FOOD-GRADE GROWING MEDIUM

Abstract

The present invention relates to the use of a food-grade culture medium for the production of lactic acid bacteria intended for food or pharmaceutical use.

Description

Title of the invention: FOOD-GRADE CULTURE MEDIUM Technical field

[0001] The present invention relates to the field of the agri-food and pharmaceutical industry, and more particularly concerns the use of a culture medium for the production of lactic bacteria intended for food or pharmaceutical use. TECHNOLOGICAL BACKGROUND

[0002] Lactic acid bacteria have many applications in the food and pharmaceutical industries. They can be used as starters for fermented products, as probiotic bacteria but also for the metabolites of interest that they produce (lactic acid, bacteriocins, various proteins of interest).

[0003] The production of biomass of these strains of interest is classically carried out in culture media such as MRS medium (from De Man, Rogosa and Sharpe) used for the production of Lactobacilli (see Table 1).

[0004] In 1960, de Man, Rogosa and Sharpe developed the formulation of a medium specifically adapted to the cultures of lactobacilli from dairy products (De Man et al. 1960). This development was carried out in order to replace a variable product (tomato juice) and at the same time to provide a medium conducive to the good development of lactobacilli in general, including strains growing poorly on existing media. MRS medium is superior to Briggs tomato juice medium (Briggs M. (1953)) and to De Man tomato juice and meat extract medium. It allows a more abundant growth of all strains of lactobacilli.

[0005] MRS medium is used for the cultivation and enumeration of bacteria of the genus Lactobacillus, particularly in dairy products and other food products as well as in products intended for animal feed. This medium is generally used at a weakly acidic pH (between 6 and 6.4) and allows the cultivation of slow-growing germs such as Lactobacillus brevis and Lactobacillus fermentum. Acidified to pH 5.4, it also allows the enumeration of Lactobacillus bulgaricus in yogurts. According to the NF ISO 15214 standard for the enumeration of mesophilic lactic acid bacteria, MRS medium is used at a pH of 5.7.

[0006] This medium, however, contains compounds of animal origin (peptones and meat extract) and ingredients whose use is controversial because they are potentially dangerous for human health (Tween 80, potassium hydrogen phosphate, cf. Table 1).

[0007] Culture media and their components influence the survival of bacteria subjected to a stabilization process (Goldberg and Eschar 1977; Beal et al. 2001; Carvallio et al. 2004a; Silva et al. 2005; Corcoran et al. 2007; Siaterlis et al. 2009). For example, the type of peptone (animal or vegetable) present in the culture medium influences the fatty acid composition of the bacterial membrane, thus modifying the membrane fluidity which plays a role in cellular resistance to the stresses of dehydration processes (Carvalho et al. 2003, 2004b).

[0008] There are culture media without controversial ingredients, developed to meet biomass production criteria. However, the stress resistance criteria of dehydration processes have often been neglected. In order to ensure the preservation of the viability and functionality of the bacterial biomass produced over the long term and until its use, it must be stabilized by dehydration. The intracellular water of the bacterial cells will be removed, which will stop the cellular metabolism. Freeze-drying is the most widely used technique for preserving bacterial biomass in industry. It allows water to be removed from the bacterial cells by sublimation.A freeze-drying cycle consists of 3 phases: freezing, which consists of forming ice crystals, primary desorption, which consists of sublimating the ice by reducing pressure, and secondary desorption, which consists of removing residual water by providing energy to the system (Blond et al. 1997). However, cellular damage can be caused during the dehydration process, generally linked to the presence of reactive oxygen species (Franca et al. 2007; Lanniello et al. 2016) and to thermal and osmotic stress associated with freezing (Broeckx et al. 2016).

[0009] In this context, a new food grade culture medium has been developed in order to obtain viable biomass after maximum freeze-drying by meeting biomass production criteria and stress resistance criteria of the dehydration process. Summary of the invention

[0010] The Applicant company has found that the use of milk permeate in a process for producing lactic acid bacteria has, surprisingly, a stimulating effect on the growth of these bacteria and an effect on the resistance to the stress of the dehydration process. In this context, the inventors have developed a new food-grade culture medium, without controversial or toxic ingredients for human or animal health, developed to obtain 1) a biomass production equivalent to that obtained with the MRS culture medium; and 2) a resistance to the stress of the dehydration process (freeze-drying) equivalent or higher than that obtained with MRS culture medium.

[0011] This innovative culture medium allows the cultivation of probiotics of the lacto-bacteria type, lactic ferments, with equivalent or higher biomass titers before and after freeze-drying compared to traditional culture media.

[0012] This culture medium, intended for bacterial culture, allows growth, that is to say the development of bacterial biomass. This culture medium allows the improvement of survival during freeze-drying and the obtaining of a greater bacterial biomass after freeze-drying.

[0013] The invention relates in particular to a food-grade culture medium comprising milk permeate. It is easy to implement and inexpensive and is suitable for a vegetarian, halal or kosher diet. Milk permeate is a by-product of the cheese and milk processing industry, readily available and inexpensive. The culture medium according to the invention contains only ingredients authorized in the food industry in Europe and can thus be used as a substitute for conventional culture media which contain meat extracts, peptones of animal origin, and / or minerals or additives (tween 80) some forms of intake of which are controversial for human health.

[0014] In particular, the cultivation of lactobacteria in the food-grade culture medium according to the invention does not require rinsing the bacteria as thoroughly as current manufacturing processes, these rinses being necessary to remove compounds that are harmful or potentially harmful to human or animal health. In addition, due to the low cost of milk permeate, the cost of production of pharmaceutical or nutraceutical compositions comprising lactic acid bacteria as probiotics cultivated on the medium of the invention has a substantially lower cost than that of products obtained from conventional media.

[0015] The invention relates to the use of a food-grade culture medium for cultivating lactic acid bacteria, said medium comprising: 35 to 70% of milk permeate by dry weight relative to the total dry weight of the medium as a carbon and nitrogen source, an additional nitrogen source and a mineral source, wherein the ratio of the carbon supply relative to the total nitrogen supply in the medium is between 4 and 10.

[0016] The invention also relates to a method for producing lactic acid bacteria comprising:

[0017] a) culturing and multiplying lactic acid bacteria in a food grade culture medium comprising 35 to 70% milk permeate by dry weight relative to the total dry weight of the medium as a carbon and nitrogen source, an additional nitrogen source and a mineral source; wherein the ratio of the carbon supply compared to the total nitrogen supply in the medium is between 4 and 10,

[0018] b) the recovery of the multiplied lactic bacteria, and

[0019] c) optionally, freeze-drying or freezing the multiplied bacteria.

[0020] The invention also relates to a food-grade culture medium for lactic acid bacteria comprising 35 to 70% by dry weight of milk permeate, 25 to 55% by dry weight of yeast extract relative to the total dry weight of the medium, and a mineral source, the culture medium having a carbon ratio relative to the total nitrogen supply of between 4 and 10.

