EP0948262A1

EP0948262A1 - Fermented milk product

Info

Publication number: EP0948262A1
Application number: EP97951127A
Authority: EP
Inventors: Rodney Stuart Moreton
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA; Nestle SA
Priority date: 1996-12-24
Filing date: 1997-11-26
Publication date: 1999-10-13
Also published as: CA2274535A1; AU5478998A; WO1998027824A1; AU723405B2; IL130285A0

Abstract

The invention concerns a method for making a fermented milk composition which consists in: pasteurising the milk at a temperature and for a duration such that its redox potential at 25 DEG C is brought down to a value less than 450 mvolt, adding agents for stabilising the milk redox, inoculating the milk with lactic bacteria, fermenting the milk until at least 10<6> cfu/ml and an Aw higher than 0.97 is obtained. The invention also concerns the use of milk having a redox potential at 25 DEG C less than 450 mvolt and containing agents stabilising this milk redox potential, for preparing a milk product fermented with lactic bacteria. The invention further concerns milk compositions packaged in a material permeable or semipermeable to oxygen, said compositions having a redox potential less than 450 mvolt and an Aw higher than 0.97, and at least 10<6> cfu/ml of probiotic lactic bacteria.

Description

Fermented milk product

The subject of the present invention is a new process for preparing a fermented milk composition, said process making it possible to obtain food compositions having a particularly high load of lactic acid bacteria, even after packaging and preservation for prolonged periods.

State of the art

Although lactic acid bacteria are generally known to have beneficial effects on human health, only certain categories of lactic acid bacteria are actually able to adhere to human intestinal cells, to exclude pathogenic bacteria on human intestinal cells, and / or act on the human immune system by allowing it to react more strongly to external aggressions. Lactic acid bacteria are said to be "probiotic" when they have at least one of these characteristics.

To date, relatively few lactic acid bacteria are truly probiotic bacteria. For example, the strains Lactobacillus casei ATCC53103, Lactobacillus acidophilus CNCM 1-1225, Bifidobacterium breve CNCM 1-1226, Bifidobacterium infantis CNCM 1-1227 and Bifidobacteriun longum CNCM 1-1228, have thus been scientifically recognized as probiotic bacteria in the measurement where they are able to adhere to human intestinal cells, to exclude pathogenic bacteria on human intestinal cells, and to act on the human immune system (EP577904; EP577903; EPI 99535; Gut, 3_5_, 483-489, 1994 ; J. of Dairy Science, 7_8_, 491-197, 1995; Applied Env. Microb., £ 9_, 4121-4128, 1993).

Probiotic lactic acid bacteria are often also extremely sensitive to oxygen, due to their adaptation to the anaerobic living conditions found in the intestinal tract. In addition, these bacteria grow poorly in milk, which poses problems in achieving a sufficient level of lactic acid bacteria in a fermented milk product. To improve the growth of slow-growing lactic acid bacteria, EPI 54614 suggests increasing the charge of the starter culture when sowing milk, and also adding growth stimulants, such as extracts, to this milk. yeast or whey protein, for example.

Although growth problems in milk can be solved by this means, the probiotic lactic acid bacteria unfortunately still remain very sensitive to the conditions for processing and storing fermented milk. In fact, most of the plastic packaging used to package dairy products is permeable to oxygen. In addition, the subsequent transformation of a fermented milk, for example into white cheeses or liquid acidified milks, requires stirring the milk in the presence of air, which increases the oxygen level in the final product.

The present invention aims to overcome the drawbacks of the prior art, by providing a process which promotes the survival of lactic acid bacteria.

Summary of the invention

To this end, the present invention relates to a process for manufacturing a fermented milk composition in which milk is heat treated at a temperature and for a time such that its redox potential at 25 ° C is lowered to a value less than 450 mvolt, and the milk is inoculated with lactic acid bacteria.

The present invention also covers all dairy compositions packaged in an impermeable or semi-permeable oxygen material, said compositions comprising at least 10 ⁶ cfii / ml of probiotic lactic acid bacteria and a redox potential of less than 450 mvolt (cfu comes from l 'English expression "colony forming unit").

Likewise, the invention also relates to the use of a milk having a redox potential at 25 ° C. which is less than 450 mvolt, for the preparation of a dairy product comprising lactic acid bacteria. Finally, the invention also relates to the use of a fermented milk composition resulting from the present process in the preparation of a dairy product comprising lactic acid bacteria.

