Use of ST Gal(+) bacteria for the production of fermented dairy products having a relatively high stable pH

Abstract

A method for producing a fermented dairy product (such as yogurt) having a relatively high stable pH value at the end of fermentation, comprising inoculating milk with Streptococcus thermophilus (ST) Gal(+) bacteria.

Description

Technical Field

[0001] This invention relates to a method for producing fermented dairy products (e.g., cheese or yogurt) with 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 uses a large number of bacteria, especially lactic acid bacteria, to improve, for example, the taste and texture of food. In the dairy industry, lactic acid bacteria (LAB) are used extensively to acidify milk (through fermentation), but they are also used for, for example, to texturize products incorporating them.

[0003] Post-acidification control has significant commercial relevance.

[0004] In this field, the term "post-acidification" is generally described in relation to the production of lactic acid by LAB after fermentation has ended—for example, paragraph

[0005] of EP2957180B1 (Chr. Hansen A / S, Denmark) states the following:

[0005] "Even in methods that include a rapid cooling step, post-acidification has been observed, where LAB produces lactic acid after fermentation has ceased and the desired pH level has been reached. Post-acidification is considered one of the most important issues in dairy fermentation processes today. Further reduction of pH during the processing and storage of fermented dairy products can lead to increased acidity and shortened shelf life."

[0006] Many current methods for producing fermented dairy products can be characterized by the following series of steps:

[0007] (a) Fermenting milk using a starter culture containing lactic acid bacteria (or LAB) capable of metabolizing glucose obtained from lactose present in milk;

[0008] (b) Fermentation leads to the production of lactic acid, which causes the pH to drop from the initial 6.4-6.8 (for milk) to pH 3.8-4.2;

[0009] (c) Once the pH required for the fermented product in question is reached, fermentation is terminated by rapidly cooling the fermented dairy product.

[0010] For example, this method is used to produce cheese, yogurt, and yogurt drinks.

[0011] Fermented dairy products at a predetermined pH level are rapidly cooled to terminate fermentation. Fermentation will continue if the product is not cooled. However, rapid cooling can have disadvantages, such as potentially causing loss of texture.

[0012] For example, to avoid the rapid cooling step—the prior art describes many different technical solutions to improve control over post-acidification.

[0013] The commercially available product example relevant to this article is from Chr. Hansen A / S in Denmark. Cultures, which are products that "improve quality and shelf life and have excellent pH stability" (see, for example, www.chr-hansen.com).

[0014] As those skilled in the art will understand, the improved shelf life due to superior pH stability is related to improved control of post-acidification.

[0015] EP2957180B1 (Chr. Hansen A / S, Denmark) describes different technical solutions for improving post-acidification control, such as:

[0016] - Use Lactobacillus bulgaricus strains with amino acid metabolism defects and / or use specific LAB strains characterized as having weak post-acidification activity - see, for example,

[0007] ;

[0017] - Controlling buffering capacity during fermentation and maintaining buffering capacity and pH within predetermined ranges - see, for example,

[0008] ;

[0018] - Use lactose-deficient (Lac(-)) Streptococcus thermophilus (ST) and Lactobacillus delbrueckii ssp. bulgaricus strains.

[0019] As is known in the art, in Streptococcus thermophilus (ST) species, galactose is secreted via a lactose / galactose system (see this article). Figure 1 (Diagram of lac / gal metabolism). A cell can secrete 1 mol of galactose by taking up 1 mol of lactose.

[0020] As is known in the art, thermophilic streptococcal (ST) strains generally do not significantly reduce the amount of galactose secreted in milk—that is, they may be referred to in the art and herein as “ST Gal(-) bacteria”—see, for example, the article by Anbukkarasi et al. (J Food Sci Technol (September 2014) 51(9):2183–2189), which states in its abstract:

[0021] "Most Streptococcus thermophilus strains are galactose-negative (Gal-), meaning they can only metabolize the glucose portion of lactose and excrete it 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. Thus, in this study, three galactose-positive (Gal+) Streptococcus thermophilus strains... were used to prepare low-galactose yogurt."

[0022] All ST strains described above, including EP2957180B1 (Chr. Hansen A / S), and the products discussed above. These are all strains of bacteria that technicians and those that this article considers to be ST Gal(-).

[0023] A relatively high concentration of galactose can cause "browning" during the heating process of cheese, as browning is often described when mozzarella cheese, for example, is produced by Streptococcus thermophilus (ST) and used in pizza production.

[0024] Existing technologies describe that some thermophilic streptococci (ST), also known as galactose-positive strains (referred to in this paper as "STGal(+) bacteria"), can be used to reduce the potential browning problem in cheeses (e.g., mozzarella) used in processes involving important heating steps (e.g., temperatures above 70°C) (e.g., for making pizza) – see, for example, the article by 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).

[0025] As is known to those skilled in the art, “reducing potential browning” and “improving post-acidification control” are potentially distinctly different issues, for example, that may be associated with different dairy products.

[0026] For example, browning problems are often more likely to be associated with cheeses (e.g., mozzarella) used in processes involving significant heating steps (e.g., heating to temperatures above 70°C) (e.g., for making pizza).

[0027] Conversely, post-acidification issues are associated with dairy products produced without significant heating steps (such as heating to temperatures above 70°C) – such as cheese and yogurt.

[0028] The article by Derkx et al. (“The art of strain improvement of industrial lacticacid bacteria without the use of recombinant DNA technology”; Microbial Cell Factories 2014, 13(Suppl 1)) is a review article. For example, it discusses post-acidification on page 9, and states in the left column on page 9:

[0029] "In another approach to obtaining modified strains with reduced post-acidification, the importance of oligopeptide transport to the growth adaptation of Streptococcus thermophilus in milk was investigated... and it was found that mutants with altered oligopeptide transport systems had reduced acidification rates."

[0030] The article by Derkx et al. mentioned above states at the top right column on page 9:

[0031] "Furthermore, due to the growth of resident lactic acid bacteria, excessive free galactose can lead to post-acidification problems and an imbalance in the cheese microbiota. Therefore, galactose-positive wild-type strains or galactose-fermenting mutants are very interesting, especially for reducing browning in pizza cheese."

[0032] As those skilled in the art will understand, the post-acidification issue discussed in the paragraphs of the article by Derkx et al. (2014) cited above relates to the “growth of resident lactic acid bacteria”—that is, the production of lactic acid from resident LABs after fermentation has ended.

[0033] WO2011 / 026863A1 (Chr.Hansen) and WO2011 / 092300A1 (Chr.Hansen) describe a thermophilic streptococcus (ST) strain with a mutation in the galK (galactokinase) gene that produces higher viscosity in fermented milk.

[0034] WO2019 / 042881A1 (Chr. Hansen) describes some instances of ST Gal(+) bacteria.

[0035] None of these three published WO documents describe or address the "post-acidification" issues discussed in this article.

