SUCROSE NEGATIVE STREPTOCOCCUS THERMOPHILUS FOR USE IN PREPARATION OF FERMENTED PRODUCTS

Abstract

The present invention relates to novel sucrose negative (Suc−) Streptococcus thermophilus strains, compositions comprising said strains, uses thereof, methods for obtaining such strains and methods for the preparation of fermented food products using such compositions.

Claims

1. A mutant Streptococcus thermophilus strain that it is galactose positive and sucrose negative.

2. The mutant strain of claim 1, wherein the strain is a texturizing strain.

3. The mutant strain of claim 1, wherein the strain is glucose positive.

4. A S. thermophilus strain deposited at the CNCM under reference number CNCM I-5448.

5. A composition comprising the mutant strain of claim 1.

6. The composition according to claim 5, wherein the composition comprises at least 10.sup.5 CFU/g of said mutant strain.

7. The composition of claim 5, wherein said composition is a food product.

8. The composition of claim 7, wherein said food product is a dairy product or a dairy alternative.

9. A method for preparing a fermented food product comprising: i) providing a mixture comprising: a) milk, and b) at least one S. thermophilus strain of claim 1, and ii) fermenting said mixture to provide a fermented food product.

10. The composition of claim 5, wherein the composition has a viscosity of at least 800 mPa.Math.s.

11. The composition of claim 5, wherein the composition has a pH of 4.7 or lower.

12. The composition of claim 5, further comprising at least one strain of Bifidobacterium or lactic acid bacteria.

13. (canceled)

14. A method for obtaining a S. thermophilus strain according to claim 1, comprising: i) providing a first S. thermophilus strain, ii) selecting at least one galactose positive clone thereof, and iii) mutating said selected clone(s) selected in ii), and selecting at least one sucrose negative variant thereof.

15. The method of claim 14, further comprising: iv) selecting a sucrose negative variant that is the most fermentative in a milk based medium and/or subculturing a sucrose negative variant in a milk based medium.

16. The method of claim 9, wherein the fermented food product has a viscosity of at least 800 mPa.Math.s.

17. The method of claim 9, wherein the fermented food product has a pH of 4.7 or lower.

18. The method of claim 9, further comprising providing at least one strain of Bifidobacterium or lactic acid bacteria to the mixture.

19. The method of claim 14, wherein said mutating is performed by means of chemical and/or radiation.

20. The composition of claim 7, wherein the food product is a fermented food product.

21. The composition of claim 20, wherein the fermented food product is yogurt, kefir, buttermilk, quark, sour cream, or cheese.

Description

DESCRIPTION OF THE FIGURES

[0156] FIG. 1. Growth of wild type strain and Gal+ clones G9 to G16 in M17m with lactose as a sole source of carbon.

[0157] FIG. 2. Growth of wild type strain and Gal+ clones G9 to G16 in M17m with galactose as a sole source of carbon.

[0158] FIG. 3. Fermentation of semi-skimmed UHT milk by the wild type strain or by the sucrose deficient clones 1-13.

[0159] FIG. 4. Growth of wild type strain and sucrose deficient clones 1-13 in M17m with lactose as a sole source of carbon.

[0160] FIG. 5. Growth of wild type strain and sucrose deficient clones 1-13 in M17m with sucrose as a sole source of carbon.

[0161] FIG. 6. Fermentation of semi-skimmed UHT milk by the wild type strain, clone 11 or clone 11R15 (CNCM I-5448).

[0162] FIG. 7. Comparison of the genomes of the wild type strain and of clone G15. (A) Alignment of the lacS nucleotide sequences. (B) Alignment of the LacS amino acid sequences. (C) Alignment of the intergenic region between galR and galK genes. A point mutation is indicated by a box. Cre site in the galK promoter is underlined.

[0163] FIG. 8. Comparison of the genomes of clone G15 and of clone 11. (A) Alignment of the scrA nucleotide sequences. (B) Alignment of the ScrA amino acid sequences. A point mutation is indicated by a box.

[0164] FIG. 9. Comparison of the genomes of clone 11 and of clone 11R15 (CNCM I-5448). (A) Alignment of the galR nucleotide sequences. (B) Alignment of the GalR amino acid sequences. A point mutation is indicated by a box.

