<i>Streptococcus thermophtlus </i>(ST) cell to make e.g. mozzarella cheese

Abstract

Deposited Streptococcus thermophilus (ST) strains that e.g. are suitable to be used in an improved method for the manufacture of low browning mozzarella cheese.

Claims

1. A mutant Streptococcus thermophilus (ST) cell selected from: (a) a cell of Streptococcus thermophilus strain CHCC19097 deposited with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under accession number DSM 32594; (b) a cell of Streptococcus thermophilus strain CHCC19100 deposited with DSMZ under accession number DSM 32595; (c) a cell of Streptococcus thermophilus strain CHCC27912 deposited with DSMZ under accession number DSM 32596; (d) a cell of Streptococcus thermophilus strain CHCC29526 deposited with DSMZ under accession number DSM 32597; (e) a cell of Streptococcus thermophilus strain CHCC29530 deposited with DSMZ under accession number DSM 32598; (f) a cell of Streptococcus thermophilus strain CHCC29525 deposited with DSMZ under accession number DSM 32897; (g) a cell of Streptococcus thermophilus strain CHCC30963 deposited with DSMZ under accession number DSM 32898; and (h) a cell of Streptococcus thermophilus strain CHCC30964 deposited with DSMZ under accession number DSM 32900.

2. A method for obtaining a mutant Streptococcus thermophilus (ST) strain, comprising mutating a cell selected from a mutant cell of claim 1 as a starting cell, and isolating a further mutant that has retained capability of reducing release of galactose in the presence of lactose as compared to the starting cell.

3. A starter culture composition comprising viable cells of one or more mutant Streptococcus thermophilus (ST) strains of claim 1.

4. The starter culture composition of claim 3, wherein the concentration of viable cells of the one or more mutant Streptococcus thermophilus strains is in the range of 10.sup.7 to 10.sup.14 colony forming units (CFU) per gram of the composition.

5. The starter culture composition of claim 3, further comprising a cell of Streptococcus thermophilus strain CHCC14994 deposited with DSMZ under accession number DSM 25838 in a concentration in the range of 10.sup.4 to 10.sup.14 CFU per gram of the composition.

6. A method of manufacturing a food or feed product comprising adding a starter culture composition according to claim 3 to a food or feed product starting material to obtain inoculated food or feed product material, and maintaining the inoculated food or feed product material under conditions where the Streptococcus thermophilus (ST) cells are metabolically active.

7. The method of claim 6, wherein the product is a food product.

8. The method of claim 7, wherein the food product is dairy product.

9. The method of claim 8, wherein the dairy product is fermented milk, yogurt, buttermilk, fresh cheese, soft cheese, cheddar cheese, mozzarella cheese, or pasta filata cheese.

10. The method of claim 9, wherein the dairy product is soft cheese, cheddar cheese, pasta filata cheese or mozzarella cheese.

11. The method of claim 10, wherein the dairy product is mozzarella cheese or cheddar cheese.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Deposited Strains/Cells

(2) A sample of the Streptococcus thermophilus cell CHCC14994 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 25838 with a deposit date of 3 Apr. 2012. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(3) As discussed above, this deposited strain is described in WO2013/160413A1 (Chr. Hansen).

(4) A sample of the novel Streptococcus thermophilus cell CHCC19097 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32594 with a deposit date of 22 Aug. 2017. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(5) A sample of the novel Streptococcus thermophilus cell CHCC19100 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32595 with a deposit date of 22 Aug. 2017. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(6) A sample of the novel Streptococcus thermophilus cell CHCC27912 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32596 with a deposit date of 22 Aug. 2017. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(7) A sample of the novel Streptococcus thermophilus cell CHCC29526 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32597 with a deposit date of 22 Aug. 2017. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(8) A sample of the novel Streptococcus thermophilus cell CHCC29530 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32598 with a deposit date of 22 Aug. 2017. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(9) A sample of the novel Streptococcus thermophilus cell CHCC29525 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32897 with a deposit date of 16 Aug. 2018. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(10) A sample of the novel Streptococcus thermophilus cell CHCC30963 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32898 with a deposit date of 16 Aug. 2018. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(11) A sample of the novel Streptococcus thermophilus cell CHCC30964 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) under the accession number DSM 32900 with a deposit date of 16 Aug. 2018. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(12) A Starter Culture Comprising a ST Cell as Described Herein

(13) The ST cell as described herein is useful as starter culture in the production of food or feed products.

