FR2723960A1 - Prodn. of Streptococcus thermophilus cultures rich in beta-galactosidase - Google Patents

Abstract

The following are claimed:(a) a process for the prodn. of a Streptococcus thermophilus culture with high beta -galactosidase ( beta gal) activity comprises culturing an S. thermophilus strain in medium contg. \-20 g/l milk protein hydrolysate, 15-30 g/l lactose and \-1 g/l yeast extract;(b) a process for producing galacto-oligosaccharides by incubating the above culture in a medium contg. 50-500 g/l lactose;(b) the opt. dried prods. of the above processes, and(c) a culture of S. thermophilus for use in the above processes.

Description

CULTURES OF STREPTOCOCUS TBERWOPHILI; TS A ACTIVITY - GAIACTOSIN.E HIGH, THEIR PROCESS OF EXECUTION, AND THEIR USES.

 The present invention relates to obtaining food products enriched in 5-galactosidase and / or galacto-oligosaccharides.

 It is known that yogurt-type fermented products are of great interest for feeding lactose-intolerant subjects, and for infant feeding. Indeed, lactic acid bacteria, traditionally used for the manufacture of these products have a relatively high β-galactosidase activity, which improves the digestibility of lactose, and consequently, the tolerance of the food.

 Β-galactosidase is responsible for the hydrolysis of lactose to glucose and galactose. Studies on the activity of ss-galactosidase preparations have shown that, starting from a certain concentration of lactose, this enzyme also catalyzes, in parallel with the hydrolysis of lactose which results in the formation of glucose and galactose, a reaction. transgalactosylation which results in the production of a family of oligosaccharides (di- to hexasaccharides) called galacto-oligosaccharides [see for example, SMART, Appl.

Microbiol. Biotechnol., 34, 495-501 (1991)].

Galacto-oligosaccharides have been shown to be a factor stimulating the growth of Bifidobacteria, and thus allow enrichment of intestinal flora in Bifidobacteria at the expense of other microorganisms, particularly pathogenic bacteria [TANAKA et al., Bifidobacteria
Microflora, Vol. 2 (1), 17-24, (1983)].

 It has been proposed to use β-galactosidases of different origins to prepare galacto-oligosaccharides, for example to obtain milk products with a high content of galacto-oligosaccharides.

Two categories of processes tending to this end have been described
1) The first category implements the treatment of a substrate containing lactose, such as milk, with preparations of -galactosidase purified from different microorganisms.

This is known by the patent application
European Patent No 323,201 to YAKULT HONSHA KK, a process which consists in treating animal milk with a preparation of 5-galactosidase extracted from Streptococcus thermophilus or Lactobacillus bulgaricus, with the aim of transforming at least 15% of the lactose contained in this milk, in galacto-oligosaccharides.

 It has also been proposed [Z. MOZAFFAR et al., Journal of Food Science, 50, p. 1602-1606, (1985)] to produce galacto-oligosaccharides from milk using a -galactosidase extracted from Bacillus circulans.

 These methods have the disadvantage of requiring the prior purification of β-galactosidase, and pose the problems of stabilization of enzymatic activity that exist, generally with any purified enzyme preparation.

 2) A second category of methods proposes the use of bacteria exhibiting 5-galactosidase activity, without prior extraction of the enzyme.

However, there is the problem of making lass-galactosidase, which is intracytoplasmic, accessible to its substrate.

 For this, one must proceed either to the lysis of the bacteria, or at least to their permeabilization, for example by an organic solvent or a surfactant. Permeabilization, although less drastic than lysis, is, however, detrimental to the viability of bacteria and also affects β-galactosidase activity; it has therefore been proposed to treat permeabilized bacteria in order to prolong their viability and to protect β-galactosidase activity.

For example, treatments with glycerol or sorbitol have been recommended for this purpose. Another type of treatment, such as that described in the Application for
Japanese patent in the name of YAKULT HONSHA KK, published on 28
June 1991 under the number 3 151 875, implements a process where permeabilized bacteria are treated with galacto-oligosaccharides to protect the active site of the ss-galactosidase. This makes it possible to delay the inactivation of this enzyme during the conservation of the bacteria, and during the galacto-oligosaccharide formation reaction.

 None of the currently known processes uses live bacterial cultures capable of expressing a directly useful galactosidase activity for the hydrolysis of lactose, and / or for the production of galacto-oligosaccharides, without prior purification of the enzyme, and without pretreatment of the bacteria.

