P7154PC00 1 LOW POST-ACIDIFYING LACTIC ACID BACTERIA STRAINS FIELD OF THE INVENTION The present invention relates to lactic acid bacteria strains which reduces post- acidification in fermented milk products. The invention further provides compositions comprising such strains, methods of producing a fermented milk product using such strains or compositions, and the fermented milk products thus obtained, including food products. BACKGROUND OF THE INVENTION Lactic acid bacteria (LAB) have been used for decades for preparing fermented food products. During fermentation, lactic acid and other organic compounds are produced by the lactic acid bacteria, thereby reducing the pH of the food product. However, acidification by the lactic acid bacteria often continues after finalizing the production of the food product, i.e. after the target pH has been reached. This acidification during self life is called post-acidification and is due to the metabolic activity of the lactic acid bacteria in the presence of metabolizable carbohydrates which is mainly lactose in milk. Post-acidification highly impacts the shelf life of a fermented product as it leads to undesired rheological, textural as well as organoleptic changes in the product. Attempts to ameliorate and reduce post-acidification includes application of a heating or cooling step for the purpose of inhibiting or inactivating lactic acid bacteria used in the manufacture of a fermented product. However, the heat treatment has a negative effect on the quality of the fermented product as changes in flavour and texture occur. Furthermore, health benefits obtained by eating live (probiotic) bacteria are greatly diminished or lost. A rapid cooling step is necessary to stop the fermentation activity of inoculated lactic bacteria and to set the final acidity of the product. It involves a continuous agitation of the fermented mass in the tank during the transfer operation to the cooler, a pumping and a pipe transfer followed by a smoothing step using a static filter, a smoothing valve or a rotor stator machine. Cooling is usually performed by using a heat exchanger (e.g. plate, tube or scrapped surface heat exchangers) followed by a storage step in a tank before packing. This step leads to a high viscosity loss due to shear stress applied
P7154PC00 2 during tank agitation, smoothing, pumping, pipe transfer and cooling. By-passing of this energy-consuming step would not only benefit the quality of the final product but would also be an economical as well as an environmental advantage. Specific lactic acid bacteria with weakly or low-post-acidifying activity have been identified and/or developed. WO2007/147890 describes how a low-post-acidifying Lactobacillus delbrueckii subsp. bulgaricus was generated and WO2010/139765 reports weakly post-acidifying Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus strains. In WO2015/193459 the post-acidification was addressed by development of lactose-deficient Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains for use in lactose-comprising substrates. WO2020/182976 describes use of a Streptococcus thermophilus strain with a R354C mutation in the beta-galactosidase to obtain femented milk not undergoing post-acidification. WO2023/166140 describes low post-acidifying lactose-positive, sucrose-negative Streptococcus thermophilus strains carrying one or more mutations in one or more genes of the sucrose regulon and optionally one or more further mutations affecting the glucose porter encoded by glcU. Insertion of a “T” in the promoter region of glcU is described. Thus, the ability of the strains to reduced post-acidification in the production of fermented products is important, however other features such as e.g. high texturizing capacity and fast acidification are also required in the production. Therefore, there is still a need in the art for new methods and strains providing fermented products having both good texture and low post-acidification. SUMMARY OF THE INVENTION The present disclosure provides lactic acid bacteria with reduced post-acidification. In a first aspect the present disclosure provides a mutant gal(+) Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In a second aspect the present disclosure provides a composition, either as a mixture or as a kit-of-parts, comprising the strain of the present disclosure.
P7154PC00 3 In a third aspect the present disclosure provides a method for producing a food product comprising the steps: (a) adding the strain of the present disclosure or the composition of the present disclosure to a milk base; and (b) fermenting said milk base until a target pH of 4.6 or below pH 4.6 is reached; wherein the food product has reduced post-acidification and/or increased texture in comparison with a food product made with the mother strain. In a fourth aspect the present disclosure provides a fermented food product comprising the strain of the present disclosure; the composition of the present disclosure; or is obtained by the method of the present disclosure. In a fifth aspect the present disclosure provides Use of the strain of the present disclosure or the composition of the present disclosure for reducing post- acidification and/or increasing texture of a fermented food product. BRIEF DESCRIPTION OF FIGURES AND SEQUENCES Figure Figure 1 shows acidification by DSM 22934 and DSM 33572. Figure 2 shows acidification by DSM 33572 at different temperatures. Sequence SEQ ID No:1 shows the glcU gene nucleotide sequence of DSM 22934. SEQ ID No:2 shows the glucose permease amino acid sequence of DSM 22934. SEQ ID No:3 shows the glcU gene nucleotide sequence of DSM 33572. SEQ ID No:4 shows the glucose permease amino acid sequence of DSM 33572. DETAILED DESCRIPTION OF THE INVENTION Strains The continued acidification of fermented food products after production is still a challenge despite many attempts to solve it. The present disclosure relates to lactic acid bacteria which acidifies down to a target pH at which no or no significant further acidification occurs. Thus pH is stabilized or substantially stabilized. Use of such strains and strains with similar property would be an important tool for control of post-acidification.