[0036] In summary, in this field, the "post-acidification" problem is generally described as related to the resident LAB in the termination of such fermentation. back Regarding lactic acid production, many different solutions related to controlling / reducing post-acidification problems have been described in the art—for example, using STLac(-) bacteria or ST bacteria with altered oligopeptide transport systems. Summary of the Invention

[0037] The problem to be solved by the present invention is to provide a method for producing fermented dairy products (e.g., yogurt) with a relatively high stable pH value at the end of fermentation, wherein the advantage of the produced fermented dairy products (e.g., yogurt) may be, for example, lower post-acidification during the storage period of the produced fermented dairy products.

[0038] This solution is based on a surprising correlation that the inventors have established between thermophilic streptococcal (ST) galactose-positive strains (referred to herein as "ST Gal(+) bacteria") and the likelihood of obtaining a stable, relatively high pH at the end of such fermentation.

[0039] As discussed above, in this field, the "post-acidification" problem is generally described as related to the resident lactic acid bacteria (LAB) during the termination of such fermentation. back It is related to the production of lactic acid, for example, during storage after such fermentation.

[0040] Clearly, the surprising connection discussed above, and the relatively high pH at the end of this type of fermentation, is based on the role of the growth curve of Gal(+)ST bacteria in this type of fermentation.

[0041] Unrestricted by theory—the inventors are unaware of any separate prior art literature that directly and unambiguously describes the aforementioned surprising link between ST Gal(+) bacteria and the possibility of obtaining a stable, relatively high pH at the end of such fermentation.

[0042] As discussed in, for example, the embodiments herein, and as Figure 2 and Figure 3 As shown, the ST Gal(+) bacteria described in this article have a significantly higher stable pH (approximately 0.2–0.6 points higher) than the corresponding wild-type ST Gal(-) bacteria at the end of this type of fermentation.

[0043] From example Figure 2 It can be seen that using the ST Gal(+) bacteria described in this article produces a pH of 4.3-4.8 at the end of fermentation, while using the wild-type CHCC27806 ST Gal(-) produces a pH of about 4.15 (i.e., less than pH 4.3).

[0044] In other words, the use of ST Gal(+) bacteria produced a significantly higher stable final pH value (approximately 0.3–0.6 points higher) compared to the use of the corresponding wild-type CHCC27806 ST Gal(-) strain.

[0045] Furthermore, the initial acidification activity remains relatively unchanged (see example). Figure 2 or Figure 3This suggests that the observed low acidification activity (i.e., the relatively high stable pH at the end of fermentation) is not due to the overall low acidification rate.

[0046] Unbound by theory—it is believed that a higher final pH at the end of such fermentation will affect post-acidification during shelf life, which could be a significant issue for dairy products such as yogurt (see above).

[0047] Therefore, the ST Gal(+) strains discussed in this paper, which have a stable high pH at the end of fermentation, lead to lower post-acidification during storage, for example, which is a desirable trait in commercially relevant dairy products.

[0048] One might argue that the novel link identified herein between ST Gal(+) strains and the likelihood of achieving a stable, relatively high pH at the end of such fermentation can be considered a “change of behavior” for those skilled in the art—for example, if someone wanted a “low-post-acidification” yogurt culture, they would choose a suitable ST Gal(+) bacterium after the disclosure of this invention, rather than other different “low-post-acidification” cultures known in the prior art (see above).

[0049] As discussed in the examples herein, approximately 20% of the tested ST Gal(+) strains actually functioned as required herein (i.e., producing the relatively high stable pH value discussed herein at the end of fermentation).

[0050] Therefore, without the knowledge of this invention, those skilled in the art may have readily tested the ST Gal(+) strains of interest without determining the positive effect of “relatively high pH at the end of fermentation” as described herein.

[0051] However, once this invention discloses the novel link discussed herein between ST Gal(+) and the effect of “stable relatively high pH at the end of fermentation”, identifying new ST Gal(+) strains with this positive effect of “stable relatively high pH at the end of fermentation” becomes a routine screening / selection task for those skilled in the art.

[0052] For example, firstly, approximately 100 different ST Gal(+) strains are simply isolated / selected using a standard procedure, and then ST Gal(+) strains with the positive effect of “stable, relatively high pH at the end of fermentation” described herein are screened / selected from the pool of these different ST Gal(+) strains (approximately 20% positive, as described herein).

[0053] Therefore, and discussed in further detail below, it may be said that the present invention is based on the inventors’ development of a new selection method for identifying new ST Gal(+) strains that have the positive effect of “stable, relatively high pH at the end of fermentation” as described herein.

[0054] As discussed in the examples below, the inventors attempted to identify positive ST strains with a "stable, relatively high pH at the end of fermentation" based on pools of different ST Gal(-) strains, but no single positive strain / cell was identified. That is, without the knowledge of the present invention, it would be impossible (or would take a very long time) to identify ST strains with the positive effect of "stable, relatively high pH at the end of fermentation" as described herein.

[0055] Therefore, a first aspect of the present invention relates to a method for producing a fermented dairy product having a relatively high stable pH value at the end of fermentation, comprising the following steps:

[0056] (a): Inoculate at least 100 L of milk with (I):

[0057] (I): Contains 10 4 Up to 10 14 A CFU / g ST bacterial cell composition of thermophilic streptococci (ST) bacteria, characterized in that the ST bacteria are able to reduce the amount of galactose secreted in milk by at least 10% compared with reference ST CHCC4323 (DSM 32826) bacteria (referred to herein as "ST Gal(+) bacteria");

[0058] The comparative test was conducted as follows: 1% of the overnight culture of the ST bacteria was inoculated into skim milk, incubated at 37°C for 18 hours, and samples were collected at the end of fermentation to measure the galactose content in the fermented milk, thereby measuring the reduction in secreted galactose compared to the reference CHCC4323; and

[0059] (b): using the bacterial fermentation milk of (a), wherein the pH is measured during fermentation in a manner consistent with determining the pH value of this step (b), and wherein fermentation is carried out under conditions where fermentation ends at a relatively high and stable pH value (defined as pH 4.3–4.9 at the end of fermentation), and wherein the pH change during the last 2 hours of fermentation does not exceed pH 0.1, and wherein the pH reaches 4.3–4.9 24 hours before fermentation (e.g., 15 hours before); and

[0060] (c): Using the fermented milk with pH 4.3-4.9 from (b) for other appropriate steps to finally obtain the fermented dairy product produced.

