FR3033332A1 - USE OF A NUTRITIONAL COMPLEMENT IN THE MANUFACTURE OF LACTIC ACID - Google Patents

Abstract

The present invention relates to the use of a nutritional supplement comprising an active principle, in raw form or extracted form, resulting from a fermentation with a mold or a bacterium, for the manufacture, by fermentation, of lactic acid.

Description

The present invention relates to the manufacture of lactic acid.

It is known to produce lactic acid from starchy raw material by an enzymatic process comprising a step of liquefying the starch with an alpha-amylase aimed at solubilizing and hydrolyzing the starch into dextrins. This step is conventionally followed by a glucoamylase saccharification step, aimed at hydrolyzing dextrins to glucose. The glucose is then fermented during a fermentation step during which it is converted into lactic acid by a lactic acid bacterium. However, the commonly used microorganisms of the genera Lactobacillus, Bacillus and Sporolactobacillus can not grow and produce lactic acid industrially without the addition of a source of organic nitrogen composed of free or bound amino acids. oligopeptides and peptides, whether its composition is defined or whether it is a natural extract. Examples of such organic nitrogen sources conventionally used are yeast autolysates and hydrolysates, vegetable and animal protein hydrolysates (casein peptone, etc.) and also the soluble by-products of wheat and corn quenching. Nevertheless, these sources of organic nitrogen are sold at relatively high prices, which greatly increases the cost of manufacturing lactic acid. Defined organic nitrogen sources reconstituted from amino acids, vitamins and purified growth promoters can also be used but with even higher costs. Solutions have been envisaged to try to reduce the exogenous supply of nitrogenous source, and thus limit production costs. It has thus been proposed in application EP 1 205 557 to carry out physical and enzymatic treatments upstream of the fermentation of the raw material and to use nutritionally less demanding microorganisms such as Lactococcus lactis. However, the addition of additional steps to the production process also entails a significant cost and does not seem to limit the exogenous addition of nutritional sources. The application EP 1 953 234 proposes for its part to use a specific raw material (cane juice and derivatives) adapted to certain microorganisms such as Bacillus sp. and Sporolactobacillus sp., but remaining unsuitable for microorganisms belonging to other genera such as Lactobacilli. Application US 2004/203122 proposes to use thermophilic microorganisms such as Bacillus sp, less demanding, but for which the addition of nitrogen supplements is still necessary. Application WO 2011/098843 proposes for its part to use lactic acid bacteria expressing starch degrading enzymes, thus avoiding the use of commercial enzymes during the saccharification and fermentation stages, but always involving the addition nitrogen supplements. There is therefore still a great need for less expensive lactic acid production processes requiring the use of less expensive or less significant organic nitrogen sources. The inventors have found, surprisingly, that this objective could be achieved by the use of a nutritional supplement for a saccharification-fermentation medium in a process for the manufacture of lactic acid by fermentation from a starchy raw material. comprising, and preferably consisting of, an active ingredient derived from a fermentation with a mold or bacteria, this in total or partial substitution of expensive exogenous nitrogen sources commonly used such as yeast extracts and peptones. The inventors have thus found that this nutritional supplement, when added, during the process of producing lactic acid, in the medium of saccharification, fermentation or simultaneous saccharification-fermentation, resulted in the release, from the starchy raw material used, a quantity and a large variety of amino acids that are directly assimilated by lactic acid bacteria, thus allowing an increase in the production of lactic acid. In addition, the inventors have found that by using this nutritional supplement, it was possible, while increasing the amount of amino acids released during saccharification, simultaneous fermentation or saccharification-fermentation, to limit amount of free amino nitrogen (or FAN) at the end of fermentation, thus facilitating the steps generally carried out downstream of the lactic acid production process, namely the extraction and purification of lactic acid.

The subject of the present invention is therefore the use of a nutritional supplement comprising an active principle, in raw form or extracted form, resulting from a fermentation with a mold or a bacterium, for the manufacture, by fermentation, preferably in medium liquid, lactic acid. The present invention furthermore relates to the use of a nutritional supplement comprising an active ingredient, in raw form or extracted form, resulting from a fermentation in solid medium with a mold or a bacterium, in order to release, in a fermentation medium lactic acid, a renewable carbohydrate raw material, the nitrogen of said raw material, during manufacture, by fermentation, preferably in liquid medium, lactic acid from said raw material.

The present invention also relates to a process for manufacturing lactic acid from a renewable carbohydrate raw material, comprising a step of fermentation, preferably in a liquid medium, with a lactic acid bacterium in the presence of a nutritional supplement comprising an active principle, in raw form or extracted form, resulting from a fermentation in solid medium with a mold or a bacterium. The present invention also provides a composition comprising (i) a source of organic nitrogen selected from the group consisting of renewable carbohydrate raw materials, exogenous nutrient solutions, and mixtures thereof and (ii) a nutritional supplement comprising an active ingredient, in raw form or extracted form, resulting from a fermentation in a solid medium with a mold or a bacterium. The present invention also provides a kit comprising: (i) an organic nitrogen source selected from the group consisting of renewable carbohydrate raw materials, exogenous nutrient solutions, and mixtures thereof, and (ii) a nutritional supplement comprising an active principle, in raw form or extracted form, resulting from a fermentation in a solid medium with a mold or a bacterium.

DETAILED DESCRIPTION OF THE INVENTION Nutritional supplement The nutritional supplement used in the context of the invention is a nutritional supplement comprising an active principle, in raw or extracted form, resulting from a fermentation in a solid medium with a mold or a bacterium . By "solid state fermentation" is meant herein the culture of microorganisms on wet solid supports, on inert carriers or on insoluble substrates which can be used as a source of carbon and energy. The fermentation process takes place in the absence or near-absence of free water in the space between the substrate particles. Substrate The substrate used for the production of the nutritional supplement is preferably an agricultural co-product. By "agricultural co-product" is meant here a product created during the same processing process and at the same time as a main agricultural product. The substrate used for the production of the nutritional supplement may in particular be a cereal co-product, an oleaginous co-product, an oilseed crop co-product, a saccharificial co-product or a mixture thereof.

Examples of substrates that can be used to produce the nutritional supplement used in the context of the invention include wheat bran, rapeseed meal, soybean meal, beet pulp, barley root, ethanol-soluble wheat flour and mixtures thereof.

Preferably, the substrate used for the production of the nutritional supplement is selected from the group consisting of wheat bran, rapeseed meal, barley rootlet, wheat ethanol soluble sorbet, and mixtures thereof. More preferably, the substrate used for the production of the nutritional supplement is wheat bran or rapeseed cake. Most preferably, the substrate used for the production of the nutritional supplement is rapeseed cake. The substrate used for the production of the nutritional supplement can be ground beforehand. The substrate used for the production of the nutritional supplement is preferably moistened and heat-treated for pasteurization or sterilization. The heat treatment may consist of heating, for example in an autoclave, typically at 121 ° C., for example for 20 minutes or at 105 ° C., for example for 35 minutes, or pasteurisation, typically at 105 ° C., for example as a loser. min. It is also possible to heat treat the substrate used for the production of the nutritional supplement by injecting water vapor, which makes it possible to simultaneously operate the humidification of the substrate. Preferably, the pH is adjusted during wetting in a range from 4 to 5.5, preferably to 4.9, for example with nitric acid or sulfuric acid, in order to improve the effect. heat treatment and the start of fermentation in a solid medium.

Humidification is preferably determined so that the water content of the substrate used for the production of the nutritional supplement is between 40 and 60% of the total mass of the substrate and water. Mold and Bacteria The microorganism used during fermentation in a solid medium may be a mold. The mold used during fermentation in a solid medium is preferably a mold of the genus Aspergillus, more preferably of the species Aspergillus piger, Aspergillus oryzae, Aspergillus tubingensis or Aspergillus flavus. In a particularly preferred manner, the mold used during fermentation in a solid medium is Aspergillus oryzae or Aspergillus flavus. Most preferably, the mold used in the solid-state fermentation is Aspergillus oryzae, particularly the Aspergillus oryzae ATCC 12892 strain (available from the ATCC Collection). Alternatively, the microorganism used during fermentation in a solid medium may be a bacterium. The bacterium used during fermentation in solid medium is preferably a bacterium of the genus Bacillus, more preferably of the species Bacillus subtilis or Bacillus licheniformis. In a particularly preferred manner, the bacterium used during fermentation in solid medium is of the species Bacillus subtilis.

Thus, the mold or bacterium used during solid-state fermentation is preferably selected from the group consisting of Aspergillus and Bacillus bacteria. More preferably, the mold or bacteria used in the solid-state fermentation is selected from the group consisting of Aspergillus flavus strains, Aspergillus oryzae strains, Aspergillus piger strains, Aspergillus strains. tubingensis, and strains of Bacillus sp. The inventors have demonstrated that the use of certain molds or bacteria makes it possible to manufacture a nutritional supplement particularly adapted to certain lactic acid bacteria. Thus, particularly preferably, the mold or bacteria used during fermentation in a solid medium is a mold of the species Aspergillus flavus or of the species Aspergillus oryzae or a bacterium of the species Bacillus subtilis. The inventors have also demonstrated that the use of certain molds or bacteria makes it possible to manufacture a nutritional supplement allowing to more strongly limit the presence of free amino nitrogen (FAN) at the end of the lactic acid production process by fermentation.

Thus, particularly preferably, the mold or bacteria used during fermentation in a solid medium is a mold of the species Aspergillus oryzae or a bacterium of the species Bacillus subtilis. In particular, when the lactic acid bacterium used for the production of lactic acid is L. coryniformis strain DSM 20004, the mold or bacteria used during fermentation in solid medium is preferably a mold of the species Aspergillus oryzae. Similarly, when the lactic acid bacterium used for the production of lactic acid is the strain L. coryniformis DSM 20005, the mold or bacteria used during fermentation in solid medium is preferably a bacterium of the species Bacillus subtilis. Inoculation of the substrate used for the production of the nutritional supplement can be carried out with any suitable inoculum. The molds used during fermentation in solid medium can thus be previously subjected to a sporulation stage before being inoculated in the form of spores. The inoculum dose will then advantageously be at least 106 spores / g of initial dry matter. Alternatively, the molds used during fermentation in solid medium can be inoculated as mycelium. The dose of inoculum will then advantageously be between 100 and 5,300 g of liquid culture medium comprising the mycelium per kg of initial dry matter. Preferably, the bacteria used during fermentation in a solid medium are subjected to a pre-culture step in order to be in the exponential phase during inoculation. The dose of inoculum will then advantageously be between 100 and 300 g of liquid culture medium comprising the bacteria in the exponential phase per kg of initial solids. Fermentation in a solid medium Fermentation in a solid medium can be carried out in any suitable reactor. Fermentation in a solid medium is preferably conducted for controlled arrest prior to sporulation of the mold strain used and / or at reaching the stationary phase of the bacterial strain used. Fermentation in solid medium is thus preferably carried out for a period of 1 to 3 days, preferably between 30 and 60 hours, particularly preferably for 48 hours. The temperature of the medium is preferably maintained between 28 ° C and 38 ° C, which corresponds to the optimum range of activity of the mold and bacteria strains used to produce the nutritional supplement. Moisture is preferably maintained between 40 and 60% during fermentation. Aeration, preferably continuously, is preferably carried out during fermentation in a solid medium in order to provide the oxygen necessary for fermentation and to avoid the excessive accumulation of carbon dioxide produced by the fermentation. Stirring may be conducted to avoid the formation of impermeable masses. Stirring can be carried out by means of stirring arms, blades or spatula or worm. The agitation, however, preferably remains moderate.