However, the work of MOLOTOV et al. [Biotekhnologiya, 2, 33-37, (1991)] who undertook the cloning of the β-galactosidase gene from
Streptococcus thermophilus, in order to increase the number of copies, to obtain recombinant bacteria overproducing this enzyme. As part of this work, they studied the influence of various factors (growth phase of bacterial culture, lactose concentration, or sugars other than lactose) on the hydrolysis of lactose by 5-galactosidase of different strains of Streptococcus thermophilus grown on M21 medium.They found that this activity was greater in the presence of lactose than other sugars, but it decreased rapidly when the lactose concentration of the medium increased (the activity is 4 times less important at 5% lactose than 0.5%).

 On the other hand, they also report, contrary to the observations made by the majority of authors, that under certain growing conditions (long-term cultures, or cultures in the presence of glucose), there are two types of Bgalactosidase activity.

Indeed, according to their observations, although a major part of the activity remains intracellular, and therefore only accessible after lysis of the bacteria, another part of the activity would be excreted by the bacteria and recovered in a culture supernatant. .

 These observations, however, have only a limited practical application; in fact, the conditions in which the excretion of the -galactosidase activity is observed are marginal, and are far from being favorable for the optimal growth of the bacteria, or for obtaining a high 5-galactosidase activity.

 However, the inventors have now discovered that by cultivating S. thermophilus under certain conditions, it was possible to obtain cultures having, without prior treatment of permeabilization of bacteria, a very high activity -galactosidase up to 25 to 30 U / ml of culture. A -galactosidase unit corresponds to one micromole of ONPG (O-nitrophenyl-s-B-galactopyranoside) hydrolysed per minute at 37 ° C. and at pH 7.3.

 The present invention relates to a process for the preparation of a thermophilic Streptococcus culture having high 5-galactosidase activity, which process comprises a step in which a strain is cultivated. of Streptococcus thermophilus in a medium which comprises at least 20 g / l of hydrolysed milk proteins, between 15 and 30 g / l of lactose, and at least 1 g / l of yeast extract.

 According to a preferred embodiment of the present invention, the culture medium comprises between 25 and 40 g / l of hydrolysed milk proteins, and between 5 and 10 g / l of yeast extract.

 "Hydrolyzed milk protein" or "milk protein hydrolyzate" means a casein hydrolyzate, or a hydrolyzate of whole milk proteins, or a hydrolyzate of a milk protein fraction comprising casein.

 Preferably, this hydrolyzate is obtained by enzymatic hydrolysis of the milk protein preparation.

 Many proteases, for example trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), etc ... may be suitable for obtaining this hydrolyzate.

 Very advantageously, the culture medium comprises a hydrolyzate of a milk ultrafiltration retentate, the protein concentration of said retentate being between 60 and 80 g per 100 g of dry extract. This hydrolyzate of an ultrafiltration retentate provides both the milk protein hydrolyzate and all or part of the lactose.

 The present invention also relates to culture media for carrying out the process according to the invention.

 The present invention also relates to bacterial cultures obtained by cultivating a strain of Streptococcus thermophilus according to the method according to the invention.

 These cultures have an activity galactosidase, measurable without prior treatment of permeabilization of bacteria, which is much higher than that of Streptococcus thermophilus cultures obtained according to methods of the prior art.

 This activity is also not released into the culture medium, but remains bound to the cells; when these are separated from the culture medium by microfiltration on a membrane allowing the passage of β-galactosidase (1.4con pore diameter), only 1.5% of the β-galactosidase activity is found in the permeate.

 Cultures according to the invention can be obtained from different strains of S.

 Thennophilus, when grown according to the process of the invention. To obtain an β-galactosidase activity greater than 20 units / ml of culture, a strain whose initial β-galactosidase activity is high will be chosen. To select a suitable strain, one can for example proceed as follows cultivation strains to be tested as described below in Example 7, and measure the activity in the medium: after about 4:30 of culture, this activity should be of the order of 0.9 U / ml of culture.

For example, strains of S. thermo philus commercially available and which are particularly suitable for carrying out the process of the invention are strains ST 12 and ST 13 sold by the company BOLL (Le Moulin d 'Aulnay, BP 64
Saint Germain les Arpajon 91292 Arpajon Cedex France).

