Composition for increasing lactic acid bacteria biomass

Abstract

This invention relates to compositions for improving the biomass and survival rate of lactic acid bacteria. The invention also relates to the use of said compositions in culture media. Accordingly, the compositions are characterized in that they comprise a yeast extract, wherein the yeast extract comprises at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight.

Description

Technical Field

[0001] This invention relates to compositions for increasing the biomass and survival rate of lactic acid bacteria. The invention also relates to the use of said compositions in culture media. Background Technology

[0002] Ingesting live microorganisms known as "probiotics" is highly beneficial to health, especially in improving gut microbiota balance and immune system function. The World Health Organization (WHO) defined "probiotics" in 2001 as "live microorganisms that, when administered in adequate amounts, produce beneficial effects on the health of the host." Lactic acid bacteria are among the main probiotics, particularly Lactobacillus (Lactobacillus spp.). Lactobacillus ) and Bifidobacterium spp. Bifidobacterium These are strains of bacteria that prevent harmful bacteria from colonizing the gut microbiota by releasing lactic acid and bacteriocins. Therefore, they contribute to better gut comfort and protect against infection. They are naturally found in fermented foods such as yogurt, kefir, sauerkraut, and sourdough starter.

[0003] Therefore, there is a demand in both the pharmaceutical and food processing industries for adding lactic acid bacteria to their final products. Consumers suffering from digestive issues who wish to boost their immune system naturally also have this need.

[0004] However, lactic acid bacteria are microorganisms with high nutritional requirements. They are considered "auxotrophic" because they lack the ability to synthesize certain nutrients necessary for their growth; therefore, these nutrients must be present in their growth substrate. The culture medium formulation is one of the most important factors. Its composition affects the growth of lactic acid bacteria. Therefore, if any essential nutrient required for their growth is lacking or insufficient, it will significantly impact the growth and yield of these bacteria.

[0005] Patent EP2251416 describes how adding compounds involved in nucleic acid biosynthesis to yeast cultures, and under high optical density conditions (greater than 10 at OD=600 nm), can improve the biomass yield of lactic acid bacteria. In this patent, the culture medium used must contain at least one porphyrin molecule to improve oxygen transport. However, such molecules are costly and derived from animals.

[0006] Currently, there is still an urgent need to develop more economical and non-animal-derived compositions to address societal concerns.

[0007] One of the objectives of this invention is to provide a composition that can cost-effectively increase the biomass yield of lactic acid bacteria without using any animal-derived raw materials. Summary of the Invention

[0008] Surprisingly, the inventors noted that compositions containing nucleoside-rich yeast extracts could meet the nutritional needs of lactic acid bacteria and increase their biomass.

[0009] One object of the present invention is to provide a composition for increasing the biomass of lactic acid bacteria, characterized in that the composition comprises a yeast extract containing at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight.

[0010] Yeast extracts are generally known to those skilled in the art. According to the present invention, it should be understood that "yeast extract" refers to the soluble component obtained by autolysis or enzymatic hydrolysis of yeast cells using endogenous or exogenous enzymes. The soluble portion can also be obtained by plasmolysis or mechanical disruption of yeast cells using known methods (such as high-pressure homogenization, mechanical grinding, glass bead mechanical lysis, ultrasonic disruption, repeated freeze-thaw cycles, or osmotic shock). Similarly, according to the present invention, the yeast extract is preferably the soluble portion obtained after autolysis of the yeast cells, i.e., the component obtained solely by enzymatic hydrolysis using endogenous yeast enzymes. Enzymatic hydrolysis of yeast cells can also be carried out using exogenous enzymes, i.e., by adding additional enzymes, such as proteases, in particular.

[0011] Yeast extracts obtained by enzymatic hydrolysis or autolysis are also referred to by those skilled in the art as "yeast hydrolysate" or "yeast peptone".

[0012] The yeast extract according to the invention is obtained from any yeast. Preferably, the yeast strain used to prepare the extract according to the invention belongs to the genus *Saccharomyces* (Saccharomyces). Saccharomyces Kluyveromyces ( Kluyveromyces ) or Candida spp. ( Candida Also known as Pichia pastoris Pichia or Lindnerella spp. Lindnera ), especially preferred from brewing yeast ( Saccharomyces cerevisiae )kind.

[0013] "Nucleoside-rich yeast extract" should be understood as a yeast extract that contains at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight.

[0014] "Dry products" should be understood as products with a dry matter content of at least 94.0%.

[0015] Nucleic acids are linear polymers composed of chains of nucleotides. Nucleotides are linked together by phosphodiester bonds.

[0016] Each nucleotide contains a nitrogenous base and an acid group linked to a pentose sugar (a 5-carbon sugar), which is ribose in RNA and deoxyribose in DNA. In other words, each nucleotide consists of a nucleoside and one to three phosphate groups linked to the pentose sugar via phosphodiester bonds. The phosphodiester bonds can be located at the 5' or 3' end.

