HK1130631B - Low post-acidifying lactic acid bacteria - Google Patents

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 contain-ing the bacteria.

Description

Low post-acidification lactic acid bacteria

Technical Field

The present invention relates to lactic acid bacteria having low or reduced post-acidification properties, and to methods of preparing such bacteria. Furthermore, the invention relates to the use of such bacteria for the preparation of fermented dairy products, and to dairy products containing said bacteria.

Background

Strains of the species Lactobacillus delbrueckii subsp. Such strains typically produce lactic acid during the shelf life of a dairy product that is fermented with a bacterial culture, which is a component of this bacterial culture. This phenomenon is often referred to as "post-acidification". Such bacteria do not generally contribute to the improved texture of dairy products obtained by fermentation with a bacterial culture, a component of which the strain is a component.

According to regulations in most of the European Union (EU) countries, starter cultures to be used for the production of yogurt must consist of strains of Lactobacillus bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus (Streptococcus thermophilus). Furthermore, acidification of milk occurs more rapidly when the starter culture is composed of these two types of strains rather than only a single type of strain.

The trend in the fermented milk market is for such products: it has a mild to no development of acidification during the shelf life (low post-acidification) and a high texture (or viscosity). When selecting strains of the lactobacillus delbrueckii subspecies bulgaricus type, alone or as a component of a culture, for the production of fermented milks and yoghurts, the product developer has to choose between known strains having the following combination of properties:

low post-acidification and low-formation capacity; or

High post-acidification and low-quality formation ability; or

High post-acidification and high-quality formation ability,

because no such strains of lactobacillus delbrueckii subsp bulgaricus are available: the strain combines in a satisfactory manner the low post-acidification feature with the high texturizing feature.

With any of the above known strains, fermented dairy products will result in a high degree of post-acidification or a low texture due to the use of bacterial cultures. The dairy industry often chooses to process with such cultures: in this culture the Lactobacillus delbrueckii subspecies bulgaricus strain combines a low post acidification and a low texture. To increase the texture of fermented dairy products, they add thickener(s) to the milk base before fermentation takes place.

Summary of The Invention

The problem faced by the skilled person is to provide alternative strains of Lactobacillus delbrueckii subsp bulgaricus that combine a low post-acidification feature with a high texture-forming feature, thereby rendering the addition of thickener superfluous. Several scientists have attempted to obtain such strains without any success. M(2001) of ller et al, in order toMutants with less post-acidification were obtained and the Lactobacillus delbrueckii subspecies bulgaricus strain (Visby strain 231) was subjected to chemical mutagenesis (with the mutagen MNNG (N-methyl-N-nitroso-N-nitroguanidine)). The mutagenized strains were plated manually and colonies were transferred manually to microtiter plates containing milk. After incubation, the pH was measured per well for approximately 2000 isolates by using a microelectrode. Four mutants with a final pH higher than the mother strain were isolated. The mutagenesis and screening did not yield any mutants with high texture and low post-acidification.

The present inventors have surprisingly shown that it is indeed possible to provide such strains, and they have developed a method for providing such strains, which is different from MThe method of ller et al (2001) because the present inventors used high-quality strains as mother strains and used weak mutagens. By carrying out the method of the present invention, the present inventors have surprisingly identified strains with improved textural properties relative to their parent strain. Thus, the present inventors have devised a method for developing a strain of lactobacillus delbrueckii subsp bulgaricus that substantially contributes to improving the texture of a dairy product obtained by fermentation with a strain of the invention or a bacterial culture of which the strain of the invention is a component.

The resulting dairy product is characterized by having an unpleasant, mild, less sour taste than previously known products.

Detailed Description

In a first aspect, the present invention relates to a strain of lactobacillus delbrueckii subsp bulgaricus characterized in that:

a) produces less acid when present in a fermented milk product (e.g. during storage) than other high-texture strains of the same species; and/or

b) The texture of the dairy product fermented with this strain is increased to a higher level than that of the same strain with low post-acidification.