On the other hand, a strain of S. thermophilus also suitable for carrying out the invention, called LFL-01, was deposited on August 25, 1994, under number I-1470 with the CNCM (National Collection of
Cultures of Microorganisms) held by the institute
Pasteur, 25 rue du Docteur Roux, Paris.

 S. thermophilus cultures in accordance with the invention can be used to obtain a wide variety of products, in particular food products, enriched with galactooligosacharides and / or β-galactosidase.

 On the one hand, the characteristics of S. thermo philus allow its direct incorporation both into products that can be kept fresh, and in products intended to support a wide variety of treatments (dehydration for example), and can also be administered especially to infants.

Indeed, this bacterium
- produces only L (+) lactic acid, and in addition in small quantity, unlike microorganisms of the Lactobacillus type, which produce lactic acid D (-) which can not be metabolized by the infant
- is very resistant to desiccation at high temperature, which is practiced for obtaining powdered milk
- Has only a very low proteolytic activity, which limits the risk of formation of compounds having an unpleasant taste during the storage of finished products.

 In addition, because in the cultures obtained by the process according to the invention, the β-galactosidase activity is bound to the cells, it is protected by these during the various treatments resulting in obtaining a finished product ready for consumption.

 This allows, if desired, to use a culture of S. thermophilus according to the invention to obtain a product enriched in galacto-oligosaccharides, and to retain the ss-galactosidase activity in this product.

 This was not possible with the 5-galactosidase preparations of the prior art (whether it be a purified enzyme, a bacterial lysate, the enzyme present in permeabilized bacteria, or a secreted enzyme), which, being unprotected, are inactivated during the production of the finished product.

 The cultures of S. thermophilus according to the invention can be dried, to obtain a powder having a high β-galactosidase activity.

 The drying can be carried out by any method known per se, for example by atomization or lyophilization.

 For spray drying, in order to retain the maximum of β-galactosidase activity during drying, an outlet air temperature of less than 90 ° C is preferred.

 The powder resulting from the drying can be added to various dry products (for example milk milks), to enrich them in β-galactosidase.

 Very advantageously, the culture may also be mixed, before drying, and immediately before it, with a milk concentrate, or any other product which it is desired to enrich in s-galactosidase.

 The cultures of Streptococcus thermophilus according to the invention can also be used for the production of galacto-oligosaccharides from lactose.

 For this purpose, a culture of Streptococcus thermophilus according to the invention is incubated in the presence of a medium containing between 50 and 500 g / l of lactose.

 The incubation medium may be a lactose solution; preferably, it will be a solution comprising between 100 and 500 g / l, and very advantageously between 300 and 450 g / l of lactose.

 According to a preferred embodiment of the present invention, the incubation is carried out at a pH of between 6 and 8 and preferably between 7 and 7.4 and at a temperature above 30 ° C. and preferably between 45 ° C. and 45 ° C. and 55 C.

 The duration of the incubation, under these conditions, will be chosen according to the amount of galacto-oligosaccharides that one seeks to obtain; advantageously it is between 30 minutes and 5 hours, for temperatures between 45 and 55 C it can, without disadvantage, be increased for lower temperatures.

 The S. thermophilus culture may advantageously be concentrated by ultrafiltration before contacting with the incubation medium to increase the rate of the transgalactosylation reaction. This reaction rate can also be increased by the addition of magnesium chloride at a concentration of between 100 and 500 mg / l.

 The incubation medium may also be milk, or any milk-based medium; it may be for example a milk concentrate, a base for infant milk, a base for yoghurt, etc ...

 To the milk-based medium can be added the ingredients necessary to produce the ready-to-eat product that is desired. If, for example, we want to obtain an infant formula, we add lactose, malto-dextrins, minerals, vitamins, fats, ingredients to reconstitute the composition of breast milk.

If desired, the fat is incorporated and then homogenized with the solution to obtain a stable emulsion.

 The solution is then seeded with a culture of Streptococcus thermophilus according to the invention, containing 107 to 109 bacteria / ml. The optimum culture temperature is between 30 and 50 ° C.

 If it is desired to obtain a finished product enriched with galacto-oligosaccharides and β-galactosidase, the bacteria will not be separated from the incubation medium at the end thereof.

 If, on the contrary, it is desired to obtain a product enriched in galacto-oligosaccharides only, it is necessary to inactivate or eliminate β-galactosidase. The enzyme can, for example, be inactivated by heat treatment.

