Fermented milk product with a reduced content of lactose

Abstract

Acidified milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, wherein the product contains a lactase, which retains its activity at a pH of 5.0 and a temperature of 37° C. at a level of at least 5% as compared to its activity at the optimum pH of the lactase.

Claims

1. An acidified milk product, produced by a process comprising: chemically acidifying or fermenting a milk substrate with a starter culture to obtain an acidified milk product having a pH of between 3.0 and 5.0 and a lactose content of at least 1.5 mg/ml, subjecting the acidified milk product to heat treatment after chemical acidification or fermentation to achieve a level of bacteria in the acidified milk product of no more than 1×10.sup.2 CFU/g, and adding a lactase to the acidified milk product after the heat treatment, wherein the lactase exhibits an activity at a pH of 5.0 and a temperature of 37° C. that is at least 5% of its activity at its optimum pH, wherein the acidified milk product has a pH of between 3.0 and 5.0, contains the lactase, and contains no more than 1×10.sup.2 CFU/g bacteria.

2. The acidified milk product according to claim 1, wherein the lactase exhibits an activity at a temperature of 10° C. and a pH of 6.0 that is at least 10% of its activity at its optimum temperature.

3. The acidified milk product according to claim 1, wherein the product contains the lactase in an amount of between 100 and 20000 LAU per liter milk product.

4. The acidified milk product according to claim 1, wherein the product contains an acid whey product selected from the group consisting of acid whey and acid whey permeate.

5. The acidified milk product according to claim 1, wherein the lactase is a lactase originating from Bifidobacterium bifidum.

6. The acidified milk product according to claim 5, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence which is at least 95% identical to a sequence selected from the group consisting of amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and lactase active fragments of any thereof.

7. The acidified milk product according to claim 5, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence selected from amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-979 of SEQ ID NO: 1, amino acids 28 1170 of SEQ ID NO: 1, amino acids 28-1323 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 1, amino acids 28-1600 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

8. A process for producing a chemically acidified or fermented milk product, comprising subjecting a chemically acidified or fermented milk product having a pH of between 3.0 and 5.0 and a lactose content of at least 1.5 mg/ml to heat treatment to achieve a level of bacteria in the product of no more than 1×10.sup.2 CFU per g, to obtain a heat-treated chemically acidified or fermented milk product having a pH of between 3.0 and 5.0 and a lactose content of at least 1.5 mg/ml, adding a lactase to the heat-treated chemically acidified or fermented milk product, wherein the lactase exhibits an activity at a pH of 5.0 and a temperature of 37° C. that is at least 5% of its activity at its optimum pH, to obtain a lactase-containing chemically acidified or fermented milk product, and storing the lactase-containing chemically acidified or fermented milk product at a temperature of at least 2° C. for at least 1 day.

9. A process according to claim 8, wherein the lactase is a lactase originating from Bifidobacterium bifidum.

10. A process according to claim 9, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence which is at least 95% identical to a sequence selected from the group consisting of amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and lactase active fragments of any thereof.

11. A process according to claim 9, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence selected from amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-979 of SEQ ID NO: 1, amino acids 28 1170 of SEQ ID NO: 1, amino acids 28-1323 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 1, amino acids 28-1600 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

12. A process according to claim 8, wherein the product is a fermented milk product and the method further comprises, prior to the heat treatment, fermenting a milk substrate using a starter culture of lactic acid bacteria to obtain the fermented milk product which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml.

13. A process according to claim 12, wherein the lactase-containing fermented milk product is stored at a temperature of at least 15° C.

14. A process according to claim 12, wherein the lactase-containing fermented milk product is stored for at least 7 days.

15. A process according to claim 12, further comprising, after the fermenting and prior to the heat treatment, subjecting the fermented milk product to a concentration step to divide the fermented milk product into a concentrated fraction and a separated acid whey fraction, wherein the separated acid whey fraction is subjected to the subsequent steps of the process.