[0021] In particular, the culture medium does not comprise peptone of animal origin and / or meat extract.

[0022] Preferably the culture medium comprises magnesium as a mineral source, preferably magnesium sulfate, in particular 0.01% and 1% by dry weight of magnesium sulfate relative to the total dry weight of the medium.

[0023] The culture medium may also comprise manganese as a mineral source, preferably manganese sulfate, in particular 0.01% and 1% by dry weight of manganese sulfate relative to the total dry weight of the medium.

[0024] According to one aspect, the culture medium has a pH between 5.5 and 7.5, preferably a pH between 6 and 7.

[0025] According to another aspect, the culture medium does not comprise at least one compound selected from the group consisting of glucose, an antibiotic, potassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate and Tween 80.

[0026] The invention also relates to the use of a food-grade culture medium according to the invention for the preparation of a pharmaceutical or nutraceutical composition comprising lactic acid bacteria.

[0027] The invention finally relates to a method for preparing a pharmaceutical or nutraceutical composition comprising a step of adding lactic bacteria prepared according to the production method of the invention, to the other components of the pharmaceutical or nutraceutical composition. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0028] By "food grade culture medium" or "Food Grade culture medium" or "FG medium" is meant here a composition, a preparation or a support allowing the culture or multiplication of cells such as microorganisms, preferably bacteria such as lactic acid bacteria. This medium satisfies the minimum nutritional requirements of the cells or microorganisms to be cultured. To be defined as food grade, the materials used for the com position of this medium must be non-toxic and safe for human or animal consumption. Preferably, a food-grade culture medium does not include compounds considered controversial, dangerous or potentially dangerous for human or animal health, in particular by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) or the American Food and Drug Administration (FDA). Preferably, the culture medium according to the invention is a culture medium whose composition is food-grade within the meaning of European Regulation 178 / 2002 of January 28, 2002.

[0029] As used herein, the term "bacteria" refers to any prokaryotic microorganism existing as a single cell, in a group, or in an aggregate of individual cells. The term "bacteria" encompasses all variants of bacteria (e.g., bacteria in the human microbiota or environmental bacteria). These bacteria are preferably lactic acid bacteria. These bacteria are especially probiotic bacteria, most preferably probiotic lactic acid bacteria.

[0030] As used herein, the term "probiotic" refers to live or inactivated bacteria which, when administered in adequate amounts, have a beneficial effect on the host organism. These probiotic bacteria are advantageous in that they are suitable for administration to humans and other mammalian subjects. Probiotic substances contain a sufficiently high number of live and active probiotic microorganisms that can exert a balancing action on the intestinal flora by direct colonization. It should be noted that, for the purposes of this description, a probiotic is understood to mean any biologically active form of probiotic.Preferably, the probiotics according to the invention are as defined by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) in the 2001 Joint FAO / WHO Expert Consultation on the Evaluation of the Health and Nutritional Properties of Probiotics in Foods, Including Powdered Milk Containing Live Lactic Acid Bacteria. Preferably, the probiotics are lactic acid bacteria (LAB), also known as lactobacteria. These bacteria are Gram-positive, low GC genome content, anaerobic or aerotolerant, generally non-spore-forming microorganisms that produce lactic acid as the main end product of carbohydrate fermentation.

[0031] Preferably, the lactic acid bacteria that can be cultivated on the medium of the present invention belong in particular to the genus Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Tetragenococcus, Leuconostoc, Oenococcus or Bifidobacterium, preferably to the genus Lactobacillus. Lactic acid bacteria (LAB) are generally recognized as safe for human and animal health and certain species have obtained Presumption of Safety Status (QPS) from the European Food Safety Agency (EFSA).

[0032] The term "carbon source" or "carbon source" included in a culture medium particularly corresponds to a nutrient (sugar, fatty acid, amino acid, etc.) or organic matter providing the carbon necessary for the constitution of new organic molecules (anabolism), for the culture, survival and / or multiplication of bacteria. According to the invention, this carbon source is metabolizable by LAB, and is preferably milk permeate.

[0033] As used herein the term "whey" refers to the food obtained in liquid form by separating the coagulum from milk, cream or skim milk. It is one of the main by-products of cheese making. "Whey permeate" is a powder resulting from the ultrafiltration of whey, generally concentrated and then spray-dried. Compared to whey, whey permeate is generally lower in protein but higher in lactose and minerals.

[0034] The term "milk permeate" refers to the food obtained after removal of milk proteins and milk fat contained in milk, partially skimmed milk or skimmed milk by ultrafiltration.

[0035] The term "nitrogen source" or "nitrogen source" present in the culture medium refers to the set of compounds capable of providing the nitrogen necessary for the culture, growth and / or survival of the bacteria in the culture medium according to the invention. This term preferably includes inorganic compounds (ammonia, ammonium salts, nitrites, nitrates) or organic sources (yeast extract, amine groups of organic compounds). According to the invention, this nitrogen source is assimilable by the LAB, and is preferably provided by milk permeate and / or by yeast extract.

[0036] By "yeast extract" is meant the general term used to designate the different forms of yeast transformed by removing the contents of the cells.

[0037] The "total carbon / nitrogen ratio" or "ratio of carbon input to nitrogen input" as used herein corresponds to the ratio of the input of carbon compounds to the input of nitrogen compounds in a medium composition. In particular, certain elements of the composition may be used as a source of carbon and / or nitrogen. Typically, the milk permeate provides both a carbon and a nitrogen source. The calculation of the carbon / nitrogen ratio thus includes the evaluation of the carbon and nitrogen content of each compound, as well as the sum of the contents of the different compounds. For example, the carbon input of the milk permeate, and the nitrogen input of the milk permeate and the yeast extract will be taken into consideration.

[0038] “Mineral source” as used herein means mineral elements contributing to the culture, survival and / or growth of bacteria in the medium of culture according to the invention.

[0039] As used herein the term "animal product" refers to products taken or derived from living or dead animals such as fat, flesh, blood, but does not include materials produced by living animals such as milk, honey and eggs.

[0040] As used herein, the term "comprising" or "comprises" is used with reference to substances, compounds, or processes that are essential to the invention, but are open to the inclusion of non-specific elements, whether essential or not. The use of "comprising" indicates inclusion rather than limitation.

[0041] The term "and / or" as used herein should be considered a specific description of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered a specific disclosure of each of the following: (i) A, (ii) B, and (iii) A and B, as if each were presented individually.

