US 201701 35.360A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0135360 A1 GARRIGUES et al. (43) Pub. Date: May 18, 2017
(54) METHOD OF PRODUCING A FERMENTED (86). PCT No.: PCT/EP2015/063767 MLK PRODUCT WITH IMPROVED S 371 (c)(1) CONTROL OF POST ACIDIFICATION (2) Date: Dec. 9, 2016 (71) Applicant: Chr. Hansen A/S. Hoersholm (DK) (30) Foreign Application Priority Data (72) Inventors: Christel GARRIGUES, Frederiksberg Jun. 19, 2014 (EP) ...... 141731968 C (DK); Christian GILLELADEN, Jul. 10, 2014 (EP) ...... 14176627.9 Copenhagen (DK); Mirjana Dec. 3, 2014 (EP) ...... 141961 250 CURIC-BAWDEN, Brookfield, WI Feb. 20, 2015 (EP) ...... 15156O25.7 (US); Thomas JANZEN, Broenshoe Publication Classification (DK); Mimi BIRKELUND, Oelsted (DK); Gaielle Lettier BUCHHORN, (51) Int. Cl. Virum (DK); Kim Ib SOERENSEN, A23C 9/23 (2006.01) Farum (DK); Nanna CHRISTENSEN, (52) U.S. Cl. Copenhagen S (DK); Claus SVANE, CPC ...... A23C 9/1238 (2013.01); A23Y 2220/29 Rungsted Kyst (DK); Soeren RIIS, (2013.01); A23Y 2220/152240/75 (2013.01); (2013.01) A23Y Roedovre (DK); Martin Bastian PEDERSEN, Cadorago Como (IT): (57) ABSTRACT Jean-Marie ODINOT, Remereville The present invention provides methods of producing a (FR); Luciana JIMENEZ, Paris (FR): fermented milk product comprising a step wherein milk is Pascal LANCIAUX, Gron (FR): fermented, wherein: (a) the fermentation is initiated by a Duncan HAMM, Singapore (SG); starter culture, which starter culture comprises lactic acid Choon Ming SIEW, Nusajaya (MY) bacteria capable of metabolizing one or several carbohy drates present in the milk, (b) the fermentation is terminated by a decrease of the concentration of the one or several (73) Assignee: Chr. Hansen A/S. Hoersholm (DK) carbohydrates during fermentation, and (c) the decrease is at least also caused by the metabolic activity of the lactic acid (21) Appl. No.: 15/317,682 bacteria. The invention further provides respective methods comprising a step, wherein at least part of the whey is (22) PCT Filed: Jun. 18, 2015 separated from the fermented milk product. Patent Application Publication May 18, 2017. Sheet 1 of 11 US 2017/O135360 A1
CHCC6008 - limiting lactose 7
6 -o- 1% lactOSe O - -- 3% lactOSe
5
4 O 5 1 O 15 20 Time (h)
Figure 1 Patent Application Publication May 18, 2017. Sheet 2 of 11 US 2017/O135360 A1
KTAteface-TekkAE 000-23:11:00
6.6 x
s6.2. W Milk without added Sugar 60. O58, 1882 + 5: 1898 + O.5. Sucross 5.8 56. y - 5,4. 5, 1912 + 05: 119 + SiOS 5.2. - 50 x 4,8.W \ 44 s 42 40.
O +3 +10 +13) +2) OOO-OO Patent Application Publication May 18, 2017. Sheet 3 of 11 US 2017/O135360 A1
TABC-TeknikAE rictoich 175
13, 18944 + 4:... Surs
E.2 ; : F8 + 8:: S-se
E.O. Wilk+.4% Slut OSE 5.8 : 0.75%. 178E1 + 0.25%. 1844-(4% sucross
5.5 5: 88 + 8, 18342 + 3, Surse
54. 58 35& 1881 + O.F5: 13344 + 4: 88
5.0
4.
4.2 .
O. : : +5 +1 +15- +8. +25 +3) +35 +ah -O-3) Figure 3 Patent Application Publication May 18, 2017. Sheet 4 of 11 US 2017/O135360 A1
pH measurements
YF-94 Acidifix
Ending pH pH after pHD+6 (6°C) pHD+6 (25° C) thermisation
Figure 4 Patent Application Publication May 18, 2017. Sheet 5 of 11 US 2017/O135360 A1
Post acidification at long term storage at 6C
F-DVSSweety 1.0
Days Figure 5 Patent Application Publication May 18, 2017. Sheet 6 of 11 US 2017/O135360 A1
455 - 4.5 assia:F-DVSYF-L7O6 4,45 44 as F-DVS YF-L706+ 3500 NLU/L lactase 4,35 as F-DVS YoFlexMild 1.0 4,3 seaseF-DVS YoFlexMild 1.0+ 4,25 ; 3500 NLU/L lactase 42 --sh::::::::::&rs: 4,15 ------Day 0 Day 1 Day 7 Day 14 Day 21 Day 48 Patent Application Publication May 18, 2017. Sheet 7 of 11 US 2017/O135360 A1
Production of fermented milk of Thermophile and Mesophilic applications with high protein, fat and solid levels. Examples of process with concentration before fermentation and concentration by whey separation after fermentation
Fortificati Fortificatio Protein powder Separation by Separation by UFI Evaporatio a Fiatic acci fe k Centrifugation
Concetrated feeted milk products life protein Ifat ewes ratios
Figure 7 Patent Application Publication May 18, 2017. Sheet 8 of 11 US 2017/O135360 A1
70%Premium+30%SSC1712bar 4.45
70%Premium+30%SSC1.79bar 4.45
70%Premium+30%SSC178bar 4.45
70%Premium+30%SSC177bar 4.45
70%Premium+30%SSC176bar 4.45
70%Premium+30%SSC175bar 4.45
70%Premium+30%SSC17 12bar 4.60
70%Premium+30%SSC1.79bar 4.60
70%Premium+30%SSC178bar 4.60
70%Premium+30%SSC177bar 4.60
70%Premium+30%SSC17 6bar 4.60
70%Premium+30%SSC175bar 4.60 Patent Application Publication May 18, 2017. Sheet 9 of 11 US 2017/O135360 A1
Shear Stress of Mild 2.0
Mid2O 9bar 4.45
Mild2.O 8bar 4.45
Mild2.O 7bar 4.45
Mild2.O 6bar 4.45
Mild2.05 bar 4.45
Mild2.0 4bar 4.45
Mild2.O 9bar 4.60
Mid2.O 8bar 4.60
Mild2.O 7bar 4.60
Mild2.O 6bar 4.60
Mild2.05 bar 4.60
Mild2.0 4bar 4.60 Patent Application Publication May 18, 2017. Sheet 10 of 11 US 2017/0135360 A1
Complex modulus of Premium + SSC17
70%Premium+30%SSC17 12 bar 4.45
70%Premium+30%SSC1.79bar 4.45
70%Premium+30%SSC178bar 4.45
70%Premium+30%SSC177bar 4.45
70%Premium+30%SSC176bar 4.45
70%Premium+30%SSC175bar 4.45
70%Premium+30%SSC17 12 bar 4.60
70%Premium+30%SSC1.79bar 4.60
70%Premium+30%SSC178bar 4.60
70%Premium+30%SSC177bar 4.60
70%Premium+30%SSC176bar 4.60
70%Premium+30%SSC175bar 4.60
I Complex Modulus
Figure 10 Patent Application Publication May 18, 2017. Sheet 11 of 11 US 2017/0135360 A1
Mid2.O 9bar 4.45
Mid2.O 8bar 4.45
Mid2.O 7bar 4.45
Mid2.O 6bar 4.45
Mid2.05bar 4.45 Mild2.0 4bar 4.45
Mid2.O.9bar 4.60 Mild2.08bar 4.60
Mid2.O 7bar 4.60
Mild2.O 6bar 4.6O &8 SXSXS
Mid 2.05bar 4.60
Mid 2.0 4bar 4.60
Complex Modulus
Figure 11 US 2017/O 135360 A1 May 18, 2017
METHOD OF PRODUCING A FERMENTED include a heat treatment step (usually 65° C./30s) after the MLK PRODUCT WITH IMPROVED fermentation. The treatment causes a significant decrease in CONTROL OF POST ACIDIFICATION the number of bacteria used for fermentation as well as in the number of yeasts and moulds. The heating step also inhibits FIELD OF THE INVENTION the activity of enzymes present in the yoghurt. As a conse quence, the product has an extended shelf life of up to 9 0001. The present invention relates to methods of pro months, wherein no or only low post acidification and ducing a fermented milk product comprising steps, wherein flavour changes are observed. milk is fermented by a starter culture comprising lactic acid 0011. However, the heat treatment has a negative effect bacteria and wherein the fermentation is terminated by a on the quality of the yoghurt and changes in flavour and decrease of the concentration of the carbohydrates that can texture are obtained. A further negative effect of the heat be metabolized by the lactic acid bacteria. The methods of treatment is that the health benefits obtained by eating live the present invention provide an improved control of post bacteria are diminished or lost. acidification, i.e. the acidification caused by the bacteria 0012. One of the approaches for controlling post acidi after the termination of the fermentation, for example during fication resides in producing milk products with a relatively further processing and storage. acidic pH. In these methods the further growth of the LAB and the production of lactic acid is inhibited by the acidic BACKGROUND OF THE INVENTION pH. However, the further production of lactic acid is only 0002 Most of the current methods for producing fer inhibited and not completely terminated and the method is mented milk products can be characterized by the following obviously unsuitable for the production of fermented milk series of steps: products with a mild taste. 0003 (a) milk is fermented using a starter culture 0013. It was assumed that post acidification is controlled comprising lactic acid bacteria (or LAB) capable of by the metabolic activity of L. bulgaricus and caused by metabolizing glucose obtained from lactose present in peptide uptake and strains with a deficiency in amino acid the milk; metabolism were generated to control post acidification 0004 (b) the fermentation causes the production of (US2010/0021586 and WO2006/042862A1). Other lactic acid, which causes a decrease of the pH from approaches for controlling post acidification have been initially 6.4 to 6.8 (for cow milk) to a range between pH described in the prior art and include processes based on the 3.8 and 4.7: use of specific LAB strains characterized by weakly post 0005 (c) the fermentation is terminated by rapid cool acidifying activity (WO2010/139765). ing of the fermented milk product once the pH desired 0014. An alternative approach for minimizing post acidi for the fermented product at issue has been reached. fication is based on the control of the ratio of protein to 0006. This method is for example used to produce lactose, the control of the buffering capacity and the main yoghurt and yoghurt beverages. tenance of the buffering capacity and the pH within a 0007. The rapid cooling of the fermented milk product at predetermined range during fermentation (WO2013/ a predetermined pH value is carried out to terminate fer 169205). However, this approach requires the determination mentation. Without cooling of the fermented product, the of a number of process parameters during fermentation and fermentation would continue. However, rapid cooling has may require the addition of proteins or lactose or a buffer to disadvantages, as it leads to loss of texture. Avoiding a rapid the fermentation medium to ensure that the predetermined cooling step would also spare a unit operation and thus ranges are maintained during fermentation. reduce production costs. 0015 There is evidently still a need for improving meth 0008 Even in methods comprising a rapid cooling step, ods for producing fermented milk products, which methods post acidification is observed, i.e. the production of lactic provide a better control of the post acidification activity in acid by the LAB after the termination of fermentation, i.e. the fermented milk product. after the desired pH has been reached. Post acidification is considered to represent one of the most important problems SUMMARY OF THE INVENTION during fermentation of milk products today. The further 0016. This problem is now solved by the methods of the decrease of pH value during processing and storage of the present invention, which provide a milk product with fermented milk product leads to problems with elevated extremely low post acidification activity. acidity and reduced shelf life. 0017. In particular, the present invention provides meth 0009 Post acidification therefore also has a negative ods of producing a fermented milk product comprising a effect on the shelf life of yoghurt. The shelf life of a yoghurt step wherein milk is fermented, wherein: varies from 30 to 50 days depending on the country. During 0.018 (a) the fermentation is initiated by a starter this time post acidification changes the quality of the culture, which starter culture comprises lactic acid yoghurt causing a sour product and high whey separation. bacteria capable of metabolizing one or several carbo Usually the quality of yoghurt is maintained as much of hydrates present in the milk, possible by keeping the product at a temperature between 4 0.019 (b) the fermentation is terminated by a decrease and 8° C. during storage. At this temperature bacteria will of the concentration of the one or several carbohydrates only have a low activity. However, in many countries it is during fermentation, and difficult to maintain the cool-chain during the storage. 0020 (c) the decrease is at least also caused by the 0010. As a consequence, the manufacture of Extended metabolic activity of the lactic acid bacteria. Shelf Life Yoghurt (ESL yoghurt), is of high interest, in 0021. The present inventors surprisingly found that the particular in countries where it is difficult to maintain the termination of fermentation can be controlled by the con cool-chain. The prior art methods of producing ESL yoghurt centration of carbohydrates in the milk to be fermented US 2017/O 135360 A1 May 18, 2017 without significantly affecting the efficiency or time required milk product within a range of 0.1 pH units over a period of for the fermentation. This is Surprising as it was assumed 20 hours if stored at a temperature for fermentation is an that a reduction of the carbohydrates present in the milk that indication of absence of post acidification. are available for the LAB for fermentation would inhibit or 0032. In a further alternative, the present invention pro delay the fermentation process and thus result in an inefli vides methods of producing a fermented milk product com cient process that cannot be used for large scale production prising: of fermented milk products, such as yoghurt. The inventors 0033 (a) a step, wherein milk is fermented using a were further surprised to note that the fermentation process starter culture, wherein: rapidly proceeds up to a point, where essentially all the 0034 (i) the fermentation is initiated using a milk carbohydrates have been consumed by the LAB and then and a starter culture, wherein the lactose concentra terminates almost completely (FIG. 1; 1% lactose). It was tion in the milk is in the range of 5 to 100 mg/g at the expected that the fermentation process in the presence of a start of the fermentation very low concentration of carbohydrates available for the 0035 (ii) the starter culture comprises Streptococ LAB would result in a low acidification activity of the LAB cus thermophilus and Lactobacillus delbrueckii over an extended period of time. Subsp. bulgaricus, 0022. In an alternative, the present invention provides 0036 (iii) the fermentation is conducted at a tem methods of producing