EP3036318A1

EP3036318A1 - Method for growth of bacterial cells

Info

Publication number: EP3036318A1
Application number: EP13776733.1A
Authority: EP
Inventors: Thomas Janzen
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2013-08-20
Filing date: 2013-08-20
Publication date: 2016-06-29
Also published as: WO2015024593A1

Abstract

The present invention relates to a method for growth of bacterial cells, and to a growth medium for growth of bacterial cells.

Description

METHOD FOR GROWTH OF BACTERIAL CELLS

FIELD OF INVENTION

The present invention relates to a method for growth of bacterial cells, and to a growth medium for growth of bacterial cells. Further, the present invention relates to novel fast- acidifying bacterial strains.

BACKGROUND OF INVENTION

Bacteria, esp. lactic acid bacteria, are used for the production of a variety of food material, such as yoghurt, cheese, curd etc. One shortcoming of bacteria, e.g. lactic acid bacteria, with regard to their preparation on a large scale and their applicability, is to be seen in their different nutritional requirements. A medium often utilised for cultivating lactic acid bacterial strains is cow milk. This medium provides a complex natural environment and its fermentation products, e.g. yoghurt, may be directly used as food material. In some cases, bacterial growth could be improved when substances of an undefined and very complex composition, such as yeast extract or peptones of various origins, had been added to the milk. However, these additional components can often cause an off-flavour with the result that cultures growing in a medium supplemented in such a way may not be used for industrial manufacture of dairy products. Moreover, the costs involved and the sometimes varying results in repeatability of achievable bacterial counts, makes them unsuitable for a commercial manufacture of microbial strains.

Fast acidification is one of the most important parameters for dairy cultures, and there is a need for improving the acidification rate of fermented milk products. In view of this, a problem of the present invention is to provide a medium which supports the growth of bacterial strains, both with respect to cell count and production of metabolites such as lactic acid.

SUMMARY OF INVENTION

The present inventors have surprisingly discovered that addition of an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) to a milk medium will boost the growth of cells of the lactic acid bacterial species Streptococcus thermophilus, resulting in a higher rate of acidification of the milk medium. The invention gives the possibility to i) improve the growth rate of the bacterial cells ii), improve the acidification rate, iii) increase the production speed of fermented milk products, and iv) use cultures which hitherto have been considered to have a too slow growth and/or acidification rate, for production of fermented milk products.

Besides, the present inventors have surprisingly discovered that the lag phase is reduced for cultures growing in a medium with an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid). A long lag phase is a general disadvantage for DVS cultures (Direct Vat Set cultures; cultures for direct inoculation) compared to bulk starter systems.

In accordance with this surprising finding, the present invention relates to a method for growth of bacterial cells, wherein the bacterial cells are grown in a growth medium comprising an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid), especially to a method for increasing the production of a bacterial metabolite (e.g. lactic acid), wherein the bacterial cells are grown in a growth medium comprising an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid).

In a preferred embodiment, the bacterial cells belong to the species Streptococcus

thermophilus strain, more preferred the cells belong to a urease negative mutant of S.

thermophilus. The advantage of using urease negative mutants for fermenting milk is the avoidance of acidification delay due to the buffering effect of the released ammonia, as a result of the Streptococcos thermophilus urease activity. This is seen especially at high milk urea concentrations. Due to variations in the natural milk urea concentration this pH delay can vary, so that urease negative mutants show more stable acidification activity (acidification activity independent of the urea concentration). In addition, ammonia can be considered as off flavour in several dairy applications, and it is not desired to have a too high ammonia concentration in the whey. However, the present inventors found that, although urease negative mutants are

characterized by reduced lag phase and a faster initial pH drop, it takes typically longer time to reach the end pH, especially in pasteurized milk, which is the milk used in dairy

fermentations.

