Method of producing a fermented milk product with improved control of post acidification

Abstract

The present invention provides methods of producing a fermented milk product comprising a step wherein milk is fermented, wherein: (a) the fermentation is initiated by a starter culture, which starter culture comprises lactic acid bacteria capable of metabolizing one or several carbohydrates present in the milk, (b) the fermentation is terminated by a decrease of the concentration of the one or several carbohydrates during fermentation, and (c) the decrease is at least also caused by the metabolic activity of the lactic acid bacteria. The invention further provides respective methods comprising a step, wherein at least part of the whey is separated from the fermented milk product.

Claims

1. A method of producing a fermented milk product with a stable pH, comprising: (a) initiating milk fermentation by adding to milk a starter culture comprising lactose -deficient lactic acid bacteria capable of metabolizing one or more fermentable carbohydrates other than lactose, wherein the lactose-deficient lactic acid bacteria comprise lactose-deficient Streptococcus thermophilus that has completely lost the ability to use lactose as a source for cell growth or maintaining cell viability, and lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus, and wherein the milk comprises one or more fermentable carbohydrates other than lactose metabolized by the lactose-deficient lactic acid bacteria, and (b) fermenting the milk at a temperature between 22 C. and 45 C., wherein termination of the milk fermentation and the pH value of the fermented milk product at the termination of milk fermentation are controlled by the concentration of the one or more fermentable carbohydrates other than lactose present in the milk, and the milk fermentation terminates when the concentration decreases to a level that can no longer be metabolized by the lactose-deficient lactic acid bacteria to produce a significant amount of lactic acid, wherein the decrease is at least partly caused by the lactic acid bacteria metabolizing the one or more fermentable carbohydrates, thereby obtaining a fermented milk product with a stable pH.

2. The method of claim 1, wherein the pH of the fermented milk product is stable such that, upon storage at the fermentation temperature for 20 hours after termination of fermentation, the pH is maintained within a range of 0.3 pH units.

3. The method of claim 1, wherein the pH of the fermented milk product is stable such that, upon storage at the fermentation temperature for 20 hours after termination of fermentation, the pH is maintained within a range of 0.1 pH units.

4. The method of claim 1, wherein the milk comprises lactose at a concentration of from 5 to 100 mg/g at the start of the fermentation.

5. The method of claim 1, further comprising packaging the fermented milk product at a temperature between 15 and 45 C.

6. The method of claim 1, wherein the lactose-deficient lactic acid bacteria are not capable of metabolizing lactose and wherein, prior to adding the starter culture, the milk comprises fermentable carbohydrates that can be metabolized by the lactic acid bacteria at a total concentration of below 45 mg/g.

7. The method of claim 1, wherein the fermentation is carried out in the presence of lactase at an initial concentration of 500 to 5000 NLU/1.

8. The method of claim 1, wherein the fermentation temperature is between 30 and 45 C.

9. The method of claim 1, wherein the pH of the fermented milk product is stable such that upon storage after termination of fermentation for a period of 6 months, the pH is maintained within a range of 0.3 pH units.

10. The method of claim 1, further comprising at least partly separating whey from the fermented milk product, wherein the pH of the fermented milk product prior to separating whey is stable such that, upon storage, processing or maintenance at the fermentation temperature after termination of fermentation for 20 hours, the pH is maintained within a range of 0.3 pH units.

11. The method of claim 10, wherein the pH of the fermented milk product prior to separating whey is stable such that, upon storage, processing or maintenance at the fermentation temperature after termination of fermentation for 20 hours, the pH is maintained within a range of 0.1 pH units.

12. A fermented milk product obtained by the method of claim 1.

13. The fermented milk product of claim 12, wherein the fermented milk product is a yoghurt, a fruit yoghurt, a yoghurt beverage or a cheese.

14. The method of claim 1, wherein the pH of the fermented milk product is stable such that upon storage after termination of fermentation for a period of 12 months, the pH is maintained within a range of 0.3 pH units.

15. The method according to claim 1, wherein the one or more fermentable carbohydrates other than lactose comprises one or more selected from sucrose, galactose and glucose.

16. The method according to claim 1, wherein the one or more fermentable carbohydrates other than lactose comprises sucrose.

17. The method according to claim 1, further comprising adding to the milk the one or more fermentable carbohydrates other than lactose.

18. The method according to claim 1, further comprising adding sucrose to the milk.

19. The method according to claim 1, wherein the milk fermentation terminates when the concentration of the one or more fermentable carbohydrates other than lactose decreases to within a range from 5 mg/g to 0.01 mg/g.