a fermented milk product comprising perature between 22 and 45° C., a step, wherein milk is fermented using a starter culture, 0037 (iv) the fermentation is terminated by a wherein: decrease of the concentration of the lactose during 0023 (i) the starter culture comprises Streptococcus fermentation, thermophilus and Lactobacillus delbrueckii Subsp. bul 0038 (v) the decrease is at least also caused by the garicus, metabolic activity of the lactic acid bacteria, and 0024 (ii) the fermentation is conducted at a tempera 0.039 (b) a step, wherein the fermented product is ture between 22 and 45° C., packaged at a temperature between 15 and 45° C. 0025 (iii) the fermentation is terminated by a decrease 0040. Due to the low post acidification this method does of the concentration of a carbohydrate during fermen not require a cooling step after the fermentation. tation, 0041. The present invention further provides fermented 0026 (iv) the decrease is at least also caused by the milk products obtainable by these methods. These fermented metabolic activity of the lactic acid bacteria, and milk products are characterized by maintaining the pH value wherein the fermented milk product is characterized in that of the product within a range of 0.3 pH units over a period the pH value of the product is maintained within a range of of 20 hours if stored at the temperature used for fermenta 0.3 pH units if stored after termination of the fermentation tion. The products are further characterized by a very low at the temperature used for fermentation in step (ii) over a concentration of the carbohydrates that can be metabolized period of 20 hours. This does not mean that the method by the LAB used for the fermentation. Other carbohydrates necessarily includes a step, wherein the fermented milk may be present in significantly higher concentration. product is maintained over a period of 20 hours after termination of the fermentation at the temperature used for DETAILED DISCLOSURE OF THE INVENTION fermentation in step (ii). This is just a functional test that can 0042. In general the present invention provides methods be used to confirm low post acidification. Maintaining a pH of producing a fermented milk product comprising a step value pH of a fermented milk product within a range of 0.3 wherein milk is fermented, wherein: pH units over a period of 20 hours if stored at a temperature 0.043 (a) the fermentation is initiated by a starter for fermentation is an indication of very low post acidifica culture, which starter culture comprises lactic acid tion. bacteria capable of metabolizing one or several carbo 0027. In a further embodiment, the present invention is hydrates present in the milk, directed to methods for producing a fermented milk product 0044 (b) the fermentation is terminated by a decrease which are characterized by essentially no post acidification. of the concentration of the one or several carbohydrates Respective methods of producing a fermented milk product during fermentation, and comprise a step, wherein milk is fermented using a starter 0.045 (c) the decrease is at least also caused by the culture, wherein: metabolic activity of the lactic acid bacteria. 0028 (i) the starter culture comprises Streptococcus 0046. In the context of the present application, the term thermophilus and Lactobacillus delbrueckii Subsp. bul “milk' is broadly used in its common meaning to refer to garicus, liquids produced by the mammary glands of animals or by 0029 (ii) the fermentation is conducted at a tempera plants. In accordance with the present invention the milk ture between 22 and 45° C., may have been processed and the term “milk' includes 0030 (iii) the fermentation is terminated by a decrease whole milk, skim milk, fat-free milk, low fat milk, full fat of the concentration of a carbohydrate during fermen milk, lactose-reduced milk, or concentrated milk. Fat-free tation, milk is non-fat or skim milk product. Low-fat milk is 0031 (iv) the decrease is at least also caused by the typically defined as milk that contains from about 1% to metabolic activity of the lactic acid bacteria, and about 2% fat. Full fat milk often contains 2% fat or more. wherein the fermented milk product is characterized in that The term “milk' is intended to encompass milks from the pH value of the product is maintained within a range of different mammals and plant sources. Mammal sources of 0.1 pH units if stored after termination of the fermentation milk include, but are not limited to cow, sheep,goat, buffalo, at the temperature used for fermentation in step (ii) over a camel, llama, mare and deer. Plant sources of milk include, period of 20 hours. Maintaining a pH value of a fermented but are not limited to, milk extracted from Soybean, pea, US 2017/O 135360 A1 May 18, 2017 peanut, barley, rice, oat, quinoa, almond, cashew, coconut, which LAB are capable to metabolize other carbohydrates, hazelnut, hemp, Sesame seed and Sunflower seed. Such as lactose and galactose. In further alternatives the 0047. In the methods and products of the present inven present invention provides methods using LAB with a tion, milk derived from cows is most preferably used as a deficiency in lactose metabolism which LAB are capable to starting material for the fermentation. metabolize other carbohydrates, such as glucose. 0.048 Lactose-reduced milk has been used in some of the 0054 The methods of the present invention are charac examples of the present application and is commercially terized by a step, wherein milk is fermented and the fer available (for example from Select Milk Producers Inc., mentation is terminated by a decrease of the concentration of Texas, USA). Lactose-reduced milk can be produced the one or several carbohydrates during fermentation. This according to any method known in the art, including hydro means that the LAB present in the fermentation medium can lyzing the lactose by lactase enzyme to glucose and galac no longer produce significant amounts of lactic acid due to tose, or by nanofiltration, electrodialysis, ion exchange a very low concentration of carbohydrates that can be chromatography and centrifugation. metabolized. In one embodiment, termination of fermenta 0049. The tem “milk base' is broadly used in the present tion can be characterized by a pH value that is maintained application to refer to a composition based on milk or milk within a range of less than 0.3 pH units while the culture is components which can be used as a medium for growth and maintained at the temperature used for fermentation for 20 fermentation of LAB. The milk base comprises components hours. For example, if a method of producing a fermented derived from milk and any other component that can be used milk product comprising a step wherein milk is fermented as for the purpose of growing or fermenting LAB. described above is carried out, one can easily test whether 0050. In the context of the present application, the term termination of fermentation is due to a decrease of the “lactic acid bacteria' or “LAB is used to refer to food-grade concentration of the one or several carbohydrates during bacteria producing lactic acid as the major metabolic end fermentation by maintaining the product at the temperature product of carbohydrate fermentation. These bacteria are for fermentation for 20 hours. If the pH does not change by related by their common metabolic and physiological char more than 0.3 pH units during that time than termination of acteristics and are usually Gram positive, low-GC, acid fermentation was caused by the decrease of the concentra tolerant, non-sporulating, nonrespiring, rod-shaped bacilli or tion of the one or several carbohydrates during fermentation. cocci. During the fermentation stage, the consumption of 0055. The prior art methods which terminate fermenta lactose by these bacteria causes the formation of lactic acid, tion by cooling as soon as the desired pH is achieved cannot reducing the pH and leading to the formation of a protein meet this test, as the residual carbohydrates available for coagulum. These bacteria are thus responsible for the acidi metabolic activity will cause significant post acidification at fication of milk and for the texture of the dairy product. As the temperature used for fermentation (FIG. 1, 3% curve: used herein, the term “lactic acid bacteria encompasses, but FIGS. 2 and 3 lowest curve). is not limited to, bacteria belonging to the genus of Lacto 0056. The methods of the present invention can further be bacillus spp., Bifidobacterium spp., Streptococcus spp., Lac characterized in that the decrease of the concentration of the tococcus spp., Such as Lactobacillus delbrueckii Subsp. carbohydrates is at least also caused by the metabolic bulgaricus, Streptococcus thermophilus, Lactobacillus lac activity of the lactic acid bacteria. This means that the lactic tis, Bifidobacterium animalis, Lactococcus lactis, Lactoba acid bacteria contribute to the decrease of the carbohydrates, cillus paracasei, Lactobacillus plantarum, Lactobacillus although other components, e.g. enzymes such as lactase, helveticus, Lactobacillus acidophilus, Bifidobacterium may also contribute to the decrease of the carbohydrate that breve and Leuconostoc spp. can be metabolized during fermentation. 0051. The fermentation step of the process for manufac turing fermented dairy products comprises the addition of a 0057. In some embodiments the methods of the present starter culture to milk. The term "starter” or “starter culture' invention comprise a step, wherein the fermented product is as used in the present context refers to a culture of one or packaged at a temperature between 15 and 45° C. As more food-grade micro-organisms, in particular lactic acid indicated above, one of the main problems of the prior art bacteria, which are responsible for the acidification of the resides in the need of rapid cooling of the fermentation milk base. Starter cultures may be fresh, frozen or freeze product to terminate fermentation. The methods of the dried. For the production of a fermented dairy product, the present invention may comprise a step, wherein fermented starter can be added in an amount from 0.01 to 3%, prefer product is packaged at a temperature between 15 and 45° C. ably from 0.01 to 0.025% by volume of the total amount of This shows that contrary to the prior art methods, rapid milk. cooling is not absolutely necessary. 0052. The term “capable of metabolizing one or several 0.058 According to one alternative, the termination of carbohydrates present in the milk' is used in the context of fermentation can be characterized by the concentration of the present invention to describe the metabolic activity of the one or several carbohydrates that can be metabolized by LAB which causes production of lactic acid as the major the lactic acid bacteria. At the termination of fermentation metabolic end-product of carbohydrate fermentation. As will the concentration of the carbohydrate metabolized by the be explained in more detail below, the LAB may be able to lactic acid bacteria can be in the range of less than 100 mg/g, metabolize one, several or all carbohydrates present in the Such as less than 30 mg/g, including a range between 25 milk. The carbohydrates may be present in natural milk or mg/g and 0.01 mg/g, or a range between 5 mg/g and 0.01 may have been added to the milk. mg/g. 0053. In certain embodiments, the present invention pro 0059. As indicated above, the methods of producing a vides methods using LAB capable to metabolize lactose and fermented milk product according to the present invention glucose. In other embodiments, the invention provides meth may further be characterized by a particularly stable pH ods using LAB with a deficiency in glucose metabolism value during storage. The fermented product may be main US 2017/O 135360 A1 May 18, 2017