[0042] The term "about" as used herein in connection with all values ​​(including the lower and upper ends of numerical ranges) means any value having an acceptable variation of up to + / - 10% (e.g., + / - 0.5%, + / - 1%, + / - 1.5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / - 9.5%). Using the term "about" at the beginning of a list of values ​​modifies each of them (i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Also, when a list of values ​​is described (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all its intermediate and fractional values ​​(e.g., 54% or 85.4%). Culture medium

[0043] The present invention relates to a food-grade culture medium. This medium is particularly suitable for the culture of so-called probiotic bacteria, in particular lactic acid bacteria.

[0044] The culture medium according to the invention is intended for the production of a biomass of probiotic bacteria, in particular lactic acid bacteria, preferably on an industrial scale. The characteristics of a culture medium intended for the industrial production of lactobacteria, lactic ferments, largely determine the final activity of a product. Despite the nutritional requirements of lactic acid bacteria, the culture media must make it possible to obtain a concentrated biomass with good production yields. Nutrient requirements are met by providing the microorganisms with a carbon source, one or more nitrogen sources, and minerals, and in certain cases vitamins, fatty acids, nitrogenous bases and / or organic acids.

[0045] The food-grade culture medium according to the invention may be solid, semi-solid or liquid.

[0046] By "solid culture medium" or "solid medium" is meant, for example, a gelled medium. Agar is the traditional gelling agent in microbiology, but it is also possible to use gelatin, agarose or other natural or artificial gelling agents. Preferably, the gelling agent is of plant origin.

[0047] The culture medium according to the invention may alternatively be in dehydrated or dry form, in particular in powder form. This culture medium may subsequently be moistened or diluted in a liquid such as water, in particular sterilized water, to form a solid, semi-solid or liquid medium.

[0048] Preferably, the culture medium according to the invention is in liquid form. The liquid culture medium may be particularly included in a fermenter or bioreactor, to enable the culture of bacteria.

[0049] In one aspect, the food grade culture medium comprises a carbon source which is whey permeate.

[0050] Preferably, the milk permeate is derived from cow's, goat's or sheep's milk.

[0051] The culture medium comprises a base in the form of a permeate of milk as a carbon and nitrogen source. This permeate is preferably obtained by ultrafiltration of skimmed and concentrated milk then dehydrated by an atomization process giving the powder very good flow and reduced hygroscopicity.

[0052] Preferably, the milk permeate contains between 0.05 and 5% by weight of proteins, between 0.2% and 1% by weight of fat, from 76 to 95% by weight of lactose, between 5 and 8% by weight of mineral matter per 100% by weight of milk permeate. The milk permeate may further comprise approximately 300 mg of calcium per 100 g of milk permeate. Preferably, the milk permeate comprises a limited amount or an absence of lactic acid and lactates, in particular at most 0.1% by weight for powdered permeates and 0.015% by weight for milk permeates in liquid form, and an absence of glycomacropeptides. An example of a milk permeate that can be used in the culture medium according to the invention is that supplied by Eurial Ingredients under the reference DR 52:127, which comprises 2% protein, 0.5% fat, 6.9% mineral matter and 85% lactose, this milk permeate composition being at pH 6.3, the percentages being given by weight.

[0053] According to one aspect, the food-grade culture medium for lactic acid bacteria comprises 35 to 70% by dry weight of milk permeate relative to the total dry weight of the medium. Alternatively or additionally, the culture medium comprises 2 to 10% by wet weight of milk permeate relative to the total wet weight of the medium.

[0054] Preferably, the food grade culture medium for lactic acid bacteria comprises 40 to 70%, 40 to 70%, 50 to 70%, 50 to 65%, 55 to 65% or 55 to 65% by dry weight of milk permeate relative to the total dry weight of the medium.

[0055] By dry weight is meant particularly the composition of the medium in dry or dehydrated mass, for example in the form of powder. By wet weight or wet weight is meant the weight of the composition once diluted in a liquid, for example in water, in particular sterilized water.

[0056] According to one aspect, the culture medium according to the invention further comprises from 25 to 55% by dry weight of yeast extract relative to the total dry weight of the medium, as a nitrogen source. Alternatively or additionally, the culture medium comprises from 1% to 10% by wet weight of yeast extract relative to the total wet weight of the medium.

[0057] Preferably, the food grade culture medium for lactic acid bacteria comprises 25% to 50%, 30% to 50%, 35% to 50%, 40 to 50%, 25% to 45%, 30% to 45%, 35% to 45% by dry weight of yeast extract relative to the total dry weight of the medium.

[0058] According to a particular embodiment, the yeast extract used is an inactivated yeast extract, for example as supplied by Biospringer.

[0059] In particular, the culture medium according to the invention comprises a carbon source and a nitrogen source, so that their respective quantity in the medium respects a total carbon / nitrogen (C / N) ratio of between 4 and 10. Preferably, the C / N ratio is between 4 and 7, preferentially between 4 and 6.5, very particularly between 4.4 and 6.3.

[0060] In particular, this ratio is calculated from the quantities of carbon and nitrogen provided by the milk permeate and the quantity of nitrogen provided by the yeast extract.

[0061] According to a particular aspect, the C / N ratio is as defined in Tables 2 to 5 and 7.

[0062] According to one aspect, the culture medium according to the invention further comprises a mineral source. Preferably, the mineral source is chosen from: phosphorus, sulfur, sodium, potassium, magnesium, manganese, calcium, iron, or a combination thereof.

[0063] Advantageously, the culture medium contains magnesium, preferably in the form of magnesium sulfate, at a content of between 0.01% and 0.5% or 1% by dry weight relative to the total dry weight of the culture medium, preferably 0.05 to 0.6%, preferably 0.1% to 0.5% and particularly 0.2 to 0.5%. Alternatively or additionally, the culture medium contains magnesium, preferably in the form of magnesium sulfate, at a content of between 0.01% and 0.1% by wet weight relative to the total wet weight of the culture medium, preferably 0.01 to 0.05%, preferably 0.01% to 0.03% and particularly 0.02%.

[0064] Advantageously, the culture medium contains manganese, preferably in the form of manganese sulfate, at a content of between 0.01% and 0.1%, 0.5% or 1% by dry weight relative to the total dry weight of the culture medium, more preferably between 0.01 and 0.2%, and particularly 0.01 to 0.015%. Alternatively or additionally, the culture medium contains manganese, preferably in the form of manganese sulfate, at a content of between 0.001% and 0.01% by wet weight relative to the total wet weight of the culture medium, preferably 0.001 to 0.05%, particularly 0.005%.

[0065] Thus, according to a particular aspect, the medium according to the invention comprises whey, whey permeate or milk permeate as a source of nitrogen and carbon, yeast extract as a source of nitrogen, magnesium and / or manganese as a mineral source, preferably in the proportions described above.