[0017] In the context of this invention, "nucleotide" should be understood as a monophosphate nucleotide, preferably a 5'-monophosphate nucleotide.

[0018] By definition, nucleosides consist of a nitrogenous base linked to a pentose sugar (a 5-carbon sugar). The pentose sugar is ribose in RNA and deoxyribose in DNA, and the two are linked by an N-glycosidic bond. The nitrogenous base can be a purine base (i.e., adenine, hypoxanthine, or guanine) or a pyrimidine base (i.e., cytosine, thymine, or uracil).

[0019] In particular, the nucleosides contained in the compositions according to the invention may have purine bases, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine and inosine; or have pyrimidine bases, such as thymidine, deoxythymidine, uridine, deoxyuridine, cytidine and deoxycytidine.

[0020] For the purposes of this invention, the term "eukaryotic" corresponds to the sum of nucleosides naturally present in yeast extracts, i.e., nucleosides prior to any exogenous treatment (e.g., hydrolysis) of the yeast extract.

[0021] "Naturally present in yeast extract" should also be understood as free nucleosides, that is, nucleosides that exist in monomeric form and have not undergone any exogenous treatment.

[0022] In contrast, this definition does not include nucleosides that are naturally present in RNA, i.e., nucleosides in polymeric form, as well as nucleotides and nitrogenous bases.

[0023] Preferably, the yeast extract in the composition contains at least 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, or 12.0% eukaryotic acid by dry weight, preferably at least 8.0%, and particularly preferably at least 10.0%.

[0024] The compositions according to the invention comprise yeast extract containing 5.0% to 20.0% enucleosides by dry weight. Preferably, it is 5.0% to 15.0%, more particularly 8.0% to 11.0%.

[0025] For the purposes of this invention, the term "potential nucleoside" refers to the sum of all free and bound nucleotide compounds obtained after treatment such as hydrolysis, including nucleotides, nucleosides, free nitrogenous bases, and compounds existing in polymeric form (such as RNA). Potential nucleosides are expressed as 5'-monophosphate nucleotides (in 5'-XMP equivalents) and are calculated as a percentage of dry weight.

[0026] To calculate the potential nucleoside content, the molar mass ratio of the corresponding nucleotide compound to the target nucleoside should be used, and the mass concentration of each nucleoside should be converted to a 5'-XMP equivalent mass concentration.

[0027] The potential nucleoside content is calculated by converting the total nucleoside mass concentration to the 5'-XMP equivalent mass concentration.

[0028] The conversion calculation is as follows: Among them, "nucleoside" corresponds to the mass concentration of the target nucleoside.

[0029] Mw5'XMP corresponds to the molar mass of the associated 5'-monophosphate nucleotide.

[0030] Wherein, Mw nucleoside corresponds to the molar mass of the target nucleoside, expressed in g / mol.

[0031] The potential nucleoside content is necessarily higher than the euglysin content.

[0032] In this invention, the yeast extract is subjected to acid hydrolysis. This treatment converts all nucleotide compounds into nitrogenous bases. The resulting nitrogenous bases are expressed as 5'-monophosphate nucleotides and expressed as a percentage of dry weight, taking into account the molecular weights of the nitrogenous bases and their corresponding 5'-monophosphate nucleotides.

[0033] The conversion calculation is as follows: Among them, "nitrogenous bases" refers to the mass concentration of the target base.

[0034] Mw5'XMP corresponds to the molar mass of the 5' monophosphate nucleoside associated with the target nitrogenous base.

[0035] Wherein, Mw nitrogenous bases correspond to the molar mass of the target nitrogenous base, expressed in g / mol.

[0036] Preferably, the yeast extract in the composition contains at least 10.0%, at least 15.0%, preferably 10.0% to 20.0% of potential nucleosides by dry weight.

[0037] Nucleoside-rich yeast extracts can be obtained by methods known to those skilled in the art.

[0038] Preferably, a nucleoside-rich yeast extract is obtained by applying an enzyme of the phosphomonyl ester hydrolase or phosphatase (EC 3.1.3) class to a yeast extract rich in free nucleotides. Preferably, the alkaline phosphatase is a nucleotidase (EC 3.1.3.1). The phosphatase hydrolyzes the phosphate group of the nucleotide, thereby releasing the corresponding nucleoside.

[0039] Advantageously, the yeast extract in the composition according to the invention is obtained by incubating a nucleotide-rich yeast extract with a phosphatase-like enzyme outside a culture medium containing lactic acid bacteria. By incubating outside the culture medium, the enzyme uses only the yeast extract as a substrate. Therefore, this method can enrich eukaryotes and potential nucleosides in the yeast extract and improve bacterial biomass yield.

[0040] "Nucleotide-rich yeast extract" should be understood as a yeast extract containing at least 2.0% free nucleotides by dry weight, preferably at least 5.0%, and more preferably at least 6.0% free nucleotides by dry weight.