tained within a range of 0.3 pH units over a period of 20 product must not have been heated after fermentation and hours when stored at the temperature used for fermentation. the dairy raw materials must represent a minimum of 70% 0060. In a further alternative, the methods of producing a (m/m) of the finished product. fermented milk product according to the present invention 0.066 Cheese, such as Mozzarella and Pizza cheese as can be characterized by a temperature during fermentation well as Feta, can also be prepared by fermentation using a of between 22 and 45° C. This temperature range includes starter culture comprising Streptococcus thermophilus and the range used for mesophilic and thermophilic cultures. In Lactobacillus delbrueckii subsp. bulgaricus (Hoier et al. the context of the present application the term “mesophilic' (2010) in The Technology of Cheesemaking, 2" Ed. Black refers to microorganisms that grow best at moderate tem well Publishing, Oxford; 166-192). peratures, i.e. at temperatures from 15° C. to 40° C. The 0067. The methods of the present invention encompass industrially most useful mesophilic bacteria include Lacto numerous different alternatives. The following alternatives coccus spp. and Leuconostoc spp. Mesophilic dairy products will be described in some detail below: include Such dairy products as buttermilk, Sour milk, cul 0068 (A) Methods of producing a fermented milk tured milk, Smetana, Sour cream and fresh cheese, such as product using milk with low lactose content. quark, tvarog and cream cheese. In the context of the present 0069 (B) Lactose deficient LAB and methods of pro application the term “thermophilic' refers to microorgan ducing a fermented milk product using lactose deficient isms that grow best at temperatures above 40° C. The LAB. industrially most useful thermophilic bacteria include Strep 0070 (C) Methods for a fermented milk product with tococcus spp. and Lactobacillus spp. Thermophilic dairy extended shelf life products include Such dairy products as yoghurt. 0071 (D) Methods of producing a fermented milk 0061 According to one embodiment, the present inven product using glucose deficient LAB. tion provides a method as described above, wherein meso 0.072 (E) Methods of producing a fermented milk philic and thermophilic cultures are both fermented at a product adding lactase to the fermentation. temperature between 22 and 45° C. 0.073 (F) Methods of producing a strained fermented 0062. The present invention further provides fermented milk product based on a product obtained with one of milk products obtainable by the methods as described above. methods (A) to (E). The fermented milk products of the present invention are 0.074 (G) Methods of producing a pasta filata cheese preferably fermented food products, including yoghurt, fruit product using one of methods (A) to (E). yoghurt, yoghurt beverage or cheese. 0063. In its most preferred embodiment all methods of A: Methods for Producing a Fermented Milk Product Using the present invention are methods for producing a yoghurt Low Lactose Milk and the product of the present invention is yoghurt. 0064. In the context of the present application, the term 0075 According to this alternative the present invention 'yoghurt refers to products comprising Streptococcus ther provides methods of producing a fermented milk product mophilus and Lactobacillus delbrueckii Subsp. bulgaricus comprising a step wherein milk is fermented, wherein: and optionally other microorganisms such as Lactobacillus 0.076 (a) the fermentation is initiated by a starter delbrueckii Subsp. lactis, Bifidobacterium animalis Subsp. culture comprising lactic acid bacteria, which starter lactis, Lactococcus lactis, Lactobacillus acidophilus and culture comprises Streptococcus thermophilus (ST) and Lactobacillus paracasei, or any microorganism derived Lactobacillus delbrueckii ssp. bulgaricus both capable therefrom. The lactic acid strains other than Streptococcus of metabolizing lactose present in the milk, thermophilus and Lactobacillus delbrueckii Subsp. bulgari 0.077 (b) the fermentation is terminated by a decrease cus, are included to give the finished product various prop of the concentration of the lactose during fermentation, erties, such as the property of promoting the equilibrium of and the flora. As used herein, the term 'yoghurt encompasses 0078 (c) the decrease is at least also caused by the set yoghurt, stirred yoghurt, drinking yoghurt, Petit Suisse, metabolic activity of the lactic acid bacteria, wherein heat treated yoghurt, Strained or Greek style yoghurt char before addition of the starter culture the concentration acterized by a high protein level and yoghurt-like products. of the lactose in the milk is below 30 mg/g and the 0065. In particular, term “yoghurt encompasses, but is concentration of the glucose in the milk is below 15 not limited to, yoghurt as defined according to French and mg/g. In one alternative the concentration of lactose is European regulations, e.g. coagulated dairy products below 25 mg/g or below 15 mg/g and the concentration obtained by lactic acid fermentation by means of specific of glucose is below 2 mg/g. thermophilic lactic acid bacteria only (i.e. Lactobacillus 0079. In one aspect of this approach the present invention delbrueckii Subsp. bulgaricus and Streptococcus thermophi provides methods of producing a fermented milk product lus) which are cultured simultaneously and are found to be comprising a step wherein milk is fermented, wherein: live in the final product in an amount of at least 10 million 0080 (a) the fermentation is initiated by a starter CFU (colony-forming unit)/g. Yoghurts may optionally con culture comprising lactic acid bacteria, which starter tain added dairy raw materials (e.g. cream) or other ingre culture comprises Streptococcus thermophilus (ST) and dients such as Sugar or Sweetening agents, one or more Lactobacillus delbrueckii ssp. bulgaricus both capable flavouring(s), fruit, cereals, or nutritional Substances, espe of metabolizing lactose present in the milk, cially vitamins, minerals and fibers, as well as stabilizers and 0081 (b) the fermentation is terminated by a decrease thickeners. In one alternative yoghurt meets the specifica of the concentration of the lactose during fermentation, tions for fermented milks and yoghurts of the AFNOR NF and 04-600 standard and/or the codex Stan A-IIa-1975 standard. 0082 (c) the decrease is caused by the metabolic In order to satisfy the AFNOR NF 04-600 standard, the activity of the lactic acid bacteria, US 2017/O 135360 A1 May 18, 2017
wherein the method further comprises a step, wherein the mophilus strain, which strain is the strain deposited with fermented product is packaged at a temperature between 15 DSMZ-Deutsche Sammlung von Mikroorganismen und and 45° C. Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunsch 0083. In one alternative, the method can be characterized weig, on 2014 Jun. 12 under the accession no. DSM 28909. in that the pH value of the fermented product is maintained 0094. According to another aspect, the present invention within a range of 0.3 pH units or within a range of 0.1 pH relates to the following lactose deficient LAB strains: units if stored after termination of the fermentation at the 0.095 (a) a Streptococcus thermophilus strain, which temperature used for fermentation over a period of 20 hours. strain is: 0084. The lactose may be naturally present in the milk or may have been added to the milk. (0096 (i) the strain deposited with DSMZ-Deutsche 0085. In this alternative the fermentation is caused by Sammlung Von Mikroorganismen und Zellkulturen LAB capable to metabolize lactose and the termination of GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on fermentation is caused by a concentration of lactose that is 2014 Jun. 12 under the accession no. DSM 28952: so low that fermentation and production of lactic acid by the (0097 (ii) or a strain derived from DSM 28952, LAB is terminated. The present inventors surprisingly found wherein the derived strain is further characterized as that even these very low concentrations of lactose in the milk having the ability to generate white colonies on a provide sufficient carbohydrate source for rapid fermenta medium containing lactose and X-Gal; tion. It was further found that once the concentration of the 0.098 (b) a Streptococcus thermophilus strain, which lactose was reduced below a certain threshold, the fermen strain is: tation is rapidly terminated (Example 1, FIG. 1). (0099 (i) the strain deposited with DSMZ-Deutsche I0086. The initial lactose concentration in the milk may be Sammlung Von Mikroorganismen und Zellkulturen below 30 mg/g, for example between 30 mg/g and 5 mg/g, GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on or between 15 mg/g and 5 mg/g, or between 10 mg/g and 5 2014 Jun. 12 under the accession no. DSM 28953; mg/g. 0100 (ii) or a strain derived from DSM 28953, 0087 Milk with reduced lactose concentration can be wherein the derived strain is further characterized as commercially obtained or produced according to methods having the ability to generate white colonies on a well known in the art. medium containing lactose and X-Gal; 0088. In one embodiment of this alternative, the milk contains a reduced lactose content and no additional carbo 0101 (c) a Lactobacillus delbrueckii ssp. bulgaricus hydrates not naturally present in cow milk. Accordingly, the strain, which strain is: initial glucose concentration in the milk can be significantly 0102 (i) the strain deposited with DSMZ-Deutsche below 15 mg/g, such as below 2 mg/g. Fermentation may be Sammlung Von Mikroorganismen und Zellkulturen carried out on milk with a reduced lactose content and GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on without any carbohydrates added to the natural cow milk. 2014 Jun. 12 under the accession no. DSM 28910; 0089. In another embodiment of this alternative, the milk (0103 (ii) or a strain derived from DSM 28910, contains a reduced lactose content and additional carbohy wherein the derived strain is further characterized as drates not naturally present in cow milk, such as glucose or having the ability to generate white colonies on a SUCOS. medium containing lactose and X-Gal. 0104. The present invention further relates to the use of B: Lactose Deficient LAB and Methods of Producing a these strains in the methods of producing a fermented milk Fermented Milk Product Using Lactose Deficient LAB product as described herein. Further, the invention relates to 0090. In a further embodiment the present invention fermented food products, comprising one or several of the provides lactose deficient LAB. following strains: 0091. The terms “deficiency in lactose metabolism' and 0105 (a) a Streptococcus thermophilus strain, which “lactose deficient” are used in the context of the present strain is: invention to characterize LAB which either partially or 0106 (i) the strain deposited with DSMZ-Deutsche completely lost the ability to use lactose as a source for cell Sammlung Von Mikroorganismen und Zellkulturen growth or maintaining cell viability. Respective LAB are GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on capable to metabolize one or several carbohydrates selected 2014 Jun. 12 under the accession no. DSM 28952: from Sucrose, galactose and/or glucose or another ferment 0107 (ii) or a strain derived from DSM 28952, able carbohydrate. Since these carbohydrates are not natu wherein the derived strain is further characterized as rally present in milk in Sufficient amounts to Support fer having the ability to generate white colonies on a mentation by lactose deficient mutants, it will be necessary medium containing lactose and X-Gal; to add these carbohydrates to the milk. Lactose deficient and partially deficient LAB can be characterized as white colo 0.108 (b) a Streptococcus thermophilus strain, which nies on a medium containing lactose and X-Gal. strain is: 0092. As is described in detail in Example 2 below, the 0109 (i) the strain deposited with DSMZ-Deutsche present inventors isolated a number of lactose deficient Sammlung Von Mikroorganismen und Zellkulturen LAB, in particular strains of Streptococcus thermophilus GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on (ST) and Lactobacillus delbrueckii ssp. bulgaricus (LB). 2014 Jun. 12 under the accession no. DSM 28953; These lactose deficient LAB metabolize sucrose. 0110 (ii) or a strain derived from DSM 28953, 0093. The strains were derived from the strain wherein the derived strain is further characterized as CHCC15914 which is not lactose deficient. Accordingly, the having the ability to generate white colonies on a present invention provides an isolated Streptococcus ther medium containing lactose and X-Gal; US 2017/O 135360 A1 May 18, 2017
0111 (c) a Lactobacillus delbrueckii ssp. bulgaricus GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on strain, which strain is: 2014 Jun. 12 under the accession no. DSM 28952: 0112 (i) the strain deposited with DSMZ-Deutsche I0128 (ii) or a strain derived from DSM 28952, Sammlung Von Mikroorganismen und Zellkulturen wherein the derived strain is further characterized as GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on having the ability to generate white colonies on a 2014 Jun. 12 under the accession no. DSM 28910; medium containing lactose and X-Gal; 0113 (ii) or a strain derived from DSM 28910, 0.129 (b) a Streptococcus thermophilus strain, which wherein the derived strain is further characterized as strain is: having the ability to generate white colonies on a I0130 (i) the strain deposited with DSMZ-Deutsche medium containing lactose and X-Gal. Sammlung Von Mikroorganismen und Zellkulturen 0114. The fermented food products can be a yoghurt, a GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on fruit yoghurt, a yoghurt beverage or a cheese. 