20. The method of claim 1, wherein the lactose-deficient Streptococcus thermophilus strain is selected from: (i) the strain deposited with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under accession no. DSM 28952; and (ii) the strain deposited with DSMZ under accession no. DSM 28953.

21. The method of claim 1, wherein the lactose-deficient Streptococcus thermophilus strain is the strain deposited with DSMZ under accession no. DSM 28952.

22. The method of claim 1, wherein the lactose-deficient Streptococcus thermophilus strain is the strain deposited with DSMZ under accession no. DSM 28953.

23. The method of claim 1, wherein the lactose-deficient Lactobacillus delbrueckii ssp. bulgaricus strain is the strain deposited with DSMZ under accession no. DSM 28910.

Description

FIGURE LEGENDS

(1) FIG. 1 compares the acidification activity of the S. thermophilus strain CHCC6008 when used to inoculate milk containing 1 and 3% lactose.

(2) FIG. 2 compares the acidification activity of S. thermophilus CHCC15914 and Lactobacillus delbrueckii ssp. bulgaricus CHCC10019 to the acidification activity of S. thermophilus CHCC17862 and lactobacillus delbrueckii ssp. bulgaricus CHCC18944 when used to ferment milk supplemented with sucrose.

(3) FIG. 3 shows the acidification activity of different ratios of S. thermophilus CHCC17861 and Lactobacillus delbrueckii ssp. bulgaricus CHCC18944 when used to ferment milk supplemented with sucrose and compared to the acidification activity of S. thermophilus CHCC15914.

(4) FIG. 4 shows post acidification, i.e. the development of pH in two different fermented milk products after fermentation using the method of the invention (Acidifix) and a prior art method (YFL-904).

(5) FIG. 5 shows the development of pH in a culture fermented with Sweety after termination of fermentation at 6 C. for 42 days (i.e. post acidification). The Figure shows that the pH varies less than 0.1 pH units under these conditions.

(6) FIG. 6 shows the development of the pH in different cultures fermented with and without lactase during storage at cooling temperature over a period of 48 days.

(7) FIG. 7 shows an overview over methods using concentration of protein prior to fermentation (left hand side) and concentration of protein after fermentation by separation using centrifugation or ultrafiltration (right hand side).

(8) FIG. 8 shows the effect of pH on shear stress during separation of whey from a milk product after fermentation with SSC17.

(9) FIG. 9 shows the effect of pH on shear stress during separation of whey from a milk product after fermentation with Mild2.0.

(10) FIG. 10 shows the effect of pH on complex modulus or gel firmness during separation of whey from a milk product after fermentation with SSC17.

(11) FIG. 11 shows the effect of pH on complex modulus or gel firmness during separation of whey from a milk product after fermentation with Mild2.0.

LAB STRAINS

(12) The subsequent examples use CH strains, some of which have been deposited for prior patent applications of Chr. Hansen. Further information on the strains is provided by the respective patent application and the deposit as follows:

(13) Streptococcus thermophilus CHCC6008 was deposited for WO2011/000879 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 2006 Mar. 23 under the accession no. DSM 18111.

(14) Lactobacillus delbrueckii ssp. bulgaricus CHCC10019 was deposited for WO2011/000879 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D38124 Braunschweig on 2007 Apr. 3 under the accession no. DSM 19252.

(15) Streptococcus thermophilus CHCC15757 was deposited for WO2013160413 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 2012 Apr. 3 under the accession no. DSM 25850.

(16) Streptococcus thermophilus CHCC16404 was deposited for WO2013160413 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 2012 Dec. 12 under the accession no. DSM 26722.

(17) Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 was deposited for WO2013160413 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 2012 Sep. 6 under the accession no. DSM 26420.

(18) The remaining strains used in the subsequent examples have either been deposited for the present application or are commercially available from Chr. Hansen.

Example 1: Methods for Producing a Fermented Milk Product Using Low Lactose Milk

(19) Customized milk containing 1% lactose (10 g/L) was obtained (from Select Milk Producers, Inc., Texas, USA). A milk containing 3% lactose (30 g/L) was prepared by adding 2% lactose (20 g/L) to the 1% lactose milk.

(20) The milk was inoculated with S. thermophilus strain CHCC6008 (0.01% F-DVS) and maintained at a temperature of 37 C. for 20 hours. The acidification (pH value) was determined automatically over time.