16. A process for reducing lactose in an acidified milk product during storage, comprising subjecting an acidified milk product having a pH of between 3.0 and 5.0 and a lactose content of at least 1.5 mg/ml to heat treatment to achieve a level of bacteria in the acidified milk product of no more than 1×10.sup.2 CFU per g, to obtain a heat-treated acidified milk product having a pH of between 3.0 and 5.0 and a lactose content of at least 1.5 mg/ml, and adding a lactase to the heat-treated acidified milk product, wherein the lactase exhibits an activity at a pH of 5.0 and a temperature of 37° C. that is at least 5% of its activity at its optimum pH.

17. A process according to claim 16, wherein the lactase is a lactase originating from Bifidobacterium bifidum.

18. A process according to claim 17, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence selected from amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-979 of SEQ ID NO: 1, amino acids 28 1170 of SEQ ID NO: 1, amino acids 28-1323 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 1, amino acids 28-1600 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

19. A process according to claim 17, wherein the lactase originating from Bifidobacterium bifidum comprises an amino acid sequence which is at least 95% identical to a sequence selected from the group consisting of amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and lactase active fragments of any thereof.

Description

FIGURES

(1) FIG. 1 shows the levels of residual lactose in samples treated with lactase added to a yogurt downstream of post pasteurization for lactase levels of 1000, 2000 and 3000 LAU/L.

(2) FIG. 2 shows the levels of residual lactose in samples treated with lactase added to a yogurt downstream of post pasteurization for lactase levels of 200, 400, 600, 800, 1000 and 1200 LAU/L at 8, 24, 48 and 76 hours.

(3) FIG. 3 shows the data of FIG. 2 at 48 hours only.

(4) FIG. 4 shows the data of FIG. 2 at 76 hours only.

EXAMPLES

Example 1

(5) Lactase Activity-Assay in Eppendorf Tubes at 37° C., pH 6.5

(6) Principle:

(7) Lactase hydrolyses lactose into glucose and galactose. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224).

(8) LAU is defined as the amount of enzyme liberating 1 micromole of glucose per min at 37° C., pH 6.5 in M-buffer (M-buffer is defined in the description of the present patent application). Alternatively, the activity in LAU for a specific lactase may be determined by the method described here. The value obtained is compared to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Lactozym obtained from Novozymes A/S, Denmark.

(9) Solutions:

(10) Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.01% Triton X100, pH 6.5

(11) GOD-Perid solution: 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP (Horse Radish Peroxidase), 0.65 g/l ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)).

(12) Substrate:

(13) 160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mom MgCl.sub.2, pH 6.5.

(14) Standard:

(15) Lactozym (available from Novozymes A/S, Denmark) with a known activity in LAU/g is used as standard, diluted in assay buffer in the range from 0.09-0.7 LAU/g.

(16) Samples:

(17) Enzyme samples are diluted appropriately in assay buffer.

(18) Procedure:

(19) 1. 375 μl substrate is incubated 5 minutes at 37° C.

(20) 2. 25 μl enzyme diluted in assay buffer is added.

(21) 3. The reaction is stopped after 30 minutes by adding 60 μl 1 M NaOH

(22) 4. 20 μl is transferred to a 96 well microtiter plate and 200 μl GOD-Perid solution is added. After 30 minutes at room temperature, the absorbance is measured at 420 nm.

Example 2

(23) 100 ml 15 or 30% (w/w) whey permeate containing primarily lactose and ions was made by mixing 15 or 30 g spray-dried whey permeate powder (Variolac, Arla) in 85 or 70 ml ionic water respectively. The solution was poured in a flask containing a magnetic stirring bar and placed in a water bath at 37° C. After 15 min, enzyme was added. Enzymes tested were Lactozym, a commercially available lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.

(24) Dosages were 4225 LAU/I milk of Lactozym and 2025 LAU/I milk of the Bifidobacterium lactase. Milk samples were taken at regular intervals up till 5.5 hrs. and the enzyme was inactivated by heating to 99° C. for 10 min in a thermomixer. Samples were diluted appropriately and filtered through a 0.20 μm filter.

(25) Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose.

(26) Results are given below.

