Use of ST Gal(+) bacteria to produce fermented dairy products with a relatively high and stable pH.

Abstract

The problem to be solved by the present invention is to provide a method for producing a fermented dairy product (e.g., yogurt) having a relatively high and stable pH value at the end of fermentation, where the advantage of the produced fermented dairy product (e.g., yogurt) may be, for example, reduced post-acidification during storage of the produced fermented dairy product. [Solution] The present invention relates to a method for producing a fermented dairy product (e.g., yogurt) having a relatively high and stable pH value at the end of fermentation, comprising inoculating milk with Streptococcus thermophilus (ST)Gal(+) bacteria.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a fermented dairy product (e.g., cheese or yogurt) having a relatively high and stable pH value at the end of fermentation, comprising inoculating milk with Streptococcus thermophilus (ST)Gal(+) bacteria. [Background technology]

[0002] The food industry utilizes many bacteria, especially lactic acid bacteria, to improve the taste and texture of food. In the dairy industry, lactic acid bacteria (LABs) are used not only to acidify milk (through fermentation), but also to give texture to products containing them, for example.

[0003] Post-acidification management is commercially important.

[0004] In the art, the term "post-acidification" is generally used to describe the production of lactic acid by LAB after fermentation is complete. See, for example, paragraph number EP2957180B1 (Chr. Hansen A / S, Denmark).

[0005] It states the following: Even with methods involving rapid cooling, post-acidification, i.e., lactic acid production by LAB after fermentation is complete—after the so-called desired pH has been reached—is observed. Post-acidification is considered one of the most important problems occurring during dairy fermentation today. If the pH value further decreases during processing and storage of fermented dairy products, it leads to problems such as increased acidity and a shortened shelf life.

[0005] Many current laws for producing fermented dairy products are characterized by the following series of steps: (a) A process of fermenting milk using a starter culture containing lactic acid bacteria (or LAB) that can metabolize glucose obtained from lactose present in milk; (b) The fermentation process causes the production of lactic acid, which initially lowers the pH from 6.4–6.8 (in the case of milk) to a range of 3.8–4.2; (c) Once the desired pH is reached in the fermented product, the fermentation process is terminated by rapidly cooling the fermented dairy product.

[0006] This method is used, for example, in the production of cheese, yogurt, and yogurt beverages.

[0007] In fermented dairy products, fermentation is stopped by rapid cooling to a predetermined pH value. If the fermented product is not cooled, fermentation will continue. However, rapid cooling can have drawbacks, such as potentially leading to a loss of texture.

[0008] For example, to avoid the rapid cooling process, prior art describes a wide variety of technical solutions for improving the control of post-acidification.

[0009] An example of a commercially available product relevant to this application is the YOFLEX® ACIDIFIX® culture from Chr. Hansen A / S of Denmark, which is a product that "has excellent pH stability and improves quality and shelf life" (see, for example, www.chr-hansen.com). As those skilled in the art will understand in this context, the improved shelf life due to excellent pH stability is related to improved control of post-acidification.

[0010] European Patent Publication EP2957180B1 (Chr. Hansen A / S, Denmark) describes various technical solutions for improving post-acidification control, for example: - Use of L. bulgaricus (L. bulgaricus; Bulgarian bacteria) strains with defects in amino acid metabolism, and / or use of special LAB strains characterized by weak post-acidification activity—see, for example, paragraph number

[0007] ; - Controlling buffering capacity during fermentation, and maintaining buffering capacity and pH within a predetermined range—see, for example, paragraph number

[0008] ; - Use of lactose-deficient (Lac(-)) Streptococcus thermophilus (ST) strains and Lactobacillus delbrueckii spp. bulgaricus (bulgaric bacteria) strains—see, for example, claim 1.

[0011] As is well known in the art, in S. thermophilus (ST), galactose is excreted via the lactose / galactose metabolic pathway (see schematic diagram of lac / gal metabolism in Figure 1 herein). For every mole of lactose taken up by the cell, one mole of galactose may be excreted.

[0012] As is well known in the art, strains of Streptococcus thermophilus (ST) do not typically significantly reduce the amount of galactose excreted in milk. That is, they can be referred to as "ST Gal(-) strains" in this art and specification. See, for example, Anbukkarasi et al. (J Food Sci Technol (September 2014) 51(9):2183-2189). This is stated in the abstract as follows: "Most strains of *S. thermophilus* are galactose-negative (Gal-), meaning they can metabolize only the glucose portion of lactose, releasing galactose into the culture medium. This metabolic defect leads to the accumulation of free galactose in yogurt, causing galactosemia in consumers. Therefore, there is an absolute need to develop low-galactose yogurt. Accordingly, in this study, three galactose-positive (Gal+) strains of *S. thermophilus* were used to prepare low-galactose yogurt."

[0013] All ST strains described in the above European Patent EP2957180B1 (Chr. Hansen A / S) and the above product YOFLEX® ACIDIFIX® are considered by those skilled in the art to be ST Gal(-) strains as herein.

[0014] A relatively high concentration of galactose can cause cheese to "brown" during heating. This is frequently reported, for example, when mozzarella cheese is produced using *Streptococcus thermophilus* (ST) during pizza production.

[0015] The prior art describes that some Thermophilus (S. thermophilus) (ST) so-called galactose-positive strains (referred to herein as "ST Gal(+) bacteria") can be used to reduce the possible browning problem of cheese (e.g., mozzarella) used in manufacturing processes (e.g., pizza-making processes) involving important heating steps (e.g., heating to temperatures above 70°C). See, for example, Anbukkarasi et al., "Production of low browning Mozzarella cheese: Screening and characterization of wild galactose fermenting Streptococcus thermophilus strains", International of Journal of advanced research, 2013, Vol. 1, No. 5, pp. 83-96).

[0016] As is known to those skilled in the art in the current situation, "reduction of possible browning" and "improvement of control of post-acidification" are significantly different (separate) problems that can be associated with, for example, different dairy products.

[0017] For example, the problem of browning can usually be associated with cheese (e.g., mozzarella) used in manufacturing processes (e.g., pizza-making processes) involving the use of important heating steps (e.g., temperatures above 70°C).

[0018] In contrast, the problem of post-acidification is associated with dairy products manufactured without using important heating steps (e.g., temperatures above 70°C), such as cheese and yogurt.

[0019] Derkx and other literature ("The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology" ("Techniques for improving strains of industrial lactic acid bacteria without using recombinant DNA technology"; Microbial Cell Factories 2014, 13 (Supplement 1)) mentions the problem of post-acidification, for example, on page 9, and it is a review described as follows in the left column on page 9: "In another approach to obtaining improved strains with reduced post-acidification, the importance of oligopeptide transport for the growth compatibility of Streptococcus thermophilus (S. thermophilus) in milk was investigated... and mutants showing a modified oligopeptide transport system were found to have a lower acidification rate (acidity)."