P7154PC00 4 Lactic acid bacteria grow in milk where lactose is the major carbon source and utilizes β-galactosidase to cleave the disaccharide lactose into the monosaccharides glucose and galactose. In Streptococcus thermophilus glucose is utilized through the glycolysis pathway where the final step is generation of lactate catalyzed by lactate dehydrogenase (EC 1.1.1.27). The production of lactate lowers pH of the substrate resulting in coagulation of milk for production of fermented products such as e.g. yoghurt and cheese. Most Streptococcus thermophilus strains are able to ferment a limited numbers of sugars: lactose, glucose, and sucrose (van den Bogaard, P.T.C. et al 2004 System. Appl. Microbiol. 27 p10-17). The lactic acid bacteria strain of the species Streptococcus thermophilus DSM 33572 of the present disclosure was derived from the mother strain DSM 22934 (lac+, glu+, gal+, suc+). It was observed that the acidification profile of this new strain was stabilized once target pH was reached. Thus continued fermentation time did not lead to further lowering of pH and was therefore selected as a low post- acidifying strain. Analysis of which carbohydrate that were fermentable as sole source revealed that DSM 33572 had gained an improved ability to acidify using glucose as compared to DSM 22934. Two different glucose import mechanism in Streptococcus thermophilus have been described. The first and main transporter being a glucose/mannose PTS IIABCD system and the second a putative glucose permease, a non-PTS permease which has been described for Lactococcus lactis. In line with the increased fermentation on glucose, a genomic analysis of the strains revealed a change in the glcU gene of DSM 33572 in the nucleotide sequence upstream of the sequence encoding the glucose permease. Furthermore very high expression of glcU was found during exponential phase of DSM 33572. The term “glcU gene” used herein means both the sequence encoding the protein (CDS region) as well at the sequence encoding regulatory elements for expressing the gene, such as e.g. a promotor etc. The upstream region in the present disclosure is defined as positions 1-206 of SEQ ID No:1 or positions 1-207 of SEQ ID No:3. The coding sequence (CDS) for the glucose permease is defined as positions 207-1093 of SEQ ID No:1 or positions 208-1094 of SEQ ID No:3. The present disclosure provides a mutant Lactic Acid Bacteria (LAB) strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence
P7154PC00 5 resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In a first aspect the present disclosure provides a mutant gal+ Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In one embodiment the present disclosure provides a mutant gal+ suc+ Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In one embodiment the present disclosure provides a mutant gal+ suc+ glu+ Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In one embodiment the present disclosure provides a mutant gal+ suc+ glu+ lac+ Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change. In one embodiment the mutant Streptococcus thermophilus strains described above may further be fructose-negative, fru(-). In one embodiment the present disclosure provides a mutant gal+ suc+ glu+ lac+ fru- Streptococcus thermophilus strain derived from a mother strain having a glcU gene nucleotide sequence encoding a glucose permease protein amino acid sequence, wherein the mutant strain has a change in the nucleotide sequence and optionally a change in the amino acid sequence resulting in increased activity of the glucose permease as compared to the activity of the glucose permease of the mother strain not having said change.
P7154PC00 6 In one embodiment the lactic acid bacteria strain may comprise a glucose permease nucleotide sequence which is at least 80%; at least 85%; at least 90%; at least 91%; at least 92%; at least 93%; at least 94%, at least 95%; at least 96%, at least 97%; at least 98%; at least 99%; or 100% identical to SEQ ID No:1. In one embodiment the lactic acid bacteria strain may comprise a glucose permease nucleotide sequence which is at least 80%; at least 85%; at least 90%; at least 91%; at least 92%; at least 93%; at least 94%, at least 95%; at least 96%, at least 97%; at least 98%; at least 99%; or 100% identical to nucleotides 1-206 of SEQ ID No:1. In one embodiment the lactic acid bacteria strain may comprise a glucose permease nucleotide sequence which is at least 80%; at least 85%; at least 90%; at least 91%; at least 92%; at least 93%; at least 94%, at least 95%; at least 96%, at least 97%; at least 98%; at least 99%; or 100% identical to nucleotides 207-1094 of SEQ ID No:1. In one embodiment the lactic acid bacteria strain may comprise a glucose permease amino acid sequence which is at least 80%; at least 85%; at least 90%; at least 91%; at least 92%; at least 93%; at least 94%, at least 95%; at least 96%, at least 97%; at least 98%; at least 99%; or 100% identical to SEQ ID No:2. In one embodiment the present disclosure relates to the strain, wherein the change is a mutation, a deletion or an insertion. In one embodiment the present disclosure relates to the strain, wherein the change is located in a region regulating expression of the coding sequence (CDS) for the glucose permease. In one embodiment the present disclosure relates to the strain, wherein the change is located in a region upstream of the coding sequence (CDS) for the glucose permease. In one embodiment the present disclosure relates to the strain, wherein the change is located in one or more of positions corresponding to positions 1-206 of SEQ ID No:1. In one embodiment the present disclosure relates to the strain, wherein the change is located in a region corresponding to positions -80; -79; -78; -77; -76; -75 in SEQ ID No:1. In one embodiment the present disclosure relates to the strain, wherein the change is insertion of a “T”. In one embodiment the present disclosure relates to the strain, wherein the change is insertion of a “T” before the region at a position corresponding to position -81 in SEQ ID No:3. In general STs have the ability to continue to acidify, i.e. post-acidify, even below the target pH where the resulting food product provide the desired taste and texture. The mutant strain of the present disclosure seems to have wholly or partly lost this ability, and has thus a reduced post-acidification as compared to its mother strain. Post-acidification is low in the mutant strain and in some instances it may be absent
P7154PC00 7 or insignificant. In one embodiment the present disclosure relates to the strain, wherein the mutant strain has a reduced post-acidification as compared to its mother strain. The change in pH in a fermented milk product made by inoculating B-milk with 107 cfu/g of a strain is less than 0.30; less than 0.25; less than 0.20; less than 0.15 ; less than 0.10; or even less than 0.05 pH units after storage from end of fermentation at 5°C for 5 days. Storage of the fermented food product at 5°C is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; or 1, 2, 3, 4, 5, or 6 month. In one embodiment the present disclosure relates to the strain, wherein (a) pH of a fermented milk product comprising the strain has changed less than 0.3 units when pH determined after end of fermentation is compared with pH determined after storage at 5°C for 5 days of the fermented milk product made by inoculating B-milk with 107 cfu/g of the strain; or (b) pH of two or more fermented products comprising the strain stabilizes at the same target pH measured at 12 hours after onset of fermentation wherein the two or more fermented milk products are made by inoculating B-milk with 107 cfu/g of the strain each at a different temperature selected from 37°C, 40°C, and 43°C. In one embodiment the present disclosure relates to the strain, wherein the texture of the fermented milk product comprising the strain has increased when texture determined after fermentation is compared with the texture determined after storage at 5°C for 28 days of the fermented milk product made by inoculating B- milk with 107 CFU/g of the strain. Texture may be measured in several ways as disclosed in the examples. Methods such as Positive compression; Gel firmness; Cohesiveness; Viscosity index; and Complex modulus have been used to show increased texture of DSM 33572. Gel firmness is a sensory descriptor of the fermented milk texture. It correlates with instrumental measurements such as positive compression area measured by texture analyzer and to complex modulus (G*) measured by rheometer. One or more of the methods may be used to measure texture of a food product made by fermentation with one or more strains. In one embodiment the present disclosure relates to the strain, wherein texture is measured by one or more of: Positive compression; Gel firmness; Cohesiveness; Viscosity index; and Complex modulus.
