LOW POST-ACIDIFYING LACTIC ACID BACTERIA
20170099849 ยท 2017-04-13
Assignee
Inventors
Cpc classification
C12N15/01
CHEMISTRY; METALLURGY
A23C9/1238
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to lactic acid bacteria that have low or reduced post-acidification properties, and to a method for providing such bacteria. Also, the invention relates to the use of such bacteria for manufacturing of fermented dairy products, and to dairy products containing the bacteria.
Claims
1.-14. (canceled)
15. A Lactobacillus delbrueckii subsp. bulgaricus strain selected from CHCC10019, CHCC8535, CHCC7159, and mutant strains of any thereof, wherein the mutant strain (a) decreases the pH of a dairy product by less than 0.20 pH units after 7 days of storage at 8 C. and (b) has a viscosity of at least 25 Pa in 9.5% milk without the addition of a thickening agent.
16. The Lactobacillus delbrueckii subsp. bulgaricus strain of claim 15, wherein the strain decreases the pH of the dairy product less than 0.30 pH units after 14 days of storage at 8 C.
17. The Lactobacillus delbrueckii subsp. bulgaricus strain of claim 15, wherein the strain is CHCC7159.
18. A dairy product produced by a method comprising: adding a strain according to claim 15 to a milk substrate.
19. The dairy product of claim 18, wherein the method further comprises fermenting the milk after adding the strain to the milk.
20. The dairy product of claim 18, wherein the dairy product has a pH in the range of from 4.25 to 4.55 after 14 days of storage at 8 C.
21. The dairy product of claim 18, wherein the Lactobacillus delbrueckii subsp. bulgaricus strain decreases the pH of the dairy product less than 0.30 pH units after 14 days of storage at 8 C.
22. A dairy product comprising a Lactobacillus delbrueckii subsp. bulgaricus strain of claim 15.
Description
LEGENDS TO THE FIGURES
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[0048]
[0049]
[0050]
[0051]
EXAMPLES
[0052] General Methods
[0053] According to a method of the invention, a mother strain of Lactobacillus delbrueckii subsp. bulgaricus that has high texturing properties and high post-acidifying properties is treated with a chemical mutagen. The resulting strains are screened for mutants having low post-acidifying properties, and the desired mutants are thereafter screened for high texturing properties. The method is described in more details below:
[0054] Assay for Determination of Viscosity (Texturing Properties)
[0055] Frozen concentrates of the strain are used to inoculate 200 mL milk reconstituted from skimmed milk powder (the standard medium). The milk had a dry matter content of 9.5% and had been heat treated to 99 C. for 15 minutes in a batch process at ambient pressure. Frozen concentrates of Lactobacillus bulgaricus typically display a cell count between 1.Math.10.sup.9 and 1.Math.10.sup.10 cfu/g. The rate of inoculation was 1 g concentrate per 10 L milk, hence between 1.Math.10.sup.5 and 1.Math.10.sup.5 cfu/mL milk. The incubation took place at 43 C. pH reached 4.55, at which time the coagulation of the milk had taken place. The fermented milk was then cooled to 5 C. under periodically stirring (by placement in an ice bath for 20 minutes, thereafter in a refrigerator).
[0056] 20 hours after reaching pH 4.55, the fermented milk was brought to 13 C. and stirred gently by means of a stick fitted with a bored disc (diameter=3 cm) until homogeneity of the sample. The stirring consisted of 20 up and down movements of the stick in the sample. The rheological properties of the sample were assessed at 13 C. on a rheometer (StressTech, Reologica Instruments, Sweden) equipped with a bob/cup (CC25) coaxial measuring system.
[0057] The viscometry test was made with shear rates varying from 0.27 to 300 1/s in 21 steps. Shear rates were increased and then decreased and the upward and downward curves of shear stress and apparent viscosity were recorded. Delay, integration and equilibrium times were 5 s, 10 s and 5 min, respectively. For further analysis shear stress at 300 s-1 was chosen.