[0041] Therefore, nucleotides or RNA in yeast extracts are converted into nucleosides. In the context of this invention, the term "nucleoside" refers to a building block of nucleic acids.

[0042] The yeast extract in the composition according to the present invention contains nucleosides, which may be adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, thymidine, deoxythymidine, inosine, uridine, deoxyuridine, or mixtures thereof.

[0043] "Mixture" should be understood as yeast extract in the composition according to the invention, which contains at least two nucleosides from the list above.

[0044] Preferably, the yeast extract of the composition contains at least a nucleoside with a purine base. More preferably, the yeast extract of the composition contains at least one nucleoside selected from guanosine and / or inosine.

[0045] Preferably, the yeast extract contains at least cytidine, uridine, inosine, guanosine, and adenosine.

[0046] In one specific embodiment, the yeast extract according to the invention contains less than 1.0% adenosine.

[0047] Advantageously, the compositions according to the invention do not contain exogenous nucleosides. Adding exogenous nucleosides to the culture medium is costly and results in lower biomass yields compared to the presence of endogenous nucleosides. Therefore, the compositions according to the invention comprise endogenous nucleosides naturally present in yeast extracts.

[0048] Regarding the nutritional requirements of lactic acid bacteria, the inventors discovered that during their growth, they consume nucleosides present in the culture medium and release them as nitrogenous bases. Nucleosides are used as a carbon source by the lactic acid bacteria. Therefore, advantageously, the compositions according to the invention can provide the nutritional requirements necessary for the growth of lactic acid bacteria, thereby increasing their biomass.

[0049] "Lactic acid bacteria" should be understood as partially aerobic anaerobic Gram-positive bacteria that can produce lactic acid through homofermentation or heterofermentation of glucose in a strain-dependent manner. These bacteria belong to Firmicutes (…). Firmicutes ) phylum, Bacillus ( Bacilli ) class and lactobacillus ( Lactobacillales Lactic acid bacteria are a taxonomic group consisting of facultative aerobic or anaerobic, non-spore-forming Gram-positive cocci, bacilli, or coccobacilli with a guanine and cytosine (G+C) content of less than 50%. These bacteria are acidophilic, with an optimal growth pH between 3.5 and 6.5. Lactic acid bacteria comprise 18 genera, which can be Lactobacillus (…). Lactobacillus Bifidobacterium spp. Bifidobacterium ), Bacillus spp. Bacillus Leuconostoc ( ) Leuconostoc Lactococcus spp. Lactococcus ), Enterococcus spp. Enterococcus Streptococcus ( Streptococcus ), Pediococcus ( Pediococcus ), Clostridium carnivorum ( Carnobacterium ), genus *Sacchariformis* Oenococcus ), Weissella spp. Weissella ), Balloonbacteria ( Aerococcus ), Tetracoccus ( Tetragenococcus Lactobacillus ( ) Lacticaseibacillus Lactobacillus spp. Levilactobacillus Streptococcus ( Streptococcus ), Lactobacillus spp. Limosilactobacillus ) and the genus *Lactococcus* ( Vagococcus ).

[0050] Preferably, the lactic acid bacteria belong to the genus Lactobacillus (Lactobacillus). Lactobacillus Lactococcus spp. Lactococcus Lactobacillus spp. Levilactobacillus ), Tetracoccus ( Tetragenococcus Lactobacillus ( ) Lacticaseibacillus Streptococcus spp. Streptococcus ), Lactobacillus spp. Limosilactobacillus ) or Bacillus spp. ( Bacillus Preferably belonging to the genus Bacillus (). Bacillus Lactobacillus () Lactobacillus Lactococcus spp. Lactococcus Lactobacillus spp. Levilactobacillus ), Tetracoccus ( Tetragenococcus Lactobacillus ( ) Lacticaseibacillus Streptococcus spp. Streptococcus ), Lactobacillus spp. Limosilactobacillus ).

[0051] More preferably, the lactic acid bacteria belong to the genus Lactobacillus (Lactobacillus). Lactobacillus Lactobacillus spp. Levilactobacillus Lactobacillus ( ) Lacticaseibacillus ) or Streptococcus spp. Streptococcus The lactic acid bacteria mentioned belong to Lactobacillus acidophilus ( ). Lactobacillus acidophilus ), Lactobacillus short-lived ( Levilactobacillus brevis Lactobacillus paracasei ( Lacticaseibacillus paracasei Streptococcus thermophilus ( Streptococcus thermophilus ), Lactobacillus reuteri ( Limosilactobacillus reuteri ), Lactococcus lactis ( Lactococcus lactis ) or a certain type of Bacillus genus ( Bacillus sp .) bacterial strains.