(21) The result is shown in FIG. 1. The fermentation using 1% lactose shows a close to ideal acidification profile. Initially, the product is acidified at a high rate, but acidification stops abruptly at pH 4.8 after about 6 hours and the pH of the culture remained unchanged for the next 14 hours although the culture was maintained at a temperature of 37 C. This shows that acidification is completely terminated.

(22) The fermentation using 3% lactose was used as a control. Initially the product is acidified at the same rate as the fermentation based on 1% lactose. This shows that the lactose concentration of 1% (10 mg/g) is not too low to inhibit fermentation in the initial phase.

(23) But the fermentation using 3% lactose is not terminated at pH 4.8 but continues acidification until a pH of about 4.5 is reached at which state the presence of the acid produced by the bacteria inhibits further fermentation. As a consequence, the fermentation proceeds at a low rate until a pH of about 4.2 is reached.

(24) These results prove that the carbohydrate (here lactose) concentration can surprisingly be used to control the termination of fermentation. This way of proceeding allows processes, wherein the desired pH value is obtained by fermentation and no post acidification is observed afterwards.

Example 2: Methods for Producing a Milk Product Using Lactose Deficient LAB

(25) Lactose deficient mutants were isolated from the EPS positive strains S. thermophilus (ST) CHCC15914 and Lactobacillus delbrueckii ssp. bulgaricus (LB) CHCC10019. The strains were selected after UV-mutagenesis as white colonies (indicating a lactose deficient phenotype) on M17 with 1% lactose and 200 mg/ml X-Gal for CHCC15914, resp. MRS agar plates with 1% lactose and 200 mg/ml X-Gal for CHCC10019.

(26) Both wild type strains possess -galactosidase activity, and wild type colonies appeared blue due to the activity of the -galactosidase.

(27) From CHCC10019 one lactose deficient mutant was isolated and designated CHCC18944 (this mutant was identified as CHCC18994 in European Patent Application 14173196; but the internal Accession No. of the Applicant was changed and now is CHCC18944; the DSMZ Accession No. was not changed and thus still is DSM28910).

(28) From CHCC15914 two lactose deficient mutants were isolated and designated CHCC17861 and CHCC17862, respectively.

(29) The growth characteristics of the isolated lactose deficient mutants were determined as follows: Phenotype of LB CHCC18944: lac, suc, gal, glc+ Phenotype of ST CHCC17861: lac, suc+, gal+, glc+ Phenotype of ST CHCC17862: lac, suc+, gal+, glc+

(30) The complete lactose operon was sequenced for all three mutants and compared with the respective wild type strain to reveal the mutation type.

(31) Comparison with the mother strain CHCC15914 revealed that CHCC17861 had an extra T nucleotide in the beginning of the lacZ gene (coding for the -galactosidase) leading to a stop codon in the coding sequence a few nucleotides downstream of the mutation. CHCC17862 showed a deletion of one nucleotide, also interrupting the coding sequence of the lacZ gene.

(32) For CHCC18944 a mutation inside the lacZ gene was identified. This resulted in an exchange of 8 nucleotides (5-CTT CCA AGC-3 to 5-CGC TAC TAT-3) and consequently a change of 3 amino acids (Leu-Pro-Ser to Arg-Tyr-Tyr) within lacZ, explaining the lactose deficient phenotype.

(33) All mutants when used as single strains or in combination (ST+LB), acidify milk depending on the addition of a fermentable carbohydrate different from lactose. The acidification activity of the lactose deficient cultures were for example determined using over-night cultures in MRS (LB wt and LB lac-mutant); M17 with 1% lactose (ST wt); or M17 with different concentrations of sucrose (1% and 0.5%; fermentation with the ST lac-mutant). The milk was inoculated and fermentation was monitored by pH development at 37 C.

(34) The pH development under fermentation temperature over 20 and 40 hours is illustrated in FIGS. 2 and 3 respectively and shows that sucrose is metabolized by the lactose deficient strains yielding a fermentation process that is nearly as fast as the process caused by the parental LAB having the ability to metabolize lactose. The sucrose driven 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 remained stable at about pH 4.5. A very stable final pH was found when CHCC17861, CHCC17862, or CHCC18944, were used as single strains, or as a mix of one of the ST mutants together with the LB mutant (mix is shown in FIG. 3). This resembles the starter culture to be used in a fermentation method for producing a typical yoghurt.

(35) No significant difference between the strains CHCC17861 and CHCC17862 could be observed. The addition of sucrose therefore provides a very precise control of acidification activity.