[0020] The above-mentioned literature by Derkx et al. (2014) describes as follows in the upper right column on page 9: "Moreover, excessive free galactose can cause post-acidification problems and imbalances in cheese flora (microbial communities) due to the growth of indigenous lactic acid bacteria. Therefore, galactose-positive wild-type strains or galactose-fermenting mutants are of particular interest for the purpose of reducing the browning of pizza cheese."

[0021] As those skilled in the art will understand in this context, the post-acidification problem mentioned in the above-quoted paragraphs in the literature by Derkx et al. (2014) is related to the "growth of indigenous lactic acid bacteria", that is, the production of lactic acid by indigenous LAB after the end of fermentation.

[0022] The pamphlets of International Publication WO2011 / 026863A1 (Chr. Hansen) and WO2011 / 092300A1 (Chr. Hansen) describe that Streptococcus thermophilus (S. thermophilus; ST) strains having mutations in the galK (galactokinase) gene cause high viscosity in fermented milk.

[0023] The international publication WO2019 / 042881A1 (Chr. Hansen) describes several examples of ST Gal(+) bacteria. None of the three WO publications describe or relate to the "post-acidification" issues discussed herein.

[0024] In summary, the current state of this technology presents a "post-acidification" problem, generally described as being related to lactic acid production by resident LABs "after" the completion of fermentation, and the technology describes several different solutions related to controlling / reducing this post-acidification problem—for example, the use of ST Lac(-) bacteria or ST bacteria with a modified oligopeptide transport system. [Overview of the project] [Problems that the invention aims to solve]

[0025] The problem to be solved by the present invention is to provide a method for producing a fermented dairy product (e.g., yogurt) having a relatively high and stable pH value at the end of fermentation, where the advantage of the produced fermented dairy product (e.g., yogurt) may be, for example, reduced post-acidification during storage of the produced fermented dairy product.

[0026] This solution is based on the inventors' discovery of a remarkable correlation between *Streptococcus thermophilus* (ST) galactose-positive strains (referred to herein as "ST Gal(+) bacteria") and the resulting possibility of achieving a stable, relatively high pH at the end of fermentation.

[0027] As mentioned above, in this field of technology, there is a "post-acidification" problem that is usually explained as being related to the production of lactic acid by resident lactic acid bacteria (LABs) "after" the completion of fermentation, for example, during storage after fermentation.

[0028] As a result of the remarkable correlation described above, the relatively high pH at the end of fermentation is clearly an effect initiated during fermentation, based on the growth profile of Gal(+) ST bacteria.

[0029] Without being bound by theory, the inventors are unaware of any single prior art document that directly and uniquely describes the above-mentioned remarkable correlation between ST Gal(+) bacteria and the possibility of achieving a stable, relatively high pH at the end of fermentation.

[0030] For example, the use of ST Gal(+) bacteria as described herein, as discussed in the examples herein and shown in Figures 2 and 3, resulted in a significantly higher (approximately 0.2 to 0.6 points) stable pH value at the end of fermentation compared to the corresponding wild-type ST Gal(-) bacteria.

[0031] For example, as shown in Figure 2, the use of ST Gal(+) bacteria as described herein resulted in a pH of 4.3–4.8 at the end of fermentation, while the use of wild-type CHCC27806 ST Gal(-) resulted in a pH of around 4.15 (i.e., lower than pH 4.3).

[0032] In other words, using ST Gal(+) bacteria resulted in a significantly higher (approximately 0.3-0.6 points higher) stable final pH value compared to using the corresponding wild-type CHCC27806 ST Gal(-) strain.

[0033] Furthermore, the initial acidifying activity remained relatively unchanged (see, for example, Figure 2 or Figure 3). This demonstrates that the observed low acidifying activity (i.e., the relatively high and stable pH value at the end of fermentation) is not due to a generally low acidification rate (acidity).

[0034] Without being bound by theory, it is thought that a higher final pH at the end of fermentation will have a greater impact on the shelf life of post-acidification. This could be a serious problem for dairy products such as yogurt (see above).

[0035] Therefore, the ST Gal(+) strain discussed herein, which has a stable, higher pH at the end of fermentation, will result in lower post-acidification under storage conditions, for example, which is a desired characteristic of the relevant commercial dairy products.

[0036] The novel relationship established in this invention between the ST Gal(+) strain and the potential to achieve a stable, relatively high pH at the end of fermentation will, as a result, be considered a "change in (conventional) behavior" for those skilled in the art. For example, if a "low post-acidification" yogurt culture is desired, after the disclosure of this invention, those skilled in the art will select the appropriate ST Gal(+) strain (according to this invention) instead of various other known "low post-acidification" cultures of the prior art (see above).

[0037] As discussed in the examples herein, approximately 20% of the ST Gal(+) strains tested actually functioned as required herein (i.e., they gave the relatively high and stable pH values ​​at the end of fermentation as discussed herein).

[0038] Therefore, without the knowledge of the present invention, those skilled in the art would not have been able to easily test the target ST Gal(+) strain and identify the positive effect (positive result) of "stable, relatively high pH at the end of fermentation" associated with the present invention.

[0039] However, once the relationship between the novel ST Gal(+) discussed herein and the "stable, relatively high pH at the end of fermentation" effect is disclosed by the present invention, identifying ST Gal(+) strains that possess this positive effect of "stable, relatively high pH at the end of fermentation" as described herein becomes a routine screening / selection process for those skilled in the art.

[0040] For example, first, approximately 100 different ST Gal(+) strains are simply isolated / selected by routine procedures, and then from this pool of different ST Gal(+) strains (approximately 20% positive as disclosed herein), ST Gal(+) strains that have the positive effect (positive result) of “stable relatively high pH at the end of fermentation” as described herein are screened / selected.

[0041] Therefore, as will be discussed in more detail below, the present invention is based on the inventors' discovery of a novel selection method for identifying a new ST Gal(+) strain that has the positive effect (positive result) of "stable and relatively high pH at the end of fermentation."

[0042] As discussed in the examples below, the inventors attempted to identify positive ST strains having a "stable, relatively high pH at the end of fermentation" based on a pool of different ST Gal(-) strains, rather than identifying a single positive strain / cell. In other words, without the knowledge of the present invention, it would have been impossible (or would have taken a very long time) to identify ST strains having the positive effect (positive result) of "stable, relatively high pH at the end of fermentation" as described herein. [Means for solving the problem]

[0043] Therefore, a first aspect of the present invention is a method for producing a fermented dairy product having a relatively high and stable pH value at the end of fermentation, the method comprising the following steps: (a): In at least 100 L of milk, add the following: (I):10 4 ~10 14 A Streptococcus thermophilus (Thermophilus; ST) composition containing CFU / g of ST bacterial cells, characterized in that, compared to reference ST CHCC4323 (DSM 32826), the ST bacteria can reduce the amount of galactose excreted in milk by at least 10%, and Streptococcus thermophilus composition (hereinafter referred to as "ST Gal(+) bacteria") The process of inoculating; Here, the comparative test is carried out by inoculating 1% of ST bacteria from the culture into skim milk overnight, incubating at 37°C for 18 hours, taking a sample at the end of fermentation, measuring the galactose content in the fermented milk, and thereby measuring the reduction in excreted galactose compared to the reference CHCC4323; and (b): A process of fermenting milk using the bacteria of (a), wherein the pH is measured during fermentation in a manner that can reliably measure the pH value of this process (b), and the fermentation ends at a relatively high and stable pH value defined as a pH of 4.3 to 4.9 at the end of fermentation, and the pH does not change by more than 0.1 points during the last two hours of fermentation, and the pH reaches 4.3 to 4.9 before 24 hours of fermentation (for example, before 15 hours of fermentation); and (c): A process that uses fermented milk (b) having a pH of 4.3 to 4.9 to carry out further appropriate processes, ultimately leading to the production of a fermented dairy product.