[0052] Preferably, the lactic acid bacteria belong to Lactobacillus acidophilus (Lactobacillus acidophilus). Lactobacillus acidophilus ), Lactobacillus short-lived ( Levilactobacillus brevis Lactobacillus paracasei ( Lacticaseibacillus paracasei Streptococcus thermophilus ( Streptococcus thermophilus ), Lactobacillus reuteri ( Limosilactobacillus reuteri ), Lactococcus lactis ( Lactococcus lactis More preferably, the lactic acid bacteria belong to the Lactobacillus acidophilus species. Lactobacillus acidophilus ), Lactobacillus short-lived ( Levilactobacillus brevis Lactobacillus paracasei ( Lacticaseibacillus paracasei Streptococcus thermophilus ( Streptococcus thermophilus ) bacterial strains.

[0053] "Lactic acid bacteria biomass" should be understood as the total mass of live lactic acid bacteria in the culture medium.

[0054] "Increase in lactic acid bacteria biomass" should be understood as an increase of at least +0.25 Log in the total mass of live lactic acid bacteria in the culture medium compared to the mass of lactic acid bacteria obtained under culture conditions without the composition according to the present invention.

[0055] "Increased survival rate" should be understood as an increase in the number of live lactic acid bacteria cells in the culture medium containing the composition of the present invention compared to the number of live cells obtained under culture conditions without the composition of the present invention.

[0056] The yeast extract according to the present invention may further contain amino acids as a nutrient source, which may be at least alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Preferably, the yeast extract contains tryptophan and / or asparagine, and more preferably, it contains tryptophan.

[0057] The compositions according to the invention comprise yeast extract containing at least 30.0% total amino acids by dry weight.

[0058] The composition according to the invention comprises a yeast extract containing 30.0% to 70.0% total amino acids, preferably 30.0% to 50.0%, more preferably 40.0% to 50.0% total amino acids, by dry weight.

[0059] The yeast extract according to the present invention may also contain carbohydrates, lipids, minerals, vitamins, peptides and nucleotides, all of which are essential nutrients for bacterial growth.

[0060] Another object of the present invention is a method for increasing the biomass of lactic acid bacteria, comprising the following steps: a) Obtain a culture medium that does not contain porphyrins; b) Add the composition to the culture medium, the composition comprising a nucleoside-rich yeast extract, the yeast extract comprising at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight; c) Incubate the lactic acid bacteria in the culture medium; d) Collected lactic acid bacteria biomass.

[0061] The culture medium used for producing lactic acid bacteria is porphyrin-free. Surprisingly, the inventors discovered that the composition according to the invention enables the production of lactic acid bacteria even in the absence of oxygen transporters in the culture medium.

[0062] The composition added in step b) provides the elements necessary for the growth of lactic acid bacteria.

[0063] The composition added in step b) comprises yeast extract, which preferably comprises at least 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0% eukaryotic acid by dry weight, preferably at least 8.0%, and particularly preferably at least 10.0%.

[0064] The composition according to the invention comprises a yeast extract containing 5.0% to 20% enucleosides by dry weight. Preferably, it contains 5.0% to 15.0% by dry weight, more particularly 8.0% to 11.0% by dry weight.

[0065] The composition added to the culture medium in step b) comprises a yeast extract containing at least 10.0%, at least 15.0%, preferably 10.0% to 20.0% by weight of potential nucleosides.

[0066] The culture medium used in the method according to the invention comprises the composition added in step b) at a concentration of 0.5 g / L to 40 g / L, preferably 10 g / L to 20 g / L.

[0067] Another object of the present invention is the use of compositions comprising nucleoside-rich yeast extracts in culture media, preferably in lactic acid bacteria culture media.

[0068] Another object of the present invention is the use of the composition according to the invention for increasing the biomass of lactic acid bacteria.

[0069] Another object of the present invention is the use of the composition according to the invention for increasing the growth rate of lactic acid bacteria cultures.

[0070] Bacterial culture consists of six stages: - Lag phase: Corresponds to a growth rate of zero. During this phase, bacteria do not divide.

[0071] -Acceleration phase: The stage in which the growth rate increases. During this phase, bacterial cells begin to divide.

[0072] - Exponential growth phase: This corresponds to the stage where bacterial cells reproduce at the fastest rate. During this phase, the growth rate reaches its maximum (also known as the maximum growth rate).

[0073] -Deceleration phase: This phase corresponds to a decrease in growth rate and a slowdown in bacterial cell reproduction.

[0074] -Stationary phase: The stage where the number of bacterial cells no longer changes.

[0075] - Decline phase: Corresponds to the stage where the growth rate is negative.

[0076] "Growth rate" corresponds to the number of cell divisions that occur per unit of time. This rate represents the rate at which bacterial cells reproduce.

[0077] "Increased growth rate" should be understood as a reduction in the time to reach the exponential growth phase and maximum growth rate, and a reduction in the lag phase, compared to culture conditions without the use of the composition according to the invention.

[0078] Therefore, an increased growth rate will shorten the bacterial culture time.