An interesting embodiment of such a strain is a strain which:

a) the pH measured in the standard test (test to determine the post-acidification property) after 14 days and/or after 21 days and/or after 28 days of storage is in the range of 4.25 to 4.55 (e.g. in the range of 4.30 to 4.55; or in the range of 4.35-4.55); and is

b) A texture forming property measured in Pa using a test to determine viscosity is not less than 25 (e.g., not less than 30; not less than 35; not less than 39; not less than 42; not less than 44; not less than 45; not less than 46; not less than 47; not less than 48; or not less than 50);

or a strain which:

a) a decrease in pH of less than 0.30pH units (e.g., less than 0.20pH units) measured in a standard test (test to determine post-acidification properties) after 14 days of storage; and is

b) A texture forming property measured in Pa using a test to determine viscosity is not less than 25 (e.g., not less than 30; not less than 35; not less than 39; not less than 42; not less than 44; not less than 45; not less than 46; not less than 47; not less than 48; or not less than 50);

or a strain which:

a) the pH measured in the standard test (test to determine post-acidification properties) is lowered by less than 0.20pH units (e.g. less than 0.15; less than 0.11; less than 0.08; less than 0.07; less than 0.06; less than 0.05; less than 0.04; less than 0.03; or even less than 0.02pH units; ) (ii) a And is

b) A texture forming property measured in Pa using a test to determine viscosity is not less than 25 (e.g., not less than 30; not less than 35; not less than 39; not less than 42; not less than 44; not less than 45; not less than 46; not less than 47; not less than 48; or not less than 50).

The term "lowering the pH" is to be understood as a decrease of the pH relative to the starting point, i.e.pH 4.55. For example, a decrease of 0.30pH units means a final pH of 4.25.

In both interesting embodiments, the reduced acid production is not due to a significant or complete inactivation of β -galactosidase activity; and/or not due to significant or complete inactivation of lactate dehydrogenase activity. The term "not due to significant inactivation" is understood to mean a reduction in activity of less than 30% (e.g. less than 20% or even less than 10%).

Presently preferred are strains of lactobacillus delbrueckii subsp bulgaricus selected from the group consisting of: CHCC 10019; CHCC 8535; and CHCC 7159; and mutants of any of these strains (e.g. functionally equivalent mutants, such as mutants having substantially the same post-acidification and texture-formation properties), and most preferred is strain CHCC7159 or a mutant thereof, e.g. functionally equivalent mutants, such as mutants having substantially the same post-acidification and texture-formation properties.

In the present context, the term "mutant thereof" is to be understood as a strain derived from a strain of the invention by, for example, genetic engineering, irradiation and/or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, such as a mutant having substantially the same post-acidification and texture-forming properties as the mother strain. Such mutants are part of the present invention. In particular, the term "mutant thereof" refers to a strain obtained by subjecting the lactobacillus delbrueckii subspecies bulgaricus strain of the present invention (e.g. CHCC 10019; CHCC 8535; CHCC 7159; CHCC 3984; CHCC3606 or CHCC5713) to any conventionally used mutagenesis treatment, including use of a chemical mutagen such as Ethyl Methanesulfonate (EMS) or N-methyl-N' -nitroso-N-Nitroguanidine (NTG), UV light, or spontaneous mutants. The mutant strain should preferably satisfy at least one of the combinations a) and b) above. Although it is presently preferred to provide such strains by random mutagenesis or by selection of spontaneous mutants, i.e.without the use of recombinant DNA techniques, it is envisaged that mutants of Lactobacillus delbrueckii subsp. The term "substantially the same post-acidification property" should preferably be understood as that the pH value may vary up to +/-0.1pH units (e.g. 0.05, 0.01 or 0.00 units) relative to the mother strain, and the term "substantially the same texture-forming property" should preferably be understood as that the Pa value may vary up to 20% (e.g. up to 10% or up to 5%) and/or +10/-2 units (e.g. +5, +3, +/-2, +/-1.0, +/-0.5 or 0.0 units) relative to the mother strain.