Packaging of the fermented product The nutritional supplement obtained following the fermentation in solid medium above can be in raw form or extracted. By "raw form" is meant here that the nutritional supplement comprises the active principle as well as the substrate used during the fermentation in solid medium with the mildew or bacterium, optionally milled. This raw form typically contains particles having a diameter of between 50 μm and 5 mm depending on the type of ground material generated. The nutritional supplement obtained after fermentation in solid medium can also be dried or frozen for preservation.

The drying is preferably carried out at a moderate temperature so as not to affect the enzymatic activities, for example in an oven preferably at 40 ° C. Freezing is preferably carried out on the moist fermented product, for example at -20 ° C. The nutritional supplement obtained after the fermentation in solid medium can also undergo a new grinding before being conditioned. By "extracted form" is meant here that the active ingredient of the nutritional supplement is isolated from the substrate used during the fermentation in solid medium with the mold or bacteria by extraction processes that can implement any well known technique of those skilled in the art, in particular by extraction in aqueous solution, extraction in alcoholic solution, extraction by solvents, high pressure homogenization, supercritical extraction, extraction by fluidized beds, grinding, cryogenic grinding, decompression, cavitation, bubbling, extraction by ultrasound adsorption on resins or zeolites. The active ingredient in liquid form can then be purified by any technique well known to those skilled in the art, in particular by centrifugation, filtration, ultrafiltration, chromatography, use of membranes or precipitation. The present invention relates to the use of a nutritional supplement as defined in the section "Nutritional supplement" above, for the manufacture, by fermentation, of lactic acid. Another subject of the invention is a method for manufacturing lactic acid from a renewable carbohydrate raw material, comprising a step of fermenting the renewable carbohydrate raw material with a lactic acid bacterium in the presence of a nutritional supplement as defined in US Pat. section "Nutritional supplement" above. Lactic Acid By "lactic acid" or "2-hydroxypropanoic acid" is meant here alpha hydroxyl acid of formula C3H603 or its lactate ion. In the context of the invention, the term "lactic acid" covers the enantiomer (R) -lactic acid or D (-) - lactic acid and the enantiomer (S) -lactic acid or L (+) - Lactic acid. Thus, the lactic acid produced in the context of the invention may be D-lactic acid, L-lactic acid or a mixture thereof. Preferably, the lactic acid produced in the context of the invention is D-lactic acid. Lactic Bacteria As is well known to those skilled in the art, lactic acid can be obtained by fermenting a substrate with lactic acid bacteria.

By "lactic acid bacterium" is meant here a gram-positive, anaerobic, partially oxygen-tolerant bacteria, generally not producing spores, in the form of shells or sticks and capable of fermenting sugars to lactic acid. . As is well known to those skilled in the art, lactic acid bacteria belong to the order Lactobacillales, of the Bacilli class of the phylum Firmicutes, and to the family Bifidobacteriaceae of the order Bifidobacteriales. The lactic acid bacterium used in the context of the invention is preferably a lactic bacterium of the order Lactobacillales. As is well known to those skilled in the art, the order Lactobacillales includes the following families: Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae. Preferably, the lactic acid bacterium used in the context of the invention is of the Lactobacillaceae family. The family Lactobacillaceae includes bacteria of the genus Lactobacillus, Paralactobacillus, Pediococcus and Sharpea. Preferably, the lactic acid bacterium used in the context of the invention is of the genus Lactobacillus. In particular, the lactic acid bacteria used in the context of the invention may be chosen from the group consisting of L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius species. , L. amylolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animalis, L. antri, L. apodemi, L. aquaticus, L. aviarius, L. bifermentans, L. brantae, L. brevis, L. Buchneri, L. cacaonum, L. camelliae, L. capillatus, L. casei, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum L. curieae L. curvatus L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. indicus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. sunkii, L. dextrinicus, L. diolivorans, L. equi, L. equicursoris, L. equigenerosi, L. fabifermentans, L. farraginis, L. farciminis, L. fermentum, L. floricola, L. forum, L. fornicalis L. fructivorans L. frumenti L. fuchuensis L. fusaii L. gallinarum L. gasseri L. gastricus L. ghanensis L. gigeriorum L. graminis L. hammesii L. 3033332 9 hamsteri , L. harbinensis, L. hayakitensis, L. helveticus, L. hilgardii, L. hokkaidonensis, L. hominis, L. homohiochii, L. hordei, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. Johnsonis L., L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchicus, L. kimchiensis, L. kimchii, L. kisonensis, L. kitasatonis, L. koreensis, L. kunkeei, L. leichmannii, L. lindneri , L. malefermentans, L. mali, L. manihotivorans, L. mindensis, L. mucosae, L. murinus, L. nagelii, L. namurensis, L. nantensis, L. nasuensis, L. odoratitofui, L. oeni, L. oligofermentans, L. oris, L. oryzae, L. otakiensis, L. ozensis, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei, L. paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pasteurii, L. paucivorans, L. pentosus, L. perolens, L. plantarum, L. pobuzihii, L. pontis, L. porcinae, L. psittaci, L. rapi, L. rennini, L. reuteri, L. rhamnosus, L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. saniviri, L. satsumensis, L. secaliphilus, L. selangorensis, L. senioris, L. sharpeae, L. siliginis, L. similis, L. sobrius, L. spicheri, L. sucicola, L. suebicus, L. sunkii, L. suntoryeus, L. taiwanensis, L. thailandensis, L. tucceti, L. uli, L. ultimensis, L. uvarum, L. vaccinostercus, L. vaginalis , L. versmoldensis, L. vini, L. vitulinus, L. xiangfangensis, L. yonginensis, L. zeae and L. zymae. Preferably, the lactic acid bacterium used in the context of the invention is selected from the group consisting of L. mindensis, L. zeae and L. coryniformis species. More preferably, the lactic acid bacterium used in the context of the invention is selected from the group consisting of L. mindensis strain LMG 21932 (available from the BCCM / LMG Collection), L. zeae strain LMG 17315 ( available from the BCCM / LMG Collection), the strain L. coryniformis ssp. torquens DSM 20004 (available from the DSMZ Collection), and the strain L. coryniformis ssp. torquens DSM 20005 (available from the DSMZ Collection).

Lactic acid bacteria capable of producing D-lactic acid, L-lactic acid or a mixture of D-lactic and L-lactic acid are well known to those skilled in the art and are, for example, described in US Pat. Connolly and Leinnerdal (2004) Nutrafoods 3: 37-49. Thus, among the lactic acid bacteria capable of producing D-lactic acid, mention may be made of the lactic acid bacteria L. coryniformis, L. delbrueckki and L. jensenii.

Among the lactic bacteria capable of producing L-lactic acid, there may be mentioned lactic acid bacteria L. zeae, L. amylophilus, L. casei and L. rhamnosus. Among the bacteria capable of producing D-lactic acid and L-lactic acid in equivalent proportion, mention may be made of the lactic acid bacteria L. mindensis, L. acidophilus, L. brevis, L. fermentum and L. plantarum.

The inventors have shown that the nutritional supplement described above is particularly effective in improving the production of lactic acid by L. coryniformis lactic bacteria. Therefore, in a particularly preferred embodiment, the lactic acid bacterium used in the context of the invention is a L. coryniformis lactic acid bacterium, in particular a strain of Lactobacillus coryniformis ssp. torquens, preferably the strain L. coryniformis ssp. torquens DSM 20004 or strain L. 5 coryniformis ssp. Torquens DSM 20005, in particular the strain L. coryniformis ssp. Torquens DSM 20005. During the lactic acid production process, the lactic acid bacteria are introduced during the simultaneous fermentation or saccharification-fermentation step.

Preferably, the lactic acid bacteria are inoculated into the simultaneous fermentation or saccharification-fermentation medium at a dose of between 107 and 109 CFU / ml of medium, preferably at a dose of 108 CFU / ml of medium. Preferably, the lactic acid bacteria are introduced into the simultaneous fermentation or saccharification-fermentation medium after having undergone a propagation step. This propagation step allows a development of lactic acid bacteria sufficient to inoculate the fermentation medium or simultaneous saccharification-fermentation at the dose mentioned above. The propagation step is typically carried out by culturing lactic acid bacteria in a conventional culture medium for lactic acid bacteria, such as MRS medium (Man's medium, Rogosa, Sharpe), preferably with stirring, for example at 100. rpm, at a temperature adapted to the growth of lactic acid bacteria, for example at 37 ° C., at a pH preferably adjusted for example to 6. Renewable carbohydrate raw material By "renewable carbohydrate raw material" is meant waste from the food industries. inexpensive, unrefined, generally non-toxic and rich in sources of carbon and nitrogen more or less assimilable. The renewable carbohydrate raw material used in the context of the invention may in particular be chosen from the co-products of the cereal sector, such as the wheat, maize, barley or rye sector, the co-products of the sugar industry, such as the beet sector. or sugarcane, the co-products of the high carbohydrate tuberous plant sector, such as the cassava or potato industry, and the co-products of the dairy sector. These renewable carbohydrate raw materials contain both starch or glucose as a source of carbon, and high molecular weight proteins, in addition to peptides and free amino acids, as a source of more or less assimilable nitrogen.

3033332 11 Renewable carbohydrate raw materials from the cereals sector include in particular liquefied and saccharified flours of wheat, maize, barley and / or rye, more particularly liquefied and saccharified flours of abraded wheat. The renewable carbohydrate raw materials derived from the sugar industry 5 include in particular the concentrated sugar carbohydrate byproducts, more particularly beet or sugarcane molasses. The renewable carbohydrate raw materials derived from the high carbohydrate tuberous plant sector include in particular liquefied flours of tubers, more particularly liquefied flours of cassava or potato.