 In the case of the present invention, where the egalactosidase activity is bound to bacteria, it is particularly simple to eliminate it by separating the bacteria from the incubation medium; to further facilitate this separation, it is possible to incubate with a culture of Streptococcus thermophilus according to the invention, the cells of which were previously fixed on a solid support.

 If bacteria are used in free form, the separation can be carried out by filtration or by centrifugation.

 The galacto-oligosaccharides may then, if desired, be purified from the medium by any method known per se.

The galacto-oligosaccharides obtained by the process according to the invention correspond to the general formula
Gal- (Gal) n-Gl where
Gal represents a galactose residue
- G1 represents a glucose residue or a galactose residue
n is 0, 1, 2, 3, or 4.

 According to the invention the production of galacto-oligosaccharides can be carried out without fermentation of the medium by S. thermophilus in this case, the incubation is carried out under conditions unfavorable to the growth of said bacteria.

Conditions unfavorable to the growth of
Streptococcus thermophilus are for example a temperature greater than 52 C and / or a high content of the medium in dry extract (greater than about 48%).

 If it is preferred to obtain a product fermented by S. thermophilus, the incubation is carried out under conditions allowing the growth of S. thermophilus.

Advantageously, the medium can be further inoculated with lactic acid bacteria other than
Streptococcus thermophilus, for example Lactobacillus acidophilus, Lactobacillus helvetica, Lactobacillus bulgaricus, mesophilic lactic streptococci such as
Streptococcus cremoris, Streptococcus lactis,
Streptococcus diacetylactis, and / or bifido-bacteria such as Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum.

 These bacteria can be added at the beginning of fermentation by Streptococcus thermophilus, during or after it.

 The product can then undergo different treatments, the nature of which varies according to the product ready to consume that one wishes to obtain. It may for example be added texture agents, flavor, vitamin or mineral supplements, fat, etc., if they have not been added in the initial medium. It can also be concentrated, or dehydrated by drying.

 The subject of the present invention is also the various products, fermented or not, enriched with galacto-oligosaccharides and / or β-galactosidase, obtainable by one of the processes, using a Streptococcus thermophilus culture in accordance with US Pat. Invention, defined above.

 The dairy products rich in β-galactosidase according to the invention are better tolerated than the known milk products, in particular by the subjects deficient in β-galactosidase, because they contain the β-galactosidase of living lactic acid bacteria, which ensures better digestibility of the lactose they contain; in addition, the dairy products according to the invention, rich in galacto-oligosaccharides, promote the development of bifidobacteria intestinal flora in situ. They are particularly well adapted to infant feeding.

 In addition to the foregoing, the invention also comprises other arrangements which will emerge from the description which follows which refers to examples of preparation of products according to the invention, in particular products intended for infant feeding.

KIEMPLE 1 PREPARATION OF A CULTURE OF S. TEERMOPEILUS
HIGH ACTIVITY ss-GALACTOSIDASE
A retentate of milk is prepared by ultrafiltration of skimmed milk until a retentate with a protein content of 60% (grams of protein per 100 g of powder) is obtained, a concentration factor of about 1.7. . Five kilos of retentate are dissolved in water in a final volume of 100 liters. The pH of the solution is adjusted to 7.5 with a 10% potassium hydroxide solution. The preparation is heat-treated at 92 ° C for 10 minutes. The solution is cooled and maintained at 50 ° C. The hydrolysis is carried out in the presence of 135 grams of pancreatin 3NF (Industrial Laboratory of Biology, Soisy-sous-Montmorency, France), at 50 ° C. for 4h30. The pH is maintained at 7.25 with lime. After hydrolysis, 500 g of yeast extract is added, before sterilization for 25 minutes at 105 ° C. of the milk retentate hydrolyzate.

 The sterilized milk retentate hydrolyzate is inoculated with 1% (v / v) of a S. thermophilus culture made on skimmed milk enriched with 1 g / l of yeast extract and containing about 108 bacteria / ml. The strains used are the strains LFL-01, and DPch. The medium is incubated at 40 ° C. for 17 hours. The pH at the end of culture is between 5 and 5.5.

 The β-galactosidase activity measured in the medium is 25 units per milliliter of culture, with the strain LFL-01, and with the DPCH strain.