(27) TABLE-US-00001 TABLE 1 Lactose, glucose and galactose in 15% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 0 499 1 2 499 1 2 30 312 135 106 410 61 63 60 211 224 155 349 119 122 120 110 295 221 220 199 202 180 66 324 249 149 281 290 240 50 346 279 84 336 348 330 37 372 312 31 350 368

(28) TABLE-US-00002 TABLE 2 Lactose, glucose and galactose in 30% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 0 848 1 4 848 1 4 30 824 109 75 819 43 45 60 615 253 150 788 86 83 120 420 370 242 651 159 158 180 291 459 300 625 232 230 240 246 559 373 501 283 273 330 154 544 367 391 333 324 1440 54 649 545 20 727 739

(29) Also when tested at higher lactose concentrations as in 15% or 30% whey permeate no or very little galactooligosaccharides are produced. Again, the produced galactose and glucose levels are equal and match the amount of lactose hydrolyzed. For comparison, Lactozym produces less galactose than glucose, clearly showing that galactooligosaccharides have been produced.

Example 3

(30) pH profile (at 37° C.) and temperature profile (at pH 6.5) of experimental lactase from Bifidobacterium bifidum using lactose as substrate.

(31) Principle:

(32) Lactase hydrolyses lactose and glucose+galactose is formed. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.)

(33) pH profile

(34) Substrate:

(35) 167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2 and pH adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10 with NaOH.

(36) Enzyme Sample:

(37) Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.01% Triton X100.

(38) Procedure: 10 μl enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp. 90 μl substrate was added at room temp. and quickly placed in a Peltier Thermal Cycler (PCT-200, MJ research) at 37° C. and incubated for 30 min and then placed on ice. The reaction was stopped by adding 100 μl 0.25 M NaOH. 20 μl was transferred to a 96 well microtitre plate and 230 μl 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

(39) TABLE-US-00003 TABLE 3 B. bifidum lactase pH relative activity (% of activity at pH 6) 3 0 4 4 5 75 6 100 7 85 8 39 9 10 10 4

(40) Temperature Profile

(41) Substrate:

(42) 167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2 and pH adjusted to pH 6.5 with NaOH.

(43) Enzyme Sample:

(44) Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.01% Triton X100 and pH adjusted to pH 6.5.

(45) Procedure: 10 μl enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp. 90 μl preheated (in a Peltier Thermal Cycler 30-70° C.) substrate was added and incubation was performed with a temp. gradient from 30-70° C. for 30 min. and then placed on ice. The reaction was stopped by adding 100 μl 0.25 M NaOH. 20 μl was transferred to a 96 well microtitre plate and 230 μl 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

(46) TABLE-US-00004 TABLE 4 B. bifidum lactase Temp. relative activity (% of activity at ° C. 38.1° C.) 20 54 21 63 22 64 24 68 26 73 29 81 31 88 34 94 36 96 38 100 43 96 48 91 52 83 57 76 62 58 66 32 69 20 70 17

Example 4

(47) Determination of Km for lactase enzymes at 5° C.

(48) Principle:

(49) Lactase hydrolyses lactose and glucose+galactose is formed. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.)

(50) Substrate:

(51) Different lactose concentrations ranging from Km/5 to 10*Km, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2 and pH adjusted to pH 6.5 with NaOH.

(52) Enzyme Sample:

(53) Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.01% Triton X100, pH adjusted to pH 6.5 with NaOH.

(54) 12 g/l Lactozym (commercially available lactase from Novozymes A/S, Denmark) was diluted 6000 times in the same buffer.

(55) Procedure: 10 μl enzyme sample (5° C.) was added to a 96 well microtitre plate on ice. 90 μl substrate (5° C.) was added and incubated for 2 hours at 5° C. The reaction was stopped by adding 100 μl 0.25 M NaOH. 20 μl was transferred to a 96 well microtitre plate and 230 μl 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

(56) Km Determination:

(57) Computerized nonlinear least-squares fitting and the Michaelis-Menten equation:
v=(Vmax*S)/(Km+S)
was used. Km for the Bifidobacterium lactase and Lactozym were determined to be 8 mM and 30 mM, respectively.

