casein α _s i (see European patent EP 0 714 910), the C6-α _s i 194-199 peptide (see US patent US 6,514,941), the C7-β177-183 peptide (see US patent US 6,514,941), the C12-α _s i23 peptide -34 (see US Patent 6,514,941), caseinophosphopeptides, α- casomorphine, casein α exorphin, casokinin, β-casomorphine, caseinomacropeptides (CMP) and glycomacropeptides (GMP), casoxin, casoplatellins, fragments 50-53, β-lactorphins, lactoferroxine, peptides Val-Pro-Pro (see European patent EP 0 583 074), Lys-Val-Leu-Pro-Val-Pro-GIn (see EP 0 737 690), Tyr-Lys-Val-Pro-GIn-Leu (see 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; and Japanese patent JP 6 197 786), fragments, analogs, derived from these, proteins and / or peptides containing them, and their combinations (for a review, see in particular Danone World Newsletter N ° 17 of September 1998).

Table 1 below lists the main functional peptides released by the hydrolysis of proteins in 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 the functional peptides from milk known to date. Table 2

Table 2 (continued)

Advantageously, the food products according to the invention are functional foods.

As indicated briefly above, by "functional food" is intended to denote a food product which advantageously affects one or more target functions of the organism, independently of its nutritional effects. This may therefore result in an improvement in the state of health and / or well-being and / or a reduction in the risks of occurrence of diseases in a consumer who ingests normal amounts of the said product. By way of examples of activities of a “functional food”, there may be mentioned in particular anti-cancer, immunostimulatory, bone health promoters, anti-stress, opioids, anti-hypertension activities, which improve the bioavailability of calcium, or anti-microbial. Such functional foods can be intended for humans and / or animals.

According to one embodiment, the food products which are the subject of the invention are fresh products.

According to another embodiment, the food products targeted by the present invention are in particular chosen from: a drink; yogurt or dessert cream; a fermented milk or juice.

The culture of the strains according to the invention, pure or in combination with other strains, can also be used as a probiotic in human or animal food, or even as a lactic ferment, for example in the context of the process described above.

In addition, the present invention relates to the use of at least one natural strain of S. thermophilus AMI deficient with reduced post¬ acidification, and / or of one or more natural variants thereof, also AMI deficient (s) with reduced post-acidification, and / or their cultures and fractions of biologically pure cultures, for prepare fermented food products whose organoleptic properties are preserved during storage.

The present invention is illustrated by the following figures:

- Figure 1: kinetics of acidification curves of the functional AMI mother strain I-3299 and of the deficient AMI strain 1-3301.

Milk: milk without casein hydrolyzate; milk + N3 0.2 g / L: milk with 0.2 g of ingredient N3 (ref 211693, Difco, USA) per liter of milk.

- Figure 2: kinetics of acidification curves of the functional AMI mother strain I-3299 and of the deficient AMI strain I-3302. Milk: milk without casein hydrolyzate; milk + N3 0.2 g / L: milk with 0.2 g of N3 ingredient per liter of milk.

- Figure 3: kinetics of acidification curves of the deficient AMI strain 1-3301 in the presence of variable amounts of ingredient N3. Milk: milk without casein hydrolyzate; milk + 0.05 N3: milk with 0.05 g of N3 ingredient per liter of milk; milk + 0.1 N3: milk with 0.1 g of N3 ingredient per liter of milk; milk + 0.2 N3: milk with 0.2 g of N3 ingredient per liter of milk; milk + 0.4 N3: milk with 0.4 g of N3 ingredient per liter of milk; milk + 0.8 N3: milk with 0.8 g of N3 ingredient per liter of milk. - Figure 4: kinetics of acidification curves of the deficient AMI strain I-3302 in the presence of variable amounts of ingredient N3. Milk: milk without casein hydrolyzate; milk + 0.05 N3: milk with 0.05 g of N3 ingredient per liter of milk; milk + 0.1 N3: milk with 0.1 g of N3 ingredient per liter of milk; milk + 0.2 N3: milk with 0.2 g of N3 ingredient per liter of milk; milk + 0.4 N3: milk with 0.4 g of N3 ingredient per liter of milk; milk + 0.8 N3: milk with 0.8 g of N3 ingredient per liter of milk.