[0044] A first aspect of the present invention can also be formulated as a so-called use claim. That is, the use of Streptococcus thermophilus (ST) Gal(+) bacteria in a method for producing a fermented dairy product having a relatively high and stable pH value of 4.3 to 4.9 at the end of fermentation, wherein the method comprises the following steps: (a): In at least 100 L of milk, add the following: (I):10 4 ~10 14 A step of inoculating a Streptococcus thermophilus (ST) composition (hereinafter referred to as "ST Gal(+) bacteria") containing CFU / g of ST bacterial cells, characterized by being able to reduce the amount of galactose excreted in milk by at least 10% compared to reference ST CHCC4323 (DSM 32826); Here, the comparative test is carried out by inoculating 1% of the ST strain from the culture into skim milk overnight, incubating at 37°C for 18 hours, taking a sample at the end of fermentation, measuring the galactose content in the fermented milk, and thereby measuring the reduction in excreted galactose compared to the reference CHCC4323; and (b): A process of fermenting milk using the bacteria of (a), wherein the pH is measured during fermentation in a manner that can reliably measure the pH value of this process (b), and the fermentation ends at a relatively high and stable pH value defined as a pH of 4.3 to 4.9 at the end of fermentation, and the pH does not change by more than 0.1 points during the last two hours of fermentation, and reaches a pH of 4.3 to 4.9 before 24 hours of fermentation; and (c): A step to obtain the final fermented milk product by using the fermented milk from (b) having a pH of 4.3 to 4.9 in order to carry out further appropriate processes.

[0045] The ST Gal(+) bacteria in "(a)(I)" of the first embodiment can be considered a standard test related to the present invention that can be routinely performed by those skilled in the art.

[0046] Many of the ST Gal(+) bacteria described in the prior art described above are expected to comply with the ST Gal(+) test. In other words, based on the prior art and the technical information provided herein, obtaining ST Gal(+) bacteria that comply with the comparative test in "(a)(I)" of the first aspect can be considered a relatively routine task.

[0047] Example 1 of this specification describes a method for obtaining a different variety of ST Gal(+) bacteria, in accordance with the comparative test of step (a)(I) of the first embodiment, i.e., the comparative test of step (a)(I) of the first embodiment is preferably carried out according to Example 1.

[0048] As stated above, the inventors are unaware of any single prior art document that directly and unambiguously explains the aforementioned surprising relationship between ST Gal(+) bacteria and the resulting possibility of obtaining a stable and relatively high pH at the end of fermentation.

[0049] Therefore, step (b) is a novel step in itself. That is, the prior art does not directly and uniquely describe a method for inoculating milk with ST Gal(+) bacteria according to step (a) of the first embodiment, and then monitoring / measuring pH in fermentation step (b) as required in step (b) of the first embodiment.

[0050] One reason for this is that, prior to the present invention, those skilled in the art were completely unaware of the possibility that ST Gal(+) bacteria could have the positive effect (positive result) of "stable, relatively high pH at the end of fermentation" as described herein. Therefore, those skilled in the art did not consider monitoring / measuring pH as required in step (b) of the first embodiment in order to control / monitor this positive effect.

[0051] As will be understood by those skilled in the art in relation to the current situation, step (b) of the first embodiment requires at least some kind of pH monitoring / measurement, which is sufficient to determine that the pH at the end of fermentation is between 4.3 and 4.9, and that the pH does not change by more than 0.1 points for at least two hours of fermentation, and that the pH reaches 4.3 to 4.9 before 24 hours of fermentation.

[0052] As those skilled in the art will understand, this pH monitoring / measurement can be carried out in various ways that allow for the objective determination / evaluation of the relevant pH value.

[0053] For example, it may not be required to measure the pH exactly two hours before the end of fermentation. For example, if measurements are taken three hours before, one hour before, and at the end of fermentation, and all three pH values ​​are within the correct range, a person skilled in the art will objectively understand that the pH is precisely within the requirements of step (b) relating to the last two hours of fermentation.

[0054] In the examples provided herein (see, for example, Figure 2 herein), pH was continuously monitored / measured, which may be a preferred procedure.

[0055] Step (b) of the first embodiment is: "The pH at the end of fermentation" is understood.

[0056] Those skilled in the art will understand that when fermentation is complete, it essentially refers to the point at which the pH can no longer fall or decrease significantly.

[0057] As is well known in this industry, fermentation ends / stops when the fermentation medium no longer contains sufficient relevant nutrients (e.g., sugars such as lactose and galactose) for bacterial growth / metabolism, or by changing the temperature to a temperature significantly different from the optimal temperature for bacterial growth.

[0058] Alternatively, fermentation inevitably terminates with an increase in the concentration of lactic acid or other growth-inhibiting compounds.

[0059] The fermentation conditions in step (b) can typically be standard, appropriate ST fermentation conditions related to the ST bacterium of interest—for example, around 37°C as used in the examples herein.

[0060] As those skilled in the art will understand in this context—and this is related to the fact that the pH at the end of fermentation is essentially due to the inherent characteristics of the ST Gal(+) bacteria used as described herein—that is, ST bacteria that do not function here would, for example, give a final pH at the end of fermentation of around pH 4.1 under standard ST fermentation conditions.

[0061] Given the technical disclosures herein and general knowledge, it is a routine task for those skilled in the art to select / identify positive (positive-effective) / useful ST Gal(+) strains and find suitable conditions for compliance with the requirements of step(b) of the first embodiment.

[0062] Step (c) of the first embodiment can be understood as an everyday operation for those skilled in the art. That is, those skilled in the art know how to produce the fermented dairy product of the choice (e.g., cheese, or e.g., yogurt).

[0063] As those skilled in the art will understand in this context, in step (a) of the first embodiment, the milk may be inoculated with other, for example, lactic acid bacteria (LAB) of interest for the production of yogurt—for example, Lactobacillus bulgaricus (L. bulgaricus) (the fermented dairy product is, for example, yogurt).

[0064] A second aspect of the present invention is a method for screening and isolating novel Streptococcus thermophilus (ST) cells, comprising the following steps: (i): From a pool of individual ST bacteria, A step of selecting and isolating a novel selected pool of ST bacteria (referred to herein as "ST Gal(+) bacteria") characterized by being able to reduce galactose as required in step (a)(I) of the first embodiment; and (ii): A step of selecting and isolating novel isolated ST Gal(+) cells from the selected pool of ST Gal(+) bacteria in step (i) that can provide a relatively high and stable pH value at the end of fermentation as required in step (b) of the first embodiment. Regarding methods including

[0065] Embodiments of the present invention are described below merely as examples. [Brief explanation of the drawing]

[0066] [Figure 1]Figure 1 is a schematic diagram of lac / gal metabolism.