P7154PC00 8 In one embodiment the present disclosure relates to the strain, wherein the target pH of the fermented milk product is in the range from 4.0 to 5.0; from 4.1 to 5.0; from 4.1 to 4.9; from 4.2 to 4.9; from 4.2 to 4.8; from 4.3 to 4.8; from 4.3 to 4.7; from 4.4 to 4.7; from 4.4 to 4.6; or from 4.5 to 4.6. In one embodiment the present disclosure relates to the strain, wherein the strain at the end of acidification of a milk base reaches a target pH at which said strain is stabilized and after which no further change in pH takes place. Low post-acidification may be due to changes in the beta-galactosidase such as e.g. with a R234C mutation as previously described. The strains of the present disclosure may have further changes in the beta-galactosidase. In one embodiment the present disclosure relates to the strain, wherein the strain has a change in the beta-galactosidase. In one embodiment the present disclosure relates to the strain, wherein the strain has a change in the beta-galactosidase corresponding to the R234C mutation as described in WO2020/182976. It has been shown in the present disclosure that a change in beta-galactosidase is not required for obtaining low post-acidification. Thus, in one embodiment the present disclosure relates to the strain, wherein said strain has no change in the beta-galactosidase. In one embodiment the present disclosure relates to the strain, wherein the strain has no change in the beta-galactosidase including no change corresponding to the R234C mutation as described in WO2020/182976. In one embodiment the present disclosure relates to the strain, wherein the species of the strain is selected from Streptococcus ssp such as e.g. S. thermophilus, and Lactobacillus ssp such as L. delbrueckii subsp. bulgaricus. In one embodiment the present disclosure relates to the strain, wherein the strain is of the species Streptococcus thermophilus and the mother strain is DSM 22934. In one embodiment the present disclosure relates to the strain, wherein the strain is DSM 33572, and mutants or variants derived thereof. In one embodiment the present disclosure relates to the strain, wherein the strain is Lactobacillus delbrueckii subsp. bulgaricus and the mother strain is DSM 19252. In one embodiment the present disclosure relates to a strain, wherein the strain is derived from the mother strain DSM 19252 and/or selected from DSM 34855, and mutants or variants derived thereof. In one embodiment the present disclosure relates to the strain, wherein the strain is selected from DSM 34855, and mutants or variants derived thereof.
P7154PC00 9 The lactic acid bacteria strain of the species Lactobacillus delbrueckii subsp. bulgaricus DSM 34855 of the present disclosure is derived from the mother strain DSM 19252. Like for the Streptococcus thermophilus strain described supra it was observed that the acidification profile of this new LB strain was stabilized once target pH was reached. Thus continued fermentation time did not lead to further lowering of pH and was therefore selected as a low post-acidifying strain. In the context of present disclosure, the term “lactic acid bacteria” or “LAB” is used to refer to food-grade bacteria producing lactic acid as the major metabolic end- product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram-positive, low-GC, acid tolerant, non-sporulating, non-respiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of dairy product. As used herein, the term “lactic acid bacteria” encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium breve and Leuconostoc spp. The term “mutant” should be understood as a strain derived from a strain of the invention, for example by means of e.g. genetic engineering, radiation and/or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties in particular in relation to the effects on inhibiting post-acidification as the deposited strain. Respective mutants represent embodiments of the present invention. The term “mutant” in particular refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl- N’-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even
P7154PC00 10 less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain. Compositions Lactic acid bacteria may be used in the form as a composition comprising one or more strains and optionally further ingredients that may promote fermentation and/or the formulation of the composition. The composition may be provided as a a mixture or as a kit-of-part. In a second aspect the present disclosure provides a composition, either as a mixture or as a kit-of-parts, comprising the strain of the present disclosure. LAB are commonly added to a milk base in the form of a starter culture. The term “starter” or “starter culture” as used herein refers to a culture of one or more food- grade microorganisms, in particular to lactic acid bacteria, which are responsible for the acidification of the milk base. Starter cultures may be fresh, but are most frequently frozen or freeze-dried. These products are also known as “Direct Vat Set” (DVS) cultures and are produced for direct inoculation of a fermentation vessel or vat for the production of a dairy product, such as a fermented milk products such as e.g Yogurt and cheese. In one embodiment the present disclosure relates to the composition, wherein the composition is a starter culture. The composition of the present disclosure may further comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition may be in frozen or freeze-dried form. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and/or nutrients, more preferably cryoprotectants, lyoprotectants and/or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectant are known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic. compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and/or flavorants. In