2014 Jun. 12 under the accession no. DSM 28953; 0115 According to this approach the present invention I0131 (ii) or a strain derived from DSM 28953, also provides methods of producing a fermented milk prod wherein the derived strain is further characterized as uct comprising a step wherein milk is fermented, wherein: having the ability to generate white colonies on a 0116 (a) the fermentation is initiated by a starter medium containing lactose and X-Gal; culture, which starter culture comprises Streptococcus 0.132 (c) a Lactobacillus delbrueckii ssp. bulgaricus thermophilus with a deficiency in lactose metabolism strain, which strain is: and Lactobacillus delbrueckii ssp. bulgaricus with a (0.133 (i) the strain deposited with DSMZ-Deutsche deficiency in lactose metabolism, Sammlung Von Mikroorganismen und Zellkulturen 0117 (b) a carbohydrate is added to the milk that can GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on be metabolized by the Streptococcus thermophilus and 2014 Jun. 12 under the accession no. DSM 28910; the Lactobacillus delbrueckii S.Sp. Bulgaricus as I0134 (ii) or a strain derived from DSM 28910, defined in (a), wherein the derived strain is further characterized as 0118 (c) the fermentation is terminated by a decrease having the ability to generate white colonies on a of the concentration of the carbohydrates added to the medium containing lactose and X-Gal. milk, and C: Methods for a Fermented Milk Product with Extended 0119 (d) the decrease is at least also caused by the Shelf Life metabolic activity of the Streptococcus thermophilus 0.135 According to one embodiment of the present inven and the Lactobacillus delbrueckii S.Sp. Bulgaricus as tion, the methods for controlling post acidification described defined in (a). above, in particular the methods described in section B. 0120 In some embodiments the method is further char above, are used to generate a fermented milk product with acterized as comprising a step, wherein the fermented prod extended shelf life. uct is packaged at a temperature between 15 and 45° C. 0.136 Respective method of producing a fermented milk 0121. In other alternatives, the method can be character product may for example comprise a step wherein milk is ized in that the pH value of the fermented product is fermented, wherein: maintained within a range of 0.3 pH or within a range of 0.1 0.137 (a) the fermentation is initiated by a starter pH units if stored after termination of the fermentation at the culture, which starter culture comprises lactic acid temperature used for fermentation over a period of 20 hours. bacteria capable of metabolizing one or several carbo 0122) The total concentration of carbohydrates that can hydrates present in the milk, be metabolized by the Streptococcus thermophilus and the 0.138 (b) the fermentation is terminated by a decrease Lactobacillus delbrueckii S.Sp. Bulgaricus as defined in (a) of the concentration of the one or several carbohydrates can be in the range of 30 mg/g to 2 mg/g, or in the range of during fermentation, and 20 mg/g 3 mg/g or in the range of 10 mg/g to 4 mg/g. 0.139 (c) the decrease is at least also caused by the 0123. This way of proceeding has the advantage that metabolic activity of the lactic acid bacteria, normal milk with a lactose concentration of around 5% (50 wherein the fermented product is characterized in that the mg/g) can be used in the production process and that the pH value of the product is maintained within a range of 0.3 fermentation can be precisely controlled by the amount of pH units if stored after termination of the fermentation for a carbohydrate added. The concentration of carbohydrate to period of 6 months. In a particularly preferred embodiment, be added can be determined in trials testing desired fermen the fermented product is characterized in that the pH value tation conditions, including final pH, temperature, starter of the product is maintained within a range of 0.3 pH units culture, etc. if stored after termination of the fermentation for a period of 0.124. In a preferred embodiment of this alternative, the 12 months. The product may be stored at a temperature fermentation is carried out using LAB that are able to between 2° C. and room temperature. Storage at refrigera metabolize Sucrose (Suc-) and Sucrose is added to the milk tion temperature, i.e. between 4 and 8°C., is preferred. before fermentation. In one alternative the concentration of 0140. As indicated above, it is not required to store the Sucrose is in the range of 30 mg/g to 2 mg/g, or in the range product for several months. The method for producing a of 20 mg/g to 3 mg/g or in the range of 10 mg/g to 4 mg/g. fermented milk product according to the present invention is 0.125. In one aspect step (a) of the above method is characterized by a product obtainable by the method that carried out using of one or several of the following strains maintains stability over the time period indicated. In one 0.126 (a) a Streptococcus thermophilus strain, which aspect the present invention provides a method of producing strain is: a fermented milk product comprises a step wherein milk is (O127 (i) the strain deposited with DSMZ-Deutsche fermented using a starter culture, wherein the product Sammlung Von Mikroorganismen and Zellkulturen obtainable by the method is stored for 6 or 12 months. US 2017/O 135360 A1 May 18, 2017
0141. In an alternative embodiment, the method of pro (0160 (ii) or a strain derived from DSM 28953, ducing a fermented milk product comprises a step wherein wherein the derived strain is further characterized as milk is fermented using a starter culture, wherein: having the ability to generate white colonies on a 0.142 (i) the starter culture comprises Streptococcus medium containing lactose and X-Gal; thermophilus and Lactobacillus delbrueckii Subsp. bul 0.161 (c) a Lactobacillus delbrueckii ssp. bulgaricus garicus, strain, which strain is: 0.143 (ii) the fermentation is conducted at a tempera 0162 (i) the strain deposited with DSMZ-Deutsche ture between 22 and 45° C., Sammlung Von Mikroorganismen and Zellkulturen 0144 (iii) the fermentation is terminated by a decrease GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on of the concentration of a carbohydrate during fermen 2014 Jun. 12 under the accession no. DSM 28910; tation, (0163 (ii) or a strain derived from DSM 28910, 0145 (iv) the decrease is at least also caused by the wherein the derived strain is further characterized as metabolic activity of the lactic acid bacteria, and having the ability to generate white colonies on a wherein the fermented product is characterized in that the medium containing lactose and X-Gal. pH value of the product is maintained within a range of 0.3 0164. For the fermentation of yoghurt, it is preferred that pH units if stored after termination of the fermentation for a a starter culture is used that comprises a Streptococcus period of 6 or 12 months. thermophilus strain and a Lactobacillus delbrueckii ssp. 0146 In a further alternative, methods of producing a bulgaricus strain as described above. fermented milk product are provided which comprise the 0.165. The method may further comprise a heat treatment following steps: after the fermentation. The heat treatment can for example 0147 (a) a step, wherein milk is fermented using a be carried out at a temperature in the range of 30 to 65° C. starter culture, wherein: for 10 to 30 seconds and is preferably carried out at a 0148 (i) the fermentation is initiated using a milk temperature in the range of 40 to 55° C. for 10 to 25 seconds. and a starter culture, wherein the lactose concentra 0166 The present invention also provides fermented tion in the milk is in the range of 5 to 100 mg/g at the milk products obtainable by the methods described above. start of the fermentation The fermented milk product can be a yoghurt, a fruit 0149 (ii) the starter culture comprises Streptococ yoghurt, a yoghurt beverage or a cheese. The invention cus thermophilus and Lactobacillus delbrueckii therefore provides set yoghurt, stirred yoghurt, drinking Subsp. bulgaricus, yoghurt, Petit Suisse, heat treated yoghurt, strained or Greek 0150 (iii) the fermentation is conducted at a tem style yoghurt characterized by a high protein level and perature between 22 and 45° C. yoghurt-like products obtainable by these methods. 0151 (iv) the fermentation is terminated by a decrease of the concentration of the lactose during D. Methods of Producing a Fermented Milk Product Using fermentation, Glucose Deficient LAB 0152 (v) the decrease is at least also caused by the 0167. In this alternative, the present invention provides metabolic activity of the lactic acid bacteria, and methods of producing a fermented milk product comprising 0153 (b) a step, wherein the fermented product is a step wherein milk is fermented, wherein: packaged at a temperature between 15 and 45° C. 0168 (a) the fermentation is initiated by a starter wherein the fermented product is characterized in that the culture, which starter culture comprises Streptococcus pH value of the product is maintained within a range of 0.3 thermophilus with a deficiency in glucose metabolism pH units if stored after termination of the fermentation for a and Lactobacillus delbrueckii ssp. bulgaricus with a period of 6 or 12 months. deficiency in glucose metabolism, 0154 According to one embodiment these methods use 0.169 (b) the fermentation is terminated by a decrease lactic acid bacteria for fermentation that are not capable to of the concentration of lactose during fermentation, and metabolize lactose. The lactic acid bacteria may be capable 0170 (c) the decrease is at least also caused by the to metabolize sucrose. In a particularly preferred embodi metabolic activity of the lactic acid bacteria. ment these methods make use of a starter culture comprising 0171 In a preferred aspect, this method can further be one or several of the following strains: characterized as comprising a step, wherein the fermented 0155 (a) a Streptococcus thermophilus strain, which product is packaged at a temperature between 15 and 45° C. strain is: 0.172. In other alternatives, the method can be character 0156 (i) the strain deposited with DSMZ-Deutsche ized in that the pH value of the fermented product is Sammlung Von Mikroorganismen und Zellkulturen maintained within a range of 0.3 pH or within a range of 0.1 GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on pH units if stored after termination of the fermentation at the 2014 Jun. 12 under the accession no. DSM 28952: temperature used for fermentation over a period of 20 hours. (O157 (ii) or a strain derived from DSM 28952, 0173 The term “deficiency in glucose metabolism' and wherein the derived strain is further characterized as the term “glucose deficient are both used in the context of having the ability to generate white colonies on a the present invention to characterize LAB which either medium containing lactose and X-Gal; partially or completely lost the ability to use glucose as a 0158 (b) a Streptococcus thermophilus strain, which Source for cell growth or maintaining cell viability. A strain is: respective deficiency in glucose metabolism can for example 0159 (i) the strain deposited with DSMZ-Deutsche be caused by a mutation in a gene inhibiting or inactivating Sammlung Von Mikroorganismen und Zellkulturen expression or activity of the glucokinase protein or the GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on glucose transporter protein. LAB with a deficiency in glu 2014 Jun. 12 under the accession no. DSM 28953; cose metabolism can be characterized as increasing the US 2017/O 135360 A1 May 18, 2017
glucose concentration in a culture medium, when grown on tation. Accordingly the present invention also provides lactose as carbohydrate source. The increase of glucose is methods of producing a fermented milk product comprising caused by glucose secretion of the glucose deficient LABs. a step wherein milk is fermented, wherein: Increase of glucose concentration in a culture medium can 0.184 (a) the fermentation is initiated by milk and a be determined by HPLC analysis, for example using a starter culture, wherein before addition of the starter Dionex CarboPac PA 203* 150 mm column (Thermo Fisher culture the concentration of the lactose in the milk is Scientific, product number 060142). below 30 mg/g and wherein the starter culture com 0.174 Several forms of glucose LAB with a deficiency in prises Streptococcus thermophilus with a deficiency in glucose metabolism have been described in the prior art, glucose metabolism and Lactobacillus delbrueckii ssp. including LAB capable of metabolizing lactose and galac bulgaricus with a deficiency in glucose metabolism, tose (WO 2013/160413, Pool et al., 2006). Respective LAB 0185 (b) the fermentation is terminated by a decrease may digest the main carbohydrate present in milk, lactose, of the concentration of the lactose during fermentation, thus generating glucose and galactose. The glucose deficient and strains. Such as the glucose deficient Streptococcus thermo 0186 (c) the decrease is at least also caused by the philus strain described in WO 2013/160413, can metabolize metabolic activity of the lactic acid bacteria. galactose and further appears to secrete glucose. Alternative 0187. The initial lactose concentration, i.e. the concen strains are described in WO 2013/160413, such as a 2-de tration before the addition of the starter culture, in the milk oxyglucose resistant Lactobacillus delbrueckii mutant LAB may be between 50 mg/g and 5 mg/g, or between 25 mg/g strain, which cannot take up glucose or galactose present in and 5 mg/g. the medium. However, the strain can take up lactose, digest 0188 Milk with reduced lactose concentration can be lactose and use the glucose obtained from lactose digestion commercially obtained or produced according to methods for growth and acid production. The publication of Pool et well known in the art. al., 2006 discloses a further method to obtain LAB with a deficiency in glucose metabolism. Using the general prin E: Methods of Producing a Fermented Milk Product Adding ciples described in these publications numerous other LAB Lactase to the Fermentation with the same or a similar deficiency in glucose metabolism can be generated by one of ordinary skill. 0189 In this alternative, the present invention provides 0175 Since the taste of glucose is much sweeter than the methods of producing a fermented milk product comprising taste of galactose, fermentation of milk using these glucose a step wherein milk is fermented, wherein: deficient mutants will increase the sweetness of the fer 0.190 (a) the fermentation is initiated by a starter mented product, while significantly decreasing the lactose culture, which starter culture comprises lactic acid concentration and without increasing the total concentration bacteria capable of metabolizing one or several carbo of carbohydrates (as described in great detail in WO2013/ hydrates present in the milk, 160413). 