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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
  • A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

• 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.
• Bacteria esp. lactic acid bacteria
• Bacteria are used for the production of a variety of food material, such as yoghurt, cheese, curd etc.
• 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.
• 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.
• 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.
• 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 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.
• 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).
• 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.
• 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).
• 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
• an ammonium salt e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of
• 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
• 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
• 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
• 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.
• 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.
• 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).
• 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 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).
• a bacterial metabolite e.g. lactic acid
• 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.
• 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 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.
• 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
• an ammonium salt of organic acid such as ammonium formate and ammonium citrate
• an ammonium salt of an inorganic acid may be added to the medium during the growth of the bacterial cells.
• 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.
• the milk substrate may be milk.
• 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.
• the bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus.
• the bacterial cells belongs the species Streptococcus thermophilus.
• 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.
• 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).
• 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.
• 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.
• 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
• 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
• 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
• optionally bacterial cells optionally bacterial cells.
• 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.
• 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
• 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).
• 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 bacterial cells are lactic acid bacterial cells, e.g. belonging to a genus selected from the group consisting of: Lactobacillus, Lactococcus, and Streptococcus.
• the bacterial cells belongs the species Streptococcus thermophilus.
• 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.
• 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).
• the milk based growth medium should comprise both a strain or strains belonging to the genus
• Lactobacillus e.g. Lactobacillus bulgaricus
• a strain or strains belonging to the species S. thermophilus e.g. Lactobacillus bulgaricus
• the present invention relates to the use of the composition of the invention for the production of a fermented dairy product, e.g. yoghurt.
• 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.
• 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 booster e.g. growth booster or acidification booster
• bacterial cells such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells.
• 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
• 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.
• the source of ammonium releases ammonium into a growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium.
• the ammonium source is exogenous.
• ammonium is not provided by the dairy substrate. It should of course be understood that ammonia may be added instead of ammonium salt.
• the term ammonium salt comprises ammonia (NH3), NH40H, NH4+, and the like.
• the present invention relates to the strains used in the examples, as well as mutants and variants thereof.
• 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".
• 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.
• 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.
• 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.
• 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.
• lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria bifidobacteria, i.e. Bifidobacterium spp.
• bifidobacteria i.e. Bifidobacterium spp.
• 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
• lactic acid bacteria belonging to genus Pediococcus.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• yellow in relation to that ur (-) clones form yellow colonies should be understood as the skilled person would understand it in the present context.
• a mutant/clone may consume urea partially and still be what skilled person would measure as an ur (-) clone in this plate assay.
• 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.
• 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.
• Example 2 was carried out as example 1, with the exception that ammonium citrate is used instead of ammonium formate.
• 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).
• 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.

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