P7154PC00 11 one embodiment the present disclosure relates to the composition, further comprising cryoprotectants, lyoprotectants, antioxidants and/or nutrients. In one embodiment the present disclosure provides a composition in the form of a solid frozen or freeze-dried starter culture comprising lactic acid bacteria in a concentration of at least 109 colony forming units (cfu) per g of frozen material or in a concentration of at least 1010 cgu/g of frozen material or in a concentration of at least 1011 cfu/g of frozen material. In one embodiment the present disclosure relates to the composition, wherein the composition is in a frozen, freeze-dried, or liquid form. Method for producing a fermented food product In a third aspect the present disclosure provides a method for producing a food product comprising the steps: (a) adding the strain of the present disclosure or the composition of the present disclosure to a milk base; and (b) fermenting said milk base until a target pH of 4.6 or below pH 4.6 is reached; wherein the food product has reduced post-acidification and/or increased texture in comparison with a food product made with the mother strain. The term “milk” herein is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals or from plants. In accordance with the present disclosure the milk may have been processed and the term “milk” includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose- reduced milk, or concentrated milk. Fat-free milk is non-fat or skim, milk product. Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat. Full fat milk often contains 2% fat or more. The term “milk” is intended to encompass milks of different mammalian and vegetable origin. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, lama, mare and deer. Vegetable sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. In particular milk of vegetable origin comprising glucose either as a monosaccharide or as a component in a disaccharide such as e.g. sucrose is preferred. Suitable sugars may also be added. In the methods and products of the present disclosure, milk derived from cows is most preferably used as a starting material for the fermentation. The term “milk” also includes fat-reduced and/or lactose-reduced milk products. Respective products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and
P7154PC00 12 galactose, or by nanofiltration, electrodialysis, ion exchange chromatograph and centrifugation. The term “milk material” or “milk base” is broadly used herein to refer to a starting material based on milk or milk components which can be used as a medium for growth and fermentation of LAB. The milk material or base comprises components derived from milk and any other component that can be used for the purpose of growing or fermenting LAB. Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art. "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices. "Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow. In one embodiment the present disclosure relates to the method, wherein the milk base is of mammalian and/or vegetable origin. In one embodiment the present disclosure relates to the method wherein the milk base is of mammalian origin derived from any of cow, sheep, goat, buffalo, camel, lama, mare, deer, or any mixture thereof. In one embodiment the present disclosure relates to the method wherein the milk base is of vegetable origin derived from any of soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed, sunflower seed, or any mixture thereof. Fermented food product In a fourth aspect the present disclosure provides a fermented food product comprising the strain of the present disclosure; the composition of the present disclosure; or is obtained by the method of the present disclosure. Fermentation is carried out to produce food products. The terms "fermented food product" or "food product” refer to products obtainable by the fermentation methods of the present disclosure and include cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais
P7154PC00 13 and cream cheese. The term food further encompasses other fermented food products, including fermented meat, such as fermented sausages, and fermented fish products. The term "cheese" is understood to encompass any cheese, including hard, semi- hard and soft cheeses, such as cheeses of the following types: Cottage, Feta, Cheddar, Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheeses, brine cheeses, Camembert and Brie. The person skilled in the art knows how to convert the coagulum into cheese, methods can be found in the literature, see e.g. Kosikowski, F. V., and V. V. Mistry, "Cheese and Fermented Milk Foods", 1997, 3rd Ed. F. V. Kosikowski, L. L. C. Westport, CT. As used herein, a cheese which has a NaCl concentration below 1.7% (w/w) is referred to as a "low- salt cheese". The term "yoghurt" as used herein refers to products comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived therefrom. The lactic acid strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, are included to give the finished product various properties, such as the property of promoting the equilibrium of the flora. As used herein, the term "yoghurt" encompasses set yoghurt, stirred yoghurt, drinking yoghurt, Petit Suisse, heat treated yoghurt, strained or Greek style yoghurt characterized by a high protein level and yoghurt-like products. In particular, term "yoghurt" encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million cfu (colony-forming unit) per g final product. Yoghurts may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavoring(s), fruit, cereals, or nutritional substances, especially vitamins, minerals and fibers, as well as stabilizers and thickeners. Optionally the yoghurt meets the specifications for fermented milks and yoghurts of the AFNOR NF 04-600 standard and/or the codex StanA-IIa-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not
P7154PC00 14 have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m/m) of the finished product. In one embodiment the present disclosure relates to the food product of the present disclosure, selected from Yogurt of set type, stirred type or drinkable type; Ymer; Buttermilk; Kefir; Dahi; sour cream; crème fraiche; or Cheese such as fresh cheese, cottage cheese, soft cheese, and white cheese. Use In a fifth aspect the present disclosure provides Use of the strain of the present disclosure or the composition of the present disclosure for reducing post- acidification and/or increasing texture of a fermented food product. The background for using the strains of the present disclosure for reducing pos- acidification and/or increasing texture of a fermented food poduct has been described in the other parts of the present disclosure. The description in the different parts of the present disclosure are not limited to the part in which it is described but can be used in combinations with features described in other parts. The headings are for guidance only. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be constructed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. DEPOSIT & EXPERT SOLUTION The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted. Table 1: Deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany. Strain Accession No. Deposit date Streptococcus thermophilus DSM 33572 2020.07.08
P7154PC00 15 Lactobacillus delbrueckii subsp. bulgaricus DSM 34855 2023.11.28 EXAMPLES Material & Methods The medium used for Streptococcus thermophilus was M17 medium with 20g/L lactose added sterile as carbon source. M17 agar medium composition per litre H20: agar, 12.75 g ascorbic acid, 0.5 g casein peptone (tryptic), 2.5 g disodium β-glycerophosphate penta hydrate, 19 g magnesium sulfate hydrate, 0.25 g meat extract, 5 g meat peptone (peptic), 2.5 g soyapeptone (papainic), 5 g yeast extract, 2.5 g final pH 7.1 ±0.2 (25°C) M17 broth composition per litre H20: ascorbic acid, 0.5 g magnesium sulfate, 0.25 g meat extract, 5 g meat peptone (peptic), 2.5 g sodium glycerophosphate, 19 g soya peptone (papainic), 5 g tryptone, 2.5 g yeast extract, 2.5 g final pH 7.0±0.2 (25°C) The medium used for Lactobacillus delbrueckii subsp. bulgaricus was MRS medium. MRS agar medium composition per litre H20: Bacto Proteose Peptone no. 3, 10 g/l Bacto Beef Extract 10 g/l Bacto Yeast Extract 5 g/l
P7154PC00 16 Dextrose 20 g/l Sorbitan Monooleate Complex 1 g/l Ammonium Citrate 2 g/l Sodium acetate 5 g/l Magnesium sulphate 0.1 g/l Manganese sulphate 0.05 g/l Potassium Phosphate Dibasis 2 g/l Bacto Agar 15 g/l final pH 6.5 ± 0.2 (25°C.) The MRS broth medium composition per litre H20: Bacto Proteose Peptone no. 3 10 g/l Bacto Beef Extract 10 g/l Bacto Yeast Extract 5 g/l Dextrose 20 g/l Sorbitan Monooleate Complex 1 g/l Ammonium Citrate 2 g/l Sodium acetate 5 g/l Magnesium sulphate 0.1 g/l Manganese sulphate 0.05 g/l Potassium Phosphate Dibasis 2 g/l final pH 6.5 ± 0.2 (25°C.) B-Milk was made from skim milk powder to a level of dry matter of 9.5% (w/v) reconstituted in distilled water and pasteurized at 99°C for 30 min, followed by cooling to 30°C. The following strains were used in the examples: DSM 19252 described in WO2007/147890; DSM 22586 described in WO2011/000879; DSM 22934 described in WO2011/026863; DSM 28910 described in WO2015/193459; and DSM 34235 described in WO2023/222575. Example 1 - Isolation of a low-post-acidifying strain of Streptococcus thermophilus. DSM 33572 is a new 2-Nitrophenyl 1-thio-beta-D-galctopyranoside (TONPG) resistant mutant strain derived from a galactose-fermenting Streptococcus thermophilus mother strain DSM 22934. Stock solution of TONPG (2-Nitrophenyl 1-thio-β-D-galactopyranoside, Sigma no. N2509-100MG): 100mg was dissolved in 1mL 96% ethanol (≈333mM). Stock