Renewable carbohydrate raw materials from the dairy sector include lactose-rich dairy co-products, particularly whey. The inventors have demonstrated when the nutritional supplement as defined above was used in processes for producing lactic acid from renewable raw carbohydrate material from the wheat or maize cereal sector, or from the sugarcane sugar sector. , it made it possible to reduce the addition of expensive exogenous nutrient solutions, as defined in the "Exogenous nutrient solutions" section below, by at least 20-25% for the same yield of lactic acid. Preferably, the renewable carbohydrate raw material used in the context of the invention is therefore chosen from the group consisting of renewable carbohydrate raw materials derived from the wheat cereal sector, in particular saccharified liquefied flours (musts) of abraded wheat, the renewable carbohydrate raw materials derived from the corn cereal sector, in particular the saccharified liquefied flours (musts) of maize and the renewable carbohydrate raw materials derived from the sugar cane sugar sector, in particular sugar cane molasses. The inventors have more particularly demonstrated that the same yield of lactic acid could be obtained from a renewable raw material of wheat from the cereal wheat sector, when a costly solution rich in nitrogenous elements of the MRS type was completely replaced by the nutritional supplement as defined above.

In a particularly preferred manner, the renewable carbohydrate raw material used in the context of the invention is therefore a renewable carbohydrate raw material derived from the wheat cereal sector, in particular abraded wheat must. The renewable carbohydrate raw material used in the context of the invention may be preconditioned so as to prepare a grind. In particular, for the renewable carbohydrate raw materials derived from the cereal wheat, maize or cassava sector, a grinding stage, for example using a hammer mill, makes it possible to obtain a milling mill. in other words, flour. Exogenous nutrient solutions 5 Exogenous nutrient solutions can be added to the fermentation medium to meet the needs of lactic acid bacteria, including their need for assimilable nitrogen elements. Nutrient solutions which can be used in the context of the present invention comprise or consist of elements commonly found in MRS broth (from Man, Rogosa, Sharpe), such as yeast extracts, casein peptones, meat, potassium phosphate dibasic, sodium acid, ammonium citrate, magnesium sulphate, manganese sulphate and Tween and mixtures thereof. These can vary in concentration, to ensure varied contributions to the fermentation medium. Exogenous nutrient solutions of the MRS type can thus be conventionally added as a source of organic nitrogen at a dose corresponding to 5 g of yeast extract, 10 g of casein peptones, 10 g of meat extract, 2 g of dibasic potassium phosphate, 5 g of sodium acetate, 2 g of ammonium citrate, 0.2 g of magnesium sulphate, 0.05 g of manganese sulphate and 1 g of Tween, per liter of fermentation broth.

The exogenous nutrient solutions that may be used in the context of the invention may in particular be selected from the group consisting of solutions A, B, C, D, E, F and G, the compositions of which are described in Table 1. Table 1: Composition of the exogenous nutrient solutions A, B, C, D, E, F and G. The element concentrations are those found in the fermentation medium. Solution Solution Solution Solution Solution Solution Solution Fermentation (g / 1) ABCDEFG Yeast Extract Casein Peptones 0 0.25 0.5 1.25 2.5 3.75 5 Meat Extract Potassium Dibasic Phosphate Acetate sodium Ammonium citrate Magnesium sulphate Tween 0 0.5 1.0 2.5 5.0 7.5 10 0 0.5 1.0 2.5 5.0 7.5 10 0 0.1 0.2 0.5 1.0 1.5 2 0 0.25 0.5 1.25 2.5 3.75 5 0 0.1 0.2 0.5 1.0 1.5 2 0 0.01 0, 02 0.05 0.1 0.15 0.2 0 0.05 0.1 0.25 0.5 0.75 1 3033332 13 Manganese sulphate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 The addition of solution G in the fermentation medium will ensure a nutrient supply similar to MRS broth (excluding carbon source).

Manufacturing Process The fermentation step carried out in the lactic acid production process according to the invention is preferably a step of saccharification-simultaneous fermentation. The step of saccharification-simultaneous fermentation consists in bringing the renewable raw material, optionally liquefied and pre-saccharified, with a lactic acid bacterium in order to produce the metabolism of interest, namely lactic acid. Preferably, the characteristics of the step of saccharification-simultaneous fermentation of the process according to the invention are as follows: the medium is maintained at a constant temperature throughout the stage, preferably between 30 and 37.degree. particularly preferably at 37 ° C; maintaining the pH preferably between 5.5 and 6, for example by adding NaOH during the step or by initial addition in calcium carbonate buffer; agitation ranging from 100 to 350 rpm; - dry matter level of the medium between 10 and 30%.

The simultaneous fermentation or saccharification-fermentation step is preferably carried out for 48 to 72 hours, particularly preferably for 48 hours. The nutritional supplement used in the context of the invention advantageously makes it possible to assimilate the nitrogen present in the renewable raw carbohydrate material, which could, in the absence of this nutritional supplement, be very little and hardly assimilated by lactic acid bacteria Therefore, in the context of the invention, the first renewable carbohydrate matrix is a source of organic nitrogen for lactic acid bacteria. This source of organic nitrogen can be supplemented by other sources of "exogenous" organic nitrogen (different from the renewable carbohydrate raw material as defined in the section "Carbohydrate raw material" above) conventionally used in fermentation or saccharification-simultaneous fermentation. Such "exogenous" organic nitrogen sources are well known to those skilled in the art and typically include exogenous nutrient solutions as described in the "Exogenous Nutrient Solutions" section above. Thus, in a particular embodiment, the simultaneous fermentation or saccharification-fermentation step is carried out in the presence of an organic nitrogen source derived from a renewable carbohydrate raw material as defined in the section " Renewable carbohydrate raw material "above and, optionally, exogenous nutrient solutions, as described in the" Exogenous Nutrient Solutions "section above. Other additives conventionally used in lactic acid production processes may further be introduced into the simultaneous saccharification, fermentation or saccharification-fermentation medium in order to improve the lactic acid production yield. The nutritional supplement as defined in the section "Nutritional supplement" above is preferably introduced during the stage of simultaneous saccharification, fermentation or saccharification-fermentation The nutritional supplement as defined in the section "Nutritional supplement" above is preferably introduced during the pre-saccharification, saccharification, fermentation or saccharification-simultaneous fermentation step at a dose of 1.3 to 5 kg / t of renewable carbohydrate raw material, more preferably Preferred at a dose of 5 kg / t of renewable carbohydrate raw material. The inventors have demonstrated that the use of the nutritional supplement as defined above makes it possible to partially or totally substitute the addition of exogenous nutrient solutions as defined in the section "Exogenous nutrient solutions" above, in the processes for the production of lactic acid from renewable carbohydrate raw material derived from cereal-flavored or non-amlylated cereals or sugar. Thus, in a particular embodiment, the saccharification, fermentation or simultaneous saccharification-fermentation step is carried out in the presence of the nutritional supplement as defined in the section "Nutritional supplement" above, preferably a dose of 1.3 to 5 kg / t of renewable carbohydrate raw material, more preferably at a dose of 5 kg / t of renewable carbohydrate raw material, and an "exogenous" organic nitrogen source, preferably a solution exogenous nutrient as defined in the section "Exogenous nutrient solutions" above, at a dose corresponding to intakes in the fermentation broth of 1 to 1.25 35 g of yeast extract, 2 to 2.5 g of casein peptones, 2 to 2.5 g of meat extract, 0.4 to 0.5 g of dibasic potassium phosphate, 1 to 1.25 g of sodium acetate, 0.4 to 0.5 g of Ammonium citrate, 0.04 to 0.05 g of magnesium sulphate, 0.05 g of sulphate, manganese fats and 0.2 to 0.25 g Tween per liter of sheep. The inventors have also demonstrated that the same yield of lactic acid can be obtained from a wheat-derived renewable raw material, when expensive, nitrogen-rich, exogenous nutrient solutions are completely replaced by wheat. nutritional supplement as defined above. Thus, in a particular embodiment, the step of saccharification, fermentation or saccharification-simultaneous fermentation is carried out in the presence of the nutritional supplement as defined in the section "Nutritional supplement!" Above, preferably at a dose of 1.3 to 5 kg / t of renewable carbohydrate raw material, more preferably at a dose of 5 kg / t of renewable carbohydrate raw material, and in the absence of an "exogenous" organic nitrogen source, particularly in the absence of the exogenous nutrient solutions as defined in the "Exogenous nutrient solutions" section above, containing assimilable nitrogen source elements. lactic acid according to the invention may comprise a step of liquefying the renewable carbohydrate raw material before the simultaneous saccharification, fermentation or saccharification-fermentation step A liquefaction step may in particular be carried out when the carbohydrate raw material A starch mill is a starch mill, and the starch mill liquefaction stage makes it possible to hydrolyze the starch into dextrins. It is advisable to mix the milling with water, acid and liquefying enzymes, so as to form a liquefied paste. Preferably, the grind is incorporated in the water so as to obtain a mixture, preferably homogeneous, having a solids content of between 20 and 30%. Preferably, the thus-formed mash is stirred between 250 and 350 rpm. Depending on the liquefaction enzyme used, the pH may be adjusted with a solution of acid, for example sulfuric acid. The pH can conventionally be adjusted between 5 and 6.5. Depending on the enzyme used, a calcium salt may also be added. The liquefaction is preferably carried out by applying the temperature kinetics shown in FIG. 1. This kinetics typically includes a temperature rise phase of preferably 15 minutes, which makes it possible to reach a temperature of 55 ° C., a step of duration. for example 30 min at 55 ° C, ure second phase of temperature rise preferably 15 min to reach a temperature of 85 ° C, a second step of a duration for example of 2 h at 85 ° C and a cooling phase of the liquefied paste at ambient temperature. The 55 ° C. plateau typically corresponds to the pre-liquefaction phase and the 85 ° C plateau typically corresponds to the liquefaction phase. The liquefaction enzymes used preferably include an alpha-amylase, such as Lyvanol Liquide Plus® alpha-amylase marketed by Lyven, and possibly a hemicellulolytic complex, such as Lyvanol Devisco Plus® sold by Lyven. The alpha-amylase is typically added between 0.17 and 0.36 kg / t dry matter and the gutting hemicellulolytic complex is typically added between 0.17 and 0.36 kg / t dry matter. Liquefaction enzymes are preferably added after the first temperature rise phase at 55 ° C. Preferably, at the end of the liquefaction stage, the liquefied pasteurized mull has a dry matter content of about 20-25%, preferably a pH of between 5.5 and 6, and preferably viscosity of the order of 50 mPa.s-1, the viscosity being typically measured using a rotational rheometer, for example model Kinexus Pro® 15 marketed by Malvern, at a temperature of 35 ° C, the viscosity preferably being measured at 180 rpm for 5 min, after a first homogenization phase of the product with a stirring speed at 960 rpm for 15 s, the value taken into account being typically the average of the last 30 seconds of measurement. The process for producing lactic acid according to the invention may further comprise a step of pre-saccharification of the liquefied products. This step makes it possible to hydrolyze the dextrins into fermentable sugars. During this step, the liquefied beetle is placed in the presence of a glucoamylase, such as the Lyvanol GA 480® glucoamylase marketed by Lyven. Glucoamylase is typically added between 0.89 and 1.45 kg / t dry matter. Other enzymes may be further used in this pre-saccharification step according to the renewable carbohydrate raw material used. Thus, invertase can be used to hydrolyze sucrose to fructose and glucose. Typically, sugarcane and beet molasses can be placed in the presence of an invertase, such as the Inverlyve P® marketed by Lyven. Invertase is typically added at 50 g / t feedstock. Preferably, during the pre-saccharification step the liquefied meal is stirred at 250 to 350 rpm. The pH can also be adjusted with a solution of acid, for example sulfuric acid. The pH can conventionally be adjusted between 4 and 5, preferably between 4.4 and 4.6. The pre-saccharification is preferably carried out by heating at a temperature of from 50 to 60 ° C, preferably at 55 ° C, for less than 24 hours, preferably for 1 hour.