[0067] [Figure 2] Figure 2 illustrates that the ST Gal(+) strains described herein have a significantly higher and more stable pH (approximately 0.3–0.6 points higher) at the end of fermentation than the corresponding wild-type CHCC27806 ST Gal(-) strain. This figure shows, for example, that the novel ST Gal(+) strains deposited here for the first time (CHCC28380=DSM 33158; CHCC32045=DSM 33159) result in a very good and stable relatively high pH at the end of fermentation. For further details, please refer to the examples described herein.

[0068] [Figure 3] Figure 3 shows that the ST Gal(+) strain described herein has a significantly higher and more stable pH (approximately 0.2 to 0.5 points higher) at the end of fermentation compared to the corresponding wild-type CHCC4426 ST Gal(-) strain. For further details, please refer to the examples described herein. [Modes for carrying out the invention]

[0069] Deposited bacterial strains / cells A sample of Streptococcus thermophilus cells CHCC4323 was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) on ​​June 5, 2018, under accession number DSM 32826. This deposit was made under the conditions of the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Proceedings.

[0070] The following deposited strains are strains deposited for the first time in connection with this application, and therefore are novel strains in themselves.

[0071] A sample of the novel Streptococcus thermophilus cell CHCC28380 was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) on ​​June 12, 2019, under accession number DSM 33158. This deposit was made under the conditions of the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Proceedings.

[0072] A sample of the novel Streptococcus thermophilus cell CHCC32045 was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig) on ​​June 12, 2019, under accession number DSM 33159. This deposit was made under the conditions of the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Proceedings.

[0073] As discussed in the examples herein, the newly deposited strains themselves have a very good and stable relatively high pH at the end of fermentation.

[0074] Accordingly, another aspect of the present invention relates to Streptococcus thermophilus cells CHCC28380 deposited under registration number DSM 33158 or Streptococcus thermophilus cells CHCC32045 deposited under registration number DSM 33159.

[0075] Accordingly, further aspects of the present invention relate to Streptococcus thermophilus cells sharing the functional characteristics of CHCC28380 deposited under registration number DSM 33158, or Streptococcus thermophilus cells sharing the functional characteristics of CHCC32045 deposited under registration number DSM 33159. In related aspects, functional characteristics mean that the ST strain can reduce the amount of galactose excreted in milk by at least 10% compared to the reference ST CHCC4323 (DSM 32826) strain (referred to herein as "ST Gal(+) strain").

[0076] Another aspect of the present invention is as follows: - A mutant strain of Streptococcus thermophilus cell CHCC28380 deposited under registration number DSM 33158; or - A mutant strain of Streptococcus thermophilus cell CHCC32045 deposited under registration number DSM 33159. The present invention relates to a method for obtaining a new mutant strain, comprising using the deposited strain as a starting stock, creating a mutant strain of the deposited strain, and isolating the new mutant strain, wherein the mutant strain retains the property of being ST Gal(+) of the deposited strain.

[0077] Fermented dairy products The milk in step (a) of the first embodiment, and the milk in the resulting fermented dairy product of the first embodiment, may be, for example, soy milk or animal milk (e.g., goat, buffalo, sheep, horse, camel, or cow's milk). Preferably, the milk is cow's milk.

[0078] Fermented dairy products are preferably dairy products such as yogurt, cheese, kefir, or buttermilk.

[0079] The cheese may preferably be, for example, a fresh cheese product, a soft cheese product, cheddar, continental cheese, pasta filata cheese, pizza cheese, or mozzarella cheese.

[0080] The product may preferably be yogurt.

[0081] Milk administration - Step (a) of the first embodiment As described above, in step (a) of the first aspect, it is also possible to inoculate milk with another, for example, target lactic acid bacterium (LAB) - for example, L. bulgaricus (Bulgarian bacterium) for yogurt production.

[0082] In step (a) of the first aspect, 10 4 ~10 14 CFU / g of Lactobacillus bacteria cells (for example, Lactobacillus delbrueckii subsp. bulgaricus, etc.) may also preferably be inoculated into the milk. This may be particularly relevant when the fermented milk product is, for example, yogurt.

[0083] In step (a) of the first aspect, 10 4 ~10 14 CFU / g of Lactococcus bacteria cells (for example, Lactococcus lactis, etc.) may also preferably be inoculated into the milk. This may be particularly relevant when the fermented milk product is, for example, cheese.

[0084] In step (a) of the first aspect, 10 4 ~10 14 CFU / g of Leuconostoc bacteria cells may also preferably be inoculated into the milk. This may be particularly relevant when the fermented milk product is, for example, cheese.

[0085] Step (a) of the first aspect may preferably relate to inoculating at least 200 L of milk or inoculating at least 1000 L of milk.

[0086] ST Gal(+) bacteria in step (a)(I) of the first embodiment In Example 1 of this specification, methods for obtaining various ST Gal(+) bacteria in accordance with the comparative test of step "(a)(I)" of the first aspect are described.

[0087] As shown in Table 1 of Example 1, by using the special method for isolating galactose-over-fermenting mutants from *S. thermophilus* described in this example, it was possible to obtain ST strains (see, for example, CHCC27912 and CHCC29526) that could reduce the amount of galactose excreted in milk by approximately 50% compared to the reference ST strain CHCC4323.

[0088] ST bacteria that can reduce the amount of galactose excreted in milk by at least 20% compared to the reference STCHCC4323 bacteria are sometimes referred to as ST Gal(++) bacteria in this specification.

[0089] Without being bound by theory, the method for isolating the galactose-over-fermenting ST Gal(++) mutant strain from *S. thermophilus* as described in Example 1 can be considered a special method because the level of galactose reduction is dramatically increased compared to the Gal(+) strain named herein. As described in Example 1, the galactose reduction level of CHCC14993, a Gal(+) mutant of CHCC4323, is 17% compared to wild-type CHCC4323, while the galactose reduction level of CHCC14994, a Gal(+) mutant of CHCC4323, is 30%. The galactose reduction level of CHCC29526, a Gal(++) mutant of CHCC4459, is as high as 52% compared to the reference CHCC4323.

[0090] Therefore, by using the method of subculturing in M17-gal broth as in Example 1, it was possible to isolate a galactose over-fermentation mutant strain with a unique galactose reduction ability.

[0091] Preferably, the ST strain of step (a)(I) of the first embodiment is an ST strain characterized by being able to reduce the amount of galactose excreted in milk by at least 20% (e.g., at least 25%, more preferably at least 30%, and even more preferably at least 40%) compared to the reference ST CHCC4323 strain.

[0092] Preferably, the Streptococcus thermophilus (ST) cell is at least one cell selected from the group consisting of: (a): Streptococcus thermophilus cells CHCC28380 deposited under registration number DSM 33158; and (b): Streptococcus thermophilus cells CHCC32045 deposited under registration number DSM 33159.