0191 (b) the fermentation is terminated by a decrease 0176 According to one alternative, the lactic acid bac of the concentration of the one or several carbohydrates teria with a deficiency in glucose metabolism may comprise during fermentation, and a glucose deficient but galactose-fermenting Streptococcus 0.192 (c) the decrease is at least also caused by the thermophilus strain and/or a Lactobacillus delbrueckii Strain metabolic activity of the lactic acid bacteria, with deficiency in glucose uptake. The Lactobacillus del and wherein the fermentation is carried out in the presence brueckii Strain with deficiency in glucose uptake can of lactase in an initial concentration of 500 to 5000 NLU/I. metabolize lactose and utilize glucose derived from intrac 0193 In a preferred aspect, this method can further be ellular lactose hydrolysis. characterized as comprising a step, wherein the fermented 0177. The starter culture may comprise additional LABs product is packaged at a temperature between 15 and 45° C. or may consist of a mixture of these strains. 0.194. In other alternatives, the method can be character 0178. Accordingly, the present invention provides meth ized in that the pH value of the fermented product is ods of producing a fermented milk product comprising a maintained within a range of 0.3 pH or a range of 0.1 pH step wherein milk is fermented, wherein: units if stored after termination of the fermentation at the 0179 (a) the fermentation is initiated by a starter temperature used for fermentation over a period of 20 hours. culture, which starter culture comprises one or more 0.195 Lactase is an enzyme that hydrolyzes lactose and Streptococcus thermophilus strains selected from produces glucose and galactose. Lactase is commercially CHCC16731, CHCC15757 and CHCC16404 and the available from many sources and can be prepared according Lactobacillus delbrueckii ssp. bulgaricus strain to methods well known in the art. Lactase activity may be CHCC16159, determined as units of neutral lactase activity (NLU). In 0180 (b) the fermentation is terminated by a decrease accordance with the present invention the lactase activity is of the concentration of lactose during fermentation, and determined using the method as described by the Food 0181 (c) the decrease is at least also caused by the Chemicals Codex (FCC) IV method. metabolic activity of the lactic acid bacteria. F: Methods of Producing a Strained Fermented Milk Product 0182. Using these glucose deficient LABs in the methods Based on Product Obtained with One of Methods (A) to (E) of the invention provides the additional advantage that the 0196. The methods A to E described above are particu fermented milk product thus produced has a Sweet taste. larly advantageous for the production of strained fermented 0183 This embodiment of the present invention may use milk products, such as strained yoghurt (Greek yoghurt, a milk product with reduced lactose concentration for fer Labneh), quark, fromage frais and cream cheese. Strained mentation in order to terminate the fermentation due to a fermented milk products are characterized by a production decrease of the concentration of the lactose during fermen step, wherein whey is removed from the fermented milk by US 2017/O 135360 A1 May 18, 2017
separation and/or filtration after fermentation resulting in Further the texture of the fermented milk product increases relatively thick products with a high protein concentration. as a consequence of the drop in pH value from 4.60 to 4.45. 0.197 Concentrating fermented milk using ultrafiltration 0207. The culture Premium 1.0+SSC17 generated higher or using a separator after conventional fermentation pro shear stress and complex modulus when compared to Mild cesses will lead to post acidification during the concentration 2.0, at both pH levels. step. Post acidification will impact several factors prior to 0208. The texture decreased significantly when back and during the separation process. First of all, post acidifi pressure was applied, between 4 and up to 9 or from 4 up to cation will cause a further decrease of the pH. As a conse 12 bars. quence of the pH decrease, gel firmness will increase. As a 0209. The proof-of-principle experiments therefore show further consequence of the pH decrease, the particle size in that it will be possible and advantageous to separate whey the yoghurt will increase. Finally, the shear stress will from the fermented milk product using less shear stress and increase due to a continuing production and secretion of complex modulus with the methods for controlling post exopolysaccarides (or extracellular polymeric Substances or acidification as described above. The texture depends on the EPS) by the LAB. type of culture, the pH and the back pressure applied during 0198 As a result, the fermented milk produced with this process prior to inlet into the separator or UF. It was not method will lack homogeneity. This will impact the major possible to reduce the texture at pH 4.45 down to the texture parameters of the separation process directly related to at 4.60 even if high back pressure has been applied to the Stokes's law, as e.g. particle size, particle water binding yoghurt curd. capacity, Viscosity of the serum phase and immobilized 0210. The methods to control post acidification thus water in the protein matrix. Due to the further reduction in provide the possibility to ferment milk using LAB cultures pH, for example from e.g. 4.65 down to pH 4.30, a lower to a specific pH depending on the carbohydrate added for product yield will be obtained and variation in the product fermentation. These methods provide an improved control quality in terms of taste, acidity and texture will be observed. of the pH in the yoghurt during post-fermentation processing In the prior art, strained fermented milk products are there and will thus in particular improve the separation step. In fore produced using methods comprising method steps, addition, the speed can be optimized as cooling is unneces which Suppress post acidification, most frequently using a sary and yield of production increases. cooling step. However, the cooling step requires a significant 0211. According to this aspect of the invention the fol amount of energy and has negative effects on the further lowing embodiments are amongst others provided: processing of the product. 0212 1. A method of producing a strained fermented 0199 An overview over different methods of producing milk product comprising a step wherein milk is fer fermented milk products using a step of protein concentra mented, wherein: tion is provided by FIG. 7. The left hand part of FIG. 7 0213 (a) the fermentation is initiated by a starter shows methods using a step of concentration prior to fer culture, which starter culture comprises lactic acid mentation and the right hand part of FIG. 6 shows methods bacteria capable of metabolizing one or several carbo using a step of concentration after fermentation by separa hydrates present in the milk, tion using centrifugation or ultrafiltration. 0214 (b) the fermentation is terminated by a decrease (0200 FIGS. 8 to 11 show results of proof of principle of the concentration of the one or several carbohydrates experiments illustrating the advantages that can be achieved during fermentation, by combining the methods of reducing post acidification as 0215 (c) the decrease is at least also caused by the described above with methods for producing a fermented metabolic activity of the lactic acid bacteria, milk product that comprise a step, wherein whey is removed 0216 (d) at least part of the whey is separated from the from the fermented milk by separation and/or filtration fermented milk product. resulting in relatively thick yoghurt with a high protein 0217 2. A method of producing a strained fermented concentration. These results were obtained with the follow milk product comprising a step wherein milk is fermented ing experimental set-up: using a starter culture, wherein: 0201 The following two batches of commercial cultures 0218 (i) the starter culture comprises Streptococcus were fermented in the same type of milk base: thermophilus and Lactobacillus delbrueckii Subsp. bul (0202 Mild 2.0 (from Chr. Hansen, Denmark); garicus, (0203 Culture type Premium 1.0 (70%)+Culture type 0219 (ii) the fermentation is conducted at a tempera SSC17 (30%) (both from Chr. Hansen, Denmark). ture between 22 and 45° C., 0204. The milk base used was skimmed milk; fermenta 0220 (iii) the fermentation is terminated by a decrease tion was carried out at a temperature of 40° C. of the concentration of a carbohydrate during fermen 0205 Both batches were fermented to pH 4.60. Samples tation, were taken for texture analysis and fermentation was con 0221 (iv) the decrease is at least also caused by the tinued until a pH of 4.45 was reached. Again, the texture of metabolic activity of the lactic acid bacteria, the product was analyzed. The four yoghurt curds (two 0222 (v) at least part of the whey is separated from the different cultures at two different pH values, 4.60 and 4.45) fermented milk product, and are processed in a Post Treatment Unit (FH Scandinox) after 0223 wherein the fermented milk product prior to reaching the specified pH value. Different back pressures separation of the whey is characterized in that the pH were applied to the cultures, between 4 to 9 or from 4 to 12 value of the product is maintained within a range of 0.3 bars. Shear stress and complex modulus were determined pH units or maintained within a range of 0.1 pH units using a rheometer. if stored, processed or maintained after termination of 0206 FIGS. 8 to 11 show that both shear stress and the fermentation at the temperature used for fermenta complex modules are higher at pH 4.45 than at pH 4.60. tion in step (ii) over a period of 20 hours. US 2017/O 135360 A1 May 18, 2017
0224 3. A method of producing a fermented milk product typically spiral filters or ceramic filters. In a further alter comprising: native plate and frame filters are used for applications with 0225 (a) a step, wherein milk is fermented using a high Solid protein and fat content, e.g. full fat cream cheese. starter culture, wherein: 0244. The strained yoghurt is characterized by a protein 0226 (i) the fermentation is initiated using a milk content of 6 to 13%. The product may further have a fat and a starter culture, wherein the lactose concentra content of 0 to 10%. tion in the milk is in the range of 5 to 100 mg/g at the 0245 Alternatively, the fermented product may be a start of the fermentation quark or fromage frais. Quark and fromage frais are gener 0227 (ii) the starter culture comprises Streptococ ally characterized by a protein content of 6 to 10%. Quark cus thermophilus and Lactobacillus delbrueckii and fromage frais may further be characterized by a fat Subsp. bulgaricus, content of 6 to 10%. 0228 (iii) the fermentation is conducted at a tem 0246. In a further alternative, the fermented product is perature between 22 and 45° C. cream cheese. Cream cheese may be characterized by a 0229 (iv) the fermentation is terminated by a protein content of 6 to 13%. Cream cheese may further be decrease of the concentration of the lactose during characterized by a fat content of 0 to 35%. fermentation, 0247. In preferred embodiments of this aspect of the 0230 (v) the decrease is at least also caused by the present invention, the separation of at least part of the whey metabolic activity of the lactic acid bacteria, and from the fermented milk product is carried out without 0231 (b) a step, wherein at least part of the whey is active cooling of the fermented milk product. This does not separated from the fermented milk product at a tem mean that the product is maintained at the temperature used perature between 15 and 45° C., for fermentation, but simply that the product is not subject 0232 (c) a step, wherein the fermented product is to cooling. packaged at a temperature between 15 and 45° C. 0248. In a further embodiment, the above method of 0233 wherein the fermented milk product is charac producing a strained fermented milk product is carried out terized in that the pH value of the product is maintained using a starter culture comprising a lactose negative strain. within a range of 0.3 pH units or maintained within a As explained in part B above, the use of lactose negative range of 0.1 pH units if stored after termination of the strains for controlling post acidification is particularly fermentation at the temperature used for fermentation advantageous. Accordingly, methods of producing a strained in step (ii) over a period of 20 hours. fermented milk product are provided which use a starter 0234 4. The method of any one of embodiments 1 to 3, culture comprising one or more LAB selected from Strains wherein the separation is carried out by filtration, includ CHCC18944, CHCC17861 and CHCC17862. ing filtration using a nozzle separator, centrifuge or ultra 0249. This embodiment is particularly suitable for the filtration with spiral filters or ceramic filters. production of Greek yoghurt, quark or cream cheese. 0235. 5. The method of any one of embodiments 1 to 4, wherein the strained fermented milk product is charac (G) Methods of Producing a Pasta Filata Cheese Product terized by a protein content of 6 to 13% and a fat content Using One of Methods (A) to (E). of 0 to 10%. 