In a second aspect, the present invention relates to a method for providing a strain of lactobacillus delbrueckii subsp bulgaricus that produces less acid when present in a fermented milk product (e.g. during storage of the final product) compared to the mother strain, the method comprising treating the strain of lactobacillus delbrueckii subsp bulgaricus (mother strain) with a mutagen (preferably a mild or weak mutagen) such as a mutagen selected from the group consisting of: ethyl methanesulfonate (e.g. 1%); nitrous acid (e.g., 0.05M); methyl methanesulfonate (e.g., 20 mM); nitrosoguanidine (e.g., 25M); and ICR-170 acridine mustard (e.g., 5g/ml), such as X-rays (e.g., 2000 r/min); or UV radiation (e.g. 600erg/mm per minute)²). Currently preferred is the mutagen EMS (ethyl methanesulfonate).

It is presently preferred that the mother strain is a strain of lactobacillus delbrueckii subsp bulgaricus having high texture formation after growth in milk and having high post-acidification, such strain being selected from the group of strains having the following properties:

a) high texture forming characteristics, such as Pa of not less than 25 (e.g., not less than 30; not less than 35; not less than 39; not less than 42; not less than 44; not less than 45; not less than 46; not less than 47; not less than 48; or not less than 50); and

b) high post-acidification properties, such as a reduction in pH of more than 0.20 (e.g. more than 0.30 or 0.40, or even more than 0.50) pH units measured in a standard test (test to determine post-acidification properties) after 7 days of storage.

Examples of such strains are CHCC3984, CHCC3606 and CHCC5713 and mutants of any of these (e.g. functionally equivalent mutants such as mutants having substantially the same post-acidification and texture-forming properties). These strains, as well as the Lactobacillus delbrueckii subsp.

In an interesting embodiment, the method of the invention further comprises:

incubating the single mutant in a culture medium (e.g.on a pH indicator plate; in a microtiter plate containing e.g.a medium supplemented with a pH indicator; or in a test tube containing e.g.a medium supplemented with a pH indicator); and is

-selecting mutants having a final pH of the medium after incubation that is significantly higher than the mother strain.

Conveniently, the pH is measured by a pH electrode or by using a pH indicator such as bromophenol violet or bromophenol green and the culture medium is milk, such as a standard culture medium.

In a further aspect, the invention relates to the use of a strain of lactobacillus delbrueckii subsp.

The invention also relates to a process for preparing a dairy product comprising mixing a strain according to the invention with milk, and to a dairy product obtainable by said process. The resulting dairy product is characterized by having an unpleasant, mild, less sour taste than previously known products.

In a still further aspect, the invention relates to a method of propagation of a strain of lactobacillus delbrueckii subsp bulgaricus comprising mixing a strain of the invention or a composition comprising said strain with a growth medium, such as milk (e.g. a standard medium).

In a final aspect, the present invention relates to a composition useful for fermenting milk comprising a strain of lactobacillus delbrueckii subsp. An example of such a composition is a starter culture, which in addition to the strain of the invention further comprises the strain used for preservation or an additive. The composition may be in a dry, lyophilized, frozen or liquid form.

Definition of

In the present context, the term "milk (milk)" includes fat-free milk, low-fat milk, full-fat milk, lactose-free milk (produced by hydrolyzing lactose to glucose and galactose with lactase or other methods), concentrated milk, or powdered milk. Fat-free milk is a fat-free (non-fat) or skim (skim) milk product. Low fat milk is typically defined as milk containing from about 1% to about 2% fat. Whole milk often contains 2% or more fat. As used herein, the term "milk" is also intended to include milk from animal and plant sources. Milk of animal origin includes, but is not limited to, human, bovine, ovine, caprine, buffalo, camel, llama, mare and deer. Milk of vegetable origin includes, but is not limited to, milk extracted from soybeans and oats. Furthermore, the term "milk" refers not only to whole milk, but also to skim milk or any liquid component derived therefrom.

