Streptococcus Thermophilus strain, fermentation process using such strain and product obtained

Abstract

The invention relates to a strain of Streptococcus thermophilus deposited at Collection Nationale de Cultures de Microorganismes (CNCM) No. I-1477 and a mutant strain that has similar milk acidification characteristics. Also disclosed is the use of the S. thermophilus strains to produce fermented milk products and the products produced thereby.

Description

The present invention relates to a Streptococcus thermophilus strain, to the use of this strain for the production of fermented dairy products and to the products obtained using this strain.

The choice of lactic acid bacteria for the production of fermented dairy products calls into play various criteria, especially the acidifying activity and the formation of flavor components which provide the organoleptic properties of the product and the production of thickening agents which play a role in the texture and the unctuousness.

The acidifying activity is characterized essentially by three parameters: the kinetic of acidification, the titratable acidity and the final fermentation pH which determines the organoleptic characteristics of the product and its preservation quality, and the post-acidification which develops during preservation of the product.

A high rate of acidification makes it possible to reduce the period during which the milk-based product is sensitive to contaminants (pH>4.7) and thereby to reduce the risk of bacterial contamination.

An increase in the rate of acidification also enhances the economics of the process by increasing the productivity and the flexibility of the industrial material.

The post-acidification properties of the strains are particularly important for the preservation of the products. Indeed, fresh fermented products are preserved at temperatures of between about 4.degree. C. and 8.degree. C. for a period in general not exceeding 4 weeks; but if the metabolic activity of the bacteria is reduced by preservation at low temperature, it is not blocked and it brings about the production of lactic acid from lactose, which has as a consequence a reduction in pH and an increase in the acid taste which damages the organoleptic properties of the product.

In addition to the criteria retained for their contribution to the quality of the products, other elements more specifically linked to the process play a role in the choice of the strains, such as the fermentation temperature, the rate of acidification and the resistance to phages.

The resistance to phages is a very important criterion in the choice of the strains for reducing the risk of phage incidents during production which may block for a longer or shorter period the entire production for decontamination.

No S. thermophilus is so far known which exhibits a high rate of acidification and a high degree of acidity combined with a very low or practically zero post-acidification activity.

Indeed, it is generally observed that cultures of S. thermophilus which have a reduced post-acidification activity often show a limited acidifying activity during the fermentation, which does not allow the desired degree of acidity to be achieved in the products. In addition, these strains often have a slow growth and they require high inoculation levels and a longer incubation period which are incompatible with the economics of an industrial process.

The subject of the present invention is an S. thermophilus strain which has a rapid acidification kinetic allowing a high degree of acidity to be achieved and which does not post-acidify fresh fermented products during preservation.

More particularly, the present invention relates to an S. thermophilus strain DN-001 116 deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) under No. I-1477 and mutants thereof which have similar milk acidification characteristics.

Mutant strains having milk acidifying characteristics similar to the deposited strain is intended to designate strains which may be obtained especially by mutation and selection from the reference strain and/or by genetic transformation with the aid of vectors.

The acidifying characteristics are an end-of-incubation pH of 4.6 in less than 4 hours and a pH variation at 28 days of less than 0.2 pH unit, preferably less than 0.1 unit.

The mutant strains may be obtained from the reference strain, especially by mutation and selection according to their acidifying properties. The mutation techniques are known, as well as the tests which make it possible to control the acidifying properties (especially Spinnier H. E., Corrieu G. "Automatic method to quantify starter activity based on pH measurement" J. of Dairy Research, 56 (1989) 755-764); by Roissart H. and Luquet F. M. Edition Lorica ISBN: 2-9507477-0-1).

These strains are more particularly advantageous for the preparation of all types of fermented milk products.

Accordingly, the present invention relates to a process for the preparation of fermented dairy products in which a milk substrate is fermented with at least one S. thermophilus strain.

It is also possible to provide, in this process, for the use of a combination of one or more bacterial strains, especially with other lactic acid bacteria such as Lactobacillus bulgaricus, lactobacillus acidophilus, Lactobacillus casei or Bifidobacterium.

Preferably, the milk substrate is natural or reconstituted milk, skimmed or otherwise, or milk-based media or media based on products of dairy origin.

This substrate may comprise items commonly used for the preparation of milk desserts, solid items such as fruits, chocolate chips or cereals for example, but also sweetened products or liquid chocolates. The strain according to the invention in fact has the advantage of being resistant to sugars, which makes it possible to ferment sweetened products containing espeially sucrose.

The present invention also relates to the ferented milk products obtained using this process, especially fresh fermented cheese, and yoghurts for example.

The products obtained using this strain have a mild taste and their organoleptic properties are preserved during storage.

The culture of the strains according to the invention, pure or in combination with other strains, may also be used as probiotic for human consumption or animal feed and also as ferment to be used in the process according to the invention.

Other characteristics and advantages of the present invention will appear on reading the examples below.