[0066] Preferably, the food grade culture medium comprises 40 to 75% by dry weight of milk permeate as a source of nitrogen and carbon, 25 to 55% by dry weight of yeast extract as a source of nitrogen, 0.01 to 1% by dry weight of magnesium, preferably in the form of magnesium sulfate and 0.01% to 0.5% by dry weight of manganese, preferably in the form of manganese sulfate, as a mineral source, per 100% by dry weight of the total composition of the culture medium.

[0067] According to one embodiment, the composition of the culture medium according to the invention is as defined in Table 6 or Table 8.

[0068] Alternatively, the food grade culture medium comprises 2 to 10% wet weight of milk permeate as a nitrogen and carbon source, 1 to 5% wet weight of yeast extract as a nitrogen source, 0.01 to 1% wet weight of magnesium, preferably in the form of magnesium sulfate and 0.001 to 0.05% wet weight of manganese, preferably in the form of manganese sulfate, as a mineral source, per 100% wet weight of the total composition of the culture medium, i.e. after dilution of the respective powders in a liquid, for example water, preferably sterilized water.

[0069] The culture medium is preferably weakly acidic, which can inhibit the growth and reproduction of most bacteria, but is conducive to the growth and reproduction of lactic acid bacteria. According to a particular aspect, the culture medium has a slightly acidic pH, preferably between 5.5 and 7, preferably between 5.5 and 6.5, more preferably between 6 and 6.5.

[0070] The present invention preferably relates to a culture medium whose composition is food within the meaning of European regulation 178 / 2002.

[0071] The medium according to the invention does not comprise any toxic, dangerous or harmful products, or products whose effect is controversial for human or animal health, in particular as established by the World Health Organization (WHO). In particular, the medium according to the invention does not comprise at least one compound selected from the group consisting of glucose, an antibiotic, potassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate and Tween 80. In a particular aspect, the medium according to the invention does not comprise potassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate or Tween 80, or any combination thereof.

[0072] According to one aspect, the culture medium according to the invention does not comprise products of animal origin. In particular, the culture medium according to the invention does not contain animal products or by-products other than milk permeate.

[0073] Preferably, the culture medium does not comprise peptone of animal origin and / or meat extract. In particular, the culture medium does not comprise peptone and / or meat extract.

[0074] According to a particular aspect, the culture medium according to the invention does not contain glucose.

[0075] According to another particular aspect, the culture medium according to the invention does not contain an antibiotic. Additional ingredients

[0076] According to a particular embodiment, the culture medium further comprises amino acids, minerals, growth factors, purine and pyrimidine bases, salts, vitamins, antioxidants, trace elements, metabolic regulators or pH regulators, or any combination thereof. Preferably, these compounds are not of animal origin and / or are not considered dangerous or potentially dangerous for human or animal health.

[0077] The amino acids are natural or synthetic and are preferably of plant origin. The amino acids may in particular be chosen from asparagine, proline, serine and L-cysteine, or a mixture thereof.

[0078] The vitamins, minerals and / or trace elements may for example be chosen from: a vitamin for example thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9) and cyanocobalamin (B12), and / or vitamins C, A, D, E, K1 and K2; magnesium, calcium, iron, iodine, copper, zinc, selenium, chromium, molybdenum, boron or a mixture thereof.

[0079] The antioxidants can for example be chosen from superoxide dismutase (SOD), Tubiquinol / ubiquinone (Coenzyme Q10), zinc sulfate, resveratrol, catechins (catechin, epicatechin and gallate derivatives), flavanols, flavanones or even anthocyanins.

[0080] According to one aspect, the medium additionally comprises a buffer with a pH buffering capacity, preferably capable of buffering the culture medium to a pH between 5 and 7, preferably between 5.5 and 6.5, more preferably between 6 and 6.5. In particular, the medium may comprise a pH regulator. Preferably, the pH regulator is chosen from: citric acid, acetic acid, tartaric acid and ascorbic acid or any combination thereof. Use

[0081] The invention also relates to the use of a food-grade culture medium according to the invention as a culture medium for lactic acid bacteria. The culture media as described above may advantageously be used in lactic acid fermentation processes, in particular for the preparation of probiotic ingredients intended for the agri-food or pharmaceutical industry, preferably on an industrial scale.

[0082] Advantageously, the culture medium according to the invention makes it possible to obtain biomass concentrations before freeze-drying which are equivalent to or greater than those obtained using a conventional culture medium, such as MRS medium.

[0083] Advantageously, the culture medium according to the invention makes it possible to obtain biomass concentrations after freeze-drying which are equivalent to or greater than those obtained using a conventional culture medium, such as MRS medium.

[0084] According to a particular aspect, the invention relates to the use of food-grade culture medium according to the invention for the preparation of a pharmaceutical or nutraceutical composition based on lactic bacteria.

[0085] The invention also relates to the use of lactic acid bacteria cultured in the food-grade culture medium according to the invention, for the preparation of a pharmaceutical or nutraceutical composition intended for human or animal use. In particular, the pharmaceutical or nutraceutical composition can be prepared according to the method described below.

[0086] The term "nutraceutical" refers to a composition or product made from food substances, but made available in the form of a tablet, powder, potion or other galenic forms not usually associated with food, and having a beneficial or protective physiological effect against animal or human disorders or diseases. This definition includes, in particular, food supplements, certain group-specific foods or meal replacements. In particular, the nutraceutical composition as understood herein is based on lactic acid bacteria, preferably as the main ingredient.

[0087] As used herein, a "pharmaceutical composition" means a preparation of one or more of the active agents with other optional chemical components such as physiologically appropriate carriers and / or excipients. The compositions of the present invention may be presented in a form suitable for any conventional route of administration or use. The pharmaceutical composition according to the invention encompasses pharmaceutical compositions used in human medicine and pharmaceutical compositions used in animal medicine, i.e. veterinary compositions. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition as understood herein is based on lactic acid bacteria, preferably as the main active ingredient.

[0088] In particular, the lactic acid bacteria are chosen from bacteria of the genus Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Tetragenococcus, Leuconostoc, Oenococcus or Bifidobacterium, and all combinations thereof, preferably Lactobacillus, Lactococcus and / or Bifidobacterium and in particular Lactobacillus.

[0089] Preferably, the bacteria that can be cultivated in the medium according to the invention are bacteria of the genus Lactobacillus, preferably chosen from: L. plantarum, L. rhamnosus, L. casei, L. paracasei, L. caucasicus, L. lactis, L. crispatus, L. reuteri, L. brevi, L. thermophilus, L. gasseri, L. helveticus, L. bifermentans, L. hominis, L. aci-dophilus, L. bulgaricus and all combinations thereof.

[0090] The compositions of the present invention find use in the manufacture of probiotics as starters or ingredients but also in the manufacture of any other ingredient, food, food, nutraceutical or pharmaceutical composition containing a lactic acid bacterium.