The term "high-quality forming strain" is to be understood as a strain which when used at an incubation temperature of 43 ℃ is at 1x10⁵-1x10⁶Inoculation of the standard medium up to pH4.55 with a ratio cfu/g results in a shear stress of the fermented milk of more than 25Pa, which is instrumentally determined 20 hours after the end of the fermentation/coagulation as described in the "viscometry". In contrast, the term "low-quality forming strain" is to be understood as a strain which, when used at an incubation temperature of 43 ℃, is 1x10⁵-1 x 10⁶Inoculation of the standard medium up to pH4.55 with the ratio cfu/g results in a shear stress of the fermented milk of less than 25Pa, which is instrumentally determined 20 hours after the end of the fermentation/coagulation, as described in the "determine viscosity test".

The term "high post-acidification strain" should be understood as a strain which, when grown according to the test for determining post-acidification properties, lowers the pH by more than 0.2pH units (the pH is lowered from 4.55 to 4.35 or less) after 7 days of storage. In contrast, the term "low post-acidification strain" should be understood as a strain which, when grown according to the test for post-acidification properties, lowers the pH by not more than 0.20pH units after 7 days of storage (the pH is lowered from 4.55 to 4.35 or to a pH value in the range of 4.55 to 4.35, both endpoints inclusive).

The term "standard medium" is to be understood as milk reconstituted from skim milk powder, having a dry matter content of 9.5% and having been heat treated to 99 degrees celsius for 15 minutes in a batch process.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language, provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Drawings

FIG. 1 depicts a pH-time plot of mutant strain CHCC8535 and parent strain CHCC5713 at 43 ℃. Reference is made to example 1.

FIG. 2 depicts the pH vs. time plot of mutant strain CHCC7159 and parent strain CHCC3606 at 43 ℃. Reference is made to example 1.

FIG. 3 depicts the average of the shear stresses measured on three separate samples produced with different strains of Lactobacillus delbrueckii subspecies bulgaricus. The error bars cross 2 standard deviations around the mean. Reference is made to example 1.

Figure 4 depicts a pH-time plot of mutant strain CHCC10019 and parent strain CHCC3984 at 43 ℃. Reference is made to example 2.

Figure 5 depicts the average of the shear stresses measured on three separate samples produced with the mutant strain CHCC10019 and the mother strain CHCC 3984. The error bars cross 2 standard deviations around the mean. Reference is made to example 2.

Examples

Conventional methods

According to the method of the present invention, a mother strain of Lactobacillus delbrueckii subspecies bulgaricus having high-quality formation properties and high post-acidification properties is treated with a chemical mutagen. Mutants with low post-acidification properties are selected from the resulting strains, and subsequently the desired mutants with high quality formation properties are selected. The method is described in more detail below:

test for measuring viscosity (texture Forming Properties)

The frozen concentrate of this strain was used to inoculate 200mL milk reconstituted from skim milk powder (standard medium). The milk has a dry matter content of 9.5% and is formed at atmospheric pressureThe batch had been heat treated to 99 ℃ for 15 minutes. Frozen concentrates of Lactobacillus bulgaricus typically exhibit a cell number between 1 and 10⁹And 1.10¹⁰Between cfu/g. The inoculation rate was 1g concentrate per 10L milk, thus in the range 1.10⁵And 1.10⁶Between cfu/mL milk. The incubation was carried out at 43 ℃ until a pH of 4.55 was reached, at which point the milk had coagulated. The fermented milk was then cooled to 5 ℃ with regular stirring (by placing in an ice bath for 20 minutes, then in a refrigerator).

After 20 hours when ph4.55 was reached, the fermented milk was warmed to 13 ℃ and gently stirred by a rod fitted with a perforated disc (diameter 3cm) until the sample was homogeneous. Stirring consisted of 20 up and down movements of the rod on the sample. The rheological properties of the samples were determined at 13 ℃ on a rheometer (StressTech, Reologica Instruments, Sweden) fitted with a rotor/quilt (CC25) coaxial measuring system.