(36) In some fermentations the formation of a shoulder was observed within the pH-curves of the mixed cultures indicating that the metabolism shifted before the acid production completely stopped (FIG. 3). To investigate this further the ratio of ST:LB was changed and this led to a change in the shoulder formation. Decrease of the concentration of the lac-ST and increase of the lac-LB strain resulted in a reduction of the pH shoulder and an even more horizontal pH-curve.

(37) Interestingly, the use of 100% ST CHCC17861, on the other hand, leads to a completely interrupted acid production when sucrose is depleted and eliminates the pH shoulder, but the pH drop stops at a 0.3 point higher pH value (FIG. 3).

(38) In all cases the pH remained stable at 4.75 up to the end of the fermentation period (48 hours).

(39) In ST/LB co-fermentation the Lactobacillus delbrueckii subsp. bulgaricus part is responsible for the final pH drop and also for a major part of the post acidification. For this reason the concentration of LB is lower than the concentration of ST in most yoghurt fermentation processes.

(40) This shows that the pH value can be completely controlled by the concentration of the added sucrose or another fermentable carbohydrate, as the Lb. Bulgaricus can easily be increased.

(41) The lac-culture will not only result in a higher final pH after e.g. 6 hours, but will also have a significantly lower post acidification and thus extended shelf life.

(42) In some experiments it was observed that the pH was very stable for about 5 hours after termination of fermentation and then slightly declined over the next 10 hours (data not shown). This is apparently due to spontaneous revertants, i.e. LAB gaining the ability to utilize lactose by spontaneous mutation.

Example 3: Methods of Producing a Fermented Milk Product with an Extended Shelf Life

(43) Yoghurt with an extended shelf life (ESL yoghurt) was produced by fermenting milk with the commercially available FD-DVS YFL-904 and in a separate fermentation with the new culture F-DVS Acidifix 2.0 (containing Streptococcus thermophilus CHCC17861 and Lactobacillus delbrueckii subsp. bulgaricus CHCC18944, described in Example 2).

(44) TABLE-US-00001 TABLE 1 Recipe ESL yoghurt: Milk Base Ingredients Specification Dosage Fresh Milk Meiji, 2 L bottle 97.95% Thermex Modified starch, Ingredion 1.00% LM 106-AS-YA Pectin, CP Kelco 0.15% Kelcogel YSS Gellan Gum, CP Kelco 0.03% Sugar Refined sugar, Phoon Huat 0.87% Cultures FD-DVS-YF-L904 100 u/mt F-DVS-Acidifix 100 u/mt Milkoscan analysis: Fat: 3.70% Protein: 3.05% 12% sugar syrup ws added to the white mass after fermentation

(45) The following parameters were used for fermentation and processing:

(46) Mixing temperature: 45 C.-50 C.

(47) Hydration time: 20 minutes

(48) Process:

(49) Homogenization pressure: 150 bar+30 bar (total 180 bar) Pasteurization condition: 95 C./4 min cooling temperature: less than 10 C. Fermentation: Fermentation temperature: 43 C. for YF-L904 and 39 C. for Acidifix End pH: 4.350.05 Break the curd manually when desired pH is achieved. Add 12% of sugar syrup into 88% of fermented milk base. Manually stir to mix it well. Thermise the yoghurt through GEA pilot plant: Homo pressure: 0 bar Thermisation condition: 65 C./30 s Filling into bottles (filling temperature: 28 C.-33 C.).

(50) The post acidification activity was analyzed for both products directly after fermentation, after thermisation and after 6 days storage at different temperatures. Results are illustrated in FIG. 4 and show high stability of pH of the product obtained with Acidifix during processing and storage.

Example 4: Methods of Producing a Fermented Milk Product Using Glucose Deficient LAB

(51) Material and Methods

(52) Milk base (1.0% fat & 4.5% protein):

(53) Ingredients: Mix of commercial milk (1.5% fat+0.5% fat+skim milk powder to reach the required fat and protein level) 9.5% w/w skimmed milk powder+90.5% tap water

(54) Commercial milk: Arla Harmonie minimlk (0.5% fat) and Arla Harmonie letmlk (1.5% fat)

(55) Skimmed milk powder: Arla Foods, Gin 990214

(56) Procedure: Mix milk and powder stir, heat treat 90 C., 20 min

(57) Lactose:

(58) Product: Lactosemonohydrate (Gin 500449, Batch 0005078607)

(59) Producer: German Lac-Sachsenmilch

(60) Added as a 19% w/w solution to the yoghurt base, to obtain the right level as prescribed