[0079] Another object of the present invention may be the use of the composition according to the invention for shortening the culture time of lactic acid bacteria.

[0080] Advantageously, the composition according to the invention enables the maximum culture rate to be reached more quickly and reduces the culture time by 30 minutes to 5 hours, preferably by 1 hour to 4 hours, compared to conventional bacterial cultures. Attached Figure Description

[0081] The following embodiments and accompanying drawings illustrate the present invention, but do not constitute a limitation on the scope of protection claimed by the present invention: Figure 1 This indicates that the composition of the present invention is effective against Lactobacillus acidophilus ( Lactobacillus acidophilus A schematic diagram illustrating the effect of the DSM20242 growth curve.

[0082] Figure 2 This indicates that the composition according to the present invention, compared with the reference product, is effective against Lactobacillus acidophilus (…). Lactobacillus acidophilus A schematic diagram illustrating the impact of DSM 20242 strain on biomass and survival rate after growth.

[0083] Figure 3 This indicates that the composition according to the present invention, compared with the reference product, is more effective against Lactobacillus acidophilus (in the presence or absence of exogenous nucleosides) Lactobacillus acidophilus A schematic diagram illustrating the effects of DSM 20242 strain growth and post-growth survival rate. "Exogenous nucleosides" should be understood as nucleosides added to the culture medium in pure form.

[0084] Figure 4 This indicates that the composition according to the present invention is effective against Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Schematic diagram of the effect of SB 303 growth curve.

[0085] Figure 5 This indicates that the composition according to the present invention, compared with the reference product, is effective against Lactobacillus paracasei (…). Lacticaseibacillus paracasei A schematic diagram illustrating the impact of SB 303 strain on biomass and survival rate after growth.

[0086] Figure 6 This indicates that the composition according to the present invention, compared with the reference product, is effective against Streptococcus thermophilus (Streptococcus). Streptococcus thermophilus A schematic diagram illustrating the impact of SB 138 strain on biomass and survival rate after growth.

[0087] Figure 7This indicates that the two compositions according to the present invention are effective against *Lactobacillus brevis* (…). Levilactobacillus brevis ) Schematic diagram of the effect of SB 305 growth curve.

[0088] Figure 8 This indicates that the two compositions according to the present invention are effective against *Lactobacillus brevis* (…). Levilactobacillus brevis ) Schematic diagram of the effect of SB 291 growth curve. Detailed Implementation

[0089] Example 1 : Increase Lactobacillus acidophilus ( Lactobacillus acidophilus DSM 20242 Biomass Reality Example In this embodiment, it was demonstrated that, thanks to the composition according to the invention, Lactobacillus acidophilus ( Lactobacillus acidophilus The biomass of lactic acid bacteria in strain DSM 20242 was increased.

[0090] Composition: Two compositions containing yeast extracts were prepared. The first composition (referred to as the "nucleoside" prototype in Example 1 and "prototype 1" in Examples 2 and 4) is a composition according to the present invention, containing a nucleoside-rich yeast extract. This prototype yeast extract is derived from *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomycescerevisiae The nucleoside prototype yeast extract was prepared from a strain of NuCel® 582 yeast. This extract was obtained by incubating NuCel® 582 yeast extract with an alkaline phosphatase. NuCel® 582 yeast extract is a nucleotide-rich yeast extract. This incubation step was performed outside of a culture medium containing lactic acid bacteria. Therefore, after this incubation step, the nucleoside prototype composition was added to the culture medium used for lactic acid bacteria growth.

[0091] The second composition (referred to as "NuCel® 582") was used as a reference product and contained NuCel 582 yeast extract, marketed by Procelys, a company under Lesaffre. Unlike the "nucleoside" prototype yeast extract, NuCel® 582 yeast extract was not treated with alkaline phosphatase enzymes. Table 1 corresponds to the composition of the yeast extract contained in the different compositions, and the specific compositions are shown in Tables 2 and 3 below: [Table 1]

[0092] [Table 2]

[0093] These compositions were added to porphyrin-free culture media.

[0094] Then, Lactobacillus acidophilus (Lactobacillus acidophilus The DSM 20242 strain was incubated in a medium containing one of these two compositions.

[0095] Lactobacillus acidophilus ( Lactobacillus acidophilus Culture conditions for DSM 20242 The amounts of each component of the culture medium are shown in Table 3 below: [Table 3]

[0096] Lactobacillus acidophilus ( Lactobacillus acidophilus The culture conditions for DSM 20242 are shown in Table 4 below: [Table 4]

[0097] Result Lactobacillus acidophilus ( Lactobacillus acidophilus Examples of DSM 20242 grown in the culture medium for this strain, with or without the composition according to the invention.

[0098] Figure 1 The results show the effects on two test products: NuCel® 582 and nucleoside prototype, Lactobacillus acidophilus ( Lactobacillus acidophilus Growth kinetics of DSM 20242.