[0061] Alternatively, the first aspect of the invention can be stated as a so-called use claim—namely, the use of *Streptococcus thermophilus* (ST) Gal(+) bacteria in a method for producing a fermented dairy product having a relatively high stable pH of 4.3-4.9 at the end of fermentation, wherein the method comprises the following steps:

[0062] (a): Inoculate at least 100 L of milk with (I):

[0063] (I): Contains 10 4 Up to 10 14 A CFU / g ST bacterial cell composition of thermophilic streptococci (ST) bacteria, characterized in that the ST bacteria are able to reduce the amount of galactose secreted in milk by at least 10% compared with reference ST CHCC4323 (DSM 32826) bacteria (referred to herein as "ST Gal(+) bacteria");

[0064] The comparative test was conducted as follows: 1% of the overnight culture of the ST bacteria was inoculated into skim milk, incubated at 37°C for 18 hours, and samples were collected at the end of fermentation to measure the galactose content in the fermented milk, thereby measuring the reduction in secreted galactose compared to the reference CHCC4323; and

[0065] (b): using the bacterial fermentation milk of (a), wherein pH is measured during fermentation in a manner consistent with determining the pH value of this step (b), and wherein fermentation is carried out under conditions where fermentation ends at a relatively stable pH value (defined as pH 4.3–4.9 at the end of fermentation), and wherein the pH change during the last 2 hours of fermentation does not exceed pH 0.1, and wherein the pH reaches 4.3–4.9 before 24 hours of fermentation; and

[0066] (c): Use the fermented milk with pH 4.3-4.9 from (b) for other appropriate steps to finally obtain the fermented dairy product produced.

[0067] The ST Gal(+) bacterial test of “(a)(I)” in the first aspect can be regarded as a standard test related to this article that can be routinely performed by those skilled in the art.

[0068] It is believed that many ST Gal(+) bacteria described in the prior art discussed above would meet the ST Gal(+) test—that is, obtaining ST Gal(+) bacteria that meet the comparative test of the first aspect “(a)(I)” based on the prior art and the technical information provided herein can be considered a relatively routine task.

[0069] In Example 1 of this document, a method for obtaining different ST Gal(+) bacteria that meet the first aspect step “(a)(I)” is described—that is, the first aspect step “(a)(I)” is preferably performed according to Example 1.

[0070] As discussed above, the inventors are unaware of any separate prior art literature that directly and unambiguously describes the aforementioned surprising link between ST Gal(+) bacteria and the possibility of obtaining a stable, relatively high pH at the end of such fermentation.

[0071] Consistent with this is step (b), which is itself a novel step—that is, the prior art does not directly and unambiguously describe a method in which milk is inoculated with ST Gal(+) bacteria according to step (a) of the first aspect, and then pH is monitored / measured in fermentation step (b) as required by step (b) of the first aspect.

[0072] One reason for this is that prior to this invention, those skilled in the art were simply unaware of the possibility that ST Gal(+) bacteria could have the positive effect of “a relatively high pH at the end of fermentation” as described herein – and therefore, they would not have considered monitoring / measuring pH to control / monitor this positive effect as required by step (b) of the first aspect.

[0073] As understood by those skilled in the art, step (b) of the first aspect requires at least some form of pH monitoring / measurement sufficient to determine that “the pH at the end of fermentation is 4.3-4.9, and wherein the pH change during the last 2 hours of fermentation does not exceed 0.1, and wherein the pH reaches 4.3-4.9 before 24 hours of fermentation.”

[0074] As technicians understand it, this pH monitoring / measurement can be performed in different ways to objectively determine / evaluate the relevant pH value.

[0075] For example, it may not be necessary to strictly measure the pH 2 hours before the end of fermentation—for example, if the pH is measured 3 hours before the end of fermentation, 1 hour before the end of fermentation, and at the end of fermentation, and the pH values ​​at all three determinations are within the correct range, then the technician will objectively understand that the pH is also correctly within the requirements of step (b) in the last 2 hours of fermentation.

[0076] In the embodiments described herein (see, for example, the embodiments described herein), Figure 2 Continuous monitoring / measurement of pH – this could be a preferred procedure.

[0077] Step (b) of the first aspect states "pH at the end of fermentation".

[0078] Technicians know when fermentation is complete, which in the context of this invention can be essentially considered as relating to when the pH no longer decreases significantly.

[0079] As is known in the art, fermentation can be terminated / stopped when, for example, the fermentation medium no longer contains sufficient relevant nutrients (e.g., sugars such as lactose, galactose, etc.) for bacterial growth / metabolism, or fermentation can be terminated / stopped by changing the temperature (e.g., by rapid cooling) to a temperature significantly different from the optimal temperature for bacterial growth.

[0080] Alternatively, fermentation may end naturally with an increase in the concentration of lactic acid or other growth-inhibiting compounds.

[0081] The fermentation conditions in step (b) can typically be standard suitable ST fermentation conditions associated with the ST bacteria of interest—such as, for example, about 37°C as used in the examples herein.

[0082] As those skilled in the art will understand—the reason for doing so relates to the inherent characteristics of the ST Gal(+) bacteria used herein, such as the pH at the end of fermentation—that ST bacteria, which are not effective in this document, would, for example, produce a final pH of approximately pH 4.1 at the end of fermentation under standard ST fermentation conditions.

[0083] Given the technical disclosure and common knowledge of this article—selecting / identifying the positive / useful ST Gal(+) strains in this article and finding suitable conditions that meet the requirements of step (b) of the first aspect is routine work for a technician.

[0084] Step (c) of the first aspect can be regarded as routine work of the technicians—that is, the technicians know how to make fermented dairy products of interest (such as cheese or yogurt).

[0085] As will be understood by those skilled in the art, in step (a) of the first aspect, milk may be inoculated with other lactic acid bacteria (LAB) of interest, such as Lactobacillus bulgaricus, to produce, for example, yogurt (i.e., fermented dairy products are, for example, yogurt).

[0086] A second aspect of the present invention relates to a method for screening and isolating novel thermophilic streptococcal (ST) bacterial cells, comprising the following steps:

[0087] (i): Selecting and isolating newly selected pools of ST bacteria from pools of individual ST bacteria, characterized in that the ST bacteria are capable of reducing galactose (referred to herein as "ST Gal(+) bacteria") as required in step (a)(I) of the first aspect;

[0088] (ii): Select and isolate new isolated ST Gal(+) bacterial cells from the ST Gal(+) bacterial pool selected in step (i), which are capable of producing a relatively high stable pH at the end of fermentation as required in step (b) of the first aspect.

[0089] The embodiments of the present invention are described below by way of example only. Attached Figure Description

[0090] Figure 1 Schematic diagram of lac / gal metabolism

[0091] Figure 2 The figure shows that the ST Gal(+) bacteria described herein exhibit a significantly higher stable pH (approximately 0.3–0.6 points higher) at the end of this type of fermentation compared to the corresponding wild-type CHCC27806 ST Gal(-) bacteria. The figure also shows that novel ST Gal(+) strains, for example, newly deposited in this paper (CHCC28380 = DSM 33158; CHCC32045 = DSM 33159), exhibit very good stable relatively high pH at the end of this type of fermentation. For further details, see the examples herein.

[0092] Figure 3 The figures show that the ST Gal(+) bacteria described herein exhibit a significantly higher stable pH (approximately 0.2–0.5 points higher) at the end of this type of fermentation compared to the corresponding wild-type CHCC4426 ST Gal(-) bacteria. See the examples in this article for further details. Detailed Implementation

[0093] Preserved strains / cells

[0094] A sample of *Streptococcus thermophilus* cells CHCC4323 has been deposited at DSMZ (German Center for Microbial Collection, 7B Inhoffenstr., D-38124, Braunschweig) with accession number DSM 32826 and a deposit date of June 5, 2018. The deposit was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0095] The strains deposited below are the first strains deposited with respect to this application—that is, they are novel strains in themselves.