[0093] Preferably, in step (a)(I) of the first embodiment, 10 per gram of milk 4 ~10 15 cfu (or 10 4 ~10 14 cfu) (colony-forming units), at least 10 per gram of milk 5 cfu, for example, at least 10 6 cfu / g milk, for example, at least 10 7 cfu / g milk, for example, at least 10 8 cfu / g milk, for example, at least 10 9 cfu / g, for example, at least 10 10 cfu / g milk, for example, at least 10 11 Viable ST cells containing cfu / g milk were inoculated into milk.

[0094] ST bacterial cells may be a mixture of various ST strains (for example, a mixture of CHCC28380 and CHCC32045 as described herein)—for example, one ST strain (e.g., CHCC28380) 10 8 cfu / g milk + another ST strain (e.g., CHCC32045) 108 It is acceptable for the milk to be cfu / g. This totals 2 × 10⁻⁶ units of milk. 8 This means that viable ST cells from cfu / g milk are inoculated.

[0095] Typically, bacteria (e.g., starter culture compositions) are in concentrated forms, including frozen, dried, or freeze-dried concentrates.

[0096] As discussed in the examples herein, not all of the ST Gal(+) strains tested actually functioned as required by the present invention (i.e., they did not provide the relatively high and stable pH values ​​described herein at the end of fermentation).

[0097] Accordingly, as discussed in the examples herein, genomic analysis was performed to identify common structural elements among well-functioning, preferred positive ST Gal(+) strains.

[0098] The results showed that the majority of preferred ST Gal(+) strains had mutations in the -10 region / box of the galactokinase gene (galK) promoter.

[0099] Such galK mutants are described, for example, in the international publication WO2011 / 026863A1 (Chr. Hansen). As mentioned above, this WO publication does not describe / is unrelated to the issues of “post-acidification” discussed herein.

[0100] The following is a diagram from page 10 of the internationally published pamphlet WO2011 / 026863A1 (Chr. Hansen).

[0101] [ka]

[0102] As shown in the diagram above and explained in the internationally published brochure WO2011 / 026863A1, the wild-type / common sequence in the -10 region / box is "TACGAT," and the strain designated "CHCC11379" contains one mutation in the -10 region / box.

[0103] The wild-type / common promoter sequence of the galactokinase gene (galK) is shown in the figure above as Sequence ID No. 8, which is identical to Sequence ID No. 8 as used herein.

[0104] In other words, a person skilled in the art can routinely determine whether the ST Gal(+) strain of interest has a mutation in the -10 region / box of the promoter of the galactokinase gene (galK).

[0105] Accordingly, in a preferred embodiment, the ST Gal(+) bacterium of step (a)(I) of the first embodiment is preferably a bacterium having one mutation in the -10 region of the promoter sequence (SEQ ID NO: 8) of the galactokinase gene (galK), where the mutation replaces one or both of C and G in the wild-type -10 region (TACGAT, SEQ ID NO: 1) with a nucleotide independently selected from the group consisting of A and T.

[0106] Preferably, the mutation results in a -10 region having the nucleotide sequence TATGAT (SEQ ID NO: 2 – see, for example, the significantly positive results for CHCC28380 and CHCC32045 discussed below) or TACTAT (SEQ ID NO: 4 – see, for example, the positive results for CHCC29248 discussed below). Most preferably, the mutation results in a -10 region having the nucleotide sequence TATGAT (SEQ ID NO: 2).

[0107] As described in the examples herein, the newly deposited ST Gal(+) strains (CHCC28380=DSM 33158; CHCC32045=DSM 33159) in the present invention themselves have a very stable and relatively high pH at the end of fermentation. These deposited strains include the mutation "TATGAT" (SEQ ID NO: 2), and therefore these strains are most preferred herein.

[0108] Without being limited to theory, higher levels of galK gene expression than wild-type expression are likely to result in the positive effect discussed herein, which is to obtain a relatively high and stable pH at the end of fermentation. Therefore, in a preferred embodiment, the ST Gal(+) bacterium of step (a)(I) of the first embodiment is preferably a bacterium having a higher level of galK gene expression than wild-type, and the ST Gal(+) bacterium has one mutation in position -35, position -10, or in the ribosome binding site (RBS) of Sequence ID No. 8.

[0109] Fermenting milk with bacteria - Step (b) of the first embodiment Step (b) of the first embodiment relates to fermenting milk with the bacteria of step (a).

[0110] As described above, the fermentation conditions in step (b) can generally be standard, appropriate ST fermentation conditions related to the ST bacterium of interest. For example, about 37°C, as used in the examples herein.

[0111] As mentioned above, this is related to the inherent characteristics of the ST Gal(+) bacteria used as described herein, which, for example, cause the pH at the end of fermentation. For example, under standard fermentation conditions, it gives a final pH of around 4.1 at the end of fermentation.

[0112] Those skilled in the art know how to ferment milk with relevant bacteria to produce a desired fermented dairy product (e.g., cheese). Therefore, it is not necessary to explain this in detail in this context.

[0113] According to this technical invention, depending on the ST used, for example, the fermentation temperature may be, for example, 25°C to 48°C, for example, 35°C to 48°C, or for example, 36°C to 38°C.

[0114] According to the art, the fermentation time in step (b) of the first embodiment may be 2 to 96 hours, for example 3 to 72 hours, or for example 4 to 48 hours. Preferably, the fermentation time in step (b) of the first embodiment may be 2 to 30 hours, for example 3 to 24 hours.

[0115] Step (b) of the first embodiment is interpreted as "pH at the end of fermentation."

[0116] As discussed above, those skilled in the art know when fermentation ends, and this can essentially be considered to be related to the point in time when the pH is no longer significantly decreasing in this context.

[0117] As is known in the art, fermentation will terminate / stop when, for example, the fermentation medium no longer contains enough relevant nutrients (e.g., sugars such as lactose and galactose) for bacterial growth / metabolism, or by changing the temperature to a temperature significantly different from the optimal temperature for bacterial growth (e.g., by rapid cooling). Alternatively, fermentation may stop spontaneously due to an increase in the concentration of lactic acid or other growth-inhibiting compounds.

[0118] The pH at the end of fermentation is preferably between 4.3 and 4.8, for example, 4.4 to 4.8 or 4.4 to 4.7.

[0119] In a preferred embodiment, the pH remains unchanged above pH 0.05 during the last two hours of fermentation.

[0120] In a preferred embodiment, a pH of 4.3 to 4.9 is reached before 15 hours of fermentation (more preferably before 10 hours of fermentation, and even more preferably before 8 hours of fermentation).

[0121] In relation to the mass fermentation of milk, which is relevant to the present invention, it is known in the art that the fermentation of milk can sometimes be completed within about 5 hours.

[0122] As discussed herein, ST strain CHCC4323 (DSM 32826) can be considered an ST reference strain equivalent to the relevant ST Gal(-) strain currently in commercial use for, for example, the production of dairy products related to the present invention.