(61) Recipe: 0.6 kg lactose monohydrate+2.4 kg tap water

(62) Process: Heat while stirring, before pasterurization (95 C., 5 min)

(63) Starter Cultures:

(64) F-DVS Sweety 1.0 consists of a blend of the following strains

(65) 2-deoxy-glucose resistant strains Streptococcus thermophilus CHCC16731 (hyper-lactose fermenting and glucose secreting mutant of CHCC11976). Streptococcus thermophilus CHCC15757 (2-deoxyglucose resistant mutant of CHCC14944). Streptococcus thermophilus CHCC16404 (hyper-lactose fermenting and glucose secreting mutant of CHCC15757). Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 (2-deoxyglucose resistant mutant of CHCC10019).
Low Post Acidification Properties of Hyper-Lactose Fermenting and Glucose Secreting Mutants of Streptococcus thermophilus and Lactobacillus delbrueckii Subsp. Bulgaricus.

(66) Selection of a hyper-lactose fermenting and glucose secreting mutant of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were carried as described in WO2013/160413 entitled Use of lactic acid bacteria for preparing fermented food products with increased natural sweetness.

(67) To show the low post acidification properties of the culture in a standard setup for yoghurt production, 0.024% of a Frozen Direct Vat Set culture (F-DVS) of Sweety 1.0 culture blend was used to inoculate 3 l of milkbase and the milk was fermented at 43 C.

(68) Acidification was followed with a CINAC pH logger (Alliance instruments) with standard pH electrodes and CINAC 4.0 software. When the pH reached 4.55 at 43 C. coagulation of the milk had taken place. The yoghurt was subsequently cooled and incubated at 71.5 C. for 42 days. The pH development was subsequently followed over a period of 42 days (FIG. 5). As can be seen from FIG. 5 the pH development after 42 days of storage of 71.5 C. is less than 0.1 pH units confirming an extremely low level of post acidification.

(69) This data show that yoghurt blends of the glucose secreting strains and the use of the methods of the present invention will coagulate milk, maintain the final pH very stable for 43 days at 71.5 C. and increase the sweetness of the fermented product.

(70) Mixed cultures of glucose secreting Streptococcus and Lactobacillus delbrueckii subsp. bulgaricus metabolize all or almost all lactose present in milk during fermentation. As a consequence, the LAB no longer cause post acidification.

Example 5: Low Post Acidification when Using Lactase in the Fermentation of Yoghurt

(71) It was surprisingly found that the addition of lactase to a method of preparing a yoghurt significantly reduces post acidification in comparison to a yoghurt made without lactase.

(72) Materials and Methods

(73) Milk base (0.1% fat & 4.5% protein):

(74) Ingredients: Mix of commercial milk (0.1% fat) and skimmed milk powder to reach the protein level.

(75) Commercial milk: Arla skummetmlk (0.1% fat)

(76) Skimmed milk powder: Milex 240, Arla Foods, lot 990214

(77) Procedure: Mix powder and milk, hydration at 5 C. overnight, heat treatment 90 C./20 min.

(78) Lactase:

(79) Product: Ha-Lactase 5200 (Gin 450805)

(80) Producer: Chr. Hansen A/S

(81) Starter Cultures:

(82) F-DVS YF-L706, Gin 685141

(83) F-DVS YF-L901, Gin 685142

(84) F-DVS YoFlex Mild 1.0, Gin 702897

(85) F-DVS YoFlex Creamy 1.0, Gin 706168

(86) F-DVS YoFlex Premium 1.0, Gin 706161

(87) All from Chr. Hansen A/S

(88) The fermentations were done in 3 l scale. The cultures were inoculated at 0.02%, and the samples were fermented at 43 C. Lactase in a dosage of 3500 NLU/I was added to the milk base together with the culture. Acidification was monitored with a pH-Meter 1120 (Mettler-Toledo AG, 52120653), and fermentation stopped at pH 4.55. The fermented milk products were stirred in a standardized way, and further pressurized and cooled (2 bar, 25 C.), before storing at 6 C. pH of the samples were then monitored at 1, 7, 14, 21 and 48 days using a pH-Meter 1120 (Mettler-Toledo AG, 52120653). Concentrations of lactose, glucose and galactose (mg/g) in yoghurt on day 1 after fermentation were determined using HPLC with a Dionex CarboPac PA 20 3*150 mm column (Thermo Fisher Scientific, product number 060142).