0250. The methods of the present invention as described 0236 6. The method of any one of embodiments 1 to 5, in sections (A) to (E) above are particularly suitable for the wherein the strained fermented milk product is charac preparation of a pasta filata cheese. A pasta filata cheese is terized by a protein content of 6 to 10%. a cheese produced by a method comprising a heat treatment 0237 7. The method of any one of embodiments 1 to 6, step of the curd. The heat treatment can be carried out in a wherein the strained fermented milk product is a yoghurt, number of different ways, including steeping the curds in hot a Greek yoghurt, Labneh, Quark, fromage frais or cream water or whey. In another alternative steam is injected into cheese. the curds. The heat treatment step imparts the finished 0238 8. The method of any one of embodiments 1 to 7, cheese with a fibrous structure and particular stretching wherein the separation of at least part of the whey from properties. Typical pasta filata cheeses are MoZZarella and the fermented milk product is carried out without active Provolone, Caciocavallo, Pallone di Gravina, and Scamorza. cooling of the fermented milk product. 0251 According to this aspect of the invention the fol 0239) 9. The method of any one of embodiments 1 to 8, lowing embodiments are amongst others provided: wherein the starter culture comprises a lactose negative 0252) 1. A method of producing a pasta filata cheese strain. comprising a step, wherein milk is fermented, wherein: 0240 10. The method of any one of embodiments 1 to 9, 0253 (a) the fermentation is initiated by a starter wherein the starter culture comprises one or more LAB culture, which starter culture comprises lactic acid selected from strains CHCC18944, CHCC17861 and bacteria capable of metabolizing one or several carbo CHCC17862. hydrates present in the milk, 0241 11. Fermented milk product obtainable by a 0254 (b) the fermentation is terminated by a decrease method of any one of embodiments 1 to 10. of the concentration of the one or several carbohydrates 0242 12. Fermented milk product according to embodi during fermentation, and ment 11, wherein the fermented milk product is a yoghurt, 0255 (c) the decrease is at least also caused by the a fruit yoghurt, a yoghurt beverage or a cheese. metabolic activity of the lactic acid bacteria; 0243 The separation of the whey may be carried out 0256 (d) the fermented product is subjected to a heat using a nozzle separator (centrifuge) commonly used in the treatment, for example by placing the fermented prod art to separate whey from fermented milk products. Alter uct in hot water or injecting steam into the fermented natively or additionally, ultra-filtration systems can be used, product. US 2017/O 135360 A1 May 18, 2017 11
0257 2. A method of producing a pasta filata cheese 0274 6. Method of producing a pasta filata cheese comprising a step, wherein milk is fermented, wherein: according to one of embodiments 1 to 3, wherein the 0258 (i) the starter culture comprises Streptococcus lactic acid bacteria are capable to metabolize lactose and thermophilus and Lactobacillus delbrueckii Subsp. bul galactose but glucose deficient. garicus, 0275 7. Method of producing a pasta filata cheese 0259 (ii) the fermentation is conducted at a tempera according to one of embodiments 1 to 6, wherein the ture between 22 and 45° C., fermentation is carried out in the presence of lactase in an 0260 (iii) the fermentation is terminated by a decrease initial concentration of 500 to 5000 NLU/I. of the concentration of a carbohydrate during fermen 0276 8. Method of producing a pasta filata cheese tation, according to one of embodiments 1 to 7, wherein the 0261 (iv) the decrease is at least also caused by the termination of fermentation is caused by a decrease of the metabolic activity of the lactic acid bacteria, and total concentration of the one or several carbohydrates 0262 wherein the fermented milk product is charac that can be metabolized by the lactic acid bacteria to a terized in that the pH value of the product is maintained value of less than 30 mg/g, including a range between 25 within a range of 0.3 pH units or maintained within a mg/g and 0.01 mg/g, or a range between 5 mg/g and 0.01 range of 0.1 pH units if stored after termination of the mg/g. fermentation at the temperature used for fermentation 0277 9. Method of producing a pasta filata cheese in step (ii) over a period of 20 hours; and wherein the according to one of embodiments 1 to 8, wherein the pH fermented product is subjected to a heat treatment, for value of the fermented product is maintained within a example by placing the fermented product in hot water range of 0.3 pH units over a period of 20 hours when or injecting steam into the fermented product. stored at the temperature for fermentation. 0263. 3. A method of producing a pasta filata cheese 0278 10. Method of producing a pasta filata cheese comprising a step, wherein milk is fermented, wherein: according to one of embodiments 1 to 9, wherein the 0264 (a) a step, wherein milk is fermented using a fermentation is carried out at a temperature between 30 starter culture, wherein: and 45° C. 0265 (i) the fermentation is initiated using a milk and a starter culture, wherein the lactose concentra 0279 11. Method of producing a pasta filata cheese tion in the milk is in the range of 5 to 100 mg/g at the according to one of embodiments 1 to 10, wherein after start of the fermentation termination of fermentation the fermented product is 0266 (ii) the starter culture comprises Streptococ packaged at a temperature between 15 and 45° C. cus thermophilus and Lactobacillus delbrueckii 0280 12. Method of producing a pasta filata cheese Subsp. bulgaricus, according to one of embodiments 1 to 11, wherein the 0267 (iii) the fermentation is conducted at a tem fermented product perature between 22 and 45° C. 0281 is characterized in that the pH value of the 0268 (iv) the fermentation is terminated by a product is maintained within a range of 0.3 pH units if decrease of the concentration of the lactose during stored after termination of the fermentation for a period fermentation, of 6 months or is characterized in that the pH value of 0269 (v) the decrease is at least also caused by the the product is maintained within a range of 0.3 pH units metabolic activity of the lactic acid bacteria, and if stored after termination of the fermentation for a 0270 (b) a step, wherein the fermented product is period of 12 months. packaged at a temperature between 15 and 45° C. 0282) 13. Fermented milk product obtainable by a 0271 wherein the fermented milk product is charac method of any one of embodiments 1 to 12, wherein the terized in that the pH value of the product is maintained milk product is a pasta filata cheese. within a range of 0.3 pH units or maintained within a 0283. 14. Fermented milk product according to embodi range of 0.1 pH units if stored after termination of the ment 13, wherein the pasta filata cheese is selected from fermentation at the temperature used for fermentation Mozzarella, Provolone, Caciocavallo, Pallone di Gravina, in step (ii) over a period of 20 hours; and wherein the and Scamorza. fermented product is subjected to a heat treatment, for 0284. In more detail, the method of producing a pasta example by placing the fermented product in hot water filata cheese of the present invention may comprises the or injecting steam into the fermented product. following method steps: 0272 4. Method of producing a pasta filata cheese 0285 obtaining a milk product with standardized pro according to one of embodiments 1 to 3, wherein the tein, fat and Solids; lactic acid bacteria are capable to metabolize lactose and 0286 adjusting temperature to coagulation tempera glucose and wherein before addition of the starter culture ture (32-38° C.); the concentration of the lactose in the milk is below 200 mg/g, for example between 100 mg/g and 5 mg/g, or 0287 addition of the Acidifix culture (Streptococcus between 50 mg/g and 5 mg/g, or between 25 mg/g and 5 thermophilus CHCC17861 and Lactobacillus del mg/g. brueckii subsp. bulgaricus CHCC18944); 0273 5. Method of producing a pasta filata cheese 0288 addition of coagulant; according to one of embodiments 1 to 3, wherein the 0289 coagulation of the milk in 15-60 minutes, cutting lactic acid bacteria are not capable to metabolize lactose and stirring of the curd, and wherein before addition of the starter culture the total 0290 optionally heating (scalding) of the curd: concentration of carbohydrates that can be metabolized by 0291 acidification of the curd (either covered by whey the lactic acid bacteria in the milk is below 45 mg/g. or drained from whey); US 2017/O 135360 A1 May 18, 2017
0292 stretching of the curd in hot water or by steam FIGURE LEGENDS injection (in a dry stretcher) or by other means of 0325 FIG. 1 compares the acidification activity of the S. heating and mechanically working the curd, thermophilus strain CHCC6008 when used to inoculate milk 0293 forming and cooling the cheese; containing 1 and 3% lactose. 0294 cooling and salting the cheese; 0326 FIG. 2 compares the acidification activity of S. 0295 packing the cheese. thermophilus CHCC15914 and Lactobacillus delbrueckii 0296. In an alternative embodiment, the method of pro ssp. bulgaricus CHCC10019 to the acidification activity of ducing a pasta filata cheese of the present invention may S. thermophilus CHCC17862 and lactobacillus delbrueckii comprises the following method steps: ssp. bulgaricus CHCC18944 when used to ferment milk 0297 obtaining a milk product with standardized pro Supplemented with Sucrose. tein, fat and Solids; 0327 FIG. 3 shows the acidification activity of different 0298 addition of sucrose sufficient for the acidification ratios of S. thermophilus CHCC17861 and Lactobacillus culture; delbrueckii ssp. bulgaricus CHCC18944 when used to fer 0299 adjusting temperature to coagulation tempera ment milk Supplemented with Sucrose and compared to the ture (32-38° C): acidification activity of S. thermophilus CHCC15914. 0300 addition of the Acidifix culture (Streptococcus 0328 FIG. 4 shows post acidification, i.e. the develop thermophilus CHCC17861 and Lactobacillus del ment of pH in two different fermented milk products after brueckii subsp. bulgaricus CHCC18944); fermentation using the method of the invention (Acidifix) and a prior art method (YFL-904). 0301 addition of coagulant; 0329 FIG. 5 shows the development of pH in a culture 0302) coagulation of the milk in 15-60 minutes, cutting fermented with Sweety after termination of fermentation at and stirring of the curd, 6° C. for 42 days (i.e. post acidification). The Figure shows 0303 optionally heating (scalding) of the curd: that the pH varies less than 0.1 pH units under these 0304 acidification of the curd (either covered by whey conditions. or drained from whey); 0330 FIG. 6 shows the development of the pH in differ 0305 cooling of the acidified curd: ent cultures fermented with and without lactase during 0306 stretching of the curd in hot water or by steam storage at cooling temperature over a period of 48 days. injection (in a dry stretcher) or by other means of 0331 FIG. 7 shows an overview over methods using heating and mechanically working the curd, concentration of protein prior to fermentation (left hand 0307 forming and cooling the cheese; side) and concentration of protein after fermentation by 0308 cooling and salting the cheese; separation using centrifugation or ultrafiltration (right hand side). 0309 packing the cheese. 0332 FIG. 8 shows the effect of pH on shear stress during 0310. In an alternative embodiment, the method of pro separation of whey from a milk product after fermentation ducing a pasta filata cheese of the present invention may with SSC17. comprises the following method steps: 0333 FIG.9 shows the effect of pH on shear stress during 0311 obtaining a milk product with standardized pro separation of whey from a milk product after fermentation tein, fat and Solids; with Mild2.0. 0312 addition of sucrose sufficient for the acidification 0334 FIG. 10 shows the effect of pH on complex modu culture; lus or gel firmness during separation of whey from a milk 0313 adjusting temperature to coagulation tempera product after fermentation with SSC17. ture (32-38° C): 0335 FIG. 11 shows the effect of pH on complex modu 0314 addition of the Acidifix culture (Streptococcus lus or gel firmness during separation of whey from a milk thermophilus CHCC17861 and Lactobacillus del product after fermentation with Mild2.0. brueckii subsp. bulgaricus CHCC18944); 0315 addition of coagulant; LAB STRAINS 0316 coagulation of the milk in 15-60 minutes, cutting 0336. The subsequent examples use CH strains, some of and stirring of the curd, which have been deposited for prior patent applications of 0317 optionally heating (scalding) of the curd: Chr. Hansen. Further information on the strains is provided 0318 acidification of the curd (either covered by whey by the respective patent application and the deposit as or drained from whey); follows: 0319 storing the acidified curd at a temperature 0337 Streptococcus thermophilus CHCC6008 was between 6 and 25° C.; deposited for WO2011/000879 with DSMZ-Deutsche Sam 0320 stretching of the curd in hot water or by steam mlung von Mikroorganismen und Zellkulturen GmbH, injection (in a dry stretcher) or by other means of Inhoffenstr. 7B, D-38124 Braunschweig on 2006 Mar. 23 heating and mechanically working the curd, under the accession no. DSM 18111. 0338 Lactobacillus delbrueckii ssp. bulgaricus 0321 forming and cooling the cheese; CHCC10019 was deposited for WO2011/000879 with 0322 cooling and salting the cheese; DSMZ-Deutsche Sammlung von Mikroorganismen und 0323 packing the cheese. Zellkulturen GmbH, Inhoffenstr. 7B, D38124 Braunschweig 0324. In all embodiments the cheese may be sliced, on 2007 Apr. 3 under the accession no. DSM 19252. shred, chunk, dice or portioned by other means before 0339 Streptococcus thermophilus CHCC15757 was packing. deposited for WO2013160413 with DSMZ-Deutsche Sam US 2017/O 135360 A1 May 18, 2017 mlung von Mikroorganismen und Zellkulturen GmbH, 0350 Both wild type strains possess B-galactosidase Inhoffenstr. 