The surprising finding of this invention was that the addition of an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) leads to a significant increase of acidification activity in S. thermophilus and even more in urease negative mutants from S. thermophilus. In example 1 it is shown that for a urease positive S. thermophilus strain it is not relevant whether sodium formate or ammonium formate is added to the milk. However, for urease negative mutant the booster effect is maximised by adding ammonium formate whereas sodium formate is not sufficient to get the same effect. It can therefore be concluded that the ammonium part of the formate is critical for the booster effect in urease mutants. The reason for this is likely to be the use of the ammonia for the cell metabolism, especially the amino acid metabolism of S. thermophilus.

It is also surprising that although ammonium is added the present inventors did not observe the same pH buffering activity as it is observed when ammonium is released by the activity of the S. thermophilus urease. The addition of ammonium formate to an S. thermophilus urease mutant acidifying milk combines therefore the two features - reduction of pH delay and shorter lag phase and - the delivery of ammonia which stimulates cell growth by boosting the cell metabolism, especially amino acid synthesis.

DETAILED DISCLOSURE

In a first aspect, the present invention relates to a method for growth of bacterial cells, wherein the bacterial cells are grown in a growth medium comprising an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid).

In an interesting embodiment, the invention relates to a method for increasing the production of a bacterial metabolite (e.g. lactic acid), wherein the bacterial cells are grown in a growth medium comprising an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid).

The ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) may be added to the growth medium before addition of the cells, or it may be added together with the cells, or it may be added before and/or during the growth of the bacterial cells.

The medium may comprise from 0.01 to 10 g/l (e.g. from 0.05 to 1.0 g/l) ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid), or the same amount of ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) may be added to the medium during the growth of the bacterial cells.

In a presently preferred embodiment, the growth medium comprises milk, e.g. cow's milk, preferably the medium comprises more that 90% (vol/vol) milk. The term "growth medium" comprises the a milk substate. "Milk substrate", in the context of the present invention, may be any raw and/or processed milk material. Useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk substrate may be milk. The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk.

In an embodiment, the bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus. In an interesting embodiment, the bacterial cells belongs the species Streptococcus thermophilus.

In a specific embodiment, the bacterial cells are urease negative or substantially urease negative, and it presently preferred that the bacterial cells are specific embodiment, the bacterial cells are urease negative cells belonging to the species S. thermophilus.

Especially when the growth medium is based on milk, and the resulting product of the fermentation is a dairy product (such as yoghurt, fermented milk, cheese) to be sold to consumers, the medium may further comprise cells of an other species (e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus). In case of yoghurt, the milk based growth medium should comprise both a strain belonging to the genus Lactobacillus (e.g. Lactobacillus bulgaricus), and a strain belonging to the species S. thermophilus.

In a second aspect, the present invention relates to a composition (e.g. a growth medium for bacterial cells), which comprises an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid), and optionally bacterial cells.

In an embodiment of this aspect, the composition comprises milk and/or an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) in an amount sufficient to promote growth of the bacterial cells.

The composition may be in the form of a mixture, or in the form of "a kit of parts", which comprises bacterial cells and an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid).

In an embodiment, the bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus. In a interesting embodiment, the bacterial cells belongs the species Streptococcus thermophilus. In a specific embodiment, the bacterial cells are urease negative or substantially urease negative, and it presently preferred that the bacterial cells are specific embodiment, the bacterial cells are urease negative cells belonging to the species S. thermophilus. This skilled person will know how to assess whether a cell is urease negative, he might use the assay disclosed in US patent 6962721.

Especially when the growth medium is based on milk, and the resulting product of the fermentation is a dairy product (such as yoghurt, fermented milk, cheese), the medium may further comprise cells of an other species (e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus). In case of yoghurt, the milk based growth medium should comprise both a strain or strains belonging to the genus

Lactobacillus (e.g. Lactobacillus bulgaricus), and a strain or strains belonging to the species S. thermophilus.

In a further aspect, the present invention relates to the use of the composition of the invention for the production of a fermented dairy product, e.g. yoghurt.