[0123] Therefore, in a preferred embodiment of step (b) of the first embodiment, the pH at the end of fermentation is 0.1 to 0.8 points (preferably 0.2 to 0.8 points, for example 0.2 to 0.6 points) higher than the corresponding comparative pH at the end of fermentation obtained by using reference ST CHCC4323 (DSM 32826) bacteria carried out under equivalent comparative fermentation conditions.

[0124] Those skilled in the art are well aware of how to conduct such comparative experiments. Specifically, the fermentation in step (b) is carried out using the ST Gal(+) strain according to the first embodiment, then repeated under the same conditions as the reference ST strain CHCC4323 (DSM 32826), and then the final pH values ​​are compared.

[0125] Further suitable steps for producing the target fermented dairy product—Step (c) of the first embodiment Step (c) of the first embodiment relates to carrying out further appropriate steps to ultimately obtain the desired fermented dairy product.

[0126] As discussed above, those skilled in the art know how to produce the fermented dairy product of interest (e.g., cheese or yogurt). Therefore, it is not necessary to explain this in detail in this context.

[0127] Storage of manufactured fermented dairy products – Optional first-stage step (d) As mentioned above, a higher final pH at the end of fermentation is likely to increase the impact of post-acidification during storage. This could be a serious problem for dairy products such as yogurt (see above).

[0128] Therefore, the ST Gal(+) strains with higher, more stable pH discussed herein would result in lower post-acidification, for example, a desirable characteristic of commercially available dairy products.

[0129] Therefore, in a preferred embodiment of the method of the first aspect, the method also includes additional steps relating to the following: (d): Storage of the fermented dairy product produced in process (c) for a storage period of at least one day (for example, at least one week, at least two weeks, at least one month, or at least two months), wherein the pH of the product at the end of the storage period is between 4.3 and 4.9.

[0130] Preferably, during storage, the pH does not change by more than 0.3 (preferably, not by more than 0.2, or more preferably, not by more than 0.1).

[0131] Storage may be at the location of dairy producers and / or retailers / shops that sell fermented dairy products (e.g., yogurt).

[0132] In relation to process (d)—if the storage period of process (d) is, for example, at least one day, and the pH of the product is measured, it is determined that the product has a pH within the range of 4.3 to 4.9 of process (e.g., pH 4.4), then one step (d) is performed—this also applies if the product can be stored for a long period (e.g., one year), and the product after one year will have a pH lower than pH 4.3.

[0133] Those skilled in the art know how to store the fermented dairy product of the purpose that has been manufactured. For example, yogurt can be stored at, for example, 2°C to 10°C, or around 5°C.

[0134] Those skilled in the art know how to measure the pH of a stored fermented dairy product of interest, and can therefore routinely determine whether the conditions of step (d) are met.

[0135] A method for screening and isolating novel ST strains—Part 2

[0136] As described above, a second aspect of the present invention relates to a method for screening and isolating cells of a novel Streptococcus thermophilus (ST) bacterium, comprising the following steps: (i): A step of selecting and isolating a novel pool of selected ST bacteria (referred to herein as "ST Gal(+) bacteria") from a pool of individual ST bacteria, characterized by the ability to reduce galactose as required in step (a)(I) of the first embodiment; (ii): A step of selecting and isolating novel isolated ST Gal(+) bacterial cells from the pool of selected ST Gal(+) bacteria of step (i) that can give a relatively high stable pH value at the end of fermentation as required in step (b) of the first embodiment.

[0137] Step (i) of the method in the second embodiment is understood to be "selection and isolation of individual ST bacteria from a pool."

[0138] As is well known, creating / making such pools of individual bacterial cells is a routine task for those skilled in the art.

[0139] For example, it can be produced from suitable and preferred initiating cells, and by subjecting those initiating cells to appropriate mutagenesis (e.g., using chemical mutagens or UV mutagenesis), a pool of mutants of the initiating cells can be created, that is, a pool of individual bacterial cells can be created.

[0140] As discussed herein, in consideration of the technical disclosure herein and ordinary general knowledge, selecting / identifying positive / useful ST strains herein by the screening and isolation method of the second aspect is a routine task for those skilled in the art. [Examples]

[0141] Example 1:ST Gal(+) bacteria—can significantly reduce galactose release (as in milk) even in the presence of large amounts of lactose—that is, ST Gal(+) bacteria in step "(a)(I)" of the first embodiment

[0142] Reference stock: • ST strain CHCC4323: This strain may be referred to as the galK natural wild-type sequence (hereinafter referred to as GalK(-)) and has a sequence that can be seen as an ST reference strain corresponding to related ST strains that are commercially used today, for example, to produce cheese. • ST strain 4323-2 (CHCC14993): This strain contains a mutation in the galK (galactokinase) gene (referred to here as Gal(+)) and can be considered a reference strain corresponding to the strains produced according to the descriptions of WO2011 / 026863A1 (Chr. Hansen) and WO2011 / 092300A1 (Chr. Hansen) above.

[0143] Deposited shares: This is disclosed in CHCC14994:DSM 25838 ST stock-WO2013 / 160413A1 (Chr. Hansen). CHCC19097:DSM 32594 ST strain CHCC19100:DSM 32595 ST strain CHCC27912:DSM 32596 ST strain CHCC29526:DSM 32597 ST strain CHCC29530:DSM 32598 ST strain

[0144] Some of the deposited ST Gal(+) strains are discussed in the International Publication WO2019 / 042881A1 (Chr. Hansen) brochure. As stated above, this International Publication (WO) does not describe / relate to the issues related to “post-acidification” as described herein.

[0145] Isolation of a galactose-over-fermenting mutant strain from *Streptococcus thermophilus*: Before isolating the mutant strains, the strains were streaked onto M17 agar plates containing 2% galactose (M17-gal plates). The wild-type (wt) strain did not grow significantly on galactose as the sole carbohydrate source.

[0146] Next, the overnight cultures were smeared onto M17-gal plates and grown at 37°C for 2 days, after which several colonies could be isolated. Several mutants were purified on M17-gal plates and retested in M17 broth containing 2% galactose as the sole carbohydrate. From the purified galactose-positive mutants, second-generation galactose-over-fermentable mutants were isolated by re-inoculating 1% daily from fully grown overnight cultures and subculturing in M17-gal broth. Incubation was performed at 37°C. After dilution smearing, 100 single colonies were isolated from M17-gal plates and inoculated into microtiter plates using M17-gal broth. OD was tracked using an OD reader, and clones showing a good increase in OD compared to the wild type during 16 hours of incubation at 37°C were further purified and characterized.