[0096] A sample of *Streptococcus thermophilus* cells CHCC28380 has been deposited at DSMZ (German Centre for Microbial Collections, 7B Inhofenstrasse, Braunschweig, D-38124) with accession number DSM 33158 on June 12, 2019. The deposit was made in accordance with the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings.

[0097] A sample of *Streptococcus thermophilus* cells CHCC32045 has been deposited at DSMZ (German Centre for Microbial Collections, 7B Inhofenstrasse, Braunschweig, D-38124) with accession number DSM 33159 on June 12, 2019. The deposit was made in accordance with the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings.

[0098] As discussed in the examples herein, the novel preserved strains described herein exhibit a very good, stable, and relatively high pH at the end of such fermentations.

[0099] Therefore, a single aspect of the present invention relates to thermophilic streptococcal cells CHCC28380 deposited under accession number DSM 33158 or thermophilic streptococcal cells CHCC32045 deposited under accession number DSM 33159.

[0100] Therefore, another aspect of the present invention relates to thermophilic streptococcal cells having the functional characteristics of CHCC28380, deposited under accession number DSM 33158, or thermophilic streptococcal cells having the functional characteristics of CHCC32045, deposited under accession number DSM 33159. In this relevant aspect, the functional characteristics mean that, compared with reference ST CHCC4323 (DSM 32826) bacteria, ST bacteria are able to reduce the amount of galactose secreted in milk by at least 10% (referred to herein as "ST Gal(+) bacteria").

[0101] A particular aspect of the present invention relates to a method for obtaining the following:

[0102] - A mutant strain of Streptococcus thermophilus cell CHCC28380, deposited with accession number DSM 33158; or

[0103] - A mutant strain of Streptococcus thermophilus cell CHCC32045, deposited with accession number DSM 33159.

[0104] This includes using a preserved strain as a starting strain, preparing mutants of the preserved strain, and isolating new mutant strains, wherein the mutant strains retain the characteristics of the preserved strain as ST Gal(+).

[0105] Fermented dairy products

[0106] The milk in step (a) of the first aspect and the milk from which the first aspect ferments dairy products can be, for example, soy milk or animal milk (e.g., goat milk, buffalo milk, sheep milk, horse milk, camel milk or cow milk).

[0107] Preferably, the milk is cow's milk.

[0108] Fermented dairy products are preferably dairy products such as yogurt, cheese, kefir or buttermilk.

[0109] Preferred cheeses may be, for example, fresh cheese products, soft cheese products, cheddar cheese, continental cheese, pasta filata cheese, pizza cheese, or mozzarella cheese.

[0110] Perhaps the preferred product is yogurt.

[0111] Inoculation with milk – Step (a) of the first aspect

[0112] As discussed above, in step (a) of the first aspect, milk can also be inoculated with other lactic acid bacteria (LAB) of interest—such as Lactobacillus bulgaricus—to produce, for example, yogurt (i.e., fermented dairy products are, for example, yogurt).

[0113] Preferably, in step (a) of the first aspect, 10 4 Up to 10 14 Milk is inoculated with CFU / g Lactobacillus bacterial cells (e.g., Lactobacillus delbrueckii subsp. bulgaricus) – this may be particularly relevant when fermented dairy products are, for example, yogurt.

[0114] Preferably, in step (a) of the first aspect, 10 4 Up to 10 14 Milk is inoculated with CFU / g of Lactococcus bacteria (e.g., Lactococcus lactis) – this is particularly relevant when the fermented dairy product is, for example, cheese.

[0115] Preferably, in step (a) of the first aspect, 10 4 Up to 10 14 Milk inoculated with CFU / g of Leuconostoc bacteria cells—this is particularly relevant when fermented dairy products are, for example, cheese.

[0116] Preferably, step (a) of the first aspect involves inoculating at least 200L of milk or at least 1000L of milk.

[0117] The first aspect step "(a)(I)" ST Gal(+) bacteria

[0118] In Example 1 of this document, a method for obtaining different ST Gal(+) bacteria that meet the first aspect step “(a)(I)” is described.

[0119] As can be seen from Table 1 of Example 1, by using the specific method described in this example for isolating galactose superfermentation mutants from Streptococcus thermophilus, ST bacteria (see, for example, CHCC27912 and CHCC29526) that can reduce the amount of galactose secreted in milk by about 50% compared to the reference ST CHCC4323 bacteria.

[0120] ST bacteria that can reduce the amount of galactose secreted in milk by at least 20% compared to the reference ST CHCC4323 bacteria may be referred to as ST Gal(++) bacteria in this paper.

[0121] Unrestricted by theory—the method described in Example 1 for isolating the ST Gal(++) mutant of galactose from *Streptococcus thermophilus* can be considered specific because the galactose reduction level is significantly increased compared to the strain referred to herein as Gal(+). As described in Example 1, the galactose reduction level of CHCC14993 (the Gal(+) mutant of CHCC4323) is 17% compared to wild-type CHCC4323, while the galactose reduction level of CHCC14994 (the Gal(++) mutant of CHCC4323) is 30%. The galactose reduction level of CHCC29526 (the Gal(++) mutant of CHCC4459) is even 52% compared to the reference CHCC4323.

[0122] By using the method of subculturing in the M17-gal broth of Example 1, a galactose superfermentation mutant with a unique galactose reduction ability can be isolated.

[0123] Preferably, the ST bacteria in the first aspect step “(a)(I)” are ST bacteria characterized by being able to reduce the amount of galactose secreted in milk by at least 20% (e.g., at least 25%, more preferably at least 30%, or even more preferably at least 40%) compared to the reference STCHCC4323 bacteria.

[0124] Preferably, the thermophilic streptococcal (ST) bacterial cells are at least one type of cell selected from:

[0125] (a): Streptococcus thermophilus cells CHCC28380, deposited with accession number DSM 33158; and

[0126] (b): Streptococcus thermophilus cells CHCC32045 deposited with accession number DSM 33159.

[0127] Preferably, in step "(a)(I)" of the first aspect, 10 mg of milk per gram is used. 4 Up to 10 15 CFU (or 10) 4 Up to 10 14 CFU (colony-forming units) live ST bacterial cells inoculated into milk, including at least 10 5 CFU / g milk, 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 milk, for example, at least 10 10 CFU / g milk or, for example, at least 10 CFU / g milk. 11 cfu / g milk.

[0128] ST bacterial cells can be a mixture of different ST strains (e.g., a mixture of CHCC28380 and CHCC32045 discussed in this paper) – for example, 10 8 A CFU / g milk strain of ST strain (e.g., CHCC28380) +10 8 Another ST strain of cfu / g milk (e.g., CHCC32045), which generally means using 2 x 10 8 Live ST bacterial cells inoculated into milk at cfu / g.