Against all expectations, lactic acid bacteria which are known to be sensitive to the conditions of fermentation and storage of a milk, for example sensitive to the presence of air, in fact become more resistant to these conditions, and in particular become tolerant to presence of air, as soon as the redox potential of the milk in which they live is less than about 450 mvolt. This resistance results in better bacterial development during the fermentation of milk, and better survival of bacteria during the conservation of fermented milk.

Although this redox potential can be adjusted by various means, it has been found that prolonged pasteurization of milk is sufficient to achieve a required redox potential. Indeed, a prolonged heat treatment makes it possible to break certain proteins in milk and thus to release reducing groups. In addition, this treatment makes it possible to react the proteins and sugars of the milk so that it forms reducing compounds resulting from Maillard reactions.

This pasteurization has other advantages. Firstly, prolonged treatment of milk at high temperatures promotes degassing of the milk, and therefore a low oxygen content in the milk. Second, this treatment converts part of the lactose in milk to lactulose, which is known to stimulate the growth of certain lactic acid bacteria.

Brief description of the figures

- Figure 1 shows, after fermentation, the number of cells of the strain CNCM 1-1225 (cfu / ml) having grown in different milks, said milks having undergone different heat treatments before fermentation, said fermented milks having been kept for 1 or 28 days at refrigeration temperatures, and said fermented milks having also been packaged in glass or polystyrene packaging. - Figure 2 shows, after fermentation, the number of cells of the strain CNCM 1-1225 (cfu / ml) having grown in different milks, said milks having a redox potential of the order of 500 mvolt or -50 mvolt before fermentation , and said fermentation being carried out under aerobic or anaerobic conditions.

- Figure 3 represents the number of cells of the strain CNCM 1-1225 (cfu / ml) having grown in different milks, as well as the redox potential at 25 ° C of these fermented milks, as a function of the storage time of these milks ferments at refrigeration temperatures.

Detailed description of the invention

In the context of the present invention, the name "rennet" is given to the coagulating extract coming from abomasum of young ruminants slaughtered before weaning. It will be recognized that rennet also includes veal rennet substitutes, such as animal pepsins; coagulating preparations from the plant kingdom extracted from artichoke, thistle, ficin, latex, fig, papain, for example; coagulating preparations from the microbial kingdom extracted from bacteria of the genus Bacillus and Pseudomonas, and molds belonging to the species Endothia parasitica, Mucor pusillus and Mucor miehei, for example.

By milk is meant, on the one hand, a milk of animal origin, such as milk from cows, goats, sheep, buflesse, zebu, mare, donkey, camel, etc. This milk may be a milk in the native state, a reconstituted milk, a skimmed milk, or a milk supplemented with compounds necessary for the growth of bacteria or for the treatment of milk such as fats, yeast extract, peptone, ascorbic acid and / or a surfactant, for example. Preferably, these milks have a pH of the order of 6.4-7, in particular pH 6.6-6.8.

The term milk also applies to what is commonly called vegetable milk, that is to say an extract of plant materials treated or not, such as legumes (soy, chickpea, lentil, ect ...) or oilseeds (rapeseed, soya, sesame, cotton, ect ...), extract which contains proteins in solution or in colloidal suspension, coagulable by chemical action, by acid fermentation and / or by heat. These vegetable milks could undergo heat treatments analogous to those of animal milks. They may also have had their own treatments, such as discoloration, deodorization, and treatments for removing unwanted taste. Finally, the word milk also designates mixtures of animal milks and vegetable milks. Preferably, these milks have a pH of the order of 6.4-7, in particular pH 6.6-6.8.

Against all expectations, it has been found that the growth and survival of certain lactic acid bacteria are also influenced by the milk water activity (Aw), that is to say by the ratio between the partial vapor pressure of the water on the surface of the powder and the vapor pressure of pure water at the same temperature. The best survivals can be obtained when the Aw of milk at 20 ° C is greater than 0.97, preferably between 0.988-0.983, for example. By way of indication, the Aw can be determined by measuring the relative equilibrium humidity reached in a closed enclosure at constant temperature. For this, a sample of a few g of milk is enclosed in a sealed container placed in a room thermostatically controlled at 20 ° C. The empty space around this sample reaches equilibrium, after 30-60 min, the same Aw value as the sample. An electronic sensor, mounted in the closure lid of the container, then measures the humidity of this empty space by means of an electrolytic resistance.