[0147] The original wild-type (wt) *Streptococcus thermophilus* strain from which the galactose-over-fermenting mutant was isolated was as follows: CHCC9861 CHCC4459 CHCC4426 CHCC4323 CHCC7018 CHCC3050

[0148] The following galactose over-fermentation mutants exhibited significantly higher galactose fermentation capacity and reduced galactose excretion into the culture medium (mutant / wild strain): CHCC27912 / CHCC9861 CHCC29526 / CHCC4459 CHCC29530 / CHCC4426 CHCC14994 / CHCC4323 CHCC19100 / CHCC7018 CHCC19097 / CHCC3050

[0149] This example also includes a typical galactose-positive strain, named CHCC14993, isolated as a first-generation mutant from CHCC4323. CHCC14993 showed a 17% reduction in typical galactose in milk (a reduction in galactose excretion in milk compared to wild-type (wt) CHCC4323).

[0150] Milk fermentation The mutant strain was inoculated at a concentration of 1% from the culture into skim milk overnight and incubated at 37°C for 24 hours. The acidification activity of the mutant strain was similar to that of the wild-type (WT) strain. At the end of fermentation, samples were taken to measure the galactose content in the fermented milk, and the reduction in excreted galactose compared to the galactose-negative reference strain CHCC4323 was measured using these samples.

[0151] Results—Analysis of acidification and excreted galactose in fermented milk All ST strains tested had similar acidification profiles; that is, the deposited ST strains had not lost their ability to acidify in milk.

[0152] The galactose excretion amounts of the various bacterial strains tested are shown in Table 1 below.

[0153] Table 1 shows the amount of galactose in fermented skim milk and the galactose reduction compared to the reference CHCC4323. The typical gal+ mutant CHCC14993 showed a galactose reduction of less than 20%, while the over-fermentable mutant showed an extremely high reduction of up to 52%. This means that, for example, when producing pizza cheese using the new mutant, the amount of free galactose will be much lower, leading to reduced browning during baking.

[0154] [Table 1]

[0155] conclusion The results above indicate that the deposited strain was able to reduce galactose release to a certain extent (similar to that in milk) even in the presence of large amounts of lactose, and this was significantly improved compared to the reference strain mentioned above.

[0156] Example 2: ST Gal(+) bacteria - pH value at the end of fermentation strain All ST Gal(+) strains considered in this example are ST Gal(+) strains that comply with requirement (a) of the first aspect of this specification (i.e., claim 1), and the comparative test is carried out according to Example 1 described above.

[0157] Newly deposited shares: The following newly deposited stocks were deposited for the first time in connection with the present invention.

[0158] CHCC28380:DSM 33158 ST strain CHCC32045:DSM 33159 ST strain

[0159] Milk fermentation ST Gal(+) mutants and reference / wild-type ST Gal(-) strains were inoculated into 1% skim milk cultured overnight in M17 medium containing 2% lactose, and incubated at 37°C for 24 hours. pH was continuously monitored and measured during fermentation (Intab PC logger, EasyView® software).

[0160] result Figure 2 of this specification shows that the ST Gal(+) strains CHCC28380, CHCC32045, and CHCC32046 described herein have a significantly higher stable pH (approximately 0.3 to 0.6 points higher pH) at the end of fermentation than the corresponding wild-type ST Gal(-) strain CHCC27806.

[0161] Figure 3 of this specification shows that the ST Gal(+) strains CHCC29249 and CHCC29529 described herein have a significantly higher and more stable pH (approximately 0.2 to 0.5 points higher pH) than the corresponding wild-type ST Gal(-) strain CHCC4426 itself at the end of fermentation. Furthermore, while the pH of CHCC4426 continuously decreases at the end of fermentation, the pH of the mutant strains CHCC29249 and CHCC29529 appeared to be more stable.

[0162] The relevant pH results for other test strains are shown in the table below.

[0163] [Table 2]

[0164] As shown in the table above, in various examples of ST Gal(+) bacteria, the stable pH at the end of fermentation is significantly higher (approximately 0.2 to 0.6 points) than that of the corresponding wild-type ST Gal(-) bacteria.

[0165] As shown in the table above, some of the tested ST Gal(+) strains did not function as required by the present invention (i.e., they did not provide a relatively high and stable pH value at the end of fermentation). An example is the ST Gal(+) mutant strain 4459-GAL6, a galactose-fermentable mutant of CHCC4459. However, its final pH after 24 hours of incubation was similar to that of the wild-type strain after 24 hours.

[0166] The mutant strains that exhibited a relatively high pH at the end of fermentation were generally the so-called galactose over-fermenting ST Gal(++) mutant strains. That is, (as described above) these strains were able to reduce the amount of galactose excreted in milk by at least 20% (e.g., at least 25%, more preferably at least 30%, and even more preferably at least 40%) compared to the reference ST CHCC4323 strain in Example 1 above.

[0167] Although data for all non-functional ST Gal(+) strains tested are not shown, approximately 20% of the tested ST Gal(+) strains actually functioned as required in this invention (i.e., they gave relatively high and stable pH values ​​at the end of fermentation as discussed herein).

[0168] Conclusion: The above results showed that various examples of ST Gal(+) bacteria had a significantly higher and more stable pH (approximately 0.2 to 0.6 points higher pH) at the end of fermentation compared to their corresponding wild-type ST Gal(-) bacteria.

[0169] For many different ST Gal(+) bacteria tested, milk fermentation yielded relatively high and stable pH values ​​at the end of fermentation, according to step(b) of the first aspect of this specification.

[0170] The data also showed that approximately 20% of the ST Gal(+) strains tested actually functioned as required herein (i.e., they gave relatively high and stable pH values ​​at the end of fermentation as discussed herein).

[0171] The results also demonstrate that, based on the technical teachings herein and common general knowledge, identifying novel ST Gal(+) strains with the positive (useful) "stable relatively high pH at the end of fermentation" described herein is a routine screening / selection task for those skilled in the art.

[0172] None of the ST Gal(-) strains tested were positive. In other words, none of them gave the pH value at the end of fermentation according to step (b) of the first aspect of the present invention.

[0173] Example 3 : Genomic analysis of the tested ST Gal(+) strain As described above, not all of the ST Gal(+) strains tested actually functioned as required by the present invention (i.e., they provided the relatively high and stable pH discussed herein at the end of fermentation).

[0174] Therefore, genomic analysis was performed to identify common structural elements among favorable, well-functioning positive ST Gal(+) mutants.

[0175] result: The following table shows the mutations in the -10 region of the promoter of the galactokinase gene (galK) in several strains discussed in Example 2 above—that is, both the positive and negative (non-functional) strains of Example 2.

[0176] Table 2. DNA sequences of the -10 region of the promoter of the galactose-positive mutant strains compared to the wild-type strain. The corresponding gal+ mutants in the wild-type strain are shown below each wild-type strain.

[0177] [Table 3]

[0178] Conclusion: The results demonstrate that the well-functioning ST Gal(+) strains related to the present invention are preferably ST Gal(+) strains having a mutation in the -10 region of the promoter sequence (SEQ ID NO: 8) of the galactokinase gene (galK). This mutation replaces one or both of the C and G nucleotides in the wild-type -10 region (TACGAT, SEQ ID NO: 1) with a nucleotide independently selected from the group consisting of A and T.

[0179] More preferably, the mutation results in a -10 region having the nucleotide sequence TATGAT (SEQ ID NO: 2 – see, for example, the significantly positive results for CHCC28380 and CHCC32045) or TACTAT (SEQ ID NO: 4 – see, for example, the significantly positive results for CHCC29248).