In a further aspect, the present invention relates to the use of an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g. growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells. The term "ammonium salt", "ammonium formate", etc, should be understood as a source of the salt or a combination of the ions. The term "source" of eg. "ammonium formate" or "ammonium salt" refers to a compound or mix of compounds that when added to a culture of cells, provides ammonium formate or an ammonium salt. In some embodiments, the source of ammonium releases ammonium into a growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, ammonium is not provided by the dairy substrate. It should of course be understood that ammonia may be added instead of ammonium salt. Thus, the term ammonium salt comprises ammonia (NH3), NH40H, NH4+, and the like.

In a last aspect, the present invention relates to the strains used in the examples, as well as mutants and variants thereof. DEFINITIONS

In the present context, the term "urease negative" or "ur(-)" refers to bacteria which are not able to degrade urea, or bacteria which substantially are not able to degrade urea. A suitable plate assay to test for urease activity is provided in Example 1 of US6962721 (see column 4). The plate assay is simple and the skilled person can routinely identify if a S. thermophilus strain is ur(-) or ur(+) - as said in Example 1 of US6962721, lines 52-54: "The ur(+) clones form blue-coloured halos owing to the production of ammonia, whereas the ur(-) clones form yellow colonies". Accordingly, a ur(-) bacterium may herein be understood as a bacterium that is a ur(-) strain in the Petri Dishes plate assay of Example 1 of US6962721 (reproduced herein as working Example 1) - i.e. a ur(-) bacterium is forming a yellow colony in the Petri Dishes plate assay.

As known to the skilled person one may routinely select/identify herein suitable ur(-) bacteria by use of e.g. the above discussed plate assay - for instance one may start from a suitable ur(+) wild-type strain, mutate this by e.g. use of a mutagen and use the plate assay to select/identify ur(-) clones/strains. As evident to the skilled person, such selected/identified random ur(-) mutants may comprise mutation(s)/deletion(s) at many herein relevant places of the bacterial genome - e.g. within a gene encoding for the urease enzyme or e.g. in relevant promoters outside the coding part of a gene as such. In the present context, it may be seen as relatively irrelevant, where such relevant mutation(s)/deletion(s) could be in a ur(-) bacterium - the main point is that such ur(-) mutants can routinely be made as discussed herein.

As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes

Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are frequently used as food cultures alone or in combination with other lactic acid bacteria.

Preferably, the lactic acid bacteria are lactic acid bacteria selected from the group consisting of: lactic acid bacteria belonging to genus Lactobacillus, such as Lactobacillus helveticus, Lactobacillus delbruekii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus salivarius or Lactobacillus rhamnosus; lactic acid bacteria belonging to genus Lactococcus, such as

Lactococcus lactis; lactic acid bacteria belonging to genus Streptococcus, such as

Streptococcus thermophilus; lactic acid bacteria belonging to genus Leuconostoc, such as Leuconostoc lactis; lactic acid bacteria belonging to genus Bifidobacterium, such as Bifidobacterium longum, bifidobacterium animalis, or Bifidobacterium breve; lactic acid bacteria belonging to genus Propionibacteria; lactic acid bacteria belonging to genus

Enterococcus, such as Enterococcus faecum; and lactic acid bacteria belonging to genus Pediococcus.

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 (e.g ., "such as") 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. LEGENDS TO THE FIGURE

Figure 1 depicts acidification curves for S. thermophilus CHCC4325 and the urease-negative mutant CHCC9908 in pasteurised milk at 37 °C with and without the addition of 0.2 g/l ammonium formate, resp. sodium formate; 1% inoculation from over-night culture. Figure 2 depicts acidification curves for S. thermophilus CHCC4325 and the urease-negative mutant CHCC9908 in pasteurised milk at 37 °C with and without the addition of 0.2 g/l ammonium citrate; 1% inoculation from over-night culture.

EXPERIMENTAL

Method of Culturing Ur(-) Bacteria on Petri Dishes

An agar-based medium whose composition is shown below is prepared and poured into Petri dishes of diameter equal to 9 cm. Composition of the culture medium.