Likewise, it has been found that the addition of at least one agent which promotes bacterial growth in milk makes it possible to significantly enhance the growth and survival of certain lactic acid bacteria. Among these agents, there may in particular be a sugar such as glucose and sucrose, an amino acid such as cysteine and glutathione, a yeast extract in particular an extract comprising large amounts of purine and pyrimidine bases as well as their phosphate derivatives (adenosine , thymine, guanine, cytosine and uracil) and / or hydrolysates of animal or vegetable protein materials (soy), for example. In particular, the milk can comprise approximately 0.1-1% of yeast extract and / or approximately 0.25-1% of peptones.

Since it has been realized that the redox potential of a dairy product comprising lactic acid bacteria is likely to increase appreciably during storage of the dairy product at refrigeration temperatures, it is also preferable to add compounds to the milk. who are likely to stabilize their potential redox. Among these compounds, one can count all food reducing agents such as ascorbic acid, vitamin E and / or their derivatives, usable at 0.01-1% by weight, for example.

Compounds that stabilize redox potential and agents that promote bacterial growth can be added to milk before pasteurization. However, since some of these compounds are liable to be destroyed or even modified, following prolonged heat treatment, it is also possible to envisage adding them to the milk after pasteurization and / or after fermentation, in the form of a solution. sterile, for example.

All the devices intended for pasteurizing milk can be used by a person skilled in the art. It is thus possible to heat treat milk at least 90 ° C for at least 30 min, preferably at 95-130 ° C for 30-120 min, so as to obtain a redox potential of less than 450 mvolt, in particular less than 400 mvolt, or even less than 350 mvolt if one wants to obtain maximum growth and survival of lactic acid bacteria, for example.

Then, the pasteurized milk is inoculated with at least one strain of lactic acid bacteria so as to obtain directly in the milk from 10 ³ to 10 ⁸ cfu / ml. The milk can be inoculated with a culture of fresh lactic acid bacteria, with a concentrated and frozen culture, or even with a culture dried by lyophilization or by spraying under a flow of hot air (see US389730), for example.

The strain of lactic acid bacteria can be chosen from the Lactococcus lactis species, in particular L. lactis subsp. cremoris, L. lactis subsp. lactic biovar diacetylactis, and L. lactis; Streptococcus thermophilus; acidophilic bacteria including L. acidophilus, L. crispatus, L. amylovorous, L. gallinarum, L. gasseri, L. johnsonii; Lactobacillus fermentum; Lactobacillus casei including L. casei subsp. casei; Lactobacillus delbruckii, in particular L. delbruckii subsp lactis; L. delbruckii subsp. helveticus; L. delbruckii subsp. bulgaricus; bifidobacteria including Bifidobacterium infantis, Bifidobacterium breve; Bifidobacterium longum; and finally Leuconostoc mesenteroides, in particular L. mesenteroides subsp cremoris, for example (Bergey's Manual of Systematic Bacteriology, vol 2, 1986; Fujisawa et al., Int. Syst. Bact, 42, 487-491, 1992). Preferably, lactic acid bacteria which are sensitive to oxygen are used, in particular all bifidobacteria, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus and Lactobacillus helveticus.

Probiotic bacteria are of particular interest in the context of the present invention. These bacteria are in fact capable of adhering to human intestinal cells, of excluding pathogenic bacteria on human intestinal cells, and of acting on the human immune system by allowing it to react more strongly to external aggressions (ability to immunomodulation), for example by increasing the phagocytosis capacities of granulocytes derived from human blood (J. of Dairy Science, 7 £, 491-197, 1995: immunomodulation capacity of the strain La-1 which has been deposited at the Institut Pasteur under the number CNCM 1-1225).

As an example, the Lactobacillus acidophilus CNCM I-1225 strain described in EP577904 can be used. This strain was recently reclassified among Lactobacillus johnsonii, following the new taxonomy, proposed by Fujisawa et al, which is now an authority on the taxonomy of acidophilic lactobacilli (Int. J. Syst. Bact., 42, 487-791, 1992). Other probiotic bacteria are also available, such as those described in EPI 99535 (Gorbach et al.) Or in US5296221 (Mitsuoka et al.), For example.

The dairy composition obtained by the process according to the invention can also be traditionally fermented until at least 10 ⁶ cfu / ml, in particular 10? -10 ⁹ cfu / ml, for example, are obtained. When these milk compositions comprise probiotic lactic acid bacteria, in particular the strain L. johnsonii CNCM 1-1225, it is preferable to carry out the fermentation in the absence of oxygen, for example under an atmosphere of carbon dioxide.