The viscosity measurement was carried out with 21 steps of shear rate from 0.27 to 300 l/s. The shear rate increased and then decreased, and the upward and downward curves of shear stress and apparent viscosity were recorded. The delay, integration and equilibration times were 5s, 10s and 5min, respectively. The shear stress at 300s-1 was chosen for further analysis.

Shear stress is measured in Pa. By definition, viscosity is equal to shear stress divided by shear rate. The viscosity is therefore proportional to the shear stress and they represent the same behavior, assuming that the shear stress is measured at a fixed shear rate of 300 l/s.

Test for determining post-acidification Properties

The frozen concentrate of this strain was used to inoculate 200mL milk reconstituted from skim milk powder (standard medium). The milk had a dry matter content of 9.5% and had been heat treated to 99 ℃ for 15 minutes in a batch process.

Frozen concentrates of Lactobacillus bulgaricus typically exhibit a cell number between 1 and 10⁹And 3.10¹⁰Between cfu/g.The inoculation rate was 1g concentrate per 10L milk, thus in the range 1.10⁵And 3.10⁶Between cfu/mL milk. The incubation was carried out at 43 ℃ until a pH of 4.55 was reached, at which point the milk had coagulated. The fermented milk was then cooled to 5 ℃ with regular stirring (by placing in an ice bath for 20 minutes, then in a refrigerator). At about 1 point of storage; 7; 14; after 21 days, the pH of the cooled fermented milk was measured with a pH electrode.

Method for preparing standard culture medium

Skim milk powder (Ara Ingredients, Denmark-Milex 240 medium temperature milk powder) was dissolved in water for no more than 30 min. The goal of making milk was 9.5% (w/w) dry matter.

The milk should be sterilized by the following temperature profile:

temperature (deg.C) time (min)

Heating- >99 +/-1 <20

Sterilizing 99 +/-115 +/-1

Cooling to 99 plus or minus 1- >40 plus or minus 5 <40 >

Subsequently, the milk should be stored at <7 ℃. The milk should not be used the next day ago.

Propagation of bacteria

Strains of Lactobacillus delbrueckii subspecies bulgaricus that increase texture in fermented milk and are post-acidified are well known. Such strains are preferably used as mother strains. The lactic acid bacteria strains are usually added to milk as liquid solutions, as freeze-dried powders or as frozen particles to allow fermentation. The bacteria count of the liquid, powder and granules can be at 1E per gram using standard cell counting techniques⁶To 1E¹²Individual colony forming unit (1. 10)⁶To 1.10¹²cfu/g). TheLiquids, powders and granules are added to milk at a level of about 0.0005 to 1%.

Mutagenesis

Bacteria such as lactobacillus delbrueckii subspecies bulgaricus strains, which produce high texture after growth in milk and have high post-acidification, are preferably subjected to chemical mutagenesis with mild mutagens such as EMS (ethyl methanesulfonate). EMS results in random base pair substitutions on the genetic material (chromosomes and plasmids) of the mutagenized bacteria. After mutagenesis the mutagenized cultures were plated on medium.

The preferable scheme is as follows: the mother strains in frozen stocks were inoculated in MRS medium and cultured anaerobically at 37 ℃ for 24 hours. For mutagenesis, overnight cultures were vortexed at maximum speed for 1 minute to separate potential cell chains and treated with EMS (10ml/ml) at 37 ℃ for 2 hours. EMs result in killing more than 99% of the cells. EMS-treated cultures were frozen at-80 ℃ in 20% glycerol and the stocks were used for screening.

Screening

Several thousand colonies were picked by a colony picking robot into microtiter plates containing 200 microliters of milk supplemented with, for example, a pH indicator (bromophenol violet/bromophenol green). Each well in the microtiter plate is then subjected to a model milk fermentation. The pH of milk in wells containing the mutants was compared to corresponding wells containing wild type strains (lactobacillus delbrueckii subsp bulgaricus strains which produce high texture after growth in milk and have high post-acidification, e.g. lactobacillus delbrueckii subsp bulgaricus strains CHCC3984, CHCC3606 and CHCC 5713). Mutants with a significantly higher final pH of the milk after acidification at 43 ℃ than the wild type strain were identified, for example by colorimetric assessment (using a pH indicator) or using a pH electrode. To confirm that the mutant resulted in lower post-acidification, larger scale (200ml) acidification was performed by recording pH in a recorder system with standard pH electrodes.