Tryptone 2.5 g,

Pepsic meat peptone 2.5 g

Papainic soya peptone 5 g Autolytic yeast extract 2.5 g

Meat extract 5 g

Sugar (glucose, lactose or saccharose) 5 g

Sodium glycerophosphate.6H20 19 g

Magnesium sulphate 0.25 g

Ascorbic acid 0.5 g

Agar 15 g

Distilled water 1 liter If needed, a cofactor of urease can be added to this medium. Adjust the pH to 7.0 and autoclave for 15 minutes at 115°C.

The cells to be analyzed are seeded on this medium so as to obtain around 100 colonies per Petri dish. The culture take place under anaerobic conditions at a temperature of 35-45°C, preferably 37-42°C.

After two days of culture, there is poured over each Petri dish around 20 ml of an agar-based solution prepared as follows: dissolve by heating 15 g of agar in 1 liter of a potassium phosphate buffer solution at 50 mM (pH 6) supplemented with 100 mg/l of bromothymol blue, cool the solution to 50°C, add 10 g of urea and acidify the medium with hydrochloric acid until a yellowish-orange color is obtained.

After solidification of the agar, the Petri dishes are incubated for 1 hour at 37°C.

The ur(+) clones form blue-colored halos owing to the production of ammonia, whereas the ur (-) clones form yellow colonies.

When the ur(-) mutants are sought, the clones not forming a blue halo are recovered and tested again on the same culture medium in order to confirm the ur(-) characteristic. It should also be verified that these mutants do not consume urea (or consume it only partially) when they are cultured in milk.

The term "yellow" in relation to that ur (-) clones form yellow colonies should be understood as the skilled person would understand it in the present context.

As described above - a mutant/clone may consume urea partially and still be what skilled person would measure as an ur (-) clone in this plate assay.

Accordingly, it may be that the color of a specific mutant/clone could be what may be termed light green - i.e. a color that it clearly significantly closer to yellow than blue - such a clone would by the skilled person be understood as a ur (-) clone in the present context.

Example 1 :

Different urease negative mutants were isolated from the fast acidifying commercial S.

thermophilus strain CHCC4325. The most promising of these mutants, CHCC9908, showed a significantly increased acidification activity and a reduced lag phase in the presence of ammonium formate.

The addition of 0.2 g/l ammonium formate in combination with the urease-negative mutant CHCC9908, showed indeed a reduced lag phase, and a pH after 4 hours of 5.2 compared to 6.1 for mother strain CHCC4325 and mutant without addition of any ammonium formate. There was no difference in acidification between CHCC4325 and CHCC9908 in the presence of ammonium formate, both strains showed a similar increased acidification activity. However, CHCC9908 was significantly slower in acidification in the presence of sodium formate compared with ammonium formate. Therefore, the addition of ammonium has a much better boosting effect for the urease-negative mutant. This is due to the non-availability of ammonium which, in the case of CHCC4325 wt, is produced by the strain itself.

Compared to the mother strain CHCC4325 there was a gain of 0.8 pH values (pH difference after 5 hours), and pH 5.2 is reached ca. 1.5 hours earlier (see Figure 1).

Example 2 :

Example 2 was carried out as example 1, with the exception that ammonium citrate is used instead of ammonium formate. The addition of 0.2 g/l ammonium citrate to pasteurised milk has a similar effect on acidification as the addition of ammonium formate.

CHCC9908 reached a pH of 5.0 two hours faster when ammonium citrate was added to the milk (see Fig. 2).

There was also an increase in acidification activity within the first six hours for CHCC4325 in the presence of ammonium citrate.

The acidification activity increase due to ammonium citrate addition was obvious, but relatively low for CHCC4325, but much more pronounced for CHCC9908. Thus, the addition of ammonium has an even better boosting effect for the urease-negative mutant. This is due to the non-availability of ammonium, which, in the case of CHCC4325 wt, is produced by the strain itself.

It can be concluded that the boosting effect for a urease negative mutant is mainly due to the ammonium component of the added compound, but, as in the case of ammonium formate, can be furthermore increased by the citrate component.