EXAMPLE 2 PREPARATION OF A POWDER RICH IN 3-GALACTOSIDASE
A culture of S. thermophilus prepared according to the procedure described in Example 1 is concentrated by membrane microfiltration (porosity 1.4 Mm). 100 liters of retentate are obtained at 580 U / ml of ss-galactosidase activity. Before drying the retentate is mixed with milk concentrate in a ratio of 1: 2 (w / w). Drying is done directly, and the resulting powder contains 480 U-galactosidase per gram. This powder, mixed with 1/1000 in a skimmed milk powder makes it possible to obtain a milk, which reconstituted at 100 g / l, has an activity equivalent to 0.5 U B-gal per ml of preparation.

This powder mixed with 1 to 3/1000 in a skimmed milk powder makes it possible to obtain a milk, which reconstituted at 100 g / l, has an ß-galactosidase activity equivalent to that of a yoghurt.

 The contact time in a culture / milk concentrate mixture should be minimum to prevent the formation of galacto-oligosaccharides.

These conditions of reduced contact time are achieved for example by continuously injecting the culture in the concentrate just before atomization.

EXAMPLE 3 PREPARATION OF A POWDER RICH IN 13-GALACTOS I DASE AND IN OLIGOSACCEARIDES
Variant 1
A culture of S. thermophilus with a volume of 258 liters, prepared according to the procedure described in Example 1, is concentrated by ultrafiltration, at 40 ° C., on a 100,000 cut-off membrane. 100 liters of retentate are obtained. at 500 U / ml of -galactosidase activity.

The retentate is incubated in the presence of milk concentrate in a ratio of 1: 2 (w / w) for 17 hours at 6 C and a 120-D acidity (a degree Dornic (-D) corresponds to 1 mg of acid lactic acid / 10 ml of milk).

 Drying is done directly. The galacto-oligosaccharides are purified and assayed as described below in Example 4. The resulting powder contains 460 U-galactosidase per gram, and 6.3% trisaccharides, 1.4% tetrasaccharides, 0.45% of pentasaccharides, ie a total of 8.1% (w / w) of galacto-oligosaccharides assayed.

 This powder mixed 1/1000 in a skimmed milk powder makes it possible to obtain a milk which reconstituted at 100 g / l possesses a 5-galactosidase activity of approximately 0.48 U / ml.

Variant 2
The culture of S. thermophilus obtained according to the procedure described in Example 1 is brought into contact with a skimmed milk concentrate containing 42% solids, at a rate of 5 ml of culture per 150 g of milk concentrate. The solution is at pH 6.42. After lh30 of reaction at 53 ° C., 9.5 g / l of galactooligosaccharides is obtained, ie approximately 2 g per 100 g of solids. The resulting powder contains 2.3 U B-galactosidase / gram.

= EMPLIE 4: PRODUCTION OF GALACTO-OLIGOSACCHARIDES
A culture of S. thermophilus carried out according to the procedure described in Example 1, and containing 25 μg / ml is supplemented with lactose to obtain a final lactose content of between 350 and 450 g / l.

The pH is maintained between 6 and 8 with a 10% potassium hydroxide solution. The incubation is carried out with stirring at a temperature between 45 and 55 C. The addition of magnesium chloride (300 mg / l) makes it possible to accelerate the transgalactosylation reaction.

 In the following example, lactose was added directly to the S. thermophilus culture.

 Lactose is added to give a final concentration of 453 g / l. The pH is adjusted to 7.10, the temperature is maintained at 55 C. 10% (w / w) of conversion of lactose to galactooligosaccharides is obtained in less than 1 hour and 30% of conversion in 4:30 of reaction. The production of galacto-oligosaccharides as a function of time is illustrated in Table I below.

TABLE I

<tb> Time <SEP> O <SEP> 1h <SEP> 1h30 <SEP> 2h <SEP> 2h30 <SEP> 3h <SEP> 3h30 <SEP> 4h30
<tb> Quantity
<tb> of oligo- <SEP>
<tb> saccha- <SEP> 2 <SEP> 49.2 <SEP> 69.3 <SEP> 85 <SEP> 100.5 <SEP> 107 <SEP> 115.2 <SE> 137.7
<tb> wrinkles
<tb> (g / l)
<Tb>
The transgalactosylation product is fractionated on an HPLC column (Fractogel TSK HW40, MERCK). The fractions are then purified by thin layer chromatography on silica in a solvent system n-butanol: acetic acid water, 2: 1: 1. The molecular weight of each purified fraction is determined directly by FAB-MS after methanolysis. Each fraction is also treated with potassium borohydride and then methylated in order to analyze the products which were not directly detected by direct FAB-MS analysis.