(58) In a similar test performed at 37° C., Km for the Bifidobacterium lactase and Lactozym were determined to be 13 mM and 30 mM, respectively.

Example 5

(59) Production of post-pasteurized yogurt containing lactase added after pasteurization

(60) Milk Substrate

(61) Fat level 2.8%*

(62) Protein level 2.8%*

(63) Lactose 3.0%

(64) Sucrose 5.0% (added)

(65) Modified Starch E1442 Cargill type 75720 1.50%

(66) Pectin type LMA CP Kelco type LM 106 AS-YA 0.25%

(67) Gellan Gum type Kelcogel YSS 0.05%

(68) * Level in final product, i.e. after heat treatment, addition of the ambient storage strain and storage for 150 days.

(69) Starter Culture

(70) YoFlex® starter culture type FD-DVS YF-L904 containing the two strains Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

(71) Lactase

(72) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2 and an activity of 295 LAU/g.

(73) Procedure for Producing Product 1. Dispersing the dry ingredients into the milk 2. Resting for 3 hours with gentle stirring 3. Heating the milk until a temperature of 65° C. is reached 4. Homogenization at 150 Bar 5. Heat treatment to 95° C. for 5 min. 6. Cooling to fermentation temperature 43° C. 7. Pump the milk into fermentation vat 8. Inoculation of YoFlex Culture type YF-L904. 9. Fermentation until pH reaches 4.30. 10. Break the curd and stir until smooth structure is obtained 11. Heat treatment at 75° C. for 30 sec. 12. Cooling to 25° C. 13. Adding the lactase at a level of 1420 LAU/L yogurt product and gently mixing the yogurt so as to distribute the lactase evenly in the yogurt. 14. Filling the yogurt into containers. 15. Storing the yogurt at a temperature of 20° C. for a period of 7 days.

Example 6

(74) Downstream Addition of Lactase to Post Pasteurized Yogurt

(75) The purpose of the experimental work carried out was to show that it is possible to add a suitable lactase after final heat treatment of post pasteurized yogurt to reach residual lactose levels of <0.01%.

(76) Post pasteurized yogurt with a composition of 2.9% protein and 2.8% fat was produced. The milk substrate was fermented to pH of 4.30 with YoFlex Culture type YF-L904. After reaching a pH of 4.30 the yogurt was cooled down to 15° C. in a plate heat exchanger and kept in insulated buffer tank at 15° C. for three hours before performing the final heat treatment. The final heat treatment was done at 74° C., 20 s. in a plate heat exchanger, and the product was filled at 25° C. into sterile beakers. The samples were stored at ambient temperature (22° C.) for one day prior to addition of sterile lactase.

(77) Milk Substrate

(78) TABLE-US-00005 Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Arla Foods 74.15%  Water 16.6% Sucrose  7.0% Whey Protein Nutrilac YO-7830, Arla Food Ingredients 0.60% Concentrate Modified starch Clearam CJ 5025, Roquette 1.50% LM Pectin LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/ metric tons

(79) Milkoscan analysis: Fat level 2.8% Protein level 2.9%

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

(81) Mixing temperature: 10° C.

(82) Hydration time: 3 hours with gentle stirring

(83) Lactase

(84) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

(85) Starter Culture

(86) YoFlex® Culture type YF-L904 from Chr. Hansen YoFlex® product range containing the two species Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

(87) Process for Producing Yogurt

(88) Homogenization pressure: 150 bar

(89) Pasteurization condition: 95° C., 5 minutes

(90) Fermentation temperature: 43° C.

(91) End pH: 4.30

(92) Break the curd manually and stir until smooth structure is obtained.

(93) Cooling to 15° C. in Plate heat exchanger

(94) Thermisation in plate heat exchanger, flow 414 L/h

(95) Homogenization pressure: 0 bar

(96) Termisation condition: 74° C., 20 s

(97) Filling into sterile 100 ml cups, filling temperature 25° C.