When the method for manufacturing lactic acid according to the invention comprises a step of pre-saccharification, the nutritional supplement as defined in the section "Nutritional supplement" above is preferably added at the beginning of this step. Also, the exogenous organic nitrogen additives and sources defined in the "Exogenous nutrient solutions" section above may be added during the pre-saccharification step, but the nutritional supplement may also be introduced during the pre-saccharification step. saccharification, fermentation or saccharification-fermentation step After the simultaneous fermentation or saccharification-fermentation step, the lactic acid produced can be separated from the fermentation or saccharification-simultaneous fermentation medium by any known conventional technique. for separating lactic acid from aqueous solutions The particles of raw material or bacteria Lactics can be eliminated with separation to increase the efficiency of the separation. The separation may be carried out for example by centrifugation, filtration, flocculation, flotation or membrane filtration. After separation of the fermentation medium or simultaneous saccharification-fermentation, the lactic acid produced by the process according to the invention may also be subjected to one or more purification steps such as extraction, distillation, crystallization, filtration, etc. .

The inventors have demonstrated that the use of the nutritional supplement as defined above during the lactic acid production process makes it possible to reduce the presence of free amino nitrogen (FAN) at the end of fermentation or saccharification-simultaneous fermentation. . However, the presence of FAN at the end of fermentation can cause problems during the subsequent steps of extraction and purification of lactic acid.

Therefore, the use of the nutritional supplement as defined in the section "Nutritional supplement" above advantageously makes it possible to increase the performance of the subsequent lactic acid extraction and purification steps. The inventors have furthermore demonstrated that the increase in the production of lactic acid observed during the use of the nutritional supplement as defined above was linked to the improvement of the nitrogen source available in the fermentation broth. For the lactic bacterium, the performance of the nutritional supplement according to the invention can therefore be related to its ability to release soluble nitrogen in the fermentation medium or simultaneous saccharification-fermentation.

The present invention therefore also relates to the use of the nutritional supplement as defined in the section "Nutritional supplement" above, for releasing, in a lactic acid fermentation medium, a renewable carbohydrate raw material, as defined. above, the nitrogen of said raw material, during manufacture, by fermentation, preferably in liquid medium, of lactic acid from said raw material Composition and kit The inventors have demonstrated that adding to the fermentation broth a combination consisting of the nutritional supplement according to the invention and a reduced amount of an exogenous organic nitrogen source, in particular an expensive exogenous organic nitrogen source such as exogenous nutrient solutions defined in the "Exogenous Nutrient Solutions" section above, it was possible to obtain the same lactic acid production performance as in It is only a significant amount of expensive exogenous organic nitrogen source. The present invention thus also relates to a composition comprising: (i) a source of organic nitrogen selected from the group consisting of renewable carbohydrate raw materials as defined in the section "Renewable carbohydrate raw material" above, exogenous nutrient solutions as defined in the section "Exogenous nutrient solutions" and mixtures thereof, (ii) a nutritional supplement as defined in the "Nutritional supplement" section above The subject of the present invention is also a kit comprising: (I) a source of organic nitrogen selected from the group consisting of renewable carbohydrate raw materials as defined in the "Renewable Carbohydrate Raw Material" section above, exogenous nutrient solutions as defined in the section "Exogenous Nutrient Solutions" ", and mixtures thereof, (ii) a nutritional supplement as defined in the section "Nutritional supplement" above, and (iii) optionally a package. The inventors have in particular demonstrated that by adding to the fermentation broth a combination consisting of the nutritional supplement according to the invention and the exogenous nutrient solution B as defined in the section "Exogenous nutrient solutions", it was possible to obtain the same performance of lactic acid production as using the exogenous nutrient solution G alone (corresponding to a nitrogen source intake similar to that of a culture broth type MRS). Thus, in a preferred embodiment, the organic nitrogen source (i) of the composition according to the invention or of the kit according to the invention corresponds to a contribution in the 5 ml of fermentation of 0.25 to 2.5 g / I of yeast extract, 0.5 to 5 g / I of casein peptones, 0.5 to 5 g / I of meat extract, 0.1 to 1 g / I of dibasic potassium phosphate, 0 25 to 2.5 g / I sodium acetate, 0.1 to 1 g / I ammonium citrate, 0.01 to 0.1 g / I magnesium sulfate, 0.05 g / I of manganese sulfate, and 0.05 to 0.5 g / I Tween 80. In a particularly preferred embodiment, the organic nitrogen source 10 (i) of the composition according to the invention or the kit according to The invention corresponds to a feed in the fermentation broth of 0.25 g / l of yeast extract, 0.5 g / l of casein peptones, 0.5 g / l of meat extract, 0.1 g / I potassium dibasic phosphate, 0.25 g / I sodium acetate, 0.1 g / I ammonium citrate, 0.01 g / I magnesium sulfate, 0.05 g / I of knew Manganese acetate, and 0.05 g / I Tween 80.

The present invention will be further illustrated by the figures and examples below.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 represents the temperature kinetics preferably applied to the liquefaction stage of the renewable carbohydrate raw material, in particular to the pasted wheat flour. A: addition of liquefaction enzymes; B: first temperature level at 55 ° C corresponding to the pre-liquefaction; C: second temperature level at 85 ° C corresponding to the liquefaction of the mout. Figure 2 shows the granulometric profile of an abraded wheat mill obtained after grinding with a hammer on a 0.8 mm grid.

3 represents the impact of the addition of exogenous nutrient solutions or of the nutritional supplement according to the invention on the lactic yields obtained in a vial after 48 hours of fermentation of an abraded wheat mill (A), of molasses. of sugarcane (B), maize (C) or cassava (D) by the strain Lactobacillus coryniformis ssp. Torquens 35 DSM 20005 inoculated at 108 CFU / ml in Example 2.

FIG. 4 represents the impact of the addition of exogenous nutrient solutions or of the nutritional supplement according to the invention on the lactic yields obtained in a vial after 48 hours of fermentation of a lamb consisting of a liquefied abraded wheat. saccharified (13% MS), by one of the 4 lactic acid strains tested, namely the strain Lactobacillus coryniformis 5 ssp. torquens DSM 20004 (A), Lactobacillus coryniformis ssp. torquens 20005 (B), Lactobacillus mindensis LMG 21932 (C), or Lactobacillus zeae LMG 17315 (D), inoculated at 107 CFU / ml in Example 3. Figure 5 shows the impact of the strain used for the production of nutritional supplement (CN) according to the invention on the lactic yields obtained in flask after 72 hours of fermentation of a liquefied wheat flour, saccharified and supplemented with the nutrient solution B, with L. coryniformis DSM 20004 inoculated with 1 x107 CFU / ml in example 5. Dotted band: zone of yields obtained with a millet supplemented with the solution G.

FIG. 6 represents the positioning of each nutritional supplement (CN) tested in Example 5 as a function of the final concentrations of FAN and lactic acid (after 72 hours of fermentation of a liquefied, saccharified wheat flour and adding the solution B, by L. coryniformis DSM 20004 inoculated at 1 × 10 7 CFU / ml). The plotted curve corresponds to the linear regression.

FIG. 7 represents the positioning of each nutritional supplement tested in Example 5 as a function of the FAN coefficients per kg of nutritional supplement and of the lactic yields obtained after 72 hours of fermentation of a liquefied, saccharified wheat flour and adding the solution B, by L. coryniformis DSM 20004 inoculated at 1x 107 25 CFU / ml. The plotted curve corresponds to the linear regression. FIG. 8 represents the impact of the type of nutritional supplement used on the lactic yields obtained in the reactor after the 50 h of fermentation of a saccharified liquefied wheat flour supplemented with the nutrient solution B, by L. coryniformis DSM 20004 Or DSM 20005 inoculated at 109 CFU / ml, in example 6. FIG. 9 represents the relative distribution of free amino acids (AAL) released by the nutritional supplements according to the invention as a function of the genera and species of the strains placed in The AAL profile is determined after 72 hours of incubation at 30 ° C. of a liquefied and saccharified wheat mill with 13% DM, adjusted to pH 5.5, in the presence of 30% of the alcohol. sodium azide and a solubilized nutritional supplement and filtered at 0.2 pm, incorporated at a dose of 5 kg / t of renewable carbohydrate raw material. Figure 10 represents the positioning of each tested nutritional supplement as a function of the final concentrations of FAN or AAL and lactic acid, after 50 h of fermentation of a saccharified liquefied wheat broth supplemented with the solution B, by the L. coryniformis strain. DSM 20004 or DSM 20005 inoculated at 109 CFU / ml, in Example 6. Figure 11 shows the comparison of commercial enzymes (types, doses) and the nutritional supplement (CN) according to the invention on the lactic yields obtained. in a reactor at the end of 48 hours of fermentation of a saccharified liquefied wheat broth supplemented with solution B, by L. coryniformis DSM 20005 inoculated at 109 CFU / ml, in example 6. FIG. free amino acids (AAL) released by the enzymes Fermgen, Prolyve BS concentrated and the nutritional supplement according to the invention produced with the strain 029 on rapeseed cake, in example 6. The AAL profile is determined born after 72 hours of incubation at 37 ° C. liquefied wheat and saccharified to 13% DM, adjusted to pH 5.5, in the presence of sodium azide and a solubilized and filtered 0.2 pm, incorporated in the dose of 5 kg / t of renewable carbohydrate raw material. Figure 13 represents the impact of the type of nutritional supplement used in the presence of nutrient solution (A or B) on the lactic acid bacteria population (discontinuous traits) and lactic acid production (continuous traits) during the 50 h fermentation of liquefied and saccharified wheat must at 20% MS by L. coryniformis DSM 20005 inoculated at 109 CFU / ml, as described in Example 7.

