Patent Grant
11690384
This application is the U.S. national stage of PCT/EP2018/072865, filed Aug. 24, 2018, and claims priority to European application 17188105.5, filed Aug. 28, 2017.
The present invention relates to 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.
The food industry uses numerous bacteria, in particular lactic bacteria, in order to improve e.g. the taste and the texture of foods. In the case of the dairy industry, lactic acid bacteria are used intensively in order to bring about the acidification of milk (by fermentation) but also in order to e.g. texturize the product into which they are incorporated.
Starter culture composition/mixture to make mozzarella cheese generally comprises the lactic acid bacterium (LAB) Streptococcus thermophilus (ST)—it may sometimes also comprise e.g. relevant Lactobacillus strains.
A relatively high concentration of galactose can result in “browning” during heating of cheeses as it is often described when e.g. mozzarella cheese is produced by S. thermophilus (ST) for e.g. pizza production.
The browning phenomenon is believed to be due to the Maillard reaction where galactose as reducing sugar is reacting with amino acids/peptides.
Beside this major problem during pizza cheese production, excess amounts of free galactose can also lead to post acidification problems and imbalance in the flora of other dairy product, such as e.g. soft cheeses.
The article of Mukherjee et al. (1994, J Dairy Sci 77:2839-2849) describes the use of so-called galactose non-releasing Streptococcus as starter cultures in the manufacture of low browning mozzarella cheese.
The article of Hassan et al. (International Journal of Food Microbiology 64 (2001) 199-203) describes that the ability of some strains of S. thermophilus (ST) to use galactose in capsule production could reduce browning of mozzarella cheese during baking by removing a source of reducing sugar.
WO2011/026863A1 (Chr. Hansen) and WO2011/092300A1 (Chr. Hansen) describe that S. thermophilus (ST) strains with mutations in the GalK (galactokinase) gene generate a higher viscosity in fermented milk. None of these two WO publications describe above discussed browning of mozzarella cheese related problem.
WO2013/160413A1 (Chr. Hansen) describes herein discussed Streptococcus thermophilus (ST) CHCC14994 strain, which was deposited on 3 Apr. 2012 under the accession No. DSM 25838. The CHCC14994 strain is described as a so-called “mother strain” suitable to be used as a starting strain for being mutated in order to obtain novel mutated strains with “mutation in the DNA sequence of the gIcK gene”. Accordingly, the CHCC14994/DSM25838 strain is not disclosed to be present/comprised in a starter culture composition nor to be used in the manufacturing of a food or feed product of interest (e.g. a dairy product).
The problem to be solved by the present invention is to provide novel 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.
The solution relates to herein discussed novel deposited Streptococcus thermophilus (ST) strains.
As shown in working examples herein—the herein discussed deposited ST strains are capable of extraordinary reducing the release of galactose also in the presence of high amounts of lactose (as in milk).
As discussed above, the prior art describes use of so-called galactose non-releasing Streptococcus as starter cultures in the manufacture of low browning mozzarella cheese.
Accordingly, since the herein deposited strains are capable of providing an extraordinary reduction of the release of galactose they may e.g. be used in an improved method for the manufacture of low browning mozzarella cheese.
As understood by the skilled person in the present context—the herein deposited strains may also be used for making other milk based (e.g. cow milk based) food product, such as e.g. soft cheeses or cheddar.
The herein deposited strains are mutants of relevant parent/mother strains made in the laboratory of the present inventors—i.e. they are novel not “natural” strains comprising novel not earlier described mutations in their respective relevant DNA sequences.
In short, the herein deposited mutant strains were obtained after several mutagenesis/selection rounds, where e.g. the selections were performed via novel combinations of relevant selection criteria—said in other words, the deposited strains were not obtained by what may be termed standard prior art routine mutagenesis/selection work (see e.g. working example herein).
As can be seen in Table 1 of Example 1, by use of the in this Example described special method for isolation of galactose hyper-fermenting mutants from S. thermophilus it was possible to obtain ST bacteria able to reduce by around 50% (see e.g. CHCC27912 and CHCC29526) the amounts of excreted galactose in milk as compared to reference ST CHCC4323 bacteria.
ST bacteria able to reduce by at least 20% the amounts of excreted galactose in milk as compared to reference ST CHCC4323 bacteria may herein be termed ST Gal(++) bacteria.
Without being limited to theory—the in Example 1 described special method for isolation of galactose hyper-fermenting ST Gal(++) mutants from S. thermophilus may be considered special due to the fact that the galactose reduction level is dramatically increased compared to a herein termed Gal(+) strain. As described in Example 1, the galactose reduction level for CHCC14993, a Gal(+) mutant of CHCC4323, is 17%, whereas the galactose reduction level of CHCC14994, a Gal(++) mutant of CHCC4323, is 30%, compared to the wild type CHCC4323. The galactose reduction level of CHCC29526, a Gal(++) mutant from CHCC4459, is even 52% compared to the reference CHCC4323.