(98) Lactase Addition

(99) The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages: a) 0 LAU/L-Ref b) 1000 LAU/L c) 3000 LAU/L d) 10000 LAU/L

(100) The samples were stored at room temperature (22° C.) and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 1 week.

(101) Results

(102) In FIG. 1 the residual lactose of the samples with lactase addition is shown. The residual lactose of the reference sample was measured to be 2.4% residual lactose. As will appear from FIG. 1 the lactose concentration decreased to a level of about 0.005% for lactase concentrations 3000 LAU/L and 10000 LAU/L after 24 hours from addition. Also, the lactose concentration decreased to a level of about 0.005% for all three lactase concentrations after 48 hours and after 1 week from addition.

Example 7

(103) Downstream Addition of Lactase to Post Pasteurized Yogurt

(104) The purpose of the experimental work carried out was to show that it is possible to add a suitable lactase after final heat treatment of post pasteurized yogurt to reach residual lactose levels of <0.1%.

(105) Post pasteurized yogurt with a composition of 2.87% protein and 2.89% fat was produced. The milk substrate was fermented to pH of 4.30 with YoFlex Culture type YF-L904. After reaching a pH of 4.30 the yogurt was cooled down to 15° C. in a plate heat exchanger and kept in insulated buffer tank at 15° C. for three hours before performing the final heat treatment. The final heat treatment was done at 74° C., 20 s. in a plate heat exchanger, and the product was filled at 25° C. into sterile beakers. The samples were stored at 25° C. for one day prior to addition of sterile lactase.

(106) Milk Substrate

(107) TABLE-US-00006 Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Arla Foods 74.15%  Water 16.6% Sucrose  7.0% Whey Protein Nutrilac YO-7830, Arla Food Ingredients 0.60% Concentrate Modified starch Clearam CJ 5025, Roquette 1.50% LM Pectin LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/ metric tons

(108) Milkoscan analysis: Fat level 2.87% Protein level 2.89%

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

(110) Mixing temperature: 10° C.

(111) Hydration time: 3 hours with gentle stirring

(112) Lactase

(113) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

(114) Starter Culture

(115) YoFlex® Culture type YF-L904 from Chr. Hansen YoFlex® product range containing the two species Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

(116) Process for Producing Yogurt

(117) Homogenization pressure: 150 bar

(118) Pasteurization condition: 95° C., 5 minutes

(119) Fermentation temperature: 43° C.

(120) End pH: 4.30

(121) Break the curd manually and stir until smooth structure is obtained.

(122) Cooling to 15° C. in Plate heat exchanger

(123) Thermization in plate heat exchanger, flow 414 L/h

(124) Thermization condition: 74° C., 20 s

(125) Filling into sterile 100 ml cups, filling temperature 25° C.

(126) Lactase Addition

(127) The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages: a) 0 LAU/L-Ref b) 200 LAU/L c) 400 LAU/L d) 600 LAU/L e) 800 LAU/L f) 1000 LAU/L g) 1200 LAU/L

(128) The samples were stored at 25° C. and samples were taken for residual lactose analysis at the following time points; 8 hours, 24 hours, 48 hours and 76 hours.

(129) Results

(130) In FIGS. 2-4 the residual lactose of the samples with lactase addition is shown. The residual lactose of the reference sample was measured to be 2.4% residual lactose. The horizontal lines at a Residual Lactose level of 0.1 and 0.5 signifies the level of lactose, which according to food regulatory legislation qualifies as lactose-free in the EU and China, respectively.

(131) As will appear from FIG. 2-4 the lactose concentration at 24 hours decreased to a level of about 0.355% for a lactase concentration of 400 LAU/L and to a level of below 0.01% for higher concentrations of lactase. At 48 hours, the lactose concentration had dropped to a level of 0.015% for a lactase concentration of 400 LAU/L and to a level of below 0.01% for higher concentrations of lactase. At 76 hours, the lactose concentration had dropped to a level of below 0.01% for a lactase concentration of 400 LAU/L and higher concentrations of lactase.