The milk composition obtained by the process according to the invention can also be transformed into unripened fresh cheeses which are commonly called in Anglo-Saxon countries "quarg" or "cottage-cheese" and in Germany "quark", by example. For this, the milk inoculated with lactic acid bacteria can be fermented, but not necessarily. Rennet is generally added to it, of the order of 0.01 to 0.15% by volume / volume, so as to pass the casein from one phase colloidal to a precipitated phase, this passage being accompanied by the formation of a whey. Then, the whey is separated by centrifugation or ultrafiltration.

The invention also covers all dairy compositions packaged in an impermeable or semi-permeable oxygen material, said compositions comprising at least 10 ⁶ cfu / ml of probiotic lactic acid bacteria and a redox potential of less than 450 mvolt, preferably less than 400-350 mvolt if one wants compositions in which the viability of lactic acid bacteria is stabilized at an acceptable level

Preferably, the dairy compositions according to the invention are packaged in a material which allows less than 0.01 cm ³ of air to pass per day and per cm ² under an external pressure of 0.21 bar, for example a material impermeable to air like glass or ethyl vinyl alcohol (EVOH), or a semi-permeable material like polystyrene (PS), polypropylene (PP), polyethylene terepthalate (PET), ethyl vinyl alcohol (EVOH), high density polyethylene (HDPE), or a mixture of these materials, for example.

Since the lactic acid bacteria which are present in a milk treated according to the present process become particularly resistant to stressful situations, the milk composition obtained by the present process can also be used to prepare other fermented milk products, in particular as starter for large-scale milk fermentation, for example.

The present invention is described in more detail below with the aid of the additional description which follows, which refers to examples of preparation of fermented milk products, as well as to the description of a test for measuring the potential. redox. Percentages and parts are given by weight unless otherwise indicated. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention of which they do not in any way constitute a limitation.

Redox potential measurement

The measurement of the redox potential is carried out in accordance with the publication by Buhler H. et al. (Ingold AG, Germany). For this, a pH / mvolt meter is used combined with a redox electrode (Ingold n ° 105053288). The pH / mvolt meter is calibrated using a standard redox buffer. The milk samples at pH 6.4-7 are previously incubated in a 25 ° C bath. The redox potential is measured after 3 min of stability, and the redox potential is calculated by adding 244.4 mvolt to the displayed redox value.

Example 1

Several milk samples are prepared consisting of 10% of a skimmed milk powder, 1% of yeast extracts and 0.5% of glucose. In order to obtain redox potentials lower than 450 mvolt, these milks are heat treated, respectively, for 30 min at 63 ° C. in a hot water bath, for 30 min at 95 ° C. in a hot water bath, for 15 min at 121 ° C in an autoclave, or for 60 min at 121 ° C in an autoclave. These milks are inoculated with the probiotic strain Lactobacillus johnsonii CNCM 1-1225 which was deposited at the Institut Pasteur, 25 rue du docteur Roux, Paris, June 30, 1992. These milks are fermented without stirring until obtaining '' a pH of the order of 4.6, each is packaged in two impermeable (glass) or semi-permeable packages, each packaged milk is stored for 1 day or 28 days at refrigeration temperatures, then after storage, the number of lactic acid bacteria that survive.

The results presented in FIG. 1 clearly show that the prolonged heat treatment of milk appreciably improves the survival of lactic bacteria, even in the presence of oxygen. Furthermore, it is also observed that the redox potentials of heat-treated milks are inversely correlated with the time and the degree of temperature applied to the milk. In other words, the more the heat treatment of the milk, the more the redox potential of the milk is lowered. In this regard, it should be noted that the lower the redox potential of milk, the more manifest the resistance of lactic acid bacteria.

Example 2

The redox potentials are adjusted at 25 ° C of two MRS artificial media, respectively to about 500 mvolt and -50 mvolt, by adding an appropriate amount of potassium ferricynide or DTT. These two media are inoculated with an inoculum of the strain Lactobacillus johnsonii CNCM 1-1225, they are ferments at aerobic. For aerobic conditions, sterile air bubbles are introduced into the fermentation media. Finally, the number of colonies of bacteria that have grown in these milks is listed.