(14) Typically, such a starter culture composition comprises the bacteria in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells, which is in the range of 10.sup.4 to 10.sup.14 cfu (colony forming units) per gram of the composition including at least 10.sup.4 cfu per gram of the composition, such as at least 10.sup.5 cfu/g, e.g. at least 10.sup.6 cfu/g, such as at least 10.sup.7 cfu/g, e.g. at least 10.sup.8 cfu/g, such as at least 10.sup.9 cfu/g, e.g. at least 10.sup.10 cfu/g, such as at least 10.sup.11 cfu/g.

(15) The composition may as further components contain cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and/or vitamins.

(16) As it is normal in the production of lactic acid bacterial fermentation processes to apply mixed cultures of lactic acid bacteria, the composition will in certain embodiments comprise a multiplicity of strains either belonging to the same species or belonging to different species.

(17) For instance, it may be preferred that the composition also comprises other cells such as e.g. Lactobacillus cells and/or Lactococcus cells.

(18) A Method of Manufacturing a Food or Feed Product

(19) As said above, an aspect of the invention relates to a method of manufacturing a food or feed product comprising adding a starter culture composition as described herein to a food or feed product starting material and keeping the thus inoculated starting material under conditions where the ST cells are metabolically active.

(20) Useful food product starting materials include any material which is conventionally subjected to a lactic acid bacterial fermentation step such as milk (e.g. soy milk or cow milk, preferably cow milk), vegetable materials, meat products, fruit juices, must, doughs and batters.

(21) The fermented products, which are obtained by the method, include as typical examples dairy products such as fermented milk, yogurt, cheese including fresh cheese products, soft cheese products, cheddar, mozzarella or buttermilk.

(22) In a preferred embodiment, the dairy product is soft cheese, cheddar cheese, pasta filata cheese or mozzarella cheese—more preferably, the dairy product is pasta filata cheese, cheddar cheese or mozzarella cheese—most preferably the dairy product is mozzarella cheese or cheddar cheese (preferably used for making pizza).

(23) In further embodiments, the substrate material is a starting material for an animal feed such as silage, e.g. grass, cereal material, peas, alfalfa or sugar-beet leaf, where bacterial cultures are inoculated in the feed crop to be ensiled in order to obtain a preservation hereof, or in protein rich animal waste products such as slaughtering offal and fish offal, also with the aims of preserving this offal for animal feeding purposes.

EXAMPLES

Example 1: Deposited Streptococcus thermophilus (ST) Strains—Capable of Extraordinary Reducing the Release of Galactose Also in the Presence of High Amounts of Lactose (as in Milk)

(24) Reference Strains: ST strain CHCC4323: It has what may be termed a GalK natural wildtype sequence (herein termed GalK(−)) and may be seen as a ST reference strain that corresponds to a today commercially relevant used ST strains for making mozzarella cheese; ST strain 4323-2 (CHCC14993): It comprises a mutation in the GalK (galactokinase) gene (herein termed Gal(+)) and may be seen as a reference strain that corresponds to a strain made according to the description of above discussed WO2011/026863A1 (Chr. Hansen) and WO2011/092300A1 (Chr. Hansen).

(25) Deposited Strains of the Present Invention:

(26) CHCC14994: DSM 25838 ST strain—disclosed in WO2013/160413A1 (Chr. Hansen).

(27) CHCC19097: Novel DSM 32594 ST strain as described herein.