[0091] This type of nutraceutical or pharmaceutical composition has the advantage of not containing any traces or residues of compounds of animal origin other than those derived from milk, of compounds which are subject to controversy, potentially dangerous or hazardous to human or animal health, and is particularly suitable for subjects following a vegetarian, kosher or halal diet. Cultivation method

[0092] The present invention also relates to a method for producing or preparing lactic acid bacteria in the food-grade culture medium according to the invention.

[0093] According to one aspect, the method of culturing lactic acid bacteria comprises:

[0094] a) the cultivation and multiplication of lactic acid bacteria in a food-grade culture medium as defined above, in particular comprising 35 to 70% of milk permeate by dry weight relative to the total dry weight of the medium as a carbon and nitrogen source, an additional nitrogen source and a mineral source; in which the ratio of total carbon / nitrogen input in the medium is between 4 and 10,

[0095] b) the recovery of the multiplied lactic acid bacteria, and

[0096] c) optionally, freeze-drying or freezing the multiplied bacteria.

[0097] In particular, the method of culturing lactic acid bacteria comprises:

[0098] a) humidifying a food-grade culture medium according to the invention, preferably in sterilized water to form a food-grade culture medium,

[0099] b) the cultivation and multiplication of lactic acid bacteria in this culture medium,

[0100] c) recovery of the multiplied lactic acid bacteria,

[0101] d) optionally, the concentration of the multiplied lactic acid bacteria,

[0102] e) optionally, the preservation and / or conservation of the multiplied bacteria, preferably by freeze-drying or freezing.

[0103] The culture method may preferably comprise a step of sterilizing the medium after humidifying the medium (step a) and before culturing the lactic acid bacteria (step b).

[0104] The multiplication of lactic acid bacteria (step b) can be carried out aerobically or anaerobically, depending on the required conditions. Preferably, the culture of lactic acid bacteria is carried out aerobically.

[0105] The culture of the bacteria of step b) can be carried out according to a batch, fed-batch or continuous feeding mode. Preferably, the culture of the lactic acid bacteria is carried out according to a batch feeding mode.

[0106] A person skilled in the art knows how to determine the appropriate culture conditions according to the type of bacteria to be cultivated and the type of yield envisaged.

[0107] According to a particular aspect, the multiplication or growth of the bacteria or the increase in biomass of step b) is carried out in a fermenter or bioreactor, preferably of industrial size, for example having a capacity of 20 liters, 50 liters, 100 liters, 200 liters, 500 liters, 1000 liters or 2000 liters.

[0108] As used herein, the term "bioreactor" or "fermenter" is an apparatus in which microorganisms (in particular here lactic acid bacteria) are multiplied for the production of biomass, or for the production of a metabolite or even the bioconversion of a molecule of interest.

[0109] According to one aspect, the method according to the invention comprises a step of concentrating the multiplied bacteria.

[0110] This step may in particular be followed by a step of rinsing the bacteria. According to a particular aspect, the method according to the invention comprises a step d) in which the bacteria are concentrated, the culture medium removed and the bacteria rinsed with a rinsing solution. A person skilled in the art knows how to determine the solution and the rinsing conditions according to the type of bacteria cultivated.

[0111] According to another aspect, the method according to the invention does not comprise a rinsing step, in particular following the recovery of the multiplied bacteria or following the concentration of the multiplied bacteria. Thus, the method according to the invention may comprise the preservation or conservation of the bacteria without the need for prior rinsing of the bacteria. The method may thus comprise a step d) in which the bacteria are concentrated and the supernatant culture medium removed, followed directly by a step e) of preservation and / or conservation of the multiplied bacteria, preferably by lyophilization.

[0112] According to one embodiment, the method according to the invention comprises a step of lyophilization or freezing of the cultured bacteria for the purpose of preserving and / or conserving or storing them. Preferably, this step comprises suspending the bacteria in a preservation solution. In particular, the method according to the invention further comprises adding one or more cryo- and / or lyoprotectants to the cultured bacteria before freezing or lyophilization, for example such as glycerol, sucrose, monosaccharides, disaccharides or polysaccharides.

[0113] In particular, the bacteria which can be cultivated according to the method of the invention are chosen from bacteria of the genus Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Tetragenococcus, Leuconostoc, Oenococcus or Bifidobacterium, and all combinations thereof, preferably Lactobacillus, Lactococcus and / or Bifidobacterium, and in particular Lactobacillus.

[0114] Preferably, the lactic acid bacteria that can be cultivated according to the method of the invention are bacteria of the genus Lactobacillus, preferably chosen from: L. plantarum, L. rhamnosus, L. casei, L. paracasei, L. caucasicus, L. lactis, L. crispatus, L. reuteri, L. brevi, L. thermophilus, L. gasseri, L. helveticus, L. bifermentans, L. hominis, L. acidophilus, L. bulgaricus and all combinations thereof.

[0115] The invention also relates to a method for preparing lactic acid bacteria comprising the steps described above, with a view to their use in a pharmaceutical or nutraceutical composition.

[0116] The invention also relates to a method for preparing a pharmaceutical or nutraceutical composition comprising a step of adding lactic acid bacteria prepared according to the above method. In particular, this method comprises adding the lactic acid bacteria to the other components of the pharmaceutical or nutraceutical composition.

[0117] The pharmaceutical or nutraceutical compositions obtained by the method according to the invention have the advantage of not having any traces or residues of compounds of animal origin other than those derived from milk, which are controversial, potentially dangerous or hazardous to human or animal health, and are particularly suitable for subjects following a vegetarian, kosher or halal diet. EXAMPLES RESULTS

[0118] Tables 9 and 10 present, for the three strains and for each culture medium, the biomass produced, the freeze-drying survival rate and the biomass after freeze-drying (CFU / mL and CFU / g of freeze-dried material). Different letters indicate a significant difference (HSD test, p<0.05).

[0119] For all strains, the skimmed milk-based medium with the ratio balanced with that of the MRS medium could not be considered an effective culture medium. Indeed, the high quantity of milk proteins in this medium and the high production of lactic acid by the bacteria caused the coagulation of the milk. This medium was therefore not compared with the MRS and the rest of the media in the case where the C / N ratios were balanced with that of the MRS (i.e. a C / N ratio of 4.4). Biomass produced

[0120] Depending on the three species of lactobacilli (plantarum, casei and rhamnosus), the biomass produced with the culture medium containing milk permeate is equivalent or greater than that produced on the MRS medium (p<0.05, ANOVA).

[0121] For L. rhamnosus, the biomass produced in the milk permeate-based medium (concentration 2) is significantly higher than the MRS and milk permeate (concentration 1) media (p<0.05, ANOVA). For the other two species (L. casei and L. plantarum), the biomass produced in the milk permeate-based medium is equivalent to that produced with the MRS medium.