- Figure 5: acidification kinetics curves obtained from the fermentation of milk after addition of amino acids with the functional AMI mother strain 1-1630 (deposited at the CNCM on 10/24/95) and the AMI strain deficient 1-3211. Name of the strain without further details: fermentation in milk; name of the strain + aa: fermentation carried out in milk supplemented with 20 amino acids (corresponding to 1/40 ^th of the quantity determined for the chemically defined medium adapted to S. thermophilus (Letort et a /., 2001).

- Figure 6: kinetic curves obtained in milk supplemented with ingredient N3.

- Figure 7: kinetics of acidification curves made with different mixtures of ingredients. Other characteristics and advantages of the present invention will appear on reading the examples appearing in the experimental part below and given purely by way of illustration.

EXPERIMENTAL PART

I. Materials and Methods:

1.1. Obtaining deficient AMI variants:

a) Evaluation of the biodiversity of resistance to aminopterin in

S. thermophilus

First, the sensitivity of the S. thermophilus strains to aminopterin was checked. To do this, 200 μg of aminopterin were deposited on a disc placed on a petri dish containing Elliker medium on which 100 μl of bacterial suspension were deposited. If the strain is sensitive, an inhibition diameter appears around the disc after an overnight incubation. The mother strains 1-1630 (deposited with the CNCM on 10/24/95) and I-3299 (deposited with the CNCM on 9/16/04) were selected because they have interesting texture properties. The strain 1-1630 does not have a wall protease, while the strain I-3299 in has one. These two strains therefore have very different growth properties.

Table 3

A different sensitivity was observed depending on the strains.

In Table 3, the deficient AMI variants obtained are indicated in gray in the shaded boxes, where appropriate, in association with their respective parent strain.

Resistance to aminopterin is higher for the variants than for the corresponding mother strain.

The wild strain 1-2774 exhibits a natural resistance to aminopterin, reflecting its deficiency in the AMI transport system.

b) Obtaining deficient AMI variants The protocols for selecting deficient variants for transporting the peptides, using a toxic peptide analog, have already been described (Higgins and Gibson, 1986).

Briefly, a range of aminopterin from 0 to 200 μg / mL was carried out in a chemically defined medium (MCDaa) adapted to the growth of S. thermophilus and containing the 20 free amino acids as a source of nitrogen. From a preculture produced in MCDaa, all the tubes in the range were seeded at 1%. After a three-day incubation at 37 ^° C., 100 μL of each culture tube was used to make a bacterial mat on an agar of Elliker medium. A disc on which 200 μg of aminopterin are deposited was placed in the center of the box. The dishes were then incubated for three days at 37 ^° C. anaerobically. The presence of clones in the inhibition zone was then observed, and about twenty clones were removed in order to inoculate 5 ml of Elliker medium. The clones which are distributed in this medium and gave a bacterial suspension after 4:00 pm of incubation at 37 ^° C. allowed the production of a mat on the surface of an agar of Elliker medium on which a disc impregnated with 200 μg of aminopterin. All the clones obtained were then tested in fermentation in milk. The kinetics of acidification provided information as to the capacity of these variants to acidify milk.

1.2. Milk preparation:

The milk was reconstituted with 120 g of skimmed milk powder in 930 mL of water. After 30 minutes of hydration, the milk was pasteurized for 30 minutes at 95 ^° C.

The ingredient N3 was added from a 10% solution made in water and then sterilized by filtration at 0.22 μm. The other ingredients tested were added to the milk before pasteurization.