[0099] In the presence of the composition according to the invention (nucleoside prototype), Lactobacillus acidophilus ( Lactobacillus acidophilus The growth of DSM 20242 was faster than that of the reference product "NuCel® 582". In the composition according to the invention, the time to reach maximum growth rate was advanced by approximately one hour, and the lag phase was shorter. Therefore, in the presence of the composition according to the invention, the incubation time was shortened.

[0100] Nucleoside prototype against Lactobacillus acidophilus ( Lactobacillus acidophilus The concentration of DSM 20242 strain also had a positive effect.

[0101] Figure 2 The prototype of the "nucleoside" was shown to have an effect on Lactobacillus acidophilus ( Lactobacillus acidophilus The impact of DSM 20242 on biomass and survival.

[0102] Bacterial biomass is expressed in colony-forming units (Log (CFU / mL)).

[0103] Active fluorescent units (AFU) also represent the number of "surviving and active" probiotics.

[0104] The only difference between these two units is the method of measurement.

[0105] In these tests, CFU was measured by coating on MRS agar (from Man, Rogosa, Sharpe), sold by BioMérieux, cultured at pH 6.2 under anaerobic conditions, and AFU was measured by flow cytometry, with bacteria traced using Syto24-propidium iodide fluorescent “label” and counted by laser as they passed through the tube.

[0106] Survival percentage was also measured by flow cytometry combined with Syto24-propidium iodide fluorescence tracing.

[0107] The results were obtained from four biological replicates.

[0108] The "nucleoside" prototype used was sterilized by autoclaving or pasteurization.

[0109] Compared to the reference product "NuCel® 582", *Lactobacillus acidophilus* was observed in the presence of the nucleoside prototype composition (autoclaved or pasteurized). Lactobacillus acidophilus The biomass yield of strain DSM 20242 was significantly increased. In the presence of pasteurized nucleoside prototypes, the yield increased by +0.35 log CFU / mL.

[0110] Compared to the reference product "NuCel® 582", Lactobacillus acidophilus was also observed in the presence of the "nucleoside" prototype. Lactobacillus acidophilus The survival rate percentage of DSM 20242 strain was significantly improved. In the presence of pasteurized nucleoside prototype composition, viable Lactobacillus acidophilus (…) Lactobacillus acidophilus The total proportion of DSM20242 strain was 88.6%, while in the presence of the reference product, the proportion was 51.8%.

[0111] The same results were observed in the presence of both autoclaved and pasteurized nucleoside prototypes. Autoclaving and pasteurization had an effect on Lactobacillus acidophilus (…). Lactobacillus acidophilus The increase in biomass and survival rate of DSM 20242 strain has no effect, and therefore has no effect on the quality and efficacy of the composition according to the present invention.

[0112] The inventors also compared the effects of adding adenosine to the culture medium on Lactobacillus acidophilus ( ​ ​ The impact of DSM 20242 on biomass production ( ​ ).

[0113] It was observed that, compared with the presence of adenosine and the reference product (NuCel® 582) in the culture medium, the biomass yield and the number of active Lactobacillus acidophilus ( ) in the presence of the nucleoside-rich composition (nucleoside prototype) according to the invention were significantly higher. ​ ​ The number of DSM 20242 strains is higher.

[0114] The inventors tested the effects of adding exogenous nucleosides to the culture medium. ​ Under standard conditions, using the "nucleoside prototype" composition alone yielded better results for *Lactobacillus acidophilus* compared to using the reference product alone or in combination with exogenous nucleosides. ​ Biomass yield and survival rate of DSM 20242 strain.

[0115] Example 2: Increasing the levels of Lactobacillus paracasei ( ​ SB303 strain biology ​

[0116] In this embodiment, it was demonstrated that, thanks to the composition according to the invention, *Lactobacillus paracasei* (… ​ The biomass and survival rate of lactic acid bacteria in strain SB303 were increased.

[0117] Lactobacillus paracasei ( ​ The culture conditions for strain SB303 are shown in the table below: [Table 5]

[0118] Lactobacillus paracasei ( ​ The components of SB303 culture medium are listed in Table 6: [Table 6]

[0119] The composition of prototype 1 corresponds to the composition of the "nucleoside" prototype of Example 1.

[0120] These compositions were added to a porphyrin-free culture medium containing the components listed in Table 6. Then, *Lactobacillus paracasei* (…) ​ SB303 strain was incubated in this medium (one of the two compositions).

[0121] ​ The production kinetics of *Lactobacillus paracasei* SB303 for two test products, NuCel® 582 and Prototype 1, are shown. In the presence of the composition according to the invention (Prototype 1), *Lactobacillus paracasei* (… ​ ​The growth of SB303 is faster than that of the reference product "NuCel® 582". In the composition according to the invention, the maximum growth rate is reached approximately two hours earlier. Therefore, in the presence of the composition according to the invention, the incubation time is shortened.