7B, D-38124 Braunschweig on 2012 Apr. 3 activity, and wild type colonies appeared blue due to the under the accession no. DSM 2.5850. activity of the B-galactosidase. 0340 Streptococcus thermophilus CHCC16404 was 0351. From CHCC10019 one lactose deficient mutant deposited for WO2013160413 with DSMZ-Deutsche Sam was isolated and designated CHCC18944 (this mutant was mlung von Mikroorganismen und Zellkulturen GmbH, identified as CHCC18994 in European Patent Application Inhoffenstr. 7B, D-38124 Braunschweig on 2012 Dec. 12 14173196; but the internal Accession No. of the Applicant under the accession no. DSM 26722. was changed and now is CHCC18944; the DSMZ Accession 0341 Lactobacillus delbrueckii subsp. bulgaricus No. was not changed and thus still is DSM28910). CHCC16159 was deposited for WO2013160413 with 0352 From CHCC15914 two lactose deficient mutants DSMZ-Deutsche Sammlung von Mikroorganismen und were isolated and designated CHCC17861 and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunsch CHCC17862, respectively. weig on 2012 Sep. 6 under the accession no. DSM 26420. 0353. The growth characteristics of the isolated lactose 0342. The remaining strains used in the subsequent deficient mutants were determined as follows: examples have either been deposited for the present appli 0354 Phenotype of LB CHCC18944: lac-, suc-, gal cation or are commercially available from Chr. Hansen. glC+ 0355 Phenotype of ST CHCC17861: lac-, suc+, gal+, Example 1: Methods for Producing a Fermented glC+ Milk Product Using Low Lactose Milk 0356 Phenotype of ST CHCC17862: lac-, suc+, gal+, glC+ 0343 Customized milk containing 1% lactose (10 g/L) 0357 The complete lactose operon was sequenced for all was obtained (from Select Milk Producers, Inc., Texas, three mutants and compared with the respective wild type USA). A milk containing 3% lactose (30 g/L) was prepared strain to reveal the mutation type. by adding 2% lactose (20 g/L) to the 1% lactose milk. 0358 Comparison with the mother strain CHCC15914 0344) The milk was inoculated with S. thermophilus revealed that CHCC17861 had an extra “T” nucleotide in the strain CHCC6008 (0.01% F-DVS) and maintained at a beginning of the lacz gene (coding for the B-galactosidase) temperature of 37° C. for 20 hours. The acidification (pH leading to a stop codon in the coding sequence a few value) was determined automatically over time. nucleotides downstream of the mutation. CHCC17862 (0345. The result is shown in FIG. 1. The fermentation showed a deletion of one nucleotide, also interrupting the using 1% lactose shows a close to ideal acidification profile. coding sequence of the lac7 gene. Initially, the product is acidified at a high rate, but acidifi 0359 For CHCC18944 a mutation inside the lacZ gene cation stops abruptly at pH 4.8 after about 6 hours and the was identified. This resulted in an exchange of 8 nucleotides pH of the culture remained unchanged for the next 14 hours (5'-CTT CCA AGC-3' to 5'-CGC TAC TAT-3') and conse although the culture was maintained at a temperature of 37 quently a change of 3 amino acids (Leu-Pro-Ser to Arg-Tyr C. This shows that acidification is completely terminated. Tyr) within lacZ. explaining the lactose deficient phenotype. 0346. The fermentation using 3% lactose was used as a 0360 All mutants when used as single strains or in control. Initially the product is acidified at the same rate as combination (ST+LB), acidify milk depending on the addi the fermentation based on 1% lactose. This shows that the tion of a fermentable carbohydrate different from lactose. lactose concentration of 1% (10 mg/g) is not too low to The acidification activity of the lactose deficient cultures inhibit fermentation in the initial phase. were for example determined using over-night cultures in 0347 But the fermentation using 3% lactose is not ter MRS (LB wt and LB lac-mutant); M17 with 1% lactose (ST minated at pH 4.8 but continues acidification until a pH of wt); or M17 with different concentrations of sucrose (1% about 4.5 is reached at which state the presence of the acid and 0.5%; fermentation with the ST lac-mutant). The milk produced by the bacteria inhibits further fermentation. As a was inoculated and fermentation was monitored by pH consequence, the fermentation proceeds at a low rate until a development at 37° C. pH of about 4.2 is reached. 0361. The pH development under fermentation tempera 0348. These results prove that the carbohydrate (here ture over 20 and 40 hours is illustrated in FIGS. 2 and 3 lactose) concentration can Surprisingly be used to control the respectively and shows that sucrose is metabolized by the termination of fermentation. This way of proceeding allows lactose deficient strains yielding a fermentation process that processes, wherein the desired pH value is obtained by is nearly as fast as the process caused by the parental LAB fermentation and no post acidification is observed after having the ability to metabolize lactose. The sucrose driven wards. fermentation process is immediately terminated and enters a flat line when the sucrose was depleted. After termination of the fermentation caused by carbohydrate depletion the pH Example 2: Methods for Producing a Milk Product remained stable at about pH 4.5. A very stable final pH was Using Lactose Deficient LAB found when CHCC17861, CHCC17862, or CHCC18944, 0349 Lactose deficient mutants were isolated from the were used as single strains, or as a mix of one of the ST EPS positive strains S. thermophilus (ST) CHCC15914 and mutants together with the LB mutant (mix is shown in FIG. Lactobacillus delbrueckii ssp. bulgaricus (LB) 3). This resembles the starter culture to be used in a CHCC10019. The strains were selected after UV-mutagen fermentation method for producing a typical yoghurt. esis as white colonies (indicating a lactose deficient pheno 0362. No significant difference between the strains type) on M17 with 1% lactose and 200 mg/ml X-Gal for CHCC17861 and CHCC17862 could be observed. The CHCC15914, resp. MRS agar plates with 1% lactose and addition of sucrose therefore provides a very precise control 200 mg/ml X-Gal for CHCC10019. of acidification activity. US 2017/O 135360 A1 May 18, 2017
0363. In some fermentations the formation of a “shoul Process: der” was observed within the pH-curves of the mixed cultures indicating that the metabolism shifted before the 0372 Homogenization pressure: 150 bar-i-30 bar (total acid production completely stopped (FIG. 3). To investigate 180 bar) this further the ratio of ST:LB was changed and this led to 0373 Pasteurization condition: 95° C./4 min a change in the shoulder formation. Decrease of the con 0374 cooling temperature: less than 10° C. centration of the lac-ST and increase of the lac-LB strain 0375 Fermentation: resulted in a reduction of the pH shoulder and an even more 0376) Fermentation temperature: 43° C. for YF-L904 horizontal pH-"curve'. and 39° C. for Acidifix 0364 Interestingly, the use of 100% ST CHCC17861, on 0377 End pH: 4.35+0.05 the other hand, leads to a completely interrupted acid 0378 Break the curd manually when desired pH is production when Sucrose is depleted and eliminates the pH achieved. shoulder, but the pH drop stops at a 0.3 point higher pH 0379 Add 12% of sugar syrup into 88% of fer value (FIG. 3). mented milk base. Manually stir to mix it well. 0365. In all cases the pH remained stable at 4.75 up to the 0380. Thermise the yoghurt through GEA pilot end of the fermentation period (48 hours). plant: 0366. In ST/LB co-fermentation the Lactobacillus del (0381 Homo pressure: 0 bar brueckii subsp. bulgaricus part is responsible for the final 0382. Thermisation condition: 65° C./30s pH drop and also for a major part of the post acidification. (0383 Filling into bottles (filling temperature: 28° For this reason the concentration of LB is lower than the C-33° C). concentration of ST in most yoghurt fermentation processes. 0384 The post acidification activity was analyzed for 0367 This shows that the pH value can be completely both products directly after fermentation, after thermisation controlled by the concentration of the added sucrose or and after 6 days storage at different temperatures. Results are another fermentable carbohydrate, as the Lb. Bulgaricus can illustrated in FIG. 4 and show high stability of pH of the easily be increased. product obtained with Acidifix during processing and Stor 0368. The lac-culture will not only result in a higher final age. pH after e.g. 6 hours, but will also have a significantly lower post acidification and thus extended shelf life. Example 4: Methods of Producing a Fermented 0369. In some experiments it was observed that the pH Milk Product Using Glucose Deficient LAB was very stable for about 5 hours after termination of fermentation and then slightly declined over the next 10 Material and Methods hours (data not shown). This is apparently due to spontane (0385 Milk base (1.0% fat & 4.5% protein): ous revertants, i.e. LAB gaining the ability to utilize lactose Ingredients: Mix of commercial milk (1.5% fat--0.5% fat-- by spontaneous mutation. skim milk powder to reach the required fat and protein level) 9.5% w/w skimmed milk powder+90.5% tap water Example 3: Methods of Producing a Fermented Commercial milk: Arla Harmonie minimaelk (0.5% fat) and Milk Product with an Extended Shelf Life Arla Harmonie letmaelk (1.5% fat) 0370 Yoghurt with an extended shelf life (ESL yoghurt) Skimmed milk powder: Arla Foods, Gin 990214 was produced by fermenting milk with the commercially Procedure: Mix milk and powder stir, heat treat 90° C. 20 available FD-DVSYFL-904 and in a separate fermentation min with the new culture F-DVS Acidifix 2.0 (containing Strep tococcus thermophilus CHCC17861 and Lactobacillus del Lactose: brueckii subsp. bulgaricus CHCC18944, described in Example 2). Product: Lactosemonohydrate (Gin 500449, Batch 0005078607) TABLE 1. Producer: German Lac-Sachsenmilch Recipe ESL yoghurt: Milk Base 0386. Added as a 19% w/w solution to the yoghurt base, to obtain the right level as prescribed Ingredients Specification Dosage Recipe: 0.6 kg lactose monohydrate--2.4 kg tap water Fresh Milk Meiji, 2 L bottle 97.95% Process: Heat while stirring, before pasterurization (95°C., Thermex Modified starch, Ingredion 1.00% LM 106-AS-YA Pectin, CP Kelco O.15% 5 min) Kelcogel YSS Gellan Gum, CP Kelco O.O.3% Sugar Refined sugar, Phoon Huat O.87% Starter Cultures: Cultures FD-DVS-YF-L904 100 umt F-DVS-Acidifix 100 umt (0387 F.-DVS Sweety 1.0 consists of a blend of the following strains Milkoscan analysis: Fat: 3.70% Protein: 3.05% 12% sugar syrup ws added to the white mass after fermentation 0388 2-deoxy-glucose resistant strains 0389 Streptococcus thermophilus CHCC16731 (hy 0371. The following parameters were used for fermenta per-lactose fermenting and glucose secreting mutant of tion and processing: CHCC11976). Mixing temperature: 45° C.-50° C. 0390 Streptococcus thermophilus CHCC15757 (2-de Hydration time: 20 minutes oxyglucose resistant mutant of CHCC14944). US 2017/O 135360 A1 May 18, 2017 15
0391 Streptococcus thermophilus CHCC16404 (hy Starter Cultures: per-lactose fermenting and glucose secreting mutant of CHCC15757). F-DVS YF-L706, Gin 685141 0392 Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 (2-deoxyglucose resistant mutant of F-DVS YF-L901, Gin 685142 CHCC10019). Low Post Acidification Properties of Hyper-Lactose Fer F-DVS YoFlex(R) Mild 1.0, Gin 702897 menting and Glucose Secreting Mutants of Streptococcus thermophilus and Lactobacillus delbrueckii Subsp. Bulga F-DVS YoFlex(R) Creamy 10, Gin 706168 ricus. 0393. Selection of a hyper-lactose fermenting and glu F-DVS YoFlex(R) Premium 1.0, Gin 706161 cose secreting mutant of Streptococcus thermophilus and Lactobacillus delbrueckii Subsp. bulgaricus were carried as 04.01 All from Chr. Hansen A/S described in WO2013/160413 entitled “Use of lactic acid 0402. The fermentations were done in 3 l scale. The bacteria for preparing fermented food products with cultures were inoculated at 0.02%, and the samples were increased natural Sweetness'. fermented at 43° C. Lactase in a dosage of 3500 NLU/I was 0394. To show the low post acidification properties of the added to the milk base together with the culture. Acidifica culture in a standard setup for yoghurt production, 0.024% tion was monitored with a pH-Meter 1120 (Mettler-Toledo of a Frozen Direct Vat Set culture (F-DVS) of Sweety 1.0 AG, 52120653), and fermentation stopped at pH 4.55. The culture blend was used to inoculate 31 of milkbase and the fermented milk products were stirred in a standardized way, milk was fermented at 43° C. and further pressurized and cooled (2 bar, 25°C.), before 0395 Acidification was followed with a CINAC pH storing at 6° C. pH of the samples were then monitored at 1, logger (Alliance instruments) with standard pH electrodes 7, 14, 21 and 48 days using a pH-Meter 1120 (Mettler and CINAC 4.0 software. When the pH reached 4.55 at 43° Toledo AG, 52120653). Concentrations of lactose, glucose C. coagulation of the milk had taken place. The yoghurt was and galactose (mg/g) in yoghurt on day 1 after fermentation subsequently cooled and incubated at 7+1.5°C. for 42 days. were determined using HPLC with a Dionex CarboPac PA The pH development was subsequently followed over a 20 3* 150 mm column (Thermo Fisher Scientific, product period of 42 days (FIG. 5). As can be seen from FIG. 5 the number 060142). pH development after 42 days of storage of 7-t1.5°C. is less 0403. Results are shown in FIG. 6, which illustrates the than 0.1 pH units confirming an extremely low level of post development of pH value during storage and thus post acidification. acidification. Further results are shown table 1, below which 0396 This data show that yoghurt blends of the glucose provides an overview over the difference in the pH value secreting strains and the use of the methods of the present obtained in the yoghurts produced with and without lactase invention will coagulate milk, maintain the final pH very with different cultures. The difference is caused by post stable for 43 days at 7+1.5°C. and increase the Sweetness of acidification and was determined using F-DVS YoFlex(R) the fermented product. Mild 1.0 yoghurt as example: F-DVS YoFlex(R) Mild 1.0 0397 Mixed cultures of glucose secreting Streptococcus yoghurt with 3500 NLU/L lactase had a pH after 21 days of and Lactobacillus delbruecki Subsp. bulgaricus metabolize 4.44, while F-DVS YoFlex(R) Mild 1.0 yoghurt without all or almost all lactose present in milk during fermentation. lactase had a pH of 4.36. Fermentation was stopped at 4.55 As a consequence, the LAB no longer cause post acidifica for both yoghurts. The post acidification for the lactase tion. treated yoghurt was 4.55-4.44=0.11, for the yoghurt without lactase it was 4.55-4.36–0.19. The difference in post acidi Example 5: Low Post Acidification when Using fication between yoghurt fermented with F-DVS YoFlex(R) Lactase in the Fermentation of Yoghurt Mild 1.0 with and without 3500 NLU/L lactase is thus 0398. It was surprisingly found that the addition of O.19-0.11=0.08. lactase to a method of preparing a yoghurt significantly reduces post acidification in comparison to a yoghurt made TABLE 2 without lactase. Difference in post acidification between yoghurts produced with the cultures in the table at 0.02%, Materials and Methods with and without 3500 NLUL lactase. 