(28) CHCC19100: Novel DSM 32595 ST strain as described herein.

(29) CHCC27912: Novel DSM 32596 ST strain as described herein.

(30) CHCC29526: Novel DSM 32597 ST strain as described herein.

(31) CHCC29530: Novel DSM 32598 ST strain as described herein.

(32) Isolation of Galactose Hyper-Fermenting Mutants from S. thermophilus:

(33) Prior to the mutant isolation the strains were streaked on M17 agar plates with 2% galactose (M17-gal plates). The wild type (wt) strains did not grow significantly on galactose as sole carbohydrate source.

(34) Overnight cultures were then plated on M17-gal plates and several colonies could be isolated after two days of growth at 37° C. Several mutants were purified on M17-gal plates and retested in M17 broth containing 2% galactose as sole carbohydrate. From purified galactose positive mutants second generation galactose hyperfermenting mutants were isolated by sub-culturing in M17-gal broth with daily 1% reinoculation from the fully outgrown overnight culture; incubation occurred at 37° C. After dilution plating, 100 single colonies were isolated from M17-gal plates and inoculated in microtitre plates with M17-gal broth. The OD was followed by an OD-reader and the clones showing a better increase of OD during 16 hours of incubation at 37° C. as the wt strain were further purified and characterized.

(35) The wt S. thermophilus strains from which galactose-hyperfermenting mutants were isolated are:

(36) CHCC9861

(37) CHCC4459

(38) CHCC4426

(39) CHCC4323

(40) CHCC7018

(41) CHCC3050

(42) The galactose-hyperfermenting mutants showing an unusually high galactose fermenting ability and reduced galactose excretion into the media are (mutant/wt):

(43) CHCC27912/CHCC9861

(44) CHCC29526/CHCC4459

(45) CHCC29530/CHCC4426

(46) CHCC14994/CHCC4323

(47) CHCC19100/CHCC7018

(48) CHCC19097/CHCC3050

(49) The example includes also a typical galactose positive strain, isolated as first generation mutant from CHCC4323, named CHCC14993. CHCC14993 showed a typical galactose reduction in milk of 17% (reduction of galactose excretion in milk compared to wt CHCC4323).

(50) Fermentation of Milk

(51) Mutant strains were inoculated in milk 1% from overnight cultures and incubated for 24 hours at 37° C. The acidification activity of mutants was similar to the wt strain. At the end of fermentation samples were taken to measure galactose content in the fermented milk and with this the reduction of excreted galactose compared to the galactose negative reference strain CHCC4323 and reference strain 4323-2 (CHCC14993).

(52) Results—Analysis of Acidification and Excreted Galactose in the Fermented Milk

(53) All the tested ST strains had similar acidification profiles—i.e. the deposited ST strains of the present invention had not lost their capacity to acidify fast in milk.

(54) The amounts of excreted galactose for the different tested strains are shown in Table 1 below:

(55) Table 1 indicates the amount of galactose in milk and the reduction of galactose compared to the reference CHCC4323. Whereas the typical gal+ mutant CHCC14993 showed a galactose reduction of less than 20%, the hyper-fermenting mutants showed a much higher reduction up to 52%, meaning that the amount of free galactose is much lower when e.g. pizza cheese is produced with the new mutants, which is leading to reduced browning during baking.

(56) TABLE-US-00001 TABLE 1 Average of two measurements from carbohydrate analysis. Results are shown in mg/g. Strain Galactose Galactose reduction (%) CHCC4323 7.1 0 CHCC14993 5.9 17 CHCC27912 3.4 52 CHCC29526 3.4 52 CHCC29530 4.9 31 CHCC14994 5.0 30 CHCC19100 4.1 42 CHCC19097 5.1 28

(57) Conclusions

(58) The results demonstrated that the herein deposited strains of the present invention are capable of reducing the release of galactose also in the presence of high amounts of lactose (as in milk) to a degree, which is extraordinarily improved as compared to above discussed reference strains.