The amino acid solution was made in water and then sterilized by filtration at 0.22 μm. 1.3. Preparation of enzymes for carrying out acidification monitoring:

A preculture was carried out in sterilized milk with autolysate of yeast seeded at 1%, incubated for 18 h at 42 ^° C. Then, the ferment was carried out in 100 m L of milk sterilized with autolysate incubated at 42 ⁰ C and stopped by placing in ice water when the acidity of 8O ⁰ D was reached. The ferment was stored at 4 ^° C. until the initiation of the acidification monitoring. For the initiation of acidification monitoring, 250 ml of milk were inoculated at 1% with the ferment of S. thermophilus.

1.4. Materials:

N3: protease peptone N ° 3, ref R211693, Difco, USA (casein hydrolyzate containing peptides and, mainly, free amino acids)

Milk powder: Milex 240, ArIa Food Ingredients

Yeast autolysate: yeast extract, ref AEB171109, AES Laboratoires

CINAC for acidification monitoring: Automatic system for measuring acidifying activity developed by G. Corrieu LGMPA, YSEBAERT brand

Alaco 7014: whey protein hydrolyzate, NZMP Gmbh,

Siemens Strasse, 6-14 D-25462, Rellingen, Germany.

MPH 955: casein hydrolyzate, NZMP Gmbh, Siemens Strasse, 6-14

D-25462, Rellingen, Germany. FSD 6441: whey protein hydrolyzate, NZMP Gmbh, Siemens

Strasse, 6-14 D-25462, Rellingen, Germany.

MPH 917: casein hydrolyzate, NZMP Gmbh, Siemens Strasse, 6-14

D-25462, Rellingen, Germany.

WPH 926: whey protein hydrolyzate, NZMP Gmbh, Siemens Strasse, 6-14 D-25462, Rellingen, Germany.

MPH 948: casein hydrolyzate, NZMP Gmbh, Siemens Strasse, 6-14

D-25462, Rellingen, Germany. C 12: milk protein hydrolyzate, DMV international NCB Laan 80, PO BOX 13 5460, Ba Zeghel, Netherlands.

MPH 910: casein hydrolyzate, NZMP Gmbh, Siemens Strasse, 6-14 D-25462, Rellingen, Germany.

II. Results:

11.1. Kinetics of acidification of the functional AMI mother strain I-3299 and of deficient AMI strains derived therefrom, in the presence or absence of ingredient N3

It can be seen, in FIGS. 1 and 2, that the functional AMI mother strain I-3299 exhibits the same kinetics in the presence or in the absence of ingredient N3 in milk. On the other hand, the deficient AMI strains exhibit different kinetics.

11.2. Kinetics of acidification with variable quantities of ingredient N3. AMI strain deficient 1-3301

The strain 1-3301 has kinetics whose latency is shorter in the presence of ingredient N3. In addition, the pH obtained at the end of fermentation is higher the higher the ingredient N3 in the medium.

11.3. Kinetics of acidification with variable quantities of ingredient N3. deficient AMI strain I-3302

The kinetics of acidification is all the more rapid as there is ingredient N3 in the medium. In addition, the pH obtained at the end of kinetics is all the lower as there is ingredient N3 in the medium. 11.4. Post-acidification results in pure strain (by comparison with mother strain 1-3299)

Table 4

The deficient AMI strain makes it possible to have a pH difference (delta) of 0.29 between the pH at the end of incubation and the pH of the product at the end of life, while the mother strain gives rise to a pH delta 0.56. The advantage of using the deficient AMI strain is 0.27 pH unit if we compare the δpH under these conditions.

11.5. Acidification kinetics obtained from the fermentation of milk after addition of amino acids with the functional AMI mother strain I-1630 (deposited at the CNCM on 10/24/95) and the deficient AMI strain 1-3211 which derivative.

The stimulation produced by the addition of free amino acids in milk is the same for the mother strain and the deficient AMI strain. This result seems logical since free amino acids represent the only source of nitrogen available under these conditions for the two strains.