EXAMPLES

[0165] The Inventors aimed to identify a sucrose negative (Suc−) S. thermophilus strain suitable for the preparation of fermented dairy products, such as yogurt said strain being sucrose negative (Suc−) to ensure improved sweetness in sugar containing products with minimal post-acidification.

[0166] Additionally it was required that said strain have texturizing properties (i.e. producing EPS) and furthermore it was preferred that the strain was fructose negative (Fru−) to ensure that the improved sweetness and reduced post-acidification in products containing fruit.

[0167] Accordingly, the Inventors carried out a screening program of 655 S. thermophilus strains from the Applicant's Danone Culture Collection. Each strain was analysed using the API 50CH test after 48 hours of incubation at 37° C. with MRS API. This made it possible to determine the fermentation profile for galactose, sucrose and fructose.

[0168] Of all the strains tested 654 were Suc+, 13 were Gal+, 84 were Fru+. All Gal+ strains (13) were also Fru+. Of all the strains tested no Suc−Gal+ was identified. The sole Suc− strain identified was Glu+, Fru+, Lac+, Gal−.

I. Selection of a S. thermophilus Gal+ Variant

[0169] The wild type strain (Wt) shows the phenotype Lac+ Glu+ Suc+ Gal− Fru− and efficiently carries out the fermentation of lactose with high texturizing properties. The wild type strain was successively subcultured in modified M17 medium (M17m, the concentration of beta-glycerophosphate is 9 g/l instead of 19 g/l in M17) in the presence of galactose as the sole carbon source. After 12 subcultures, some clones were isolated on dishes containing M17m galactose. Eight clones (G9 to G16) were isolated and cultured in M17m galactose at 5 g/l.

[0170] The growth of the eight selected clones was tested and monitored using a Bioscreen C at a wavelength of 600 nm (Bioscreen C is a spectrophotometer allowing the monitoring of growth by measuring the optical density at 600 nm in microplates) in two different media. Growth was tested in the presence (i) of lactose as the sole source of carbon or (ii) of galactose as the sole source of carbon. The precultures were carried out in M17m gal for the colonies and in M17m lac for the wild type strain. The inoculation rate was 1% from the precultures.

[0171] The wild type strain and the eight isolated clones efficiently grow in the medium containing lactose (FIG. 1), whereas, in the medium containing galactose, only the eight isolated clones are able to grow (FIG. 2).

[0172] These results show the isolation of Gal+ variants from the Gal− wild type strain.

[0173] Clone G15 was selected because it showed the best growth in both galactose and lactose.

II. Mutagenesis of the S. thermophilus Gal+ Variant

[0174] Ultraviolet mutagenesis was performed on the Gal+ clone G15 derived from the wild type strain. For the mutagenesis, a dose of UV giving approximately 2% survival was used.

[0175] Four thousand clones obtained from mutagenesis were tested for their capacity to acidify the M17m medium in the presence of galactose at 5 g/l as a carbon source and to acidify the M17m medium in the presence of sucrose at 5 g/l as a source of carbon. Purple bromocresol was used as a pH indicator, which turns yellow in acidic medium while it is purple in a non-inoculated medium.

[0176] Thirteen out of 4000 tested clones showed a reduced ability to acidify the medium in the presence of sucrose while maintaining a good acidification in the presence of galactose as a carbon source.

III. Characterization of the S. thermophilus Gal+ Mutants with a Deficiency in Sucrose Metabolism

[0177] 13 clones were tested in semi-skimmed UHT milk supplemented with yeast extract at 2 g/l. The milk enriched with yeast extract was inoculated at 1% from a pre-culture made in semi-skimmed UHT milk enriched with yeast extract at 2 g/l. The cultures were incubated at 38° C. and the fermentation was stopped at pH 4.7 by cooling to 4° C.

[0178] The fermentation was monitored by the iCinac system which makes it possible to obtain the pH values continuously. All clones showed a reduced capacity to acidify the milk (FIG. 3).

[0179] The ability of the clones to grow in a medium containing sucrose as a carbon source was also evaluated by monitoring the optical density at 600 nm with Bioscreen C.

[0180] Growth monitoring was carried out in the presence (i) of lactose as the sole source of carbon or (ii) of sucrose as the sole source of carbon. The precultures were carried out in M17m gal for the colonies and in M17m Lac for the wild type strain. The inoculation rate was 1% from the precultures.