EXAMPLE 1

Comparison of the Rate of Acidification and the Post-acidification Activity of Different S. thermophilus Strains

A reconstituted skimmed milk containing 12% dry matter and enriched with 0.1% yeast extract is sterilized for 15 min at 121.degree. C. An active culture of S. thermopilus comprising 10.sup.8 cells per milliliter is used to inoculate this medium at 1% (v/v). The starter culture is obtained after about 4 hours of incubation at 44.degree. C. This starter culture is used to inoculate (1% v/v) 1 liter of reconstituted skimmed milk containing 12% dry matter, enriched with 0.1% yeast extract and previously pasteurized at 95.degree. C. for 30 min. The inoculated milk is stirred and then incubated at 44.degree. C. When the acidity reaches a pH of 4.6, the preparation is cooled for 16 hours at 4.degree. C. This fermented milk is subjected to a test of preservation at 8.degree. C. for 28 days. The pH after 28 days of preservation is compared for the different strains. The results are presented in Table 1. The strain I-1477 corresponds to the strain DN-001 116. Only the milk fermented with the S. thermophilus strain DN-001 116 has a practically zero post-acidification after 28 days of preservation at 8.degree. C. The strain DN-001 116 shows a rapid rate of acidification; a pH of 4.6 is reached in less than 4 hours of incubation at 44.degree. C.

TABLE 1______________________________________Strain Incubation End of pH atcode time incubation pH 28 days Difference______________________________________01 DN-001 003 4 h 46 min 4.60 4.31 0.2902 DN-001 004 7 h 21 min 4.62 4.26 0.3603 DN-001 013 7 h 18 min 4.65 4.25 0.4004 DN-001 015 6 h 16 min 4.65 4.30 0.3505 DN-001 022 5 h 18 min 4.60 4.15 0.4506 DN-001 023 3 h 55 min 4.59 4.19 0.4007 DN-001 025 6 h 48 min 4.64 4.41 0.2308 DN-001 031 5 h 43 min 4.60 4.40 0.2009 DN-001 032 4 h 18 min 4.61 4.51 0.1010 DN-001 047 3 h 42 min 4.60 4.18 0.4211 DN-001 054 4 h 19 min 4.60 4.16 0.4412 DN-001 064 5 h 30 min 4.60 4.10 0.5013 DN-001 067 4 h 09 min 4.60 4.10 0.5014 DN-001 094 3 h 54 min 4.61 4.08 0.5315 DN-001 107 3 h 45 min 4.62 4.52 0.1016 DN-001 111 3 h 33 min 4.60 4.11 0.4917 DN-001 116 3 h 45 min 4.61 4.58 0.0318 DN-001 138 4 h 15 min 4.60 4.12 0.4819 DN-001 143 4 h 05 min 4.59 4.18 0.4120 DN-001 145 3 h 40 min 4.60 4.17 0.4321 DN-001 147 6 h 09 min 4.64 4.11 0.5322 DN-001 156 4 h 24 min 4.60 4.12 0.4823 DN-001 162 4 h 05 min 4.60 4.14 0.4624 DN-001 171 5 h 30 min 4.58 4.08 0.5025 DN-001 181 4 h 33 min 4.61 4.11 0.5026 DN-001 223 5 h 30 min 4.60 4.21 0.3927 DN-001 225 3 h 54 min 4.60 4.25 0.3528 DN-001 228 4 h 35 min 4.61 4.16 0.4529 DN-001 230 6 h 15 min 4.64 4.20 0.4430 DN-001 236 5 h 30 min 4.63 4.10 0.5331 DN-001 242 3 h 51 min 4.61 4.11 0.5032 DN-001 276 5 h 25 min 4.60 4.18 0.4233 DN-001 277 3 h 35 min 4.61 4.20 0.4134 DN-001 280 6 h 00 min 4.62 4.10 0.5235 DN-001 289 4 h 50 min 4.61 4.06 0.5536 DN-001 342 5 h 30 min 4.60 4.53 0.0737 DN-001 343 3 h 55 min 4.62 4.25 0.37______________________________________

EXAMPLE 2

Comparison of the Post-acidification Activity of the S. thermopilus Strain 001 116 with other S. thermophilus Strains

The starter culture is prepared as in Example 1. This starter culture is used to inoculate (1% v/v) 1 liter of reconstituted skimmed milk containing 12% dry matter, enriched with 0.1% yeast extract and previously pasteurized for 30 min at 95.degree. C. The inoculated milk is stirred and then incubated at 4.degree. C. When the acidity reaches 80.degree. D, the preparation is cooled for 16 hours at 4.degree. C. This fermented milk is subjected to a test of preservation at 8.degree. C. for 28 days. Table 2 presents the incubation time and the variation of the pH and of the Dornic acidity during storage at 8.degree. C. for 28 days.

TABLE 2______________________________________ Dornic Dornic acidity Dornic Dornic acidity and pH at acidity acidity and pHStrain Incubation end of and pH at and pH at differencecode time incubation 24 h 28 days for 28 days______________________________________001 116 3 h 25 min 80.degree. D, 86.degree. D, 92.degree. D, +12.degree. D, 4.80 4.77 4.63 -0.17ST25 5 h 30 min 80.degree. D, 86.degree. D, 95.degree. D, +15.degree. D, 4.75 4.77 4.57 -0.20ST44 3 h 55 min 80.degree. D, 88.degree. D, 111.degree. D, +21.degree. D, 4.75 4.71 4.35 -0.40______________________________________

The post-acidification by the strain ST25 is comparable to that of the strain DN-001 116 but its acidification rate is much slower. The strain ST44, which exhibits a satisfactory acidification rate, shows a very high post-acidification.