EXAMPLES EXAMPLE 1 Manufacture of Nutritional Supplement This example describes the production of the nutritional supplement used in the context of the invention. The method of manufacturing the nutritional supplement by solid fermentation comprises the following different stages: the cereal co-products (wheat bran, rapeseed meal, etc.) used as raw material are conditioned and pretreated. In parallel, the molds or bacteria that will be used for fermentation are sporulated or cultured for inoculation. These molds or bacteria are then brought into contact with the pretreated cereal co-products in a fermentation step in a solid medium. The product resulting from the fermentation can then be treated or not, for example be ground or extracted.

In this example, the inventors used as a cereal co-product a rapeseed meal and as a mold Aspergillus flavus strain. The nutrient medium was thus constituted by rapeseed cake in flour pre-moistened with 60% dry matter and autoclaved for 35 min at 105 ° C. After cooling, the medium was inoculated with a spore solution of Aspergillus flavus in order to obtain a concentration of 1 × 10 7 spores per gram of dry matter and an initial humidity of 45%. The pH was adjusted to 4.9 by addition of sulfuric acid. The culture medium thus obtained was divided into flasks of Erlenmeyer flask with 10 g of dry matter per flask. The Erlenmeyer flasks were then incubated at 33 ° C under aerobic conditions in the dark for 48 hours without shaking. Moisture remained between 40 and 60% during the fermentation. Aeration allowed the supply of oxygen necessary for fermentation and avoided the accumulation of carbon dioxide produced by the fermentation. The fermentation was stopped at the appearance of the first spores in the culture medium. The mixture obtained at the end of fermentation can be dried and used as such, that is to say in raw form, in the lactic acid production process or be extracted in 0.1 M sodium acetate buffer. (ratio 10% w / v nutritional supplement / buffer) then filtered to 0.2 lm to be added in liquid form.

EXAMPLE 2 Effect of Nutritional Supplement on Different Raw Materials Used to Produce Lactic Acid This example shows that the nutritional supplement according to the invention can be used with many renewable carbohydrate raw materials for making acid. lactic. The inventors have studied in a comparative way the quantity of lactic acid produced during a lactic acid production process implementing a step of saccharification-simultaneous fermentation (SSF) of different renewable raw materials (wheat, maize, cassava, sugar cane) by introducing either an exogenous nutrient solution more or less concentrated in elements such as extracts of meat, yeast and casein peptones, or the nutritional supplement obtained in example 1. Materials and methods 15 Description general of the lactic acid production process The lactic acid production method used comprises the following steps: the renewable raw material (wheat, corn, etc.) is packaged and then liquefied before being subjected to a stage pre-saccharification. In parallel, the lactic acid bacteria used for lactic fermentation are pre-fermented in a pre-fermenter. This lactic acid bacterium is then contacted with the raw material in a SSF step. The lactic acid produced during this SSF step can then be extracted and purified. Conditioning of raw material The raw material was conditioned to obtain a grind. For wheat, maize and cassava, a milling step resulted in milling, ie flour. For wheat, the abraded wheat grains were ground using a hammer mill (Electra). The size of the grid used was set at 0.8 mm. The grind obtained has a moisture content of 10% (± 2%) and a grain size profile as represented in FIG. 2. According to the same principle, corn mills were obtained by hammer milling. In the form of flours, these raw materials are stored away from light in a dry place at room temperature.

Liquefaction stage The liquefaction stage of the generated millings makes it possible to hydrolyze the starch into dextrins. The principle is to mix the wheat flour (for example) with water, acid and liquefying enzymes, so as to form a liquefied paste paste.

The pasted meal was prepared by incorporating the flour into the water so as to obtain a homogeneous mixture having a solids content of about 20 to 30%. This well thus constituted was distributed in reactor and agitated between 250 and 350 rpm. The temperature kinetics applied to wheat wheat is presented in Figure 1.

At the beginning of the rise in temperature, the pH of the sheep was adjusted to 5.4 (by addition of H2SO4) in order to optimize the work of the liquefaction enzymes added after reaching 55 ° C. Two liquefaction enzymes were used, one corresponding to a deviscosante (Lyvanol Devisco Plus® - LYVEN) added to 0.24 kg / t of dry matter, the other corresponding to an alpha-amylase (Lyvanol Liquide Plus® - LYVEN) also added to 0.24 kg / t dry matter. After 30 minutes pre-liquefaction at 55 ° C, the mice were liquefied at 85 ° C for 2 h in an add-on reactor. At the end of the liquefaction, the roast was cooled to room temperature. Wheat and corn feeds had a solids content of 13% and 30% respectively, a pH of 5.5-6 and a viscosity of about 50 mPa.s-1.

Pre-saccharification step The pre-saccharification step of the liquefied meats hydrolyses the dextrins into fermentable sugars (glucose, maltose, maltotriose). An amyloglucosidase enzyme (Lyvanol GA 480®-LYVEN) was used at 0.890 kg / t dry matter: the liquefied meats of wheat and maize were divided into 250 ml baffled flasks agitated between 250 and 350 ml. rpm, each containing 100 ml of sheep, supplemented with Lyvanol GA 480® at 0.890 kg / t of dry matter and CaCO3 (at 30 g / l of sheep). An exogenous nutrient solution, rich in nitrogenous elements, or the nutritional supplement obtained according to Example 1, have also been added where appropriate. The pH was adjusted to acidic pH (around 4.4 and 4.6) and the flasks were held at a temperature of 50 ° C for 2 h. Stage of propagation of the lactic bacteria At the end of the 2 hours of pre-saccharification, the mouts were inoculated with a lactic bacterium. The lactic acid bacterium used is a bacterium of the genus Lactobacillus, a producer of D-lactic acid, the strain Lactobacillus coryniformis ssp. torquens DSM 20005.

This strain was introduced into simultaneous saccharification-fermentation medium (SSF) after being cultured in rich broth medium Man, Rogosa and Sharpe (MRS). This propagation step allows a development of the sufficient strain necessary for the inoculation of SSF medium at 108 CFU / ml of sheep. The propagation conditions were as follows: stirring at 100 rpm, temperature set at 37 ° C. and keeping the pH at 6 by adding NaOH. Saccharification Step-Simultaneous Fermentation The SSF step involves bringing a pre-saccharified mice with a lactic acid bacterium together to produce the metabolism of interest: lactic acid. To do this, the mouts were inoculated with 108 CFU of lactic bacteria obtained in the propagation step per ml of sheep. The mice were stirred at 100 rpm. The temperature was kept constant throughout this step at 37 ° C. Maintaining pH between 5.5 and 6 was ensured by the initial addition of calcium carbonate buffer. The fermentation lasted 48 hours. Fermentation Mets Fermentation meats are composed of one of the liquefied, or saccharified, renewable carbohydrate materials, together with one of the exogenous nutrient solutions defined in Table 2, or the nutritional supplement according to the invention: - Mout + A: carbohydrate renewable material with addition of the solution A - Mout + D: renewable carbohydrate material with addition of the solution D - Mout + E: renewable carbohydrate material with addition of the solution E 25 - Mout + F: renewable carbohydrate material with addition of the solution F - Mout + G: renewable carbohydrate material with addition of the solution G - Mout + A + nutritional supplement: renewable carbohydrate material with addition of solution A and the nutritional supplement obtained from a rapeseed cake ( TC) fermented by the Aspergillus flavus strain, following the method described in Example 1. Table 2: Composition of exogenous nutrient solutions Example 2 Concentration in g / l of sheep Solution A Solution D Solution E Solution F Solution G Yeast extract 0 1.25 2.5 3.75 5 Casein peptones 0 2.5 5 7.5 Meat Extract 0 2.5 5 7.5 10 3033332 26 Concentration in g / l of sheep Solution A Solution D Solution E Solution F Solution G Potassium phosphate dibasic 0 0.5 1 1.5 2 Sodium acetate 0 1, 25 2.5 3.75 5 Ammonium citrate 0 0.5 1 1.5 2 Magnesium sulphate 0 0.05 0.1 0.15 0.2 Tween 0 0.25 0.5 0.75 1 Sulfate manganese 0.05 0.05 0.05 0.05 0.05 Potassium glucose potential: between 100 and 150 g / I of sheep (variable depending on the raw material). Contribution of the nutritional supplement in the fermentation mash between 1.3 and 5 kg / t of raw material); Initial pH of the mouts: between 5.5 and 6.

Results As shown in Figure 3, the production of lactic acid is improved by the addition of exogenous nutrient solutions more or less concentrated in nitrogen elements. The yield gain is variable according to the renewable carbohydrate material used.

On abraded wheat, the maximum yield in flasks is reached with the addition of solution G (0.64 g / g of glucose), or with a nitrogen element supply for the lactic strain similar to that which it may have. in a culture broth type MRS. On sugar cane molasses, the lactic strain is unable to produce lactic acid without the addition of a nutrient solution rich in nitrogen elements. The maximum yield of flasks of 0.415 g / g of glucose is obtained with an addition of the solution F. On corn, the addition of the solution D makes it possible to improve the production yield of lactic acid in flasks. a factor of 5 (0.28 g / g against 0.05 g / g on M + A). The addition of more concentrated nutrient solutions increases the yield to a maximum of 0.36 g / g in vials. Finally, on cassava, the maximum yield in flasks can be reached with the addition of the solution D (0.33 g / g of glucose), or with a contribution in nitrogenous elements for the lactic strain similar to that which it could have in a culture broth type MRS diluted to a quarter. The addition of the nutritional supplement according to the invention allows, as well as nutrient solutions, but at lower costs, to improve the production yields of lactic acid by the strain Lactobacillus coryniformis ssp. Torquens DSM 20005. Its effect is also variable depending on the renewable carbohydrate material used. Thus, on abraded wheat, the nutritional supplement can totally replace the addition of so-called expensive nutrient solutions (eg solution G) and guarantee the maintenance of the lactic yield. On other renewable carbohydrate materials, the nutritional supplement may partly replace the addition of nutrient solutions. On sugars and maize molasses, the nutritional supplement makes it possible to achieve the same yield as with the addition of the solution D. By allowing a better assimilation of the nitrogen potential of the carbohydrate 3033332 27 renewable materials by the strain, the complement can substitute a costly input of yeast extract, meat extract and peptones of 1.25, 2.5 and 2.5 g / I respectively. On cassava, the nutritional supplement makes it possible to improve the lactic efficiency of 30% compared to the only one.