[0129] Typically, bacteria (such as starter culture compositions) are in concentrated forms, including freeze-dried, dried, or lyophilized concentrates.

[0130] As discussed in the examples herein, not all tested ST Gal(+) strains actually functioned as required herein (i.e., producing the relatively high stable pH discussed herein at the end of fermentation).

[0131] Therefore, and as discussed in the embodiments herein, genomic analysis is performed to identify common structural elements in the preferred, well-functioning positive ST Gal(+) strains.

[0132] The results showed that most of the superior ST Gal(+) strains had mutations in the -10 region / box of the galactokinase gene (galK) promoter.

[0133] Such galK mutants are described, for example, in WO2011 / 026863A1 (Chr.Hansen) – as discussed above, this WO disclosure does not describe / address the “post-acidification” related issues discussed herein.

[0134] The following figure is shown on page 10 of WO2011 / 026863A1 (Chr. Hansen):

[0135]

[0136] As shown in the figure above and discussed in WO2011 / 026863A1, the wild-type / common sequence of the -10 region / box is “TACGAT”, and the strain named “CHCC11379” contains the mutation within the -10 region / box.

[0137] The wild-type / common promoter sequence of the galactokinase gene (galK) is shown in SEQ ID NO:8 in the figure above, which is the same as SEQ ID NO:8 in this article.

[0138] In short, technicians can routinely determine whether the ST Gal(+) strain of interest has a mutation in the -10 region / box of the galactokinase gene (galK) promoter.

[0139] Therefore, in a preferred embodiment, the ST Gal(+) bacteria of the first aspect step “(a)(I)” are preferably bacteria with a mutation in the -10 region of the galactokinase gene (galK) promoter (SEQ ID NO:8), wherein the mutation results in one or both of the C and G in the wild-type -10 region (TACGAT, SEQ ID NO:1) being replaced by nucleotides independently selected from the group consisting of A and T.

[0140] Preferably, the mutation results in the -10 region having the nucleotide sequence TATGAT (SEQ ID NO:2 – see, for example, very positive results of CHCC28380 and CHCC32045 discussed below) or TACTAT (SEQ ID NO:4 – see, for example, positive results of CHCC29248 discussed below) – most preferably, the mutation results in the -10 region having the nucleotide sequence TATGAT (SEQ ID NO:2).

[0141] As discussed in the examples herein, the novel preserved ST Gal(+) strains (CHCC28380 = DSM33158; CHCC32045 = DSM 33159) exhibit very good stability at a relatively high pH at the end of such fermentations. These preserved strains contain the mutant “TATGAT” (SEQ ID NO:2) and are therefore the preferred strains in this study.

[0142] Without being bound by theory, it is believed that higher expression of the galK gene than the wild type will produce the positive effect of obtaining a relatively high stable pH at the end of fermentation, as discussed herein. Therefore, in the preferred embodiment, the ST Gal(+) bacteria in the first aspect step “(a)(I)” are preferably bacteria with higher expression of the galK gene than the wild type, and wherein the ST Gal(+) bacteria preferably have a mutation in the -35, -10 or ribosome binding site (RBS) of SEQ ID NO:8.

[0143] Fermenting milk with bacteria – Step (b) of the first aspect

[0144] Step (b) of the first aspect involves fermenting the milk with the bacteria from (a).

[0145] As discussed above, the fermentation conditions in step (b) can typically be standard, suitable ST fermentation conditions associated with the ST bacteria of interest—for example, about 37°C as used in the examples herein.

[0146] As discussed above, the reason for doing so relates to the inherent characteristics of the ST Gal(+) bacteria used in this paper, such as the pH at the end of fermentation—that is, ST bacteria that do not function in this paper would, for example, produce a final pH of about 4.1 at the end of fermentation under standard ST fermentation conditions.

[0147] Those skilled in the art know how to use relevant bacteria to ferment milk to prepare fermented dairy products of interest (e.g., cheese) – therefore, this need not be described in detail in the context of this invention.

[0148] According to existing technology and depending on, for example, the ST used, the fermentation temperature can be, for example, 25°C to 48°C, such as 35°C to 48°C or 36°C to 38°C.

[0149] According to the art, the fermentation time in step (b) of the first aspect can be 2-96 hours, for example 3-72 hours or for example 4-48 hours.

[0150] Preferably, the fermentation time in step (b) of the first aspect can be 2-30 hours, for example 3-24 hours.

[0151] Step (b) of the first aspect states: "pH at the end of fermentation".

[0152] As discussed above, those skilled in the art know when fermentation is complete, which in the context of this invention can be essentially considered as relating to when the pH no longer decreases significantly.

[0153] As is known in the art, fermentation can be terminated / stopped when, for example, the fermentation medium no longer contains sufficient relevant nutrients (e.g., sugars such as lactose, galactose, etc.) for bacterial growth / metabolism, or fermentation can be terminated / stopped by changing the temperature (e.g., by rapid cooling) to a temperature significantly different from the optimal temperature for bacterial growth.

[0154] Alternatively, fermentation may end naturally with an increase in the concentration of lactic acid or other growth-inhibiting compounds.

[0155] Preferably, the pH at the end of fermentation is 4.3-4.8, for example, 4.4-4.8 or 4.4-4.7.

[0156] In a preferred embodiment, the pH change during the last 2 hours of fermentation does not exceed pH 0.05.

[0157] In a preferred embodiment, the pH reaches 4.3-4.9 15 hours before fermentation (more preferably 10 hours, or even more preferably 8 hours before fermentation).

[0158] Regarding large-scale milk fermentation, which is relevant to this paper, it is known in the art that milk fermentation can sometimes be completed in about 5 hours.

[0159] As discussed in this paper, the ST strain CHCC4323 (DSM 32826) can be considered as the ST reference strain corresponding to the ST Gal(-) strains currently used in commercial production, such as the dairy products discussed in this paper.

[0160] Therefore, in a preferred embodiment of step (b) of the first aspect, the pH at the end of fermentation is 0.1-0.8 points (preferably 0.2-0.8 points, e.g. 0.2-0.6 points) higher than the corresponding comparative pH at the end of fermentation obtained by using reference ST CHCC4323 (DSM 32826) bacteria under comparable and identical fermentation conditions.

[0161] Technicians certainly know how to conduct such a comparative experiment—that is, fermentation in step (b) is carried out using the STGal(+) strain of the first aspect, and then repeated under the same conditions with the reference ST CHCC4323 (DSM 32826) bacteria, and then the final pH values ​​are compared.

[0162] Other appropriate steps for making fermented dairy products of interest - Step (c) of the first aspect

[0163] Step (c) of the first aspect involves performing other appropriate steps to ultimately obtain the fermented dairy product of interest produced.

[0164] As discussed above, those skilled in the art know how to make fermented dairy products of interest (such as cheese or yogurt) – therefore, it is unnecessary to describe this in detail in the context of this invention.