[0180] The newly deposited ST Gal(+) strains (CHCC28380=DSM 33158; CHCC32045=DSM 33159) described herein exhibit a very good, stable, and relatively high pH at the end of fermentation, and these deposited strains possess the mutation "TATGAT" (SEQ ID NO: 2), and are therefore most preferred here.

[0181] References 1. EP2957180B1 (Chr. Hansen A / S, Denmark) 2. YOFLEX® and ACIDIFIX® are registered trademarks of Chr. Hansen A / S. 3. Anbukkarasi et al. (J Food Sci Technol (September 2014) 51(9):2183-2189) 4. Anbukkarasi et al. (“Production of low browning Mozzarella cheese: Screening and characterization of wild galactose fermenting Streptococcus thermophilus strains”, International Journal of advanced research, 2013, vol. 1, no. 5, pp. 83-96) 5. Derkx et al. (“The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology”; Microbial Cell Factories 2014, 13 (Suppl 1)) 6. WO2011 / 026863A1 (Chr. Hansen) 7. WO2011 / 092300A1 (Chr. Hansen)

Claims

1. A method for producing a fermented dairy product having a relatively high and stable pH value at the end of fermentation, comprising the following steps: (a): In at least 100 L of milk, add the following: (I): A Streptococcus thermophilus (ST) composition characterized by being able to reduce the amount of galactose excreted in milk by at least 10% compared to the reference ST CHCC4323 (DSM 32826), 10 4 ~10 14 A step of inoculating a Streptococcus thermophilus (ST) bacterial composition containing ST bacterial cells at CFU / g; Here, the comparative test involves inoculating 1% of the ST strain from the overnight culture into skim milk, incubating at 37°C for 18 hours, taking a sample at the end of fermentation, measuring the galactose content in the fermented milk, and thereby measuring the reduction in excreted galactose compared to the reference CHCC4323; and (b): A step of fermenting milk using the bacteria of (a), wherein the pH is measured during fermentation in a manner that can reliably measure the pH value of step (b), and the fermentation is defined as ending at a relatively high and stable pH value where the pH at the end of fermentation is between 4.3 and 4.9, and the pH does not change by more than 0.1 points during the last two hours of fermentation, and reaches a pH of 4.3 to 4.9 before 24 hours of fermentation; and (c): A process that uses fermented milk from (b) having a pH of 4.3 to 4.9 to carry out further appropriate steps, ultimately leading to the production of a fermented milk product. A method that includes this.

2. The method according to claim 1, wherein the milk in step (a) of claim 1 is cow's milk, and the fermented dairy product in step (c) of claim 1 is yogurt, cheese, kefir, or buttermilk.

3. The method according to claim 2, wherein the fermented dairy product in step (c) of claim 1 is yogurt.

4. In step (a) of claim 1, 10 4 ~10 14 The method according to claim 3, further comprising inoculation of Lactobacillus cells (for example, Lactobacillus delbrüeckii subsp. bulgaricus) at a CFU / g level.

5. The method according to any one of claims 1 to 4, wherein the ST bacterium in step (a)(I) of claim 1 is an ST bacterium characterized by being able to reduce the amount of galactose excreted in milk by at least 25% compared to the reference ST CHCC4323 bacterium.

6. The aforementioned Streptococcus thermophilus (ST) bacteria, (a): Streptococcus thermophilus cells CHCC28380 deposited under registration number DSM 33158; and (b): Streptococcus thermophilus cells CHCC32045 deposited under registration number DSM 33159 The method according to any one of claims 1 to 5, wherein the cell is at least one cell selected from the group consisting of the following.

7. The method according to any one of claims 1 to 6, wherein the ST Gal(+) bacterium in step (a)(I) of claim 1 is a bacterium having a mutation in the -10 region of the galactokinase gene (galK) promoter sequence (sequence number 8), wherein the mutation causes the substitution of one or both of C and G in the wild-type -10 region (TAC-GAT, sequence number 1) by a nucleotide independently selected from the group consisting of A and T.

8. The method according to claim 7, wherein the mutation results in a -10 region having the nucleotide sequence TATGAT (SEQ ID NO: 2).

9. -The pH value of step (b) of claim 1 is continuously measured until the end of fermentation; -The fermentation temperature in step (b) of claim 1 is 25°C to 48°C; -The pH at the end of fermentation in step (b) of claim 1 is 4.4 to 4.8; - The pH does not change by more than pH 0.05 during the last two hours of fermentation in step (b) of claim 1; and -In step (b) of claim 1, the pH is reached to 4.3 to 4.9 before 10 hours of fermentation. The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 9, wherein in step (b) of claim 1, the pH at the end of fermentation is 0.2 to 0.8 points higher than the corresponding comparative pH at the end of fermentation obtained by using reference ST CHCC 4323 (DSM 32826) under the same fermentation conditions.

11. The method according to claim 1, (d) Storage of fermented dairy products produced in process (c) during a storage period of at least one week. The method according to any one of claims 1 to 10, comprising the additional step of having a pH of 4.3 to 4.9 at the end of the storage period.

12. The method according to claim 11, wherein the storage temperature is 2°C to 10°C, and the fermented dairy product is yogurt or cheese.

13. A method for screening and isolating novel Streptococcus thermophilus (ST) bacterial cells, comprising the following steps: (i): A step of selecting and isolating a newly selected pool of ST bacteria (referred to herein as "ST Gal(+) bacteria") from a pool of individual ST bacteria, wherein the ST bacteria are characterized by being able to reduce galactose as required in step (a)(I) of claim 1; (ii): A step of selecting and isolating novel isolated ST Gal(+) bacterial cells from the selected pool of ST Gal(+) bacteria of step (i), which can obtain a relatively high and stable pH value at the end of fermentation as required in step (b) of claim 1. A method that includes this.

14. Streptococcus thermophilus cells CHCC28380 deposited under registration number DSM 33158, or Streptococcus thermophilus cells CHCC32045 deposited under registration number DSM 33159.

15. Streptococcus thermophilus cells that share the functional characteristics of Streptococcus thermophilus cells CHCC28380 deposited under registration number DSM 33158 or Streptococcus thermophilus cells CHCC32045 deposited under registration number DSM 33159.

16. The aforementioned functional characteristics mean that the ST cells can reduce the amount of galactose excreted in milk by at least 10% compared to the reference ST CHCC4323 (DSM 32826) strain, a mutant strain of Streptococcus thermophilus cells (referred to herein as "ST Gal(+) bacteria").

17. - A mutant strain of Streptococcus thermophilus cell CHCC28380 deposited under registration number DSM 33158; or - A mutant strain of Streptococcus thermophilus cell CHCC32045 deposited under registration number DSM 33159. A method for obtaining a deposit strain, comprising using the deposit strain as a starting strain, creating a mutant strain of the deposit strain, and isolating a novel mutant strain, wherein the mutant strain retains the characteristic of being ST Gal(+) of the deposit strain.

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