[0122] ​ The composition (prototype 1) according to the invention was shown to be effective against Lactobacillus paracasei (NuCel® 582) compared to two reference products (NuCel® 582). ​ The effects of SB303 strain on biomass and survival rate.

[0123] Bacterial biomass is expressed in colony-forming units (Log (CFU / mL)). Active fluorescent units (AFU) also indicate the number of "live and active" probiotics.

[0124] The only difference between these units is the method of measurement. In these tests, CFU is measured by spreading it onto MRS medium under anaerobic conditions, and AFU is measured by flow cytometry. Bacteria are traced using a Syto24-propidium iodide fluorescent "label" and counted by laser as they pass through the tube.

[0125] Survival percentage was also measured by flow cytometry combined with Syto24-propidium iodide tracer.

[0126] On the one hand, compared with the reference product NuCel 582, *Lactobacillus paracasei* was observed in the presence of prototype 1. ​ SB303 cell survival rate improved.

[0127] On the other hand, compared with the reference product, the biomass of active Lactobacillus paracasei cells increased by +0.29 Log to +0.32 Log in the presence of prototype 1.

[0128] Therefore, the composition according to the present invention is effective against Lactobacillus paracasei ( Lacticaseibacillus paracasei The survival rate and biomass of strain SB303 have a positive impact.

[0129] Examples 3 and 4 For the following embodiments, a third composition according to the invention (referred to as "prototype 2") was used, which comprises a nucleoside-rich yeast extract. The yeast extract of this prototype is derived from *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae The strain was prepared. The "Prototype 2" yeast extract was obtained by incubating a nucleotide-rich yeast extract (NuCel® 780) with an alkaline phosphatase. This incubation step was performed outside the culture medium. Therefore, the "Prototype 2" composition was added to the culture medium for lactic acid bacteria growth after this incubation step.

[0130] The fourth composition (referred to as "NuCel® 780") was used as a reference product and contained NuCel® 780 yeast extract, marketed by Procelys, a company under Lesaffre. Unlike "Prototype 2" yeast extract, NuCel® 780 yeast extract was not treated with alkaline phosphatase enzymes.

[0131] Table 7 corresponds to the composition of the "Prototype 2" yeast extract and the reference product NuCel® 780: [Table 7]

[0132] Example 3: Enhancing the levels of thermophilic streptococci (Streptococcus thermophilus) Biomass of SB138 strain Example : This embodiment demonstrates that, in the presence of the composition according to the invention, *Streptococcus thermophilus* (… Streptococcus thermophilus The biomass and survival rate of strain SB138 were increased.

[0133] Table 8 lists the compositions tested in Example 3: [Table 8]

[0134] Streptococcus thermophilus ( Streptococcus thermophilus The culture conditions for strain SB138 are shown in the table below: [Table 9]

[0135] Streptococcus thermophilus ( Streptococcus thermophilus SB138 culture medium Streptococcus thermophilus ( Streptococcus thermophilus The components of SB138 medium are listed in Table 10: [Table 10]

[0136] These compositions were added to a porphyrin medium containing the components listed in Table 11. Then, *Streptococcus thermophilus* (…) Streptococcus thermophilus The SB138 strain was incubated in this medium (one of the two compositions was present).

[0137] Figure 6 The composition according to the invention (prototype 2) showed its effectiveness against Streptococcus thermophilus (Streptococcus thermophilus) compared to the reference product (NuCel® 780). Streptococcus thermophilus The effects of SB138 strain on biomass and survival rate.

[0138] Bacterial biomass is expressed as AFU / mL. Active fluorescent units (AFU) also indicate the number of “live and viable” probiotics. AFU is measured by flow cytometry, with bacteria traced using a Syto24-propidium iodide fluorescent “label” and counted by laser as they pass through the tube. Survival percentage is also measured by flow cytometry combined with Syto24-propidium iodide tracer.

[0139] Compared to the reference product, in the presence of the composition according to the invention (prototype 2), it is effective against Streptococcus thermophilus (…). Streptococcus thermophilus The SB138 strain showed an increase in bacterial biomass of approximately +0.51 Log. Furthermore, bacterial cell viability was also improved in the presence of prototype 2 compared to the reference product.

[0140] Example 4: Improving the growth of two short-lived Lactobacillus strains ( Levilactobacillus brevis SB 305 and SB 291 Examples of strain growth rate and shortened culture time: In this embodiment, it was demonstrated that, thanks to the composition according to the invention, *Lactobacillus shortsum* (… Levilactobacillus brevis The lactic acid bacteria growth rate of strains SB 305 and SB 291 was increased and the culture time was shortened.

[0141] Table 12 lists the compositions tested in Example 4: [Table 12]

[0142] The composition of prototype 1 corresponds to the composition of the "nucleoside" prototype in Examples 1 and 2.