11.6. Kinetics obtained in milk with the N3 ingredient added

The functional AMI mother strain 1-1630 (deposited at the CNCM on 10/24/95) is clearly stimulated by the addition of ingredient N3 in milk, while the deficient AMI strain which derives from it is not. not. The peptides of the hydrolyzate N3 can be used by the mother strain, but are not used by the deficient AMI strain. 11.7. Acidification kinetics performed with different ingredients with the AMI deficiency strain 1-3211

The hydrolyzates MPH955 and WPH926 best stimulate the fermentation of the AMI strain deficient in milk. These are also the two hydrolysates with the highest levels of small peptides and free amino acids.

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Claims

1. Natural strain of Streptococcus thermophilus AMI deficient with reduced post-acidification, and its natural variants AMI deficient with similar milk acidification characteristics, as well as their cultures and fractions of biologically pure cultures.

2. Strain according to claim 1, characterized in that it is resistant to a toxic oligopeptide analog transported by the AMI system.

3. Strain according to claim 2, characterized in that said toxic analog is aminopterin.

4. Strain according to claim 3, characterized in that the inhibition diameter observed in the presence of a paper disc soaked with 200 μg of aminopterin is less than or equal to approximately 2 cm, preferably less than or equal to approximately 1 , 8 cm, on a box seeded with 100 μl of saturated bacterial suspension.

5. Strain according to any one of claims 1 to 4, characterized in that it is chosen from:

- the strain deposited at the CNCM on 05/10/04 under the number 1-3211;

- the strain deposited at the CNCM on 09/16/04 under the number 1-3301; - the strain deposited at the CNCM Ie16 / 09/04 under the number I-3302;

- the strain deposited at the CNCM on 24/01/02 under the number I-2774.

6. Strain according to any one of claims 1 to 5, characterized in that said deficient natural AMI variants are obtained by mutation and selection of their acidifying properties.

7. A method of preparing a fermented food product in which a substrate is fermented with at least one living strain according to any one of claims 1 to 6.

8. Method according to claim 7, characterized in that the fermentation is carried out in the presence of at least one other strain of living bacteria.

9. Method according to claim 8, characterized in that said other strain of bacteria is a lactic acid bacterium.

10. Method according to claim 9, characterized in that said lactic acid bacterium is chosen from: Streptococcus spp. ; Lactobacillus spp, especially L. bulgaricus, L. acidophilus and L. casei; Lactococcus spp. and Bifidobacterium spp.

11. Method according to any one of claims 7 to 10, characterized in that said substrate is chosen from: milk substrates, in particular milk; substrates based on vegetable raw materials, such as fruits, cereals and soybeans.

12. Method according to claim 11, characterized in that said substrate contains solid elements.

13. Method according to claim 12, characterized in that said solid elements are chosen from: fruits, chocolate products and cereals.

14. Fermented food product comprising at least one living strain according to any one of claims 1 to 6.

15. Fermented food product capable of being obtained by a process according to any one of claims 7 to 13.

16. Fermented food product according to claim 14 or 15, characterized in that it is a dairy or vegetable product.

17. Fermented food product according to any one of claims 14 to 16, characterized in that it contains at least one bioactive food ingredient.

18. Fermented food product according to claim 17, characterized in that said bioactive food ingredient is chosen from proteins, peptides, analogs or derivatives thereof.

19. Fermented food product according to claim 17 or 18, characterized in that it is a functional food.

20. Fermented food product according to any one of claims 14 to 19, characterized in that it is a fresh product.

21. Fermented food product according to any one of claims 14 to 20, characterized in that it is chosen from: a drink; yogurt or dessert cream; a fermented milk or juice.

22. Fermented food product according to any one of claims 14 to 21, characterized in that its organoleptic properties are preserved during storage.

23. Use of at least one strain according to any one of claims 1 to 6 as a lactic ferment.

24. Use of at least one strain according to any one of claims 1 to 6 as a probiotic in human or animal nutrition.

25. Use of at least one strain according to any one of claims 1 to 6 for preparing fermented food products whose organoleptic properties are preserved during storage.