P7154PC00 17 solution of IPTG (Isopropyl β-D-1-thiogalactopyranoside, SIGMA 16758) 100mM in H2O. The mother strain DSM 22934 was inoculated into M17 broth with 2% lactose and incubated overnight at 37°C under anaerobic conditions. The cells were centrifugated down at 4000g for 5 minutes, followed by washing in a 0.9% solution of sodium chloride. The cells were resuspended in 2mL M17 with 5mM TONPG and 2mM IPTG and incubated over weekend at 37°C under anaerobic conditions. The resulting TONPG culture was plated out in dilutions from 10-2 to 10-6 of the culture on M17 agar plates with 2% lactose. Plates were incubated anaerobically at 37°C. Twelve colonies were picked and streaked to single colonies 3 times before finally testing their acidification. The mother strain and the mutant were inoculated at in B-Milk and acidification at 40oC were followed for approximately 23 hours using a CINAC system or an ICINAC system (AMS alliance), see figure 1. DSM 33572 was found to have an arrest of acidification at approx. pH 4.55 whereas the mother strain acidifies to a lower pH at approx. 4.35. At the end of fermentation the difference in pH measured for the samples comprising the mother strain DSM 22934 and the samples comprising the mutant strain DSM 33572 was 0.2 units. After storage of the fermented samples for 5 days at 5°C the difference in pH was 0.2 units, i.e. unchanged. Because target pH for fermented food products such as e.g. yogurt is about pH 4.5 the DSM 33572 strain do not further acidify thereby providing virtually no post-acidification. The mother strain DSM 22934 on the other hand will continue to acidify down to about pH 4.35 which will be apparent as udesired post-acidification. Example 2 - Characterization of low-post-acidification mutant DSM 33572. The glcU gene sequences of DSM 22934 (SEQ ID No:1) and DSM 33572 (SEQ ID No:3) shown below were compared. The genomic analysis of DSM 33572 identified an insertion at position -81 of a “T” in the region upstream of the glcU gene sequence encoding the glucose permease protein. The extra T insertion is shown in bold and underlined and the start and stop codons are underlined in the nucleotide sequence below (SEQ ID No:3). The protein coding sequences (CDS) of SEQ ID No:1 is nucleotide 206..1093, and the CDS of SEQ ID No:3 is 207..1094. The “T” is inserted in a region already comprising six “T”s and it is thus difficult to identify at which position the additional “T” is inserted. Therefore the “T” may be
P7154PC00 18 inserted at a position corresponding to any one of the positions -81; -80; -79; -78; -77; -76; or -75 in SEQ ID No: 3. SEQ ID No:1 – DSM 22934 glcU gene (1..1093): ATCACGCCAT AACATGACAA AGACGGCTAC CCAAATGGAG AACCGCCTCA AATGATAAAT TTATTATTCA ACACCAGTTG ACGTAGAACC ACAGTTATGG TTCTTGTGTA TTTTTTTATC TCTTG_TTTT TTCCCGAAAT AAGAGTAATA TAGAGCTATG CTTAATTTTT TAGGTAAAAA ATTATTTTAA AGAGGTAAAT ATAAACATGC AAGGAGTTCT TTTCGCGCTT GTTCCAATTT TTGCTTGGGG TGCTGTCGGA TTGGTAGCTA ATATACTTGG TGGTGATCCT AATCAACAAA CACTGGGAAT GACTTTGGGC GCTTTTGTTG TTGCACTTAT TGTTTCCTTA TTCCGCATGC CAACGTTGAC ATGGCAAATT TTCTTAATTG GATTTATTGG TGGATTGTTT TGGGTAATTG GACAATTTGG TCAGTTTAAT TCAATGAAAT ACATGGGTGT TTCAGTAGCG AGTCCACTTT CAGCAGGAAG TCAATTAGTA TTTGGTGTAT TGCTTGGGGT TTTTGCTTTC CACGAATGGA CAAAACAAAT TCAATTTATT ATCGGATTTA TTGCGATGGC TCTTTTGGTA GTTGGGTTCT ATTTCTCAGC TAAACGTGAC CCAGAAAATG CAGTTGTTAA AGAAGGACGT AATTATACTA AAGGATTGAT TGCTTTAACT TACTCAACTT TGGGATATGT TCTCTATGTT ATTCTTTTTA ATAACTTAGC AGTACTTTGG TTCAATGTTC ATTTTGATAC ACTGACAATT ATCTTGCCAA TGTCAGTTGG AATGATCTTT GGAGCACTTG TGATGGGTCG TTTCAAAATT AAAATGGAAA AATATGTTTA TCGAAATATA ATTGATGGAG TAATGTTTGG TGTAGGTAAT ATCTTTATGC TTATGGCTGC AAGCGCTGCT GGTAACGCAA TTGCCTTTTC ATTCGCACAA TTAGGTGTTA TCATTTCAAC TATTGGAGGA ATTCTCTTCC TTGGTGAAAA GAAAACCAAA AAAGAATTGG TTTATGTTGG AATTGGAAGT GTTCTGTTCG TAACAGGTGC AATTTTACTT GCAATTGTAA AATCTAAAGG ATAA SEQ ID No:2 – DSM 22934 Glucose permease (1..295): MQGVLFALVP IFAWGAVGLV ANILGGDPNQ QTLGMTLGAF VVALIVSLFR MPTLTWQIFL IGFIGGLFWV IGQFGQFNSM KYMGVSVASP LSAGSQLVFG VLLGVFAFHE WTKQIQFIIG FIAMALLVVG FYFSAKRDPE NAVVKEGRNY TKGLIALTYS TLGYVLYVIL FNNLAVLWFN VHFDTLTIIL PMSVGMIFGA LVMGRFKIKM EKYVYRNIID GVMFGVGNIF MLMAASAAGN AIAFSFAQLG VIISTIGGIL FLGEKKTKKE LVYVGIGSVL FVTGAILLAI VKSKG SEQ ID No:3 – DSM 33572 glcU gene (1..1094): ATCACGCCAT AACATGACAA AGACGGCTAC CCAAATGGAG AACCGCCTCA AATGATAAAT TTATTATTCA ACACCAGTTG ACGTAGAACC ACAGTTATGG
P7154PC00 19 TTCTTGTGTA TTTTTTTATC TCTTGTTTTT TTCCCGAAAT AAGAGTAATA TAGAGCTATG CTTAATTTTT TAGGTAAAAA ATTATTTTAA AGAGGTAAAT ATAAACATGC AAGGAGTTCT TTTCGCGCTT GTTCCAATTT TTGCTTGGGG TGCTGTCGGA TTGGTAGCTA ATATACTTGG TGGTGATCCT AATCAACAAA CACTGGGAAT GACTTTGGGC GCTTTTGTTG TTGCACTTAT TGTTTCCTTA TTCCGCATGC CAACGTTGAC ATGGCAAATT TTCTTAATTG GATTTATTGG TGGATTGTTT TGGGTAATTG GACAATTTGG TCAGTTTAAT TCAATGAAAT ACATGGGTGT TTCAGTAGCG AGTCCACTTT CAGCAGGAAG TCAATTAGTA TTTGGTGTAT TGCTTGGGGT TTTTGCTTTC CACGAATGGA CAAAACAAAT TCAATTTATT ATCGGATTTA TTGCGATGGC TCTTTTGGTA GTTGGGTTCT ATTTCTCAGC TAAACGTGAC CCAGAAAATG CAGTTGTTAA AGAAGGACGT AATTATACTA AAGGATTGAT TGCTTTAACT TACTCAACTT TGGGATATGT TCTCTATGTT ATTCTTTTTA ATAACTTAGC AGTACTTTGG TTCAATGTTC ATTTTGATAC ACTGACAATT ATCTTGCCAA TGTCAGTTGG AATGATCTTT GGAGCACTTG TGATGGGTCG TTTCAAAATT AAAATGGAAA AATATGTTTA TCGAAATATA ATTGATGGAG TAATGTTTGG TGTAGGTAAT ATCTTTATGC TTATGGCTGC AAGCGCTGCT GGTAACGCAA TTGCCTTTTC ATTCGCACAA TTAGGTGTTA TCATTTCAAC