[0058] The shear stress is measured in Pa units. By definition, viscosity equals shear stress divided by shear rate. Viscosity and shear stress are therefore directly proportional and express the same properties given that shear stress values are determined at a fixed shear rate of 300 1/s.
[0059] Assay for Determination of Post-Acidifying Properties
[0060] Frozen concentrates of the strain are used to inoculate 200 mL milk reconstituted from skimmed milk powder (the standard medium). The milk had a dry matter content of 9.5% and had been heat treated to 99 C. for 15 minutes in a batch process.
[0061] Frozen concentrates of Lactobacillus bulgaricus typically display a cell count between 1.Math.10.sup.9 and 3.Math.10.sup.10 cfu/g. The rate of inoculation was 1 g concentrate per 10 L milk, hence between 1.Math.10.sup.5 and 3.Math.10.sup.10 cfu/mL milk. The incubation took place at 43 C. until pH reached 4.55, at which time the coagulation of the milk had taken place. The fermented milk was then cooled to 5 C. under periodically stirring (by placement in an ice bath for 20 minutes, thereafter in a refrigerator). The pH of the cooled fermented milk was measured using a pH electrode, appropriately after 1; 7; 14; 21 and 28 days of storage.
[0062] Method for Preparing the Standard Medium
[0063] Dissolve the skimmed milk powder (Arla Ingredients, DenmarkMilex 240 medium heat milk powder) in water for not more than 30 min. The aim for the preparation of milk is a dry matter of 9.5% (w/w).
[0064] The milk should be sterilized by the following temperature profile:
TABLE-US-00001 Temperature ( C.) Time (min) Heating >99 1 <20 Sterilization .sup.99 1 15 1 Cooling 99 1 > 40 5 <40
[0065] Subsequently, the milk should be stored at <7 C. The milk should not be used before the next day.
[0066] Propagation of the Bacteria
[0067] Strains of the species Lactobacillus delbrueckii subsp. bulgaricus that increase texture fermented milks, and that post-acidify, are well known. Such strains are preferably used as mother strains. The lactic acid bacterial strains are usually added as a liquid solution, as a freeze-dried powder or as frozen pellets to the milk to be fermented. The bacterial count of the liquid, powder and pellets may vary from 1E.sup.6 to 1E.sup.12 colony forming units per gram (1.Math.10.sup.6 to 1.Math.10.sup.12 cfu/g), applying standard cell count techniques. The liquid, powder or pellets are usually added at levels of around 0.0005-1% to the milk.
[0068] Mutagenesis
[0069] The bacterium, such as of a strain of Lactobacillus delbrueckii subsp. bulgaricus, which gives high texture upon growth in milk, but also has a high post-acidification, is mutagenised chemically, preferably with a mild mutagen, e.g. with EMS (Ethyl methane sulphonate). EMS gives rise to random base pair substitutions in the genetic material (the chromosome and plasmids) of the mutagenised bacterium. After the mutagenesis the mutagenised cultures is plated on media.
[0070] Preferred protocol: The mother strains were inoculated from frozen stocks in MRS broth (Difco) for 24 hours at 37 C., anaerobically. For the mutagenesis, the overnight cultures were vortexed at maximum speed for 1 minute to separate putative chains of cells and treated with EMS (10 ml/ml) for 2 hours at 37 C. The EMS treatment resulted in more than 99% of killing of the ceils. The EMS treated cultures were frozen at 80 C. in 20% gycero and this stock used for the screenings.