[0165] Storage of produced fermented dairy products – Optional step (d) of the first aspect

[0166] As discussed above, it is believed that a higher final pH at the end of such fermentation can affect post-acidification during the shelf life, which could be a significant problem for dairy products such as yogurt (see above).

[0167] Therefore, the ST Gal(+) strains with stable higher pH discussed in this paper result in lower post-acidification, which is a desired trait, for example, in commercially relevant dairy products.

[0168] Therefore, in a preferred embodiment of the first aspect of the method, the method further includes additional steps related to the following:

[0169] (d): The fermented dairy product produced in step (c) is stored for a storage period of at least 1 day (e.g., at least 1 week, at least 2 weeks, at least 1 month or at least 2 months), and the pH of the product at the end of the storage period is 4.3-4.9.

[0170] Preferably, the pH change during storage does not exceed pH 0.3 (preferably not exceeding pH 0.2 or even more preferably not exceeding pH 0.1).

[0171] Storage can be done at dairy producers and / or at retailers / stores that sell fermented dairy products (such as yogurt).

[0172] Regarding step (d) – if the storage period of step (d) is, for example, at least one day and the pH of the product has been measured to determine that the pH of the product after one day is within the pH range of 4.3-4.9 in step (b) (e.g., pH 4.4), then step (d) has been performed – even if the product may be stored for a longer period (e.g., one year) and, for example, the pH of the product after one year is below 4.3 (i.e., outside the range of step (d)).

[0173] Technicians know how to store the fermented dairy products of interest produced—for example, yogurt can be stored at, for example, 2°C to 10°C—for example, around 5°C.

[0174] Technicians know how to measure the pH of the stored fermented dairy product of interest, and thereby routinely determine whether the conditions of step (d) are met.

[0175] Methods for screening and isolating novel ST bacteria – Part Two

[0176] As discussed above, a second aspect of the present invention relates to a method for screening and isolating novel Streptococcus thermophilus (ST) bacterial cells, comprising the following steps:

[0177] (i): Selecting and isolating new selected ST bacterial pools from individual ST bacterial pools, characterized in that the ST bacteria are capable of reducing galactose (referred to herein as "ST Gal(+) bacteria") as required in step (a)(I) of the first aspect;

[0178] (ii): Selection and isolation – from the ST Gal(+) bacterial pool selected in step (i) – newly isolated ST Gal(+) bacterial cells that are capable of producing a relatively high stable pH at the end of fermentation as required by step (b) of the first aspect.

[0179] The second aspect of the method, step (i), is described as “selection and isolation from individual ST bacterial pools”.

[0180] As is well known, preparing / producing such individual bacterial cell pools is routine work for technicians.

[0181] It can be prepared, for example, from suitable preferred starting cells that can be appropriately mutagenized (e.g., using chemical mutagens or UV mutagenesis) to prepare a mutant pool of said starting cells—that is, a pool of individual bacterial cells.

[0182] As discussed in this article, given the technical disclosure and common knowledge presented here—selecting / identifying the positive / useful ST strains in this article through the screening and isolation methods of the second aspect is routine work for technicians.

[0183] Example

[0184] Example 1: ST Gal(+) bacteria – significantly reducing galactose release even in the presence of high levels of lactose (e.g., in milk) – i.e., the ST Gal(+) bacteria of the first inventive step “(a)(I)”.

[0185] Reference strain:

[0186] -ST strain CHCC4323: It has a naturally wild-type galK sequence (referred to as GalK(-) in this paper) and can be regarded as the ST reference strain corresponding to the ST strains currently used in commercial applications for making, for example, cheese.

[0187] -ST strain 4323-2 (CHCC14993): It contains a mutation in the galK (galactokinase) gene (referred to as Gal(+) in this paper) and can be regarded as a reference strain corresponding to the strains prepared according to the descriptions of WO2011 / 026863A1 (Chr.Hansen) and WO2011 / 092300A1 (Chr.Hansen) discussed above.

[0188] Preserved strains:

[0189] CHCC14994: DSM 25838 ST strain - published in WO2013 / 160413A1 (Chr.Hansen).

[0190] CHCC19097: DSM 32594ST strain

[0191] CHCC19100: DSM 32595ST strain

[0192] CHCC27912: DSM 32596ST strain

[0193] CHCC29526: DSM 32597ST strain

[0194] CHCC29530: DSM 32598ST strain

[0195] Some preserved ST Gal(+) strains are discussed in WO2019 / 042881A1 (Chr.Hansen) – as mentioned above, this WO discloses issues related to “post-acidification” that are not described or addressed in this paper.

[0196] Isolation of galactose superfermentation mutants from Streptococcus thermophilus:

[0197] Prior to mutant isolation, the strain was streaked onto M17 agar plates containing 2% galactose (M17-gal plates). Wild-type (wt) strains showed little growth when galactose was the sole carbohydrate source.

[0198] The overnight culture plates were then inoculated onto M17-gal plates, and several colonies were 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 the sole carbohydrate.

[0199] The second-generation galactose superfermentation mutant was isolated from the purified galactose-positive mutant by subculturing in M17-gal broth with daily re-inoculation of 1% from the fully grown overnight culture; incubation was carried out at 37°C.

[0200] After dilution plate inoculation, 100 single colonies were isolated from the M17-gal plates and inoculated into microtiter plates containing M17-gal broth. OD was followed by OD reading, and clones exhibiting better OD increase during incubation at 37°C for 16 hours (e.g., wt strains) were further purified and characterized.

[0201] The *Streptococcus thermophilus* strain from which the galactose-superfermentation mutant was isolated is:

[0202] CHCC9861

[0203] CHCC4459

[0204] CHCC4426

[0205] CHCC4323

[0206] CHCC7018

[0207] CHCC3050

[0208] The galactose hyperfermentation mutant exhibiting abnormally high galactose fermentation capacity and reduced galactose secreted into the culture medium is (mutant / wt):

[0209] CHCC27912 / CHCC9861

[0210] CHCC29526 / CHCC4459

[0211] CHCC29530 / CHCC4426

[0212] CHCC14994 / CHCC4323

[0213] CHCC19100 / CHCC7018

[0214] CHCC19097 / CHCC3050

[0215] This embodiment also includes a typical galactose-positive strain, named CHCC14993, which was isolated from CHCC4323 as a first-generation mutant. CHCC14993 exhibits a typical 17% reduction in galactose in milk (compared to wt CHCC4323, galactose secretion in milk is reduced).

[0216] Milk fermentation

[0217] The mutant strain was inoculated into skim milk at 1% of the overnight culture and incubated at 37°C for 24 hours. The acidification activity of the mutant was similar to that of the wt strain. At the end of fermentation, samples were collected to measure the galactose content in the fermented milk, and the reduction in secreted galactose was compared with that of the galactose-negative reference strain CHCC4323.

[0218] Results - Analysis of acidified and secreted galactose in fermented milk

[0219] All tested ST strains exhibited similar acidification curves—meaning that the preserved ST strains did not lose their acidification ability in milk.