[0122] In comparison, the biomass produced in a guar, skim milk or whey-based medium was significantly lower or equivalent to that produced on the MRS medium. In particular, a significantly lower biomass was observed:

[0123] - with a medium containing guar for L. casei and L. rhamnosus,

[0124] - with a medium containing skimmed milk for L. plantarum and L. rhamnosus, and

[0125] - with a medium containing whey for L. rhamnosus. Survival rate in the freeze-drying process

[0126] Depending on the three species of lactobacilli (plantarum, casei and rhamnosus), the survival rate in the freeze-drying process with the culture medium containing milk permeate is equivalent to that of the bacteria produced on the MRS medium (p<0.05, ANOVA).

[0127] For L. plantarum, the survival rate to the freeze-drying process after cultivation in MRS medium is lower than that after cultivation in milk permeate-based media (p<0.05, ANOVA). For the other two species (L. casei and L. rhamnosus), the survival rate to the freeze-drying process after cultivation in milk permeate-based media is equivalent (not significantly different) to the survival rate obtained after culture in the MRS environment.

[0128] In comparison, the survival rate in guar, skim milk or whey permeate based medium was significantly lower or equivalent to that produced on MRS medium. Notably, a significantly lower survival rate was observed:

[0129] - with a medium containing guar for L. plantarum,

[0130] - with a medium containing skimmed milk for L. plantarum, and

[0131] - with a medium containing whey permeate for L. plantarum. Biomass present after freeze-drying

[0132] The biomass present after freeze-drying results from the biomass produced and the survival of the bacterial cells in the freeze-drying process.

[0133] Thus, for all species of lactobacilli, the biomasses after freeze-drying are equivalent or greater than those obtained after culture in MRS when the cultures were carried out in media based on milk permeate.

[0134] Furthermore, for L. rhamnosus, the biomass present after freeze-drying when the culture was carried out in the milk permeate-based medium (concentration 2) is significantly higher than the biomass present after freeze-drying when the culture was carried out in the milk permeate-based medium (concentration 1).

[0135] When the C / N ratios are not balanced with that of the MRS, for the L. plantarum strain, the biomass after freeze-drying is thus 4.1011 CFU / g of freeze-dried material, for L. casei it is 2.2.1011 CFU / g of freeze-dried material and for L. rhamnosus it is 2.4.1011 CFU / g of freeze-dried material.

[0136] In comparison, the biomasses after freeze-drying in a guar, skimmed milk or whey permeate-based medium were significantly lower, equivalent or higher than those produced on the MRS medium. In particular, significantly lower biomasses after freeze-drying were observed:

[0137] - with a medium containing guar for L. rhamnosus,

[0138] - with a medium containing skimmed milk for L. rhamnosus and L. plantarum, and

[0139] - with a medium containing whey permeate for L. plantarum. Conclusions

[0140] Food grade media made from milk permeate provide a biomass after freeze-drying equivalent to MRS medium (balanced C / N ratios) for all strains (Table 9). When the C / N ratios of food grade media are increased compared to MRS, food grade media made from milk permeate provide a biomass after freeze-drying equivalent to or greater than MRS for all strains (Table 10). Milk permeate is a by-product of the cheese and cheese processing industry. Milk, readily available and less expensive than whey. Milk permeate is thus chosen as a food-grade source of nitrogen and carbon for the production of lactobacillus strain biomass. In addition, it is suitable for vegetarian, kosher, or halal diets. MATERIALS AND METHODS Strains

[0141] To evaluate the developed culture medium, three strains of Lactobacilli were used: Lactobacillus plantarum (Lpl), Lactobacillus casei (Lc03) and Lactobacillus rhamnosus (Lr04). The strains were stored at -80°C as aliquots containing 1 mL of stationary phase culture suspended in MRS medium supplemented with 20% glycerol (v / v) (G7893, Honeywell, USA). Culture media MRS environment (Table 1)

[0142] MRS medium (Lactobacillus Broth acc. to De Man, Rogosa and Sharpe, 69966, Sigma-Aldrich) was prepared according to the manufacturer's instructions and sterilized at 120°C for 20 minutes after the addition of 0.1% (v / v) Tween 80 (P4780, Sigma-Aldrich, CAS: 9005-65-6). Its final composition is therefore: peptone 10 g / L, meat extract 8 g / L, yeast extract 4 g / L, D (+)-Glucose (CAS: 50-99-7) 20 g / L, potassium hydrogen phosphate (CAS: 7758-11-4) 2 g / L, sodium acetate trihydrate (CAS: 6131-90-4) 5.0 g / L, triammonium citrate (CAS: 3458-72) 2 g / L, magnesium sulfate heptahydrate (CAS: 10034-99-8) 0.2 g / L, manganese sulfate tetrahydrate (CAS: 10101-68-5) 0.05 g / L and tween 80 ImL / L. The final pH is 6.2 + / -0.2 at 25°C. Food grade culture media

[0143] Four sources of nitrogen and carbon were evaluated for biomass production and survival in the freeze-drying process of Lactobacillus strains: skimmed milk, whey, milk permeate, whey permeate and guar peptone. Guar peptone is obtained from the legume Cyamoposis tetragonolobus which has been purified by mechanical treatment (removal of the guar gum attached to the germ). Tables 2 to 8 show in particular the compositions and C / N ratios used.

[0144] The components of animal origin and / or controversial present in the MRS medium have been replaced by food raw materials, i.e. authorized in the European regulatory sense. The components considered to be toxic substances have been eliminated from the composition.

[0145] The composition of food grade culture media is based on that of the simplified MRS medium: a nitrogen source (or a nitrogen and carbon source) ali supplementary, yeast extract (NuCel 582 MG, Procelys Lesaffre Fermentation Nutrients, France), a carbon source (glucose, D9434, Sigma-Aldrich), magnesium (magnesium sulfate heptahydrate, CLOO, 1324.0250, Chem-Lab NV, Belgium) and manganese (manganese (II) sulfate monohydrate, 221287, Honeywell, USA).

[0146] The dairy nitrogen and carbon sources were supplied by Eurial Ingrédients & Nutrition (France) and the plant nitrogen source was supplied by Organotechnie (France).

[0147] Skim milk, whey, milk permeate and whey permeate provide nitrogen and carbon (lactose) whereas guar peptone provides only nitrogen.

[0148] The skimmed milk, whey, milk permeate and whey permeate powders were added after sterilization of the medium (120°C - 20 minutes) at a temperature between 65°C and 75°C (milk pasteurization temperature). The guar peptone was added at the same time as the other components of the culture medium and then the latter was sterilized as described previously. The pH of the media was then adjusted to 6.2 ± 0.2.