Example 2: Deposited Streptococcus thermophilus (ST) Strains—Capable of Extraordinary Reducing the Release of Galactose Also in the Presence of High Amounts of Lactose (as in Milk)

(59) Reference Strains:

(60) Same as Example 1 above.

(61) Deposited Strains of the Present Invention:

(62) CHCC29525: Novel DSM 32897 strain as described herein.

(63) CHCC30963: Novel DSM 32898 strain as described herein.

(64) CHCC30964: Novel DSM 32900 strain as described herein.

(65) Isolation of Galactose Hyper-Fermenting Mutants from S. thermophilus:

(66) Prior to the mutant isolation the strains were streaked on M17 agar plates with 2% galactose (M17-gal plates). The wild type (wt) strains did not grow significantly on galactose as sole carbohydrate source.

(67) Overnight cultures were then plated on M17-gal plates and several colonies could be isolated after two days of growth at 37° C. Several mutants were purified on M17-gal plates and retested in M17 broth containing 2% galactose as sole carbohydrate.

(68) From purified galactose positive mutants second generation galactose hyperfermenting mutants were isolated by sub-culturing in M17-gal broth with daily 1% reinoculation from the fully outgrown overnight culture; incubation occurred at 37° C. After dilution plating, 100 single colonies were isolated from M17-gal plates and inoculated in microtitre plates with M17-gal broth. The OD was followed by an OD-reader and the clones showing a better increase of OD during 16 hours of incubation at 37° C. as the wt strain were further purified and characterized.

(69) The wt S. thermophilus strains from which galactose-hyperfermenting mutants were isolated are:

(70) CHCC4458

(71) CHCC4459

(72) The galactose-hyperfermenting mutants showing an unusually high galactose fermenting ability and reduced galactose excretion into the media are (mutant/wt):

(73) CHCC30963/CHCC4458

(74) CHCC30964/CHCC4458

(75) CHCC29525/CHCC4459

(76) The example includes also a typical galactose positive strain, isolated as first generation mutant from CHCC4323, named CHCC14993. CHCC14993 showed a typical galactose reduction in milk of 17% (reduction of galactose excretion in milk compared to wt CHCC4323).

(77) Fermentation of Milk

(78) Same as Example 1 above.

(79) Results—Analysis of Acidification and Excreted Galactose in the Fermented Milk

(80) All the tested ST strains had similar acidification profiles—i.e. the deposited ST strains of the present invention had not lost their capacity to acidify fast in milk.

(81) The amounts of excreted galactose for the different tested strains are shown in Table 2 below:

(82) Table 2 indicates the amount of galactose in milk and the reduction of galactose compared to the reference CHCC4323. Whereas the typical gal+ mutant CHCC14993 showed a galactose reduction of less than 20%, the hyper-fermenting mutants showed a much higher reduction up to 57%, meaning that the amount of free galactose is much lower when e.g. pizza cheese is produced with the new mutants, which is leading to reduced browning during baking.

(83) TABLE-US-00002 TABLE 2 Average of two measurements from carbohydrate analysis. Results are shown in mg/g. Strain Galactose Galactose reduction (%) CHCC4323 7.0 0 CHCC14993 5.9 16 CHCC29525 3.0 57 CHCC30963 3.7 47 CHCC30964 4.8 31

(84) Conclusions

(85) The results demonstrated that the herein deposited strains of the present invention are capable of reducing the release of galactose also in the presence of high amounts of lactose (as in milk) to a degree, which is extraordinarily improved as compared to above discussed reference strains.

REFERENCES

(86) 1. Mukherjee et al (1994, J Dairy Sci 77:2839-2849) 2. Hassan et al. (International Journal of Food Microbiology 64 (2001) 199-203) 3. WO2011/026863A1 (Chr. Hansen) 4. WO2011/092300A1 (Chr. Hansen) 5. WO2013/160413A1 (Chr. Hansen)