0399 Milk base (0.1% fat & 4.5% protein): Culture Day 21 Day 48 Ingredients: Mix of commercial milk (0.1% fat) and F-DVSYF-L706 O.OO O.OO skimmed milk powder to reach the protein level. F-DVSYF-L901 O.04 O.04 F-DVS YoFlex (RCreamy O.10 O.O7 Commercial milk: Arla skummetmaelk (0.1% fat) F-DVSYoFlex (RPremium 1.0 O.O8 O.08 Skimmed milk powder: Milex 240, Arla Foods, lot 990214 F-DVS YoFlex (RM 1.0 O.O8 O.11 Procedure: Mix powder and milk, hydration at 5° C. over night, heat treatment 90° C./20 min. 04.04 The data presented shows that select Chr. Hansen Lactase: commercial cultures in combination with lactase can be used to obtain yoghurt with reduced post acidification. F-DVS YoFlex(R) Mild 1.0, F-DVSYoFlex(R) Creamy 1.0 and F-DVS Product: Ha-Lactase 5200 (Gin 450805) YoFlex(R) Premium 1.0, all show low post acidification. The 0400 Producer: Chr. Hansen A/S reduction of post acidification is less pronounced in the US 2017/O 135360 A1 May 18, 2017
culture YF-L901 and no reduction of post acidification is ganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 observed for F-DVS YF-L706. Braunschweig, on 2014-06-12 under the accession no. DSM 28953. TABLE 3 0413. The deposit was made according to the Budapest treaty on the international recognition of the deposit of Concentrations of lactose, glucose and galactose (mg/g) in yoghurt produced with and without 3500 NLUL lactase microorganisms for the purposes of patent procedure. On day 1 after fermentation determined using HPLC. REFERENCES Galactose Glucose Lactose Culture (mg/g) (mg/g) (mg/g) 0414 US2010/0021586 F-DVSYF-L706 9.2 0.4 423 0415 WO2006/042862A1 F-DVSYF-L706 + 3500 NLU lactase 26.2 21.8 2.0 0416 WO2010/139765 F-DVSYF-L901 10.6 1.6 39.4 0417 WO2013/169205 F-DVSYF-L901 + 3500 NLU lactase 26.1 21.4 2.1 F-DVS YoFlex (RCreamy 1.0 7.7 O.O 43.4 0418 WO2011/000879 F-DVS YoFlex (RCreamy 1.0 + 23.1 21.2 5.3 0419 Pool et al., Metabolic Engineering, vol. 8, 2006, 3500 NLU lactase 456-464 F-DVSYoFlex (RPremium 1.0 7.7 O6 41.7 F-DVSYoFlex (RPremium 1.0 + 25.1 23.8 2.0 1-18. (canceled) 3500 NLU lactase 19. A method of producing a fermented milk product F-DVS YoFlex (RM 1.0 7.7 O6 42.2 comprising a milk fermentation step, comprising: F-DVSYoFlex (RMild 1.0 + 25.4 24.1 2.9 3500 NLU lactase (a) initiating milk fermentation by adding to milk a starter culture comprising lactic acid bacteria capable of metabolizing one or more carbohydrates present in the 04.05 Table 3 shows that the cultures fermented in the milk, and presence of lactase have very low residual lactose but higher wherein the milk fermentation is terminated by a decrease residual glucose and galactose concentrations. The fact that in concentration of the one or more carbohydrates, post acidification is observed for the strains can thus be wherein the decrease is at least partly caused by the explained by the high total residual amount of carbohydrates lactic acid bacteria metabolizing the one or more car available for the metabolism of the LAB. bohydrates. 0406. The extent of post acidification is still very low and thus Surprising in view of the residual concentrations of 20. The method of claim 19, wherein carbohydrates. The low post acidification appears to be (i) the starter culture comprises Streptococcus thermophi caused by the need for a metabolic shift of the carbohydrate lus and Lactobacillus delbrueckii Subsp. bulgaricus, Source in the microorganisms. In other words, the cultures and showing low post acidification contain strains that struggle (ii) the milk fermentation is conducted at a temperature with the shift from lactose to glucose as the carbohydrate between 22 and 45° C., SOUC. wherein the pH of the fermented milk product is stable Such that, upon storage for 20 hours after termination of Deposit and Expert Solution fermentation at the fermentation temperature, the pH is 0407. The applicant requests that a sample of micro maintained within a range of 0.3 pH units. organisms deposited for the present application as described 21. The method of claim 20, wherein the pH of the below may only be made available to an expert, until the fermented milk product is stable such that, upon storage for date on which the patent is granted. 20 hours after termination of fermentation at the fermenta 0408 Streptococcus thermophilus CHCC16731 was tion temperature, the pH is maintained within a range of 0.1 deposited with DSMZ-Deutsche Sammlung von Mikroor pH units. ganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 22. The method of claim 20, wherein the milk comprises Braunschweig, on 2014-06-04 under the accession no. DSM lactose at a concentration of from 5 to 100 mg/g at the start 28889. of the fermentation. 04.09 Streptococcus thermophilus CHCC15914 was 23. The method of claim 20, further comprising packag deposited with DSMZ-Deutsche Sammlung von Mikroor ing the fermented milk product at a temperature between 15 ganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 and 45° C. Braunschweig, on 2014-06-12 under the accession no. DSM 24. The method of claim 19, wherein the lactic acid 28909. bacteria are capable of metabolizing lactose and glucose, 0410 Lactobacillus delbrueckii ssp. bulgaricus and wherein, prior to adding the starter culture, the milk CHCC18944 was deposited with DSMZ-Deutsche Sam comprises lactose at a concentration selected from below mlung von Mikroorganismen und Zellkulturen GmbH, 200 mg/g, between 100 mg/g and 5 mg/g, between 50 mg/g Inhoffenstr. 7B, D-38124 Braunschweig, on 2014 Jun. 12 and 5 mg/g, and between 25 mg/g and 5 mg/g. under the accession no. DSM 28910. 25. The method of claim 19, wherein the lactic acid 0411 Streptococcus thermophilus CHCC17861 was bacteria are not capable of metabolizing lactose and deposited with DSMZ-Deutsche Sammlung von Mikroor wherein, prior to adding the starter culture, the milk com ganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 prises carbohydrates that can be metabolized by the lactic Braunschweig, on 2014-06-12 under the accession no. DSM acid bacteria at a total concentration of below 45 mg/g. 28952. 26. The method of claim 19, wherein the lactic acid 0412 Streptococcus thermophilus CHCC1 7862 was bacteria are capable of metabolizing lactose and galactose deposited with DSMZ-Deutsche Sammlung von Mikroor but are glucose deficient. US 2017/O 135360 A1 May 18, 2017
27. The method of claim 19, wherein the fermentation is 39. An isolated LAB strain selected from: carried out in the presence of lactase at an initial concen (a) a Streptococcus thermophilus strain selected from: tration of 500 to 5000 NLU/1. (i) the strain deposited with DSMZ-Deutsche Sam 28. The method of claim 19, wherein the fermentation is mlung Von Mikroorganismen und Zellkulturen terminated by a decrease in total concentration of the one or GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on more carbohydrates that can be metabolized by the lactic 2014-06-12 under the accession no. DSM 28952: acid bacteria to a level select from less than 30 mg/g, from and 25 mg/g to 0.01 mg/g, and from 5 mg/g to 0.01 mg/g. (ii) strains derived from DSM 28952, wherein the 29. The method of claim 19, wherein the pH of the derived strains have the ability to generate white fermented milk product is stable such that, upon storage for colonies on a medium containing lactose and X-Gal; 20 hours after termination of fermentation at the fermenta (b) a Streptococcus thermophilus strain selected from: tion temperature, the pH is maintained within a range of 0.3 (i) the strain deposited with DSMZ-Deutsche Sam pH units. mlung Von Mikroorganismen und Zellkulturen 30. The method of claim 19, wherein the fermentation GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on temperature is between 30 and 45° C. 2014-06-12 under the accession no. DSM 28953; 31. The method of claim 19, wherein, after termination of and fermentation, the fermented milk product is packaged at a (ii) strains derived from DSM 28953, wherein the temperature between 15 and 45° C. derived strains have the ability to generate white 32. The method of claim 19, wherein the pH of the colonies on a medium containing lactose and X-Gal; fermented milk product is stable such that: (c) a Lactobacillus delbrueckii ssp. bulgaricus Strain (i) upon storage after termination of fermentation for a selected from: period of 6 months, the pH is maintained within a range (i) the strain deposited with DSMZ-Deutsche Sam of 0.3 pH units, or mlung Von Mikroorganismen und Zellkulturen (ii) upon storage after termination of fermentation for a GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on period of 12 months, the pH is maintained within a 2014-06-12 under the accession no. DSM 28910; range of 0.3 pH units. (ii) strains derived from DSM 28910, wherein the 33. The method of claim 19, further comprising at least derived strains have the ability to generate white partly separating whey from the fermented milk product, colonies on a medium containing lactose and X-Gal. wherein the pH of the fermented milk product prior to 40. A fermented food product, comprising one or more separating whey is stable Such that, upon storage, strains selected from: processing or maintenance after termination of fermen (a) a Streptococcus thermophilus strain selected from: tation for 20 hours at the fermentation temperature, the (i) the strain deposited with DSMZ-Deutsche Sam pH is maintained within a range of 0.3 pH units. mlung Von Mikroorganismen und Zellkulturen 34. The method of claim 33, wherein the pH of the GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on fermented milk product prior to separating whey is stable 2014-06-12 under the accession no. DSM 28952: Such that, upon storage, processing or maintenance after and termination of fermentation for 20 hours at the fermentation (ii) strains derived from DSM 28952, wherein the temperature, the pH is maintained within a range of 0.1 pH derived strains have the ability to generate white units. colonies on a medium containing lactose and X-Gal; 35. The method of claim 20, further comprising at least (b) a Streptococcus thermophilus strain selected from: partly separating whey from the fermented milk product, (i) the strain deposited with DSMZ-Deutsche Sam wherein the pH of the fermented milk product prior to mlung Von Mikroorganismen und Zellkulturen separating whey is stable Such that, upon storage, GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on processing or maintenance after termination of fermen 2014-06-12 under the accession no. DSM 28953; tation for 20 hours at the fermentation temperature, the and pH is maintained within a range of 0.3 pH units. (ii) strains derived from DSM 28953, wherein the 36. The method of claim 20, further comprising at least derived strains have the ability to generate white partly separating whey from the fermented milk product, colonies on a medium containing lactose and X-Gal; wherein the pH of the fermented milk product prior to (c) a Lactobacillus delbrueckii ssp. bulgaricus Strain separating whey is stable Such that, upon storage, selected from: processing or maintenance after termination of fermen (i) the strain deposited with DSMZ-Deutsche Sam tation for 20 hours at the fermentation temperature, the mlung Von Mikroorganismen und Zellkulturen pH is maintained within a range of 0.1 pH units. GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 37. A fermented milk product obtained by the method of 2014-06-12 under the accession no. DSM 28910; claim 19. (ii) strains derived from DSM 28910, wherein the 38. The fermented milk product of claim 37, wherein the derived strains have the ability to generate white fermented milk product is a yoghurt, a fruit yoghurt, a colonies on a medium containing lactose and X-Gal. yoghurt beverage or a cheese. k k k k k