[0071] Screening
[0072] Several thousand colonies randomly picked by a colony picking robot into microtiter plates containing 200 microL milk e.g. supplemented with a pH indicator (such as bromophenol purple/bromophenol green). Each of the wells in the microtiter plates then corresponds to a model milk fermentation. The pH of the milk in wells containing mutants are compared to the corresponding wells containing the wild type strain (a strain of Lactobacillus delbrueckii subsp. bulgaricus, which gives high texture upon growth in milk, but also has a high post-acidification, such as Lactobacillus delbrueckii subsp. bulgaricus strains, CHCC3984, CHCC3606 and CHCC5713). Mutants with significant higher end-pH of the milk after acidifications at 43 C. than the wild type strains are identified, e.g. by colorimetric assessment (using a pH indicator) or by use of a pH electrode. Acidifications in a larger scale (200 mi) are performed by recording pH in a logger system with standard pH electrodes in order to verify that the mutants give rise to less post-acidification.
[0073] In order to control that the obtained mutants had the same texturing potential as the mother strain, samples of milk were fermented with the mutants and the mother strains to same final pH values and the rheological properties of the obtained fermented milks were assessed and compared by standard techniques.
Example 1
Preparation and Properties of LB Strains CHCC7159 and CHCC8535
[0074] CHCC7159 and CHCC8535 are lactic acid bacteria of the type Lactobacillus bulgaricus. These strains are able to acidify milk under industrially relevant conditions and are characterised by a low post acidification. The strain CHCC7159 is a mutant obtained from CHCC3606, to which it was compared. CHCC8535 is a mutant of CHCC5713, to which it was compared. Mutagenesis (preferred protocol) and screening was carried out as described above.
[0075] Frozen concentrates of CHCC7159, CHCC8535, CHCC3606 and CHCC5713 were used to inoculate 200 mL milk reconstituted from skimmed milk powder. The milk had a dry matter content of 9.5% and had been heat treated to 99 C. for 15 minutes in a batch process. Frozen concentrates of Lactobacillus bulgaricus typically display a cell count between 1.Math.10.sup.9 and 3.Math.10.sup.10 cfu/g. The rate of inoculation was 1 g concentrate per 10 L milk, hence between 1.Math.10.sup.5 and 3.Math.10.sup.6 cfu/mL milk. The incubation took place at 43 C. until pH reached 4,55, at which time the coagulation of the milk had taken place. The fermented milk was then cooled to 5 C.
[0076] A pH vs time plot shows that CHCC8535 reached pH 4.60 in less than 20 hours, proving that the strains are able to acidify milk under conditions of inoculation and incubation that are industrially relevant (
[0077] A pH vs time plot shows that CHCC7159 reached pH 4.60 in around 21 hours, proving that the strains are able to acidify milk under conditions of inoculation and incubation that are industrially relevant (
[0078] CHCC7159 and CHCC8535 were throughout the fermentation less acidifying than their respective mother strains. CHCC8535 showed a pH after 20 hours of 4.17, which was 0.32 pH units higher than the mother strain. CHCC7159 showed a pH after 20 hours of 4.64, which was 0.50 pH units higher than the mother strain.
[0079] CHCC7159 and CHCC8535 are Able to Generate an Elevated Texture in a Fermented Milk
[0080] The day after incubation, the fermented milk was brought to 13 C. and stirred gently by means of a stick fitted with a bored disc until homogeneity of the sample. The rheological properties of the sample were assessed on a rheometer (StressTech, Reologica Instruments, Sweden) equipped with a C25 coaxial measuring system. The viscometry test was made with shear rates varying from 0.27 to 300 1/s in 21 steps. Shear rates were increased and then decreased and the upward and downward curves of shear stress and apparent viscosity were recorded. Delay and integration times were 5 s and 10 s, respectively. For further analysis shear stress at 300 s-1 was chosen. Fermented milks incubated with concentrates of CHCC7159 and CHCC8535 were more texturing than fermented milks produced with their respective mother strains (
[0081] Conclusion
[0082] The strains CHCC7159 and CHCC8535 combine the following properties: [0083] are able to generate a high texture in a fermented milksuperior to 40 Pa under the experimental conditions tested [0084] are able to acidify milk to pH 4.60 in less than 24 hours [0085] reach a pH after 20 hours, which is superior to 4.15 under the experimental conditions tested
Example 2
Preparation and Properties of LB Strain CHCC10019
[0086] CHCC10019 is a lactic acid bacterium of the type Lactobacillus bulgaricus. CHCC10019 is able to acidify milk under industrially relevant conditions and is characterised by a low post acidification. The strain CHCC10019 is a mutant obtained from CHCC3984, to which it was compared. Mutagenesis and screening was carried out as described above (preferred protocol). The mutagen used was EMS.