[0220] The amount of galactose secreted by different test strains is shown in Table 1 below:

[0221] Table 1 shows the amount of galactose in fermented skim milk and the reduction in galactose compared to the reference CHCC4323. The typical gal+ mutant CHCC14993 shows a galactose reduction of less than 20%, while the super-fermented mutant shows a much greater reduction of up to 52%. This means that when producing pizza cheese, for example, with the new mutant, the amount of free galactose will be much lower, leading to less browning during baking.

[0222] 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

[0223] Table 1. Average of two measurements in carbohydrate analysis. Results are expressed in mg / g.

[0224] in conclusion

[0225] The results showed that the preserved strain was also able to reduce the release of galactose to a certain extent in the presence of large amounts of lactose (such as in milk), which was significantly improved compared to the reference strain discussed above.

[0226] Example 2: ST Gal(+) bacteria – pH value at the end of fermentation

[0227] strain

[0228] All ST Gal(+) strains discussed in this embodiment are ST Gal(+) strains that meet the requirements of the first aspect (a) of this document, wherein comparative tests were performed according to Example 1 above.

[0229] New preserved strain:

[0230] The following new strains were preserved for the first time in connection with this invention.

[0231] CHCC28380: DSM 33158ST strain

[0232] CHCC32045: DSM 33159ST strain

[0233] Milk fermentation

[0234] ST Gal(+) mutant strain and reference / wild-type ST Gal(-) strain were inoculated into skim milk at 1% of the overnight culture in M17 containing 2% lactose and incubated at 37°C for 24 hours.

[0235] pH was continuously monitored / measured during fermentation (using an intab PC recorder and EasyView software).

[0236] result

[0237] This article Figure 2 The results show that the ST Gal(+) bacteria CHCC28380, CHCC32045 and CHCC32046 described in this paper have a significantly higher stable pH at the end of this type of fermentation than the corresponding wild-type ST Gal(-) bacteria CHCC27806 (about 0.3-0.6 points higher).

[0238] This article Figure 3 The results show that the ST Gal(+) bacteria CHCC29249 and CHCC29529 described in this paper have significantly higher stable pH values ​​(approximately 0.2–0.5 points higher) at the end of this type of fermentation compared to the corresponding wild-type ST Gal(-) bacteria CHCC4426. Furthermore, at the end of fermentation, the pH of CHCC4426 continuously decreased, while the pH of the mutants CHCC29249 and CHCC29529 appeared to be more stable.

[0239] The pH results of other test strains related to this study are shown in the table below.

[0240] Table 1. pH differences between ST Gal(-) wild-type strains and galactose-positive mutants at the end of fermentation. The corresponding gal+ mutants of the wild-type strains are shown below their respective wild-type strains.

[0241]

[0242]

[0243] As shown in the table above, at the end of this type of fermentation, the instances of different ST Gal(+) bacteria had significantly higher stable pH (approximately 0.2–0.6 points higher) than the corresponding wild-type ST Gal(-) bacteria.

[0244] As shown in the table above, some tested ST Gal(+) strains did not function as required in this paper (i.e., they did not produce the relatively high stable pH discussed in this paper at the end of fermentation). For example, this included the ST Gal(+) mutant 4459-GAL6. This strain is a galactose fermentation mutant of CHCC4459. However, the final pH after 24 hours of incubation was similar to that of the wild-type strain after 24 hours.

[0245] The mutant with the highest relative pH at the end of fermentation is typically the so-called galactose superfermentation ST Gal(++) mutant—that is, (as discussed above) they are able to reduce the amount of galactose secreted in milk by at least 20% (e.g., at least 25%, more preferably at least 30%, even more preferably at least 40%) compared to the reference ST CHCC4323 bacteria, according to Example 1 above.

[0246] Data for all ST Gal(+) strains that did not function were not shown—but approximately 20% of the ST Gal(+) strains tested actually functioned as required in this paper (i.e., producing the relatively high stable pH discussed in this paper at the end of fermentation).

[0247] in conclusion:

[0248] The results showed that at the end of this type of fermentation, the instances of different ST Gal(+) bacteria had significantly higher stable pH (about 0.2-0.6 points higher) than the corresponding wild-type ST Gal(-) bacteria.

[0249] For many different ST Gal(+) bacteria tested—according to step (b) of the first aspect of this article—the fermentation of milk produced a relatively high stable pH at the end of fermentation.

[0250] The data also indicate that approximately 20% of the tested ST Gal(+) strains actually functioned as required in this paper (i.e., producing the relatively high stable pH value discussed in this paper at the end of fermentation).

[0251] The results also demonstrate that, based on the technical teachings and common knowledge presented herein, identifying novel ST Gal(+) strains with the positive effect of “stable relative high pH at the end of the invention” as described herein is a routine screening / selection task for technicians.

[0252] None of the ST Gal(-) strains tested were positive – that is, none of these strains produced the pH value required at the end of fermentation according to step (b) of the first aspect of this document.

[0253] Example 3: Genomic analysis of the tested ST Gal(+) strain

[0254] As discussed above, not all ST Gal(+) strains tested actually functioned as required in this article (i.e., producing the relatively high stable pH discussed in this article at the end of fermentation).

[0255] Therefore, genomic analysis was performed to identify common structural elements in the preferred positive ST Gal(+) mutant strains that functioned well.

[0256] result:

[0257] The table below shows mutations in the promoter-10 region of the galactokinase gene (galK) of some strains discussed in Example 2 above—that is, some positive and negative (invalid) strains of Example 2 above.

[0258] Table 2. DNA sequences of the promoter-10 region of the galactokinase (galK) gene in galactose-positive mutants compared to wild-type strains. The corresponding gal+ mutants of the wild-type strains are shown below their respective wild-type strains.

[0259]

[0260]

[0261] in conclusion:

[0262] The results demonstrate that the ST Gal(+) strains that function well in this study are preferably ST Gal(+) bacteria with mutations in the -10 region of the galactokinase gene (galK) promoter (SEQ ID NO:8), wherein the mutation results in one or both of the C and G in the wild-type -10 region (TACGAT, SEQ ID NO:1) being replaced by nucleotides independently selected from the group consisting of A and T.

[0263] More preferably, the mutation results in the -10 region having the nucleotide sequence TATGAT (SEQ ID NO:2 – see, for example, very positive results of CHCC28380 and CHCC32045) or TACTAT (SEQ ID NO:4 – see, for example, very positive results of CHCC29248).

[0264] The novel preserved ST Gal(+) strains (CHCC28380=DSM 33158; CHCC32045=DSM33159) exhibited very good stability at a relatively high pH at the end of such fermentations—these preserved strains contain the mutant “TATGAT” (SEQ ID NO:2)—and are therefore the preferred strains in this study.

