FIELD OF INVENTION

The present invention relates to mutants of lactic acid bacteria, such asLactobacillus delbrueckiisubsp.bulgaricusandStreptococcus thermophilus, which are resistant to the antibiotic ampicillin, and which were found to give an increased texture when grown in milk while maintaining the other growth properties of the parent strain. The present invention, furthermore, relates to cultures, such as starter cultures, comprising such mutants, and to dairy products fermented with the cultures.

BACKGROUND OF INVENTION

The food industry uses numerous bacteria, in particular lactic acid bacteria, in order to improve the taste and the texture of foods but also in order to extend the shelf life of these foods. In the case of the dairy industry, lactic acid bacteria are used intensively in order to bring about the acidification of milk (by fermentation) but also in order to texturize the product into which they are incorporated.

Among the lactic acid bacteria used in the food industry, there can be mentioned the generaStreptococcus, Lactococcus, Lactobacillus, Leuconostoc, PediococcusandBifidobacterium. The lactic acid bacteria of the speciesStreptococcus thermophilusare used extensively alone or in combination with other bacteria such asLactobacillus delbrueckiisubsp.bulgaricusfor the production of food products, in particular fermented products. They are used in particular in the formulation of the ferments used for the production of fermented milks, for example yoghurts. Certain of them play a dominant role in the development of the texture of the fermented product. This characteristic is closely linked to the production of polysaccharides.

The current trend in yoghurts is for mild flavor and high texture. Today this is achieved by the use of cultures which produce a mild flavor and the addition of thickeners or protein to give the desired thickness. Yoghurt producers would like to be able to make yoghurt with these properties without the addition of thickening agents. This will help them reduce cost and give a cleaner label. One very attractive way to achieve this would be to have a starter culture which produces a high level of texture.

In order to meet the requirements of the industry, it has become necessary to provide novel texturizing strains of lactic acid bacteria, in particular ofLactobacillus delbrueckiisubspbulgaricusandStreptococcus thermophilus, for texturizing food products. Especially there is a need for novel texturizing strains ofLactobacillus delbrueckiisubspbulgaricuswhich can be used together with novel texturizing strains ofStreptococcus thermophilus.

The inventors of the present invention has previously developed a novel selection method for the identification of improved lactic acid bacteria, such asLactobacillus delbrueckiisubsp.bulgaricusandStreptococcus thermophilusbacteria, which result in high texture when used for fermenting milk substrate for fermented milk products.

The method, which is described in International (PCT) patent application No. WO 2012/052557, relates to that the present inventors have identified a surprisingly relevant link between resistance to D-cycloserine and functionally equivalent antibiotics and improved texturizing properties of the lactic acid bacteria.

D-cycloserine (D-4-amino-isoxasolidone) is an antibiotic which inhibits alanine racemase, D-alanyl-D-alanine ligase, D-alanyl-alanine synthase and D-alanine permease causing cell lysis. D-alanine racemase is essential for the production of D-alanine, an integral part of the peptidoglycan layer of the cell wall.

Ampicillin is an antibiotic of the beta-lactam class of antibiotics and is effective against many gram-positive bacteria including most lactic acid bacteria. Ampicillin is a competitive inhibitor of enzymes of the DD-transpeptidase type (EC 3.4.16.4). Inhibition of DD-transpeptidase by ampicillin prevents the formation of a peptide bond required for the formation of the bacterial cell wall and ultimately leads to lysis of the cell.

Mutants resistant to ampicillin have been described for a number of different bacteria but—to the knowledge of the present inventors—there is in the prior art not described or suggested any herein relevant link between resistance to ampicillin and improved texturizing properties.

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that a group of lactic acid bacteria mutants resistant to ampicillin generates high shear stress and/or gel stiffness when the bacteria are used for fermenting milk.

The present inventors have developed a method for obtaining such texturizing lactic acid bacteria strains which are resistant to the antibiotic ampicillin and/or another antibiotic which inhibits the enzyme DD-transpeptidase.

The herein described method for obtaining a lactic acid bacteria strain comprises the following two steps:(I) first to screen and select for lactic acid bacteria strains which are resistant to ampicillin, one may term it a resistance to ampicillin that is significantly higher than normally present in natural/wildtype lactic acid bacteria; and(ii) from the pool of ampicillin resistant strains identified in step (i) to screen and select for a lactic acid bacteria strains that has improved texturizing properties.

As shown in Example 2 herein, the present inventors found that from a pool of ampicillin resistant lactic acid bacteria strains it was relatively rapid to screen/select for a lactic acid bacteria strain that has improved texturizing properties. Essentially, the reason for this is that the present inventors have identified that a very high percentage of the ampicillin resistant lactic acid bacteria strain (selected in step (i)) also have improved texturizing properties.

Accordingly, the first screening and selection for resistance to ampicillin may be seen as a kind of pre-step to rapidly and efficiently be able to screen and select for a lactic acid bacteria strain that has improved texturizing properties.

As evident to the skilled person—a significant advantage of the herein described screening and selection method is that one relatively rapidly and efficiently is able to screen and select for a lactic acid bacteria strain that has improved texturizing properties. For instance, if one already has a lactic acid bacteria strain with commercially relevant properties in relation to e.g. low post-acidification when used in preparing fermented milk products, one can then use this strain as a starting cell for mutagenesis and then relatively rapidly select for and obtain a novel lactic acid bacteria strain that has improved texturizing properties while still maintaining its good properties with respect to e.g. low post-acidification.

As shown in Example 2 herein approximately 25% of the first selected ampicillin resistant strains also resulted in significantly higher texture as determined by an efflux time of 28 ml acidified milk from a polystyrene 25-ml pipette of at least 50 seconds.

It is submitted that without using the novel screening and selection method as described herein, it would not be possible (or it would take a very long time) to identify a lactic acid bacteria strain that results in such a long efflux time.

Without being limited to theory, a theoretical explanation for the herein surprisingly identified link between ampicillin resistance and improved texturizing properties could be that such ampicillin resistant lactic acid cells produce more of the so-called extracellular polysaccharides (EPS). It could then theoretically be that such EPS could give a kind of protection around the cell, i.e. that these EPS protect the cells against ampicillin entry into the cells and thereby give the increased ampicillin resistance.

Without being limited to theory, it could also be the increased production of EPS that result in the increased texture in milk fermented with the ampicillin resistant lactic acid bacteria.

As discussed above, the herein identified lactic acid bacteria strains are first selected for being resistant to ampicillin, i.e. resistant to ampicillin concentrations that are significantly higher than the ampicillin concentrations tolerated by natural/wildtype lactic acid bacteria.

In accordance with the above surprising findings, the present invention relates to texturizing lactic acid bacteria strains, such asLactobacillus delbrueckiisubspbulgaricusandStreptococcus thermophilusstrains, which are ampicillin resistant and/or resistant to another antibiotic which inhibits the enzyme DD-transpeptidase and a method for obtaining such strains. Furthermore, the present invention relates to cultures, such as starter cultures, comprising the mutants and to dairy products, such as fermented milk products fermented with the cultures.