[0087] Overnight cultures of CHCC10019 and CHCC3984 were prepared by inoculating 10 mL milk reconstituted from skimmed milk powder. The milk had a dry matter content of 9.5% and had been heat treated to 99 C. for 15 minutes in a batch process. Biological material was taken from frozen ampoules of the respective strains. Incubation took place at 37 C. for 24 hours.
[0088] Overnight cultures of CHCC10019 and CHCC3984 were used to inoculate of 200 mL milk reconstituted from skimmed milk powder (standard medium). The milk used was the same as described above. The rate of transfer was 1%. For each strain two bottles of milk were incubated. One bottle was used to obtain an acidification curve; the second bottle was used to obtain product for determination of rheological properties.
[0089] Overnight cultures of Lactobacillus bulgaricus typically display a cell count between 1.Math.10.sup.6 and 1.Math.10.sup.8 cfu/g. The rate of inoculation was 1 g overnight culture per 100 g milk, hence between 1.Math.10.sup.4 and 1.Math.10.sup.5 cfu/mL milk.
[0090] The incubation took place at 43 C. for 20 hours. A second bottle prepared under same conditions was incubated at 43 C. until pH reached 4.55, at which time the coagulation of the milk had taken place. This bottle was cooled to 5 C. until analysis of rheological properties the following day.
[0091] A pH vs time plot shows that CHCC10019 reached pH 4.50 in around 7 hrs30 min, proving that the strain is able to acidify milk under conditions of inoculation and incubation, that are industrially relevant, see
[0092] CHCC10019 was throughout the fermentation less acidifying than its mother strain. CHCC10019 showed a pH after 20 hours of 4.22, which was 0.64 pH units higher than the mother strain.
[0093] CHCC10019 is Able to Generate an Elevated Texture in a Fermented Milk
[0094] The day after incubation, the fermented milk was brought to 13 C. and stirred gently by means of a stick fitted with a bored disc until homogeneity of the sample. The rheological properties of the sample were assessed as described in example 1. For results see
[0095] Fermented milks incubated with CHCC10019 were more texturing than fermented milks produced with CHCC3984. Recorded shear stress values were on average 45 Pa for the mutant strain, while they were around 35 Pa for the mother strains.
[0096] CHCC7159 was deposited 8 Feb. 2006 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) and given the accession number DSM 17959. CHCC3606, CHCC5713 and CHCC8535 were deposited 30 Mar. 2006 at DSM and given the accession numbers DSM 18142, DSM 18143, and DSM 18144, respectively, CHCC10019 and CHCC3984 were deposited 3 Apr. 2007 and given the accession numbers DSM 19252 and DSM 19251, respectively. All deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
[0097] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
REFERENCES
[0098] Mller, C., Bockelmann, W., and K. J. Heller, 2001. Isolierung von Lactobacillus delbveckii subsp. bulgaricus mutagen zur herstellung eines joghurts mit milder geschmackscharakteristik, Kieler Milchwirtchaftliche forchungsberichte 53, 167-185.
[0099] WO2006/063142
[0100] EP518096A1
[0101] EP638642A
[0102] U.S. Pat. No. 5,545,554A
[0103] Guide To Short-Term Tests For Detecting, Mutagenic And Carcinogenic Chemicals. Prepared for the IPCS by the International Commission for Protection Against Environmental Mutagens and Carcinogens, WHO, 1985.
[0104] http://www.inchem.org/documents/ehc/ehc/ehc51.htm
[0105] All references cited in this patent document are hereby incorporated herein in their entirety by reference.