In order to control that the obtained mutants have the same texturizing potential as the mother strain, milk samples were fermented with the mutants and the mother strain to the same final pH value and the rheological properties of the obtained fermented milks were assessed and compared by standard techniques.

Example 1: preparation and characterization of LB strains CHCC7159 and CH8535

CHCC7159 and CHCC8535 are lactic acid bacteria of Lactobacillus Bulgaricus (LB) type. These strains are capable of acidifying lactic acid under industrially relevant conditions and are characterized by a low post-acidification. Strain CHCC7159 is a mutant obtained from CHCC3606 and compared to it. CHCC8535 is a mutant of CHCC5713 and compared thereto. Mutagenesis (preferred protocol) and screening were performed as described above.

Frozen concentrates of CHCC7159, CHCC8535, CHCC3606 and CHCC5713 were used to inoculate 200mL milk reconstituted from skim milk powder. The milk had a dry matter content of 9.5% and had been heat treated to 99 ℃ in a batch process for 15 minutes. Frozen concentrates of Lactobacillus bulgaricus typically exhibit a cell number between 1 and 10⁹And 3.10¹⁰Between cfu/g. The inoculation rate was 1g concentrate per 10L milk, thus in the range 1.10⁵And 3.10⁶Between cfu/mL milk. The incubation was carried out at 43 ℃ until a pH of 4.55 was reached, at which point the milk had coagulated. The fermented milk was then cooled to 5 ℃.

The pH-time diagram indicates that CHCC8535 reached pH 4.60 in less than 20 hours, demonstrating that the strain is capable of lactate under industrially relevant inoculation and incubation conditions (figure 1).

The pH-time diagram indicates that CHCC7159 reached pH 4.60 in about 21 hours, demonstrating that the strain was able to acidify lactic acid under industrially relevant inoculation and incubation conditions (fig. 2).

CHCC7159 and CHCC8535 were less acidified than their respective parent strains throughout the fermentation. CHCC8535 showed a pH of 4.17 after 20 hours, which was 0.32pH units higher than the mother strain. CHCC7159 showed a pH of 4.64 after 20 hours, which was 0.50pH units higher than the mother strain.

CHCC7159 and CHCC8535 are capable of producing enhanced texture in fermented milk

The day after incubation, the fermented milk was warmed to 13 ℃ and gently stirred by a rod with a disc with holes until the sample was homogeneous. The rheological properties of the samples were determined on a rheometer (StressTech, reological Instruments, Sweden) fitted with a C25 coaxial measurement system. The viscosity measurement was carried out with a shear rate of 0.27 to 300l/s in 21 steps. The shear rate increased and then decreased, and the upward and downward curves of shear stress and apparent viscosity were recorded. The delay and integration times were 5s and 10s, respectively. The shear stress at 300s-1 was chosen for further analysis. Fermented milks incubated with concentrates of CHCC7159 and CHCC8535 had a better texture than fermented milks produced with their respective mother strains (fig. 3). The shear stress values recorded for these two mutant strains averaged 45 and 55Pa, while their parent strain had a shear stress value of approximately 40Pa or less.

Conclusion

Strains CHCC7159 and CHCC8535 combined the following properties:

● produced high texture in fermented milk-higher than 40Pa under the conditions of the test experiment

● is capable of acidifying lactic acid to a pH of 4.60 in less than 24 hours.

● reached a pH higher than 4.15 after 20 hours under the test experimental conditions.

Example 2: preparation and characterization of LB Strain CHCC10019

CHCC10019 is a lactic acid bacterium of the lactobacillus bulgaricus type. CHCC10019 is capable of acidifying lactic acid under industrially relevant conditions and is characterized by low post-acidification. The strain CHCC10019 is a mutant obtained from CHCC3984 and compared thereto. Mutagenesis and screening were performed as described above ("preferred protocol"). The mutagen used was EMS.