Example 3: Effect of Nutritional Supplement on Different Strains of Lactic Acid Bacteria Used to Produce Lactic Acid This example shows that the nutritional supplement can be used with various types of lactic acid bacteria conventionally used to produce lactic acid. The inventors have comparatively investigated the amount of lactic acid produced in the wheat sheep SSF by different lactic acid bacteria producing D- and / or L-lactate strains by introducing either an exogenous nutrient solution. more or less concentrated in elements such as extracts of meat, yeast and casein peptones, the nutritional supplement obtained in Example 1, or a mixture of both. Materials and Methods In these tests, the inventors first carried out a liquefaction of abraded wheat flours for 2 hours at 85 ° C. in a stirred reactant having a final solids content of 13%. The liquefied wheat fl ate was divided into stirred flasks of 250 ml, each containing 100 ml of must, supplemented with Lyvanol GA 480® (amyloglucosidase at 0.890 kg / t dry matter), and CaCO 3 (at 30 g / l). of mutton). Fermentation Mets The fermentation meats are composed of liquefied abraded wheat, supplemented with one of the exogenous nutrient solutions defined in Table 3 or the nutritional supplement 30 according to the invention, or both: Mout + A: liquefied abraded wheat with addition of the A Mout + C solution: liquefied abraded wheat with the addition of the C Mout + D solution: liquefied abraded wheat with the addition of the D Mout + E solution: liquefied abraded wheat with the addition of the E 35 Mout + G solution: abraded wheat liquefied with addition of G 3033332 solution 28 Mout + A + nutritional supplement: liquefied abraded wheat with addition of solution A and the nutritional supplement obtained from rapeseed meal (TC) fermented by the Aspergillus flavus strain, according to process described in Example 1 Mout + B + nutritional supplement: liquefied abraded wheat with addition of solution B 5 and the nutritional supplement obtained from fermented rapeseed meal (TC) by the Aspergillus flavus strain, according to the method described in Example 1. TABLE 3 Composition of the exogenous nutrient solutions used in Example 3 Concentration in g / l of sheep Solution Solution Solution Solution Solution ABCDEG Extract Yeast 0 0.25 0.5 1.25 2.5 5 Casein peptones 0 0.5 1 0 2.5 5 10 Meat extract 0 0.5 1.0 2.5 5 10 Potassium dibasic phosphate 0 0, 1 0.2 0.5 1 2 Sodium acetate 0 0.25 0.5 1.25 2.5 5 Citrate 0 0.1 0.2 0.5 1 2 ammonium Magnesium sulphate 0 0.01 0 , 02 0.05 0.1 0.2 Tween 0 0.05 0.1 0.25 0.5 1 Manganese sulphate 0.05 0.05 0.05 0.05 0.05 0.05 10 L Nutritional supplement intake is 5 kg / t of raw material. Initial pH of the sheep: between 5.5 and 6. The vials thus formed were incubated at 50 ° C for 2 h. These latter have a glucose potential of 100 g / l of sheep. At the end of these 2 hours pre-saccharification, mouts were inoculated with 107 CFU of lactic bacteria per ml of sheep. The bacterial strains used in this example are: Lactobacillus mindensis LMG 21932 (L & D-lactate producer) Lactobacillus zeae LMG 17315 (L-lactate producer) Lactobacillus coryniformis ssp. Torquens DSM 20004 (D-lactate producer) Lactobacillus coryniformis ssp. Torquens DSM 20005 (D-lactate producer) The fermentation was conducted for 48 hours at 37 ° C under agitation (100 rpm).

Results As shown in FIG. 4, lactic acid production is improved by the addition of the nutritional supplement, irrespective of the strain used. The lactic yields obtained with Lactobacillus coryniformis ssp. 5 torquens DSM 20004 and DSM 20005, initially inoculated at 1x107 CFU / ml in the fermentation broth (condition mout + A), do not exceed 5%. By adding the nutritional supplement to 5 kg / t of wheat, accessibility to the nitrogen source intrinsic to the raw material used is improved, which increases the lactic yields. Thus, the yields in flasks reach 55% with the strain DSM 20004, 45% with the DSM 2000 strain. Compared with the yields obtained with the addition of nutritive solutions (solutions C, G), the nutritional supplement can, in part or totally, substitute for these expensive inputs (including yeast extracts, peptones, meat extracts, ...). For strain DSM 20005, the lactic yield obtained with the addition of the nutrient solution richest in nitrogenous elements (solution G) can be exceeded by combining the nutritional supplement with a nutrient solution whose concentration of nitrogenous elements is reduced by 95%. (solution B). Thus, the quantities and therefore the costs of inputs can be reduced by the implementation of the nutritional supplement. For the Lactobacillus mindensis LMG 21932 and Lactobacillus zeae LMG 17315 strains, the lactic yields after 48 hours of saccharified liquefied sugar fermentation of abraded wheat are rather low with 6.6% and 17%, respectively. The addition of nutrient solutions rich in nitrogenous elements makes it possible to improve these yields (here solutions C, D and E). To reach the 50% yield in flasks, it is necessary to provide a nitrogen source corresponding to solution D for strain LMG 21932, corresponding to solution C for strain LMG 17315. By its action on the raw material used, The addition of the nutritional supplement makes it possible to reduce the exogenous nutrient supply necessary for the strain while maintaining the yields at 50%. In combination with the nutritional supplement, the addition of a less expensive nitrogen source, corresponding in this example to solution B, can then be sufficient to meet the needs of lactic acid strains.

EXAMPLE 4 Effect of Nutritional Supplement in Lactic Acid Production Process in Batch Type Reactor This example shows that the nutritional supplement can be used effectively in conducting different lactic acid production processes. scales.

The inventors have compared in a comparative manner the amount of lactic acid produced during wheat sheep SSF by L. coryniformis strain DSM 20004, by introducing either an exogenous nutrient solution concentrated in such elements as meat extracts. yeast and casein peptones, the nutritional supplement obtained in Example 1, and by varying the fermentation line, namely either a fermentation line in a flask for which pH control is not optimal, or a batch reactor pipe with continuous pH maintenance. Materials and Methods In these tests, the inventors first carried out a liquefaction of abraded wheat bran for 2 hours at 85 ° C. in a stirred reactor having a final solids content of 13% for the vial condition of 20%. % for the batch type reactor condition. The liquefied wheat flour 13% dry matter (DM) was divided into stirred flasks of 250 ml, each containing 100 ml of wort, supplemented with Lyvanol GA 480® (amyloglucosidase at 0.890 kg / t dry matter) and CaCO3 (at 30 g / I of sheep). The liquified lamb wheat 20% MS was divided into reactors, each containing 3000 ml of must, supplemented with Lyvanol GA 480® (amyloglucosidase at 0.890 Kg / t DM) and put under control of the pH by the addition of acid. 5 N sulfuric acid and 7 N sodium hydroxide for a pH keeping set point of 6 throughout the fermentation.

Fermentation meats The fermentation meats are composed of liquefied abraded wheat, added with one of the exogenous nutrient solutions defined in Table 4 or the nutritional supplement according to the invention: Mout + G: liquefied abraded wheat with addition of the solution G Mout + A + nutritional supplement: liquefied abraded wheat with addition of solution A and the nutritional supplement obtained from rapeseed meal (TC) fermented by the Aspergillus flavus strain, according to the process described in Example 1, for a concentration of 5 kg / t wheat.

TABLE 4 Composition of exogenous nutrient solutions used in Example 4 Concentration in g / I of sheep Solution A Solution G Yeast extract 0 5 Casein peptones 0 10 Meat extract 0 10 Potassium dibasic phosphate 0 2 Sodium acetate 0 5 Ammonium citrate 0 2 Magnesium sulphate 0 0.2 Tween 0 1 Manganese sulphate 0.05 0.05 initial pH of the sheep: between 5.5 and 6. The vials thus formed were incubated at 50 ° C for 2 hours. The latter have a glucose potential of 100 g / l of sheep. At the end of these 2 hours of pre-saccharification, the mules were inoculated with at least 108 CFU of lactic bacteria per ml of sheep. The bacterial strain used in this example is Lactobacillus coryniformis ssp. Torquens DSM 20004. The fermentation was conducted for 48 h at 37 ° C with shaking (100 rpm).

In the reactor, the pre-saccharification was carried out at 60 ° C for 1 hour. The glucose potential is 135-145 g / I of sheep. Mouts were inoculated with at least 108 CFU of lactic bacteria per ml of sheep. The bacterial strain used in this example is Lactobacillus coryniformis ssp. Torquens DSM 20004. The fermentation was conducted for 48 h at 30 ° C with stirring (300 rpm).

Results As shown in Table 5, the scale-up of the flask with the batch reactor makes it possible to improve the performance of the strain: the best control of the fermentation parameters (temperature / agitation) as well as the continuous pH regulation makes it possible to increase the lactic acid production yields by the strain L. coryniformis DSM 20004, whatever the origin of the source of nitrogenous elements available for the strain (exogenous with the solution). nutritive G or intrinsic under the action of the nutritional supplement). The yield gain observed in scale-up for the M1 condition supplemented with the nutrient solution G is 18% (yield improvement from 78.8% to 93.4%). This gain is 71% for the condition mout supplemented with the nutritional supplement.

Table 5: Lactic yields obtained in a vial and in a batch reactor after 48 hours of fermentation of a saccharified wheat lard of abraded wheat, with Lactobacillus coryniformis DSM 20004 inoculated at 1 × 10 8 CFU / ml Lactic yield in% Gain of yield at (on glucose potential) scale-up (%) Pipeline driving in batch reactor reactor Mout + G 78.8 93.4 18.5 Mout + A + nutritional supplement 42.8 73.3 71.3 5 The change of scale, through a better control of the parameters of saccharification and fermentation, amplifies the impact of nutritional supplement on lactic yield.

EXAMPLE 5 Effect of Nutritional Supplements Produced with Different Substrates and Different Molds and Bacteria This example shows that the nutritional supplement can be obtained by means of different substrates and different molds and bacteria.

The inventors have comparatively investigated the amount of lactic acid produced in the wheat sheep SSF by L. coryniformis strain DSM 20004, by introducing either an exogenous nutrient solution concentrated in such elements as meat extracts, of yeast and peptones of casein, the nutritional supplement according to the invention, by varying the strain and the substrate used for the manufacture of the nutritional supplement. Materials and Methods In these tests, the inventors first carried out liquefaction of abraded wheat bran for 2 hours at 85 ° C. in a stirred reactor having a final solids content (DM) of 13%. The liquefied wheat flask was divided into stirred flasks of 250 ml, each containing 100 ml of must, supplemented with Lyvanol GA 480® (amyloglucosidase at 0.890 kg / t DM) and CaCO3 (at 30 g / l of sheep). .