[0181] In the presence of lactose, all clones were able to grow (FIG. 4). In the presence of sucrose, most of the clones showed a growth that was reduced as compared to the growth of wild type strain, but only clone 11 showed an inability to grow in the presence of sucrose (FIG. 5).

[0182] These results show the successful isolation of a Suc− clone from the Gal+ variant.

IV. Improvement of the Growth of the Gal+ Suc− Strain in Milk.

[0183] Clone 11 showed a clear Suc− phenotype, but also showed a reduction of its fermentative efficacy in milk. In order to restore an efficacy similar to the one of the wild type strain, 15 successive subcultures in semi-skimmed UHT milk supplemented with yeast extract at 2 g/l were carried out. Clone 11R15 was obtained and subsequently deposited as strain CNCM I-5448, as described above. The fermentation with the wild type strain, clone 11 or clone 11R15 in UHT semi-skimmed milk with 2 g/l yeast extract was measured by the iCinac system.

[0184] Clone 11R15 (CNCM I-5448) showed a capacity to acidify milk better than that of clone 11 and similar to that of the wild type strain (FIG. 6).

[0185] These results show the isolation of a Suc− variant with the same capacity to ferment milk as compared to the wild type strain.

V. Characterization of the Clone 11R15 (CNCM I-5448)

[0186] V.1. Dosage of Sucrose

[0187] Mixtures were prepared using UHT semi-skimmed milk with 2 g/l yeast extract and 55 g/l sucrose and inoculated with clone 11R15 or with the wild type strain, fermentation was carried out at 38° C. and stopped when the pH was 4.7 by rapid cooling.

[0188] As a control an unfermented acidic (pH 4.7) milk product (no strains) was prepared using UHT semi-skimmed milk with 2 g/l yeast extract and 55 g/l sucrose and glucono delta-lactone (GDL) 1.5% w/w as an acidifying agent. Said control product was heated to 38° C. for 4 hours, to ensure comparability to test products.

[0189] Products were stored for 7 days at 4° C., then the products were centrifuged at 5000 g for 5 minutes at 4° C. The determination of sucrose in the supernatants was assayed on 3 samples in 2 experiments using a Glucose-Fructose-Sucrose assay kit from Biosentec. The results are given in Table 1.

TABLE-US-00001 TABLE 1 Level of sucrose in the products after fermentation with wild type strain or with clone 11R15 (CNCM I-5448). First Standard Second Standard experiment deviation experiment deviation Wt  47.7 g/L 1.77  44.1 g/L 2.36 clone 11R15  52.9 g/L 2    53 g/L 2.15 (CNCM I-5448) GDL 1.5% 55.46 g/L 1.22 52.16 g/L 1.09 Control

[0190] In the first experiment, the product fermented with the wild type strain has lost 7.3±1.8 g whereas the product fermented with the clone 11R15 has lost only 2.1±1.8 g. In the second experiment, the product fermented with the wild type strain has lost 10.9±2 g whereas the product fermented with the clone 11R15 has lost only 2±2.1 g.

[0191] These results show that clone 11R15 (CNCM I-5448) is able to achieve fermentation while significantly maintaining the level of sucrose in the ingredients, contrary to the wild type strain. The slight reduction in sucrose during fermentation can reasonably be attributed to the inherent degradation of sucrose in the acidic pH, as it is comparable to that of the GDL control in the second experiment.

[0192] V.2. Viscosity

[0193] Mixtures were prepared using UHT semi-skimmed milk with 2 g/l yeast extract and 55 g/l sucrose and inoculated with clone 11R15, with clone 11 or with wild type strain. fermentation was carried out at 38° C. and stopped when the pH was 4.7 by rapid cooling. Products were stored for 7 days at 4° C., then the viscosity was measured by shear stress measurement.

[0194] This method consists of determining the viscosity of products, after manual stirring and incubation for 30 minutes at 4° C. Three measurements were carried out at 4° C. on three pots of milk fermented by the same strain and under the same conditions. The apparatus used for this analysis was a refrigerated MCR301 rheometer equipped with a 27 mm concentric cylinder system. This rotating system makes it possible to observe a destructurisation of the product as a function of a linear shear gradient, i.e. a stress at a given gradient. The results are obtained in the form of a continuous flow curve, rising and falling ramp between 0 and 20 s−1. The product undergoes an increasing shear gradient from 0 to 20 s−1 for 1 minute. This phase corresponds to the rising ramp. Then, it undergoes a decreasing shear gradient from 20 to 0 s−1 for 1 minute, corresponding to the downward ramp.