 To determine the branching points of the galacto-oligosaccharides, the products are subjected to methanolysis followed by re-acetylation, and analyzed by gas phase chromatography coupled to a mass spectrometer.

 The disaccharides represent 65% of the galacto-oligosaccharides assayed by the thin layer chromatography technique on silica. In this fraction, lactose represents 49.2% of these compounds, allolactose (beta-Gal (1.6) Glc): 42.6% and disaccharide beta (1.3) 8.2%.

 trisaccharides represent 28% of the carbohydrates measured by this method and the tretracaccharides 6%. The penta- and hexasaccharides are present as traces relative to other structures.

 The majority of the structures are linear but it is possible to have branched structures, in this case, it is always a substitution on galactose by bonds beta- (1,3) and beta- (1,6 ).

 In the case of linear galacto-oligosaccharides, the beta- (1,3) bond is still predominant over beta- (1,6) and beta- (1,4) bonds.

 Figure 1 shows the different structures identified in the transgalactosylation product.

EXAMPLE 5: OBTAINING FRESH FERMENTED PRODUCTS
CONTAINING GALACTO-OLIGOSACCEARIDES
A yoghurt base consisting of 1 liter of milk with 1.1% fat is enriched with dry extract by the addition of 30 g / l of skimmed milk powder. This preparation, which contains between 60 and 65 g / l of lactose, is pasteurized and then inoculated with 1% by volume of a culture of S. thermophilus obtained according to the procedure described in Example 1. It is incubated at 44 ° C. during 4h45 and a fermented product is obtained.

wswMoLE 6: PRODUCTION OF zEKSkNrs INFANT POWDER WITH s-GALACTOSIDASE AND CONTAINER ACTIVITY
GALACTO-OLIGOSACCRARIDES
The culture obtained according to the procedure described in Example 1 is used to inoculate an infant milk base with an inoculation rate of 5% (v / v). The composition of the infant milk base is given in Table II below.

 To a skimmed cow's milk, warmed to 75 C, vegetable fat and lecithin are added. The mixture is homogenized at the same temperature in two stages, the first under 200 kg / cm 2 and the second under 50 kg / cm 2. A second fraction consisting of casein or whole proteins of milk, mineral salts, and maltodextrins is added. A third part is added consisting of a solution containing the vitamins that one wishes to incorporate into the infant food. The final mixture is pasteurized at 115 ° C. and then concentrated by evaporation to 41.5% dry matter. The concentrate has the following composition by weight per 100 grams of dry matter.

TABLE II

<tb> Material <SEP> fat <SEP> vegetable <SEP> 24 <SEP> g
<tb> of which <SEP> lecithin <SEP> 0.25 <SEP> g
<tb> Proteins <SEP> of <SEP> milk <SEP> (of which <SEP> 80% <SEP>)
<tb> caseins <SEP> and <SEP> 20% <SEP> of <SEP> prot. <SEP> serum) <SEP> 12.85 <SEP> g
<tb> Lactose <SEP> 40 <SEP> g
<tb> Malto <SEP> dextrins <SEP> of which <SEP> 10% <SEP> added <SEP> to <SEP> sec <SEP> 20.4 <SEP> g
<tb> Salts <SEP> Minerals <SEP> 2,6 <SEP> g
<tb> Vitamins <SEP> 0.15 <SEP> g
<Tb>
To this composition can be added 0.25 g per 100 g of dry matter of a casein hydrolyzate.

 The preparation is incubated for 5h30 at 44 ° C., the acidity reached is 110 ° C. and the content of β-galactosidase activity is 3 U / g.

 The preparation is then spray-dried; the tower outlet temperature remains below 90 ° C.

EXAMPLE 7 COMPARISON OF DIFFERENT STRAINS OF S.

 THERNOPHILUS.

 Different strains of S. thermophilus are used according to the conditions defined in Example 6, starting from an inoculum (5%, 108 germs / ml) obtained by culturing S. thermophilus on milk enriched with extract. yeast at lg / l. The results are shown in Table III below.