[0149] Production of biomass and freeze-drying of the produced biomass

[0150] From the cryotubes, strains were prepared by inoculating 100pL of culture on Petri dishes containing MRS agar medium: MRS supplemented with 15g / L of agar (84609, VWR, Belgium). After 24h of growth in an incubator at 37°C, the strains were kept in a refrigerator at 4°C and can be used up to 3 weeks after their culture. Pre-cultures were carried out by inoculating 10 mL of MRS medium with colonies taken from these strains. Finally, after 24h of culture at 37°C, the FG culture media or the control culture medium (MRS) were inoculated at 1% (v / v) with the pre-cultures and then incubated at 37°C.

[0151] Enumeration of the biomass produced and determination of the freeze-drying survival rate

[0152] The number of cells present was estimated by the Colony Forming Unit (CFU) method. The biomass produced (CFU / mL before lyophilization) was harvested at the beginning of the stationary phase of growth. After centrifugation (4000g - 10 minutes, Eppendorf 5810 R), the pellets were resuspended in sucrose (59378, Sigma) at 5% (m / v) in PBS (Phosphate Buffered Saline, P4417, Sigma). Sucrose is added so as to concentrate the number of cells produced per mL by 10.

[0153] One milliliter of this mixture is introduced into 5 ml amber glass bottles suitable for lyophilization applications and then frozen at -80°C for 2 hours before being lyophilized for 24 hours.

[0154] At the end of the lyophilization process, the samples are rehydrated in 1 mL of MRS medium at 37°C in order to carry out a biomass count (CFU / mL after lyophilization).

[0155] Survival in the freeze-drying process (%) is determined by calculating the ratio between the biomass present after freeze-drying and rehydration (CFU / mL before freeze-drying) and the biomass present before freeze-drying (CFU / mL after freeze-drying and rehydration).

[0156] To estimate the number of cells present per gram of lyophilisate, the number of cells present in 1L of rehydrated lyophilisate was related to 50g of dry matter, i.e. divided by 20 (dry matter in the lyophilisate, i.e. dry matter of 5% sucrose in PBS or 50g / L). Statistical analysis

[0157] Analysis of variance (ANOVA) and Tukey's HSD post-hoc test (if p < 0.05) were performed to determine whether significant differences in biomass production, freeze-drying survival, or biomass present after freeze-drying existed between culture media. Analyses were performed using R software (version 3.5.1).

[0158] The experiments were carried out in biological triplicates (3 independent cultures). The results presented are the means and standard deviations for n=3. Bibliographic references

[0159] Beal C, Fonseca F, Corrieu G (2001) Resistance to Freezing and Frozen Storage of Streptococcus thermophilus Is Related to Membrane Fatty Acid Composition. J Dairy Sci 84:2347-2356.

[0160] Blond G, Simatos D, Catté M, et al (1997) Modeling of the water-sucrose State diagram below 0 °C. Carbohydr Res 298:139-145.

[0161] Briggs M (1953) An improved medium for lactobacilli. J. Dairy Res. 20, 36-4

[0162] Broeckx G, Vandenheuvel D, Claes IJJ, et al (2016) Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. Int J Pharm 505:303–318.

[0163] Carvalho AS, Silva J, Ho P, et al (2003) Effect of variant growth media on survival during storage of freeze-dried Enterococcus faecalis and Enterococcus durans. J Appl Microbiol 94:947–952.

[0164] Carvalho AS, Silva J, Ho P, et al (2004a) Effects of Varions Sugars Added to Growth and Drying Media on Thermotolerance and Survival throughout Storage of Freeze-Dried Lactobacillus delbrueckii ssp. bulgaricus. Biotechnol Prog 20:248–254.

[0165] Carvalho AS, Silva J, Ho P, et al (2004b) Relevant factors for the preparation of freeze-dried lactic acid bacteria. Int Dairy J 14:835–847.

[0166] Corcoran BM, Stanton C, Fitzgerald GF, Ross RP (2007) Growth of probiotic lac- tobacilli in the presence of oleic acid enhances subséquent survival in gastric juice. Mi-crobiology 153:291-299.

[0167] De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of Lac-tobacilli. J Appl Bact 23: 130-135.

[0168] FAO-WHO (2006) Probiotics in food: health and nutritional properties and guidelines for évaluation. Food and Agriculture Organization of the United Nations : World Health Organization, Rome.

[0169] Franca MB, Panek AD, Eleutherio EC (2007) Oxidative stress and its effects during déhydration. Comp Biochem Physiol A Mol Integr Physiol 146:621-631.

[0170] Goldberg I, Eschar L (1977) Stability of lactic Acid bacteria to freezing as related to their Fatty Acid composition. Appl Env Microbiol 33:489-496.

[0171] lanniello RG, Zotta T, Matera A, et al (2016) Investigation of Factors Affecting Aérobic and Respiratory Growth in the Oxygen-Tolerant Strain Lactobacillus casei N87. PLoS One ll:e0164065.

[0172] Siaterlis A, Deepika G, Charalampopoulos D (2009) Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Lett Appl Microbiol 48:295-301.

[0173] Silva J, Carvalho AS, Ferreira R, et al (2005) Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying. J Appl Microbiol 98:775-782.

[0174] MRS bmth components (g / L) Nitrogen sources 10 Meat extracts 8 Yeast extract 4 Carbon sources Glucose 20 Mineral sources Magnesium sulfate heplahydrate aa Manganese sulfate tetrahydrate 0.05 Potassium hydrogen phosphate 2 Others Sodium acetate trihydrate 5 Tnammonsum citrate 3 TwëÀ 80 1 mt Price / Kg 216

[0175] Composition of the MRS medium. In dark gray: components of animal origin; In light gray: ingredients listed among the substances toxic to human health or whose use is controversial.

[0176] [Tables2] Per 160g of dry matter milk, skimmed whey Whey protein Whey protein Sugar Total nitrogen (%) M 0.4 0.4 84 Protein (^*08} (¾¾) MS 11.5 2 & 2.8 8.M Total carbon (%) 815 Vitamins,? 0 lactose (42 of carbons) {g} 554 25 85 85 G

[0177] Nitrogen and carbon levels of the five food-grade nitrogen and carbon sources supplementary (per 100 grams of dry matter).

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] In IL of MRS medium C / N ratio 4.4 Total nitrogen (¾) 1.8 Peptone (10% nitrogen)' (g / l) 10 Meat extract of atote) ig / G 8 Total carbon (%) S Glucose [40% carbon) (g / L) 20 Nitrogen and carbon levels per 1 liter of MRS culture medium. [Tables 4] In the middle MRS was êoémé Lactosêntm Permést of milk Closed! of tactosermn Ratio c / N 4.4 4.4 4.4 4.4 4.4 Total nitrogen ¢4) 1.85 1.S ■u U Dairy powder [g / q 34.3 85.3 32.3 2A 3 Yeast extract (g / y / b 8.1 17.ï •j ? 1 Carbon testai (%) S qiS â SS Lactose (42% carbon) (g) 0 19 19 19 iâ Glucose (40% carbon) (g) 20 IM Û 0 0 Nitrogen and carbon levels per 1 liter of milk protein-based medium with C / N ratios balanced with MRS and the percentage of carbon fixed to that of MRS. [Table 5] In 11 of Guar medium Ratio C / N Nitrogen (g / tj 4 4 estimated (^L) tsïœose (40% âe C) (g / tjo Nitrogen and carbon levels per 1 liter of guar peptone-based FG medium with the C / N ratio balanced with that of the MRS.