By using the newly developed, not previously described method of sub-culturing in M17-gal broth it was therefore possible to isolate galactose hyper-fermenting mutants with a unique galactose reducing ability.
As discussed below—in working examples were the herein deposited strains of the invention compared to following so-called reference strains:
It is believed that above discussed reference strains may be seen as reference strains that correspond to herein relevant described strains of the prior art (see e.g. prior art discussion above).
As discussed below—working examples herein show that the herein discussed deposited strains of the 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 extraordinary improved as compared to above discussed reference strains.
Accordingly, a first aspect of the invention relates to a Streptococcus thermophilus (ST) cell, wherein the cell is at least one cell selected from the group consisting of:
(a) a Streptococcus thermophilus cell CHCC19097 deposited with registration number DSM 32594;
(b) a Streptococcus thermophilus cell CHCC19100 deposited with registration number DSM 32595;
(c) a Streptococcus thermophilus cell CHCC27912 deposited with registration number DSM 32596;
(d) a Streptococcus thermophilus cell CHCC29526 deposited with registration number DSM 32597;
(e) a Streptococcus thermophilus cell CHCC29530 deposited with registration number DSM 32598;
(f) a Streptococcus thermophilus cell CHCC29525 deposited with registration number DSM 32897;
(g) a Streptococcus thermophilus cell CHCC30963 deposited with registration number DSM 32898; and
(h) a Streptococcus thermophilus cell CHCC30964 deposited with registration number DSM 32900.
It is clear for the skilled person that by using the herein discussed deposited strains as starting material, the skilled person can routinely, by conventional mutagenesis or reisolation techniques, obtain further mutants or derivatives thereof that retain the herein described relevant features and advantages. Accordingly, the term “a mutant thereof” or “a mutant of a deposited strain” relates to a mutant strain obtained by using the deposited strain as starting material.
As discussed above, WO2013/160413A1 (Chr. Hansen) describes herein discussed Streptococcus thermophilus (ST) CHCC14994 strain, which was deposited on 3 Apr. 2012 under the accession No. DSM 25838. The CHCC14994/DSM25838 strain is not disclosed to be present/comprised in a starter culture composition nor to be used in the manufacturing a food or feed product of interest (e.g. a dairy product).
Accordingly, a second aspect of the invention relates to a method to obtain a mutant strain of a deposited cell of the first aspect or the Streptococcus thermophilus cell CHCC14994 deposited with registration number DSM 25838 comprising using at least one of the deposited strains of the first aspect or DSM 25838 as starting strain, making mutants of the deposited strain and isolating a novel mutant strain, wherein the mutant strain has retained the property of being capable of reducing the release of galactose in the presence of lactose of the deposited strain.
The property of the obtained mutant strain relating to “being capable of reducing the release of galactose in the presence of lactose” may be measured as described in working example herein—based on the description herein, this is routine work for the skilled person.
It is evident, that a mutant strain obtained according to the method of the second aspect may be used to e.g. a method of manufacturing a food or feed product as discussed herein (e.g. a method of the fourth aspect as discussed herein).
A third aspect of the invention relates to a starter culture composition comprising at least one of the Streptococcus thermophilus (ST) cells of the first aspect or the Streptococcus thermophilus cell CHCC14994 deposited with registration number DSM 25838, wherein the starter culture composition is having a concentration of viable cells of the first aspect or DSM 25838, which is in the range of 104 to 1014 cfu per gram of the composition.
As understood, the composition of the third aspect may e.g. comprise 108 cfu per gram of e.g. only one of the herein described deposited strains or it may e.g. comprise 108 cfu per gram of one deposited strain plus 108 cfu per gram of another of the deposited strains, which would give a composition with 2×108 cfu per gram of viable cells of the first aspect.
Further, the composition may comprise other cells such as e.g. Lactobacillus and/or Lactococcus cells.
A fourth aspect of the invention relates to a method of manufacturing a food or feed product comprising adding a starter culture composition according to the third aspect to a food or feed product starting material and keeping the thus inoculated starting material under conditions where the Streptococcus thermophilus (ST) cells are metabolically active.
Embodiment of the present invention is described below, by way of examples only.
Deposited Strains/Cells
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.
As discussed above, this deposited strain is described in WO2013/160413A1 (Chr. Hansen).
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.
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.
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.
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.
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.
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.
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.
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.
A Starter Culture Comprising a ST Cell as Described Herein
The ST cell as described herein is useful as starter culture in the production of food or feed products.