[0195] Each downward curve is then modeled according to the Casson model (equation I).

√{square root over (τ)}=√{square root over (τ.sub.0)}+η×D  (1)

τ: Stress (Pa)

[0196] τ.sub.0: Yield point of the product (Pa)
η: Viscosity of the product (Pa.Math.s)
D: Shear gradient (s−1)
Viscosity of the products after 7 days of storage at 4° C. are given in Table 2.

TABLE-US-00002 TABLE 2 Viscosity the products fermented with wild type strain, with clone 11 or with clone 11R15. Standard Viscosity deviation Wt 1116 mPa .Math. s 76 clone 11  557 mPa .Math. s 36 clone 11R15 1143 mPa .Math. s 42

[0197] These results show that clone 11R15 (CNCM I-5448) has similar texturizing properties as compared to the wild type strain.

[0198] V.3. API (Analytical Profile Index) Test

[0199] The fermentation profile of the clone 11R15 (CNCM I-5448) was obtained by carrying out the API 50CH test after 48 hours of incubation at 37° C. with MRS API. This made it possible to determine the fermentation profile for galactose, sucrose and fructose.

[0200] Clone 11R15 (CNCM I-5448) is galactose positive (Gal+), sucrose negative (Suc−), fructose negative (Fru−), lactose positive (Lac+), glucose positive (Glu+) as determined using said test and furthermore is highly fermentative and texturizing (significantly EPS producing).

VI. Genomic Comparisons of the S. thermophilus Variants

[0201] The complete genomes of the S. thermophilus wild strain and of its variants, i.e. clone G15, clone 11 and clone 11R15, were sequenced by NGS using Illumina NovaSeq6000 system.

[0202] The genomic sequences of all variants were compared to each other in order to identify the mutations associated to specific phenotypes which appeared step by step to the isolation of clone 11R15 (CNCM I-5448).

[0203] VI.1. Gal+ Phenotype

[0204] The comparison of the genome of the wild type strain with the genome of clone G15 showed that a mutation in the lacS gene and a mutation in the intergenic region between galR and galK were associated with the Gal+ phenotype.

[0205] In particular, the Inventors noted a substitution C/A in the lacS gene in position 1159 of SEQ ID NO: 1 (FIG. 7A), which results in a replacement of a Proline by a Threonine in position 387 of the LacS protein of SEQ ID NO: 3 (FIG. 7B), and a substitution G/T in galK promoter in position 228 of SEQ ID NO: 5 (FIG. 7C).

[0206] VI.2. Suc− Phenotype

[0207] The comparison of the genome of clone G15 with the genome of clone 11 showed that a non-sense mutation in the scrA gene was associated with the Suc− phenotype.

[0208] In particular, the Inventors noted a non-sense substitution C/T in the scrA gene in position 1393 of SEQ ID NO: 7 (FIG. 8A), which results in a stop in the ScrA protein in position 465 of SEQ ID NO: 9 (FIG. 8B).

[0209] VI.2. Rapid Growth in Milk

[0210] The comparison of the genome of clone 11 with the genome of clone 11R15 (CNCM I-5448) showed that a mutation in the galR gene was associated with the improvement of the growth of the strain in milk.

[0211] In particular, the Inventors noted a substitution T/C in the galR gene in position 281 of SEQ ID NO: 11 (FIG. 9A), which results in a replacement of a Leucine by a Proline in the GalR protein in position 94 of SEQ ID NO: 13 (FIG. 9B).

[0212] The mutations are summarized in order of appearance in Table 3.

TABLE-US-00003 TABLE 3 Summary of the step-by-step mutations between S. thermophilus variants. Clone 11R15 (CNCM I-5448) Clone Gal+ Wild Clone 11 Suc− type G15 Gal+ Rapid growth in Mutation strain Gal+ Suc− milk lacS No yes yes yes Substitution C/A (P.fwdarw.T) Intergenic region between No yes yes yes galR and galK Substitution G/T in galK promoter (cre site) scrA No No yes yes Substitution C/T (Non-sense mutation) galR No No No yes Substitution T/C (L.fwdarw.P)