Fermentation Muts The fermentation meats are composed of liquefied abraded wheat supplemented with the nutrient solution B with or without the addition of one of the nutritional supplements according to the invention listed in Table 6.

Table 6: List of various nutritional supplements (CN) used in fermentation at a value of 5 kg / t of wheat Initial pH of the sheep: between 5.5 and 6. The strain of Aspergillus oryzae 044 is the strain of Aspergillus oryzae ATCC 12892.

The vials thus formed were incubated at 50 ° C for 2 h. These latter have a glucose potential of 100 g / l of sheep. At the end of these 2 hours of pre-saccharification, the mouts were inoculated with 107 CFU of lactic bacteria per ml of sheep. The bacterial strain used in this example is Lactobacillus coryniformis ssp. Torquens DSM 20004. The fermentation was conducted for 72 h at 30 ° C with shaking (100 rpm). Results Figure 5 illustrates that nutritional supplements produced by solid-state fermentation (FMS) from different substrates and different strains can increase the lactic yield of a fermentation in a liquid medium by a strain of the genus Lactobacillus by compared to the condition Mout + solution B. The nutritional supplements obtained by FMS make it possible to increase the lactic yield of the fermentation by a factor of 1.6 to 2 compared to the simple addition of a nutritive solution B (corresponding to others to an addition in yeast extract of 0.25 g / I of sheep, in meat extract of 0.5 g / I of sheep and in peptones of 0.5 g / I of sheep). Strain Genus, species Codification Substrate Aspergillus flavus 0248 Aspergillus flavus 029 Aspergillus flavus 046 Aspergillus oryzae 030 Aspergillus oryzae 044 Aspergillus oryzae CL228 Aspergillus niger 043 Aspergillus 027 tubigensis Bacillus subtilis CL260 Bacillus licheniformis 0B62 Rapeseed meal Rapeseed meal Wheat bran and barley rootlets Rapeseed meal Rapeseed meal Rapeseed meal Rapeseed meal Water soluble rape ethanol Wheat flour Rapeseed meal CN 1 CN 2 CN 3 CN 4 CN 5 CN 6 CN 7 CN 8 CN 9 CN 10 3033332 34 This result shows that it is possible to produce effective nutritional supplements for the production of lactic acid from different types of microorganisms, sporulating bacteria such as Bacillus sp. or fungi such as Aspergillus sp.

Moreover, as shown in FIG. 6, the concentrations of FAN (Free Amino Nitrogen) present at the end of fermentation in the mues, representative of the soluble nitrogen (amino acids and small peptides), can be related to those of the lactic acid produced. From the linear regression plotted with all of the nutritional supplements produced by FMS tested here, the increase in lactic acid production, made possible by the action of nutritional supplements, is related to the improvement of availability of the intrinsic nitrogen source to the fermentation broth for the lactic acid bacterium (r2 = 88.5%). The performance of simultaneous saccharification-fermentation (SSF) nutritional supplements can therefore be related to their ability to release soluble nitrogen into the fermentation medium. Thus, FIG. 7 shows the link which exists between the capacity of the nutritional supplement to release FAN in the fermentation mole (in the absence of the lactic strain), otherwise called FAN coefficient (determined after 24 h of incubation at 35 ° C. a liquefied wheat flour saccharified at 13% DM, adjusted to pH 5.5, in the presence of sodium azide, for a solubilized nutritional supplement incorporation and filtered at 0.2 lm of 5 kg / t of raw material), and the lactic yields obtained in SSF with the strain L. coryniformis DSM 20004 under the action of the same nutritional supplement (r2 = 70.5%). By analyzing the performance of the nutritional supplements tested (FIG. 7), the inventors were able to form three distinct groups, under the conditions tested: a first group represented by the dashed circle comprising the four most basic nutritional supplements, a second one group represented by the dashed circle made up of 4 nutritional supplements with more advantageous performances, and a third group represented by the solid circle with the 2 most powerful nutritional supplements. The "solid line" group consists of 2 of 3 strains of Aspergillus flavus, the "dotted" group consists of strains of Aspergillus oryzae, and the "dashes" group includes the strains of Aspergillus niger and A. tubigensis, two closely related species, as well as the two strains of Bacillus sp. In this example, the strains of Aspergillus flavus 029 and 046 tested are the two most efficient strains to meet the nitrogen requirements of the lactic strain used in saccharification-simultaneous fermentation.

Example 6: Specific Benefits of Certain Nutritional Supplements This example shows that the use of certain mold strains or bacteria for the production of the nutritional supplement may be particularly advantageous for the production of lactic acid by certain strains of lactic acid bacteria. The inventors have studied in a comparative way the quantity of lactic acid produced during the saccharification-simultaneous fermentation of wheat wheat by the L. coryniformis DSM 20004 or L. coryniformis DSM 20005 strains, by introducing either a protease solution of variable concentration, the nutritional supplement according to the invention by varying the strain used for fermentation in solid medium. In view of the amino acid profiles released and the soluble nitrogen concentrations at the end of fermentation, the inventors evaluated the specific advantages which the nutritional supplement could confer for different strains of lactic acid bacteria, in response to their nutritional needs, by comparing to commercial proteases. Materials and Methods In these tests, the inventors first carried out liquefaction of abraded wheat flours for 2 hours at 85 ° C. in a stirred reactor with a final solids content of 20%. This liquefied beetle was divided into reactors, each containing 3000 ml of must, supplemented with Lyvanol GA 480 (amyloglucosidase at 0.890 kg / t dry matter), and placed under control of the pH by the addition of 5 N sulfuric acid and of sodium hydroxide 7 N for a set point of keeping the pH at 6 all along the fermentation. Thus, the fermentation mulls consisted of liquefied, saccharified abraded wheat, supplemented with the nutrient solution B with the addition of either one of the nutritional supplements according to the invention listed in Tables 7 and 8, or one of the commercial protease solutions listed in Table 8. Table 7: Fermentation conditions for test 1 R1 Reactor R2 R3 R4 R5 R6 R7 R8 Nutritional supplement Strain Concentration (kg / t wheat) A. flavus 029 20.0 B. subtilis CL260 20.0 A. flavus 046 20.0 A. orizae 044 20.0 A. flavus 029 20.0 B. subtilis CL260 20.0 A. flavus 046 20.0 A. orizae 044 20.0 Type CN CN CN CN CN CN CN CN Lactobacillus strain DSM 20004 DSM 20004 DSM 20004 DSM 20004 DSM 20005 DSM 20005 DSM 20005 DSM 20005 3033332 36 CN: Nutritional supplement obtained by the method described in Example 1 using the mold or bacterium strain mentioned in Table 5 Table 8: Fermentation conditions for test 2 ReactorNutritional supplement / enzyme Lactic acid bacterium Type Strain Concentration Strain (kg / t wheat) R1 Enz Fermgen Trichoderma sp 0.10 DSM 20004 R1 Enz Prolyve BS conc.2 B. subtilis 0.12 DSM 20004 R2 Enz Prolyve BS conc.2 B. subtilis 0.20 DSM 20004 R3 Enz Prolyve BS conc.2 B. subtilis 0.40 DSM 20004 R4 Enz Prolyve BS conc.2 B. subtilis 0.60 DSM 20004 R5 Enz Prolyve BS conc.2 B. subtilis 0.80 DSM 20004 R6 Enz Prolyve BS conc.2 B. subtilis 1.00 DSM 20004 R7 CN A. flavus 029 5.00 DSM 20004 protease marketed by Genencor International; 2 protease marketed by Lyven CN: nutritional supplement obtained by the method described in Example 1 using the mold or bacterium strain mentioned in Table 10 The NCs could be tested in excess (20 kg / t of wheat) in Test 1 for better determination of amino acid profiles. Different concentrations of commercial proteases could be tested in Trial 2 for a better evaluation of their potential and comparison with the nutritional supplement.

After 1h30 of pre-saccharification at 60 ° C., the sheep have a glucose potential of 150 g / l. The latter were inoculated with 109 CFU of lactic bacteria per ml of sheep. The bacterial strains used in trials 1 and 2 are Lactobacillus coryniformis ssp. Torquens DSM 20004 and DSM 20005. The fermentation was conducted for 50 h at 30 ° C with stirring (300 rp-n) and maintained at pH 6 by adding 7 N sodium hydroxide. Results Test 1 Figure 8 shows that the 4 nutritional supplements according to the invention can respond optimally and analogously to the needs of the two lactic acid strains in fermentation (when the latter have the same yields, as for example for the nutritional supplements obtained with strains 029 or 046), but also to respond particularly more favorably to the needs of one or other lactic acid strain. For example, the nutritional supplement obtained with strain CL260 allows a higher lactic yield with the strain of lactic acid bacterium DSM 20005; the nutritional supplement obtained with the strain 044 allows a higher lactic yield with the strain of lactic bacteria DSM 20004. Figure 9 shows moreover that according to the type of strain used for the manufacture of the nutritional supplement, the profile of free amino acids (AAL) released by this last 5 in the fermentation may be different and therefore specifically meet particularly advantageous the needs of lactic acid strains used in the lactic acid production process. Thus, the LAA profile for A. piger type reflects a very diverse release of ALA, mainly glutamine (13% of LAA released), but also Alanine, Glycine, Leucine, Valine, etc., in proportions between 2 and 5% of the LAA. In view of the results presented in Figure 8, this type of profile seems to be interestingly suited to the two lactic acid strains tested since the two nutritional supplements 029 and 046 (strains of A. flavus, a species similar to A. piger) have similar performance on lactic acid production in saccharification-simultaneous fermentation. The LAA profile for the A. oryzae type also reflects a large diversity of LAA released in the fermentation broth, most of which are glutamine (more than 20% of LAA), Tyrosine (6%), Proline, Leucine, etc. . In fermentation, this profile found with the nutritional supplement obtained with the strain 044 seems to be particularly advantageous for the DSM 20004 lactic strain. Finally, the LAA profile for the Bacillus subtilis type reflects a less diversified release of ALA. , mainly Tyrosine (+ 20% of ALA), Aspartic Acid, Methionine, Glutamine and Asparagine in proportions less than 10% of LAA released. In fermentation, this profile, found with the nutritional supplement obtained with strain CL260, seems to be particularly advantageous for the DSM 20005 lactic strain. The diversity of LAA profiles released in fermentation substrates by the different types of nutritional supplements is an advantage for fermentation because it makes it possible to provide the different lactic acid strains, potentially used for the production of lactic acid, of the assimilable nitrogen source corresponding more specifically to their nutritional needs. Furthermore, considering both the final concentrations of lactic acid and FAN in the fermentation broth (FIG. 10), the inventors have been able to show that, according to the lactic strain used for the production of lactic acid by saccharification. In simultaneous fermentation, the nutritional supplements obtained with certain strains of mold or bacteria proved to be particularly advantageous, both from the point of view of the capacity of the nutritional supplement to release FAN in the medium and to favor the production of fungicides. 'Lactic acid. For example with the nutritional supplement obtained with the strain 029, for the same concentration of lactic acid obtained with the lactic strain DSM 20004 and DSM 20005, there is less FAN released in the end-fermented lactic stock with the lactic acid strain. DSM 20004. Therefore, the specific use of the nutritional supplement obtained with the strain 029 in the production of lactic acid by the lactic strain DSM 20004 has an additional advantage. Since the presence of FAN at the end of fermentation can cause problems on the downstream stages of the lactic acid production process, namely the extraction and purification of lactic acid, a nutritional supplement which makes it possible to limit the the presence of FAN at the end of fermentation therefore has an additional advantage, for the same effect on the production of lactic acid. In the example with the strain L. coryniformis DSM 20004, the nutritional supplement produced with the strain 044 therefore has an additional advantage over the nutritional supplement produced with the strain 029. Likewise with the strain L. coryniformis DSM 20005, the Nutritional supplement produced with strain CL260 has an additional advantage over the nutritional supplement produced with strain 044 to FAN equivalent.

Test 2 As shown in Figure 11, the performance of the nutritional supplement added to 5 kg / t of renewable raw material on simultaneous saccharification-fermentation lactic acid production was superior to that of the two commercial proteases tested herein. It is possible to note the saturating effect of Prolyve BS conc which, added to more than 600 g / t of renewable raw material, no longer makes it possible to improve the lactic yield. Figure 12 shows the profile of LAA released in the fermentation broth under the action of the nutritional supplement produced with strain 029 or under the action of each of the two commercial proteases Prolyve BS conc. (marketed by Lyven) and Fermgen (marketed by Genencor International). The inventors have observed that the diversity of ALAs released in the presence of the nutritional supplement according to the invention is greater than with commercial proteases. For the nutritional supplement, there is the release of 19 different amino acids, including Glutamine in greater proportion, Proline and Leucine around the 5% of LAA released. Under the action of the Fermgen protease, the profile of LAA released in the medium is restricted to 8 amino acids including Glutamine and Aspartic Acid. For the protease Prolyve BS conc., The profile of the LAA released is rather diversified with 16 different amino acids but in proportions other than those found with the nutritional supplement. With the protease Prolyve BS conc., Tyrosine and Methionine are mainly found.

The advantage of adding a nutritional supplement according to the invention with respect to commercial proteases in the lactic acid production process can thus be related to the diversity of LAAs (quality and relative quantity) potentially released. in the fermentation substrate, which makes it possible to provide an assimilable nitrogen source that more specifically meets the nutritional requirements of the lactic acid strains used in fermentation. Example 7 Effect of Nutritional Supplement on Bacterial Growth This example demonstrates that the effect of nutritional supplement on increased production or lactic acid production yield is unrelated to an increase in the bacterial population. in the fermentation broth. The inventors have compared in a comparative manner the amount of lactic acid produced during the simultaneous saccharification-fermentation of wheat meal by the L. coryniformis DSM 20004 or L. coryniformis DSM 20005 strains, by introducing the nutritional supplement according to US Pat. invention by varying the strain used for the production of the nutritional supplement, supplemented with one of the two nutritive solutions A or B defined above. With regard to concentrations and yields of lactic acid and concentrations of lactic acid bacteria, they evaluated the specificity of the effects of nutritional supplements. Materials and Methods In this test, the inventors first carried out liquefaction of abraded wheat bran for 2 hours at 85 ° C. in a stirred reactant having a final dry matter content of 20%. This liquefied beetle was divided into reactors, each containing 3000 ml of must, supplemented with Lyvanol GA 480® (amyloglucosidase at 0.890 kg / t DM), and put under control of the pH by the addition of 5N sulfuric acid and sodium hydroxide. 7N for an instruction to maintain the pH at 6 throughout the fermentation. The fermentation conditions are respectively presented in Table 9, the nutritional supplements having been prepared as in Example 5.

Table 9: Fermentation Conditions of Example 7 Reactor Nutritional Supplement Solution Lactic Nutrient Bacteria Type Strain Concentration Strain (kg / t wheat) R1 CN Flavors 029 5.0 OJ OJ OJ OJ OJ DSM 20004 R2 CN A flavus 029 5.0 DSM 20004 R3 CN A. flavus 0248 5.0 DSM 20004 R4 CN A. flavus 029 5.0 DSM 20005 R5 CN Flavors 029 5.0 DSM 20005 R6 CN Flavors 0248 5.0 DSM 20005 R7 CN B. subtilis CL260 5.0 DSM 20005 After 1.5 hours of pre-saccharification at 60 ° C, the soft has a glucose potential of 150 g / l. The latter are inoculated with 109 CFU of lactic bacteria per 5 ml of sheep. The bacterial strains used in this test are Lactobacillus coryniformis ssp. torquens DSM 20004 and DSM 20005. The fermentation was conducted for 50 h at 30 ° C with stirring (300 rpm) and pH mahtien to 6 by adding 7N sodium hydroxide. The bacterial population was determined during fermentation by performing counting on Thoma cell.

Results The bacterial growth and lactic acid production dynamics are illustrated in FIG. 13 by results obtained with the DSM 20005 strain, respectively in dotted lines and in solid lines. The evolution of the bacterial population, initially inoculated at 109 CFU / ml, during saccharification-simultaneous fermentation (SSF) is similar regardless of the nutritional supplements used, in combination or not with a nitrogen source. exogenous (here provided by solution B in combination with CN 029 TC). After 24 hours of SSF, the bacterial population stabilizes around 101 20 bacteria / ml of sheep. At the end of the 50 hours of fermentation, the concentrations of bacteria in the mouts are very close for all the conditions tested. If the conditions for adding nutritional supplement and exogenous nutrient solutions do not impact the bacterial population dynamics, on the other hand, they impact the lactic acid bacteria's ability to produce lactic acid.

As shown in FIG. 13, the production of lactic acid by the lactic bacterium DSM 20005 is favored by the use of the nutritional supplement obtained with the strain CL260 or by the nutritional supplement obtained with the strain 029 associated with the solution. B.

The same is true with the lactic acid bacterium DSM 20004: the production of lactic acid is greater by the use of the nutritional supplement obtained with the strain 029 compared to the nutritional supplement obtained with the strain 0248, without this gain is related to an increase in the lactic acid bacteria population (Table 10). Table 10: Impact of the type of nutritional supplement used in combination of nutrient solutions on the lactic bacterium L. coryniformis DSM 20004 at the end of the 50 h of fermentation of a liquefied and saccharified wheat mill at 20% MS. lactic acid CN solution population (b / ml) lactic acid product (g / l) nutritive DSM 20004 0248 TC B 1.79x1010 52.49 DSM 20004 029 TC B 1.79x1010 98.52 DSM 20004 029 TC A 2.15x1010 79 Thus, the advantageous effect of adding a nutritional supplement according to the invention to the lactic acid production process can not be linked to a simple increase in lactic biomass but to an improvement in the capacities production of lactic acid by lactic acid strains used in fermentation. 10 15

Claims (8)

REVENDICATIONS1. Use of a nutritional supplement in raw or extracted form, comprising an active ingredient and resulting from a solid-state fermentation of a substrate selected from the group consisting of wheat bran, rapeseed meal, soybean meal, beet pulp, barley rootrush, ethanol soluble wheat grain, and mixtures thereof, with a mold of Aspergillus flavus or Aspergillus oryzae or with a bacterium of the species Bacillus subtilis, for the manufacture, by fermentation, of lactic acid. 2. Use of a nutritional supplement in raw or extracted form, comprising an active principle and resulting from a solid-state fermentation of a substrate selected from the group consisting of wheat bran, rapeseed meal, soybean meal , beet pulp, barley rootrush, wheat ethanol soluble grain, and mixtures thereof, with a mold of the species Aspergillus flavus or Aspergillus oryzae or with a bacterium of Bacillus subtilis species, for releasing, in a lactic acid fermentation medium of a renewable carbohydrate raw material, the nitrogen of said raw material, during the manufacture, by fermentation, of lactic acid from said raw material . 3. Process for producing lactic acid from a renewable carbohydrate raw material, comprising a step of fermenting the renewable carbohydrate raw material with a lactic acid bacterium in the presence of a nutritional supplement in raw or extracted form, comprising a principle active, and resulting from a solid-state fermentation of a substrate selected from the group consisting of wheat bran, rapeseed cake, soybean meal, beet pulp, barley rootlet, soluble ethanol sorbet wheat, and mixtures thereof, with a mold of the species Aspergillus flavus or Aspergillus oryzae or with a bacterium of the species Bacillus subtilis. 4. The process according to claim 3, wherein the fermentation step is a saccharification-simultaneous fermentation step. The method of claim 3 or 4, wherein the lactic acid bacterium is selected from the group consisting of L. mindensis, L. zeae and L. coryniformis. 6. Method according to any one of claims 3 to 5, wherein the fermentation step is carried out in the presence of a source of organic nitrogen from the renewable raw material carbohydrate and, optionally, nutrient solutions. exogenous. 7. Composition comprising (i) a source of organic nitrogen selected from group 5 consisting of renewable carbohydrate raw materials, exogenous nutrient solutions, and mixtures thereof and (ii) a nutritional supplement in raw form or extracted form, comprising an active ingredient, and resulting from a solid-state fermentation of a substrate selected from the group consisting of wheat bran, rapeseed meal, soybean meal, beet pulp, barley root wheat ethanol soluble feed, and mixtures thereof, with a mold of the species Aspergillus flavus or Aspergillus oryzae or with a bacterium of the species Bacillus subtilis. 8. A kit comprising: (i) a source of organic nitrogen selected from the group consisting of renewable carbohydrate raw materials, exogenous nutrient solutions, and mixtures thereof, and (ii) a raw form or non-starter nutritional supplement; extracted, comprising an active principle, and resulting from a solid-state fermentation of a substrate selected from the group consisting of wheat bran, rapeseed meal, soybean meal, beet pulp, root rosemary; barley, ethanol soluble wheat sorbet, and mixtures thereof, with a mold of the species Aspergillus flavus or Aspergillus oryzae or with a bacterium of the species Bacillus subtilis.