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 104 to 1014 cfu (colony forming units) per gram of the composition including at least 104 cfu per gram of the composition, such as at least 105 cfu/g, e.g. at least 106 cfu/g, such as at least 107 cfu/g, e.g. at least 108 cfu/g, such as at least 109 cfu/g, e.g. at least 1010 cfu/g, such as at least 1011 cfu/g.
The composition may as further components contain cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and/or vitamins.
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.
For instance, it may be preferred that the composition also comprises other cells such as e.g. Lactobacillus cells and/or Lactococcus cells.
A Method of Manufacturing a Food or Feed Product
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.
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.
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.
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).
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.
Reference Strains:
Deposited Strains of the Present Invention:
CHCC14994: DSM 25838 ST strain—disclosed in WO2013/160413A1 (Chr. Hansen).
CHCC19097: Novel DSM 32594 ST strain as described herein.
CHCC19100: Novel DSM 32595 ST strain as described herein.
CHCC27912: Novel DSM 32596 ST strain as described herein.
CHCC29526: Novel DSM 32597 ST strain as described herein.
CHCC29530: Novel DSM 32598 ST strain as described herein.
Isolation of Galactose Hyper-Fermenting Mutants from S. thermophilus:
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.
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.
The wt S. thermophilus strains from which galactose-hyperfermenting mutants were isolated are:
CHCC9861
CHCC4459
CHCC4426
CHCC4323
CHCC7018
CHCC3050
The galactose-hyperfermenting mutants showing an unusually high galactose fermenting ability and reduced galactose excretion into the media are (mutant/wt):
CHCC27912/CHCC9861
CHCC29526/CHCC4459
CHCC29530/CHCC4426
CHCC14994/CHCC4323
CHCC19100/CHCC7018
CHCC19097/CHCC3050
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).
Fermentation of Milk
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).
Results—Analysis of Acidification and Excreted Galactose in the Fermented Milk
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.
The amounts of excreted galactose for the different tested strains are shown in Table 1 below:
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.
Conclusions
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.
Reference Strains:
Same as Example 1 above.
Deposited Strains of the Present Invention:
CHCC29525: Novel DSM 32897 strain as described herein.
CHCC30963: Novel DSM 32898 strain as described herein.
CHCC30964: Novel DSM 32900 strain as described herein.
Isolation of Galactose Hyper-Fermenting Mutants from S. thermophilus:
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.
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.
The wt S. thermophilus strains from which galactose-hyperfermenting mutants were isolated are:
CHCC4458
CHCC4459
The galactose-hyperfermenting mutants showing an unusually high galactose fermenting ability and reduced galactose excretion into the media are (mutant/wt):
CHCC30963/CHCC4458
CHCC30964/CHCC4458
CHCC29525/CHCC4459
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).
Fermentation of Milk
Same as Example 1 above.
Results—Analysis of Acidification and Excreted Galactose in the Fermented Milk
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.
The amounts of excreted galactose for the different tested strains are shown in Table 2 below:
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.
Conclusions
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17188105 | Aug 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/072865 | 8/24/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/042881 | 3/7/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8865238 | Janzen et al. | Oct 2014 | B2 |
| 9060524 | Janzen et al. | Jun 2015 | B2 |
| 9416351 | Janzen et al. | Aug 2016 | B2 |
| 9562221 | Janzen et al. | Feb 2017 | B2 |
| 20120164275 | Janzen et al. | Jun 2012 | A1 |
| 20120301575 | Janzen et al. | Nov 2012 | A1 |
| 20150086675 | Johansen et al. | Mar 2015 | A1 |
| 20150099273 | Janzen et al. | Apr 2015 | A1 |
| 20150322415 | Janzen et al. | Nov 2015 | A1 |
| 20160227803 | Janzen et al. | Aug 2016 | A1 |
| 20170096635 | Janzen et al. | Apr 2017 | A1 |
| 20170298457 | Janzen et al. | Oct 2017 | A1 |
| 20190183160 | Gilleladen et al. | Jun 2019 | A1 |
| 20200093149 | Johansen et al. | Mar 2020 | A1 |
| 20210259266 | Janzen | Aug 2021 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-2011026863 | Mar 2011 | WO |
| WO-2011092300 | Aug 2011 | WO |
| WO-2013160413 | Oct 2013 | WO |
| WO-2015193449 | Dec 2015 | WO |
| WO-2017103051 | Jun 2017 | WO |
| WO-2017194650 | Nov 2017 | WO |
| Entry |
|---|
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| Non-Final Office Action issued in U.S. Appl. No. 17/253,851, dated Mar. 14, 2023 (8 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20200375207 A1 | Dec 2020 | US |