The acidities are expressed in D; the β-galactosidase activity after 5h15 of incubation is expressed in
U ss-gal / g of concentrate.

TABLE III

<tb> Strain <SEP> Acidity <SEP> Acidity <SEP> Activity
<tb><SEP> after <SEP> 4h <SEP> after <SEP> 5h15 <SEP> B-gal <SEP>
<tb><SEP> incubation <SEP> incubation
<tb> ST <SEP> 20 <SEP> (Boll) <SEP> 95 <SEP> 105 <SEP> 0.67
<tb> ST <SEP> 13 <SEP> (Boll) <SEP> 68 <SEP> 75 <SEP> 0.94
<tb> ST <SEP> 134 <SEP> (Boll) <SEP> 79 <SEP> 99 <SEP> 0.66
<tb> ST <SEP> 77 <SEP> (Boll) <SEP> 96 <SEP> 110 <SEQ> 0.48
<tb> ST <SEP> 9 <SEP> (Boll) <SEP> 62 <SEP> 70 <SEP> 0.49
<tb> ST <SEP> 12 <SEP> (Boll) <SEP> 94 <SEP> 100 <SEP> 0.97
<tb> ST <SEP> 143 <SEP> (Boll) <SEP> 89 <SEP> 95 <SEP> 0.51
<tb> ST <SEP> 75 <SEP> (Boll) <SEP> 87 <SEP> 106 <SEP> 0.27
<tb> ST <SEP> 7 <SEP> (Boll) <SEP> 101 <SEP> 111 <SEP> 0.76
<tb> TH <SEP> 3 <SEP> (Boll) <SEP> 81 <SEP> 96 <SEP> 0.31
<tb> LFL-01 <SEP> 101 <SEP> 106 <SEP> 1.02
<Tb>
The strains ST12 and ST13 give results equivalent to the strain LFL-01 used for the prior examples and are also adapted for the implementation of the method according to the invention.

Claims (14)

 1) Process for preparing a culture of Streptococcus thermophilus with high β-galactosidase activity, which process comprises a step in which a strain of Streptococcus thermophilus is cultured in a medium which comprises at least 20 g / l of hydrolysed milk, between 15 and 30 g / l of lactose, and at least 1 g / l of yeast extract.  2) Process according to claim 1, characterized in that the culture medium comprises between 25 and 40 g / l of hydrolysed milk proteins, and between 5 and 10 g / l of yeast extract.  3) Process according to any one of claims 1 or 2, characterized in that the proteins come from a milk ultrafiltration retentate hydrolyzate, whose protein concentration is between 60 and 80 g per 100 g of dry extract.  4) Culture medium for carrying out the method according to any one of claims 1 to 3, characterized in that it comprises at least at least 20 g / l of hydrolysed milk proteins, between 15 and 30 g / l of lactose and at least 1 g / l of yeast extract.  5) Culture of Streptococcus thermophilus, characterized in that it is obtainable by a method according to any one of claims 1 to 3.  6) Powder rich in β-galactosidase, characterized in that it is obtainable by desiccation of a culture of S. thermophilus according to claim 5.  7) A method for producing galactooligosaccharides, characterized in that it comprises a step during which incubation of a culture of Streptococcus thermophilus according to claim 5, in the presence of a medium comprising between 50 and 500 g / l lactose.  8) Method according to claim 7, characterized in that the incubation is carried out in the presence of a lactose solution comprising between 300 and 450 g / l of lactose.  9) Method according to claim 8, characterized in that the incubation of said culture of Streptococcus thermophilus is carried out at a pH between 7 and 7.5 and at a temperature between 45 and 55 C.  10) Method according to claim 7, characterized in that the culture of Streptococcus thermophilus is incubated in the presence of a milk concentrate.  11) Method according to claim 7, characterized in that the culture of S. thermophilus is incubated in the presence of a composition for infant milk diet.  12) Process according to any one of claims 10 or 11, characterized in that the culture medium is further inoculated with at least one bacterial strain selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helvetica, Lactobacillus bulgaricus, Streptococcus cremoris, Streptococcus lactis, Streptococcus diacetylactis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum.  13) Product enriched in galactooligosaccharides and / or β-galactosidase, characterized in that it is obtainable by a method according to any one of claims 7 to 12.  14) Product enriched in galactooligosaccharides and / or β-galactosidase, characterized in that it is obtainable by desiccation of a product according to claim 13.