Overnight cultures of CHCC10019 and CHCC3984 were prepared by inoculating 10mL of milk reconstituted from skim milk powder. The milk had a dry matter content of 9.5% and had been heat treated to 99 ℃ for 15 minutes in a batch process. The biological material was taken from frozen ampoules of the respective strains. Incubate at 37 ℃ for 24 hours.

An overnight culture of CHCC10019 and CHCC3984 was used to inoculate 200mL milk reconstituted from skim milk powder (standard medium). The milk used was the same as described above. The amount of transfer was 1%. Two bottles of milk were incubated for each strain. One bottle was used to obtain the acidification curve; the second bottle was used to obtain a product for determining the rheological properties.

An overnight culture of Lactobacillus bulgaricus typically exhibits a cell number between 1 and 10⁶And 1.10⁸Between cfu/g. The inoculation rate was 1g overnight culture per 100g milk, thus at 1 · 10⁴And 1.10⁶Between cfu/mL milk.

Incubate at 43 ℃ for 20 hours. The second flask, prepared under the same conditions, was incubated at 43 ℃ until a pH of 4.55 was reached, at which point the milk had coagulated. The bottle was cooled to 5 ℃ until the next day for rheological analysis.

The pH-time diagram indicates that CHCC10019 reached pH 4.50 in about 7 hours and 30 minutes, which demonstrates that the strain is capable of lactate under industrially relevant conditions of inoculation and incubation, see fig. 4.

CHCC10019 was less acidified than its mother strain throughout the fermentation. CHCC10019 showed a pH of 4.22 after 20 hours, which was 0.64pH units higher than the mother strain.

CHCC10019 is capable of producing enhanced texture in fermented milk

The day after incubation, the fermented milk was warmed to 13 ℃ and gently stirred by a rod of perforated discs until the sample was homogeneous. The rheological properties of this sample were evaluated as described in example 1. See figure 5 for results. Fermented milk incubated with CHCC10019 had a better texture than fermented milk produced with CHCC 3984. The shear stress values recorded for this mutant strain were on average 45Pa, whereas they were about 35Pa for the mother strain.

CHCC7159 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) on 8.2.2006 and was given the deposit number DSM 17959. CHCC3606, CHCC5713 and CHCC8535 were deposited at DSM on month 3 and 30 of 2006 and are given deposit numbers DSM 18142, DSM 18143 and DSM18144, respectively. CHCC10019 and CHCC3984 are deposited on month 4, 03 of 2007 and are given deposit numbers DSM19252 and DSM 19251, respectively. All deposits were made under the Budapest treaty on the preservation of microorganisms internationally recognized for patent procedures.

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.

Reference to the literature

Mller，C.，Bockelmann，W.，and K.J.Heller，2001.Isolierung vonLactobacillus delbrueckii subsp.bulgaricus mutanten zur herstellung einesjoghurts mit milder geschmackscharakteristik.Kieler Milchwirtchaftlicheforchungsberichte 53，167-185.

WO2006/063142

EP518096A1

EP638642A

US5545554A

Guide To Short-Term Tests For Detecting Mutagenic And CarcinogenicChemicals.Prepared for the IPCS by the International Commission forProtection Against Environmental Mutagens and Carcinogens.WHO，1985.

http://www.inchem.org/documents/ehc/ehc/ehc51.htm

All references cited in this patent are incorporated herein by reference in their entirety.

Claims (3)

1. Lactobacillus delbrueckii subsp. bulgaricus strain selected from the group consisting of: CHCC10019, CHCC8535, and CHCC 7159; these strains were deposited as DSM19252, DSM18144 and DSM17959, respectively.

2. Lactobacillus delbrueckii subsp. bulagrickius strain CHCC7159, deposited as DSM 17959.

3. A dairy product obtained by mixing the strain according to claim 2 or 3 with milk.