[0143] Lactobacillus short-lived ( Levilactobacillus brevis The culture conditions for strains SB 305 and SB 291 are shown in the table below: [Table 13]

[0144] Lactobacillus short-lived ( Levilactobacillus brevis The components of SB 305 and SB 291 culture media are listed in Table 14: [Table 14]

[0145] These compositions were added to a porphyrin-free culture medium containing the components listed in Table 13. Then, *Lactobacillus shortii* (…) Levilactobacillus brevis SB 305 or SB 291 bacteria are incubated in this medium (one of four compositions is present).

[0146] Figure 7 The results show that for four test products: NuCel® 582, NuCel® 780, Prototype 1, and Prototype 2, *Lactobacillus shortii* (…) Levilactobacillus brevis Production dynamics of SB 305.

[0147] In the presence of the compositions according to the invention (prototype 1 and prototype 2), *Lactobacillus shortsum* ( Levilactobacillus brevis The growth of SB 305 is faster than that of the reference products “NuCel® 582” and “NuCel® 780”. The maximum growth rate is reached approximately four hours earlier in both compositions (Prototype 1 and Prototype 2) according to the invention, relative to the two reference products.

[0148] Figure 8 The results show that for four test products: NuCel® 582, NuCel® 780, Prototype 1, and Prototype 2, *Lactobacillus shortii* (…) Levilactobacillus brevis Production kinetics of SB 291. Therefore, compared to the reference product, the incubation time is shortened in the presence of the compositions according to the invention (prototypes 1 and 2).

[0149] In the presence of the compositions according to the invention (prototype 1 and prototype 2), *Lactobacillus shortsum* ( Levilactobacillus brevis The growth of SB 291 is faster than that of the reference products “NuCel® 582” and “NuCel® 780”. The maximum growth rate is reached approximately two hours earlier in the two compositions (Prototype 1 and Prototype 2) according to the invention, relative to the two reference products. Therefore, the incubation time is shortened in the presence of the compositions (Prototype 1 and Prototype 2) according to the invention compared to the reference products.

Claims

1. A composition for increasing the biomass of lactic acid bacteria, characterized in that, The composition comprises a yeast extract containing at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight.

2. The composition according to claim 1, characterized in that, The yeast extract is obtained from yeast strains of the genus *Saccharomyces*, preferably *Saccharomyces cerevisiae*.

3. The composition according to any one of claims 1 or 2, characterized in that, The nucleosides are selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, inosine, deoxycytidine, thymidine, deoxythymidine, uridine, deoxyuridine, or mixtures thereof.

4. The composition according to claim 3, characterized in that, The yeast extract contains at least one nucleoside selected from guanosine and / or inosine.

5. The composition according to any one of claims 1 to 4, characterized in that, The yeast extract contains at least 30.0% total amino acids by dry weight.

6. The composition according to any one of claims 1 to 5, characterized in that, The yeast extract contains 30.0% to 70.0% total amino acids by dry weight, preferably 30.0% to 50.0% by dry weight, and more preferably 40.0% to 50.0% by dry weight.

7. The composition according to any one of claims 1 to 6, characterized in that, The yeast extract contains tryptophan and / or asparagine, preferably tryptophan.

8. The composition according to any one of claims 1 to 6, characterized in that, The lactic acid bacteria belong to the genera *Lactobacillus*, *Lactococcus*, *Lactobacillus*, *Tetracoccus*, *Caseobacillus*, *Streptococcus*, *Lactobacillus mucinus*, *Lactococcus*, or *Bacillus*.

9. The composition according to claim 8, characterized in that, The lactic acid bacteria are a species of Lactobacillus acidophilus, Lactobacillus short-lived, Lactobacillus paracasei, Streptococcus thermophilus, Lactobacillus reuteri, Lactococcus lactis, or Bacillus.

10. A method for increasing the biomass of lactic acid bacteria, the method comprising the following steps: a) Obtain a culture medium that does not contain porphyrins; b) Add the composition to the culture medium, the composition comprising a nucleoside-rich yeast extract, the yeast extract comprising at least 5.0% eukaryotic acid and at least 10.0% potential nucleoside by dry weight; c) Incubate the lactic acid bacteria in the culture medium; d) Collected lactic acid bacteria biomass.

11. The method for increasing lactic acid bacteria biomass according to claim 10, characterized in that, The culture medium contains the composition added in step b) at a concentration of 0.5 g / L to 40 g / L, preferably 10 g / L to 20 g / L.

12. Use of the composition according to any one of claims 1 to 7 in a culture medium.

13. Use of the composition of claim 12 in a lactic acid bacteria culture medium.

14. Use of the composition according to any one of claims 1 to 7 for increasing the biomass of lactic acid bacteria.

15. Use of the composition of any one of claims 1 to 7 for shortening bacterial culture time.

16. The use as described in claim 15, characterized in that, The culture time is shortened by 30 minutes to 5 hours, preferably 1 to 4 hours.

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