[0184] [Tableauxô] Osm XL à® mHlnn Suwr Lslt skimmed Lsctnsèrum Milk permsët Whey pêméat Powder® feitiwës «« WM 21.4 343 253 22.3 223 Extract d® tevwrç 4 0 ¢.1 13.1 174 Glucose (g / td 17 16.7 û Û 8 Magnesium (g / M ¢3 83 as u.?. 03 Manganese (g / Lj 0.05 0.05 035 0.05 6.2 + 03 63 * 0.2 53 103 83 1$3 63 + 83

[0185] Example of composition of Food Grade culture media (balanced C / N ratios).

[0186] [Tables?] In IL of MRS medium Skimmed milk Whey Milk permeate Whey permeate C / N ratio 4.4 28.6 28.2 6.3 6.3 Protein (N*6.3S) % 18 8.36 5.4 23.5 23.5 Total nitrogen (%J 1.8 0.84 0.85 3.7 ÿ1 Milk powder [g / L] / 15.6 47.1 64 64 Added yeast extract (g / LJ / 0 0 34 34 Total carbon (%) 8 24 24 22.8 22.8 Lactose [42% carbon] (g) 0 8.6 35.3 54.4 54.4 Glucose [40% carbon) [gl 28 51 23 Û û

[0187] Nitrogen and carbon levels per 1 liter of milk protein-based medium with the C / N ratios higher than that of the MRS. In IL of medium Skimmed milk Whey Closed»! of hit Closed»! of whey Powder Vegetable matter WU X5.6 474 64 64 Yeast extract [g / Ü 4 4 33 38 Glucose (g / L) 81 23 0 9 Magnesium (g / i) 9.2 9.2 42 0.2 Manganese [g / Q 0.i05 iW 0.08 OS pH 6.2 f. 0.2 6.2 + 0.2 6.2 .♦ 0.2 6.2 4 0.2

[0189]

[0190] Example of composition of Food Grade culture media (C / N ratios not balanced with MRS). 5 &: « 1 «S: i Kï! <<$ -¾ ; >■$ s$ « ss 1-^ 1 4 3 3|è iy L< $ ''J *1" ■< 1 :<< II «------- | 1 £ 5¾ >$ ^'1 s * ■X AX \ *ÿ <•* X^" li <•: * 1 ;> « S o i «j; | <s | 1 â s SS jfS S « 33 *0 b, K *' <x s>\ 3::J| 3 5v । yy <<<: xxx;Ÿ xxs V 1 x' j xÿ >3: x> *«.| ..v ;“y?:< / s> <y>•\vv | $ | ..." ->> SS 1 , *J.. ?.> ^-5, ♦* <-x •X' ^'l ** \ ix <*X ' ' ■ J to K $ M & < S' | >■ $ î x 1 g 3 ■'S' « -» S ® a â * « £S § « < / §• a

[0191] Biomass produced, freeze-drying survival rate and biomass after freeze-drying as a function of the culture medium (C / N ratio of the culture media balanced with that of MRS). Letters represent a significant difference between culture media with the HSD test (p < 0.05). This table also indicates the efficiency of the tested media in comparison with the MRS medium: Significantly lower than MRS: <; Equivalent to MRS: =; Significantly higher than MRS: >.

[0192]

[0193] Biomass produced, freeze-drying survival rate and biomass present after freeze-drying as a function of the culture medium (C / N ratios of FG culture media not balanced with that of MRS). Letters represent a significant difference between culture media with the HSD test (p < 0.05). This table also indicates the effectiveness of the tested media in comparison with the MRS medium: Significantly lower than MRS: < ; Equivalent to MRS: = ; Significantly higher than MRS:< / y> ​

Claims

Claims

1. Use of a food-grade culture medium for cultivating lactic acid bacteria, said medium comprising: 35 to 70% of milk permeate by dry weight relative to the total dry weight of the medium as a carbon and nitrogen source, 25 to 55% by dry weight of yeast extract relative to the total dry weight of the medium, and a mineral source, wherein the ratio of the amount of carbon to the amount of nitrogen in the medium is between 4 and 10.

2. A method for producing lactic acid bacteria comprising: a) culturing and multiplying lactic acid bacteria in a food-grade culture medium comprising 35 to 70% milk permeate by dry weight relative to the total dry weight of the medium as a carbon and nitrogen source, 25 to 55% by dry weight of yeast extract relative to the total dry weight of the medium, and a mineral source; wherein the ratio of the amount of carbon to the amount of nitrogen in the medium is between 4 and 10, b) recovering the multiplied lactic acid bacteria, and c) optionally, freeze-drying or freezing the multiplied bacteria.

3. A food grade culture medium for lactic acid bacteria comprising 35 to 70% by dry weight of milk permeate, 25 to 55% by dry weight of yeast extract relative to the total dry weight of the medium, and a mineral source, the culture medium having a ratio of the amount of carbon to the amount of nitrogen in the medium of between 4 and 10.

4. Use according to claim 1, method according to claim 2 or culture medium according to claim 3, wherein the culture medium does not comprise peptone of animal origin and / or meat extract.

5. Use according to claim 1 or 4, method according to claim 2 or 4, or culture medium according to claim 3 or 4, wherein the culture medium comprises magnesium as a mineral source, preferably magnesium sulfate, in particular 0.01% and 1% by dry weight of magnesium sulfate relative to the total dry weight of the medium.

6. Use according to claim 1, 4 or 5, method according to claim 2, 4 or 5 or culture medium according to claim 3, 4 or 5, wherein the culture medium comprises manganese as a mineral source, preferably manganese sulfate, in particular 0.01% and 1% by dry weight of manganese sulfate relative to the total dry weight of the medium.

7. Use according to any one of claims 1 and 4-6, method according to any one of claims 2 and 4-6, or culture medium according to any one of claims 3-6, wherein the culture medium has a pH between 5.5 and 7.5, preferably a pH between 6 and 7.

8. Use according to any one of claims 1 and 4-7, method according to any one of claims 2 and 4-7 or culture medium according to any one of claims 3-7, wherein the culture medium does not comprise at least one compound selected from the group consisting of glucose, an antibiotic, potassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate and Tween 80.

9. Use of a food grade culture medium according to any one of claims 3-8 for the preparation of a pharmaceutical or nutraceutical composition comprising lactic acid bacteria.