(132) Thus, the present experiments show that it is possible to meet a target of residual lactose of 0.01% or below with a lactase dose of 600 LAU/I at 48 hours and a lactase dose of 400 LAU/I at 76 hours.

Example 8

(133) Downstream Addition of Lactase to Post Pasteurized Yogurt at Different pH Levels (from 4.0 to 4.4)

(134) The purpose of experimental work carried out was to show that in a process for removing lactose in Post Pasteurized Yogurt (PPY) using lactase it is possible to obtain a desired lactose removal for PPY with different pH in the range of 4.0 to 4.4.

(135) Milk Substrate

(136) TABLE-US-00007 Dosage Standard Dosage Ingredients Specification Lactose High Lactose Fresh milk 3.5% fat, commercial 74.30% 74.0% milk, Arla Foods Water 16.45%  15.95%  Sucrose  7.0%  7.0% Whey Protein Nutrilac YO-7830, 0.60% 0.60% Concentrate Arla Food Ingredients Modified starch Clearam CJ 5025, Roquette 1.50%  1.5% LM Pectin LM 106-AS YA, CP Kelco 0.12% 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% 0.03% Cultures FD-DVS YF-L904 200 units/ 200 units/ metric tons metric tons

(137) Milkoscan Analysis:

(138) Standard Lactose milk base: Fat level 2.88%, Protein level 2.91%

(139) High Lactose milk base: Fat level 2.91%, Protein level 2.94%

(140) Lactase

(141) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

(142) Process for Producing Yogurt

(143) Homogenization pressure: 150 bar at 60° C.

(144) Pasteurization condition: 95° C., 5 minutes

(145) Fermentation temperature: 43° C.

(146) End pH for standard lactose milk base: 4.0, 4.1, 4.2, 4.3 and 4.4

(147) End pH for high lactose milk base: 4.3

(148) Break the curd manually and stir until smooth structure is obtained.

(149) Cooling to 15° C. in Plate heat exchanger

(150) Thermisation in plate heat exchanger, flow 414 L/h

(151) Termisation condition: 74° C., 20 s

(152) Filling into sterile 100 ml cups, filling temperature 25° C.

(153) Lactase Addition

(154) The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages: a) 0 LAU/L-Ref b) 200 LAU/L c) 400 LAU/L d) 600 LAU/L e) 800 LAU/L

(155) The samples were stored at 15° C., 20° C., 25° C. and 30° C. and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 72 hours.

(156) Results

(157) For reasons of brevity only results for samples stored at 25° C. are shown. Corresponding results were obtained for samples stored at 15° C., 20° C. and 30° C.

(158) TABLE-US-00008 TABLE 5 Residual lactose levels for samples stored at 25° C. Dosage of Lactase 24 Hours 48 hours 72 Hours Milk Base End pH (LAU/L) (%) (%) (%) Standard 4.4 200 0.918 0.220 0.034 Standard 4.4 400 0.106 0.004 0.003 Standard 4.4 600 0.077 0.003 0.003 Standard 4.4 800 0.009 <0.002 0.002 Standard 4.4 1000 0.004 0.002 0.002 Standard 4.3 200 1.322 0.567 0.223 Standard 4.3 400 0.566 0.043 0.008 Standard 4.3 600 0.063 0.005 0.004 Standard 4.3 800 0.015 0.003 0.003 Standard 4.3 1000 0.007 <0.002 0.002 Standard 4.2 200 1.422 0.0803 0.356 Standard 4.2 400 0.671 0.071 0.013 Standard 4.2 600 0.177 0.008 0.004 Standard 4.2 800 0.128 0.005 0.003 Standard 4.2 1000 ND ND ND Standard 4.1 200 1.719 1.177 0.808 Standard 4.1 400 0.923 0.266 0.051 Standard 4.1 600 0.572 0.066 0.019 Standard 4.1 800 0.245 0.010 0.005 Standard 4.1 1000 0.108 0.005 0.003 Standard 4.0 200 1.725 1.196 0.933 Standard 4.0 400 1.236 0.589 0.265 Standard 4.0 600 0.773 0.187 0.039 Standard 4.0 800 0.502 0.049 0.010 Standard 4.0 1000 ND ND ND High 4.3 200 2.011 1.139 0.584 High 4.3 400 0.876 0.121 0.018 High 4.3 600 0.250 0.009 0.005 High 4.3 800 0.053 0.006 0.003 High 4.3 1000 0.017 0.004 0.003