EXAMPLE 3

Stability of a Fermented Ultrafiltered Milk Prepared with the Strain DN 001 116

A mixture composed of an ultrafiltered milk retentate containing 6% protein (NT-NPN.times.6.38) and cream containing 40% fat is prepared. The proportion of each of the two constituents is 82% retentate and 18% cream. 0.03% (w/w) casein hydrolysate is added. The content of the mixture is 5.3% protein and 7.2% fat. The preparation is subjected to a preheating at 75.degree. C., a homogenization at 75.degree. C. and at 200 bar, and then to a pasteurization at 95.degree. C. for 8 min before cooling to 40.degree. C. The mixture is then inoculated with a frozen concentrated preparation of the strain DN-001 116 in an amount of 30 g/100 l of a starter culture at 9.times.10.sup.9 CFU/g. The inoculated mixture is incubated at 40.degree. C. until a pH of about 4.7 is obtained. After settling of the curd and after discharging the curd, it is made into a smooth paste in a suitable valve before cooling to 20.degree. C. in a plate exchanger. The smoothed and cooled product is then packaged in 1 kg pots. These pots, after palletization, pass inside a cold-air tunnel so as to cool the product to 4.degree. C. The product is then subjected to a test of preservation at 4.degree. C. and at 10.degree. C. The results are presented in Table 3.

TABLE 3______________________________________ Preservation at 4.degree. C. Preservation at 10.degree. C.Number of days pH Tasting pH Tasting______________________________________0 4.70 (+++) 4.70 (+++)3 4.66 (+++) n.d. (+++)12 4.66 (+++) 4.66 (+++)24 4.63 (++) 4.58 (++)______________________________________ (+++) good, very mild (++) good, mild

These results confirm that the S. thermophilus strain DN-001 116 exhibits a very reduced post-acidification at 4.degree. C. and at 10.degree. C.

EXAMPLE 4

Phage-resistance Character of the S. thermophilus Strain DN-001 116

60 phages of S. thermopilus were isolated over 10 years of production and in factories in different geographical areas. Analysis of their host spectrum made it possible to classify them into 7 subgroups. The resistance of the strain DN-001 116 was tested for phages representative of these 7 subgroups (.phi.15, .phi.57, .phi.47, .phi.77, .phi.76, .phi.29, .phi.65), the titer of the phages exceeded 10.sup.7 Plaque Forming Units/ml (The phages underlined have been described in the publication Biochimie (1990) 72, 855-862). The strain DN-001 116 is resistant to all the phages tested.

EXAMPLE 5

Resistance to Sugars

The procedure is carried out as in Example 3, but by adding to a medium to be fermented varied quantities of sucrose; the time for the target pH to be obtained is measured:

medium containing 9% sucrose: pH 4.7 620 min medium containing 13% sucrose: pH 4.7 920 min

The Streptococcus thermophilus strain has been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) under No. I-1477 on the date of Sep. 22, 1994

Claims

1. An isolated culture of Streptococcus thermophilus strain deposited at Collection Nationale de Cultures de Microorganismes (CNCM) under No. I-1477.

2. An isolated culture of mutant strain of the S. thermophilus strain according to claim 1, which has milk acidification characteristics comprising an acidification of milk products to a pH of 4.6 in less than 4 hours and a pH variation of no more than 0.03 unit following storage of said milk products at 8.degree. C. for 28 days.

3. The mutant strain according to claim 2, wherein the mutant strain is obtained by mutation and selection for its acidification characteristics.

4. A process for preparing fermented dairy products, wherein said process comprises fermenting a milk substrate with at least one S. thermopilus strain according to claim 1 or 2.

5. The process according to claim 4, wherein the fermentation is carried out in the presence of at least one other strain of bacterium.

6. The process according to claim 5, wherein the other strain of bacterium is a lactic acid bacterium.

7. The process according to claim 6, wherein the lactic acid bacterium is selected from the group consisting of:

Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium.

8. The process according to claim 4, wherein the milk substrate is milk.

9. The process according to claim 4, wherein the substrate contains solid items.

10. The process according to claim 9, wherein the solid items comprise fruits, chocolate products, or cereals.

11. Fermented products obtained using the process according to claim 4.

12. The Streptococcus thermophilus strain according to claim 1 or 2, wherein the strain is phage-resistant.

13. Fermented products obtained using the process according to claim 5.

14. Fermented products obtained using the process according to claim 6.

15. Fermented products obtained using the process according to claim 7.

16. Fermented products obtained using the process according to claim 8.

17. Fermented products obtained using the process according to claim 9.

18. Fermented products obtained using the process according to claim 10.

19. A process for preparing fermented dairy products, wherein said process consists of fermenting a milk substrate with a S. thermopilus strain according to claim 1 or 2.