[0265] References

[0266] 1.EP2957180B1(Chr.Hansen A / S,Denmark)

[0267] 2. of Chr.Hansen A / S

[0268] 3. Anbukkaasi et al. (J Food Sci Technol (September 2014) 51(9):2183–2189)

[0269] 4.Anbukkaasi et al. ("Production of low browning Mozzarella cheese: Screening and characterization of wild galactose fermenting Streptococcusthermophilus strains", International Journal of advanced research, 2013, vol.1, no.5, pp.83-96)

[0270] 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(Suppl1))

[0271] 6.WO2011 / 026863A1(Chr.Hansen)

[0272] 7.WO2011 / 092300A1(Chr.Hansen)

[0273] 8.WO2019 / 042881A1(Chr.Hansen)

[0274]

[0275] sequence list <110> K.H.S. Ltd. <120> ST Gal(+) bacteria are used in the production of fermented dairy products with a relatively high stable pH. <130> P6482EP00 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> DNA <213> Streptococcus thermophilus <400> 1 tacgat 6 <210> 2 <211> 6 <212> DNA <213> Streptococcus thermophilus <400> 2 tatgat 6 <210> 3 <211> 6 <212> DNA <213> Streptococcus thermophilus <400> 3 tattat 6 <210> 4 <211> 6 <212> DNA <213> Streptococcus thermophilus <400> 4 tactat 6 <210> 5 <211> 70 <212> DNA <213> Streptococcus thermophilus <400> 5 aaaatattga ttttccatgt gaaaggggtt atgatttcag tataaacaaa aagaataagt 60 gagatacatc 70 <210> 6 <211> 70 <212> DNA <213> Streptococcus thermophilus <400> 6 aaaatattga ttttccatgt gaaaggggtt acgatttcag tataaacaaa aagaataagt 60 gagatacatc 70 <210> 7 <211> 70 <212> DNA <213> Streptococcus thermophilus <400> 7 aaaatattga ttttccatgt gaaaggggtt acgatttcag tataaacaaa aagaataagt 60 gagatacatc 70 <210> 8 <211> 70 <212> DNA <213> Streptococcus thermophilus <400> 8 aaaatattga ttttccatgt gaaaggggtt acgatttcag tataaacaaa aagaataagt 60 gagatacatc 70

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): inoculating at least 100 L of milk with (I): (I): Comprising 10 4 to 10 14 CFU / g of Streptococcus thermophilus (ST) bacterial cells in an ST bacterial composition, said ST bacterial cells being STGal(+) bacteria, characterized in that, compared to the reference STCHCC4323 bacteria deposited under accession number DSM 32826, said ST Gal(+) bacteria are capable of reducing the amount of galactose secreted in milk by at least 10%; wherein the ST Gal(+) bacterium is a bacterium having a mutation in the -10 region of the galactokinase gene (galK) promoter as shown in SEQ ID NO:8, wherein the mutation results in the substitution of one or both of the C and G in the wild-type -10 region sequence TACGAT as shown in SEQ ID NO:1 with nucleotides independently selected from the group consisting of A and T; A comparative test is carried out in the following manner: inoculating the ST bacterium into skim milk at 1% of an overnight culture, incubating at 37 °C for 18 hours, and collecting a sample at the end of the fermentation to measure the galactose content in the fermented milk, thereby measuring the reduction in secreted galactose compared to the reference CHCC4323; and (b): fermenting the milk with the bacterium of (a), wherein during the fermentation, the pH is measured in such a way as to ensure determination of the pH value of this step (b), and wherein the fermentation is carried out under the condition that the fermentation ends with a relatively high and stable pH value, the relatively high and stable pH value being defined as a pH of 4.3 - 4.9 at the end of the fermentation, and wherein the change in pH during the last 2 hours of the fermentation does not exceed pH 0.1, and wherein the pH reaches 4.3 - 4.9 before 24 hours of the fermentation; and (c): using the fermented milk with a pH of 4.3 - 4.9 of (b) for other appropriate steps to finally obtain the produced fermented dairy product.

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. The method according to claim 3, wherein in step (a), the milk is also inoculated with 10 4 to 10 14 CFU / g of Lactobacillus bacterial cells.

5. The method according to claim 4, wherein the Lactobacillus bacterium cells are Lactobacillus delbrueckii subsp. bulgaricus cells.

6. The method according to claim 1, wherein the ST Gal(+) bacterium is an ST Gal(+) bacterium characterized in that compared with the reference ST CHCC4323 bacterium, the ST Gal(+) bacterium can reduce the amount of galactose secreted in the milk by at least 25%.

7. The method according to claim 1, wherein the ST Gal(+) bacterium cells are cells of at least one selected from the group consisting of: (a): Streptococcus thermophilus cells CHCC28380 deposited under the accession number DSM 33158; and (b): Streptococcus thermophilus cells CHCC32045 deposited under the accession number DSM 33159.

8. The method according to claim 1, wherein the mutation results in the -10 region having the nucleotide sequence TATGAT as shown in SEQ ID NO:

2.

9. The method according to claim 1, wherein: - the pH value of step (b) of claim 1 is continuously measured until the end of the fermentation; - the fermentation temperature in step (b) of claim 1 is 25°C to 48°C; - the fermentation time in step (b) of claim 1 is 2 - 30 hours; - the pH at the end of the fermentation in step (b) of claim 1 is a pH of 4.4 - 4.8; - in step (b) of claim 1, the change in the pH during the last 2 hours of the fermentation does not exceed 0.05; and - in step (b) of claim 1, the pH reaches 4.3 - 4.9 before 10 hours of fermentation.

10. The method according to claim 1, wherein in step (b), the pH at the end of the fermentation is a pH that is 0.2 - 0.8 points higher than the corresponding comparative pH at the end of the fermentation obtained by performing the fermentation under comparable identical fermentation conditions using the reference ST CHCC4323 bacteria.

11. The method according to claim 1, wherein the method comprises the following additional step: (d): storing the fermented milk product produced in step (c) for a storage period of at least 1 week, and wherein the pH of the product at the end of the storage period is a pH of 4.3 - 4.

9.

12. The method according to claim 11, wherein the storage temperature is 2°C to 10°C, and wherein 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): selecting and isolating a new selected pool of ST bacteria from a pool of individual ST bacteria, the new selected pool of ST bacteria being a pool of ST Gal(+) bacteria, characterized in that the ST Gal(+) bacteria are capable of reducing galactose as required in step (a)(I) of claim 1; (ii): selecting and isolating - from the selected pool of ST Gal(+) bacteria of step (i) - new isolated STGal(+) bacterial cells, the bacterial cells being capable of producing a relatively high and stable pH value at the end of the fermentation as required in step (b) of claim 1.

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

15. Streptococcus thermophilus cells having the functional characteristics of CHCC28380 deposited under accession number DSM 33158, or Streptococcus thermophilus cells having the functional characteristics of CHCC32045 deposited under accession number DSM 33159, wherein the functional characteristics mean that the ST bacteria are capable of reducing the amount of galactose secreted in milk by at least 10% compared to the reference ST CHCC4323 bacteria deposited under accession number DSM 32826.

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