(89) Results are shown in FIG. 6, which illustrates the development of pH value during storage and thus post acidification. Further results are shown table 1, below which provides an overview over the difference in the pH value obtained in the yoghurts produced with and without lactase with different cultures. The difference is caused by post acidification and was determined using F-DVS YoFlex Mild 1.0 yoghurt as example: F-DVS YoFlex Mild 1.0 yoghurt with 3500 NLU/L lactase had a pH after 21 days of 4.44, while F-DVS YoFlex Mild 1.0 yoghurt without lactase had a pH of 4.36. Fermentation was stopped at 4.55 for both yoghurts. The post acidification for the lactase treated yoghurt was 4.554.44=0.11, for the yoghurt without lactase it was 4.554.36=0.19. The difference in post acidification between yoghurt fermented with F-DVS YoFlex Mild 1.0 with and without 3500 NLU/L lactase is thus 0.190.11=0.08.

(90) TABLE-US-00002 TABLE 2 Difference in post acidification between yoghurts produced with the cultures in the table at 0.02%, with and without 3500 NLU/L lactase. Culture Day 21 Day 48 F-DVS YF-L706 0.00 0.00 F-DVS YF-L901 0.04 0.04 F-DVS YoFlexCreamy 0.10 0.07 F-DVS YoFlexPremium 1.0 0.08 0.08 F-DVS YoFlexMild 1.0 0.08 0.11

(91) The data presented shows that select Chr. Hansen commercial cultures in combination with lactase can be used to obtain yoghurt with reduced post acidification. F-DVS YoFlex Mild 1.0, F-DVS YoFlex Creamy 1.0 and F-DVS YoFlex Premium 1.0, all show low post acidification. The reduction of post acidification is less pronounced in the culture YF-L901 and no reduction of post acidification is observed for F-DVS YF-L706.

(92) TABLE-US-00003 TABLE 3 Concentrations of lactose, glucose and galactose (mg/g) in yoghurt produced with and without 3500 NLU/L lactase on day 1 after fermentation determined using HPLC. Galactose Glucose Lactose Culture (mg/g) (mg/g) (mg/g) F-DVS YF-L706 9.2 0.4 42.3 F-DVS YF-L706 + 3500 NLU/lactase 26.2 21.8 2.0 F-DVS YF-L901 10.6 1.6 39.4 F-DVS YF-L901 + 3500 NLU/lactase 26.1 21.4 2.1 F-DVS YoFlexCreamy 1.0 7.7 0.0 43.4 F-DVS YoFlexCreamy 1.0 + 23.1 21.2 5.3 3500 NLU/lactase F-DVS YoFlexPremium 1.0 7.7 0.6 41.7 F-DVS YoFlexPremium 1.0 + 25.1 23.8 2.0 3500 NLU/lactase F-DVS YoFlexMild 1.0 7.7 0.6 42.2 F-DVS YoFlexMild 1.0 + 25.4 24.1 2.9 3500 NLU/lactase

(93) Table 3 shows that the cultures fermented in the presence of lactase have very low residual lactose but higher residual glucose and galactose concentrations. The fact that post acidification is observed for the strains can thus be explained by the high total residual amount of carbohydrates available for the metabolism of the LAB.

(94) The extent of post acidification is still very low and thus surprising in view of the residual concentrations of carbohydrates. The low post acidification appears to be caused by the need for a metabolic shift of the carbohydrate source in the microorganisms. In other words, the cultures showing low post acidification contain strains that struggle with the shift from lactose to glucose as the carbohydrate source.

(95) Deposit and Expert Solution

(96) The applicant requests that a sample of micro-organisms deposited for the present application as described below may only be made available to an expert, until the date on which the patent is granted.

(97) Streptococcus thermophilus CHCC16731 was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014-06-04 under the accession no. DSM 28889.

(98) Streptococcus thermophilus CHCC15914 was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014-06-12 under the accession no. DSM 28909.

(99) Lactobacillus delbrueckii ssp. bulgaricus CHCC18944 was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014 Jun. 12 under the accession no. DSM 28910.

(100) Streptococcus thermophilus CHCC17861 was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014-06-12 under the accession no. DSM 28952.

(101) Streptococcus thermophilus CHCC1 7862 was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 2014-06-12 under the accession no. DSM 28953.

(102) The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

REFERENCES

(103) US2010/0021586 WO2006/042862A1 WO2010/139765 WO2013/169205 WO2011/000879 Pool et al., Metabolic Engineering, vol. 8, 2006, 456-464