(159) TABLE-US-00009 TABLE 6 Residual lactose level with no addition of lactase. Dosage of Lactase 24 Hours Milk Base pH (LAU/L) (%) Standard 4.4 0 2.6 Standard 4.3 0 2.7 Standard 4.2 0 2.6 Standard 4.1 0 2.5 Standard 4.0 0 2.5 High 4.3 0 3.2 Standard 0 3.3 (before fermentation) High (before 0 4.0 fermentation)

(160) As will appear from Table 5 and 6, the lower the end pH of the yogurt, the higher the residual lactose content is. The results further show that at the lowest end pH it is possible to reach a target of residual lactose of below 0.1% with a lactase dose of 800 LAU/L at 48 hours. Furthermore, at an end pH of 4.1 it is possible to reach a residual lactose of 0.108% with a lactase dose of 1000 LAU/L at 24 hours.

Example 9

(161) Downstream Addition of Lactase to Post Pasteurized Yogurt at Low Levels of Added Lactase

(162) The purpose of experimental work carried out was to test the effectiveness of low dosages of lactase in a process for removing lactose in Post Pasteurized Yogurt (PPY) using lactase in order to establish the minimum dose required to obtain a desired target of residual lactose.

(163) Milk Substrate

(164) TABLE-US-00010 Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Arla Foods 74.30%  Water 16.45%  Sucrose  7.0% Whey Protein Nutrilac YO-7830, Arla Food 0.60% Concentrate Ingredients Modified starch Clearam CJ 5025, Roquette 1.50% LM Pectin LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/ metric tons

(165) Milkoscan Analysis:

(166) Milk Base: Fat Level 2.81%, Protein Level 3.11%

(167) Lactase

(168) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

(169) Process for Producing Yogurt

(170) Homogenization pressure: 150 bar at 60° C.

(171) Pasteurization condition: 95° C., 5 minutes

(172) Fermentation temperature: 43° C.

(173) End pH for standard lactose milk base: 4.15, 4.20, 4.25 and 4.30

(174) End pH for high lactose milk base: 4.3

(175) Break the curd manually and stir until smooth structure is obtained.

(176) Cooling to 15° C. in Plate heat exchanger

(177) Thermisation in plate heat exchanger, flow 414 L/h

(178) Termisation condition: 74° C., 20 s

(179) Filling into sterile 100 ml cups, filling temperature 25° C.

(180) Lactase Addition

(181) The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages: a) 0 LAU/L-Ref b) 50 LAU/L c) 100 LAU/L d) 200 LAU/L e) 300 LAU/L

(182) The samples were stored at 25° C. and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 72 hours.

(183) Results

(184) For reasons of brevity only results for samples stored at 25° C. are shown. Corresponding results were obtained for samples stored at 15° C., 20° C. and 30° C.

(185) TABLE-US-00011 TABLE 7 Residual lactose levels for samples Dosage of Lactase 24 Hours 48 hours 72 Hours Milk Base End pH (LAU/L) (%) (%) (%) Standard 4.30 50 2.1 1.8 1.5 Standard 4.25 100 1.8 1.3 0.9 Standard 4.20 100 1.9 1.4 1.1 Standard 4.15 100 1.9 1.5 1.2 Standard 4.30 200 1.2 0.4 0.087 Standard 4.25 200 1.1 0.5 0.119 Standard 4.20 200 1.4 0.7 0.237 Standard 4.15 200 1.4 0.7 0.317 Standard 4.30 300 0.7 0.055 0.010 Standard 4.25 300 0.8 0.102 0.017 Standard 4.20 300 0.9 0.2 0.037 Standard 4.15 300 1.0 0.3 0.081

(186) The pasteurized yogurt base with a pH of 4.3, 4.25, 4.20 and 4.15 and with no added lactase had a lactose level of 2.57, 2.52, 2.47 and 2.40, respectively.