TATTGGAGGA ATTCTCTTCC TTGGTGAAAA GAAAACCAAA AAAGAATTGG TTTATGTTGG AATTGGAAGT GTTCTGTTCG TAACAGGTGC AATTTTACTT GCAATTGTAA AATCTAAAGG ATAA SEQ ID No:4 – DSM 33572 Glucose permease (1..295): MQGVLFALVP IFAWGAVGLV ANILGGDPNQ QTLGMTLGAF VVALIVSLFR MPTLTWQIFL IGFIGGLFWV IGQFGQFNSM KYMGVSVASP LSAGSQLVFG VLLGVFAFHE WTKQIQFIIG FIAMALLVVG FYFSAKRDPE NAVVKEGRNY TKGLIALTYS TLGYVLYVIL FNNLAVLWFN VHFDTLTIIL PMSVGMIFGA LVMGRFKIKM EKYVYRNIID GVMFGVGNIF MLMAASAAGN AIAFSFAQLG VIISTIGGIL FLGEKKTKKE LVYVGIGSVL FVTGAILLAI VKSKG Example 3 - Characterization of milk fermented with low-post-acidification mutant DSM 33572. Fermentation of B-Milk with DSM 22934 and DSM 33572 were conducted as described in Example 1. The fermented milks including a B-milk control were analysed after approximately 23 hours. The concentration of selected metabolites are shown in the table below. It appears that fermentation of B-milk (lactose) by DSM 33572 as compared to DSM 22934 is not metabolizing as much lactose, is not
P7154PC00 20 excreting as much galactose and is not generating as much lactic acid. This result is in line with figure 1 described in Example 1. Table 2: Concentration of selected metabolites in fermented milk (g/L). Sample Lactose Glucose Galactose Lactic Acid DSM 22934 31.6 0 5.9 6.9 DSM 33572 33.6 0.4 5.0 6.1 B-Milk 45.3 0 0 0 The strains were tested for their ability to metabolize different sugars. Acidification in the presence of individual carbohydrates: glucose, galactose, fructose, lactose, and sucrose by the mother strain DSM 22934 and the mutant DSM 33572 were investigated. Comparison of the acidification curves of DSM 22934 and DSM 33572 showed similar results for all carbohydrates tested except for glucose. Acidification in the presence of glucose as sole source using DSM 22934 was very slow and did not reach pH 4.5 within 24 hours whereas acidification by DSM 33572 reached pH 4.5 within the first 6 hours. Because no changes were found in the genes of the glucose transporter system mainly used by ST strains, the glucose/mannose PTS IIABCD system, when comparing the mother and the mutant strain the change in glucose fermentation is attributed to the change found upstream of the glcU gene. Its appears that the the change in the nucleotide sequence upstream of the glcU gene has activated the glucose permease. In fact, a very high expression of glcU was found in DSM 33572 in particular during growth, i.e. exponential phase and to a lesser degree during stationary phase. Example 4 – Acidification by DSM 33572 at different temperatures Milk acidification curves for DSM 33572 were obtained in B-milk at 37ºC, 40ºC and 43ºC in 200mL scale using 1% inoculum (2mL in 200mL milk), which was incubated at 37°C in M17 +2% lactose overnight. Milk acidification curves of DSM 33572 at different temperatures are shown in figure 2. The acidification rate increases with increasing temperature. The acidification stabilizes at a target pH which are the same for all temperatures at pH 4.55. Example 5 – Reduced post-acidification of compositions comprising DSM 33572.
P7154PC00 21 Milk acidifications took place at 43°C in two formats: 2mL microtiter plates and 100mL cups. The overnight inoculum was prepared as in Example 4. Milk was fermented using a combination of ST and LB strains; the inoculum contained 0.9 ST : 0.1 LB. When two ST strains were combined with an LB strain, the inoculum contained 0.7 ST1 : 0.2 ST2 : 0.1 LB. Two milk bases were used: "Plain" milk base contained 3.4% protein and 1.4% fat; "Sweet" milk base contained 3.4% protein, 1.4% fat and 8.5% sucrose. The fermentations stopped when the pH reached 4.55. The samples were cooled down for approx. 30 min on ice and then kept at room temperature for 7 days to assess post-acidification. The result in the tables below show that fermentation by different compositions comprising the mutant strain DSM 33572 lead to reduced post-acidification both in plain as well as in sweet yogurt. Table 3a: pH in plain yogurt on day 1 and 7 after end of fermentation. ST1 ST2 LB 0.9 - 0.1 2mL Day1 2mL Day7 Δ Day1-Day7 100mL Day7 DSM 22586 4.37 4.14 0.23 4.14 1 e DSM 19252 4.37 4.15 0.22 4.13 T S n o N DSM 28910 4.36 4.17 0.19 4.16 DSM 34855 4.36 4.17 0.19 4.18 2 DSM 22586 4.49 4.39 0.10 4.44 75 3 e n DSM 19252 4.49 4.36 0.13 4.41 3 o MS N DSM 28910 4.49 4.37 0.12 4.44 D DSM 34855 4.50 4.39 0.11 4.43 Table 3b: pH in sweet yogurt on day 1 and 7 after end of fermentation. ST1 ST2 LB 2mL Day1 2mL Day7 Δ Day1-Day7 100mL Day7 0.9 - 0.1 +Sucrose +Sucrose +Sucrose +Sucrose DSM 22586 4.45 4.08 0.37 4.06 1 e DSM 19252 4.48 4.12 0.36 4.08 T S n o N DSM 28910 4.43 4.13 0.20 4.11 DSM 34855 4.47 4.13 0.34 4.12 M DSM 22586 4.51 4.19 S 75 n 0.32 4.22 D 3 o 3 N e DSM 19252 4.52 4.35 0.17 4.30