The results presented in FIG. 2 show that milks having a redox potential at 25 ° C. of the order of -50 mvolt give the best growth scores, whether in the absence or in the presence of air. Consequently, when we lower the redox potential of a milk, we then promote the growth of certain lactic acid bacteria.

Example 3

Two starters of the Lactobacillus johnsonii CNCM 1-1225 strain and of the Streptococcus thermophilus CNCM 1-1421 strain, which was deposited at the Institut Pasteur, 25 rue du docteur Roux, Paris, on May 18, 1994, are prepared 10% of a skimmed milk powder, 1% yeast extract and 0.5% glucose, said milk having previously been heat treated at 95 ° C for 30 min.

Skimmed pasteurized milk is conventionally inoculated at 115 ° C. for 20 min with 5% of the starter of the strain Lactobacillus johnsonii CNCM 1-1225 and 0.5% of the starter of the strain Streptococcus thermophilus CNCM 1-1421. When the pH of fermented milks reaches pH 4.5, 0.1% w / v of vitamin C is added, the milks are packed in semi-permeable jars and kept at refrigeration temperatures for 1 , 14 or 28 days, after which the redox potential of the fermented milks is measured and the number of Lactobacillus johnsonii CNCM 1-1225 bacteria which have survived in these fermented milks is listed.

For the comparison, a milk is fermented under the same conditions with the difference that no vitamin C is added.

The results presented in Figure 3 show that if we lower and stabilize the redox potential of a fermented milk less than 450 mvolt, we obtain at least 50% survival of lactic bacteria after 28 days of storage; while if the redox potential of fermented milk is greater than 450 mvolt, less than 1% survival is obtained after 28 days of storage. Example 4

Two milks made of 10% of a skimmed milk powder and different, yeast extract concentrations are prepared, these media are heat treated at 11 ° C for 15 min, they are inoculated with 5% of a fresh culture of the Lactobacillus johnsonii CNCM 1-1225 strain, they are incubated at 40 ° C for 1 to 28 days, and the number of lactic acid bacteria having survived these storage conditions is determined.

For comparison, under the same conditions, fermented milks stored for 1 to 28 days are analyzed, said milks not comprising yeast extracts.

The results show that the user of 0.1 to 1% of yeast extracts in the culture medium promotes the survival of lactic acid bacteria during the prolonged conservation of this medium. The best results are obtained for milks having the order of 1% yeast extracts.

Example 5

Several milks are prepared containing 10% of a skimmed milk powder and different concentrations of additives, these media are heat treated at 115 ° C. for 15 min, they are inoculated with 5% of a fresh culture of the Lactobacillus johnsonii strain. CNCM 1-1225, they are incubated at 40 ° C to pH 4.5, cooled at 4 ° C for 28 days in air-permeable or semi-air-permeable pots, and the number of lactic acid bacteria that survived these storage conditions. The experimental conditions are given in Table 1 below. However, it should be noted that vitamin C is added after fermentation and before storage, in the form of a sterile solution.

The results presented in table 1 below show that the peptone extracts, the yeast extracts and / or vitamin C, make it possible to improve the survival of the probiotic lactic acid bacteria after 28 days of storage at refrigeration temperatures, and even in the presence of air.

Claims

1 Process for manufacturing a fermented milk composition in which a milk is pasteurized at a temperature and for a time such that its redox potential at 25 ° C. is lowered to a value below 450mvolt, agents stabilizing the redox potential are added milk, the milk is inoculated with lactic acid bacteria, the milk is fermented until at least 10 ⁶ cfu / ml is obtained and an Aw greater than 0.97.

2. The method of claim 1 wherein, the milk further comprises agents promoting bacterial growth.

3. Method according to one of claims 1 or 2, in which the milk is fermented by lactic acid bacteria capable of adhering to human intestinal cells, of excluding pathogenic bacteria on human intestinal cells, and of acting on the human immune system by allowing it to react more strongly to external aggressions.

4. Use of a milk having a redox potential at 25 ° C lower than 450mvolt and containing agents stabilizing this redox potential of milk, for the preparation of a dairy product fermented by lactic bacteria.

5. Use according to claim 4, for the preparation of a dairy product conditioned in a material impermeable or semi-permeable to oxygen.

6. Dairy compositions conditioned in a material impermeable or semi-permeable to oxygen, said compositions having a redox potential less than 450 mvolt and an Aw greater than 0.97, and at least 10 ⁶ cfu / ml of probiotic lactic acid bacteria.