(187) As will appear from Table 7, it is possible to reach a target of residual lactose of below 0.5% with a lactase dose of 200 LAU/L at 48 hours for samples with a pH of 4.3 and 4.25 and with a lactase level of below 0.3 with a lactase dose of 300 LAU/L at 48 hours for all samples tested.

Example 10

(188) Production of Acid Whey Beverage with Low Level of Lactose by Addition of Lactase

(189) The objective of the experimental work carried out was to show that it is possible to remove lactose from an acid whey permeate and investigate the effect of various lactase dosages, reaction times and temperatures.

(190) Milk Substrate

(191) TABLE-US-00012 Dosage Ingredients Specification (g/1000 g) Acid whey Acid whey permeate (side-product from 800 production of Skyr) Sucrose Nordic Sugar 70 Water (ionized) 90 HM Pectin YM-115-H, CP-Kelco 4.5 Sum 979.5

(192) Acid whey permeate (from ultrafiltration) is a side-product from production of Skyr.

(193) The acid whey permeate had the following composition:

(194) Protein 0.2%

(195) Sugars 3.7%

(196) Fat 0.01%

(197) Ash 0.8%

(198) Moisture 94.7%

(199) pH 4.27

(200) Lactase

(201) Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

(202) Process 1. Heat acid permeate to 72° C./2 min. 2. Cooling to a. 5° C. (2×1000 ml) b. 40° C. (3×1000 ml) 3. Dosing of lactase: a. 5° C.: 2500 and 5000 LAU/L (2×1000 ml) b. 40° C.: 500, 1000 and 2500 LAU/L (3×1000 ml) 4. Samples for analyzing lactose: a. Before addition of lactase b. At 40° C. and 2500 LAU/L: every 1 hour from 3 to 7 hours and 24 hours c. Other samples: 24 hours. 5. Processing of beverage based on acid whey a. Addition of HM pectin solution, flavor and sucrose. b. Adjust pH to 3.9 by citric acid c. Heat to 80° C. 2 min. d. Homogenization at 150 bar at 80° C. e. Filling into bottles.

(203) Results

(204) TABLE-US-00013 TABLE 8 Residual lactose levels (g/L) for test samples Temp. 5° C. 5° C. 40° C. 40° C. 40° C. Lactase (LAU/L) 0 2500 5000 0 500 1000 2500 0 hours 2.7235 2.7235 1 Hours 2 Hours 3 Hours 1.7793 4 Hours 0.5527 5 Hours 0.3117 6 Hours 0.1681 7 Hours 0.1376 24 Hours  0.5646 0.1018 0.4381 0.0830 0.0378

(205) As will appear from Table 8, it was possible to reduce the level of lactose to levels around 0.1 g/L for test samples at a temperature of 40° C. and for lactase concentrations of 1000 and 2500 LAU/L as well as at a temperature of 5° C. for a lactase concentration of 5000 LAU/L. For the two other samples it was possible to reduce the level of lactose to levels below 0.5 g/L.

SEQUENCE LISTING

(206) SEQ ID NO.: 1 shows the sequence of a mutant of SEQ ID NO. 4. SEQ ID NO.: 2 shows the sequence of a mutant of SEQ ID NO. 4. SEQ ID NO.: 3 shows the sequence of a lactase from Bifidobacterium bifidum DSM20215. SEQ ID NO.: 4 shows the sequence of a lactase from Bifidobacterium bifidum NCIMB41171, the nucleotide sequence of which is listed in NCBI with the accession number DQ448279. SEQ ID NO: 4 is discussed in the following references, wherein it is referred to as bbgIII: Appl Microbiol Biotechnol (2007) 76:1365-1372, T K Goulas et al. Appl Microbiol Biotechnol (2009) 82:1079-1088, T Goulas et al. Appl Microbiol Biotechnol (2009) 84:899-907, T Goulas et al.