P7154PC00 22 DSM 28910 4.53 4.35 0.18 4.32 DSM 34855 4.53 4.37 0.16 4.33 Table 3c: pH in plain yogurt on day 1 and 7 after end of fermentation. ST1 ST2 LB 0.7 0.2 0.1 2mL Day1 2mL Day7 Δ Day1-Day7 100mL Day7 5 DSM 22586 4.42 4.17 0.25 4.18 3 1 2 DSM 19252 4.40 4.16 0.2 T 4 4 4.17 S 3 MS DSM 28910 4.39 4.19 0.20 4.19 D DSM 34855 4.41 4.17 0.24 4.19 2 DSM 22586 4.49 4.33 0.16 4.28 7 5 5 3 3 2 DSM 19252 4.46 4.29 0.17 4.29 3 4 3 MS MS DSM 28910 4.48 4.33 0.16 4.31 D D DSM 34855 4.48 4.33 0.15 4.33 Table 3d: pH in sweet yogurt on day 1 and 7 after end of fermentation. ST1 ST2 LB 0.7 0.2 0.1 2mL Day1 2mL Day7 Δ Day1-Day7 100mL Day7 5 DSM 22586 4.41 4.12 0.29 4.09 3 1 2 4 DSM 19252 4.43 4.14 0.29 4.11 T S 3 MS DSM 28910 4.43 4.13 0.30 4.12 D DSM 34855 4.46 4.14 0.32 4.13 2 5 DSM 22586 4.50 4.25 0.25 4.21 75 3 3 2 4 DSM 19252 4.51 4.30 0.21 4.27 3 3 MS M DSM 28910 4.52 4.31 0.21 4.28 D S D DSM 34855 4.51 4.31 0.20 4.29 Example 6 – Isolation of a low post-acidifying strain of Lactobacillus delbrueckii subsp. bulgaricus. The strain DSM 34855 is a hippuric acid resistant mutant derived from a Lactobacillus delbrueckii subsp. bulgaricus mother strain DSM 19252. The Lactobacillus delbrueckii subsp. bulgaricus mother strain DSM 19252 was inoculated into MRS broth and grown anaerobic overnight at 37°C. From this
P7154PC00 23 culture, a new culture was inoculated in MRS +5g/L hippuric acid and incubated with shaking 80 rpm. After overnight anaerobic growth at 37°C, the culture was spread in dilutions on MRS agar plates with +5g/L hippuric acid. The plates were then incubated 48 hours under anaerobic conditions, and subsequently 24 hours outside the anaerobic chamber. Colonies appeared after the first 48 hours of incubation. 19 colonies were picked and streaked to single colonies 3 times before analysing the acidification profile in B-milk. B-milk was inoculated 1% with an overnight culture made by inoculating 10mL M17 +2% lactose or MRS with the strain. Acidification was followed for approximately 106 hours of acidification at 40°C using a CINAC system or an ICINAC system (AMS alliance). Several isolates were found to have an arrest of acidification at a higher pH than the mother strain DSM 19252 (Data not shown). One such isolate deposited as DSM 34855 was showing virtually no post-acidification. Example 7 – Increased texture of fermented products by DSM 33572. Milk acidifications of the mutant strain DSM 33572 at three different temperatures were performed as described in Example 4. Several replicates were made for each of the temperatures tested, both in 100mL cups and in 200mL baby bottles. The fermentations were stopped after different time points, but all of the samples had the same target pH of 4.58, as the mutant strain DSM 33572 did not post-acidify. The samples were cooled down for approx. 30 min on ice and then kept at 4°C prior to texture measurements using rheometer or texture analyzer. The results in the tables below show that prolonged fermentation of milk by the mutant strain DSM 33572 leads to increased texture without a further decrease of pH. Thus, by increasing fermentation time with DSM 33572, it is possible to obtain a higher gel firmness, without generating more acidity in the fermented product produced. Gel firmness was expressed either as positive compression area [g.sec] measured with texture analyzer, which is typically used for set types of yoghurts, or as complex modulus [Pa] measured with rheometer, which is typically used for stirred types of yoghurts. Table 4a: Texture of milk fermented with DSM 33572 measured using texture analyzer TAXT-2 using standard method. Positive compression area [g.sec] measured with texture analyzer in fermented milk samples produced in 100mL cups was used to correlate with gel firmness measured by sensorial analysis.
P7154PC00 24 Temp. Fermen- Positive Gel Cohesive- Index of (Celcius) tation com- firmness ness Viscosity Time pression (g) (g) (g.sec) (hour) area (g.sec) 37oC 11h 163 24 -14 -32 14h 182 28 -16 -46 17h 204 31 -18 -58 40oC 10h 182 27 -16 -45 12h 202 31 -17 -55 17h 219 33 -19 -65 43oC 10h 195 30 -17 -54 12h 217 33 -19 -65 17h 244 37 -20 -83 Table 4b: Texture expressed of milk fermented with DSM 33572. Complex modulus [Pa] measured with rheometer in 200mL samples is used to correlate with gel firmness measured by sensorial analysis. Fermen- Temp Frequency [Hz] tation Time (Celcius) (hour) 0.50 0.87 1.52 2.64 4.59 8.00 37oC 11h 63.3 69.3 75.7 83.2 88.7 96.8 14h 82.5 89.9 97.9 107 117 127 17h 93.3 102 111 121 133 146 40oC 10h 68.1 74.7 81.8 89.4 98 108 12h 86.6 94.5 103 113 124 135 17h 103 112 122 134 146 161 43oC 10h 81.4 89.4 98.1 107 117 128 12h 95.8 105 115 126 139 154 17h 122 133 146 161 176 195 Example 8 – Sugar fermentable by DSM 33572. For assessment of growth in different single sugars as sole carbon source, strains were cultivated in Chemically Defined Medium (CDM) containing 2% (w/v) lactose (Markakiou et al 2023 Microbiol. Spectr. doi:10.1128/spectrum.00668-23.) using crimp-top serum bottles flushed with a gas mixture of 80% N2 and 20% CO2 to ensure anaerobic conditions. After overnight incubation at 40°C, the cultivated cells were used for the inoculation of 200 ml CDM containing 2% lactose, 2% sucrose, 1% glucose, 1% galactose or 1% fructose to an initial optical density at 600 nm
P7154PC00 25 (OD600) of 0.05. Cultures were incubated at 40°C under anaerobic conditions as described above, and growth was monitored through measurement of optical density at 600 nm (OD600). Table 5: Growth of DSM 22934 and DSM 33572 in different cartbon sources. DSM 22934 DSM 33572 Lactose (lac) ++ ++ Glucose (glu) + ++ Galactose (gal) + + Sucrose (suc) ++ ++ Fructose (fru) - -