The difference in acidification between a urease positive wild type strain and its urease negative mutant is even higher when additional amounts of urea are added to the pasteurised milk (to mimick a milk with a naturally higher urea concentration), since then the buffering activity (pH increase due to released ammonium) of the urease enzyme activity is leading to a more pronounced delay in acidification (data not shown). 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

EP1211947B1

EP1048215B1

US6962721

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

Claims

1. A method for growth of bacterial cells, wherein the bacterial cells are grown in a growth medium comprising an ammonium salt.

2. A method of claim 1, wherein the ammonium salt is selected from the group consisting of an ammonium salt of organic acid or an ammonium salt of an inorganic acid.

3. A method of claim 1 or 2, wherein the ammonium salt is selected from the group consisting of ammonium formate and ammonium citrate.

4. A method for growth of bacterial cells, wherein the bacterial cells are grown in a growth medium comprising ammonium formate.

5. A method for increasing the production of a bacterial metabolite (e.g. lactic acid), wherein the bacterial cells are grown in a growth medium comprising an ammonium salt.

6. A method of claim 5, wherein the ammonium salt is selected from the group consisting of an ammonium salt of organic acid or an ammonium salt of an inorganic acid.

7. A method of claim 4 or 5, wherein the ammonium salt is selected from the group consisting of ammonium formate and ammonium citrate.

8. A method for increasing the production of a bacterial metabolite (e.g. lactic acid), wherein the bacterial cells are grown in a growth medium comprising ammonium formate.

9. The method of any preceding claim, wherein the medium comprises from 0.01 to 10 g/l (e.g. from 0.05 to 1.0 g/l) of the ammonium salt, or wherein the same amount is added to the medium during the growth of the bacterial cells), and/or wherein the growth medium comprises milk, e.g. cow's milk, such as more than 90% (vol/vol) milk.

10. The method of any preceding claim, wherein the medium comprises from 0.01 to 10 g/l (e.g. from 0.05 to 1.0 g/l) ammonium formate, or wherein the same amount is added to the medium during the growth of the bacterial cells), and/or wherein the growth medium comprises milk, e.g. cow's milk, such as more than 90% (vol/vol) milk.

11. The method of any preceding claim, wherein the bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus, such a bacterial cells belonging the species Streptococcus thermophilus.

12. The method of any preceding claim, wherein the bacterial cells are urease negative or 5 substantially urease negative.

13. A composition, e.g. in the form of "a kit of parts", which comprises bacterial cells (e.g. more than 10E5 cell forming units pr mg) and an ammonium salt, such as a composition which comprises at least 1 mg ammonium salt per 50 g of bacterial cells (eg in frozen or freeze dried

10 form).

14. A composition of claim 13, wherein the ammonium salt is selected from the group consisting of an ammonium salt of organic acid or an ammonium salt of an inorganic acid.

15 15. A method of claim 13 or 14, wherein the ammonium salt is selected from the group

consisting of ammonium formate and ammonium citrate

16. A composition, e.g. in the form of "a kit of parts", which comprises bacterial cells (e.g. more than 10E5 cell forming units pr mg) and ammonium formate.

20

17. The composition of any of claims 13 to 16, wherein the bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus, such a bacterial cells belonging the species Streptococcus thermophilus.

25

18. The composition of any of claims 13 to 17, wherein the bacterial cells are urease negative or substantially urease negative.

19. Use of the composition of any of claims 13 to 18 for the production of a fermented dairy 30 product, e.g. yoghurt or cheese.

20. Use of an ammonium salt as a booster (e.g. growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells.

35

21. Use of claim 20, wherein the ammonium salt is selected from the group consisting of an ammonium salt of organic acid or an ammonium salt of an inorganic acid.

22. Use of claim 20 or 21, wherein the ammonium salt is selected from the group consisting of 40 ammonium formate and ammonium citrate.

23. Use of ammonium formate as a booster (e.g. growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells.